Aquatic Animal Nutrition, A Mechanistic Perspective From Individuals To Generations (VetBooks - Ir)

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ir
Christian E. W. Steinberg
Aquatic
Animal
Nutrition
A Mechanistic Perspective from
Individuals to Generations
Aquatic Animal Nutrition
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Aquatic Animal Nutrition

A Mechanistic Perspective from Individuals
to Generations
Department of Biology
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Humboldt University at Berlin

Berlin, Germany
ISBN 978-3-319-91766-5 ISBN 978-3-319-91767-2 (eBook)

https://doi.org/10.1007/978-3-319-91767-2
Library of Congress Control Number: 2018953153
© Springer Nature Switzerland AG 2018

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of
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Preface
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Never attend an expedition to Vietnam that is devoted to the study of freshwater and
marine fishes! However, if you do, you run the risk of making good new friends in
the commercial and ornamental fish business, sharing some (too many!) Tiger beers
and promising to spread new information about the nutrition of fish and other
aquatic animals. I took that risk, went to Vietnam, made new friends, and fell in love
with South (East) Asia. Several years have passed since then, and experts, as well as
laypersons, have had to tolerate and survive several seminars on aquatic animal
nutrition, given by me. Now, it is time to keep my promise.
A freshwater ecologist by education and a stress ecologist by preference, my
primary interest has not been to write a book that discusses higher productivity in
the aquaculture industries or reviews recipes for more effective functional aquafeeds
to increase survival, reproduction or productivity of farmed animals. Instead, I am
more interested in answering the question of how certain dietary ingredients influ-
ence the life history traits not only of the consumers but also of their succeeding
generations. In evolutionary-ecological terms: How do dietary components impact
the Darwinian fitness of populations and thereby influence their long-term persis-
tence in the ecosystem? The ecologist in me always noticed gaps in the more prag-
matic experimental approaches of raising aquatic animals referenced in this book.
To identify the gaps, I had to sometimes crawl ashore, since I felt obliged to borrow
information about new developments from terrestrial or laboratory model animal
studies. Nevertheless, I hope that my raised forefinger will encourage the develop-
ment of new experimental setups for the aquaculture community.
The content for the originally planned one-volume book on “Aquatic Animal
Nutrition,” however, turned out to be so voluminous that I split it into two volumes.
I thank Springer Publishing Company for this courtesy. My sincere appreciation
goes particularly to “my Springer ladies” in Dordrecht, namely Alexandrine
Cheronet and Judith Terpos, who were always very supportive and never hesitated
to answer my questions, even if they were simple.
Furthermore, I am thankful to all the photographers and artists who allowed me
to use their wonderful images free of charge. Doubtless, they contributed to an
attractive appearance of this book. We all agree that good illustrations can often
v
vi Preface
explain complex ideas much better than thousands of words. Nevertheless, also
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good words count, and I thank Sarah L. Poynton for the excellent word crafting that
resulted in the title.
Even to a book, space limitation applies. Due to this circumstance, I would like
to apologize in advance to all individuals whose research was not cited or whose
papers have not been discussed in full but whose work has certainly advanced the
understanding of this complex field of research, practice, and education.
This book is dedicated to my bright grandkids, Anna S. and Paul N., who like
watching colorful and intriguing fishes in a living room tank. Since they started this
business at a much younger age than I did, I am certain that one, or both of them,
will be inclined to write brilliant books on this fascinating subject.
Berlin, Germany Christian E. W. Steinberg

Contents
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1 Introduction – ‘You Are What You Eat’................................................... 1

Appendix.................................................................................................... 6
Technical Note....................................................................................... 6
References.................................................................................................. 7
2 Diets and Digestive Tracts – ‘Your Food Determines
Your Intestine’........................................................................................... 9
2.1 Digestive Tract.................................................................................. 9
2.2 Digestion........................................................................................... 14
2.2.1 Protein Digestion.................................................................. 15
2.2.2 Lipid Digestion..................................................................... 16
2.2.3 Carbohydrate Digestion........................................................ 16
2.3 Ontogenesis and the Intestine........................................................... 17
2.3.1 Fishes.................................................................................... 17
2.3.2 Invertebrates.......................................................................... 26
2.4 Herbivory, a Disadvantageous Acquization Strategy?...................... 34
2.4.1 Fishes.................................................................................... 37
2.4.2 Invertebrates.......................................................................... 40
2.5 Starvation and Gut Morphology....................................................... 42
2.6 Trophic Positions: An Omnivores’ Dilemma?.................................. 45
2.7 Concluding Remarks......................................................................... 53
References.................................................................................................. 54
3 The Intestinal Microbiota – ‘Your Eating Feeds a Plethora
of Guests’ and ‘This Plethora of Guests Determines
Who You Are and How Well You Do’........................................................ 61
3.1 Invertebrates...................................................................................... 67
3.1.1 Hydrozoa............................................................................... 67
3.1.2 Mollusks................................................................................ 68
3.1.3 Echinoderms......................................................................... 72
3.1.4 Crustaceans........................................................................... 73
vii
viii Contents
3.2 Fishes................................................................................................ 80

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3.2.1 Microbiome Ontogenesis...................................................... 81

3.2.2 Does a Core Microbiome Exist?........................................... 85
3.2.3 Zebrafish as Witness of Microbiome Development.............. 111
3.2.4 Control Functions by Gut Microbiota................................... 115
References.................................................................................................. 128
4 Dietary Restriction, Starvation, Compensatory
Growth – ‘Short-Term Fasting Does Not Kill You:
It Can Make You Stronger’....................................................................... 137
4.1 Indicators of Starvation..................................................................... 143
4.2 Starvation Tolerance and Starvation Impact..................................... 148
4.2.1 Cnidarians............................................................................. 162
4.2.2 Rotifers.................................................................................. 163
4.2.3 Mollusks................................................................................ 166
4.2.4 Echinoderms......................................................................... 168
4.2.5 Crustaceans........................................................................... 170
4.2.6 Fishes.................................................................................... 192
4.2.7 Summary of Starvation Effects............................................. 198
4.2.8 Starvation: Point-of-no-Return............................................. 199
4.3 Compensatory Growth...................................................................... 201
4.3.1 Invertebrates.......................................................................... 206
4.3.2 Fishes.................................................................................... 214
4.4 Compensatory Growth in Populations.............................................. 228
4.5 Regulation of Compensatory Growth............................................... 230
4.5.1 Appetite-Regulating Hormones............................................ 233
4.5.2 Neuropeptides....................................................................... 236
4.5.3 Transcription of Growth Regulators..................................... 243
4.6 Concluding remarks.......................................................................... 252
References.................................................................................................. 255
5 Chrononutrition – ‘The Clock Makes Good Food’................................ 289
5.1 How Does a Biological Clock Work?............................................... 293
5.1.1 Fishes.................................................................................... 294
5.1.2 Invertebrates.......................................................................... 302
5.2 Food and Circadian Gene Transcription........................................... 308
5.2.1 Major Nutrients..................................................................... 308
5.2.2 Xenobiotic or Antinutritional Compounds........................... 315
References.................................................................................................. 325
Contents ix
6 Transgenerational Effects – ‘Your Offspring Will Become

What You Eat’............................................................................................ 333
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6.1 Parental Effects................................................................................. 335

6.1.1 Maternal Effects.................................................................... 344
6.1.2 Paternal Effects..................................................................... 373
6.2 What Is Epigenetics?......................................................................... 378
6.2.1 Time Scales of Epigenetic Inheritance.................................. 380
6.2.2 Epigenetic Mechanisms........................................................ 381
References.................................................................................................. 416
7 Trophic Diversification and Speciation – ‘Your Eating
Fuels Evolution’........................................................................................ 431
7.1 Individual Specialization.................................................................. 437
7.2 Underlying Mechanisms of Speciation............................................. 438
7.3 Trophic Speciation............................................................................ 441
7.3.1 Ancient Lakes....................................................................... 441
7.3.2 Rivers.................................................................................... 451
7.3.3 Coral Reefs............................................................................ 452
7.4 Convergent Evolution....................................................................... 454
7.5 Rapid Speciating Taxa...................................................................... 458
7.5.1 Pumpkinseed Sunfish............................................................ 458
7.5.2 Cyprinodon........................................................................... 459
7.5.3 Terapontidae (Grunters)........................................................ 460
7.5.4 Three-Spined Stickleback..................................................... 460
7.5.5 Arctic Charr.......................................................................... 462
7.6 Time Span of Trophic Speciation..................................................... 465
References.................................................................................................. 467
Abbreviations and Glossary
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AAs amino acids

abcb4 gene encoding multidrug resistance protein 3, a membrane-
bound transporter
abcg2 gene encoding ATP-binding cassette subfamily G member
2, a membrane-bound tranporter
Acrophase time at which the peak of a rhythm occurs
AgRPs agouti-related proteins: neuropeptides produced in the
brain with an appetite-stimulating potential; activated by
the hormone ghrelin, inhibited by the hormone leptin
AHR anti-hydroxyl radical
AhR aryl-hydrocarbon receptor, Ah receptor: a ligand-activated
transcription factor involved in the regulation of biological
responses to planar aromatic (aryl) hydrocarbons, includ-
ing xenobiotic compounds
ahr2 gene encoding the aryl-hydrocarbon receptor 2 found in
zebrafish
AL ad libitum = “at one’s pleasure”
Alternative splicing a regulated process during gene expression that results in a
single gene coding for multiple proteins
ambra1a, ambra1b autophagy-regulating genes
AMPK AMP-activated protein kinase: 5’adenosine
monophosphate-activated protein kinase, an enzyme cen-
tral in cellular energy homeostasis
anadromous fish migrate from the sea up into freshwater to spawn;
examples are salmon and striped bass
anorexigen appetite-suppressing drug or food constituent
apaf-1 encodes apoptotic protease-activating factor 1, a pro-
aptotic protein
arα, arβ encode androgen receptor α and β
ASA anti-superoxide anion
xi
xii Abbreviations and Glossary
Autocrine (signaling) a form of cell signaling in which a cell secretes a signaling

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chemical that binds to receptors on that same cell

Autophagy natural, regulated, destructive mechanism of the cell that
disassembles unnecessary or dysfunctional components
BAX Bcl-2-associated X protein: functions as part of an apop-
totic activator
BBS bombesin-like peptides: a large family of peptides initially
isolated from amphibian skin; peptides with neuroendo-
crine and neuromodulator function in fish
Bcl2 B-cell leukemia/lymphoma-2: protein regulating cell death
(apoptosis), by either inducing (pro-apoptotic) or inhibit-
ing (anti-apoptotic) apoptosis
bd β-defensins: antimicrobial peptides implicated in the resis-
tance of epithelial surfaces to microbial colonization
beclin autophagy-regulating gene
Bioaccumulation the process which causes chemical concentrations in the
tissues of an aquatic organism to exceed those in the water,
due to uptake by all exposure routes
bmal(1,2..) encode transcription factor(s) involved in the circadian
rhythm (clock gene); together with clock the positive loop
of the molecular clock
bmp15 encodes bone morphogenetic factor 15, mainly involved in
folliculogenesis
C3 complement component 3, a protein of the immune sys-
tem, central in the activation of the complement system; a
connecting link between innate and acquired immunity
CART cocaine and amphetamine regulated transcript: a neuro-
peptide that produces similar behavior in animals to
cocaine and amphetamine having roles in reward, feeding,
and stress
caspase-3…9 members of the cysteine-aspartic acid protease (caspase)
family: sequential activation of caspases plays a central
role in the execution-phase of cell apoptosis
CAT catalase: enzyme that catalyzes the dismutation of hydro-
gen peroxide (H2O2) to water (H2O) and oxygen (O2)
Catch-up growth attainment of control size
CCK cholecystokinin: peptide hormone of the gastrointestinal
system stimulating the digestion of fat and protein
cenpf1...3 encode centromere proteins F, involved in physical cell
division
c-fos FBJ murine osteosarcoma viral oncogene homolog, a
proto-oncogene involved in signal transduction, cell prolif-
eration and differentiation
CFU colony-forming units
CG compensatory growth: faster than usual growth rate
Abbreviations and Glossary xiii
Chaperon protein stabilizing new proteins to ensure correct folding

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or helping to refold proteins that were damaged by cell

stress
chordin early developmental gene ruling the dorsalizing process
CK creatine kinase: enzyme that catalyzes the conversion of
creatine and utilizes adenosine triphosphate (ATP) to cre-
ate phosphocreatine (PCr) and adenosine diphosphate
(ADP)
clk, clock encodes transcription factor(s) involved in the circadian
rhythm (clock gene); together with bmal(1,2..) the positive
loop of the molecular clock
Complement factor B an acute-phase protein increasing during inflammation
Cosinor analysis analysis of biologic time series that demonstrate pre-
dictible rhythms
cox-2 encodes cyclooxygenase forming prostanoids from PUFAs
CRH corticotropin-releasing hormone
cry encodes cryptochrome, blue light-sensitive flavoproteins
involved in the circadian rhythm; with per2 and tim the
negative loop of the molecular clock
CSF-1R colony-stimulating factor 1 receptor (also macrophage
colony-stimulating factor receptor): receptor for a cytokine
called colony-stimulating factor 1, which controls the pro-
duction, differentiation, and function of macrophages
CTR calcitriol, increasing the uptake of calcium from the gut
into the blood
Curcumin complex polyphenol in the rhizome of turmeric, Curcuma
longa
CuZn-SOD copper/zinc superoxide dismutase; enzyme that catalyzes
the dismutation of superoxide radical (•O2−) to water (H2O)
and oxygen (O2)
cyc encodes cycle protein; genetic transcription-translation
feedback loop that generates circadian rhythms
cyp17-II cytochrome P450 c17II; a monooxygenase affecting
growth, gonad differentiation and development, and other
reproductive traits of fish
cyp1a gene encoding cytochrome P450, family 1, subfamily A, is
involved in phase I xenobiotic and drug metabolism
Cytokine cell signaling protein
DAF-16 ortholog of the FOXO family of transcription factors in the
nematode C. elegans; it is the primary (but not the only)
transcription factor required for lifespan extension
dazl deleted in azoospermia-like: This gene encodes a member
of the depleted in azoospermia-like (DAZL) protein
family
xiv Abbreviations and Glossary
dbt encodes DOUBLETIME protein; a kinase that phosphory-

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lates PER protein that regulates the molecularly driven,

biological clock controlling circadian rhythm
DE digestible energy
DGGE denaturing gradient gel electrophoresis; method for identi-
fying genes from natural ecosystems
Dicer a key initiative protein of the RNA interference (RNAi)
pathway: (= endoribonuclease dicer) an enzyme cleaves
double-stranded RNA (dsRNA) and pre-microRNA (pre-
miRNA) into short double-stranded RNA fragments called
small interfering RNA
DM dry matter
DNMT DNA methyltransferase
dpf days past fertilization
dph days past hatch
DR dietary restriction
dsRNA double-stranded (ds)RNA, central in RNAi
Dysbiosis microbial imbalance or maladaptation
EF-1α eukaryotic translation elongation factor 1 alpha responsi-
ble for the enzymatic delivery of aminoacyl tRNAs to the
ribosome
EFA essential fatty acid; EFA requirements vary qualitatively as
well as quantitatively among different animal species
EGCG epigallocatechin-3-gallate, the most active ingredient in
green tea
ELOVL1 FA elongase elongates saturated and monounsaturated C20-
C26 acyl-CoAs
ELOVL2 FA elongase elongates C20-C22 polyunsaturated acyl-CoAs
ELOVL3 FA elongase elongates saturated and unsaturated C16-C22
acyl-CoAs
ELOVL4 FA elongase for the synthesis of ULCFAs (C ≥ 26)
ELOVL5 FA elongase elongates C18-C20 polyunsaturated acyl-CoAs
ELOVL6 FA elongase elongates C12:0-C16:0
ELOVL7 FA elongase elongates saturated and unsaturated C16-C22
acyl-CoAs
endozoochory dispersal of spores or seeds in the gut of animals
Enterocytes intestinal absorptive cells: simple columnar epithelial cells
found in the small intestine
EPA eicosapentaenoic acid, an omega-3 fatty acid with the
chemical formula C20H30O2
ER estrogen receptor
FA fatty acid
FAA food anticipatory activity
fas encodes fatty acid synthase
Abbreviations and Glossary xv
fMHC fast myosin heavy chain, ATP-dependent motor protein,

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involved in muscle contraction and other motility pro-

cesses in eukaryotes
FoxOs forkhead box proteins, a family of transcription factors
regulating the expression of genes involved in cell growth,
proliferation, differentiation, and longevity
fsh encodes follicle-stimulating hormone
fshr encodes follicle-stimulating hormone receptor
GABA γ-aminobutyric acid, a non-proteinogenic amino acid:
inhibitory neurotransmitter
GAL (Gal) galanin: neuropeptide involved in feeding and growth
Garcinol polyisoprenylated benzophenone derivative isolated from
kokum, Garcinia indica
gdh (also gldh) encodes glutamate dehydrogenase
Geldanamycin antitumor antibiotic inhibiting the function of HSP90
Genistein isoflavonoid phytoestrogen
germ-free animals animals that have no microorganisms living in or on them
GF germ free, gnotobiotic
GH growth hormone that activates AgRP, thus increasing appe-
tite; is opposed by the hormone leptin; furthermore, as
pleotropic hormone GH is involved in growth, stress
response, energy homeostasis, reproduction
GHR growth hormone receptor; a protein that is a transmem-
brane receptor for growth hormone
GHra, GHrb growth hormone receptors a & b
GHRH growth hormone-releasing hormone: a releasing hormone
of growth hormone (GH) that stimulates GH production
and release by binding to the GHRH Receptor (GHRHR)
on cells in the anterior pituitary
Ghrl also ghrelin: growth hormone release inducing: the “hun-
ger hormone” is a peptide hormone regulating appetite and
the distribution and rate of use of energy. Ghrelin acts as
orexigenic hormone
GHS-R growth hormone secretagogue receptor, or ghrelin recep-
tor, is a ghrelin-binding receptor and plays a role in energy
homeostasis and regulation of body weight
GI gastrointestinal (tract)
GIFT genetically improved farmed tilapia by conventional
breeding
GLP glucagon-like peptide, a neuropeptide
GnRHs gonadotropin-releasing hormones; among other functions:
decreases quantity and frequency of food consumption
GO gene ontology
goosecoid early developmental gene ruling the dorsalizing process
GPx glutathione peroxidase
xvi Abbreviations and Glossary
GR glutathione reductase
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GRP gastrin-releasing peptide: a neuropeptide, stimulating the

release of gastrin from the G cells of the stomach
GST glutathione transferase
gstr1 gene encoding glutathione transferase rho1
HCRT hypocretin (orexin) neuropeptide precursor regulating
appetite
Heterochrony developmental change in the timing or rate of events, lead-
ing to changes in size and shape
hpf hours past fertilization
HSFs heat shock factors: transcription factors regulating the
expression of heat shock proteins
hsl hormone-sensitive lipase gene
HSP90 heat shock protein 90 (atomic mass approximately 90 kDa),
a chaperone protein assisting other proteins to fold prop-
erly, stabilizing proteins against stress; it also canalizes
phenotypic variability
HSPs heat shock proteins: family of proteins produced by cells in
response to exposure to stressful conditions. Many HSPs
perform chaperone function
HYP hypothalamus
IGF-1…3 insulin-like growth factor 1…3, hormones similar in
molecular structure to insulin
IgM immunoglobulin M
InAP intestinal alkaline phosphatase
irf7 encodes interferon regulatory factor 7, a transcription fac-
tor, central in the transcriptional activation of virus-
inducible cellular genes, including the type I interferon
genes
Isothiocyanate chemical group –N=C=S; mustard oils contain
isocyanates
IU international units: amount of a drug, hormone, vitamin,
enzyme, etc., that produces a specific effect as defined by
an international body and accepted internationally
KEGG Kyoto Encyclopedia of Genes and Genomes, a database
resource for understanding high-level functions and utili-
ties of the biological system
kiss1, kiss2 encode kisspeptins
Kisspeptins peptides that stimulate gonadotropin release
lc3 autophagy-related gene
LDLR low-density lipoprotein receptor, mediates the endocytosis
of cholesterol-rich LDL and thus maintains the plasma
level of LDL
Abbreviations and Glossary xvii
Lecithotrophy nourishment and development of the embryo only via the

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yolk originally contained within its egg; opposite:

matrotrophy
LEP leptin, a hormone that regulates appetite to achieve energy
homeostasis by inhibiting hunger; it is opposed by the hor-
mone ghrelin
lh gene encoding the luteinizing hormone
lhcgr gene encoding the luteinizing hormone/choriogonadotro-
pin receptor
Lipofuscin finely granular yellow-brown pigment granule indicative
of aging cells
lpl encodes lipoprotein lipase
LPS lipopolysaccharides (also lipoglycans, endotoxins): large
molecules consisting of a lipid and a polysaccharide; found
in Gram-negative bacteria and cyanobacteria and elicit
strong immune responses in animals
Lysozyme c glycoside hydrolase, an enzyme that damages bacterial cell
walls
Matrotrophy form of maternal care during organism development, in
which the embryo is supplied with additional nutrition
from the mother, e.g. through a placenta; opposite:
lecithotrophy
MC4R melanocortin-4 receptor, a protein involved in feeding
behavior, suppressing hunger
mch encodes melanin-concentrating hormone: a cyclic orexi-
genic hypothalamic peptide originally isolated from the
pituitary gland of teleost fish
Melatonin N-acetyl-5-methoxy tryptamine, a hormone that antici-
pates the daily onset of darkness; hormone affecting the
modulation of wake/sleep patterns
mgst3a gene encoding microsomal glutathione transferase 3a (in
zebrafish), central in phase II detoxification
mhc2a (also mhcIIa) encodes mhc class 2A chain (major histocompability com-
plex IIα), molecules found on antigen-presenting cells,
important in initiating immune responses
Microbiota resident microbial communities in fishes and invertebrates
microRNA miRNA, about 22 nucleotides, functioning in RNA silenc-
ing and posttranscriptional regulation of gene expression
Mn-SOD manganese superoxide dismutase; an enzyme that cata-
lyzes the dismutation of superoxide radical (•O2−) to water
(H2O) and oxygen (O2)
MyoD one myogenic regulatory factor
myog encodes myogenin
xviii Abbreviations and Glossary
Myogenin (= myog, myogenic factor 4) transcription factor involved

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in the coordination of skeletal muscle development or

myogenesis and repair
Myostatin a myokine: protein produced and released by myocytes
inhibiting muscle cell growth and differentiation, member
of the TGF-β superfamily
myp encodes major yolk protein
NAD nicotinamide adenine dinucleotide
Neurolipofuscin lipofuscin in the nervous system
NF-κB nuclear transcription factor-κB: protein complex that con-
trols transcription of DNA, cytokine production and cell
survival; found in almost all animal cell types and involved
in cellular responses to stimuli such as stress, cytokines,
free radicals, ultraviolet irradiation, oxidized low-density
lipoprotein, and bacterial or viral antigens
NPY neuropeptide Y: with slight variations, a neurotransmitter
in the brain and in the autonomic nervous system of ani-
mals; one of the strongest orexigenic signals
opn encodes osteopontin, an important mediator of bone (re)
modeling
Orexigen drug, hormone, or compound that stimulates appetite
osr-1/unc-43/sek-1 pathway that promotes resistance to osmotic stress; osr-1
is coupled to SEK-1 (a MAK kinase) through UNC-43
(Ca2+/calmodulin-dependent protein kinase II)
osx encodes osterix, a transcription factor for osteoblast dif-
ferentiation, mediates antitumor activity in murine
osteosarcoma
OTU operational taxonomic units
OX orexin, a neuropeptide that regulates arousal, wakefulness,
and appetite
Oxidative burst refer to respiratory burst
Oxidative stress states where the balance between generation and elimina-
tion of ROS is disturbed in favor of the generation of ROS
p38 encodes P38 mitogen-activated protein kinase; is activated
by a variety of cellular stresses
paqr8 encodes progestin and adipoQ receptor family
Paracrine (signaling) a form of cell-cell communication in which a cell produces
a signal to induce changes in nearby cells; opposite:
autocrine
PBS phosphate-buffered saline (buffer)
PC phosphatidylcholine
pepT1…2 encode oligopeptide transporters (members of the solute
carrier family 15) encode solute carriers localized to the
brush border membrane of the intestinal epithelium and
mediate the uptake of di- and tripeptides
Abbreviations and Glossary xix
per1,2 encoding the period circadian regulators 1,2; with cry1 and
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tim the negative loop of the molecular clock

PG phosphatidylglycerol
PI phosphatidylinositol
Pineal organ pineal body, epiphysis cerebri, epiphysis or the “third eye”:
small endocrine gland producing melatonin
PIT pituitary or hypophysis, an endocrine gland. Its hormones
help control: growth, blood pressure, and certain functions
of the sex organs, thyroid glands, metabolism, and
reproduction
POA preoptic area, part of the hypothalamus; responsible for
thermoregulation and receives nervous stimulation from
thermoreceptors in the skin, mucous membranes, and
hypothalamus itself
POMC proopiomelanocortin, a precursor polypeptide which
cleavage gives rise to several peptide hormones
pparα…γ encode peroxisome proliferator-activated receptors α, β, γ;
nuclear receptor proteins functioning as transcription fac-
tors; central in the regulation of cellular differentiation,
development, and metabolism (carbohydrate, lipid,
protein)
PSM plant secondary metabolites
Procyanidin a condensed tannin
proPO prophenoloxidase: part of the major innate defense system
in invertebrates via melanization of pathogens and dam-
aged tissues
PUFA polyunsaturated fatty acid containing two or more ethyl-
enic bonds, such eicosapentaenoic acid (EPA, 20:5n─3 or
20:5ω─3)
PY peptide Y, a NPY-related peptide
PYY peptide YY is a member of the neuropeptide Y (NPY)
family
QTL quantitative trait locus: section of DNA, the locus that cor-
relates with variation in a phenotype, the quantitative trait
Quercetin a flavonoid polyphenol
Rapamycin also sirolimus; chemical immune suppressant
RAS recirculating aquaculture systems
Respiratory burst rapid production and release of reactive oxygen species
Resveratrol a stilbenoid polyphenol
retinoic acid a metabolite of vitamin A (retinol) that mediates the func-
tions of vitamin A required for growth and development
Ribotype molecular bacterial identification using information from
rRNA-based phylogenetic analyses
xx Abbreviations and Glossary
RNAi RNA interference or RNA silencing: biological process in

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which RNA molecules inhibit gene expression or transla-

tion, by neutralizing targeted mRNA molecules
rnf213 encodes E3 ubiquitin-protein ligase RNF213, involved in
protein ubiquitination
ROS reactive oxygen species: chemical reactive molecules con-
taining oxygen, such as peroxides, superoxide, hydroxyl
radical, and singlet oxygen (also see electrophilic stress
and oxidative stress)
RXR retinoid receptors: nuclear receptors that bind to retinoids;
when bound to a retinoid, they act as transcription factors
SAH S-adenosyl-l-homocysteine: amino acid derivative, inter-
mediate in the synthesis of cysteine and adenosine; SAH is
formed by the demethylation of S-adenosyl-l-methionine
(SAM)
SAM S-adenosyl-l-methionine: involved in methyl group
transfers
SCN suprachiasmatic nucleus of the hypothalamus in mammals,
a neuronal structure defining the circadian rhythms
SGR specific growth rate
SIRT1 sirtuin (silent mating type information regulation 2 homo-
log) 1, an enzyme deacetylating proteins and thereby regu-
lating reaction to stress and longevity
SMADs structurally similar proteins that are the main signal trans-
ducers for receptors of the transforming growth factor-beta
(TGF-β) superfamily
SOD superoxide dismutase: enzyme that catalyzes the dismuta-
tion of superoxide (•O2−) radicals into ordinary molecular
oxygen (O2) and hydrogen peroxide (H2O2)
SOD1 Cu-Zn superoxide dismutase
SOD2 Mn superoxide dismutase = superoxide dismutase 2
Somatomedins group of hormones that promote cell growth and division
in response to stimulation by growth hormone (GH)
Sulforaphane organosulfur compound (isothiocyanate) in cruciferous
vegetables
sult2_st2 gene encoding the cytosolic sulfotransferase 2 (in
zebrafish)
T3 thyroid hormone; more active than T4 by a factor of 3 to 5
TAG triglyceride or triacylglycerol: ester derived from glycerol
and three fatty acids
TGF-β transforming growth factor β: multifunctional cytokine.
The TGF-β superfamily includes endogenous growth-
inhibiting proteins, for instance, with anti-inflammatory
function
Abbreviations and Glossary xxi
tim a, b, h encode timeless proteins; essential in regulating the circa-

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dian rhythm; part of a transcription-translation negative

feedback loop involving the period (per) and cry genes and
their proteins
TLRs Toll-like receptors: class of proteins central in the innate
immune system that recognize structurally conserved mol-
ecules derived from microbes
toll encodes members of the Toll-like receptor class of
proteins
TOR target of rapamycin: highly conserved, nutrient-sensitive
protein kinase, a central controller of protein synthesis,
cell growth, cell proliferation, cell motility, cell survival,
autophagy, transcription, and aging
TRH thyrotropin-releasing hormone: a releasing hormone, pro-
duced by the hypothalamus that stimulates the release of
thyrotropin
Trp tryptophan: 1 of the 22 standard amino acids and an essen-
tial in diets; distinguishing structural feature is the indole
functional group
TSH thyroid-stimulating hormone: a pituitary hormone that
stimulates the thyroid gland to produce thyroxine (T4)
usp5 encodes ubiquitin carboxyl-terminal hydrolase 5, a deu-
biquitinating enzyme
Veliger planktonic larva of many sea snails and freshwater snails,
as well as most bivalve mollusks
ZT zeitgeber time, time from any external or environmental
cue on that entrains or synchronizes an organism’s biologi-
cal rhythms to the Earth’s 24-hour light/dark cycle and
12-month cycle. The time of the cue is ZT 00:00
β-diversity ratio between regional and local species diversity
β-endorphin endogenous opioid neuropeptide and peptide hormone that
is produced in certain neurons within the central nervous
system and peripheral nervous system; even protists, such
as Tetrahymena, produce this hormone
β-oxidation catabolic process by which fatty acid molecules are broken
down in the mitochondria in eukaryotes to generate acetyl-
CoA, which enters the citric acid cycle, and NADH and
FADH2, which are coenzymes used in the electron trans-
port chain
Chapter 1
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Introduction – ‘You Are What You Eat’
Abstract The trivial word ‘You are what you eat’ (YAWYE) actually applies also
to fish and aquatic invertebrates; however, not literally, but in a more hidden, subtle
manner. This introductory chapter will briefly address the content of Volume I of
Aquatic Animal Nutrition: Chapter 2 recalls basic textbook knowledge and dis-
cusses dietary impacts on morphology and functioning of the intestine. Chapter 3
focuses on the central significance of the intestinal microbiota, the forgotten ecosys-
tem. Central in Chap. 4 is the message that dietary restriction and starvation are
natural occurrences and do not necessarily kill the individuals which often respond
with compensatory growth and improved Darwinian fitness – even of their off-
spring. Chapter 5 discusses the circadian rhythmicity of digestive and biotransfor-
mation gene transcription and questions the paradigm of ‘antinutritional factors’.
Chapter 6 addresses transgenerational dietary effects including starvation resis-
tance; and the last Chapter shows that diets can be the basis for sympatric speciation
whereby not only genetical, but also epigenetical mechanisms likely apply.
‘You are what you eat!’

This trivial, hackneyed word is certainly a consensus promoting empty phrase.
However, does its frequent usage increase the plausibility of it’s meaning? Is this
phrase really true, or is it just the wishful thinking of some zealous nutritionists who
want the majority of the human population to live healthier or take up diets such as
vegetarianism or veganism? Is it more than a consensus creating, but empty phrase?
‘Are you really what you eat?’
Let’s therefore briefly switch to a bizarre aquatic creature: the black swallower
(Chiasmodon niger), which has attracted the attention of many marine biologists.
This common species of fish has a worldwide distribution in tropical and subtropi-
cal waters, at depths of 700–2745 m (Coad and Reist 2004). The black swallower
feeds on bony fishes, which it swallows whole. Incredibly, its highly distensible
© Springer Nature Switzerland AG 2018 1

C. E. W. Steinberg, Aquatic Animal Nutrition,
https://doi.org/10.1007/978-3-319-91767-2_1
2 1 Introduction – ‘You Are What You Eat’
stomach allows it to swallow prey over twice its length and 10 times its mass
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(Fig. 1.1) (Jordan 1905).

Provided that “you are what you eat” (YAWYE) is applied literally, one could
hypothesize that the black swallower, following the consumption of prey larger than
itself, should grow and grow and eventually reach the size and appearance of its
prey. Does this actually happen? If so, the black swallower could be a real-world
equivalent to the abstract conceptualization of a frog metamorphosing into a fly – a
literal interpretation of YAWYE, as cleverly depicted by American artist, Sarah
DeRemer (Fig. 1.2).
Of course, Sarah’s peculiar creature is pure fiction, and animals generally do not
metamorphose into their dietary prey. If YAWYE is really valid as opposed to being
a handy phrase for nutritionists, it must take place at a more subtle, cryptic level.
To identify the impacts of dietary sins, different diets, or healthy eating, we have
to leave the simple phenotypic level and must become acquainted with the micro-
biological, biochemical, and biomolecular levels of the consumer – levels which, as
Fig. 1.1 A black swallower containing a fish much larger than itself. (From Günther 1880, cour-
tesy of the Biodiversity Heritage Library)
Fig. 1.2 A fly-eating frog

metamorphoses into a fly,
as depicted by the artist
Sarah DeRemer (©Sarah
DeRemer)
1 Introduction – ‘You Are What You Eat’ 3
we shall see, are not only affected by present diet, but the diets of past generations,
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even spanning through evolutionary time.

We shall see that YAWYE is certainly applicable at these levels, with complex
effects that have far reaching impacts, surpassing the individual consumer and span-
ning across generations. In this context, we can refine this trivial phrase, and instead
claim:
• ‘Your food determines your intestine’
We must briefly recall textbook knowledge about the tools by which aquatic
animals can make use of their diets. The intestine of herbivores, omnivores, and
carnivores differ significantly. Here we learn that aquatic animals – fish in
particular – are very flexible; if necessary, fish can change the morphology of the
intestine with a change in trophic niche;
• ‘Your eating feeds a plethora of guests’
A diet does not only feed the individual consumer by supplying energy, macro-,
and micronutrients, but also feeds an extremely high number of diverse microorgan-
isms in the intestine. Most of these microorganisms provide additional metabolic
pathways and, thus, utilize dietary intake more efficiently than microbe-free ani-
mals ever could. These microorganism communities can be considered as an addi-
tional and effecacious organ;
• ‘This plethora of guests determines who you are and how well you do’
By several mechanisms, beneficial microorganisms suppress adverse bacteria,
cyanobacteria, and yeasts and in addition, provide signal molecules that strengthen
immunity as well as resistance to pathogens and parasites. More surprisingly, the
composition of the intestinal microbiotia has the potential to even determine gen-
der – at least in certain invertebrates;
• ‘Short-term fasting does not kill you – it can make you stronger’
In natural habitats, food is not always available in sufficient quantities or quali-
ties. Dietary restriction (food shortage) and starvation are regular occurrences for
many animals in a variety of ecosystems. They have had to develop coping mecha-
nisms for these situations – with surprising results. For instance, parental short-term
starvation can increase disease resistance in offspring and can even have beneficial
impacts on population growth;
• ‘The clock makes good food’
From studies of terrestrial invertebrates and mammals, there is accumulating evi-
dence that biotransformation activity on the biomolecular and biochemical levels is
subject to circadian rhythmicity. Depending on the time of day, adverse effects from
identical exposure to natural or synthetic xenobiotic compounds can differ signifi-
cantly. Since this rhythmicity appears to be evolutionarily conserved, it can be
hypothesized that it will also apply to aquatic animals. The confirmation of this
hypothesis will surely challenge the paradigm of the so-called anti-nutritional

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factors in animal feed in general, and for aquafeed in particular;

• ‘Your offspring will become what you eat’
Recent studies, most of which concerning terrestrial vertebrates and inverte-
brates, have discovered transgenerational mechanisms of how diet-mediated prop-
erties of the parental generation are passed not only to the next filial generation, but
also to the grand and great grand generations. These phenomena have been detected
in terrestrial invertebrates as well as in mammals, and appear to be conserved
throughout evolutionary history; there is no reason to assume that corresponding
mechanisms do not apply to fishes and aquatic invertebrates;
• ‘Your eating fuels evolution’
For a long period of time, speciation was thought to only happen between habi-
tats that are geographically isolated. However, the high diversity of fish species
flocks in coral reefs, of cichlid species flocks in Lake Victoria, Lake Tanganyika,
and Lake Malawi, and of sculpin and gammarid species flocks in Lake Baikal ques-
tioned this paradigm and shed light onto food source diversity as a potential evolu-
tionary driver. Food-based bottom-up effects may even lead to sympatric speciation
in fishes and invertebrates (i.e. speciation occurring within the same habitat). This
type of speciation can happen within a few generations. Simply put, this is evolution
in action.
The subject of this book is the nutrition of both wild and farmed finfishes and
aquatic invertebrates, as of course, both farmed aquatic organisms and their wild
counterparts must meet their fundamental needs – replenishing energy and refilling
both inorganic and organic nutrient reserves – through feeding. However, though it
may sound trivial, it must be stated that the scientific approaches and methodologies
of aquaculture and aquatic ecology do differ. Furthermore, theoretical backgrounds
and practical methodologies contrast between the two disciplines in numerous
instances. Throughout the history of science (for more details, refer to Kuhn 2012),
it is well understood that backgrounds and methodologies have a huge impact on the
scientific outcome of an experiment or of a study, even following scientific review.
Therefore, it can be expected that studies either in aquaculture or in the field, even
when concerning identical species, may significantly differ in their results. This is
by no means a drawback. Instead, both aquatic disciplines have a great potential for
scientific cross-fertilization. Aquaculture may learn from aquatic ecology and evo-
lution, whereas ecology may learn from the biochemical and biomolecular path-
ways in individual farmed species.
The background of aquaculture is, according to the Food and Agriculture
Organization (FAO), ‘the farming of aquatic organisms, including fish, mollusks,
crustaceans, and aquatic plants. Farming implies some form of intervention in the
rearing process to enhance production, such as regular stocking, feeding, protection
from predators, etc. Farming also implies individual or corporate ownership of the
stock being cultivated. This definition clearly shows that the focus of aquaculture is
put on individuals of a given species in order to increase production. Relationships
1 Introduction – ‘You Are What You Eat’ 5
to and interactions with the environment are only of interest when identifying and
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reducing potential constraints of production, and keeping it as productive, efficient,

and economical as possible.
In order to avoid endless trial and error, knowledge of the environment, trophic
level, and possible evolutionary relationships of a species is central to understand-
ing the nutritional requirements of that species (this may not be universally appli-
cable however, as not all aquaculture species can be studied in depth in their natural
environment). A striking example of how the ecology of a species can affect its diet,
is the requirement of essential fatty acids in fish species. It is well understood that
the dietary preference for n-3 or n-6 polyunsaturated fatty acids (PUFAs) is deter-
mined by the specific dietary environments. For example, PUFA sources in marine
environments predominantly consist of long-chain n-6 PUFAs. As marine fish
inhabit an environment rich in long chain PUFAs, there is no evolutionary pressure
to retain the ability to endogenously produce long-chain PUFAs by elongation and
desaturation. Conversely, the higher prevalence of shorter PUFAs in freshwaters has
maintained this evolutionary pressure in freshwater fish. Current biomolecular stud-
ies and meta-analyses try to understand the evolution of fatty acid elongation and
desaturation pathways in fishes and invertebrates. This topic will be revisited in
detail in Volume 2.
This PUFA example may be the most notable case of combining dietary require-
ments with the ecological niches of species under consideration. However, there
exists a plethora of examples for so-called trophic bottom-up effects, which demon-
strate that the nutritional basis of lower trophic levels controls the higher ones: for
instance, the diets of herbivores controls the development and well-being of their
predators. A few classical examples may highlight this phenomenon and, further-
more, show that the feedback between ecological niche and species is rather subtle
and even cryptic. Nevertheless, this feedback may serve as a stimulus for aquacul-
turists to study ecological reports more intensively, and vice versa. Crossing the
disciplines of aquaculture and aquatic ecology still has room for improvement, and
could have the potential for new discoveries.
Dietary inorganic bottom-up effects in ecosystems are textbook examples of ter-
restrial and aquatic ecology. Disparities between plant resources and their herbi-
vores in terms of nutrient content not only have major consequences on the success
of the herbivores themselves and their offspring, but also translate to higher trophic
levels and even to ecosystem functioning. For instance, stoichiometric mismatches
between food and consumers affect consumer-driven recycling of limiting nutrients,
which, in turn, functions as a positive or negative feedback on food quality depend-
ing on other sources of nutrient supply. Without doubt, in production-based aqua-
culture, this ecology-based perspective of nutrition is truncated – only one trophic
level is considered.
Food-based bottom-up effects may even lead to sympatric speciation in fishes
and invertebrates within relatively short periods of time. This kind of sympatric
speciation points out the extremely high significance of nutrition to aquatic animals,
though we are only beginning to understand the underlying mechanisms. Doubtless,
genetic variation has often, but not always, been identified as the basis for this kind
of speciation. Alternatively, the action of microRNAs and other epigenetic

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processes are beginning to be taken into consideration. In short-lived animals, such

as protists, nematodes, and fruit flies, it is well documented that epigenetic changes
happen directedly, rather than stochastically, and can be almost as stable as muta-
tions. These animals may serve as guides for future studies with fishes and aquatic
invertebrates, since it can be expected that, by considering diet-driven epigenetic
modifications, at least the broodstocks for aquaculture purposes may be altered and
even improved.
With the still-preliminary total number of marine, brackish, and freshwater fish
species approximated at 29,000 (Lévêque et al. 2008) and the quote ‘A fish is a fish
like a cow is a canary’ from marine biologist Christoffer Schander (Pittman et al.
2013) in mind, I have to confess that ‘Aquatic Animal Nutrition’ can only focus on
a few selected fish and aquatic invertebrate species. Most of those examined are
phylogenetically distant from each other, with inherently wide variations in devel-
opmental strategy. The discrepancy between the number of studied and existing
aquatic invertebrates appears to be even larger than with fishes.
This book does not purely focus on the physiology or ontogenetic development
of fish and aquatic invertebrates, or on increasing productivity in aquaculture.
Numerous textbooks are available that cover these aspects in depth. ‘Aquatic Animal
Nutrition’ comprises two volumes. Volume One presents basic and recent discover-
ies of major feeding mechanisms, as well as long-term effects of diet – from the
individual to the generational level. Volume Two will cover the effects of individual
food ingredients on phenotypic, physiological, and biomolecular levels in the con-
sumers themselves and, where applicable, in their offspring.
Even in two volumes, the following treatise has to be fragmentary and may raise
more issues than fill existing gaps. If, however, the raised issues will initiate new
and innovative studies, this book will have served its purpose.
Appendix
Technical Note
Throughout the books, names and abbreviations of genes are written in lower-case
italics and the abbreviations of the corresponding proteins in capital letters. The
taxonomy of fishes follows ‘fishbase’ and that of invertebrates ‘Encyclopedia of
Life’ and ‘World Register of Marine Species’. If necessary, additional recent revi-
sions were used. Nevertheless, it cannot be guaranteed that no outdated scientific
name has sneaked into this treatise.
References 7
References
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Coad BW, Reist JD (2004) Annotated list of the Arctic marine fishes of Canada. Can Manuscr Rep
Fish Aquat Sci 2674:iv–112
Günther ACLG (1880) An introduction to the study of fishes. Adam & Charles Black, Edinburgh.
https://doi.org/10.5962/bhl.title.54205
Jordan DS (1905) A guide to the study of fishes. H. Holt and Company, New York. https://doi.
org/10.5962/bhl.title.914
Kuhn TS (2012) The structure of scientific revolutions. 50th anniversary, 4th edn. University of
Chicago Press, Chicago
Lévêque C, Oberdorff T, Paugy D, Stiassny MLJ, Tedesco PA (2008) Global diversity of fish (Pisces)
in freshwater. Hydrobiologia 595(1):545–567. https://doi.org/10.1007/s10750-007-9034-0
Pittman K, Yúfera M, Pavlidis M, Geffen AJ, Koven W, Ribeiro L, Zambonino-Infante JL,
Tandler A (2013) Fantastically plastic: fish larvae equipped for a new world. Rev Aquacult
5(SUPPL.1):S224–S267. https://doi.org/10.1111/raq.12034
Chapter 2
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Diets and Digestive Tracts – ‘Your Food

Determines Your Intestine’
Abstract This chapter is an inventory of basic digestive mechanisms and of how

dietary sources determine the forms of the digestive tract of fishes and aquatic inver-
tebrates. Due to high dietary quality and energy provision, carnivores have the
shortest digestive tracts, whereas low dietary quality, low energy, but high fibre
content, determine long intestines of herbivores. However, taken all ecological traits
together, herbivory appears to be a successful, rather than disadvantageous nutri-
tional strategy. The simple relationship of dietary source and intestine length pos-
sesses poor statistical significance and is, therefore, often questioned and apparent
exceptions of this relationship are available. Nevertheless, ontogenetic changes of
intestine length and morphology as well as seasonal changes of the intestine after
dietary alterations support the hypothesis. One major reason for the poor signifi-
cance of this relationship likely are coarse or even incorrect trophic classifications;
consequently, this chapter presents selected examples that species classified as her-
bivores or carnivores are omnivores in reality – the omnivore’s dilemma. Omnivores
have a rather flexible foraging strategy.
2.1 Digestive Tract
To understand the short-term, as well as the long-term and even transgenerational

effects of diets and nutrients, some basic features of the digestive tract have to be
briefly mentioned. This consideration will stay cursory and focus on the dietary
impact on the morphology of the intestine, which can best be observed during onto-
gentic development and metamorphosis, or annual shifts of the trophic niches.
Like other organisms, fishes and aquatic invertebrates require an energy source
to fuel their body systems with fundamental processes, including growth, metabo-
lism and reproduction. Different species have evolved feeding structures and diges-
tive mechanisms that allow them to exploit a vast array of plant and animal food

https://doi.org/10.1007/978-3-319-91767-2_2
10 2 Diets and Digestive Tracts – ‘Your Food Determines Your Intestine’
sources. Consequently, the digestive tract of fishes and aquatic invertebrates has
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incorporated numerous adaptations for the efficient breakdown and absorption of

essential nutrients, including appropriate digestive enzymes and absorptive surface
areas. Since the dietary requirements of larvae are different from those of juveniles
or adults, larval nutrition should always be considered along with the organization
and functionality of the digestive system, nutritional needs and the behavior of lar-
vae at different developmental stages. In addition to being the site of nutrient diges-
tion and absorption, the digestive organs provide a barrier to environmental toxins,
confer essential immune function and have important roles in metabolism and salt
and water absorption (Lazo et al. 2011). For more details about the histological and
physiological ontogeny of the digestive tract in fishes, the audience is referred to
this comprehensive, textbook-like presentation.
As a first approximation, the length of the intestine appears to be a function of
the quality and quantity of the available diet (Davis et al. 2013; Karasov and del Rio
2007; Karasov and Douglas 2013; Kramer and Bryant 1995a, b; Wagner et al.
2009). It changes in concert with histological and physiological modifications, sig-
nificantly during the ontogenetic shift from one dietary source to another; for
instance, from insectivory to frugivory, from herbivory to carnivory, or during star-
vation (German et al. (2010); see also Fig. 2.22).
Benavides et al. (1994) presented one of the first experimental demonstrations in
fishes that an increased capability to digest macroalgae is associated with an increase
in relative gut length. This allows larger fish to meet their energetic demands by
consuming algae, owing to their improved capability to digest low-quality food.
Even carnivorous fishes can increase their gut lengths, but herbivores tend to show
a more rapid increase (Kramer and Bryant 1995a). We shall revisit this issue.
Ontogenetic increases in gut length are well known in many, but not all, marine
and freshwater herbivorous fishes (Kramer and Bryant 1995a; Montgomery 1977;
Drewe et al. 2004; Zihler 1981; Ribble and Smith 1983; Stoner and Livingston
1984; Gallagher et al. 2001). More recently, Wagner et al. (2009) reported that diet
quality predicts the intestine length in Lake Tanganyika’s cichlid fishes independent
of their trophic position (Fig. 2.1). The authors tested the effect of trophic positions
on intestine length across 32 species. Trophic positions were inferred from nitrogen
stable isotopes (δ15N), which provide a temporally integrated, quantitative
perspective on the complex diets. Trophic position explains 51% of size-standard-
ized variation in intestine length. Thus, diet is a strong predictor of intestine length
at both intra- and interspecific scales, indicating that fish adjust their phenotype to
balance nutritional needs against energetic costs.
In a meta-study, Karachle and Stergiou (2010) summarized existing literature
data with respect to gut length and Zihler index1 in relationship to the trophic types
(Fig. 2.2). Despite large standard deviations, which will be discussed below, the
trend became clear: herbivores have the longest intestine and carnivores the
shortest.
1

Zihler index (Zihler 1981) is the relation between gut length and body mass: gut
length×(10 × bodymass1/3)−1
2.1 Digestive Tract 11
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Fig. 2.1 Mean size-corrected intestine length and trophic position in the food web (based on mean
δ15N) for 32 species of Lake Tanganyikan cichlids. Regression lines were derived from generalized
least squares regression where (a) λ = 0 (red dashed line; y = 2.4890 ± 0.1640x; r2 = 0.5902),
equivalent to ordinary least squares regression without phylogenetic correction, (b) λ = 0.7180
(ML estimate; black solid line; y = 2.2572 ± 0.1287x; r2 = 0.5093) and (c) λ = 1 (blue dotted line;
y = 2.0371 ± 0.0907x; r2 = 0.4036). Species are color-coded by trophic guild inferred from gut
contents. (From Wagner et al. 2009, courtesy of Wiley)
H H
Functional trophic group
Functional trophic group
OV OV
OA OA
CD CD
CC CC
0 10 20 0 25 50 75
Mean relative gut length Mean Zihler index
Fig. 2.2 Box-plots of mean relative gut length and Zihler’s index values provided by the original
authors for the different functional trophic groups of marine and freshwater fishes, where H herbi-
vores, OA omnivores with preference to animal material, OV omnivores with preference to vegeta-
ble material, CD carnivores with preference to decapods and fish and CC carnivores with preference
to fish and cephalopods. The central box indicates the range of values representing 50% of cases
around the median (vertical lines), the whiskers show the range of the values (horizontal lines), and
cross indicates the mean value (+). (From Karachle and Stergiou 2010, courtesy of the Acta
Ichthyologica et Piscatoria) (Although the authors use trophic classifications, the risk of pure nom-
inal classifications or even misclassifications cannot be excluded; see also ‘Trophic Positions: An
Omnivores’ Dilemma?’ below. Nominal, rather than empirically observed, classifications surely
increase the standard deviation.)
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Fig. 2.3 Relationship between phylogenetically independent contrasts of intestinal length residu-
als and contrasts of arcsine transformed proportion of animal material in diet. Numbers refer to
different grunter species. (From Davis et al. 2013, courtesy of Biomed Central Ltd.)
So far, the relationship between trophic position and length of the intestine and
its evolutionary derivation have received little attention from a phylogenetic per-
spective. Davis et al. (2013) documented the phylogenetic development of intestinal
length variability, and resultant correlation with dietary habits, within a molecular
phylogeny of 28 species of terapontid fishes. They found that the shorter the
intestine, the higher the share of animal preys in the diet (Fig. 2.3). The Terapontidae
(grunters), an ancestrally euryhaline-marine group, is the most trophically diverse
of Australia’s freshwater fish families, with widespread shifts away from
animal-prey-dominated diets occurring since their invasion of freshwaters. The
ontogenetic development of intestinal complexity appears to represent an important
functional innovation underlying the extensive trophic differentiation, specifically
facilitating the pronounced shifts away from the carnivorous (including inverte-
brates and vertebrates) diets evident across the family. The capacity to modify intes-
tinal morphology and physiology appears to be an important facilitator of trophic
diversification during the phyletic radiations – not only within the grunters.
Several striking examples prove that the intestine length varies in a single spe-
cies, depending on the available diet source. Investigating how the diet and intesti-
nal length of a persistent and generalist fish species (Bryconamericus iheringii,
Characidae) responds to riparian modifications in 31 subtropical streams in south-
ern Brazil, Dala-Corte et al. (2017) showed that the generalist and locally persistent
fish species responded to environmental alterations caused by riparian degradation
2.1 Digestive Tract 13
by consuming a greater proportion of autochthonous material (algae and aquatic

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macrophytes) instead of allochthonous material (terrestrial plant fragments and ter-

restrial invertebrates). These findings indicate that plasticity in intestinal length is an
important characteristic to determine whether fish populations can persist in a vari-
ety of habitat conditions and cope with the digestion of a greater proportion of low-
quality and low-protein food items in human-altered environments.
The intestine length also varies on an annual basis. In the Trinidadian guppy,
Zandona et al. (2015) showed that, even in omnivorous fish, gut length adapted to
different diets, being more evident when the magnitude of difference between ani-
mal and plant material in the diet was very large. The authors sampled guppies from
sites with low (LP) and high predation (HP) pressure in the Aripo and Guanapo
Rivers in Trinidad. They collected fish during the dry and wet seasons and assessed
their diet and gut length. During the dry season, guppies from HP sites fed mostly
on invertebrates, while guppies in the LP sites fed mainly on detritus. During the
wet season, the diet of LP and HP populations became very similar.
This study indicates that guppies have a broad range of variation in the propor-
tion of invertebrates and detritus in their diet, which changed with season and was
associated to local adaptation. These variations in diet were correlated with the gut
length (Fig. 2.4). Guppies that showed higher levels of carnivory also had the
Fig. 2.4 Mean proportion of invertebrates in diets vs. mean relative gut length. Only guppies
between 14 and 20 mm were included. Each data point represents one site (Aripo HP and LP for
both dry and wet season, and Guanapo HP and LP from the dry season). Relative gut length was
calculated as the gut length divided by fish length. An average value was assigned for the propor-
tion of invertebrates for each site, which was the estimated marginal mean obtained from the diet
analysis. (From Zandona et al. 2015, courtesy of the Public Library of Science)
shortest guts, and vice versa, as those with higher levels of herbivory had longer
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guts. The flexibility of the digestive system is an important adaptation that enables
guppies (and other fish species) to respond favorably to changes in food sources and
maximize nutrient absorption and energy extraction from different food types.
Different gut types can also be observed during the ontogenetic development, as
displayed, for instance, in Brycon guatemalensis (Drewe et al. 2004), a Neotropical
characid fish. It consumes an entirely terrestrial diet, shifting from eating insects as
juveniles to fruits and leaves as adults. Juveniles and larger-sized fishes were stud-
ied to test the hypotheses that, with ontogeny, (1) relative gut length increases, (2)
pyloric ceca arrangement and number remain unchanged and (3) pepsin, trypsin and
lipase activities decrease, while α-amylase activity increases. These hypotheses
were supported in that larger fish had longer guts, unchanged pyloric ceca arrange-
ment, and lower pepsin and trypsin activities, but higher α-amylase activities than
the juveniles. This study supports the view that B. guatemalensis is specialized mor-
phologically and biochemically to function first as a carnivore and then as an herbi-
vore during its life history.
2.2 Digestion
One major function of the intestine is digestion. This is the process of hydrolysis
and solubilization of ingested nutrient polymers into molecules and elements suit-
able for transport across the intestinal wall. The digestive enzymes secreted from
the stomach and exocrine pancreas are of major importance for enzymatic hydroly-
sis of complex food polymers, such as proteins, fats and carbohydrates, into smaller
fragments. The resulting smaller fragments are further digested at the epithelium of
the intestinal tract by the enzymes located in the brush border membrane of the
enterocytes, releasing molecules small enough for absorption, i.e. small peptides
and amino acids, monosaccharides, and fatty acids. This process is summarized in
Fig. 2.5. However, the contribution of exogenous digestive enzymes present in the
natural diet to total digestive capacity has most likely been largely underestimated.
A recent review focuses on exogenous contributions to digestion in fishes (see
Kuz’mina (2008) and Functional Aquafeed, Volume 2) and the following will there-
fore focus on endogenous gastrointestinal digestion processes. Considering the
importance of providing cultured fish with highly digestible formulated feeds for
rapid, cost-efficient fish growth and low waste released to the environment, the vast
majority of the investigations on digestive processes and factors that affect nutrient
digestibility have been carried out on production fish.
Fish have a digestive enzyme apparatus qualitatively similar to that of other ani-
mals with very similar substrate specificities across taxonomic groups. Although
molecular characterizations are now being published with increasing frequency,
knowledge is still limited regarding more specific characteristics of various
digestive enzymes for most fish species. Species-specific isoforms of the various
enzymes exist with differences in, for example, molecular-weights, specific activi-
2.2 Digestion 15
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Fig. 2.5 Schematic drawing of the digestive processes along the digestive tract of fish. The loca-
tion of various enzymes and other digestive components and the respective processes in the lumen,
as opposed to the intestinal mucosa, are indicated. FFA free fatty acids, FSVit fat soluble vitamins
Design: F. Venold. (From Bakke et al. 2010 with permission from Elsevier)
ties, pH-optima and efficiencies towards different bonds. Fish enzymes typically
show higher specific activity and substrate affinities than those in homeothermic
animals, presumably representing an evolutionary adaptation to function at lower
temperatures. For example, trypsin from Atlantic cod has a 17-times higher catalytic
efficiency than bovine trypsin when measured at the same temperature range.
2.2.1 Protein Digestion
In fish species with stomachs, the low pH from HCl secretion denatures most of the
proteins as they are solubilized, opening the structure for easier access by the pro-
teolytic enzyme pepsin. Pepsinogen and pepsin from several fish species have been
characterized. The enzyme is present in fishes in more than one form, and the dif-
ferent forms show different activation rates, pH optima (varying between 1 and 5),
specific activities and substrate specificities. Pepsins are endopeptidases, i.e. they
hydrolyze peptide bonds, with a high affinity for hydrophobic bonds involving
amino acids (AAs), such as tyrosine (Tyr) and phenylalanine (Phe). The partial
hydrolysis of the proteins increases the solubility and dissolution of other food com-
ponents, and prepares the diet—after this stage called chyme—for entry into the
intestine through the pyloric sphincter.
Proteins and peptides entering the intestine, with or without prior processing in a
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stomach, are diluted and dissolved in alkaline secretions from the liver, pancreas
and/or gut wall. The actions of the pancreatic endopeptidases trypsin, chymotrypsin
and elastases I and II, as well as the exopeptidases carboxypeptidase A and B, result
in a mixture of free AAs and smaller peptides (Fig. 2.5). The final steps of peptide
hydrolysis take place at the brush border of the enterocytes by aminopeptidases, or
by intracellular peptidases following peptide transport across the membrane.
However, some proteins and peptides entering the intestine either from the diet,
gastrointestinal or pancreatic secretions, may resist proteolysis and reach the distal
intestine more or less intact.
2.2.2 Lipid Digestion
Efficient lipid digestion requires emulsifiers in the mixture of food—mainly pro-

teins and phospholipids—as well as from endogenous bile acid and phospholipid
secretion in the proximal part of the digestive tract. The emulsifiers orient them-
selves on the surface of lipid droplets that form as dietary lipid is released during the
physical, chemical and enzymatic degradation of the food. If the emulsifying capac-
ity is deficient, the digestion of released lipids may be hindered. The main source of
lipolytic enzymes in fish is the acinar cells of theexocrine pancreas. Lipase activity
differs between fish species, as illustrated by the difference between related species,
such as Atlantic salmon and rainbow trout. Active fishes such as mackerel (family
Scombridae) and scup (family Sparidae) are among the species that have especially
high activities. Knowledge of characteristics and specificities of fish lipases is far
from complete. Freshwater fishes may have mainly co-lipase-dependent pancreatic
lipase (PL), whereas marine fishes have bile-acid-dependent carboxyl ester lipase
(CEL). PL has higher specificity and digestive efficiency for triglycerides than the
CEL. The latter hydrolyses a broader range of lipids, including wax esters (Bakke
et al. (2010) with reference).
2.2.3 Carbohydrate Digestion
Carbohydrates in natural fish diets and formulated feeds range from the highly sol-
uble and digestible mono-, di- and oligosaccharides, glycogen and starch, to only
marginally soluble and digestible chitin, hemicelluloses and celluloses. Fish species
vary greatly in their capacity to digest and absorb even soluble carbohydrates. Some
may have developed intestinal structures, functions and microbiota that enable
hydrolysis of a greater variety of carbohydrates, although this appears to be variable
even among herbivorous species. Fish have two categories of endogenous enzymes
2.3 Ontogenesis and the Intestine 17
involved in carbohydrate digestion: pancreatic α-amylase and disaccharidases in the

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brush border membrane of the intestinal epithelial cells (Fig. 2.5).
2.3 Ontogenesis and the Intestine
2.3.1 F
ishes
According to the classical and illustrative paper by Dabrowski (1984), the ontoge-
netic changes in digestive tract development of freshwater fishes during the larval–
juvenile transition can be categorized into three types:
1. Stomachless fish with an increase in complexity of the coiling pattern (mainly
cyprinids) (Fig. 2.6). These fishes remain stomachless throughout life. However,
in this group the coiling pattern of the intestine undergoes ontogenetic changes;
2. Stomachless larvae which develop a stomach structure after ingestion of food
(coregonids, silurids, serrasalmids) (Fig. 2.7); and
3. Alevin and juvenile stages of fish capable of ingesting the first food when the
stomach is present as a distinguished feature (salmonids, cichlids) (Fig. 2.8). In
other words, salmonids appear to have a functional stomach, before changing
from endogenous to external food. In the ontogeny of the cichlid digestive tract,
the small stomach is visible before yolk-sac absorption and, as the fishes take
their first external food, the stomach appears as a sizeable blind pouch. In spite
of the extremely different feeding habits of this species group (algae, plants,
fruits, detritus, insects, or fish), the overall appearance of the digestive tract
remains the same.
Morphological features of the digestive system are of great consequence in
respect to the diet type that larval/juvenile fish are able to utilize, especially at the
high growth rates during early ontogenetic development [50% per day in larval
common carp (Cyprinus carpio), 30–50% per day in the African catfish (Clarias
gariepinus) larvae]. Cichlids are exceptional, as their digestive gastrointestinal tract
appears to be completely formed with a functional stomach and an elongated intes-
tine prior to the use of yolk sac reserves. Unlike most other teleosts, cichlid juveniles
pass through an extended period of “mixed” feeding of endogenous (yolk sac) and
exogenous feeding. This modulation shifts the focus to maternal–offspring nutrient
transfer in juveniles, rather than a sole dependence on external food intake and its
quality (nutrient presence and availability) for larval fish. Juvenile, first feeding Nile
tilapia, for instance, were able to grow on phytoplankton (especially, coccal green
algae) provided during the first several weeks of life (Dabrowski and Portella
(2005)).
In addition to the well-documented freshwater species, Fig. 2.9 exemplifies the
ontogenetic development of the digestive system in the orange clownfish, a species
Swim bladder inflates

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Trypsin activity rises

exogenous feeding
Chymotrypsin activity appears

weight (mg)
Amino peptidase activity increases
1000
100
10
1
15 30
age (days)
Fig. 2.6 Ontogenetic development of the cyprinid fish digestive tract, exemplified by common
carp. (From Dabrowski 1984, courtesy of Editions scientifiques medicales Elsevier, Paris)
with an advanced alimentary canal at hatching. These larvae can immediately start
exogenous feeding.
A second marine example, Elbal et al. (2004) reported light and electron micro-
scopic studies of the digestive tract of the gilthead sea bream (Sparus aurata,
Fig. 2.10) from hatching to 69 days. Five significant phases were established. Phases
I and II comprise the lecitotrophic period. During phase I, the yolk sac was large
and the uniform digestive tract showed a layer of squamous epithelial cells with
Exogenous feeding
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Trypsin + chymotrypsin activity present

Guanine in skin (metamorphosis)
weight (mg)
Glyco-glycogeno-lysis as energy

Morphologically functional
stomach
Stomach acid pH
pepin digestion
150
100
50
10
30 60
age (days)
Fig. 2.7 Ontogenetic development of the corregonid fish digestive tract, exemplified by Coregonus
pollan. (From Dabrowski 1984, courtesy of Editions scientifiques medicales Elsevier, Paris)
numerous free ribosomes. Phase II was characterized by the opening of the anus and
the differentiation of three digestive regions: the esophagus, with a stratified epithe-
lium; the presumptive stomach, whose cuboid epithelial cells had some apical pro-
cesses and clear vesicles; and the intestine, with large intercellular spaces among
prismatic epithelial cells that had a periodic acid Schiff reagent-positive striated
border. Phase III, or lecitoexotrophic period, began with the opening of the mouth,
where absorption of the yolk sac started and the intestine became differentiated into
two regions separated by a valve. Intestinal epithelial cells showed basal lamellar
structures and lipoprotein particles. Some columnar cells appeared inside the epi-
thelium of the esophagus. Phases IV and V comprise the exotrophic period, where
phase IV begins with the disappearance of the yolk sac; mucous cells containing
Functional pancreas and gastric glands

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Exogenous feeding
Trypsin activity increases

weight (mg)
200
100
30 60
age (days)
Fig. 2.8 Ontogenetic development of the salmonid fish digestive tract, exemplified by rainbow
trout. (From Dabrowski 1984, courtesy of Editions scientifiques medicales Elsevier, Paris)
sulphomucin-type acid mucosubstances appeared in the esophagus and goblet cells,

with acid and neutral mucosubstances appeared in the intestine. The epithelial cells
of the first and posterior intestinal segments showed large lipid droplets and heavy
pinocytosis, with large supranuclear vesicles and numerous lysosomes, respectively.
Phase V was marked by the appearance of neutral mucosubstances in the esopha-
geal mucous cells and in the stomach epithelial cells, and the differentiation of
pyloric ceca and gastric glands. The ultrastructural features of glandular cells indi-
cated that they secrete both pepsinogen and hydrochloride acid. The epithelial cells
of the first intestinal segment showed large lipid droplets, often close to mitochon-
dria, at the beginning of this phase. These lipid droplets decreased in size, while the
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Juvenile
Growth and Differentitation
Metamorphosis
e
Digestion becomes
siz extracellular
n
s i ar
mn cell der
s
se mn Larva
gastric gland
colu ach bor
a
s
re olu
inc s c
sh
en cell
ru
ar
lum gut tb
gu
m
d
sto
nd
hin
Hi
Hatching
Mouth opened Onset of exogenous feeding
Jaws ossified Digestion is pinocytotic Embryo
Alimentary canal
differentiated
Liver and spleen
differentiated
Six days 1 3 5 5 5 7
after
fertilisation Age (Days after hatch)
Fig. 2.9 Left: Steps in the ontogeny of the digestive system of the orange clownfish (Amphiprion
percula). The various structures of the alimentary canal grow and differentiate at different rates,
but are completed and functional at the same time, enabling the larvae to undergo rapid metamor-
phosis from one stage to another. The steps in A. percula development have been divided into
embryonic, larval, and juvenile stages. There is no eleutheroembryo stage as the larvae begin
exogenous feeding immediately after hatching and before the yolk sac is fully absorbed. (From
Gordon and Hecht 2002, with permission from Wiley); right: Breeding A. percula (courtesy of
Haplochromis, Wikimedia)
Fig. 2.10 Images of Sparus aurata and Gadus morhua. (From Bloch 1785–1790, courtesy of the
Biodiversity Heritage Library)
rough and smooth endoplasmic reticulum and Golgi complex, related to lipoprotein
synthesis, progressively developed during this phase. Pinocytotic and large supra-
nuclear vesicles disappeared from epithelial cells of the posterior intestinal
segment.
Overall, in the early development stages of S. aurata, lipid absorption occurs in
the epithelial cells of the first intestinal segment, while the absorptive cells of the
posterior intestinal segment are able to take up proteins by pinocytotic mechanisms.
The appearance of the first gastric glands improves extracellular protein digestion,
and the supranuclear inclusions in the absorptive cells of the posterior intestinal
segment disappear. This event probably encourages lipoprotein formation and the
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exportation of lipids in the form of chylomicrons. These findings are of considerable

importance for the evaluation of the digestive tract functionality, and will be useful
for establishment of optimal rearing techniques and artificial food for S. aurata
larvae for the commercial production of this species.
Pedersen and Falk-Petersen (1992) compared the development of Atlantic cod
(Gadus morhua, Fig. 2.10) during transition from larva to juvenile with that of four
other teleosts, namely red seabream (Pagrus major), yellowtail (Seriola quinquera-
diata), spot croaker (Leiostomus xanthurus) and olive flounder (Paralichthys oliva-
ceus) (Fig. 2.11). The differentiation of the alimentary tract during the early stages
of juvenile development, into a stomach originating from the posterior part of the
esophagus, and pyloric ceca2 developing from the anterior part of the intestine, is in
accordance with the general scheme seen in other teleosts. In cod, the developmen-
tal changes in the alimentary tract occur at a later stage than the disappearance of
the median finfold, the development of the median fins and the appearance of the
vertebrae. Compared with some other fish species, alimentary tract development
appears to be delayed in cod. In red sea-bream, spot and olive flounder, the stomach
and pyloric ceca develop at the time of the disappearance of the median finfold
(Fig. 2.11). In yellowtail and cod, the development of stomach and pyloric ceca
takes place later than the development of characters associated with locomotion
(Fig. 2.11). In cod, the food-storing capacity of the stomach seems to develop in
individuals larger than 20 mm and this capacity is well-developed in individuals
larger than 40 mm. In fish of 40 mm and over, the vast majority of identifiable cope-
pods in the alimentary system were found in the stomach.
The strategy of expanding intestinal absorptive surface is taking place in both
cold water and tropical fishes. In the cold-water salmonid rainbow trout, which pos-
sesses on average 70 pyloric ceca, the total length of the pyloric ceca is 6-fold larger
than the total intestine. Several tropical characid fishes also possess numerous
pyloric ceca. A generalization predicts that diets composed of voluminous food,
algae and detritus should result in elongation of the intestine. Thus, the relative
length of the intestine seems to be an indicator of diversified use by the digestive
tract of food of high or low nutrient concentrations (Fig. 2.12, Dabrowski and
Portella (2005)). For instance, the Lake Tanganyika cichlid Petrochromis polyodon
possesses a particularly long intestine (6–10-fold body length), because it ingests
unicellular microalgae by scraping the rock biocover (Takamura 1984). In contrast
to this fish species, the carnivorous channel catfish (Ictalurus punctatus) is charac-
terized by the shortest relative intestine length (Fig. 2.12). Its diet consists primarily
of small fish, crustaceans (e.g. crayfish), clams and snails; it feeds, also, on aquatic
insects, even small mammals, but seldom plant material (Tyus and Nikirk 1990).
The concept of nutrient requirement and morphology of the intestine has been
questioned several times. For instance, there are strict stomachless carnivorous fish
2
Pyloric ceca are blind appendages attached to the proximal intestine of many fish. Buddington
and Diamond (1987) provided evidence that ceca are an adaptation for increasing the intestinal
surface area without increasing the length or thickness of the intestine itself.
Species Standard length (mm)

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character 0 10 20 30 40 50
Gadus morhua1
eleutheroembryo I
median finfold
median fins
stomach
pyloric caeca
Pagrus major 2
eleutheroembryo I
median finfold
median fins
stomach
pyloric caeca
gastric glands
Seriola quinqueradiata 3
eleutheroembryo I
median finfold
median fins
stomach
pyloric caeca
gastric glands
Leiostomus xanthurus 4
eleutheroembryo I
median finfold
median fins
stomach
pyloric caeca
Paralichthys alivaceus 5
eleutheroembryo I
median finfold
median fins
stomach
pyloric caeca
Fig. 2.11 The development scheme of Atlantic cod (Gadus morhua) during transition from larva
to juvenile, compared with those of some other teleost species. (From Pedersen and Falk-Petersen
1992, with permission from Wiley)
(the cyprinid Leuciscus aspius) and herbivores with short digestive tract, 1.5 × body
length (Horn 1989). Another example underlines this concern, but does not suspend
the general trend displayed in Fig. 2.1 and in the meta-study referred to in Fig. 2.2.
Rather, it contributes to the understanding of the huge standard deviations in the
latter figure. In particular, German and Horn (2006) measured relative gut length,
body mass (Zihler’s index) and relative gut mass in four species of prickleback
fishes, a group of fish which will serve as witness for the considerations about
herbivory.
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Fig. 2.12 Changes in the relative length of intestine (expressed in body lengths) in several fish
species. (From Dabrowski and Portella 2005, with permission from Elsevier)
The authors were interested in the effects of ontogeny, diet and phylogeny on
these gut dimensions (Fig. 2.13). Of the four species, Cebidichthys violaceus and
Xiphister mucosus shift to herbivory with growth, whereas X. atropurpureus and
Anoplarchus purpurescens remain carnivores. A. purpurescens belongs to a carniv-
orous clade, and the three other species belong to an adjacent, herbivorous clade.
The comparison of the gut dimensions in three feeding categories of the four species
revealed:
1. Small, wild-caught juveniles representing the carnivorous condition before two
species shift to herbivory;
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Fig. 2.13 Upper graph: Non-metric multidimensional scaling plot of all three gut dimension
parameters combined for each species as a function of ontogeny (w30–40 and W60–75 categories – the
subscripts refer to body lengths in mm) in Cebidichthys violaceus (Cv), Xiphister mucosus (Xm),
X. atropurpureus (Xa) and Anoplarchus purpurescens (Ap). Arrows indicate magnitude of ontoge-
netic shifts in gut dimensions. The stress value indicates that the plot fits well (i.e., values <0.1)
into two-dimensional space. Lower graph: Non-metric multidimensional scaling plot of all three
gut dimension parameters combined for each species as a function of diet (W60–75 and L60–75 catego-
ries) in C. violaceus (Cv), X. mucosus (Xm), X. atropurpureus (Xa) and A. purpurescens (Ap).
Arrows indicate magnitude of diet-related changes in gut dimensions. The stress value indicates
that the plot fits well (i.e., values <0.1) into two-dimensional space. (From German and Horn 2006,
with permission from Springer)
2. Larger, wild-caught juveniles representing the natural diet condition of the two
carnivores and the two species that have shifted to herbivory; and
3. Larger, laboratory raised juveniles produced by feeding an artificially high-
protein diet to small juveniles until they have reached the size of the larger, wild-
caught juveniles.
Comparisons of the gut dimensions in categories (1) versus (2) tested for an
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ontogenetic effect, in (2) versus (3) for a dietary effect, and within each category for
a phylogenetic effect. C. violaceus and X. mucosus increased gut dimensions with
an increase in body size, and did not change the ontogenetic trajectory in gut dimen-
sions on the artificially high-protein diet, indicating that they are genetically pro-
grammed to develop relatively large guts associated with herbivory. X. atropurpureus
increased its gut dimensions with an increase in size similar to its sister taxon, X.
mucosus, indicating a phylogenetic influence, but decreased gut dimensions on the
artificially high-protein diet, showing phenotypic plasticity. Nevertheless, X. atro-
purpureus displayed a larger gut than A. purpurescens, further evidence that it
evolved in an herbivorous clade. A. purpurescens possessed a relatively small gut
that was less affected by ontogeny or diet. Overall, ontogeny and phylogeny, more
than diet, appear to influence gut dimensions in the four species, thus favoring
genetic adaptation over phenotypic plasticity as the major force acting on digestive
system features in the two prickleback clades. This contrasts the metastudy in
Fig. 2.2.
Further details of ontogeny and physiology of the digestive system of marine and
freshwater fish larvae and adults can be found in recent monographs and reviews
(Bucking 2015; Zambonino Infante et al. 2008; Portella and Dabrowski 2008;
Yúfera and Darias 2007; Zhang et al. 2017; Grosell et al. 2010; Cortés et al. 2008).
2.3.2 Invertebrates
Dietary impact on the development of the intestines in aquatic invertebrates is much

less well documented than in fish species. We shall focus on selected examples from
echinoderms and crustaceans.
2.3.2.1 E
chinoderms
Sea urchins exhibit dramatic changes in the ontogenetic niche between newly set-
tled recruits, that may occupy cryptic habitats and feed on microalgae or detritus,
and mature adults, that may catch drift kelp or actively graze on macroalgae and
invertebrates. Wing and Wing (2015) examined patterns in the ontogenetic niche
development of Evechinus chloroticus within the New Zealand fjords, a system with
a series of strong environmental and benthic productivity gradients. E. chloroticus,
also known as “kina” (from the Māori name), is a sea urchin endemic to New
Zealand (Fig. 2.14). Using data from 15 long-term monitoring sites, the authors
examined relationships between the diet of juvenile and adult sea urchins and the
morphology of their preferred food, the kelp Ecklonia radiata. Stable isotope analy-
sis (δ13C and δ15N) of E. chloroticus stomach contents, muscle tissue and samples
along the blades of E. radiata revealed evidence of large variation in nutritional
conditions. The contribution of E. radiata to the diet of E. chloroticus declined from
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Fig. 2.14 Left: Trophic level of adult (>100 mm test diameter, ●) and juvenile (<40 mm test
diameter, ○) sea urchins along a gradient between outer coast wave-exposed sites and inner fjord
wave-sheltered sites. Distances (km) are from the entrance of each fjord. Error bars: ±1
SE. Numbers refer to sampling sites. (From Wing and Wing 2015, with permission from Inter-
Research, ©Inter-Research 2015.). Right: Kina urchins (Evechinus chloroticus), South East Bay,
Manawa Tawhi Three Kings Islands, attacking Ecklonia radiata (courtesy of Peter Southwood,
Wikimedia)
the wave-exposed sites near the entrances of fjords to the fully wave-sheltered sites
of the inner fjords for adults, but not for juveniles (Fig. 2.14). Coincident with the
decline in prevalence of E. radiata as a food source, the trophic level of E. chloroti-
cus increased at inner fjord sites, indicating prey switching to grazing on inverte-
brates. Along this gradient, the authors observed a divergence in the trophic levels
of adult E. chloroticus from those of juveniles, indicating that adults maintain a
higher trophic level at sites where their preferred food resources are scarce. These
results contribute to the understanding of how sea urchin populations persist across
strong gradients in primary productivity, and their role in subtidal food webs.
2.3.2.2 C
rustaceans
Due to their commercial value, decapode crustaceans are in the scientific focus and
will be dealt with here. In addition to decapods, in the model crustacean genus
Daphnia, intriguing transgeneral diet-mediated effects on food acquisition have
recently been identified. Therefore, this small crustacean and its intestine deserve
description. This is particularly true because comparable studies of decapods are not
yet available, but could be helpful in improving the quality of broodstocks.
Decapods
In his paper on the greentail prawn (Metapenaeus bennettae), Dall (1967) described
the functional anatomy of the intestine tube. He states that structure and function of
the proventriculus is similar to that of a number of other Decapoda, such as Astacus

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sp., Cancer sp., Nephrops norvegicus, Galathea sp., Caridina laevis and Litopenaeus
setiferus. Two distinct cell types occur in the digestive gland: a secretory type and a
mucopolysaccharide-containing type. The digestive gland has no intrinsic muscles,
and depends on extrinsic muscles. The midgut extends to the sixth abdominal
somite, and fecal material is contained in a peritrophic membrane. The rectal lining
is formed into six longitudinal pads, which are used to expel long sections of peri-
trophic membrane containing feces. Food is found to begin leaving the proventricu-
lus almost as soon as it is filled, but complete emptying takes 6–12 h. Defecation is
at a peak 5–8 h after food ingestion, but continues up to 20 h.
In their review, McGaw and Curtis (2013) confirmed that the crustacean gut is
essentially an internal tube opening at the esophagus and ending as the anus in the
telson of the abdomen, and present some more generalizing details. It is divided into
the foregut, midgut and hindgut (Fig. 2.15a, b). The mouth opens into a short esoph-
agus, the walls of which are lined with tegumental glands. These glands secrete
mucus that lubricates the food as muscular waves propel it towards the foregut. The
foregut is lined with cuticle and separated into an anterior cardiac chamber, with a
smaller pyloric chamber lying posteriorly. The cardiac chamber is a large distensi-
ble sac that functions as an area for storage and processing of food. The internal
structure of the cardiac chamber varies between species. In most decapods, it is
lined with numerous calcified ossicles which function in mechanical digestion.
Filtration grooves in the walls of the cardiac chamber channel the food towards the
posterior of the chamber which contains the gastric mill apparatus. The gastric mill
is composed of large ossified teeth which masticate the food. The processed food is
then pushed posteriorly towards to the pyloric chamber by rhythmic contraction of
the muscles of the foregut.
At the junction of the two chambers, cardiopyloric setae, in conjunction with the
cardiopyloric valve, regulate movement of digesta between the cardiac and pyloric
chambers. Any coarse unprocessed material captured by the cardiopyloric setae is
pushed back into the cardiac chamber and gastric mill for further mechanical
digestion. The smaller pyloric chamber is situated posteriorly and ventrally to the
cardiac chamber (Fig. 2.15). Its primary function is sorting of food for subsequent
transport into the midgut region. Gland filters in the pyloric region filter out particu-
late matter, so that only the liquid form of digesta reaches the hepatopancreas. The
rhythmic pumping of the pyloric sac also functions in propelling food along the
midgut region (McGaw and Curtis (2013) and references therein).
The midgut starts at the junction with the pyloric sac and ends in a coiled tube,
known as the posterior midgut cecum at the junction between the carapace and
abdomen (Fig. 2.15a, b). Unlike the foregut and hindgut which are lined with cuti-
cle, the walls of the midgut are lined with glandular columnar epithelium. The mid-
gut varies in length depending on species. It tends to be short in some species of
crayfish, but extends into the abdomen in the brachyuran crustaceans. Ducts arise at
the junction of the midgut and pyloric sac, and branch extensively as blind-ending
tubules within the digestive gland or hepatopancreas. The hepatopancreas (also
known as the digestive gland) is a large organ occupying most of the dorsal region
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Fig. 2.15 (a) Anatomy of the digestive system of a decapod crustacean, showing the foregut,
midgut, and hindgut regions. (b) Schematic diagram of digestive processes occurring in the deca-
pod crustacean gut. (From McGaw and Curtis 2013, with permission from Springer)
of the cepaholathorax, and may extend backwards into the abdomen. It is here that
enzymatic digestion continues and absorption of food occurs.
The hepatopancreatic ducts contain musculature indicating that their contraction
aids in movement of the digesta through the hepatopancreas. Inert particles are fil-
tered out at the entrance of the ducts, and only liquid and particles less than 100 nm
in diameter enter the hepatopancreas. The hepatopancreas has a number of special-
ized cells (B, E, F and R cells) within the walls of the tubules. These are involved
with enzyme production and recycling, and absorption of nutrients and water. It is
here that intracellular digestion and protein synthesis begin and can continue for up
to 2–3 days. Most nutrients are absorbed across the tubules of the hepatopancreas,
although some may be absorbed directly across the midgut wall. In some crustaceans,
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blind ending extensions of the midgut, the anterior and posterior midgut ceca are
evident. Their small size suggests that they only play a minor role in digestion, pos-
sibly activation or production of some enzymes, maintenance of pH balance or
accommodation of volume changes as the foregut contracts. They may also play a
role in ion and water regulation. The hindgut arises behind the posterior midgut
cecum and runs the length of the abdomen to the anus. It is usually a simple cuticle
lined tube, surrounded by outer layers of longitudinal and circular striated muscle.
It functions in expelling the mucoperitrophic membrane and its contents by rhyth-
mic contractions along its length. Tegumental glands along the length of the hindgut
secrete mucus to lubricate the walls. Some digesta may remain in the hindgut and is
not voided until a subsequent meal is ingested (McGaw and Curtis (2013) and refer-
ences therein).
Studying comparatively the gut morphology (tooth, epithelia surface, hindgut
lining) of seven mud shrimp (Thalassinidea) species, Pinn et al. (1999) reported
that, as to be expected and applicable to further groups of decapods, the general
scheme of digestive-tract morphology is modified to some extent by feeding mode
and diet. The authors found that deposit feeders, such as burrowing ghost shrimp
(Callianassa subterranean) and burrowing lobster (Axius stirhynchus) have a diges-
tive system with a robust gastric mill. The studied Upogebiidae (mud shrimps) have
a gastric mill designed for dealing with smaller particles obtained by suspension-
feeding. Jaxea nocturna has a digestive system intermediate between these two
opposites, as might be expected from its resuspension method of feeding. The gut
of Calocaris macandreae is designed to deal with material of a higher organic con-
tent than would be encountered by deposit-feeding alone.
For more information about the functional anatomy and physiology of nutrition
and digestion in crustaceans, the reader is referred to reviews by Saborowski (2015),
and specific monographs by Mikami and Takashima (2008) or Spicer and Saborowski
(2010).
In marine planktonic food webs, the size of decapod larvae determines the tro-
phic position. Decapods have adopted a full range of reproductive strategies from
the release of large numbers of small eggs (Penaeoidea) to the release of relatively
low numbers of large advanced larvae (Nephropidae). The larvae exploited all food
sources from phyto- to zooplankton, with many species changing trophic position
during ontogenetic development. Comparative studies on digestive enzymes, levels
of activity and changes during ontogeny, together with measurements of gastroevac-
uation rates and food energy values, appear to reveal a general pattern. While her-
bivorous decapod larvae adapt to low food energy values with high enzyme activity
levels, rapid food turnover and low assimilation efficiency, carnivorous larvae
exhibit low levels of enzyme activity, but compensate by extending retention time
of high-energy food to maximize assimilation efficiency (Le Vay et al. 2001).
In particular, Fig. 2.16 shows an overview of trypsin content in larvae of a
wide range of crustacean species, indicating a trend in trypsin content related to
the trophic level (Le Vay et al. 2001). The overall trend is for a higher trypsin
content in species and developmental stages which are dependent on digestion of
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Fig. 2.16 Comparison of trypsin-like content (×10─5 μg─1 dry wt) in a various crustacean larvae.
In all cases, enzyme activity was assayed using N-α-p-toluenesulphonyl-l-arginine as the sub-
strate. Annotations: A adult, N nauplius, PZ protozea, M mysis, PL postlarva, St stage, Z zoea;
white bars carnivorous, black herbivorous, gray omnivorous. (From Le Vay et al. 2001, with per-
mission from Elsevier)
phytoplankton, declining through the omnivores and with the lowest activity in
carnivorous feeders.
Within a species, omnivorous larvae may exhibit a wide variation in protease
content, depending on food composition. For example, Marsupenaeus japonicus
mysis larvae feeding on protein-rich zooplankton show a very marked reduction in
protease content compared to that in larvae fed an algal food of low-protein content.
Figure 2.17 shows the results of a similar experiment with Litopenaeus vannamei
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Fig. 2.17 Specific trypsin content (×10─5 μg─1 dry wt) in Litopenaeus vannamei nauplius and
protozoea larvae, comparing the effect of herbivorous and carnivorous dietary regimes. Larvae
were cultured in 2-L round-bottom flasks at an initial density of 100 larvae L─1 in 35 ppt seawater.
In the control flasks, larvae were fed a 70:30 mixture of the microalgae Chaetoceros muelleri and
Isochrysis galbana at 50 cells μL─1 from protozoea 1 to protozoea 3 (PZ1─PZ3). The treatment
flasks were switched to carnivorous feeding (2 Artemia nauplii mL─1) at PZ2. Larvae were sampled
at each stage for dry weight estimation and assay of trypsin-like activity using N-α-p-
toluenesulphonyl-l-arginine as substrate. Annotations: N nauplius, PZ protozea. (* indicates sig-
nificant difference between treatments within that larval stage, P < 0.05). (From Le Vay et al. 2001,
with permission from Elsevier) (Image courtesy of FAO)
protozoeal (PZ) larvae. Shrimp of this genus differ from the Asian penaeids in
having only five nauplius stages and, in the case of L. vannamei, in being able to
successfully adapt to carnivorous feeding at an earlier developmental stage (PZ2).
Trypsin content at the PZ1 stage was found to be higher than that measured in other
penaeid species, indicating a strong adaptation to herbivory at first feeding (Le Vay
et al. 2001).
Daphnia
Returning to freshwater, most species of the planktonic cladocerans are strictly filter
feeders and very effective at removing various kinds of suspended microparticles.
As specialized filter feeders, they have developed a specific thoracic limb morphol-
ogy, that works together as a perfect pumping and filtering apparatus in which every
part has its specific function. Filtering apparatus and intestine determine the trophic
niche of cladoceran individuals and populations (Smirnov (2014) and references
therein).
The intestine consists of a stomodeum (foregut or esophagus) passing into the
mesenteron (midgut) and proctodeum (hindgut). A blind cecum may be present on
the ventral side of the midgut, before the beginning of the hindgut. Food is trans-
ferred from the midgut to the blind cecum, and accumulates there before being
finally evacuated. The food collected in the intestine is surrounded by a peritrophic
membrane. The tubular peritrophic membrane surrounds the food and extends
through the midgut and hindgut (Smirnov (2014)).
Food stays in the cladoceran intestine for only a few minutes, and the digestion
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appears to be very efficient. This high efficiency may depend on the following facts:
1. The dense cover of microvilli in the intestinal lumen greatly increases the gut
surface;
2. The inner surface of the intestine is folded, which further increases the inner
surface;
3. The efficiency is further increased through intensive mixing of the food by peri-
stalsis; and
4. Dialysis occurs through the peritrophic membrane (Smirnov (2014) and refer-
ences therein).
Macháček and Seda (2016) added intriguing morphological aspects to the filter-
ing efficiency of Daphnia. Their experiments showed that the filtering setae number
in the filtering screens in D. galeata is constant in the postembryonic period of the
individual development. Furthermore, the filtering setae number is phenotypically
variable due to transgenerational plasticity, and it is related to the body size at the
first juvenile instar. The food-dependent plasticity of the relative filtering setae
length is in the range from ~15% to ~20% of the carapace length, irrespective of the
filtering setae number. This means that individuals with low filtering setae number
are more restricted in the size of the filtering screen area in limited food than indi-
viduals with the high filtering setae number. The results of this study indicate that
transgenerational and ontogenetic plasticity substantially determine the morphol-
ogy of the filtering structures in Daphnia.
Three papers cover the transgenerational issue, one induced by an abiotic
(Macháček and Seda 2013) and two by a biotic trigger (Garbutt and Little 2014;
Coakley et al. 2017). Macháček and Seda (2013) reported that a field investigation
of seasonal variation of filtering setae number in the filtering screens of D. galeata
showed a clear tendency toward low filtering setae number in animals with low
temperature and food levels (an over-wintering population and a deep-hypolimnion-
inhabiting part of the population). Laboratory experiments were carried out to
uncouple the effects of temperature and food level on the size of neonates and filter-
ing setae number in their filtering apparatus. It became obvious that temperature
was the main factor inducing the changes in filtering setae number, and the induc-
tion takes place during embryogenesis. This effect was passed to the succeeding
generation. Eggs from the same clutch incubated in vitro at temperatures of 19, 10
and 6 °C developed into neonates with smaller carapace length with decreasing tem-
perature. Smaller neonates had lower filtering setae number in their filtering screens.
However, this apparent disadvantage can be overcompensated, if the mother was
well-fed, since Garbutt and Little (2014) documented that maternal food quantity
affects offspring feeding rate, with low quantities of food triggering mothers to
produce slow-feeding offspring. Such a change in the rate of resource acquisition
has broad implications for population growth or dynamics, and for interactions with
predators and parasites, for example.
This maternal effect can also explain the previously puzzling situation that the
offspring of well-fed mothers, despite being smaller, grow and reproduce better than
the offspring of food-starved mothers. As an additional source of variation in

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resource acquisition, this maternal effect may also influence relationships between
life history traits, i.e. trade-offs, and thus put constraints on adaptation.
Several uncertainties in the ecological significance of the transgenerational
effects, however, remain. Coakley et al. (2017) detected numerous maternal effects
in their D. magna clone. Notable findings included the large size of offspring from
poorly fed or older mothers (see also ‘Transgenerational Effects’) and the early age
at first reproduction of offspring born to older mothers. The adaptive nature of these
effects are not yet fully clear. Other genotypes, or other traits, may respond differ-
ently to the same treatments. At the same time, the production of larger offspring
with different reproductive features will itself alter the competitive environment, a
scenario that can more fully reveal the consequences of maternal effects.
2.4 Herbivory, a Disadvantageous Acquization Strategy?
Herbivory is not the most common mode of energy and nutrient acquisition. In most
cases, nutrient composition does not exactly match the nutrient requirements and,
due to the high fiber contents, the energy provision is comparably low and requires
long intestines. Therefore, herbivory appears to be a relatively rare case in fish com-
munities. In contrast to fishes, in crustaceans plant feeding promotes diversification,
as recently reported by Poore et al. (2017) (see below). If one considers herbivory
from an ecological point, it has many (hidden) advantages, even for the herbivores
themselves. Even more striking, and in contrast to expectations, herbivory has
evolved multiple times in some fish clades, indicating the usefulness of this kind of
acquisition strategy. Here, herbivory will be considered under this bias.
Recently, Sanchez and Trexler (2016) provided an interesting eco-evolution-
based hypothesis that removes “herbivory” from the pure productivity perspective
and puts it into a broad framework. Although herbivory is thought to be nutritionally
inefficient relative to carnivory and omnivory, the authors argue that herbivory
evolved from carnivory in many terrestrial and aquatic lineages, indicating that
there are advantages of eating plants. Herbivory has been well-studied in both ter-
restrial and aquatic systems, and there is abundant information on feedbacks
between herbivores and plants, coevolution of plant and herbivore defenses, mecha-
nisms for mediating nutrient limitation, effects of nutrient limitation on herbivore
life history, and, more recently, the origins of the herbivorous diet. Researchers have
sufficiently defined the ecological context and evolutionary origins of the herbivo-
rous diet, and these main areas of research have laid the groundwork for studying
herbivory as an adaptation. However, one has yet to synthesize this information in a
way that allows the establishment of a framework of testable adaptive hypotheses.
To understand the adaptive significance of this diet transition, Sanchez and Trexler
(2016) present five interesting hypotheses on the evolution of herbivory from car-
nivory (Table 2.1) – worth being tested in depth in studies to come:
1. Intake efficiency: Herbivores use part of their food source as habitat, thus mini-
mizing the energy/time spent searching for food and avoiding predators;
2.4 Herbivory, a Disadvantageous Acquization Strategy? 35
Table 2.1 Description of the proposed hypotheses for the adaptive evolution of herbivory in
freshwaters
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Hypothesis
(short) Explanation Assumptions References
I. Intake Aquatic herbivores may use Freshwater herbivores Brönmark and
efficiency all or part of their food are relatively small and Vermaat (1998)
source as habitat. require refuge from
Herbivory may allow an predators, usually in the
organism to maximize the form of submerged
intake energy by aquatic vegetation.
minimizing the time spent Submerged aquatic
searching for food, energy vegetation is associated
consumed during prey with more palatable
capture, and energy costs plants like algae that are
avoiding predators. consumed by herbivores.
II. Suboptimal Herbivory may allow Herbivores are able to Proulx and
habitat organisms to invade detect the food Mazumder (1998)
suboptimal or recently availability and/or
disturbed habitats. Such quality of the current
habitats are often habitat and make
characterized by having dispersal decisions
high primary production accordingly.
relative to consumer
biomass.
III. Heterotroph Herbivory may be adaptive Heterotrophic microbes Martin-Creuzburg
facilitation because herbivores (heterotrophic bacteria, et al. (2008)
supplement their diets by protozoa, etc.) are in
indirectly consuming close association with
heterotrophic microbes that freshwater primary
are associated with algae. producers and herbivores
These heterotrophs can consume them indirectly.
provide nutrients that are
not attainable by eating
algae alone.
IV. Lipid Some freshwater algae are At least some essential Brett and
allocation sources of essential lipids lipids come from Müller-Navarra
and herbivorous organisms freshwater primary (1997), Karasov
consume large quantities of producers. and del Rio (2007)
these lipids relative to and
animal-consuming species. Sharathchandra
Because aquatic organisms and Rajashekhar
use lipids for energy (2011)
storage and reproduction
consuming a diet rich in
fatty acids may result in
greater reproductive
allocation. Herbivory may
be adaptive because higher
lipid consumption leads to
higher reproductive
allocation and thus,
increased fitness.
(continued)
Table 2.1 (continued)
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Hypothesis
(short) Explanation Assumptions References
V. Disease Animal prey may serve as Parasites and prion Covich et al.
avoidance intermediate hosts and diseases are only (1999) and
facilitate transmission of transmitted via animal Marcogliese
parasites or prions through vectors. (2002)
the diet. Herbivory may be
adaptive because it reduces
animal-facilitated disease
transmission.
From Sanchez and Trexler (2016), courtesy of the Ecological Society of America; complete refer-
ences added for the sake of understandability
2. Suboptimal habitat: Herbivory allows organisms to invade and establish popula-

tions in habitats that have high primary production but low abundance of animal
prey;
3. Heterotroph facilitation: Herbivory is adaptive, because herbivores consume
microbes and fungi associated with producers;
4. Lipid allocation: Herbivory is adaptive, because producers are rich in fatty acids,
which fuel reproduction and storage; and
5. Disease avoidance: Herbivory minimizes animal-facilitated disease transmission.
Due to the extensive literature, the authors have limited their review to herbivory
in freshwater systems. To my knowledge, no prior work has compiled a comprehen-
sive list of conditions that favor an herbivorous diet in nature. With backgrounds in
both theoretical and experimental ecology, the incorporation of these hypotheses to
the current literature will provide information about diet evolution, where it is cur-
rently lacking.
The proposed hypotheses represent a research framework that may lead to more
comprehensive studies of diet evolution not only in freshwater, but also in other
aquatic systems. In turn, herbivory, in general, and frugivory, in particular, contrib-
ute to the dispersal of endozoochorous terrestrial and aquatic plants (Agami and
Waisel 1988; Horn 1997; Mannheimer et al. 2003; Correa et al. 2007; Weiss et al.
2016; Boedeltje et al. 2015; Galetti et al. 2008; Gottsberger 1978; Anderson et al.
2011). Interestingly, the passage through the gut accelerates or delays the germina-
tion speed of certain plant species (Boedeltje et al. 2016). The authors compared
carp and Nile tilapia and found that carp’s efficient mastication affects the germina-
tion speed of plant species after the gut passage, whereas stomach-passage in tilapia
generally does not influence the germination speed. In addition, a delayed seed
germination of a floodplain tree species was observed by freshwater Pantanal sar-
dines (Yule et al. 2016). This aspect deserves more and intensive attention with
respect to advantages of the consuming fishes and aquatic invertebrates.
2.4.1 Fishes
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The eco-evolutionary meaningfulness of herbivory can best be figured out in closely

related animals belonging to the different trophic categories: herbivores, omnivores
and carnivores. This is the case, for instance, in the prickleback fishes (Stichaeidae,
Fig. 2.18), which have been extensively studied in the German laboratory, at the
University of California, Irvine. Their series of papers reveals intriguing results, and
shall here be displayed and discussed in detail. In particular, German et al. (2004)
measured the activities of eight digestive enzymes in four species of herbivorous
and carnivorous prickleback fishes, and determined the effects of ontogeny, diet and
phylogeny on these enzyme activities. Of the four species, C. violaceus and X.
mucosus shift to a more herbivorous diet as they grow, whereas X. atropurpureus
and Anoplarchus purpurescens remain carnivores throughout life. Digestive enzyme
activities of small carnivorous juveniles were compared with those of larger wild-
caught juveniles that had consumed a natural diet and larger juveniles raised on a
high-protein animal diet. C. violaceus and both species of Xiphister showed
Fig. 2.18 Phylogenetic relationships of the family Stichaeidae based on 2100 bp of cytb, 16 s, and
tomo4c4 genes. Bayesian posterior probabilities are indicated on nodes. The species used in this
study are in bold script and indicated with **. H herbivory, O omnivory and C carnivory. Evolution
of herbivory (- - - -) and omnivory (.......) are shown. Numbers in parentheses show the number of
taxa evaluated at that branch. Boxes highlight alleged tribes within the polyphyletic subfamily
Xiphisterinae, with Esselenichthyini (top), Xiphisterini (middle), and Alectriini (bottom) all high-
lighted. Relative gut length [RGL = gut length (mm)/standard length (mm)] values are shown for
the studied fish species on right side. RGLvaried significantly among the species (P < 0.001), and
those values sharing a superscript letter are not significantly different. (From German et al. 2015,
with permission from Elsevier)
ontogenetic changes in digestive enzyme activities, whereas A. purpurescens did

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not. Despite dietary differences between X. atropurpureus and X. mucosus, these

sister taxa displayed the most similar digestive enzyme activities from ontogenetic
and dietary perspectives (high α-amylase and lipase and low trypsin and aminopep-
tidase activities), and both were more similar to C. violaceus, a member of the same
largely herbivorous clade, than either was to A. purpurescens, a member of an adja-
cent, carnivorous clade. The results support the hypothesis that phylogeny influ-
ences digestive enzyme activities in these fishes. A. purpurescens, a carnivore with
a diverse diet, showed great plasticity in enzyme activity, especially trypsin and
aminopeptidase, which were elevated in this species to the highest level among the
four species after consuming the high-protein diet. These results support the hypoth-
esis that fishes with relatively broad diets can modulate digestive enzyme activities
in response to changes in dietary composition.
Later, German et al. (2015) demonstrated that there was more than one way to be
an herbivore. There has been a convergent evolution of herbivory in this family.
Herbivory has evolved within a spectrum of digestive strategies, with two extremes
on opposite ends: (i) a rate-maximization strategy characterized by high intake,
rapid throughput of food through the gut, and little reliance on microbial digestion
or (ii) a yield-maximization strategy characterized by measured intake, slower
transit of food through the gut, and more of a reliance on microbial digestion in the
hindgut.
In the Stichaeidae, convergent evolution of herbivory occurred in C. violaceus
and X.mucosus (Fig. 2.18), and despite nearly identical diets, these two species have
different digestive physiologies. The authors found that C. violaceus has more
digesta in its distal intestine than in other gut regions, has comparatively high con-
centrations of short-chain fatty acids (SCFA, the endpoints of microbial fermenta-
tion, see also Aquatic Animal Nutrition, Volume II) in its distal intestine and a spike
in β-glucosidase activity in this gut region. These findings indicate that, when cou-
pled with long retention times of food in the guts of C. violaceus, a yield-maximizing
strategy is present in this species. X. mucosus showed none of these features and
was more similar to its sister taxon, the omnivorous X. atropurpureus, in terms of
digestive enzyme activities, gut content partitioning and concentrations of SCFA in
their distal intestines. Furthermore, the authors contrasted these herbivores and
omnivores with other sympatric stichaeid fishes, P. chirus (omnivore) and A. pur-
purescens (carnivore), each of which had digestive physiologies consistent with the
consumption of animal material.
This study showed that rate- and yield-maximizing strategies can evolve in
closely related fishes, and proves that resource partitioning plays out on the level of
digestive physiology in sympatric, closely related herbivores. It also shows that C.
violaceus and X. mucosus play different roles within their shared communities.
These two herbivorous species may optimize protein or energy consumption at dif-
ferent times of the year from each other based on what algal species are available for
consumption, and these differences may have underpinnings on the digestive level.
Fishes that pursue yield- or rate-maximization strategies, in turn, will have different
contributions to ecosystem fluxes based on what, and how much, they eat, but also
based on what they excrete back into the environment. Yield and rate maximizers
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will excrete differently, both in terms of fecal and ionic contributions (German et al.
(2015)).
German et al. (2014) tested the hypothesis that an ontogenetic dietary shift from
carnivory to herbivory or omnivory, and concomitant changes in the gut facilitating
digestion of algae, are synapomorphies3 of the tribes Xiphisterini and Esselenichthyini
in the Stichaeidae. Previous investigations have revealed that two xiphisterine prick-
lebacks – X. mucosus and X. atropurpureus (Fig. 2.18) – become herbivorous or
omnivorous, respectively, as their bodies grow larger, and that their guts show
related changes in length and function. In this study, German et al. found that, with
an increase in size, the basal member of the Xiphisterini, P. chirus, showed an
increased proportion of algae in its diet, increased activity of α-amylase and
decreased activity of aminopeptidase, all of which support the synapomorphy
hypothesis. C. violaceus, an herbivore in the Esselenichthyini, shows similar onto-
genetic changes in diet and digestive tract length and physiology, but these features
were not observed in two derived carnivores within the clade (Dictyosoma burgeri
and D. rubrimaculatum). These results indicate that herbivory is isolated to C. vio-
laceus within the Esselenichthyini. Allometric relationships of the gut length as a
function of body size generally follow diet within the Xiphisterini and
Esselenichthyini, with herbivores having the longest guts. The guts become dispro-
portionately longer than body size as the fishes grow, with omnivores to intermedi-
ate gut lengths, and carnivores the shortest. A carnivore from an adjacent clade, A.
purpurescens, had the shortest gut, which did not change in length relative to body
length as the fish grew (Fig. 2.19). Overall, these results clarify the patterns of
dietary evolution within the Stichaeidae, and lay the foundation for more detailed
studies of dietary and digestive specialization in fishes in this family.
Most young fishes lack the ability to function as herbivores. Day et al. (2011)
addressed the inevitable question: Why can’t young fish eat plants? Neither diges-
tive enzymes nor gut development preclude herbivory in the young of a stomachless
marine herbivorous fish. This fact has been attributed to two aspects of the digestive
system: elevated nitrogen demand and a critical gut capacity. Day et al. (2011) com-
pared the digestive morphology and biochemistry of two size classes of a marine
herbivore, the Eastern river garfish (Hyporhamphus regularis ardelio), to determine
what limits the onset of herbivory: preontogenetic trophic shift (pre-OTS, <100 mm)
and post-ontogenetic trophic shift (post-OTS, >100 mm) (Fig. 2.20). Two gut-
somatic indices comparing the gut length to body length (relative gut length) and
body mass (Zihler’s Index) demonstrated a significant decrease in gut length rela-
tive to body size (Fig. 2.20). There was little difference in enzyme activity between
the two classes, with juveniles showing similar levels of carbohydrase and lipase
and less protease compared with adults. This indicated that the juveniles did not
preferentially target nitrogen and were as capable of digesting an herbivorous diet.
Applies to apomorphic (evolutionary advanced) features possessed by two or more taxa in com-
3
mon (Allaby 2015)

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Fig. 2.19 Regressions of relative gut length (gut length×standard length−1) as a function of stan-
dard length (left column, plots a–c) and Zihler’s index [gut length×(10 × bodymass1/3)−1] as a
function of standard length (right column, plots d–f). This study included seven species of prick-
leback fishes with different diets from three different clades: Esselenichthyini (plots a and d),
Xiphisterini (plots b and e) and the Alectriini (plots c and f). H herbivory, O omnivory, and C
carnivory. (From German et al. 2014, with permission from Elsevier)
These findings point out the fact that neither digestive enzyme activity, nor the
length of the gut, are the best predictors of the onset of herbivory in this stomachless
fish. This lends further support to the hypothesis that halfbeak herbivory is limited
primarily by the development of the pharyngeal mill to mechanically process plants.
It is open to future studies as to whether, or not, similar phenomenona apply to other
herbivores fish species in their pre-OTS state.
2.4.2 Invertebrates
About half of the world’s animal species are arthropods associated with plants, and
the ability to consume plant material has been proposed to be an important trait
associated with the spectacular diversification of terrestrial insects. Correspondingly,
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Fig. 2.20 Left: Pre-OTS (65 mm SL) and post-OTS (187 mm SL) Hyporhamphus regularis arde-
lio, below and above respectively, with excised intestines. Intestines are positioned approximately
according to where they lie in the visceral cavity. Gut region divisions for digestive enzyme analy-
ses are marked with arrowheads. These sections are referred throughout as posterior, mid and distal
segments. Right: Morphometric analyses of gut length in relation to fish length and mass. (a)
Relative gut length (RGL) and (b) Zihler Index (ZI) in Hyporhamphus regularis ardelio. Mean
RGL in pre-OTS fish was 0.59 ± 0.01and significantly decreased to 0.49 ± 0.01 in post-OTS fish.
Mean ZI in pre-OTS fish was 3.24 ± 0.06 which significantly decreased to a mean of 2.44 ± 0.05
(n = 10) in post-OTS fish. (From Day et al. 2011, with permission from Elsevier)
Poore et al. (2017) reviewed the phylogenetic distribution of plant feeding in
Crustacea, the other major group of arthropods that commonly consume plants, to
estimate how often plant feeding has arisen, and to test whether this dietary transi-
tion is associated with higher species numbers in extant clades. They present evi-
dence that at least 31 lineages of marine, freshwater, and terrestrial crustaceans
(including 64 families and 185 genera) have independently overcome the challenges
of consuming plant material. These plant-feeding clades are, on average, 21-fold
more speciose than their sister taxa, indicating that a shift in diet is associated with
increased net rates of diversification – or, herbivory as evolutionary advantageous
strategy. In contrast to herbivorous insects, most crustaceans have very broad diets,
and the increased richness of taxa that include plants in their diet likely results from
access to a novel resource base, rather than host-associated divergence.
2.5 Starvation and Gut Morphology

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The gastrointestinal (GI) tract and associated organs are some of the most metabolically
active tissues in an animal. Hence, when facing food shortages or poor food quality,
an animal may reduce the size and function of their GI tract to conserve energy.
German et al. (2010) investigated the effects of prolonged starvation and varying
food quality on the structure and function of the GI tract in a detritivorous catfish,
Pterygoplichthys disjunctivus (Fig. 2.21), native to the Amazonian basin, which
experiences seasonal variation in food availability. After 150 days of starvation, or
consumption of a wood-diet too low in quality to meet their energy needs, the fish
reduced the surface area of their intestines by 70 and 78%, respectively. They also
reduced the microvilli surface area by 52 and 27%, respectively, in comparison to
wild-caught fish consuming their natural diet and those raised in the laboratory on a
high-quality algal diet (Fig. 2.22). Intake and dietary quality did not affect the pat-
terns of digestive enzyme activity along the guts of the fish, and the fish on the low-
quality diet had similar mass-specific digestive enzyme activities to wild-caught
fish, but lower summed activity when considering the mass of the gut. Overall, P.
disjunctivus can endure prolonged starvation and low food quality by downregulat-
ing the size of its GI tract.
The diet did not only affect the overall mass of the gut, but the ultrastructure as
well. The wild-caught fish and those that ate algae in the laboratory had larger intes-
tinal folding patterns than the fish consuming wood, or those that were starved in the
laboratory (Fig. 2.22).
The Japanese common sea cucumber (Apostichopus japonicus) undergoes sea-
sonal inactivity phases (estivation, a unique strategy of marine invertebrates for
summer survival in response to hot conditions) and ceases feeding during the sum-
mer months (Hoyoux et al. 2009). Gao et al. (2008) used this sea cucumber species
as a model in which to examine phenotypic plasticity of the digestive tract in
response to food deprivation. They measured the body mass, gross gut morphology
and digestive enzyme activities of A. japonicus before, during and after the period
of inactivity to examine the effects of food deprivation on the gut structure and func-
tion of this animal. At the peak of inactivity and lack of feeding, A. japonicus
Fig. 2.21 Vermiculated
sailfin catfish
(Pterygoplichthys
disjunctivus). (Courtesy of
FiMSeA/ffish.asia)
2.5 Starvation and Gut Morphology 43
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Fig. 2.22 Cross-sections of proximal, mid and distal intestinal tissue of Pterygoplichthys disjunc-
tivus consuming different diets. Tissues were stained with a modified Masson’s trichrome. Scale
bars are labeled on each photograph. (From German et al. 2010, with permission from Elsevier)
decreased its body mass, gut mass and gut length by 50%, 85%, and 70%,
respectively, in comparison to values for these parameters preceding the inactive
period (Fig. 2.23, left). The activities of amylase, cellulase and lipase decreased
significantly, whereas pepsin activity increased twofold during the inactive phase.
Alginase and trypsin activities were variable and did not change significantly across
the experiment. Principal Component Analysis (PCA), utilizing the digestive
enzyme activity and body size index data, divided the physiological state of this
cucumber into four phases (Fig. 2.23, right): an active stage, prophase of inactivity,
peak inactivity and a reversion phase. A. japonicus clearly exhibits phenotypic plas-
ticity – or life cycle staging – of the digestive tract during its annual inactive period,
elicited by elevated water temperature. Further work is necessary to evaluate why
specific enzymes behaved in different fashions (e.g., why pepsin activity spikes in
the feeding cessation period, and why lipase activity drops during reversion), and to
further characterize the abiotic triggers of periods of inactivity in this species.
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Fig. 2.23 Left: The digestive tracts and respiratory trees of Apostichopus japonicus (a) in active
phase and (b) in inactive phase, showing the significant atrophy of the digestive tract in periods of
inactivity. Right: Loadings from a corresponding Principal Component Analysis (PCA) model,
showing the correlation between the digestive enzymes, relative gut mass (RGM), relative gut
content mass (RGCM) and Zihler’s index (ZI) in A. japonicus. (From Gao et al. 2008, with permis-
sion from Elsevier)
Fig. 2.24 Venn diagrams showing the overlap in differential protein expression between the three
groups of sea cucumbers (NA non-estivation, DA deep estivation and AA arousal after estivation).
Shown is the total number of significantly differentially expressed proteins, as well as the numbers
of upregulated and downregulated proteins. (From Chen et al. 2016, with permission from Elsevier)
To figure out the underlying pathways behind the estivation of A. japonicus,

Chen et al. (2016) used a tandem mass tag-coupled LC-MS/MS approach to identify
and quantify the global proteome expression profile over the estivation-arousal
cycle. From the intestine of the sea cucumber, a total of 3920 proteins were identi-
fied. Among them, 630 proteins showed significant differential expression when
comparing three conditions of the animals: non-estivating (active), deep-estivation
(at least 15 days of continuous estivation) and arousal after estivation (renewed
moving and feeding). Sea cucumbers in deep estivation showed substantial differen-
tially expressed proteins (143 upregulated and 267 downregulated proteins com-
pared with non-estivating controls) (Fig. 2.24). These differentially expressed
proteins indicate that protein and phospholipid are likely major fuel sources during
hypometabolism, and a general attenuation of carbohydrate metabolism was
observed during deep estivation.
2.6 Trophic Positions: An Omnivores’ Dilemma? 45
Differentially expressed proteins also provided the first global picture of a shift
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in protein synthesis, protein folding, DNA binding, apoptosis, cellular transport and
signaling, and cytoskeletal proteins during deep estivation in sea cucumbers. A
comparison of arousal from estivation with deep estivation revealed a general rever-
sal of the changes that occurred in estivation for most proteins. Western blot detec-
tion further validated the significant upregulation of HSP70 and downregulation of
methyltransferase-like protein 7A-like in deep-estivation. Overall, these papers
indicated that there is substantial post-transcriptional regulation of proteins during
the estivation arousal cycle in sea cucumbers. However, the authors do not discuss
the function, especially, of the methyltransferase. This enzyme is involved in epi-
genetic processes, which are central in the development of individuals, but also in
the transgenerational non-genetic inheritance.
Recently, Xu et al. (2018) confirmed that cell loss by apoptosis is one major
mechanism in the intestinal degeneration that occurs during the estivation in the
Japanese sea cucumber. Furthermore, cell death by apoptosis is extremely rapid in
sea cucumbers undergoing thermal stress, and is usually completed within 20 days.
2.6 Trophic Positions: An Omnivores’ Dilemma?
Trophic classification is frequently based on functional morphology, gut content

analyses and foraging observation – sometimes even exclusively on functional mor-
phology. There are, however, many examples that these classification of trophic
niches (feeding categories) are often too rigid or too coarse (Choat and Clements
1992), because many species are facultative herbivores or facultative carnivores
with strong tendencies to omnivory. In other words, omnivory occurs much more
frequently in ecosystems, but is classified as carnivory. Similarly, the functional role
of omnivores as predators has often been underestimated in complex food webs, as
hypothesized by Hellmann et al. (2013). These authors studied the omnivores
Gammarus pulex and Hydropsyche spp. and found that the trophic positions of
Gammarus and Hydropsyche shifted between winter and summer. The stable iso-
tope values of the 2 omnivores were aligned with secondary consumers in late win-
ter, whereas the omnivores were similar to primary consumers with trophic position
values of nearly two in summer (Fig. 2.25). Gammarus pulex is often described as
a shredder and grazer, and previous stable-isotope analysis results indicated that
Gammarus spp. is a primary consumer. Nevertheless, results of laboratory studies
showed a strong potential for predation and cannibalism. Hellmann et al. (2013)
found only six probable resources for G. pulex: chironomid and plecopteran larvae,
gammarids, fine particulate organic matter, alder leaves and periphyton (Fig. 2.26,
left). Gut-content analysis of Hydropsyche spp. showed that Chironomidae and lar-
vae of small stonefly taxa and Heptageniidae were potential prey taxa. Fine particu-
late organic matter and filamentous green algae also were found in the guts
(Fig. 2.26, right).
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Fig. 2.25 Mean stable-isotope values of the strict invertebrate predators (Rhyacophila fasciata,
Isoperla grammatica, and Dugesia gonocephala) and the omnivores Hydropsyche spp. and
Gammarus pulex in winter (black symbols) and summer (white symbols). The lines indicate the
trophic position (TP) of different consumer levels (TP 2 = primary consumers, TP 3 and
higher = nonprimary consumers). (From Hellmann et al. 2013, courtesy of The University of
Chicago Press)
The preference of omnivores for animals as prey depends on the season or the
specific situation of the food web structure in each ecosystem – or “an open door
may tempt a saint”. One of many tempted saints, Nile tilapia (Oreochromis nilotica)
does not scorn Daphnia as food. Okun et al. (2008) studied trophic cascades in a
tropical water body. Trophic cascades occur when predators in a food web suppress
the abundance or alter the behavior of their prey, thereby releasing the next lower
trophic level from predation (or herbivory, if the intermediate trophic level is an
herbivore). For example, if the abundance of large piscivorous fish is increased in a
lake, then the abundance of their prey, such as smaller fish that eat zooplankton,
should decrease. The resulting increase in zooplankton should, in turn, cause the
biomass of its prey, phytoplankton, to decrease. Based on the fact that strong trophic
cascades have been well documented in pelagic food webs of temperate lakes, Okun
et al. (2008) hypothesized that, because of limited available evidence, strong cas-
cades are less typical in tropical lakes. Therefore, the authors measured the effects
of omnivorous Nile tilapia on planktonic communities and water transparency of a
small man-made tropical lake. They found that omnivorous tilapia significantly
decreased the abundance of large Cladocerans, increased the abundance of small
algae and decreased water transparency, as predicted by trophic cascade theory.
Therefore, omnivory was not a sufficient factor to prevent a trophic cascade in this
tropical pelagic community, although the cascade effect was weaker than reported
from many north temperate, nutrient-rich lakes.
Even the classification of herbivores is often too coarse, or even incorrect. In a
comprehensive nutritional survey of herbivorous reef fishes, several nominally her-
bivores prefer carnivorous food. The species with diets dominated by planktonic
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Fig. 2.26 Mean stable-isotope signatures of the consumer Gammarus pulex (left) and Hydrospyche
spp. (right) and their possible resources measured by stable-isotope analysis (SIA) in April (A) and
October (B). All resource values were corrected for trophic fractionation (D13C = 1.3%and
D15N = 2.2%). Consumer values (□) were not corrected. The black lines connect G. pulex and
Hydrospyche with its probable resources (according to Euclidean distances). FPOM = fine particu-
late organic matter. In right A, note similar chironomid, plecopteran and consumer signatures.
(From Hellmann et al. 2013, courtesy of The University of Chicago Press)
animal material comprise Naso annulatus (Fig. 2.27), N. brevirostris, N. hexacanthus

and N. vlamingii (Fig. 2.27) (Choat et al. 2002).
Adult silver mylossoma (Mylossoma duriventre, Fig. 2.29) in the Amazonian
floodplain is often classified as frugivorous (Hamlett (1992); Correa et al. (2016)),
due to high share of fruits and seeds in the stable isotope analyses and the stomach
content (Fig. 2.28). However, a closer look shows that the stomach content was also
comprised of a visible content of terrestrial and aquatic invertebrates, as well as
some zooplankter (Fig. 2.28 below), indicating that M. duriventre is, in fact, an
omnivore.
Analyzing Mylossoma from other várzea lakes of the huge Amazone floodplain,
Oliveira et al. (2006) and Rebelo et al. (2010) classified M. duriventre as omnivo-
rous. For instance, Oliveira et al. (2006) found that during receding and low water
situations, the stomachs contained high shares of insects (Fig. 2.29). In addition,
Rebelo et al. (2010) identified fishes and zooplankton during the low water situation
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Fig. 2.27 Images of whitemargin unicornfish (Naso annulatus) and bignose unicornfish (Naso
vlamingii) (courtesy of Petersen and Kubica, Wikimedia)
in 12.5% of the stomachs. Again and unequivocally, the trophic position of

Mylossoma is omnivorous. The same oversimplification can be found with the char-
acins Colossoma macropomum and Piaractus brachypomus, which are classified as
frugivore (Anderson et al. 2011; Lucas 2008), whereas in the analyses by Rebelo
et al. (2010), the gut content consisted to high percentage of spiders, insects and
fishes during low flood situations. Oliveira et al. (2006) added zooplankton and
mollusks to the menu of C. macropomum. These examples clearly show that these
fishes are omnivorous, but with seasonally varying preferences for specific food
sources, such as plant, wood and seed during high flow situations when these food
items are available. The line of evidence can arbitrarily be prolonged. However,
these examples suffice to point out the omnivores’ dilemma. Based on a seasonal
bias of the study, or a scientific bias based on the background of the researcher,
omnivores are often incorrectly classified as herbivores, frugivores or carnivores.
Simply, they are omnivores. Furthermore, one can easily predict that imprecise, or
even wrong, classifications of trophic positions of marine and freshwater fishes are
one major cause for the high standard deviations in Fig. 2.2.
Similar concerns apply to herbivorous marine fishes as with the Amazonian fish
species. Clements et al. (2017) conclude that the above nominal classification of
trophic positions for herbivorous fishes of coral reefs is too coarse. Coral reef eco-
systems are remarkable for their high productivity in nutrient-poor waters. A high
proportion of primary production is consumed by the dominant herbivore assem-
blage, teleost fishes, many of which are the product of recent and rapid diversifica-
tion (refer to Chapter 7 ‘Trophic Diversification an Speciation’). The synthesis of
the trophodynamics of herbivorous reef fishes indicates that current models under-
estimate the level of resource partitioning, and thus trophic innovation, in this
diverse assemblage. The authors also examined several lines of evidence, including
feeding observations, trophic anatomy, and biochemical analyses of diet, tissue
composition and digestive processes to show that the prevailing view of parrotfishes
as primary consumers of macroscopic algae is incompatible with available data.
Instead, the data were consistent with the hypothesis that most parrotfishes are
microphages that target cyanobacteria and other protein-rich autotrophic microor-
ganisms that live on (epilithic), or within (endolithic), calcareous substrata, are epi-
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Fig. 2.28 Above: Carbon and nitrogen stable isotope ratios (mean ± SD) of a frugivorous fish
(Mylossoma duriventre) and potential food sources (abundant primary producers) in the flooded
forest (várzea) of the Tarapoto Lakes complex, western Amazonia. Small black diamonds repre-
sent isotopic signatures of individual fish. Fish positions were adjusted to account for trophic
fractionation. Dashed polygon connects mean source values; dotted oval encloses sources within
approximately one standard deviation of the mean. Below: Diet of Mylossoma duriventre assessed
from the volumetric analysis of stomach contents of 55 individuals caught during the rising waters
season of 2007 in the flooded forest (várzea) of the Tarapoto Lakes complex, western Amazon
Basin. (From Correa et al. 2016, courtesy of biota neotropica)
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Fig. 2.29 Left: Frequency (%) of the main food items identified in the stomachs of Mylossoma
duriventre. Numbers of analyzed fish are given on top of the set of bar graphs of each season: ris-
ing, high, receding and low. Columns from left: Plant material, fuits/seed, algae, insects, zooplank-
ton and fish. (From Oliveira et al. 2006 with permission from Springer). Right: Image of
Mylossoma duriventre. (From Francis de Castelnau: Expédition dans les parties centrales de
l’Amérique du Sud, de Rio de Janeiro à Lima et de Lima au Para sous la direction 1856)
phytic on algae or seagrasses, or endosymbiotic within sessile invertebrates. This

novel view of parrotfish feeding biology provides a unified explanation for the
apparently disparate range of feeding substrata used by parrotfishes, and integrates
parrotfish nutrition with their ecological roles in reef bioerosion and sediment trans-
port. Accelerated evolution in parrotfishes can now be explained as the result of: (1)
the ability to utilize a novel food resource for reef fishes, i.e. microscopic autotrophs
and (2) the partitioning of this resource by habitat and successional stage. The closer
look to the trophic niche most likely does not apply only to parrotfishes, but to many
more aquatic animals.
The trophic function of omnivorous invertebrate is less well documented than in
fishes. Nevertheless, it turns out that being omnivorous is highly advantageous. For
instance, Snyder et al. (2015) reported recently that diet-switching by omnivorous
freshwater shrimps (Macrobrachium carcinus and M. olfersi) diminishes differ-
ences in nutrient recycling rates and body stoichiometry across a food quality gradi-
ent. In particular, the P content of shrimp did not change despite large differences in
P content of their likely food resources (Fig. 2.30). Also, shrimp P recycling rates
did not increase in high-P streams with P-enriched food resources. Stable isotope
results combined with a change in body nutrient content indicate that shrimp show
different diet choices over the P gradient, feeding at a higher trophic level in low-P
streams. This dietary shift may partially compensate for the lower P content in a
given food resource in the low-P streams. Therefore, diet switching appears to be an
important strategy for omnivorous shrimps in correcting the stoichiometric imbal-
ance between food resource and consumer biomass.
Human economy and nutrition do equal ecology. As omnivores, the most unselec-
tive eaters, humans are faced with a wide variety of food choices, resulting in a
dilemma, intriguingly described by Pollan (2006). In nature, by contrast, above the
herbivore trophic level, food webs are characterized as a tangled web of omnivores,
as identified in a meta-analysis (Thompson et al. 2007). Omnivory has been assumed
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Fig. 2.30 Log–log relationship between phosphorus (P) excretion and dry mass of individual
shrimp of two species (Macrobrachium carcinus and Macrobrachium olfersi) in four study streams
with different levels of soluble reactive P (SRP) at La Selva Biological Station, Costa Rica.
Excretion rate was measured as lg TDP shrimp 1 h1, and biomass was measured in grams. The
symbols refer to sites: triangles = Sabalo, plusses = Salto-5, crosses = Sura-30 and diamonds = Sura-
60. (From Snyder et al. 2015, with permission from Wiley)
to stabilize consumer-resource interactions, diffuse top-down influences through

food webs and alter the expression of top-down control. Trophic-level-based con-
cepts such as trophic cascades may apply to systems with short food chains, but they
become less valid as food chains lengthen. Some recent examples may illustrate this
issue.
To reconstruct historical trophic interactions patterns along a gradient of
resources, Yao et al. (2016) used stable isotope analysis of archived scales of two
pelagic single-chain omnivorous fish species, bighead carp (Hypophthalmichthys
nobilis) and silver carp (Hypophthalmichthys molitrix). The authors reported that,
although bighead carp and silver carp utilize similar resources from the pelagic food
chain, they can coexist and persist not only by regulating their trophic position and
trophic dissimilarity, but also by regulating trophic niche width (Fig. 2.31). This
figure presents a schematic illustration of trophic interaction in a pelagic food
web dominated by bighead and silver carps. These coexisting omnivores in the
pelagic food chain regulate their trophic dissimilarity (from A to B), or niche width
a
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Trophic level 3 Bighead
silver carp
Zooplankton
Trophic level 2
Phytoplankton
Trophic level 1
Silver carp Bighead

b Trophic level 3
A B C
Trophic level 2
Fig. 2.31 Schematic illustration of trophic interaction in a pelagic food web dominated by big-
head and silver carps. In this diagram, phytoplankton represent trophic level 1 and zooplankton
trophic level 2, which pass to bighead carp and silver carp through two distinct trophic transfers.
The authors hypothesized that these coexisting omnivores in the pelagic food chain regulate their
trophic dissimilarity (from A to B), or trophic niche width (from B to C) to accommodate inter-
specific resource sharing (i.e. zooplankton and phytoplankton), but these two regulatory mecha-
nisms may not be mutually exclusive (from A to C). The dots represent the isotope position of the
fish. (From Yao et al. 2016, with permission from the Ecological Society of Japan)
(from B to C), to adapt to inter-specific resource sharing (i.e. zooplankton and

phytoplankton), which may not be mutually exclusive (from A to C). These possible
mechanisms suggest that variable dietary contributions of phytoplankton and zoo-
plankton would regulate their trophic niche partitioning, and facilitate the coexis-
tence of both omnivorous fish.
Omnivorous fish often exhibit flexible foraging strategies, which is closely
related to the availability of ecologic context. Yao et al. (2016) found a positive
relationship between trophic dissimilarity and zooplankton density, which may
indicate that the competitive interactions induce strong top-down effects on zoo-
plankton, and/or that high zooplankton availability releases the between-population
trophic interaction through bottom-up effect. The trophic niche width of bighead
2.7 Concluding Remarks 53
carp was positively related with zooplankton availability, probably reflecting that
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the niche of an omnivore at a higher trophic position is more sensitive to high qual-
ity resources. The results indicate how different aspects of the trophic partitioning
of coexisting omnivores may be regulated by different ecological contexts. These
alternatives are not mutually exclusive, and further theoretical work should include
both these mechanisms to re-evaluate the effects of omnivory on food web proper-
ties. It can be hypothesized that this phenomenon of resource-sharing of omnivores
is more wide-spread than currently anticipated; omnivory may contribute to food
web dynamics not only through the regulation of trophic dissimilarity in a fine scale
niche, but also through the regulation of trophic niche properties, such as niche
segregation and niche width.
In several papers, Crossman and coworkers characterized the nominal herbivores
of coral fishes, and detected that detritus and plankton are valuable food sources to
“grazing” fishes on coral reefs (Crossmann et al. (2005; 2001)). These fishes are
omnivores, planktivores or even detrivores. Crossman et al. (2005) investigated the
composition of dietary nutrients targeted by nominally herbivorous acanthurids
(surgeonfishes) and scarids (parrotfishes). The most significant finding of this study
was that there are major differences in the dietary macronutrients targeted by nomi-
nally herbivorous fishes on coral reefs. Those feeding on detrital aggregates con-
sume and assimilate a diet high in total protein amino acids, and appear to have a
limited capacity for fermentation. This is in contrast to algivorous species, which
consume and assimilate diets high in carbohydrate, and appear to have a high capac-
ity for fermentation. This study indicates that there are major differences in the food
resources targeted by detritivorous and algivorous fish species on coral reefs.
Does a dilemma of aquatic omnivores exist? The answer simply is that yes, it
does. However, in contrast to Pollan’s book and postulate, the dilemma does not lie
in the consumer himself. Rather it lies within the perception of the aquatic consum-
ers and reflects different biases and an incomplete understanding of the trophic
structure and trophodynamics of aquatic food webs. Omnivores often hide behind
nominal herbivores or nominal carnivores. Omnivory appears to be the prevailing
trophic type in many (Thompson et al. 2007), if not all, aquatic systems with spe-
cies- or ontogenesis-depending tendencies to herbivory, detritivory or carnivory,
depending on the specific circumstances in each system and the season.
2.7 Concluding Remarks
The diet determines gut length and structure, as well as the performance of the
animals.
• Due to apparently low food qualities, herbivores fishes and aquatic invertebrates
have the longest intestine; carnivorous species, on the other hand, the shortest
ones. Due to high fiber contents and low energy provision, herbivory requires
long intestines, and appeared to be a minor nutritional strategy.
• This simple relationship, however, is often overlain by coarse, or even incorrect,

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trophic classifications. Therefore, many published relationships are somewhat

fuzzy, with poor statistical significances. Most animals belong to omnivores.
A reclassification is indispensable, combined with the recalculation of the cor-
responding regressions.
• Taken the ecological frame into consideration, herbivory no longer appears to be
the minor nutrient and energy acquisition strategy. So far, this can be seen with
both freshwater invertebrates and reef fishes. Studies to come will show that this
hypothesis will prove valid for further biocenoses.
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enzymes nor gut development preclude herbivory in the young of a stomachless marine herbivo-
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Drewe KE, Horn MH, Dickson KA, Gawlicka A (2004) Insectivore to frugivore: ontogenetic
changes in gut morphology and digestive enzyme activity in the characid fish Brycon gua-
temalensis from Costa Rican rain forest streams. J Fish Biol 64(4):890–902. https://doi.
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Elbal MT, Hernandez MPG, Lozano MT, Agulleiro B (2004) Development of the digestive tract
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Chapter 3
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The Intestinal Microbiota – ‘Your Eating

Feeds a Plethora of Guests’ and ‘This Plethora
of Guests Determines Who You Are and How
Well You Do’
Abstract The gastrointestinal tract of fishes and aquatic invertebrates provides

home to an abundant and highly numerous and diverse community of microbes
(bacteria, cyanobacteria, yeasts). This microbiota can be considered as a ‘forgotten
organ’ or even a symbiotic ecosystem that significantly expands the ecological
niche of the germfree conspecifics. The gut microbiota provides the host with a
wide range of catabolic capabilities, improves growth performance, increases envi-
ronmental stress resistance and longevity, elevates the innate immunity, likely
impacts the circadian clock, and aids reproduction. Furthermore, it is required for
normal neurobehavioral development and has the potential to determine the gender.
The composition of the microbiota is controlled by species-specific genetic factors
of the host, ontogenetic developmental stage, diets, and environmental factors. The
question of whether there exists a core microbiome in the intestine of fishes and
aquatic invertebrates is still controversially debated; however, the majority of
reports point out the existence of a core microbiome. Several papers demonstrate a
reduced microbiome diversity in captivity-raised as compared to wild animals.
Bacterial populations are initiated with a few founding bacteria and grow to many
thousands, whereby the growth exhibits dynamics typical of resource-limited
growth. The host’s gut environment produces distinct spatiotemporal patterns.
Many bacterial products and metabolites may themselves be directly or indirectly
causative of behavioral change; for instance, dietary supplementation with
Lactobacillus plantarum reduces the anxiety-related behavior. Similarly, there is a
strong guess that the gut microbiota determines the success of invasive species.
Since it is evident that the microbiota has enormous potential for health of farmed
animals, strategic considerations are accumulating as to how microbial communi-
ties can be applied to mitigate emerging diseases in aquaculture.
For the sake of improved readability, not only the names of genera and species, but also of bacterial
phyla, classes, and families are written in italics.

https://doi.org/10.1007/978-3-319-91767-2_3
62 3 The Intestinal Microbiota – ‘Your Eating Feeds a Plethora of Guests’
All animals on Earth have developed in a pre-existing microbial world and thus
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have co-evolved in constant interaction with microbes. In particular, alimentary

canals of all animals are colonized by different microbial cells (i.e., gut or intestinal
microbiota) (Yoon et al. 2015). The gut microbiota is a fascinating phenomenon. A
simple definition is that it is a complex community of microorganisms that live in
the digestive tract of animals. The gut microbiome refers to the genomes of the gut
microbiota. For a long time, the gut and its inhabitants were not well perceived.
Rather, they were considered more or less dirty and not worthy of being studied in
detail unless a certain gut disease affected the host. The excrement was to be diverted
as organic waste. The gut microbiome was not regarded as providing a surplus of
fast growing microbes with the potential to benefit the host.
To date, the intestinal microbiota can be considered as a specific ecosystem,
namely a symbiosis. The host provides shelter, an appropriate climate, water, and
substrates to digest aerobically, as well as anaerobically. The microbiota, in turn,
provides energy from the fermentation of undigested carbohydrates and subse-
quently short-chain fatty acids and other functional compounds, such as vitamins
and most likely neuro-active metabolites (Phelps et al. 2017). In short, the gastroin-
testinal tract is home to an abundant and highly diverse community of microbes that
have evolved important nutritional and physiological dependencies among them-
selves and with the host.
To facilitate the understanding of gut microbiome structure and function, Shapira
(2016) proposed a multilayered structure model (Fig. 3.1): at one end, a core micro-
biota of host-adapted microbes reproducibly included in gut microbiota assembled
from diverse environments and determined by genetic factors; at the other end, a
flexible pool of microbes dependent on environmental diversity and on external
conditions. Between the two ends occur a range of intermediates, including microbes
associated to varying degrees with the host, and variably affected by the environ-
ment. In gut microbiota, the abundance of certain bacterial families is strongly influ-
enced by host genetics representing members of the core microbiota (Li et al.
2017a). Others are affected by diet and environment, indicating that it is part of the
flexible microbiota. It should also be noted that, while the composition of the micro-
biota flexible pool is proposed here to be shaped by environmental conditions, it
could equally be shaped by drift, increasing inter-individual variability before any
selection by environmental factors (Bordenstein and Theis 2015). As we shall see
below, further factors shaping the flexible microbiota are host determinants and
host’s ontogenetic stage.
The host carries microorganisms which exceed the number of cells of the host by
several orders of magnitude. The metabolic activities performed by these bacteria
resemble those of an organ, a ‘forgotten’ organ as O’Hara and Shanahan (2006)
accurately termed it. To get an impression of this forgotten organ, we are going to
consider a striking recent experiment with mammals.
The assumption that fearful or courageous behavior of an individual is a more or
less stable trait is outdated; rather, it appears to be a function of the gut microbiome.
Bercik et al. (2011) carried out a convincing experiment – an umbrella experiment.
They authors tested one mouse strain (BALB/c) displaying more timid and anxious
3 The Intestinal Microbiota – ‘Your Eating Feeds a Plethora of Guests’ and ‘This… 63
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Fig. 3.1 The multilayered hologenome. A schematic of the proposed model showing the host and
its gut microbes, each with its own genome, and colored according to the degree of host adaptation.
The gut microbiota could be divided to three major parts: (1) vertically transmitted core taxa,
which are coevolved with the host and important for basic host functions, such as developmental
programs, or essential adaptations (e.g., herbivory); (2) members of the flexible microbe pool,
which could be acquired through horizontal transmission and exchanged with the environment, but
are better adapted for host colonization than environmental strains, and can facilitate host adapta-
tion to new niches or to environmental conditions; and (3) environmentally acquired microbes,
which are advantageous in facilitating host adaptation to new conditions, but are not adapted to the
host. (From Shapira 2016, with permission from Elsevier)
behavior compared to another, braver (NIH-Swiss) strain. They investigated

whether the behavioral phenotype of a mouse was altered by transferring intestinal
microbiota from another mouse strain with a different behavioral profile.
Astonishingly, after a given time of microbial colonization, the braver strain dis-
played substantially less exploratory behavior than their untreated peers; the time
taken (latency) to step down from a small podium significantly increased (Fig. 3.2),
but did not reach that of the fearful mice. In contrast, the more timid and anxious
strain, now colonized by the microbiota from the braver strain, displayed markedly
more exploratory behavior than their untreated peers; their latency time decreased,
but remained clearly greater than that of the brave mice.
This experiment demonstrates the existence and significance of a gut–microbi-
ota–brain axis. The intestinal microbiota strongly influences the behavior independently
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Fig. 3.2 Intestinal microbiota transfer differentially affects behavior of recipient mice. Step-down
test from a podium measured as period of latency in NIH Swiss and BALB/c mice at 3 weeks after
colonization. SPF: specific pathogen-free; GF+: germ-free mice colonized either with NIH Swiss
(white columns) or BALB/c (grey columns) microbiota. (From Bercik et al. 2011, with permission
from Elsevier)
of the autonomic nervous system. This notable study has prompted several studies
with fish that will be discussed below (Sect. 3.2.4.2 Behavior). Furthermore, the gut
microbiota has strong influence on gastrointestinal-specific neurotransmitters and
inflammation. In other words, the borrowed genes and metabolic pathways of the
numerous bacteria, protozoa, and yeasts in the gut of the hosts clearly expand the
autochthonous properties of the host itself. They are fueled by the ingested food.
Therefore, many more surprises can be expected concerning how the microbiota
influences life history traits. Consequently, it can already be concluded from this
umbrella experiment that the intestinal microbiota impacts on further traits, not only
in mammals, but also in other animals. These critical traits are the immune and
metabolic functions of the host; the corresponding body of evidence for fish,
although still comparably small, has been fast growing over the past few years. As
a poorly functional gut microbiota can be detrimental to host survival and fitness,
this internal ecosystem is emerging as a ‘novel’ trait under strong natural selection.
Furthermore, we have to keep in mind that the ecological niches of fish and aquatic
invertebrates are much more diverse than those of small laboratory rodents; there-
fore, a much more detailed picture of the intestinal microflora and its impact on
aquatic animals than for laboratory model animals may emerge.
The gut microbiota provides the host with a wide range of catabolic capabilities
for the degradation of plant-derived polysaccharide biomass that are absent (Flint
3 The Intestinal Microbiota – ‘Your Eating Feeds a Plethora of Guests’ and ‘This… 65
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Fig. 3.3 Intestinal concentration of short-chain fatty acids (SCFA) in marine and freshwater fish
compared to gut transit time. Although gut transit time varies with both temperature and fish size,
it is clear that species with high levels of gastrointestinal fermentation are mainly herbivorous
marine species. Species line drawings are scaled to the relative length of fish used in each study,
and points are color-coded by diet category. Species identities are as follows: (1) Mozambique
tilapia (Oreochromis mossambicus), (2) Nile tilapia (O. niloticus), (3) American gizzard shad
(Dorosoma cepedianum) (4) Central stoneroller (Campostoma anomalum) (5) Largescale
stoneroller (C. oligolepis) (6) Bluefin stoneroller (C. pauciradii) (7) Grass carp (Ctenopharyngodon
idella) (8) Common carp (Cyprinus carpio) (9) River chub (Nocomis micropogon), (10) Parore/
Black bream (Girella tricuspidata), (11) Zebra-perch (Hermosilla azurea), (12) Silver drummer
(Kyphosus sydneyanus), (13) Butterfish (Odax pullus), (14) Steephead parrots (Chlorurus micro-
rhinos), (15) Hypostomus pyrineusi, (16) Dusky Panaque (Panaque nocturnus), (17) Royal pan-
aque (P. cf. nigrolineatus), (18) Vermiculated sailfin catfish (Pterygoplichthys disjunctivus), (19)
Monkeyface prickleback (Cebidichthys violaceus). (From Clements et al. 2014, with permission
from Wiley)
et al. 2008). In particular, herbivorous marine fish species with high intestinal
short-chain fatty acid (SCFA) concentrations rely on gut microbes to convert unas-
similable algal constituents, such as mannitol, to metabolically useful SCFAs, and
these fish display metabolic specialization to hindgut fermentation. The compari-
son of different feeding types shows that herbivorous and omnivorous freshwater
fish tend to display shorter gut transit times and thus lower levels of SCFA in the
gut than some of their marine counterparts (Fig. 3.3). This may reflect the differ-
ences in herbivorous diets between the two systems, especially in relation to carbo-
hydrate composition, but to date no freshwater herbivorous fish species have been
identified that appear to rely on hindgut fermentation to the extent that marine
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Fig. 3.4 Locomotor hyperactivity in axenic zebrafish larvae relative to conventionalized controls
at 10 days past fertilization (dpf). Axenic (AX) and conventionalized (AC1) larvae were subjected
to behavioral testing at 10 dpf. Line graphs showing distance moved (cm) during each 2-min
period. White and black bars along the x-axis represent the light and dark periods, respectively, and
represent mean movement during each 10-min light or dark period. Different letters indicate sig-
nificance (P < 0.05). Axenic (blue) and conventionalized (green) data are shown. Error bars repre-
sent SEM. (From Phelps et al. 2017, courtesy of Scientific Reports)
herbivorous species do (Clements et al. (2014) and references therein). In fact, the
literature provides little evidence that gastrointestinal fermentation supplies a major
component of daily energy requirements in freshwater fish and, in particular, that
cellulose is a major substrate for gut microbiota in these animals (Clements et al.
2014; German et al. 2014).
Another major function of the gut microbiota is modulation of the susceptibility
to infections and parasites, with a significant reduction of susceptibility in most
cases. Hereby, colonization of the indigenous microbiota on the surface of the intes-
tine plays an important role in the formation of a commensal community and colo-
nization resistance against invading pathogens (Yoon et al. 2015).
Consistent with findings in mammals (Cryan and Dinan 2012; Erny et al. 2015),
Phelps et al. (2017) recently showed that microbial colonization during early life is
also required for normal neurobehavioral development in fish. Axenic zebrafish
exhibited hyperactivity compared to conventionally colonized controls (Fig. 3.4).
Colonization of axenic embryos at 1 dpf with individual bacterial species Aeromonas
veronii or Vibrio cholerae was sufficient to block locomotor hyperactivity at 10 dpf;
exposure to heat-killed bacteria was not sufficient to block hyperactivity in axenic
larvae. Impairment of host colonization using antibiotics also caused hyperactivity
in conventionally colonized larvae. This finding supports the concept that antibiot-
ics and other environmental chemicals may exert neurobehavioral effects via dis-
ruption of host-associated microbial communities.
Most gut microbiota of vertebrates are composed of approximately 500–1000
different bacterial species, over 98% of which are represented by two dominant
phyla, Bacteroidetes and Firmicutes. The structure of gut microbiota depends on the
external environment, internal environmental conditions, feeding source and habit,
host phylogeny and genetics, and other factors such as infections (Li et al. 2017a;
Yoon et al. 2015 and references therein) (Fig. 3.5).
In contrast to vertebrates, relatively lower bacterial diversity is usually found in
invertebrates; insect gut microbiota is dominated by Proteobacteria and Firmicutes
representing approximately 60% and 20% of all phylotypes, respectively. Gut
3.1 Invertebrates 67
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Fig. 3.5 A combination of biotic and abiotic factors (red arrows), such as genotype, fish physio-
logical status (including properties of the innate and adaptive immune systems), fish pathobiology
(disease status), fish lifestyle (including diet), fish environment and the presence of transient popu-
lations of microorganisms affect the composition, function and metabolic activity of the fish gut
microbiota. These changes affect processes involved in growth, performance, energy storage and
health in fish. (From Ghanbari et al. 2015 with permission from Elsevier)
environmental conditions, such as gut morphology, pH variation in different gut

compartments, and oxygen availability, are considered to be important factors that
induce the variation in host-specific gut microbiota (Yoon et al. 2015 and refer-
ences therein).
Now, major driving forces that structure the gut microbiota in both fish and
aquatic invertebrates will iteratively be disentangled by reporting and discussing
representative studies.
3.1 Invertebrates
Due to the high diversity of aquatic invertebrates and their various ontogenetic
stages, stock taking of their gut microbiota is still in its infancy.
3.1.1 Hydrozoa
Hydra-associated microbiota is known to aid development, budding, reproduction,

and maintenance of epithelial homeostasis. Furthermore, it is required even for
innate defenses (Bosch 2012; Fraune et al. 2015). Very often, the microbial gut
composition of laboratory-reared model organisms significantly differ from their
PCoA - PC1 vs PC2

0.25
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0.20
0.15
0.10
PC1-26.77%
0.05
0.00
-0.05
-0.10
-0.15
-0.2 -0.1 0.0 0.1 0.2 0.3 0.4

PC2-59.33%
Fig. 3.6 Principal coordinates analysis plot of microbial communities of Hydra vulgaris.
Microbial communities belonging to the same geographic locality clustered together, while micro-
bial communities from different geographic locations formed a distinct cluster. Red triangle:
Laboratory-maintained (LM), Orange circles: Wild population collected from Talegaon Dabhade
(Maharashtra, India), Blue square: Wild population collected from Naukuchital (Uttarakhand,
India). (From Gaikwad et al. (2016) with permission from Springer; inserted image ©CEW
Steinberg)
natural counterparts. Controlled environment in the laboratory versus dynamic con-

ditions in the wild is likely to be one of the reasons for such differences. Therefore,
Gaikwad et al. (2016) explored the microbiota of H. vulgaris collected from two
localities in India, as well as laboratory-maintained populations. All samples were
dominated by the phylum Proteobacteria; however, at a deeper taxonomic level,
microbial structure of the samples collected from different localities showed nota-
ble differences between them and the laboratory-maintained population (Fig. 3.6).
To summarize, this study indicates the influence of local environmental factors on
the microbial community structure of the Hydra. Apparently, Hydra possesses a
core microbiome only at a high taxonomic level.
3.1.2 Mollusks
King et al. (2012) analyzed the stomach and gut microbiomes of the eastern oyster
(Crassostrea virginica) from different sites of coastal Louisiana, USA. Stomach
microbiomes in oysters from Hackberry Bay were overwhelmingly dominated by
Mollicutes most closely related to Mycoplasma; a richer community dominated by

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Planctomyctes occurred in Lake Caillou oyster stomachs. Gut communities of oys-

ters from both sites differed from stomach communities, and harbored a relatively
diverse assemblage of phylotypes. Phylotypes most closely related to Shewanella
and a Chloroflexi strain dominated the Lake Caillou and Hackberry Bay gut micro-
biota, respectively. While many members of the stomach and gut microbiota
appeared to be transients or opportunists, a putative core microbiome was identified
based on phylotypes that occurred in all stomach or gut samples only. The putative
core stomach microbiome comprised 5 operational taxonomic units (OTUs) in 3
phyla, while the putative core gut microbiome contained 44 OTUs in 12 phyla.
These results collectively revealed novel microbial communities within the oyster
digestive system; the functions of the oyster microbiome are largely unknown. A
comparison of microbiomes from Louisiana oysters with bacterial communities
reported for other marine invertebrates and fish indicates that molluscan microbi-
omes were more similar to each other than to microbiomes of polychaetes, deca-
pods, and fish.
In contrast to the eastern oyster, Trabal Fernández et al. (2014) reported that
Proteobacteria was the most abundant phylum in three other oyster species
(Crassostrea corteziensis, C. gigas, C. sikamea). Bacteroidetes was the second most
common phylum. The site of rearing influenced the bacterial community composi-
tion of C. corteziensis and C. sikamea adults. Many of the taxa that were found in
the bacterial populations associated with the three oysters were described for the
first time, but the physiological and ecological significance of these populations
remains unknown.
Exploring an interesting hypothesis, Takacs-Vesbach et al. (2016) tested whether
different reproductive strategies and the transition to asexuality may be associated
with microbial symbionts. Such a link within mollusks has never been evaluated
previously. The authors performed pyrosequencing of bacterial 16S rRNA genes
associated with Potamopyrgus antipodarum, a New Zealand freshwater snail. A
diverse set of 60 tissue collections from P. antipodarum that were genetically and
geographically distinct and either obligate sexual or asexual were included, which
allowed the evaluation of whether the reproductive mode was associated with a
particular bacterial community. A total of 2624 unique OTUs (97% DNA similar-
ity) were detected, which were distributed across ~30 phyla (Fig. 3.7). Significant
differences in bacterial community composition and structure were detected
between sexual and asexual snails, as well as among snails from different lakes and
genetic backgrounds. The main dissimilarity of the bacterial communities between
the sexual and asexual P. antipodarum was driven by the presence of Rickettsiales
in sexual snails and Rhodobacter in asexual snails. This study shows that there is
most likely a link between reproductive mode and the bacterial microbiome of P.
antipodarum, although a causal connection requires further study.
The common octopus (Octopus vulgaris) is an attractive species for aquaculture;
however, several challenges limit sustainable commercial production. Little is
known about the early paralarval stages in the wild, including diet and intestinal
microbiota, which are likely to play a significant role in the development and vitality
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Fig. 3.7 Phylum-level taxonomy of the New Zealand mudsnail (Potamopyrgus antipodarum)
populations expressed as the percentage of total sequences (prior to rarefaction and filtering
Cyanobacteria OTUs). This figure includes only the eight most abundant phyla; remaining OTUs
are compressed into the ‘other’ category. (From Takacs-Vesbach et al. 2016, courtesy of the Public
Library of Science)
of this important life stage. Roura et al. (2017) characterized the gastrointestinal
microbiome of wild O. vulgaris paralarvae collected from two different upwelling
regions off the coast of north-west Spain and Morocco. These microbiomes were
compared to that of paralarvae reared with Artemia for up to 25 days in captivity
(Fig. 3.8). In addition to the Octopus microbiomes, the gastrointestinal microbiome
of zooplankton prey (crabs, copepod and krill) was also analyzed to determine
whether the microbial communities present in wild paralarvae are derived from
their diet. Paralarvae reared in captivity with Artemia showed a depletion of bacte-
rial diversity, particularly after day 5, when almost half the bacterial species present
on day 0 were lost and two bacterial families (Mycoplasmataceae and Vibrionaceae)
dominated the microbial community. In contrast to captive-raised paralarvae, bacte-
rial diversity increased in wild paralarvae as they developed in the oceanic realm of
both upwelling systems; this is likely to be due to the exposure of new bacterial
communities via ingestion of a wide diversity of prey (Fig. 3.9). Remarkably, the
bacterial diversity of recently hatched paralarvae in captivity was similar to that of
wild paralarvae and zooplankton, thus suggesting a marked effect of the diet on both
the microbial community species diversity and evenness.
The similarity of the microbial communities found in wild zooplankton and of
paralarvae growing near the coast is striking (Fig. 3.9). Although only four zoo-
plankton species were analyzed, the observed similarities support a close relation-
ship between the microbial gut communities of predator and prey. Wild paralarvae
continuously diversify their core gut microflora with a diverse diet, which provides
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Fig. 3.8 Principal coordinate analysis (PCO) plot showing the microbial communities found in
Octopus vulgaris paralarvae collected in the wild (green) and reared in captivity (dark blue), as
well as their zooplankton prey (light blue). (a) Axes PCO1 vs. PCO2 showing the main drivers
(vectors) of variation in the microbial communities. (b) Axes PCO1 vs. PCO3. Overlaid variable
vectors represent the strength of the correlations with the different PCO axes obtained with the
distance linear model, with the circle considered as the unity. DML, dorsal mantle length; H′,
Shannon’s diversity index; Reads, total reads passing filter; Species, number of bacterial species;
Sucker, number of suckers. (From Roura et al. 2017, courtesy of Frontiers of Physiology)
Fig. 3.9 Relative abundance of the main bacterial families showing the ontogenic changes in the
microbial communities of Octopus vulgaris paralarvae reared in captivity for 25 days and in two
upwelling regions of the North Eastern Atlantic: NW Spain and Morocco. The numbers present in
the two upwelling regions represent the sucker number. The microbial community found in the
zooplankton prey (Zoo) is represented together with the paralarvae collected in the same coastal
sample (asterisk). (From Roura et al. 2017, courtesy of Frontiers of Physiology)
a natural source of allochthonous bacteria. This diverse microbiota likely serves a

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variety of functions in the nutrition and health of the host by promoting nutrient
supply, preventing the colonization of infectious agents, energy homeostasis and
maintenance of normal mucosal immunity (Roura et al. 2017).
3.1.3 Echinoderms
Analyzing Lytechinus variegatus, Nelson et al. (2010) found that captive-raised sea
urchins contained a limited number of representative bacterial genera. Most com-
monly identified genera included Pseudomonads, Vibrio, and various epsilon and
γ-Proteobacteria. Hakim et al. (2015) specified that order Campylobacterales had
the highest relative abundance among the ε-Proteobacteria. Further analysis of the
Campylobacterales at a lower taxonomic level identified Arcobacter sp.,
Sulfuricurvum sp., and Arcobacter bivalviorum. Consistently, Hakim et al. (2016)
revealed an enrichment of Campylobacteraceae in the gut lumen of urchins from
their natural habitats, similar to that observed in L. variegatus kept in culture and
fed formulated diets.
Holothurians are a prominent group of deposit-feeders. They rework considerable
amounts of sediment, their activity may influence microbial processes at the sediment-
water interface and, vice versa, this feeding behavior may be the source of the micro-
biome in the intestine. To investigate this, Gao et al. (2014) surveyed the bacterial
community composition in the habitat surface sediment, the foregut and hindgut con-
tents of Apostichopus japonicus by pyrosequencing. The bacterial richness and
Shannon diversity index were both higher in the ambient sediments than in the gut
contents (Fig. 3.10). Proteobacteria was the predominant phylum in both the gut con-
tents and sediment samples. The predominant classes in the foregut, hindgut, and
ambient sediment were Holophagae and γ-Proteobacteria, δ-Proteobacteria and
γ-Proteobacteria, and γ-Proteobacteria and δ-Proteobacteria, respectively. The poten-
tial probiotics, including sequences related to Bacillus, lactic acid bacteria
(Lactobacillus, Lactococcus, and Streptococcus) and Pseudomonas were detected in
the gut of A. japonicus. The principle component analysis showed that the foregut,
hindgut, and ambient sediment, respectively, harbored different characteristic bacterial
communities. Selective feeding of A. japonicus is likely to be the primary reason for
the different bacterial communities in the foregut contents and ambient sediments.
Contrasting results, however, were obtained by Sawabe et al. (2016), so that a
general impact of the external microbial community cannot yet be ruled out. The
authors detected that Rhodobacterales was the most significantly abundant bacterial
group in the larger specimens of A. japonicus. Further detailed studies revealed a
significant abundance of microbiome retaining polyhydroxybutyrate (PHB) metab-
olism genes in the largest individual. The PHB metabolism reads were potentially
derived from Rhodobacterales. These results imply a possible link between micro-
bial PHB producers and potential growth promotion of sea cucumber individuals.
For instance, the link between dietary PHB and improvement of the growth
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Fig. 3.10 Principal component analysis results showing the relatedness of bacterial community in
the different samples of Apostichopus japonicus. GCA and GCP, respectively, represent the con-
tent of foregut and hindgut in the five sampled sea cucumbers. For example, GCA1 represents the
foregut content in the sea cucumber No. 1. SED represents the four surface sediment samples.
(From Gao et al. 2014, courtesy of the Public Library of Science)
performance, modulation of the intestinal digestive and immune function, increase

of intestinal SCFA content, and body composition in Litopenaeus vannamei has
been shown (Duan et al. 2017) and applies also to mollusks and other aquatic ani-
mals (see also, Volume 2 Functional Aquafeed).
3.1.4 Crustaceans
Numerous crustacean species have been analyzed in terms of gut microbiome com-
position and function. For instance, Sison-Mangus et al. (2015) identified the sig-
nificance of microbiota for survival, growth, and reproduction of Daphnia sp. The
authors tested Daphnia originating from both laboratory-reared parthenogenetic
eggs of a single genotype and genetically diverse field-collected resting eggs and
showed that bacteria-free hosts are smaller, less fecund and have higher mortality
than those with microbiota (Fig. 3.11). Consistent with experiments involving other
animal taxa, this study showed that Daphnia suffers significant losses in fitness
when deprived of bacteria and that these losses are prevented when bacteria are
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Fig. 3.11 Survival curves of bacteria-supplemented (E-Bac-Suppl), untreated (E-untreated), and

bacteria-free (E-BacFree) Daphnia hatched from ephippial eggs. Mantel-Cox log-rank test indi-
cates that bacteria-supplemented Daphnia lived longer than untreated and bacteria-free Daphnia
(P < 0.001). (From Sison-Mangus et al. (2015), courtesy of the Nature Publishing Group)
restored or replaced. This example shows most strikingly the significance of the gut
microbiota in an aquatic animal.
Chen et al. (2015) found that the Chinese mitten crab (Eriocheir sinensis) har-
bors diverse, novel and specific gut bacteria, which are likely to live in close rela-
tionships with their host. The analysis of cloned 16S rRNA genes revealed that the
majority of the bacteria belonged to four phyla, Tenericutes, Bacteroidetes,
Firmicutes, and Proteobacteria (Fig. 3.12).
In several papers, Dash and coworkers showed that the dietary supplementation
of probiotic bacteria, Bacillus licheniformis and Lactobacillus plantarum, can
manipulate the gut microbiota and increase the growth and immune response of M.
rosenbergii (Dash et al. 2014; Ranjit Kumar et al. 2013). The improved immune
competence was tested by challenges with Vibrio alginolyticus and Ae. hydrophila
and proven by increased survival after the challenge.
One illustrative example of how ontogenetic development impacts the diversity
of the gut microbiota has been elaborated by Mente et al. (2016). The authors stud-
ied whether there was a succession in the resident bacterial community structure
along the molting process in the giant freshwater prawn (M. rosenbergii) (Fig. 3.13)
and reported that major structural changes in the gut bacterial community coincide
with molting stage C, that comprises the most significant differences in animal mor-
phometry. These gut bacterial community changes were due to the increased num-
ber of OTUs, most of them being newly introduced in the gut habitat and
characterized as specialists. The majority of the bacteria belonged to the
Actinobacteria and unaffiliated groups, indicating the unique gut conditions that
prevail at stage C. Moreover, the inferred ecophysiological role of these OTUs is
related to food material processing, and the bacteria originate from gut environments
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3.1 Invertebrates
Proteobacteria, 15.6%
Bacteroidetes, 16.4%
Firmicutes, 10.1% Actinobacteria, 2.0%
Chloroflexi, 0.2%
Acidobacteria, 0.3%
Cyanobacteria, 0.8%
Others, 1.4%
Tenericutes, 53.3%
Fig. 3.12 Relative abundance of bacterial species on phylum level in the Chinese mitten crab. (From Chen et al. 2015, courtesy of the Public Library of
Science)
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Fig. 3.13 Above: Venn diagrams showing the number of unique and (below) the number of total
(red line) and newly introduced shared operational taxonomic units (OTUs) of gut bacteria between
the four molting stages (A, B, C, D) in Macrobrachium rosenbergii. (From Mente et al. 2016, with
permission from Elsevier)
of other aquatic animals. The presence of 13 OTUs, with most of them being related
to the γ-Proteobacteria and Firmicutes, was found to be unaffected by the molting
stage, pointing at a core gut biome and indicating the role of these bacteria in the
general gut ecophysiology.
Similarly, Huang et al. (2016) monitored the development of the intestinal micro-
biome during the growth in a marine counterpart, namely L. vannamei. Bacterial
members belonging to Commamonadaceae dominated the intestinal bacterial
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Fig. 3.14 Intestinal bacterial community composition of Litopenaeus vannamei during the stages
from 14 days postlarvae (L14) to 3-month old juveniles (J3) classified at the phylum level. HG and
WG denote water samples. P1 and P7 denote shrimp cultured in a pond. (From Huang et al. 2016,
with permission from Wiley)
community during the early growth stages of the shrimp, most likely because of
transfer from Artemia nauplii. The community became dominated by
Flavobacteriaceae during the middle growth stages and Vibrionaceae during the
late growth stages. Rhodobacteraceae and Flavobacteriaceae were present in all
growth stages and are likely to form the intestinal core microbiome (Fig. 3.14). The
intestinal bacterial community of L. vannamei underwent dynamic changes at the
OTU level during the growth stages.
In the black tiger shrimp (P. monodon), the succession of the intestine microbi-
ome during ontogenesis has been studied by Rungrassamee et al. (2013). Bacteroides,
Firmicutes, and Proteobacteria were found at all four studied growth stages, with
γ-Proteobacteria dominating. Based on a PCA (Fig. 3.15), the intestinal bacterial
communities from the three juvenile stages were more similar to each other than to
that of the 15-day old post-larval (PL) shrimp.
As reported for Hydra and Octopus, the question arises whether or not domesti-
cation and culturing also have an impact on the gut microbiome of crustaceans. To
answer this question, Rungrassamee et al. (2014) compared the intestinal microbi-
ota from three wild-caught and three captive-reared groups of adult P. monodon.
The bacterial profiles showed similar dominant genera in wild-caught and domesticated
a
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100%
Actinobacteria
% Relative abundance
80% Betaproteobacteria
Firmicutes
60%
Bacteroidetes
40% Alphaproteobacteria
Other bacteria
20% Gammaproteobacteria
0%
PL15 J1 J2 J3
Actinobacteria 0.40% 0.20% 0.05% -
Betaproteobacteria 1.30% 0.03% 0.04% -
Firmicutes 2.20% 0.20% 0.20% 0.05%
Bacteroidetes 5.10% 0.50% 0.05% 0.95%
Alphaproteobacteria 6.50% 0.50% 0.30% 9.40%
Other bacteria 3.50% 0.03% 0.60% 0.05%
Gammaproteobacteria 80.70% 98.50% 98.70% 89.50%
Growth stage
b
100%
Actinobacteria
% Relative abundance
80% Betaproteobacteria
60% Bacteroidetes
Firmicutes
40% Alphaproteobacteria
20% Gammaproteobacteria
0%
PL15 J1 J2 J3
Actinobacteria 0.17% 0.20%
- - -
Betaproteobacteria 0.03% - - 0.07%
Bacteroidetes 0.18% 2.42% 0.07% 0.74%
Firmicutes 0.25% 1.97% 1.33% 1.68%
Alphaproteobacteria 28.04% 0.50% 24.90% 34.66%
Gammaproteobacteria 71.33% 94.97% 73.69% 62.85%
Growth stage
1.2
1 PL15
Component2 (24%)
0.8
0.6
0.4
0.2
0
J3 J1
J2
-0.2
0 0.2 0.4 0.6 0.8
Component1 (75%)
Fig. 3.15 Above: Frequency distribution of phylogenetic groups in intestines of different growth
stages of the black tiger shrimp: 15-day old post-larva (PL15) and 1-, 2- and 3-month old juveniles
shrimp, proving the occurrence of a core bacterial population (Fig. 3.16). Five
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phyla, Actinobacteria, Fusobacteria, Proteobacteria, Firmicutes, and Bacteroidetes,

were found in all shrimp from both wild and domesticated environments. The prin-
cipal coordinate analysis pattern (Fig. 3.16 below) showed that bacterial popula-
tions in domesticated P. monodon showed more similarities within this group,
whereas the wild-caught shrimp had higher variation. However, the statistical analy-
sis of intestinal bacterial diversity did not show significant differences between the
wild-caught and domesticated shrimp. Overall, the observation of the composition
of the core microbiome agrees well with reports in other aquatic organisms, such as
Fenneropenaeus chinensis (Chinese shrimp), Penaeus merguiensis (banana prawn),
Nephrops norvegicus (Norway lobster), and Danio rerio (zebrafish) (Roeselers
et al. 2011; Oxley et al. 2002; Liu et al. 2011; Meziti et al. 2012).
Comparable to the Daphnia paper above, Rungrassamee et al. (2016) presented
another intriguing example of the significance of the gut microbiome. The authors
compared the resistance of the Pacific white shrimp and the black tiger shrimp chal-
lenged by the pathogenic Vibrio harveyi and found a higher infection resistance in
the Pacific white shrimp than in the black tiger shrimp. This can be explained by the
ability of the Pacific white shrimp to regain their intestinal microbial balance.
Conversely, the pathogen was able to outcompete normal intestinal bacteria in the
black tiger shrimp; hence, resulting in a higher mortality associated with V. harveyi
(Fig. 3.17). Finally, intestinal microbial management has the potential to contribute
to disease prevention in aquaculture.
Based on this and similar studies, such a strategy for high-density aquaculture
has been drafted by Xiong et al. (2016), since this form of aquaculture has led to
increasing occurrence of diseases in shrimp. The authors introduced intestinal
indicative assemblages as independent variables with which to quantitatively pre-
dict incidences of shrimp disease. Given the ignorance regarding the niches differ-
ences in the shrimp intestine throughout its developmental stages, the use of
probiotics in aquaculture has had limited success. Therefore, Xiong et al. propose
the exploration of effective probiotic bacteria from shrimp intestinal flora and the
establishment of therapeutic strategies dependent on shrimp age (Fig. 3.18).
Following ecological selection principles, the authors hypothesize that the larval
stage provides the best opportunity to establish a desired gut microbiota through
preemptive colonization of the treated rearing water with known probiotics.
Fig. 3.15 (continued) (J1, J2 and J3, respectively). (A) Percent distribution of bacterial phylum by
pyrosequencing analysis and (B) relative abundance of the six bacterial phylogenetic groups esti-
mated by real-time PCR. Below: Principal component analysis of bacterial populations in intes-
tines of different growth stages of the black tiger shrimp. The principal component analysis (PCA)
was performed using R script to compare bacterial community structures among the samples based
on relative abundance of various bacteria genera. (From Rungrassamee et al. 2013, courtesy of the
Public Library of Science)
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Fig. 3.16 Above: Shared OTU analysis of pyrosequencing reads from Penaeus monodon intes-
tines. The Venn diagram represents the unique and shared OTUs at 97% similarity level in libraries
of (A) wild-caught group (WC1, WC2, and WC3) and (B) the domesticated group (DB1, DB2 and
DB3). Below: Principal coordinate analysis (PCoA) of bacterial populations associated with the
intestines from the wild-caught and the domesticated groups. (From Rungrassamee et al. 2014,
image courtesy of the CSIRO, Wikimedia)
3.2 Fishes
After pioneering and intriguing papers on intestinal microbiota in mammals and

zebrafish (Bäckhed et al. 2005), showing that the gut microbiota is essential for
processing critical substrates, such as dietary polysaccharides, this new approach
3.2 Fishes 81
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Fig. 3.17 The survival rates of the 3-month old juveniles of the black tiger shrimp and the Pacific
white shrimp during challenge by the pathogenic Vibrio harveyi. Standard deviations were calcu-
lated from triplicate trials for each species. The survival rates of black tiger and Pacific white
shrimp under V. harveyi challenge were statistically compared by using unpaired t-tests and
showed no significant differences (P < 0.05). (From Rungrassamee et al. 2016 with permission
from Elsevier)
soon entered and conquered the aquatic phase with a focus on model fish, ornamen-
tal fish, as well as commercial fish and invertebrates in aquaculture.
In a meta-study, Gatesoupe (2016) provided a survey of microbial phyla, genera,
and species reported in available papers. Many studies have dealt with the genus
Bacillus, which is the main source of fish probiotics after lactic acid bacteria
(Fig. 3.19). Some complex consortia have been proposed to provide the widest
range of expected beneficial effects, such as live Bacillus subtilis, Lactobacillus
acidophilus, Clostridium butyricum, and Saccharomyces cerevisiae, which
improved the immune response and disease resistance in fish (Taoka et al. 2006).
Doubtless, stimulation of the immune defenses of the host is one of the most prom-
ising modes of actions (Lazado and Caipang 2014). The viability of probiotics is
essential for immunostimulation, and these specific effects should not be confused
with those of dead cells or cell components that are used as prebiotic immunostimu-
lants (Gatesoupe 2016).
3.2.1 Microbiome Ontogenesis
Recently, Llewellyn et al. (2014) reported that early promotion of nutrient metabo-
lism and innate immune response depend upon the bacterial species that colonize
the digestive tract. It is therefore of primary importance to understand the mecha-
nisms that orchestrate the early steps of colonization of the gastrointestinal tract of
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Fig. 3.18 Practical guidelines for following ecological patterns in aquaculture. During early
growth stages, studies should be primarily focused on the correlation between the intestinal micro-
biota and that of the surrounding water. This approach will enable the identification of probiotic
candidates that easily colonize the gut. Comparison of intestinal microbial composition between
healthy and diseased shrimp could help identify potential pathogens and probiotics for each growth
stage, facilitate the development of stage-specific therapeutic strategies (e.g., applying narrow-
spectrum antibiotics to target specific pathogens), and provide data for the management of the gut
microbiota via the alteration of driving factors. Finally, the relative abundance of bioindicators
could serve as independent variables for quantitatively predicting incidence of shrimp disease.
(From Xiong et al. 2016, with permission from Springer)
fish, leading to the build-up of a stable, diversified and resilient endogenous micro-
bial community. The colonization steps are summarized in Fig. 3.20. In the aquatic
environment, bacteria move easily between habitats and hosts. Thus the first steps
of interactions and colonization of fish progeny occur as soon as the eggs are laid.
The number of bacteria colonizing salmonid eggs, for example, ranges between 103
and 106 bacteria g−1 (Yoshimizu et al. 1980). The diverse microbiota that eventually
develops on the egg surface is expected to reflect the bacterial composition of the
water. However, even species-specific differences were observed in terms of bacte-
rial colonization of fish eggs between cod and halibut (Hansen and Olafsen 1999).
Such host-specific assemblages on the chorion may result from differential attrac-
tion to surface receptors to those being coded by host genotype. Once eggs hatch,
sterile larvae are rapidly colonized by ova debris and microbiota present in the envi-
ronment (Hansen and Olafsen 1999). Passage of surface bacteria into the gut is
expected to colonize the larval gut as soon they are begin to ingest their liquid
3.2 Fishes 83
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Fig. 3.19 Venn diagrams constructed from a non-exhaustive selection of 475 references, corre-
sponding to the publication of experiments that dealt explicitly with probiotics for finfish or live
food organisms. The references were collected until the end of June 2014 and selected by search-
ing for titles, keywords, and abstracts. The numbers correspond to the total articles referring to one
or several types of probiotics (a and b), or effects (c and d). (a) Half of the references dealt with
lactic acid bacteria, and only 12% with yeast; (b) the other sources of probiotic bacteria were
mainly the genus Bacillus and the phylum Proteobacteria; (c) in total, 444 articles dealt with at
least one class of effects which concerned mainly the host, while 19% considered only the direct
antagonism to pathogens, and 4.5% attended to the application of probiotics to improve water
quality; and (d) among the 350 papers dealing with the effects on host health, 42% considered
immunological parameters and 10% focused on the activity of digestive enzymes, while the other
references reported only the general rearing performances, or sometimes particular applications.
(From Gatesoupe 2016, with permission from Elsevier)
medium (Lauzon et al. 2010), and the alimentary tract of first-feeding fries is colo-
nized with bacteria associated with food (Blanch et al. 1997; Korsnes et al. 2006;
Reid et al. 2009). The process of recruitment of taxa to the developing microbiome
clearly has to work with those bacteria present in the immediate environment.
Romero and Navarrete (2006) identified dominant bacterial populations associ-
ated with early life stages of coho salmon. They focused on three developmental
stages (eggs, first-feeding fry and juvenile) and documented environmental bacte-
rial communities (surrounding water and pelletized feed) in order to determine the
putative origin of dominant intestine tract strains. Interestingly, a dominant
Pseudomonas sp. found in the juvenile gastrointestinal tract was also present on
eggs, but not in the water or in the food. This may indicate a vertical transmission of
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Fig. 3.20 Teleost microbiome during development. The figure shows a schematic of the general-
ized life cycle of a teleost and accessory indigenous bacteria (different taxa represented by colored
elipses). (1) Bacteria colonize the chorion of the egg. Taxonomic differences of bacteria between
fish species indicate specific early interactions, perhaps through precursors of innate immunity
(symbolized by squares and triangles on the chorion surface). (2) Egg hatches. The larva is colo-
nized by environmental bacteria as well as those originally present on the chorion. (3) Early diges-
tive tract colonization occurs when larva commences feeding. Bacterial taxa strongly resemble
those associated with food source. (4) Microbiome develops, accumulates diversity and matures.
(5) Adult microbiome is diverse assemblage of microbial taxa. Differences exist between surface
mucosal and intestinal communities. Intestinal communities also become compartmentalized/spe-
cialized to niches within the alimentary tract. Question mark indicates possible vertical transmis-
sion of microbiome components to eggs during oviposition. (From Llewellyn et al. 2014, courtesy
of Frontiers in Microbiology)
a pioneering strain, which is commonly observed as a dominant genus in gut micro-

biota of mature fish (Hansen and Olafsen 1999; Jensen et al. 2004; Navarrete et al.
2008). Overall, pioneering communities harbor very few ribotypes, those encoun-
tered important shifts, at least in terms of taxonomic diversity, between eggs, first-
feeding fry, and juvenile steps. The authors conclude that the early steps of the gut
microbiota colonization by bacterial strains do not reflect a stable microbiota, which
would be established after the first feeding stages, by recruiting its major compo-
nents from water and prey. Furthermore, the temporal pattern in which gut micro-
biota evolves is characterized by a remarkable inter-individual variation. Over time,
3.2 Fishes 85
microbial groups that typically dominate the adult intestinal microbiota overcome
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the early-colonizing microbes that are less adapted to the intestinal environment (El
Aidy et al. 2013).1
In addition to this general ontogenetic development of the gut microbiota,
Llewellyn et al. (2016) compared various life cycle stages and geographic origins of
the Atlantic salmon. In the case of migratory fish, successful exploitation of multi-
ple habitats may affect and be affected by the composition of the intestinal microbi-
ome. To identify to driving forces for the fish gut microbiome composition,
Llewellyn et al. collected 96 individuals from across the Atlantic encompassing
both freshwater and marine phases. Dramatic differences between environmental
and gut bacterial communities were observed. Furthermore, community composi-
tion was not significantly impacted by geography (Fig. 3.21). Instead, life cycle
stage strongly defined both the diversity and identity of microbial assemblages in
the gut, with evidence for community destabilization in migratory phases. Patterns
of Mycoplasmataceae phylotype recruitment to the intestinal microbial community
among sites and life cycle stages support a dual role of deterministic and stochastic
processes in defining the composition of the S. salar gut microbiome.
3.2.2 Does a Core Microbiome Exist?
The microbiome appears to be characterized by a high diversity among hosts

(=β-diversity) and by a common core metagenome and seems to differ flexibly
among animals with different diets (Karasov et al. 2011). Ringø and Birkbeck
(1999) enumerated five criteria required to be considered as core gut microbiota in
fishes: (1) they must be present in healthy individuals, (2) they colonize the gut at
early life stages and persist throughout the lifespan of the fish, (3) they are found in
both wild and cultured fish populations, (4) they are able to grow anaerobically, and
(5) they are associated with the stomach, foregut, or hindgut.
The existence of a core metagenome has been demonstrated by, e.g., a transplant
experiment between zebrafish and mice (Rawls et al. 2006). The gut microbiota of
these species share six bacterial divisions, although the specific bacteria within
these divisions differ. To test how factors specific to host gut habitat shape microbial
community structure, the authors performed reciprocal transplantations of these
microbiota into germ-free zebrafish and mouse recipients. The results reveal that
communities are assembled in predictable ways. The transplanted community
resembles its community of origin in terms of the lineages present (Fig. 3.22), but
the relative abundance of the lineages changes to resemble the normal composition
of the gut microbial community of the recipient host. Thus, differences in commu-
nity structure between zebrafish and mice arise in part from distinct selective pres-
sures imposed within the gut habitat of each host. Nonetheless, vertebrate responses
to microbial colonization of the gut are ancient: Functional genomic studies disclosed
1
References taken from Llewellyn et al. (2014); courtesy of Frontiers in Microbiology.
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Fig. 3.21 Taxonomic composition of the Atlantic salmon intestinal microbiome. (a) Phlyum-level
composition of total OTU abundances among distinct life cycle stages and environmental samples.
(b) Core (present in ⩾85% of individuals) 97% identity OTUs assigned to genus level from each
life cycle stage are represented by red-outlined ellipses. Black-outlined ellipses denote the pres-
ence of these ‘core’ OTUs among other life cycle stages. Ellipse area is proportional to the mean
abundance of OTUs assigned to each genus over all samples from each life cycle stage. Core
genera that occur at a mean frequency >1000 in each sample time are bold (adults), underlined
(parr) or italicized (freshwater). (c) Heat map displaying the frequency distribution of OTUs
belonging to family Mycoplasmataceae across distinct life cycle stages and countries of origin.
Genera within the Mycoplasmataceae are indicated on the maximum likelihood phylogeny (left)
on which values indicated the percentage of bootstrap support for the respective clades. A single
asterisk indicates the Candidatus OTU also recovered from a sympatric environmental sample. A
double asterisk indicates the most abundant core Mycoplasma OTU recovered from adult life cycle
stages. (From Llewellyn et al. 2016, with permission of the Nature Publishing Group)
3.2 Fishes 87
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Fig. 3.22 Similarity indices for pairwise comparisons of communities defined as assemblages of
phylotypes computed at levels of %ID ranging from 86%ID to 100%ID and compared at each %ID
threshold using the Chao–Jaccard abundance-based Similarity Index. Abbreviations: zebrafish
into mouse, CONV-R zebrafish compared to Z-mouse microbiota; mouse into zebrafish, CONV-R
mouse compared to M-zebrafish microbiota; zebrafish into zebrafish, CONV-R zebrafish com-
pared to Z-zebrafish microbiota; mouse into mouse, CONV-R mouse compared to M-mouse
microbiota. Similarity indices range from 0 (no overlap in composition) to 1 (identical communi-
ties). (From Rawls et al. 2006 with permission from Elsevier)
shared host responses to their compositionally distinct microbial communities and

distinct microbial species that elicit conserved responses.
Which factors influence the composition of the microbiome? Does a core micro-
biome exist, or do the host species coin the quality of the microbiome? We shall see
that these two approaches are not mutually exclusive. On the one hand, Li et al.
(2012) stressed the host species to be a major determinant of the intestinal microbi-
ota of fish larvae. The authors demonstrated distinct dominant intestinal microbiota
(consisting of Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, and
Cyanobacteria) in four fish larvae (silver carp, Hypophthalmichthys molitrix; grass
carp, Ctenopharyngodon idella; bighead carp, Aristichthys nobilis; and blunt-snout
bream, Megalobrama amblycephala) reared in the same environment. Therefore,
species turned out to be a strong determinant of the intestinal microbiota. The
eukaryotic taxa were almost the same in the different fish intestines, due to the
shared natural food source (zooplankton).
On the other hand, several papers stress the existence of core microbiome in
fishes. For instance, Llewellyn et al. (2016) showed that a core microbiome does exist
in the Atlantic salmon, with Mycoplasmataceae phylotypes abundantly recovered in
all life cycle stages (Fig. 3.21). Therefore, the question arises whether or not a core
microbiome in the gut of fishes is a general phenomenon or only specific to the

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Atlantic salmon and a few more species.

Roeselers et al. (2011) performed 16S rRNA gene sequence-based comparisons
of gut bacterial communities in three zebrafish (Danio rerio) collected recently
from their natural habitat and in two reared for generations in laboratory facilities in
different geographic locations. Patterns of gut microbiota structure in domesticated
zebrafish varied across different laboratory facilities, in correlation with historical
connections between those facilities. However, gut microbiota membership in the
domesticated and recently caught zebrafish was strikingly similar, with a shared
core gut microbiota. In detail, all three samples were dominated by Proteobacteria,
although the relative abundance of other phyla varied between samples. The bacte-
rial classes present in the three samples were highly similar. Roeselers et al. (2011)
detected 525 OTUs across all three samples; however, many were observed in only
one pyrosequencing data, set and only 21 of these OTUs were detected in all three
data sets (Fig. 3.23). These shared constituents of the intestinal microbiota of
domesticated and recently caught zebrafish might constitute a “core microbiota” of
the zebrafish intestine. In-depth analysis revealed that the 21 OTUs were comprised
of 12 genera within the γ-Proteobacteria, β-Proteobacteria, Fusobacteria, Bacilli,
Flavobacteria, and Actinobacteria classes, with Aeromonas and Shewanella appear-
ing as the most frequent genera. The zebrafish intestinal habitat therefore selects for
specific bacterial taxa despite radical differences in host provenance and domestica-
tion status.
Although the concept of a core gut microbiota has been explored in the context
of mammalian hosts, the presented data indicate that these concepts also apply to
bony fishes; the mechanisms and selective pressures, however, that produce a core
gut microbiota remain unresolved (Roeselers et al. 2011).2 Nevertheless, from the
study of obese and lean human twins, it can be generalized that the “core”
functional groups include several pathways that are likely to be important for life
in the gut, such as those for carbohydrate and amino acid metabolism (for exam-
ple, fructose/mannose metabolism, amino–sugar metabolism and N-glycan degra-
dation) (Turnbaugh et al. 2009). In rainbow trout, the core gut microbiota provides
a specific resilience to the individuals. This has recently been demonstrated by
Wong et al. (2013) who fed aquacultured individuals fishmeal- or grain-based
diets, and reared them under high- or low-density conditions. Variations in diet
and rearing density resulted in only minor changes in intestinal microbiota com-
position, despite the significant effects of these variables on fish growth, perfor-
mance, fillet quality, and welfare.
Even with parts of, and thereby less diverse, gut communities, such as yeasts,
a core intestinal microbiota appears to exist. For instance, Raggi et al. (2014)
investigated the composition of the intestinal yeast microbiota of wild and reared
2
Resident gut microbes that produce cobalamin are important in several freshwater fish species by
obviating the need for dietary cobalamin. One of these cobalamin-producing microbes,
Cetobacterium somerae, is ubiquitous in wild and laboratory-reared zebrafish, indicating a possi-
ble metabolic importance (Roeselers et al. 2011).
3.2 Fishes 89
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Fig. 3.23 Deep sequencing of 16S rRNA genes has revealed a core intestinal microbiota shared
among recently caught and domesticated zebrafish. India: recently caught from the wild; UW,
UNC: domesticated in aquaculture facilities at the Universities of Washington and North Carolina,
respectively. Above: Venn diagram showing the distribution of all 525 operational taxonomy units
(OTUs), revealing a shared community of 21 OTUs found in all three locations. Below: Pie charts
showing the relative abundances of bacterial phyla in the intestinal microbiota of the three zebraf-
ish strains as well as the 21 OTUs shared between all three locations, which may comprise a “core”
zebrafish gut microbiota. (From Roeselers et al. 2011 with permission from the Nature Publishing
Group)
carnivorous salmonids, croakers, and yellowtails. A high proportion of yeasts

produced aminopeptidases and lipases, which can be explained by the high pro-
portion of protein and lipids in the carnivorous diet. The core of the reared fish
was composed of eight species, in contrast to the wild fish core, which consisted
of two species (Fig. 3.24): Debaryomyces hansenii and Rhodotorula mucilagi-
nosa. It is obvious that these two species comprise the core.
Even a recent study on fish meal and fish oil replacement in juveniles of the
piscivorous sablefish (black cod, Anoplopoma fimbria) indicated that the existence
of a core gut microbiome is not unlikely. Rhodes et al. (2016) compared the effects
of a standard fish-based diet with two diets that contained primarily terrestrial plant
ingredients with flaxseed or corn oil replacing fish oil. The bacterial community
structures from corn oil fish were much less diverse than those fed on other diets, and
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Fig. 3.24 Venn diagrams of the yeast core of reared and wild zebrafish. Mv: Metschnikowia viti-
cola, Sp: Sporobolomyces sp., Dm: Debaryomyces marasmus, Ssu: Sporobolomyces sugiyamanus,
A: Aureobasidium sp., Pf: Pseudozyma fusiformata, Rv: Rhodotorula vanillica; Ap: Aureobasidium
pullulans, C: Candida sp., Cl: Cryptococcus laurentii, Rg: Rhodotorula glutinis, R: Rhodotorula
sp., Cg: Candida glabrata, Pk: Pichia kudriavzevii, Rgr: Rhodotorula graminis, Sc: Saccharomyces
cerevisiae, Ss: Saccharomycodes sinensis, Fu: Filobasidium uniguttulatum, Rb: Rhodosporidium
babjevae, Cz: Candida zeylanoides, T: Trichosporon sp., Tg: Trichosporon gracile, Tm:
Trichosporon middelhovenii, Pm: Pichia mexicana, Td: Torulaspora delbrueckii, Sd:
Saccharomyces dairenensis, K: Kazachstania sp., Cm: Cryptococcus magnus, Co: Candida
oleophila, Csa: Candida sake, Dh: Debaryomyces hansenii, Rm: Rhodotorula mucilaginosa, Cde:
Candida deformans, Wc: Wickerhamomyces ciferrii, Yl: Yarrowia lipolytica, D: Debaryomyces
sp., Tc: Trichosporon cutaneum, Cd: Candida davisiana, Cso: Candida sorbophila, P: Pichia sp.,
Tm: Trichosporon middelhovenii, Rs: Rhodosporidium sphaerocarpum, Sd: Sakaguchia dacryoi-
dea. (From Raggi et al. 2014; Roeselers et al. 2011 with permission from Wiley)
the microbiome structures from the three diets were distinctly different from each
other. The intestinal microbiome for the fish-based diet included the largest number
of families (68), and these fish also had the largest number of unique bacterial fami-
lies (11) compared to those for corn oil (two) or flaxseed oil (one) fish. Regardless of
diet, the stomach and intestinal microbiomes differed significantly from each other,
and the feed microbiome differed from all gastrointestinal communities, indicating
that food is not a significant source of gut bacterial diversity. Similar to other tele-
osts, the sablefish gastrointestinal microbiome was dominated by Bacteroidetes,
Firmicutes, and Proteobacteria. Although diversity in bacterial communities was
highest in fish receiving the reference diet, the structures of bacterial communities
were only modestly different among the dietary treatments (Fig. 3.25) pointing to the
existence of a core gut microbiome.
Following the study of Roeselers et al. (2011), the question arises: Which driving
forces shape the entire intestine microbial community in fishes – in larvae as well as
in adults? Is it the abiotic factors of the environment or is it the food? To address this
question in adult fishes, Sullam et al. (2012) performed a meta-analysis of 16S rRNA
gene sequence data from 17 data sets on teleost gut communities, including the
3.2 Fishes 91
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Fig. 3.25 Above Abundance boxplots of bacterial genera in juvenile sablefish (Anoplopoma fim-
bria) with significant differences between diet treatments in the stomach (A) or intestine (B).
Asterisks indicate: *, P < 0.05; **, P < 0.005; ***, P < 0.001. (From Rhodes et al. (2016), with
permission from Elsevier). Below: image of a juvenile sablefish. (Courtesy of D Ross Robertson)
Trinidadian guppy (Poecilia reticulata). Phylogenetic and statistical analyses revealed

that salinity, trophic level, and possibly host phylogeny shape the composition of fish
gut bacteria. When analyzed alongside bacterial communities from other environ-
ments, fish gut communities typically clustered with gut communities from mammals
and insects. Similar consideration of individual phylotypes (vs. communities) revealed
evolutionary ties between fish gut microbes and symbionts of animals, as many of the
bacteria from the guts of herbivorous fish were closely related to those of mammals.
These results indicate that fish harbor more specialized gut communities than previ-
ously recognized. They also highlight a trend of convergent acquisition of similar
bacterial communities by fish and mammals, raising the possibility that fishes were
the first to evolve symbioses resembling those found among extant gut-fermenting
mammals. The similarity among gut bacterial communities is presented in Fig. 3.26.
Bacterial communities vary along the x-axis according to their associations with
organisms. Environmental bacteria, from both aquatic and terrestrial habitats, fall on
the left-hand side of the X-axis (represented by the blue area). Bacterial communities
derived from eukaryotic animal hosts tend to fall further right along the X-axis
Carnivorous
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Cultured communities
fish gut
Omnivorous
Other vertebrate
fish gut
gut
Eukaryote-
Non-cultured communities
associated
Herbivorous
Environmental
fish gut
Free-living communities Host-associated communities
Fig. 3.26 Similarity among gut bacterial communities in fishes from different trophic levels com-
pared to non-fish-associated bacterial communities from various sources. Based on Sullam et al.
(2012). (From Wong and Rawls 2012, with permission from Wiley)
(represented by the beige area), with gut communities from mammals and other
vertebrates found furthest right (represented by the black area). Herbivorous fish gut
bacterial communities (represented by the green area), which were all derived from
marine species, are more similar to mammalian gut bacterial communities. In contrast,
omnivorous fish gut bacterial communities (represented by the yellow area) are more
similar to environmental samples, and carnivorous fish bacterial communities (repre-
sented by the red area) are more similar to those found in insects and other eukaryotic
habitats. Bacterial communities also differentiate along the y-axis depending, in part,
on whether they are derived from culture-based or culture-independent approaches.
Most past studies investigated the gut bacterial diversity under laboratory condi-
tions. Next-generation sequencing of the 16S rRNA gene as a culture-independent
molecular technique has greatly expanded the ability to obtain more comprehensive
information on complex microbial communities. Further, high-throughput DNA
sequencing has been used to explore the gut microbiota composition of some com-
mercially important fishes, including European sea bass (Carda-Diéguez et al.
2014), grass carp (Wu et al. 2012; Li et al. 2015), perch (Bolnick 2014), channel
catfish (Larsen et al. 2014), and rainbow trout (Ingerslev et al. 2014). All studies
indicated the overwhelming significance of (first) feeding and diet type for the
development of the gut microbiome. However, most of them were studied in the
rearing conditions and very little is known about the difference in the composition
3.2 Fishes 93
of gut microbiota between fish species with distinct trophic levels from natural
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environments.
Actually, Liu et al. (2016) showed how the diet category, or host trophic level, is
the major factor driving the composition and metabolism of gut microbiota
(Fig. 3.27). Even in the same fish species, the gut microbiota diversity varies when
reared in different environments. The most abundant gut microbiota of grass carp
collected from artificial ponds near the middle reaches of the Yangtze River (Wuhan,
China) were dominated by Proteobacteria, Firmicutes, Cyanobacteria, and
Actinobacteria, respectively, while grass carp from the Dongxihu Fish Farm
(Wuhan, China) were dominated by Fusobacteria, Firmicutes, Proteobacteria,
Bacteroidetes (Wu et al. 2012; Li et al. 2015). This indicates that the composition
and diversity of animal gut microbiota are influenced by many non-independent
factors. Different niches do not have the same availability of diet, which really
affects the base for comparison of gut microbiota between animal species.
In addition to Liu et al. (2016), Wu et al. (2012) concluded that the initial intes-
tinal bacteria of grass carp originate mainly from the water and sediment, and sub-
sequent food may significantly influence the composition of the gut microbiota. Liu
et al. (2016) utilized a meta-analysis of 16S rRNA gene sequences for comparison.
To minimize the influence of environmental factors, fish belonging to eight species
in four trophic levels (herbivorous, carnivorous, omnivorous, and filter-feeding,
Fig. 3.27 upper part) were collected at the same time and in the same water area.
Furthermore, the authors determined the gut content cellulase, amylase, and trypsin
enzyme activities of these eight species. Principal coordinates analyses (PCoA)
showed that gut bacterial communities of carnivorous and herbivorous fishes formed
distinctly different clusters in PCoA space. Although fish in different trophic levels
shared a large number of OTUs comprising a core microbiota community, at the
genus level a strong distinction existed. Cellulose-degrading bacteria Clostridium,
Citrobacter, and Leptotrichia were dominant in the herbivorous, while Cetobacterium
and protease-producing bacteria Halomonas were dominant in the carnivorous.
Further analyses of the metagenome function revealed that different trophic levels
of the fishes affected the metabolic capacity of their gut microbiota. Moreover, cel-
lulase and amylase activities in herbivorous fishes were significantly higher than in
the carnivorous, while trypsin activity in the carnivorous was much higher than in
herbivorous fishes. This result consistently relates to the composition of the corre-
sponding gut microbiota, clearly demonstrated by Canonical Correspondence
Analysis (Fig. 3.28).
The study by Liu et al. (2016) clarifies the importance of gut microbiota in diges-
tion, and provides evidence to understand how host trophic levels influence the
composition and metabolic capacity of gut microbiota and the enzyme activities of
gut content.
By comparing microbial communities of plant roots and animal guts in another
meta-analysis, Hacquard et al. (2015) went one step further than the papers above.
In both kingdoms of life, the microbial communities extend the host’s metabolic
repertoire, and bacteria are the overwhelmingly dominant microbial taxon.
Intriguingly, the bacterial communities in the fish gut are more closely related to
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Fig. 3.27 Above: Map of the Liangzi Lake. Eight species are herbivorous M. amblycephala and
C. idellus, carnivorous S. chuatsi and C. alburnus, omnivorous C. carpio and C. auratus, and filter-
feeding H. molitrix and H. nobilis. Below: Unique and shared OTUs in fish samples of four trophic
3.2 Fishes 95
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Fig. 3.28 Canonical Correspondence Analysis showing the correlation between the gut microbial
compositions of eight fish species and their enzyme activities. For abbreviations of species names,
refer to Fig. 3.27 upper part. (From Liu et al. 2016, courtesy of the Nature Publishing Group)
those in the root and rhizosphere samples (Fig. 3.29) than are those in the mammalian
gut, partially due to a high abundance of Proteobacteria. The authors concluded that
this shows that shared environmental and physiological features, rather than phylo-
genetic relatedness of the hosts, are decisive for community establishment.
To answer the above question of the initial microbial colonization in fishes,
Bakke et al. (2013) investigated the effect of diet and of rearing in separate tanks on
the cod (Gadus morhua) larval microbiota. Their hypothesis was that the gastroin-
testinal tract of newly hatched fish is colonized by bacteria present in the water. To
test this hypothesis, cod larvae were fed three different live-food diets, namely,
rotifers cultivated on Baker’s yeast and long-chain polyunsaturated fatty acids
(PUFAs), rotifers cultivated on Rhodomonas baltica, and copepods cultivated also
on R. baltica. All live-food diets support growth equally well (Fig. 3.30, above).
The authors found no differences in the larval microbiota due to diet after 8 day
past hatch (dph). Moreover, the larval microbiota was similar at 17 and 32 dph,
despite a change in live food at 18 dph. The larval microbiota was generally more
similar to the water microbiota than to the live-food microbiota (Fig. 3.30 below)
indicating support for the initial hypothesis. Bakke et al. (2013) furthered that rear-
ing of larvae in replicate tanks with identical diets could result in significant differ-
ences in larval microbiota. These findings indicated that diet does not entail major
Fig. 3.27 (continued) levels. (A) Venn diagram displays the number of shared and unique OTUs
among eight fish species in different trophic levels at 30% cutoff level. (B) Pie chart shows the
characteristics of shared OTUs with a frequency higher than 1%. (C) The characteristics of unique
OTUs from four trophic levels with a frequency higher than 1%. (From Liu et al. (2016), courtesy
of Nature Publishing Group)
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Fig. 3.29 Cumulative Abundance Plots: Relative abundances grouped at the phylum or class taxo-
nomic level for each sample included in the meta-analysis. The bar plots have been arranged along
the x-axis separating different host groups as well as different species and compartments. (From
Hacquard et al. 2015, with permission from Elsevier)
changes to the composition of cod larval microbiota. If comparable phenomena

apply to cod as they do to the Trinidad guppy, the sensitivity of the intestinal micro-
biota to food quality must happen during adulthood.
The study of Bakke et al. (2013) further indicated that diet may not be a good
strategy for management of larval microbiota in aquaculture – at least with larval
cod – and therefore delivery of probiotics through live food might not be the best
strategy in the larval stages. However, manipulation of the microbiota of live food
to exclude opportunistic pathogens seems to be beneficial.
The dominating role of the environment in establishing and maintaining micro-
bial gut communities obviously applies only to nascent communities, and appears
no longer valid if the fish individuals reach adulthood, not even if the microbiota is
perturbed by salinity acclimation. This has recently been demonstrated with the
euryhaline black molly (Poecilia sphenops) (Schmidt et al. 2015). To assess the
relative influence of stochastic versus deterministic processes in fish microbiome
assembly, the authors manipulated the bacterial species pool around each fish by
changing the salinity of the aquarium water. The results showed a complete and
repeatable turnover of dominant bacterial taxa in the microbiomes from individuals
of the same species after acclimation to the same salinity. Furthermore, they showed
that changes in fish microbiomes are not correlated to corresponding changes in
abundant taxa in tank water communities, and that the dominant taxa in fish micro-
biomes were rare in the aquatic surroundings, and vice versa. Obviously, bacterial
taxa were best able to compete within the unique host environment at a salinity most
appropriate to a given niche space, independent of their relative abundance in tank
3.2 Fishes 97
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Fig. 3.30 Above: Average weight (mg dw per individual) of cod larvae fed three different first-
feeding diets (CR: rotifers cultivated on Baker’s yeast and long-chain polyunsaturated fatty acids,
RR: rotifers cultivated on Rhodomonas baltica, and COP: copepods cultivated on R. baltica). Error
bars indicate standard error of the mean; image of Gadus morhua. (From Bloch (1782–1784),
courtesy of Biodiversity Heritage Library). Below: Average Bray–Curtis similarities for compari-
sons of cod larvae microbiota with water and live-food microbiota, respectively, among three rep-
licate tanks for the COP and RR rearing systems. (From Bakke et al. 2013, with permission from
Wiley)
water communities. In this experiment, deterministic processes appeared to drive

fish microbiome assembly, with little evidence for stochastic colonization. To date,
it is open whether these finding can be generalized or whether they apply only to
this individual fish species.
Another approach to answer the question of which factors determine the struc-
ture of the intestinal microbiota is to study closely related species that undergot a
rapid dietary niche radiation. This applies to African cichlid fishes, in general, and
to Tanganyika cichlid fishes, in particular, which were studied by Baldo et al. (2015).
Lake Tanganyika carries some 250 cichlid species, mostly endemic, which are cur-
rently subdivided into 14 tribes. One of these tribes, the Perissodini, has evolved a
unique feeding habit primarily based on the scales of other fishes (known as lepi-
dophagy), which makes this tribe perhaps the most specialized among cichlids.
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Fig. 3.31 Drawings of Plecodus paradoxus and of Astatotilapia burtoni. (Courtesy of GA

Boulanger, Wikimedia, and of the FAO)
Perissodini represent a relatively young monophyletic clade of nine species, which

transitioned from a mostly zooplanktivorous feeding habit (species belonging to the
genus Haplotaxodon) to a scale-eating habit (species belonging to the genera
Plecodus (Fig. 3.31) and Perissodus) following adaptation of feeding morphology
(i.e. jaw and teeth) and behavior.
The gut microbiota structure reflects both a host phylogenetic history and a sig-
nature of adaptation to the host ecological, mainly trophic, niches. The authors
surveyed the host natural intra- and interspecific variation of the gut microbiota of
five cichlid species from the monophyletic tribe Perissodini of Lake Tanganyika,
whose members transitioned from being zooplanktivorous to feeding primarily on
fish scales. The riverine species Astatotilapia burtoni (Fig. 3.31), largely omnivo-
rous, was also included in the study.
Fusobacteria, Firmicutes, and Proteobacteria represented the dominant compo-
nents in the gut microbiota of all 30 specimens analyzed, together contributing more
than 90% of the total reads per fish species (Baldo et al. 2015). Fusobacteria and
Firmicutes represented the dominant component in both libraries (with a pooled
median of 78% of reads per species, each library), while largely fluctuating in rela-
tive abundance across species. Proteobacteria were consistently less represented.
The remaining four phyla occurred at remarkably lower abundance (< 1%), although
consistently in all species (Fig. 3.32).
All members of the Perissodini tribe showed a homogenous pattern of micro-
bial diversities, with no significant qualitative differences, despite changes in diet.
The recent diet shift between zooplankton- and scale-eaters was reflected by a
significant enrichment of Clostridium taxa in scale-eaters, where they might be
involved in the scale metabolism. The comparison with the omnivorous species A.
burtoni makes it obvious that, with increased host phylogenetic distance and/or
increasing herbivory, the gut microbiota begins differentiating also at a qualitative
level. The cichlids showed the presence of a large conserved core of taxa and a
small set of core OTUs (average 13–15%), remarkably stable, and putatively
favored by both restricted microbial transmission among related hosts (putatively
enhanced by mouth-brooding behavior) and common host constraints. The three
most abundant core OTUs in cichlids were classified as Cetobacterium somerae,
Plesiomonas shigelloides, and Clostridium perfringens. All three species have
previously been associated with the intestinal tract of freshwater fishes. C. somerae,
3.2 Fishes 99
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Fig. 3.32 Relative abundance of the seven cichlid core phyla. Interquartile ranges (25th and 75th
percentiles) and whiskers show data dispersion across species averages. Medians are shown as
central horizontal lines. The two libraries returned a highly concordant pattern of core phyla abun-
dance: the cichlid gut microbiota is dominated by Fusobacteria, Firmicutes and Proteobacteria,
with the first two phyla largely fluctuating in relative abundance across species. Bacteroidetes,
Planctomycetes, Actinobacteria, and Verrucomicrobia were consistently less represented in all
species (overall contributing less than 1% of the total reads). (From Baldo et al. 2015, courtesy of
the Public Library of Science)
in particular, an obligate anaerobe putatively involved in the metabolism of vita-

min B12, was recently found as a core species in several fishes, such as channel
catfish (Ictalurus punctatus), largemouth bass (Micropterus salmoides), and blue-
gill (Lepomis macrochirus) (Larsen et al. 2014), and in Trinidadian guppies
(P. reticulata) (Sullam et al. 2012).
A similar shift of food sources can be observed in the involuntary shift from a
carnivorous to a more or less herbivorous food source that happens to commercial
fishes in aquaculture. Politically, ethically, and economically desirable, the replace-
ment of fish meal in aquaculture diets by plant proteins is not yet well understood
in terms of effects on the intestinal microbiota and fish health. Desai et al. (2012)
examined the intestinal microbiome of rainbow trout fed diets including plant
ingredients (peas, soybean, canola) at two processing levels (meal and concentrate),
or fed a fish meal (FM) control diet. In general, plant ingredient diets were associ-
ated with higher Firmicutes/Proteobacteria ratios than the controls (Fig. 3.33) and
correspondingly higher overall richness and diversity – the reasons behind this
remain obscure.
Nevertheless, the observations by Desai et al. (2012) supported the hypothesis
that changes to the structure of the intestinal microbiome are at least partially
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Fig. 3.33 Proportional abundance of bacterial phyla derived from the intestinal contents of rain-
bow trout. FM fish meal, PM pea meal, PPC pea protein concentrate, SBM soybean meal, SPC soy
protein concentrate, CM canola meal, CPC canola protein concentrate. (From Desai et al. 2012,
responsible for the adverse impacts of plant protein meal ingredients incorporated
at high inclusion rates on fish growth and health. The paper showed that the use of
the protein-concentrate forms of these ingredients reduces the impact of the ingre-
dients on the intestinal microbiome structure. The knowledge gathered from this
study is not relevant just to salmonids, but will also be useful for other carnivorous
fish, helping the aquaculture industry to reduce its dependence on fish meal.
The long-term changes observed by Desai et al. (2012) were, however, somewhat
surprising, and indicate that factors such as stress, age, developmental stage and the
selective pressure of the maintenance diet, continue to affect the microbiome over
the long term. Obviously, this type of long-term microbial succession should be
critically considered when evaluating diets and ingredients.
In a subsequent paper, Baldo et al. (2017) extended their comprehensive study on
East African cichlids and added an macroscale perspective. Ecoevolutionary dynam-
ics of the gut microbiota at the macroscale level, that is, in across-species compari-
sons, are largely driven by ecological variables and host genotype. The repeated
explosive radiations of African cichlid fishes in distinct lakes, following a dietary
diversification in a context of reduced genetic diversity (also see Chap. 7 Trophic
Diversification and Speciation), provide a natural setup to explore convergence,
divergence and repeatability in patterns of microbiota dynamics as a function of the
host diet, phylogeny and environment. Baldo et al. (2017) characterized the gut
microbiota of 29 cichlid species from two distinct lakes/radiations (Tanganyika and
Barombi Mbo) and across a broad dietary and phylogenetic range. Within each lake,
a significant deviation between a carnivorous and herbivorous lifestyle was found
(Fig. 3.34). Within each radiation, a dietary gradient can be observed at OTU level,
with a strong parallel trend at both lakes (Fig. 3.34b): distance typically increased
3.2 Fishes 101
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Fig. 3.34 Principal coordinate analysis (PCoA) of cichlid gut bacterial communities according to
lake (a and c) and diet (b and d). (a and b) Taxonomic (OTU) clustering based on unweighted
UniFrac distances. Circles represent individual specimens, with ellipses showing range of varia-
tion after multiple rarefactions to an even depth (13,000 reads). (c and d) Functional (KO) compo-
sition clustering based on binary Jaccard, after rarefaction to 1,835,713 gene counts. Barombi Mbo
(red) and Tanganyika (blue) specimens significantly separate at both the taxonomic (along PC2)
and functional level (along PC3). Given the time of divergence between the two radiations, deep
phylogenetic and geographic effects are here intrinsically linked. Within lakes, diet explains most
taxonomic and functional bacterial variance, with a clear divergence between carnivores (C) and
herbivores (H). (P planktivores; O omnivores; I insect eaters). (From Baldo et al. 2017; courtesy of
the Nature Publishing Group) UniFrac is a β-diversity measure that uses phylogenetic information
to compare environmental samples (Lozupone et al. 2011)
from carnivores to planktivores, omnivores, and herbivores. This trend alone

explained most of the taxonomic diversity among bacterial communities and signifi-
cantly discriminated between carnivores and herbivores. The functional profile sup-
ported a similar trend (Fig. 3.34d), with carnivores and herbivores discriminating
along PC1. Notably, within Tanganyika in particular, while the carnivores were
sparser in their clustering at both taxonomic and functional profiles, most herbi-
vores, omnivores, and planktivores strongly converged, beyond tribe boundaries. At
the functional level, the convergence among herbivores even spanned across lakes
(Fig. 3.34d).
In brief, herbivore species were characterized by an increased bacterial taxo-
nomic and functional diversity and converged in key compositional and functional
community aspects. Despite a significant lake effect on the microbiota structure,
this process has occurred with remarkable parallels in the two lakes. A metabolic
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signature most likely explains this trend, as indicated by a significant enrichment in

herbivores/omnivores of bacterial taxa and functions associated with fiber degrada-
tion and detoxification of plant chemical compounds. Overall, compositional and
functional aspects of the gut microbiota individually and altogether validate and
predict main cichlid dietary habits, suggesting a fundamental role of gut bacteria in
cichlid niche expansion and adaptation (Baldo et al. 2017).
3.2.2.1 M
icrobiome and Starvation
The functioning of the intestinal microbiota–host system becomes particularly obvi-

ous under starvation conditions. Starvation not only affects the nutritional and health
status of the animals, but also the microbial composition in the host’s intestine. In
the natural environment, starvation of aquatic animals occurs frequently; many ani-
mals face unpredictable food sources and periods of prolonged fasting, which are
likely to present significant challenges to gut microorganisms. This challenge can
serve as a drastic test of a core microbiota. How do hosts respond if major microbial
elicitors are missing? Which genes are upregulated? And, vice versa, which bacteria
are persevered and which die off? Recently, Xia et al. (2014) investigated changes
in the microbial composition and host–microbe interactions in the intestine of the
Asian seabass (Lates calcarifer) in response to starvation. The authors found 33
phyla, 66 classes, 130 orders and 278 families in the intestinal microbiome.
Proteobacteria (48.8%), Firmicutes (15.3%), and Bacteroidetes (8.2%) were the
three most abundant bacterial taxa. Comparative analyses of the microbiome
revealed shifts in bacteria communities, with dramatic enrichment of Bacteroidetes,
but significant depletion of β-Proteobacteria in starved intestines (Fig. 3.35).
Significantly more reads were assigned to the Bacteroidetes phylum under starva-
tion (36%) as compared to the control sample (8.2%). At the class level, Bacteroidia
(1.3% in the control sample vs. 24.4% in the starved sample) and Sphingobacteria
(1.1% in the control sample vs. 7.8% in the starved sample) contributed to higher
percentages of the microbiota in the starved sample than in the control sample. The
significant elevation of Bacteroidetes in the intestinal community of the starved sea-
bass sample is in agreement with some other studies on dietary shifts. For example,
in mice, fasting was associated with a significant increase in the proportional repre-
sentation of the Bacteroidetes. Bacteroides with a much larger genome size (e.g.,
Bacteroides fragilis Strain NCTC9343: 5,205,140 bp) are normally mutualistic in
the animal gastrointestinal flora. A large part of the proteins made by the Bacteroides
genome are able to break down polysaccharides and metabolize their sugars. They
play a fundamental role in the processing of complex molecules to simpler ones in
the host intestine. Their ability to harvest alternative energy sources from food might
allow Bacteroides to be more competitive than other bacteria in the fish intestine
during starvation (Xia et al. 2014 and references therein).
In addition, significant differences in functional categories were observed.
Genes related to antibiotic activity in the microbiome were significantly enriched
3.2 Fishes 103
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Fig. 3.35 Comparison of the taxonomic composition in the intestinal microbiome of Asian sea-
bass in response to starvation. The relative abundances (percentage) for the top 20 taxa of the
metagenomes at phylum and class levels between the control sample (Food) and the experimental
sample (Fast) are presented. Asterisks indicate significant differences (Bootstrap test:
***P < 0.001). (From Xia et al. 2014, courtesy of BioMedCentral)
in response to starvation, and host genes related to the immune response were
generally upregulated. This paper shows that long-term malnutrition or starvation
damages the mucosal barrier of the Asian seabass by increasing the permeability
of the intestinal mucosal barrier. The intestinal microflora, especially opportunistic
pathogens, can then cross the intestinal barrier when the intestinal mucosal barrier
is damaged or the normal flora has been destroyed by antibiotics or nutrition defi-
ciency. Based on prior findings from mammals, short-term stress experienced at
the time of immune activation can enhance innate and adaptive immune responses,
but long-term stress can suppress immunity by decreasing immune cell numbers
and function and/or increasing active immunosuppressive mechanisms (Xia et al.
2014 and references therein).
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Fig. 3.36 Above: phylogenetic diversity of the cecal microbial communities of fish hosts (Nile
tilapia) at different time points over prolonged fasting. Different letters above the bars indicate
significant differences. Bars represent means ± 1 SEM. Below: relative abundances of microbial
phyla in the colonic microbial communities of fish hosts at different time points over prolonged
fasting. Colored arrows represent significant changes in the abundances of microbial taxa as a
result of fasting. (From Kohl et al. 2014, with permission from the Oxford University Press)
Despite the described changes in the relative microbial composition, representatives

of all classes were present under fed and starved conditions, pointing to the exis-
tence of a core microbiome.
The findings in the Asian seabass are supported by a study of Nile tilapia (Kohl
et al. 2014). In this fish, microbial phylogenetic diversity in the colon increased as a
result of fasting, while it decreased in the cecum (Fig. 3.36). The changes in diver-
sity were driven by differential changes in the abundances of microbial taxa across
host species. For example, five bacterial phyla exhibited significant differences,
depending on feeding state (Fig. 3.36). The most drastic of the responses was an
increase, followed by a decrease, in the abundance of Fusobacteria. Additionally,
the relative abundances of Proteobacteria were five-fold higher in late-fasting tila-
pia compared to nourished tilapia. Tilapia exhibited roughly a 40–52% decrease in
3.2 Fishes 105
phylogenetic diversity between fed and fasted states (Kohl et al. 2014). Overall, a
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few species persisted during the fasting treatment and are indicative of a core intes-
tinal microbiota.
3.2.2.2 H
ost’s Impact on Microbiota
The intestinal microbiota and their dynamics can be affected by a multitude of fac-
tors, such as host physiology, developmental stage, age, food, and environmental
conditions (Ghanbari et al. 2015; Nayak 2010). To date, the hypothesis of a core
intestinal microbiota in fishes is not yet fully accepted, since contrasting reports do
exist. For instance, Li et al. (2012) observed distinct dominant intestinal microbiota
compositions in silver carp, grass carp, bighead carp, and blunt-snout bream reared
in the same environment. The same applies to juvenile paddlefish (Polyodon spath-
ula) and bighead carp, which have similar feeding strategies (Li et al. 2014b). The
authors showed that the intestinal microbiota differed between paddlefish and big-
head carp reared in the same pond when fed similar nature food. Therefore, poten-
tial host factors, such as the genetic background, gut histology and physiology are
assumed to be involved in the intestinal bacterial compositions.
In the same line of reasoning, Franchini et al. (2014) found significant differ-
ences in the microbiota of limnetic and benthic adult cichlid fish reared in common
environments. Furthermore, recent pyrosequencing analyses from three carp spe-
cies clearly revealed that the intestinal bacterial communities harbored specific
features, despite the fish cohabiting in the same environment (Li et al. 2014a).
Also, studies on rainbow trout, transgenic common carp, silver carp, and American
gizzard shad (Dorosoma cepedianum) have shown that even under a common
living/rearing/feeding environment, significant differences are present in the intes-
tinal bacterial communities of different species (Navarrete et al. 2012; Li et al.
2013; Ghanbari et al. 2015; Franchini et al. 2014).
In addition to species-specific factors impacting the microbial gut composition,
age-specific factors are also supposed to contribute to the composition. In particular,
Wei et al. (2018) worked out information about the gut microbiota and their dynam-
ics during the process of developmental stages in the large yellow croaker
(Larimichthys crocea). The intestinal microbiota at six ages changed significantly;
however, the change did not follow any trend. The large yellow croaker intestines
harbored specific bacterial communities that differed from those in both food and
water. Sequencing revealed that the diversity of intestinal bacteria in 12-day-old fish
was higher than that in 1-year-old fish, and the bacterial composition differed sig-
nificantly between them (Fig. 3.37). γ-Proteobacteria and Pseudoalteromonas sup-
plied the most abundant phylum and genus in the 12-day-old fish intestine. However,
in the 1-year-old fish intestine, Firmicutes and Clostridium were the most dominant,
respectively. The findings of this study do not really support the existence of a core
microbiome.
Bledsoe et al. (2016) showed that, in the channel catfish (Ictalurus punctatus),
bacterial phyla present in the gut throughout ontogeny include Bacteroidetes,
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Fig. 3.37 Heatmaps of taxonomic composition at phylum (a) and genus (b) levels of 12-day-old
(G12d) and 1-year-old large yellow croaker (G1y). (From Wei et al. 2018, with permission from
Wiley)
Fig. 3.38 Images of Carassius auratus gibelio and Epinephelus coioides. (From Bloch (1782–
1784), courtesy of the Biodiversity Heritage Library and courtesy of FAO)
Firmicutes, Fusobacteria, and Proteobacteria, with the species Cetobacterium

somerae and Plesiomonas shigelloides showing the highest abundance at 3 dph.
Although there is also evidence that the environmental microbiota serves as an inoc-
ulum to the fish gut, comparisons of the gut microbiota with the environmental
microbes reveals that the fish gut is maintained as a niche habitat, separate from the
overall microbial communities present in diets and water supply. The authors con-
cluded that ontogenetic development is the major force determining the diversity of
the microbiota.
In the same line of evidence, the results of Li et al. (2017b) indicated that the gut
microbial diversity increases significantly as the fish develop (gibel carp, Carassius
auratus gibelio, Fig. 3.38). The gut microbial community composition showed sig-
nificant shifts corresponding to host age, and appeared to shift at two time points,
despite consistent diet and environmental conditions, pointing to some features of
the gut microbial community possibly being determined by the host’s development.
3.2 Fishes 107
Dietary and environmental changes also seem to cause significant shifts in the fish
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gut microbial community. This study revealed that the gut microbiota of gibel carp
assembles into distinct communities at different times during the host’s develop-
ment, and that this process is less affected by the surrounding environment than by
the host’s diet and development.
3.2.2.3 E
nvironmental Impact on Microbiota
In addition to the studies above, Sun et al. (2015) reported that the bacterial commu-
nity in the rearing water of grouper Epinephelus coioides (Fig. 3.38) larvae played the
critical role in the establishment of the gut microbiota of fish larvae. The authors found
similar internal microbiota in larvae fed both fertilized oyster eggs and rotifers.
In support, Eichmiller et al. (2016) showed that the environment shapes the fecal
microbiome of invasive carp species. Although the common, silver, and bighead
carps are native and sparsely distributed in Eurasia, these fish have become abun-
dant and invasive in North America. The animal-associated microbiome plays an
important role in host health, and probably even in the invasive success. The phyla
Proteobacteria, Firmicutes, and Fusobacteria dominated carp guts, comprising
76.7% of total reads. The environment played a large role in shaping fecal microbial
community composition, and microbiomes among captive fishes were more similar
than among wild fishes. Although differences among wild fishes could be attributed
to feeding preferences, diet did not strongly affect microbial community structure in
laboratory-housed fishes. Comparison of wild and laboratory-housed invasive carp
revealed five shared OTUs that comprised approximately 40% of the core fecal
microbiome. This paper shows that the environment is a dominant factor shaping
the fecal bacterial communities of invasive carp. Captivity alters the microbiome
community structure relative to wild fish, while species differences are pronounced
within habitats. Despite the absence of a true stomach, invasive carp species exhib-
ited a core microbiota that warrants future study. The existence of a core microbi-
ome, particularly in the wild fish, may contribute to the invasive success, as will be
demonstrated below.
A core microbiome was also found in the Atlantic salmon (Dehler et al. 2017);
however, in addition to the core there was a high number of unique and variable taxa.
In the freshwater salmon, the diversity was higher than in the marine individuals.
As expected, Ramírez and Romero (2017) demonstrated that the microbiome of
Seriola lalandi of wild and aquaculture origin revealed differences in composition
and potential function. They showed that at the genus level, a total of 13 genera
were differentially represented between the two groups, all of which have been
described as beneficial microorganisms that have an antagonistic effect against
pathogenic bacteria, improve immunological parameters and growth performance,
and contribute to nutrition (Fig. 3.39). Additionally, the changes in the presump-
tive functions of the intestinal microbiota of yellowtail were identified. The most
abundant functional categories were those corresponding to the metabolism of
cofactors and vitamins, amino acid metabolism and carbohydrate metabolism,
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Fig. 3.39 Relative abundance (percentage) at phylum level for each sample in the intestinal
microbiota from wild and aquacultured Seriola lalandi. In the figure, A corresponds to aquaculture
fish (Aquaculture S. lalandi) and W corresponds to individual wild fish (Wild S. lalandi). (From
Ramírez and Romero 2017, courtesy of Frontiers in Microbiology)
revealing differences in the contribution of the microbiota depending on the origin

of the animals (Fig. 3.40).
3.2.2.4 D
iet
Many studies show that diet is one major force shaping the gut microbiome. For
instance, Schmidt et al. (2016) described that diet has a clear influence on the micro-
biome structure of the salmon intestine. The authors tested the influence of a
fishmeal-free diet on the microbial communities in recirculating aquaculture system
(RAS) water, biofilters, and salmon microbiomes. In fact, the salmon intestinal
communities varied with diet treatments, particularly within the order Lactobacillales
(lactic acid bacteria), but that these changes occurred between closely related micro-
bial taxa and did not impact fish performance. Therefore, this study provides sup-
port for the hypothesis that novel protein diets are viable alternatives to traditional
fishmeal-based diets, at least in an RAS.
Support for the diet-based hypothesis comes also from Sevellec et al. (2014),
who compared normal and dwarf lake whitefish (Coregonus clupeaformis). The
authors found that normal whitefish were characterized in all lakes by a greater
average taxonomic diversity than were dwarf whitefish, thus translating into paral-
lelism in difference of taxonomic diversity. Dwarf whitefish, and limnetic whitefish
3.2 Fishes 109
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Fig. 3.40 The general metabolic pathways of the intestinal microbiota from wild and aquacul-
tured S. lalandi. The asterisks indicate significant differences in pathways of the bacterial compo-
nents between wild and aquacultured yellowtail kingfish. This was assessed using t-test; P-values
were corrected with the Benjamini–Hochberg false discovery rate method. Values of P < 0.05 were
considered significant. (From Ramírez and Romero 2017, courtesy of Frontiers in Microbiology)
in general, feed almost exclusively on zooplankton, most often on the same taxa in
different lakes. In contrast, normal whitefish are more generalist feeders, and feed
on more diverse prey items including zoobenthos, mollusks, and fish prey, the com-
position of which varies between lakes and throughout the year.
Support for the core gut hypothesis came also from Gajardo et al. (2016) who
analyzed the bacterial microorganisms in five different compartments of the gut of
Atlantic salmon. The authors showed that bacterial populations varied substantially
between the regions of the intestine, and especially between digesta and mucosa
compartments. Proteobacteria and Firmicutes were the most abundant phyla in the
digesta, while Proteobacteria almost completely dominated in the mucosa-
associated microbiota. A core group of microbiota composed mainly of bacteria
belonging to the phylum Proteobacteria was identified (Fig. 3.41).
More generally, the processes that drive interindividual variation are still not well
understood. To address this, Burns et al. (2016) surveyed the microbial communities
associated with the intestine of the zebrafish (Danio rerio) over developmental time.
Therefore, they compared observations of community composition and distribution
across hosts with that predicted by a neutral assembly model, which assumes that
community assembly is driven solely by chance and dispersal. They found that as
hosts develop from larvae to adults, the fit of the model to observed microbial dis-
tributions decreases, indicating that the relative importance of non-neutral pro-
cesses, such as microbe–microbe interactions, active dispersal, or selection by the
host, increases as hosts mature. The authors also observed that taxa that depart in
their distributions from the neutral prediction form ecologically distinct sub-groups,
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Fig. 3.41 Venn diagrams showing compartmental core microbiota OTU distributions. (a) Digesta
compartments: 88 OTUs were identified as the core microbiota (80% of samples in each compart-
ment) for the proximal intestinal digesta (PID), mid intestinal digesta (MID) and the distal intesti-
nal digesta (DID). (b) Mucosa compartments: 32 OTUs were identified as the core microbiota
(80% of samples in each compartment) for the mid intestinal mucosa (MIM) and the distal intesti-
nal mucosa (DIM). (c) Core microbiota (80% of samples in each compartment) for all studied
compartments. Twenty two OTUs were found in all compartments. (From Gajardo et al. 2016,
courtesy of the Nature Publishing Group)
which are phylogenetically clustered with respect to the full meta-community.

These results demonstrate that neutral processes are sufficient to generate substan-
tial variation in microbiota composition across individual hosts, and show that
potentially unique or important taxa may be identified by their divergence from
neutral distributions. In other words, the distribution of microorganisms in these
systems is the result both of local factors specific to individual hosts and those pro-
cesses occurring at a broader meta-community scale linking multiple hosts.
Supporting the hypothesis of a core microbiome, Parma et al. (2016) evaluated
the gut bacterial community of gilthead sea bream (Sparus aurata) fed with increas-
ing levels of dietary soybean meal (SBM) in a low-fishmeal (FM)-based diet, in
comparison with a control diet. Five experimental diets were formulated to contain
increasing levels of SBM (0, 100, 200, and 300 g kg−1, named S0, S10, S20 and S30,
respectively) with150 g kg−1of FM, and one control diet (C) without SBM and con-
taining 350 g kg−1 of FM. The gut bacterial community of the distal intestine con-
tent was analyzed by next-generation sequencing. At the phylum level, the gut
bacterial community was dominated by Firmicutes (relative abundance 71%), while
the most represented family was Lactobacillaceae (26%). Even if no significant
3.2 Fishes 111
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Fig. 3.42 Box plot showing the relative abundance of (a) Cyanobacteria, (b) Synergistetes, (c)
Actinobacteria, and (d) Lactobacillaceae in different diets fed to gilthead sea bream (Sparus
aurata). Significance of the differences was obtained by Kruskall-Wallis test. (From Parma et al.
2016, with permission from Elsevier)
differences were detected in the α- and β-diversity of the gut bacterial community
according to the different diets (Fig. 3.42), Cyanobacteria and Lactobacillaceae
increased progressively from diet C to diet S30.
3.2.3 Zebrafish as Witness of Microbiome Development
In order to explore the dynamics of early microbial colonization of the zebrafish gut,
Jemielita et al. (2014) inoculated germ-free larval zebrafish at 5 days past fertiliza-
tion (dpf) with enhanced green fluorescent protein-expressing Ae. veronii by intro-
ducing the bacteria to flasks containing free-swimming larvae (Fig. 3.43a).
Beginning approximately 1 h after the start of inoculation, zebrafish were imaged
using light-sheet fluorescence microscopy at 25-min intervals for up to 16 h. The
rapid start and long duration of imaging post-inoculation allowed quantification of
intestinal bacterial populations that initiated with a few founding bacteria
(Fig. 3.43b) and grew to many thousands (Fig. 3.43c). The bacterial populations
robustly exhibit dynamics typical of resource-limited growth; a lag phase of 1.6–
1.7 h followed by an exponential phase and an apparent stationary phase when the
carrying capacity is reached (Fig. 3.43d, e). The stability of the population during
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Fig. 3.43 (a) Image of a larval zebrafish at 5 days post-fertilization, with the extent of the intestine
shown by a microgavage of phenol red dye. Scale bar, 250 μm. (b) Maximum intensity projection
(MIP) at 1.6 h post-inoculation with Aeromonas veronii. Several individual bacteria are visible,
and the inset shows a magnified view of a single bacterium (inset width, 22 μm). White bars indi-
cate autofluorescent sources from the zebrafish host. Scale bar, 100 μm. (c) MIP at 9.1 h post-
inoculation (same fish as that shown in panel b), showing a large bacterial population. Scale bar,
100 μm. (a–c) Orientation of the images is anterior to the left and dorsal to the top of the panel. (d)
Quantification of bacterial load over time in an individual fish (same fish as shown in panels b and
c). The dashed line shows best fit to a logistic growth model. (e) Quantification of bacterial load
for all fish imaged from this clutch of siblings. The vertical axis gives the population (N) rescaled
by the fitted carrying capacity (K). Time is measured from the moment of inoculation. Solid curves
show the best fit of each sample to a logistic growth model. (b and c) Images are composites of
multiple fields of view. (From Jemielita et al. 2014, courtesy of the American Society for
Microbiology)
3.2 Fishes 113
lag phase demonstrates that there is negligible further bacterial influx into the
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intestine from the mouth or esophagus during the imaging period while the fish
were mounted in agar, allowing population growth during imaging to be attributed
to the division of the initial colonizers and not to immigration of new individuals.
The data of Jemielita et al. (2014) yielded the first ever measurements of the
growth kinetics of a microbial species inside a live vertebrate intestine and showed
dynamics that robustly fit a logistic growth model. Intriguingly, bacteria were non-
uniformly distributed throughout the gut, and bacterial aggregates showed consider-
ably higher growth rates than did discrete individuals. The form of aggregate growth
indicates intrinsically higher division rates for clustered bacteria, rather than
surface-mediated agglomeration into clusters. Thus, the spatial organization of gut
bacteria, relative both to the host and to each other, impacts overall growth kinetics,
indicating that spatial characterizations will be an important input to predictive
models of host-associated microbial community assembly.
In a subsequent paper, Wiles et al. (2016) confirmed the importance of the spatial
organization of the gut microbiota and, probably most importantly, the impact of the
host itself. In particular, the authors monitored the dynamics of a defined two-
species microbiota within the zebrafish gut and observed that the interplay between
each population and the gut environment produces distinct spatiotemporal patterns
(Fig. 3.44). Imaging fluorescently marked variants of each species during mono-
association revealed that they have different intestinal biogeographies and behavior.
Populations of Vibrio largely comprise planktonic, highly motile cells that appear
capable of sampling all available regions within the intestine. Vibrio is most abun-
dant in the anterior bulb. By contrast, Aeromonas is most abundant in the midgut
and largely takes the form of dense aggregates with a smaller subpopulation of
motile individuals.
To identify the temporal dynamics of the two-member community, Wiles et al.
(2016) challenged established Aeromonas populations with Vibrio. They found that
Vibrio populations expanded smoothly; strikingly, sharp drops in intestinal
abundance, in which the population declined by orders of magnitude within an hour,
sporadically interrupted growth of Aeromonas. These local “collapses” appeared to
be due to Aeromonas populations being purged from the gut. The expelled microbes
may still have been alive, but were no longer residents of the zebrafish intestine. The
ratio of the Vibrio population before and after Aeromonas collapse events within the
same fish was approximately 1, corroborating observations from imaging and plat-
ing data that Vibrio is resistant to the perturbations that affect Aeromonas. Modeling
revealed that direct bacterial competition could only partially explain the observed
phenomena, showing that a host factor is also important in shaping the community.
Wiles et al. identified that the host determinant is gut motility. Overall, this study
provides evidence that host-mediated spatial structuring and stochastic perturbation
of communities can drive bacterial population dynamics within the gut, and it
reveals a new facet of the intestinal host–microbe interface by demonstrating the
capacity of the enteric nervous system to influence the microbiota.
Stephens et al. (2016) pointed out another important determinant of gut micro-
biota composition: the ontogenetic stage of the individual. The assembly of resident
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Fig. 3.44 Vibrio and Aeromonas have distinct community architectures and biogeographies within
the larval zebrafish intestine. (a) A larval zebrafish at 5 dpf; the intestine is highlighted by phenol
red dye via microgavage. Scale bar: 500 μm. (b) A maximum intensity projection (MIP) of Vibrio
in the larval intestine. Scale bar: 100 μm. (c) The probability density of Vibrio along the intestinal
axis. From (b) and (c), we see that Vibrio is predominantly localized in the anterior bulb. (d) MIP
of Aeromonas in the larval intestine. Scale bar: 100 μm. (e) The probability density of Aeromonas
along the intestinal axis. (d) and (e) show that Aeromonas is predominantly localized in the mid-
gut, with a smaller population in the anterior bulb. (From Wiles et al. 2016, courtesy of the Public
Library of Science)
microbial communities is an important event in animal development; however, the

extent to which this process mirrors the developmental programs of host tissues is
unknown. The authors surveyed the intestinal bacteria at key developmental
time points in a sibling group of 135 individuals of the zebrafish and revealed
3.2 Fishes 115
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Fig. 3.45 Significant changes in diversity of individual zebrafish intestinal communities through-
out development. (a) Number of observed taxa. (b) Faith’s phylogenetic diversity. (c) Simpson’s
diversity index. Black circles and error bars represent the means and 95% confidence intervals,
respectively. Letters above age groups indicate significant differences in the means. (From
Stephens et al. 2016, with permission of the Nature Publishing Group)
stage-specific signatures in the intestinal microbiota, and extensive interindividual

variation (Fig. 3.45), even within the same developmental stage. Microbial com-
munity shifts were apparent during periods of constant diet and environmental con-
ditions, as well as in concert with dietary and environmental change. Interindividual
variation in the intestinal microbiota increased with age, as did the difference
between the intestinal microbiota and microbes in the surrounding environment.
These results indicate that zebrafish intestinal microbiota assemble into distinct
communities throughout development, and that these communities are increasingly
different from the surrounding environment and from one another.
3.2.4 Control Functions by Gut Microbiota
Deciphering the make-up of fish gut microbiota is of great importance for under-
standing the dynamic functions of this community and the manner in which these
organisms affect their host’s physiology. This information has the potential to help
manage fish populations and their feeding and growth in captivity (Ghanbari et al.
2015). Some recent examples of the significance of the microbiome for life history
traits are listed in Table 3.1. It is widely accepted that diet modulates the species
composition of symbiotic gut microbiota in vertebrates. Studies have shown that
dietary composition is one of the most important factors shaping the fish gastroin-
testinal microbial community – at least of the variable moiety – and altering the
metabolism and population sizes of key symbiont species, resulting in biological
changes to the host.
Table 3.1 Modulation of fish gut microbiota composition and their feedback on life history traits
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Fish species Major findings

Angelfish Artemia enriched with a synbiotic (Pediococcus acidilactici and
(Pterophyllum fructooligosaccharide) improved growth performance, increased the
scalare) population of lactic acid bacteria in the intestinal microbiota, increased
environmental stress resistance, and elevated the total immunoglobulin and
lysozyme activity level of the skin mucus. The synbiotic diet was more
effective than singular enrichment with probiotics or prebiotics
Asian sea bass, The intestinal microbiota composition changed in response to starvation
barramundi towards a significant enrichment of Bacteroidetes and depletion of
β-Proteobacteria; antibiotic activity-related genes were significantly enriched
in response to starvation; host immune system-related genes were generally
upregulated
Atlantic salmon Successional dynamics over a period of 13 months in salmon hindgut
communities with respect to season and fish growth phases, but not
significantly related to differences in commercial diet
Beluga Saccharomyces cerevisiae var. ellipsoideus improved growth performance
and modulated intestinal microbiota of beluga sturgeon without detrimentally
impacting the basic hematological parameters
Caspian white Beneficial effects of dietary xylooligosaccharide on different parameters of
fish mucosal immunity (both skin, mucus and intestinal microbiota)
Common carp Fructo-oligosaccharide significantly increased leukocyte counts and
respiratory burst activity and modulated cultivable autochthonous gut
microbiota levels and stress resistance
Gilthead Dietary alginate encapsulated Shewanella putrefaciens increased lactic acid
seabream bacteria and had immunostimulant properties on humoral parameters (IgM
level and serum peroxidase activity)
Grass carp External variables, especially the abundance of food in the fish gut and food
composition, significantly affected the composition and function of the
intestinal microbiota
Perch & Genotype (sex)-by-environment (diet) interactions regulated the intestinal
Three-spined microbiota composition in fish
stickleback
Rainbow trout Variations in diet and rearing density caused minor changes in intestinal
microbiota composition, but significant effects on fish growth, performance,
fillet quality, and welfare
Rainbow trout The colonization of the intestinal microbiota in rainbow trout fry was
influenced by first feeding and diet with the marine-favored presence of
Proteobacteria and plant-based diet-favored presence of Firmicutes
Rainbow trout Dietary and pathogenic challenge-mediated differences in the composition of
the intestinal microbiota were observed; the protective effect of a plant-based
diet against Yersinia ruckeri, likely due to the higher number of bacteria from
the family Lactobacillaceae
Sea bass Administration of functional diets containing essential oils caused significant
changes in the gut microbiota of sea bass
Sea bream Different feeding modes were reflected in the gut prokaryotic community
structure in conventionally reared, organically reared, and wild S. aurata
individuals
(continued)
3.2 Fishes 117
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Fish species Major findings

Senegalese sole Administration of oxytetracycline (OTC) increased the expression of
apoptosis genes; the joint administration of OTC and Shewanella
putrefaciens induced the upregulation of anti-apoptosis genes
Siberian A combination of dietary probiotics and AXOSa resulted in a significant
sturgeon reduction in bacterial richness compared with the control diet
Siberian The administration of 2% AXOS-32–0.30, but not 4%, showed beneficial
sturgeon shifts in gut microbiota, primarily in the phylum Firmicutes, and higher
concentrations of short-chained FAs
Three-spined MHC IIbb polymorphisms contribute to interindividual variation in gut
stickleback microbiota within a single wild population of three-spined stickleback
Various marine Various yeasts, mainly Saccharomyces cerevisiae & Debaryomyces hansenii:
& freshwater Improvement of nutrient processing and absorption; stimulation of the
species immune and antioxidant systems, angiogenesis, gut maturation and fish
growth
Zebrafish Microbiota regulated the intestinal absorption and fatty acid metabolism in
the zebrafish
Zebrafish Treatment with L. rhamnosus from birth to sexual maturation seemed to
influence larval development by accelerating growth and backbone
calcification and anticipating sex differentiation. Administered to adults, L.
rhamnosus influenced the maturational competence and improved the
number of vitellogenic follicles
Zebrafish larvae Initial colonization by commensals in newly hatched zebrafish primed
neutrophils and induced several genes encoding pro-inflammatory and
antiviral mediators, increasing the resistance of larvae to viral infection
From Ghanbari et al. (2015), Carnevali et al. (2013), Navarrete and Tovar-Ramírez (2014),
Galindo-Villegas et al. (2012), Hoseinifar et al. (2011, 2014a, b), Azimirad et al. (2016), Tapia-
Paniagua et al. (2015), and Cordero et al. (2015)
a
Arabinoxylan–oligosaccharides, a prebiotic carbohydrate with a high degree of polymerization
b
Major histocompatibility complex class II (MHCII) receptors
In more details, Carnevali et al. (2013) summarized the probiotic effects of

L. rhamnosus on zebrafish reproduction. After 10 days treatment, L. rhamnosus
induced a significant increase of kiss1 and kiss2 transcription in the brain; the
increase of these two neuropeptides was concomitant with an increase in gnrh3
transcription (Gioacchini et al. 2010). Concomitantly, the daily numbers of ovulated
eggs increased significantly; the increase was evident from the second day of admin-
istration and remained significantly higher during the 10 days of treatment. The
higher mean number of ovulated eggs was associated with a better gamete quality,
as shown by the higher hatching rate and faster embryonic development (Gioacchini
et al. 2010). The contribution of probiotics in enhancing the follicle growth phase
was supported by the increase in the gonadosomatic index (GSI) along with histo-
logical studies on ovary sections, showing an increase of vitellogenic follicles
(Gioacchini et al. 2010, 2011) in the ovary of treated fish (Fig. 3.46).
The host may contribute to the bacterial community assemblage by selecting for
microbial populations that include specialized bacteria to aid in the digestion and
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Fig. 3.46 Representative micrographs of ovaries from (a–c) females fed on Lactobacillus rham-
nosus and (d–f) control females. PV previtellogenic follicles, V vitellogenic follicles. (From
Carnevali et al. 2013, with permission from Elsevier)
absorption of nutrients from a variety of food sources (i.e., protein versus chitin or
structural polysaccharides) (Roeselers et al. 2011). Recent studies based on 16S
rRNA gene sequencing in cyprinids have demonstrated a significant role for the
intestinal microbiota in the digestion of plant material within the fish intestinal tract.
van Kessel et al. (2011) analyzed the taxonomic profiles of the bacterial communi-
ties associated with the gastrointestinal contents of the common carp. Notably, bac-
terial taxa known to be involved in vitamin production and food digestion comprised
the majority of the retrieved sequences, and, interestingly, some of these bacteria
had not been previously reported in common carp. Overall, it is evident that phylo-
genetic factors, such as host physiology and gut anatomy, may interact with envi-
ronmental and ecological factors (e.g., biogeography of host fishes), such that many
factors must be considered when assessing relationships between microbiota com-
position and host biology.
3.2.4.1 C
ircadian Clock
With only a few exceptions, fishes and aquatic invertebrates and their gut microbi-
ome are still awaiting studies in terms of circadian rhythm. One of these exceptions,
Fortes-Silva et al. (2016) described the structural coincidence in the nocturnal tam-
baqui (Colossoma macropomum) that during the light period, a significantly
increased percentage of bacteria with low locomotor activity could be observed in
the gut. A functional analysis has not yet been carried out.
3.2 Fishes 119
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Fig. 3.47 Schematic showing diurnal cross talk between host and intestinal microbiota of a mam-
mal. (From Thaiss et al. 2015, courtesy of the Public Library of Science)
From mammalian studies, however, evidence is accumulating that the gut

microbiome strongly impacts even the circadian clock. Major evidence has come
from studies of the intestinal microbiota in mice, where gut microbial colonization
influences rhythmic signaling events in the ileal epithelium downstream of toll-like
receptors. This, in turn, regulates the organization of the molecular clock activity
and glucocorticoid production in the intestine. The microbiota also impacts clock
gene expression beyond the gastrointestinal tract. Germ-free mice, which were born
and raised under strict sterile conditions in the absence of microorganisms, feature
alterations in clock gene expression in the liver and the hypothalamus. These studies
indicate that microbial colonization has a previously unappreciated function in the
maintenance of circadian rhythms in eukaryotic hosts. The underlying mechanisms
for this activity remain obscure (Fig. 3.47). Recently, it has been discovered that the
circadian interactions between host and microbiota are not restricted solely to
microbial control of host clock function, but rather constitute a bidirectional cross
talk (Thaiss et al. 2015 and references therein). Subsequently, Thaiss et al. (2016)
showed that the diurnal microbial behavior drives, in turn, the global programming
of the host circadian transcriptional, epigenetic, and metabolite oscillations. In
fishes and aquatic invertebrates, there is lot to discover in studies to come.
3.2.4.2 Behavior
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The consumption of probiotics has become increasingly popular as a means to try

to improve health and well-being. Not only are probiotics considered beneficial to
digestive health, but increasing evidence shows direct and indirect interactions
between gut microbiota and the central nervous system (CNS). Specifically, in addi-
tion to direct effects on the innate immune system and on the composition of the
resident microbiota, certain strains of Lactobacillus exert a positive effect on
anxiety-related behavior and responses to stress. This appears to be the fish equiva-
lent to the mouse example above (Fig. 3.2).
Davis et al. (2016b) supplemented the diet of adult zebrafish with Lactobacillus
plantarum to determine the effects of probiotic treatment on structural and func-
tional changes of the gut microbiota, as well as on host neurological and behavioral
changes. Zebrafish exhibit numerous behaviors that have been correlated with those
seen in human neurological processes and disorders, such as anxiety, learning, fear,
sociability, and psychosis, and zebrafish neurotransmitter systems demonstrate
clear translatability to other organisms, including rodents and humans.
L. plantarum administration altered the β-diversity of the gut microbiota while
leaving the major core architecture intact. These minor structural changes were
accompanied by significant enrichment of several predicted metabolic pathways. In
addition to gut micro modifications, L. plantarum treatment also significantly
reduced anxiety-related behavior and altered GABAergic and serotonergic s ignaling
in the brain. In particular, novel tank diving is a validated behavior test for assessing
anxiety-related behavior in adult zebrafish, wherein the time spent in the top por-
tions of the tank correlates with less anxiety. To determine if L. plantarum influ-
ences anxiety-related behavior in adult zebrafish, a novel tank diving test was
performed on fish on diets with and without L. plantarum supplement. Zebrafish
from both groups displayed similar locomotor activity as measured by the total
distance traveled and average swimming speed (Fig. 3.48a, b). However, zebrafish
supplemented with L. plantarum exhibited a strong trend of more transitions to the
upper zone and spent significantly more time in the upper portion of the tank
(Fig. 3.48c–f). These results indicate that L. plantarum is effective in reducing
anxiety-related behavior in adult zebrafish. Furthermore, it protects against stress-
induced dysbiosis (Fig. 3.49).
Quantitative real-time PCR of candidate genes thought to be associated with anx-
iety-related behavior include genes encoding the GABA-A alpha 1 receptor (gabra1)
and serotonin transporter A (slc6a4a) that were upregulated in L. plantarum-treated
zebrafish. No differences were detected in neuropeptide expression levels (npy and
oxtl) in L. plantarum-treated fish compared to controls (Fig. 3.48g).
To investigate how the microbiota affects physiological responses to stress in
zebrafish larvae, an osmotic stress test was performed, and factors related to the
hypothalamic-pituitary-interrenal (HPI) axis were measured (Davis et al. 2016a).
Conventionalized and conventionally-raised larvae demonstrated the expected
response to stress, with a significant elevation in cortisol levels relative to untreated
controls (Fig. 3.50a). Interestingly, germ-free (GF) larvae did not display an
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Fig. 3.48 (a, b) No differences were detected in locomotor activity between controls and
Lactobacillus plantarum-treated zebrafish. (c–f) Zebrafish supplemented with L. plantarum
exhibit significantly less anxiety-related behavior than controls, as indicated by number of transi-
tions into the upper zone (c) and time spent in the upper portions of the tank (d–f). (g) Genes
encoding the GABA-A alpha 1 receptor (gabra1) and serotonin transporter A (slc6a4a) are upreg-
ulated in L. plantarum-treated fish. * denotes P ≤ 0.05. (From Davis et al. 2016b, courtesy of the
Nature Publishing Group)
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Fig. 3.49 (a–c) Chronic unpredictable stress (CUS) induced dramatic alterations of the gut micro-
biota in control zebrafish; however, L. plantarum treatment was shown to be protective against this
stress-induced dysbiosis. (a) The relative abundance of the core phylum, Fusobacteria (indicated
by the yellow bar), was greatly diminished in the chronically stressed control group, whereas the
major core GM remained intact in the chronically stressed L. plantarum-treated fish. (b, c)
Principal component analysis revealed significant shifts in response to CUS in the control group,
while chronically stressed L. plantarum-treated zebrafish clustered in conjunction with non-
stressed L. plantarum-treated fish. (From Davis et al. 2016b, courtesy of the Nature Publishing
Group)
elevation in cortisol production in response to the stress test (Fig. 3.50a). In agree-

ment with cortisol production, larvae raised in non-sterile environments had
increased melanocortin type 2 receptor (mc2r), 11 β-hydroxylase, and steroidogenic
acute regulatory protein (star) transcription in response to stress, while GF fish
displayed no significant changes relative to controls (Fig. 3.50b–d). This study dem-
onstrates the importance of gut microbes in mounting an appropriate response to
stress.
3.2 Fishes 123
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Fig. 3.50 Exposure to microbes allowed for characteristic activation of hypothalamic-pituitary-

interrenal (HPI) axis in response to a stressor in 6 dpf zebrafish larvae. (a) Conventionally-raised
(CV and CR) larvae demonstrated characteristic elevations of cortisol following an osmotic stress
challenge, whereas germ-free (GF) larvae exhibit a blunted response. (b-d) Expression of genes
involved inactivation of the HPI axis and cortisol production were also elevated in CV and CR
larvae following a stressor, while there was no change in GF larvae. Target gene expressions were
normalized to β-actin expression levels. Data are means ± SEM; bars above the columns denote
P ≤ 0.05. (From Davis et al. 2016a, courtesy of Elsevier)
Overall, L. plantarum supplementation provided protection against anxiety-

related behavior and stress-induced dysbiosis of the gut microbiota. These studies
underscore the influence that commensal microbes have on physiological function
in the host, and demonstrate bidirectional communication between the gut micro-
biota and the host, particularly to the CNS via the microbiome–gut–brain axis.
Further probiotic modulation of the microbiota–gut–brain axis and behavior has
recently been documented in zebrafish (Borrelli et al. 2016). After 28 days of dietary
administration with the probiotic Lactobacillus rhamnosus, the authors found dif-
ferences in shoaling behavior, brain expression levels of a growth factor (brain-
derived neurotrophic factor) and in genes involved in serotonin signaling/
metabolism, between control and treated zebrafish groups. In addition, a significant
increase of Firmicutes and a trending reduction of Proteobacteria were found in the
microbiota. This study demonstrates that selected microbes can be used to modulate
endogenous neuroactive molecules in zebrafish.
Are there indications that the above findings may apply to field conditions?
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Fig. 3.51 Gut diagrams and microbial diversity. (a) Rarefaction curve sequences showing the
microbial community complexities in the guts of the planktivorous gizzard shad (GZSD; Dorosoma
cepedianum) at the 3% distance cutoff. (b) Rarefaction curve sequences showing the microbial
community complexities in the guts of planktivorous silver carp (SVCP; Hypophthalmichthys
molitrix) at 3% distance cutoff. F foregut, H hindgut. Fish illustrations were gifts from Duane
Raver. (From Ye et al. 2014, with permission from the Nature Publishing Group)
Ye et al. (2014) compared gut microbiota of invasive Asian silver carp (SVCP)
and indigenous planktivorous gizzard shad (GZSD) in the Mississippi river basin
using 16S rRNA gene pyrosequencing. GZSD hindgut (GZSD_H) samples exhib-
ited the highest α-diversity indices, followed by SVCP foregut, GZSD foregut and
SVCP hindgut (SVCP_H) (Fig. 3.51).
3.2 Fishes 125
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Fig. 3.52 Venn diagram showing the number of shared and unique OTUs among all the four types
of GZSD and SVCP gut samples at 30% cutoff level. The percentages in the Venn diagram indicate
the ratios of the sequences that are associated to the OTUs in total sequences. (From Ye et al. 2014,
with permission from the Nature Publishing Group)
The microbiota of GZSD_H and SVCP_H were clearly separated into two
clusters: samples in the GZSD cluster were observed to vary by sampling location
and samples in the SVCP cluster by sampling date. Furthermore, distinct microbial
communities between foregut to hindgut were obtained for individual GZSD and
SVCP. Cyanobacteria, Proteobacteria, Actinobacteria and Bacteroidetes were
detected as the predominant phyla, regardless of fish or gut type. The high abun-
dance of Cyanobacteria observed was possibly supported by their role as the fishes’
major food source. Furthermore, unique and shared OTUs and OTUs in each gut
type were identified (Fig. 3.52), three OTUs from the order Bacteroidales, the genus
Bacillariophyta and the genus Clostridium were found to be significantly more
abundant in GZSD_H than in SVCP_H samples. The microflora of GZSD was
affected by location, whereas SVCP_H microflora was affected by sampling time.
These differences were presumably caused by the differences in the type of food
sources, including bacteria ingested, the gut morphology and digestion, and the
physiological behavior between GZSD and SVCP, and may be one reason for the
invasion success of SVCP.
3.2.4.3 Development and Health
The importance of the gut microbiota in the health and well-being of its hosts has
been stressed several times (for instance, in Table 3.1 and Fig. 3.5) and the dietary
monospecies supplementation will be reviewed in the Chapter Functional Aquafeed,
Volume 2. Nevertheless, a few intriguing papers need still to be discussed here.
Rolig et al. (2015) tested the hypothesis that the relative abundances of different
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taxa can predict health status. Therefore, they developed an assay in which they
monitored both the composition of the bacterial community in zebrafish and the
innate immune response in an individual fish. The authors colonized gnotobiotic
zebrafish with zebrafish-derived bacterial isolates, and measured bacterial abun-
dance and host immune responses. Surprisingly, combinations of bacteria elicited
immune responses that did not reflect the numerically dominant species. This indi-
cates that various species have different per capita immunostimulatory effects. The
authors concluded that the proportional representation of bacteria in a community
does not necessarily predict its ig. 12 capacities; a minor member can dominate the
host response with a secreted immunosuppressive factor.
In their review, Merrifield and Rodiles (2015) stressed the cross talk between
host and microbiota and its role in ontogenetic development and health mainte-
nance. For instance, zebrafish larvae reared in germ-free environments fail to
develop correctly (Bates et al. 2006). The gastrointestinal tract fails to differentiate
properly, and the intestine displays reduced levels of enteroendocrine cells and gob-
let cells, a lack of brush border intestinal alkaline phosphatase (InAP) activity, and
reduced epithelial cell turnover rates, and a loss of epidermal integrity develops.
These characteristics ultimately lead to a failure of the intestine to take up protein
macromolecules (Merrifield and Rodiles 2015).
The InAP, localized to the intestinal lumen brush border, is an enzyme involved
in mucosal tolerance due to its ability to detoxify bacterial lipopolysaccharide (LPS)
endotoxins (Fig. 3.53), as has been demonstrated by Bates et al. (2007). In particu-
lar, LPS upregulates InAP, which functions to prevent excessive intestinal inflam-
mation, a response that would be detrimental to microbiota and host alike. Although
this paper involved a high variety of mammalian studies, it remains the only one so
far dealing with aquatic animals.
Fig. 3.53 Intestinal activity of alkaline phosphatase (AP in ng Pi/min/mg tissue) in zebrafish is
upregulated by the microbiota. AP activity was quantified in homogenates of dissected intestines.
CV conventionally reared, GF germ-free, XGF ex-germ-free. Bacteria were added to originally
germ-free animals, dpf days post-fertilization. (From Bates et al. 2006, with permission from
Elsevier)
3.2 Fishes 127
In addition, Gatesoupe (2016) reported that there is a mutual shaping of the

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intestinal microbiome and the host’s transcriptome, including possible epigenetic

regulation. The early nutrition and microbial gut colonization are critical for epigen-
etic programming in man (Takahashi 2014). Two bacterial metabolites were pro-
posed as potential mediators for epigenetic regulation: folate (which may be
produced by the intestinal microbiota) and butyrate (one of the main products of
carbohydrate fermentation in fish intestine).
Galindo-Villegas et al. (2012) have recently proved the bacterial epigenetic regu-
lation of immunity in zebrafish by comparing axenic and conventionally reared ani-
mals. Using a germ-free model, the authors showed that colonization by commensals
in newly hatched zebrafish primes neutrophils and induces several genes encoding
pro-inflammatory and antiviral mediators, increasing the resistance of larvae to viral
infection. The results demonstrated that sensing of commensal microbes via both
the TLR/MyD88/NF-κB3 signaling pathway and gene-specific chromatin modifica-
tions is associated with the protection of zebrafish larvae against infectious agents
before adaptive immunity has developed, and at the same time prevents pathologies
associated with excessive inflammation during development.
The symbiosis of microbiota in the gut is very important as it controls the expres-
sion of several innate immunity genes, as Banerjee and Ray (2017) summarized.
For example, the gut microbiota plays a vital role in upregulation of serum amyloid
A1, complement component 3, C-reactive protein, angiogenin 4, myeloperoxidase,
and glutathione peroxidase. The gut microbiota is considered to be a good source of
different compounds, such as sebastenoic acid, which acts as an antimicrobial agent
against various pathogenic bacteria such as Staphylococcus aureus, Bacillus subti-
lis, Vibrio mimicus, and Enterococcus faecium. Actinobacteria happen to be perma-
nent residents in the gut of several fishes, and are reported to be good producers of
secondary metabolites. Bifidobacterium, a member of the Actinobacteria group, is
a key regulator of interleukin-10 secretion, which plays a vital role during inflam-
mation. Overall, the establishment of a healthy bacterial community in the gut is
important in terms of nutrition, physiology and immunity.
On the basis of the available literature, de Bruijn et al. (2018) pointed out the
necessity of a strategy as to how microbial communities can be applied to mitigate
emerging diseases in aquaculture. It is evident that the fish microbiome has enor-
mous potential for fish health, helping the host in its defense against pathogen
colonization and infection. A mechanistic understanding of how specific microbes
or microbial consortia, including their metabolites, act against pathogens and trig-
ger the fish immune response may provide practical means to engineer the indige-
nous fish microbiome for enhancing disease control and fish health. At present,
most achievements have been reached with probiotics (see Functional Aquafeed,
Volume 2).
3
Toll-Like Receptors (TLRs) play a critical role in the early innate immune response to invading
pathogens by a sensing microorganism. Stimulation of TLRs initiates signaling cascades leading
to the activation of transcription factors, such NF-κB. One way that TLR signaling takes place is
via a MyD88-(myeloid differentiation primary response gene 88)-dependent pathway that leads to
the production of inflammatory cytokines.

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The paramount significance of the gut microbiome in many – if not all – fishes and
aquatic invertebrates is beginning to be understood. Many endogenous and exoge-
nous factors shape the microbiome: host genetics, abiotic and biotic environment,
diet, and developmental stage being important stirring factors.
It is still a matter of controversial debate whether or not a core microbiome does
exist. From mammalian studies, it is well understood that core microbiomes are
central in many life history traits, including immunity and health, or the invasive
success of alien species. This aspect is important in the globalized species exchange,
in order to understand the mechanisms, and probably combat the phenomenon,
behind the fact that indigenous species are often outcompeted, and the fauna
becomes increasingly homogenized.
Contradictory results in core microbiome studies may be, at least partly, due to
different and sometimes insufficient methodologies. Results based on different
hypervariable regions might be inconsistent. In general, drawing conclusions based
on only one sequencing region and on one classification method should be avoided,
due to the potential for false-negative results (Guo et al. 2013; Cruaud et al. 2014).
Overall, although the hypothesis, that the shared microbiota is shaped by evolu-
tionarily conserved aspects of digestive tract anatomy, physiology and immunity, is
currently receiving increased attention, additional studies are required to confirm
this concept. Additional information on the core gut microbiota is likely to facilitate
the development of safe and effective methods for manipulating the composition of
the gut microbiota to promote the health and growth of fish (Ghanbari et al. 2015).
These studies should look behind the Venn diagrams and apply ecological princi-
ples, particularly that of combining structure and function of the core consortium.
Ecological insight into core microbiomes can be enriched by ‘omics approaches
that assess gene expression of the hosts, thereby extending the concept of the core
beyond taxonomically defined membership to community function and behavior
(Shade and Handelsman 2012) or to metabolic activity of the core members (Raggi
et al. 2014).
References
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fsi.2016.05.001
Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI (2005) Host-bacterial mutualism in
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Roura A, Doyle SR, Nande M, Strugnell JM (2017) You are what you eat: A genomic analysis of
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Chapter 4
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Dietary Restriction, Starvation,

Compensatory Growth – ‘Short-Term
Fasting Does Not Kill You: It Can Make
You Stronger’
Abstract In natural systems, short-term food deprivation (dietary restriction, DR;

calorie restriction, CR) occurs frequently and animals have developed strategies to
cope with this situation. This process has been in the focus of many ecological and
aquacultural studies and will be discussed in this chapter. In general, life history
theory assumes trade-offs in resource allocation among growth, self-maintenance
and reproduction; complimentarily, evolutionary theory expresses the general
expectation that DR will often result in improved survival probability, and extended
life span. The response to DR is species-specific and often even specific to subpopu-
lations or clones, and several aquatic species respond to DR with reduced life span
and fecundity. In the case of life span extension, one major mechanism appears to
be increased stress resistances which can even be passed to succeeding generations,
most likely via epigenetic mechanisms. In model organisms, but not yet in fishes or
aquatic invertebrates, major signaling pathways have been identified. Provided
these mechanisms are better understood in fish and invertebrate species, brood-
stocks can intentionally be improved, instead of using the stochastic genetic
approach.
After food restriction and restoration of favorable food conditions, many, but not
all, animals are capable of compensatory growth (CG), that is a period of acceler-
ated growth. A few aquatic species have been found to be able of over compensatory
growth, a phenomenon looked extensively for in aquacultural practice, for instance
by intermittent feeding. In contrast to DR-mediated increased longevity, regulatory
pathways are relatively well understood, likely due to its potential of practical appli-
cation. However, in many instances CG is not free of costs: often immunity traits are
adversely affected.
In all (aquatic) ecosystems, cycles of food deprivation (dietary restriction, DR) and
food availability occur frequently, driven by seasonal productivity changes. In the
stochastically fluctuating real world, DR and even starvation with the subsequent
opportunity for compensatory growth are common scenarios. This means that

https://doi.org/10.1007/978-3-319-91767-2_4
138 4 Dietary Restriction, Compensatory Growth – Short-Term Fasting Does Not Kill You
animals have had to develop strategies to cope with food deprivation – and they have
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successfully done so, with sometimes surprising strategies. An example of such a

challenging scenario is the dramatic climate-driven regime shift which has recently
been reported from the Arctic Ocean (Greene and Pershing 2007): in the Gulf of
Maine and Georges Bank, salinity levels decreased with subsequent increases in the
phytoplankton and small copepods, the food for larval and juvenile fishes.
Mammalian studies have shaped our understanding of the endocrine control of
appetite and body weight in vertebrates and provided the basic vertebrate model that
involves central (brain) and peripheral signaling pathways, as well as environmental
cues. The hypothalamus has a crucial function in the control of food intake, but
other parts of the brain are also involved. Endocrine signals are based on hormones
that can be divided into two groups: those that induce appetite and food consump-
tion (orexigenic), and those that inhibit it (anorexigenic). Peripheral signals origi-
nate in the gastrointestinal tract, liver, adipose tissue, and other tissues and reach the
hypothalamus through both endocrine and neuroendocrine action. While endocrine
appetite-controlling networks and mechanisms have been described for a few key
model teleosts, mainly zebrafish and goldfish, very little is known about these sys-
tems in fishes as a group. Fishes represent over 30,000 species, and there is large
variability in their ecological niches and habitats as well as life history adaptations,
transitions between life-stages and feeding behaviors (Rønnestad et al. 2017).
To summarize briefly, food intake is affected by external factors, such as tem-
perature and photoperiod, stress, predators, and food availability, as well as by inter-
nal factors, such as genetics, life-stage, gut filling, and stored energy. The
hypothalamus is the hub that controls appetite and energy balance while integrating
peripheral signals related to food intake and digestion, metabolism, and energy stor-
age (Fig. 4.1). These signals include not only endocrine signals (gut peptides), but
also other signals such as nutrient levels detected by the central nutrient sensing
systems and the presence/absence of food in the gastrointestinal (GI) tract sensed
through vagal afferents projecting to the brain (Rønnestad et al. 2017). Details of
hormonal and biomolecular regulation of CG can be found below.
At the level of the individual, food digestion is a rather stressful affair because
oxygen has to be activated for the oxidation of dietary organic carbon. This process
reduces, at least temporarily, the cellular antioxidant capacity. Any reduction of the
antioxidant capacity is oxidative stress (Steinberg 2012). And, vice versa, reduced
food consumption can save that energy for the replenishment of spontaneous anti-
oxidants, which can now be allocated to other vital processes, such as growth or
reproduction. The oxidative stress caused by food digestion can easily be illustrated.
For instance, Furuhagen et al. (2014) reported the following two linear relationships
for the water flea Daphnia magna fed a mixture of coccal green algae (Raphidocelis
subcapitata and Scenedesmus subspicatus).
Thiobarbituric acid reactive substances = 0.470 ´ feeding rate + 0.51 (4.1)

Oxygen radical quenching capacity = - 0.014 ´ feeding rate + 0.12 (4.2)

4 Dietary Restriction, Compensatory Growth – Short-Term Fasting Does Not Kill You 139
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Fig. 4.1 Key organs and signaling pathways thought to be involved in the control of appetite in
fishes. Some of the central and peripheral endocrine factors explored so far are listed. (From
Rønnestad et al. (2017), courtesy of Frontiers in Endocrinology). Abbreviations: NPY neuropep-
tide Y, AGRP agouti-related proteins, OX orexin, MCH melanin-concentrating hormone, GAL
galanin, CART cocaine and amphetamine regulated transcript, POMC proopiomelanocortin, CRH
corticotropin-releasing hormone, TRH thyrotropin-releasing hormone, LEP leptin, GHRL ghrelin,
CCK cholecystokinin, PYY peptide YY, GRP gastrin-releasing peptide, GLP glucagon-like
peptide
Thiobarbituric acid reactive substances are indicative of lipid peroxidation, one

notable symptom of oxidative stress. It is obvious that lipid peroxidation correlates
positively with the feeding rate and negatively with the antioxidant capacity of the
organisms: feeding reduces the antioxidant capacity. Therefore, it could be fruitful
(and fascinating) to work out how DR reduces the cellular oxidative stress and, in
turn, affects life history traits. Combined studies on DR or cellular oxidative status
and body maintenance, longevity or lifetime reproduction are comparably scarce.
Nevertheless, it is important to review how DR in its various forms impacts life his-
tory traits and to identify the various strategies aquatic animals have developed to
cope with this situation. These strategies shed light on many biological phenomena
and therefore deserve attention from several perspectives: evolutionary theory, life
history theory, and stress ecology, as well as aquaculture production practices.
Fishes manifest a high capacity to face short- and long-term starvation periods
under natural environmental conditions and even farmed fish undergo fasting peri-
ods associated with aquaculture practices. Food deprivation entails a mobilization
of energy stores, and, in some cases, even of body tissues, by physiological and
metabolic adjustments to meet the energy requirements for survival.
Pérez-Jiménez et al. (2011) summarized the general metabolic responses to food
deprivation as the mobilization of reserves mediated by the endocrine system
through a complex process involving several hormonal factors. Nevertheless, there
are intra- and interspecific factors that affect the metabolic response to starvation.
Food deprivation leads to hypoglycemia in most fish species. Moreover, in the first
stages of starvation the hepatic glycogenolytic pathway is activated, in order to
maintain plasma glucose levels and cover energy expenditure. Glycogen in muscle
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can be decreased or maintained at the expense of hepatic glucose release. The hypo-
glucemic status can induce a lower glucose cellular fraction by a decrease in the
activity of enzymes related to this substrate catabolism; activation of gluconeogenic
pathways occurs in response to decreased glucose levels. In parallel with glycogen
exhaustion, the activation of lipid reserves plays an important role in covering
energy demands. The analyses of different lipid body reserves under starvation con-
ditions manifests first as mobilization of perivisceral fat followed consecutively by
hepatic and muscle stores. It is remarkable that this response is mainly species-
specific. If starvation is prolonged, proteins from muscle can be mobilized.
Depending on the particular starvation conditions, fish refeeding can lead to a total,
partial, or ineffective recovery response. Usually damage is not dramatic and fish
can restore their physiological parameters. Fast weight recovery is usually linked to
an increased growth rate that exceeds the pre-starvation condition. This response is
called “compensatory growth”. It is of great interest for fish production practices,
since alternating fasting and refeeding cycles can increase production.
Life history theory assumes trade-offs in resource allocation among growth,
reproduction and self-maintenance (aging, repair and healing, defense against
pathogens and parasites) as sketched in a simplified manner in Fig. 4.2. Usually, it
is supposed that maintenance has priority over other needs and energy in excess of
maintenance can be shared between growth and reproduction. Heino and Kaitala
(1999) assumed that simultaneous allocation of energy to growth and reproduction
results in indeterminate growth. In perennial species, it is also possible that
indeterminate growth results from seasonal switching between allocation to growth
only and allocation to reproduction only. Consequently, strategies to cope with
stress by food deprivation vary widely, not only between different species, but also
body
size
W
somatic
growth
γ(a)f(W ) total
energy
intake
reproduction
?
(1-γ(a))f(W ) ?
γ(a)
maintenance
surplus
energy
f(W )
survival
Fig. 4.2 A schematic representation of an energy allocation model. Usually only the allocation of
surplus energy between reproduction and somatic growth is studied, while allocation to mainte-
nance is simply ignored. Allocation to somatic growth results in positive feedback on body size.
(from Heino and Kaitala (1999), with permission from Wiley)
4 Dietary Restriction, Compensatory Growth – Short-Term Fasting Does Not Kill You 141
within species when subject to various environmental stressors in the stochastically

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changing aquatic environment.

In addition to life history theory, evolutionary theory expresses the general
expectation that DR will often result in increased survival probability, and thus
increased life span. DR extends healthy life span in many organisms, best seen in
short-lived organisms, including budding yeast (Saccharomyces cerevisiae), the
nematode Caenorhabditis elegans, and the fruit fly (Drosophila melanogaster). The
experimental tractability of yeast and invertebrates facilitated discovery of the
(often evolutionarily conserved) mechanisms through which genetic and environ-
mental intervention improve health during aging. The mechanisms mediating the
health benefits of DR are not yet fully understood (Fontana and Partridge 2015).
One major mechanism is thought to be the increase in various forms of stress resis-
tances (Ruetenik and Barrientos 2015; Schumpert et al. 2014). Multiple, parallel
processes contribute to the increase in health during aging from DR, and the relative
contributions of these may vary between DR regimes and organisms. Interestingly,
recent work has revealed the importance of timing of food intake (also see Chap. 5
“Chrononutrition”), the role of specific nutrients, the nature of the effector mecha-
nisms, the longer-term (including transgenerational) consequences of diet, and the
key role played by the gut microbiota (Fontana and Partridge (2015) and references
therein). These issues indicate that dietary manipulations can improve the health
status of aquatic animals as well as their offspring and prevent diseases during
aging.
Further evidence regarding underlying mechanisms comes from biomedical and
biomolecular studies in model organisms, which may not readily be available to
ichthyologists, aquaculturists, or ecologists. Recently, Leonov et al. (2015) reviewed
these mechanisms comprehensively and identified 18 cellular proteins and signaling
pathways that are indispensable for their ability to prolong life span and improve
health.
(1) DAF-2, the only known receptor of the insulin/insulin-like growth factor 1
(IGF-1) signaling (IIS) pathway; this pathway defines longevity in the nema-
tode C. elegans by regulating metabolism, protein homeostasis, resistance to
many stresses, development, and reproduction;
(2) Phosphatidylinositol 3-kinase AGE-1, an essential protein component of the
IIS pathway in the nematode C. elegans;
(3) AKT-2, a serine/threonine protein kinase involved in the IIS pathway in the
nematode C. elegans;
(4) SKN-1/Nrf (master regulator of antioxidant response), one of the transcription
factors that plays an essential role in the IIS pathway;
(5) Heat-shock factors (HSFs), transcription factors involved in the IIS pathway;
(6) Transcription factor DAF-16/FOXO (forkhead box proteins), a family of tran-
scription factors regulating the expression of genes involved in cell growth,
proliferation, differentiation, and longevity and its nuclear import in the nema-
tode C. elegans and mammals—this protein is a key component of the IIS
pathway;
(7) OSR-1/UNC-43 (CaMKII)/SEK-1 (p38 MAPK), a signaling pathway that, at

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least in the nematode C. elegans, defines resistance to osmotic stress, arsenic,

and pathogen infection;
(8) Nicotinic acetylcholine receptor EAT-2, essential for longevity regulation in
the nematode C. elegans because it defines the rate of pharyngeal pumping in
this organism;
(9) NHR-8, a non-canonical nuclear hormone receptor which is essential for lon-
gevity regulation—probably because it defines resistance to xenobiotic stress
and plays essential roles in the metabolism of cholesterol, bile acids, and neu-
tral lipids in the nematode C. elegans;
(10) Mitogen-activated protein kinase (MAPK) kinase MEK-1, involved in protein
synthesis and stress-induced apoptosis and defines resistance to pathogen
infection and heavy metals;
(11) The MEV-1 subunit of succinate-coenzyme Q oxidoreductase, a component of
the mitochondrial electron transport chain that defines longevity in the nema-
tode C. elegans;
(12) The histone acetyl transferase CBP-1, a transcriptional activator which is
involved in mRNA processing and neurogenesis in the nematode C. elegans;
(13) TPH-1, a tryptophan hydroxylase enzyme involved in serotonin synthesis in
the nematode C. elegans;
(14) The sirtuins SIR1 in the yeast S. cerevisiae, SIR-2.1 in the nematode C. ele-
gans, SIR2 in the fruit fly D. melanogaster, and SIRT1 in mice on a high-
calorie diet, all of which define longevity by modulating numerous cellular
processes;
(15) The target of rapamycin complex 1 (TORC1, nutrient-sensitive protein kinase
complex), controls cell metabolism, protein synthesis, resistance to many
stresses, and autophagy in the yeasts S. cerevisiae and Sch. pombe;
(16) The nutrient-sensing protein kinases SCH9 and GCN2, define longevity by
modulating cell cycle progression, transcription, protein synthesis, responses
to various stresses, amino acid synthesis and sphingolipid synthesis in the
yeast S. cerevisiae;
(17) Cytosolic and mitochondrial superoxide dismutases SOD1 and SOD2 (respec-
tively), both play essential roles in longevity regulation by detoxifying the
superoxide radical, modulating cellular respiration, and controlling cell
response to various stresses; and
(18) The non-selective autophagy pathway for degradation of various cell organ-
elles and macromolecules in the yeast S. cerevisiae, nematode C. elegans, and
fruit fly D. melanogaster.
Many DR studies have demonstrated that life span extension tends to be accom-
panied by a reduction in female fecundity: a correlation widely interpreted as evi-
dence that DR triggers an adaptive reallocation of resources from reproduction to
somatic maintenance. To human aging studies, this approach has contributed the
plastic term ‘Disposible Soma Theory’ (DST) (Westendorp and Kirkwood 1998).
Yet, recent evidence showed that survival and fecundity need not always trade off
4.1 Indicators of Starvation 143
under DR, calling the reallocation hypothesis into question. Because the effects of
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DR on both survival and reproduction have rarely been tested in both sexes, or under
a range of ecologically relevant environments, the universality of this trade-off
remains unclear. Recently, Adler et al. (2013) examined the effects of DR on sur-
vival and reproduction in both sexes and across a range of environments (larval diet
quality and adult sex ratio) in the neriid fly Telostylinus angusticollis. The authors
found that the life span–reproduction trade-off is both context- and sex-dependent.
Although DR extended life span in both sexes by 65% and rendered females com-
pletely infertile, the costs of DR on male fecundity were subtle and apparent only in
particular environmental combinations. The findings implied that a reallocation of
resources might not underlie the life span extension response to DR. Instead, full
feeding may be associated with increased costs in comparison to DR, such that life
span extension may be achieved without increased resource investment to the soma.
Do similar concerns also apply to longevity studies? Is the trade-off between
longevity and fecundity only a laboratory artifact? Although some findings will be
presented below, the eventual answer to these questions is reserved for studies to
come.
The implied trade-off between life history traits appears to be even more signifi-
cant for the maintenance of populations. Resource allocation among the three com-
partments is assumed to have a genetic basis, but can be modified in response to
environmental challenges, including food deprivation, through phenotypic plastic-
ity (West-Eberhard 1989; Steinberg 2012). Therefore, it might be expected that food
limitation will also have cross-generational effects and resource-limited mothers
will produce smaller offspring. However, several studies have proven the opposite
(for more details, see Chap. 6 “Transgenerational Effects”), namely that resource-
limited mothers produce larger offspring. Although producing larger offspring often
comes at the cost of producing fewer individuals, this fecundity cost may be out-
weighed if larger offspring have increased fitness. Offspring size is thought to have
a greater effect on offspring fitness in low-resource environments than in high-
resource environments, this greater selective advantage would increase the optimal
offspring size in low-resource environments. Therefore, if mothers respond to
reduced food availability by producing larger offspring, then this environmental
maternal effect represents adaptive plasticity (Bashey (2006) and references
therein). This subject will be revisited below.
4.1 Indicators of Starvation
Starvation can be indicated at various biochemical and biomolecular levels. In the

ciliate Tetrahymena pyriformis, hormones are used as indicators. Short-term
(30 min) starvation hardly influenced the hormone content of cells (only serotonin
was elevated), however, long-term starvation (24 h) increased the production of
β-endorphin, adrenocorticotropin (ACTH), serotonin, histamine, insulin, and T3 by
50%. After feeding for a short time (30 min), endorphin and histamine production
returned to the basal level, ACTH and histamine levels decreased, but remained
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significantly higher than normal, and levels of insulin and T3 remained at the starva-
tion level (Csaba et al. 2007, 2008).
Nucleic acids play a major role in growth and development, and DNA that is
stable under changing environmental conditions has been used as an indicator of
biomass and cell number. RNA is directly involved in protein synthesis and larval
growth depends on available RNA. After the pioneering studies of Sutcliffe (1965),
Holm-Hansen et al. (1968), and Bulow (1970), several investigations have shown
that the whole-animal RNA/DNA ratio is correlated with nutritional condition in
both larval fish and aquatic animals (Foley et al. 2016).
Through a meta-analysis of published research studies, Foley et al. (2016)
showed that RNA:DNA ratios of larval fish are responsive to starvation stress
(Table 4.1), with effect size increasing with duration of starvation. The authors
related these results to a surrogate measure of irreversible long-term negative
impacts on fish populations, and showed that the timescale over which RNA:DNA
ratios respond to stress was long enough to reflect before-and-after entrainment
stress. Table 4.1 shows that the critical RNA:DNA ratios are highly variable, span-
ning more than one order of magnitude from 0.15 to 6.2. Foley et al. (2016) high-
lighted the diverse factors contributing to variation of RNA:DNA ratios, these
influences include ontogenetic (Gorokhova and Kyle 2002), thermal, or gender
(Chícharo et al. 2007) elements.
Several factors impacting the RNA:DNA ratios in Sprattus sprattus, particularly
body length and temperature, were studied by Peters et al. (2015). The authors
determined larval lipid content, growth rates based on RNA:DNA ratios, and fatty
acid (FA) composition during the spawning season of S. sprattus in the Central
Baltic Sea, and evaluated these in the light of feeding, mortality, and recruitment.
They estimated the starvation resistance and compared it with RNA:DNA ratios.
Starvation resistance was highest in April, with a median of up to 5 d (Fig. 4.3).
Smaller larvae in May (< 9 mm) were on average able to survive starvation for a
maximum of 2.5 d, whereas larger larvae (> 9 mm) could only starve for one day.
Starvation resistance for larvae in June and (especially) July, when water tempera-
ture was highest at ca. 14 °C, was very low, and varied between 0 and 1 d (except
for larvae <6 mm in June). Estimated mass-specific growth rates based on RNA:DNA
ratios in April and May showed a similar pattern according to larval length and
increased with size from 0 to 0.05 d−1. Specific RNA:DNA ratios increased with
size in all months. In more detail, Voss et al. (2006) found higher mean RNA:DNA
ratios in spring- than in summer-born larvae, indicating a strong selection for fast
growth in April and May. Zooplankton data revealed high concentrations of naupliar
Acartia spp. (a key dietary component of sprat) in April and May. In sum, larger
sprat (> 11 mm) in April and May 2002 were food limited and, therefore, had low
survival rates.
In Homarus gammarus, too, RNA:DNA values were positively related to food
quantity (expressed as C, Fig. 4.4) for both high (upper regression line) and low
(lower regression line) food qualities. Food with a lower C:P content resulted in
better condition of the lobster larvae regardless of the quantity of food. The reactions
Table 4.1 Critical RNA:DNA ratio indicating starvation in selected larval fish and aquatic
invertebrate
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Critical
RNA:DNA Conditions,
Species Common name ratio remarks Reference
Invertebrates
Abra ovata Bivalve mollusk 0.3–0.9* Grémare and Vétion
(1994)
Acartia tonsa Calanoid 4.6* Speekmann et al.
copepod (2006)
Artemia salina Brine shrimp ca 2* Dagg and
Littlepage (1972)
Astacus astacus Noble crayfish ca 0.6* Grimm et al. (2015)
Calanus finmarchicus Calanoid 0.46–1.48 C1 to C5 larvae Wagner et al.
copepod at 12 °C (1998)
2.1*–2.3* C4, C5 Becker et al. (2005)
Chlamys islandica Iceland scallop No change by food availabilitya Blicher et al.
(2010)
Crangon crangon Brown shrimp 0.64–0.87 Size-dependent Hufnagl et al.
(2010)
Crassostrea angulata Portuguese 2.90 ± 1.96 Chícharo et al.
oyster (2001)
C. virginica Atlantic Oyster < 1.8* Wright and Hetzel
(1985)
Daphnia galeata Water flea 6.2 Vrede et al. (2002)
Doryteuthis Opalescent 2.0– < 5.0 5 d-starvation Vidal et al. (2006)
opalescens inshore squid
Diplodon chilensis South American 0.28–0.38* Depending on Bianchi et al.
freshwater previous food (2015)
mussel source
Diporeia spp. Amphipods 0.15–0.43 In different lakes Ryan et al. (2012)
Euchaeta elongata Calanoid 1.2–1.4 Dagg and
copepod Littlepage (1972)
Eurytemora affinis Calanoid 2.14 Pommier et al.
copepod (2010)
Homarus gammarus European 0.9–1.17 Buss et al. (2012)
lobster
Litopenaeus Pacific white 2.2–4.0 Negative growth Moss (1994)
vannamei shrimp rates
Mysis diluviana Opossum < 2.0 Johannsson et al.
shrimp (2009)
Mytilus californianus California 1.95 Chícharo and
mussel Chícharo (2008)
Neocalanus cristatus Calanoid < 2.31 ± 1.13 Ikeda et al. (2007)
N. flemingeri copepods < 5.80 ± 1.82
N. plumchrus < 3.46 ± 2.21
N. robustior < 1.12
(continued)
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Critical
RNA:DNA Conditions,
Oithona davisae Cyclopoid 2.2–3.5 Yebra et al. (2011)
copepod
Ruditapes decussatus Grooved carpet 4.88 ± 2.65 Different trials Chícharo and
shell 2.1 ± 0.15 Chícharo (2008)
1.47 ± 0.18 Fathallah et al.
(2010)
Saxidomus Purplish 1.1* Females Kim et al. (2005)
purpuratus Washington 0.2* Male
clam
Sepia officinalis Common 0.0–0.5 at 17.5 °C Sykes et al. (2004)
cuttlefish 1.08–0.93 at 22.5 °C Melzner et al.
1.08–0.82 (2005)
Fishes
Ammodytes American sand < 2.0 Buckley et al.
americanus eel (1984)
A. marinus Lesser sand eel ca. 1.4* Malzahn et al.
(2007)
Clupea harengus Atlantic herring 1.0–2.5 in Illing et al.
(2016)
C. harengus pallasi Pacific herring 2.06 in Foley et al.
C. harengus, Atlantic herring 2.5 (2016)
laboratory
Gadus morhua Atlantic cod 1.5
Coregonus Houting 0.38 8 °C Malzahn et al.
oxyrhinchus 0.36 18 °C (2003)
Engraulis anchoita Argentine 0.74–2.98 Diaz et al. (2011)
anchovy
Gobiusculus Two-spotted 0.58–4.0* Temperature- Frommel and
flavescens goby dependent Clemmesen (2009)
Limanda limanda Common dab ca. 0.4* Malzahn et al.
(2007)
Melanogrammus Haddock 1.4 Negative growth Caldarone (2005)
aeglefinus rates
Oncorhynchus mykiss Rainbow trout 1.56–2.05 different tissues Sevgili et al.
at week 4 (2013b)
Paralichthys Olive flounder; < 2.0 Negative growth Gwak and Tanaka
olivaceus Japanese 3–4 rates (2002)
halibut <3 Fukuda et al.
(2001)
Tanangonan et al.
(1998)
Pleuronectes platessa European plaice 0.32 Selleslagh and
Amara (2013)
(continued)
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Critical
RNA:DNA Conditions,
Pseudopleuronectes Winter flounder 3.2–3.5 7–8 °C in Buckley (1984)
americanus
Salmo salar Atlantic salmon 2.5–4.0 Negative growth Caldarone et al.
rates (2016)
Sardina pilchardus European 1.3 in Foley et al.
pilchard (2016)
Sardinops Japanese 1.17 < 17.5 °C
melanostictus pilchard 1.32 > 17.5 °C
Sciaenops ocellatus Red drum 1.17
S. ocellatus, 2.45
laboratory
Sardinella brasiliensis Brazilian 0.4* Rossi-
sardine Wongtschowski
et al. (2003)
Solea solea Common sole 1.9–0.35* Time-dependent Richard et al.
(1991)
Sprattus sprattus European sprat 2.1–2.3 Temperature- Peters et al. (2015);
dependent Voss et al. (2006)
Takifugu obscurus Pufferfish, fugu ca 0.8* 4 to 5 dah, 1 and Kim et al. (2008)
ca 2.0* 3 psu**
ca 1.5* 25 dah, 1 psu
ca 1.8* 25 dah, 3 psu
48 dah, 1 and
3 psu
Thunnus orientalis Pacific bluefin3.73 < 5 mm, 25 °C in Foley et al.
tuna 2.83 > 5 mm, 25 °C (2016)
5.39 < 5 mm, 28 °C
1.24 > 5 mm, 28 °C
Trachurus Horse mackerel 1.05 ± 0.08 Yandi and Altinok
mediterraneus (2015)
*estimated from corresponding graphs; **practical salinity units
a
Presumably, the RNA:DNA ratio reflects the growth potential rather than the actual growth rate of
Chlamys islandica (Blicher et al. 2010)
of the lobster larvae to different qualities of food (as shown by the regression slopes)
were not significantly non-parallel (the slopes do not differ), showing that food
quality affected the larvae irrespective of quantity. This was true even for very low
quantities of food. Differences in the behavior of the fed and non-fed larvae were
also observed, with the fed animals displaying sustained bursts of swimming activ-
ity during their hunt for the prey items, while the starved animals remained inert and
did not show a great deal of activity (Schoo et al. 2012). Interestingly, starved ani-
mals exhibited higher RNA:DNA ratios than animals fed low quantities (Fig. 4.4),
probably as preconditioning for compensatory growth, should food become avail-
able again.
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Fig. 4.3 (a) Estimated sprat (Sprattus sprattus) starvation resistance based on lipid content, (b)
calculated weight-based growth rates based on RNA:DNA ratios and (c) sRNA:DNA ratios as
moving median vs. larval size, for the months of April (6 °C, dashed grey line), May (7 °C, solid
grey line), June (9 °C, dashed black line) and July (14 °C, solid black line); no growth rate or
sRNA:DNA ratios are given for June. (from Peters et al. (2015), with permission from Inter-
Research; ©Inter-Research 2015)
4.2 Starvation Tolerance and Starvation Impact
Certain key studies reveal the different states-of-the-art of starvation tolerance and
trade-offs between life history traits as well as compensatory strategies in various
aquatic animals, starting from rotifers and peaking with fishes. The various studies
demonstrate the wide variety of strategies that the animals use to cope with
4.2 Starvation Tolerance and Starvation Impact 149
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1,4
1,2
RNA:DNA
1,0
0,8
f/2
-P
Starvation
0,6
0 5 10 15 20 25 30 35
Quantity of food (µg C)
Fig. 4.4 RNA:DNA ratio of lobster larvae (Homarus gammarus) and food quantity expressed as
the amount of carbon in the diet. (from Schoo et al. (2012), courtesy of Public Library of Science).
nutrition-induced stress situations. There is no common strategy: instead, there is a

continuum between two contrasting strategies. At one end of this continuum are
animals following a slow pace-of-life strategy with a combination of traits that gen-
erally include low rates of reproduction and high levels of self-maintenance, such as
increased stress tolerance, immune response, and low rates of aging. Unsurprisingly,
at the opposite end of the spectrum, the converse is true, animals follow a fast pace-
of-life strategy. The core mechanism underlying the fast–slow continuum of life
history traits is the trade-off in the allocation of resources to competing physiologi-
cal functions, such as reproduction and somatic maintenance. Resource allocation
trade-offs imply that, since resources are finite and physiological mechanisms
require them to function properly, multiple functions cannot be maximized at the
same time for a given level of resources. Hence, if evolution acts to increase repro-
duction, investments into somatic maintenance should decrease, and vice versa.
Furthermore, when animals experience resource-poor environments, trade-offs
become exaggerated, and the negative correlation between life history traits is more
likely to be revealed (Tökölyi et al. (2016) and references therein). Responses to
increased or reduced food availability can be extremely variable. For instance, in a
study of life history responses to reduced food in ten planktonic rotifer, some spe-
cies respond to low resource levels by reducing reproduction, while others did the
opposite (Kirk 1997) (Table 4.2). Response to food variability adds a further dimen-
sion to the diversity of life history variation in animals.
Table 4.2 Impact of food availability on major life history traits in selected fish and invertebrate
species ⇧ increase; ⬇ decrease; ↔ ambigous
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Food availability
Low Intermit. High
Species Life history trait Response Reference
Cnidarians
Hydra viridissima Stress tolerance ⇧ ⇧⇧ Tökölyi et al.
(2014)
H. viridissima Budding rate ⇧ ⬇ Tökölyi et al.
H. vulgaris ⇧ ⇧⇧ (2016)
H. oligactis ⇧ ⇧
H. circumcincta ⇧ ⇧⇧
H. oxycnida ⇧ ⇧⇧
Rotifers
Keratella cochlearis Growth rate ⇧⇧ Walz (1995)
Brachionus Growth rate ↔ ⇧⇧ ⇧
angularis
Cephalodella sp. Life span ↔ ↔ ↔ Weithoff (2007)
Fecundity ↔ ↔ ↔
Elosa worallii Life span ⇧
Fecundity ⬇
Brachionus patulus Life span ⇧ ↔ ⬇ Sarma and Rao
Decreasing with increasing (1991)
temperature
Asplanchna Life span ⬇ ⇧ Kirk (1997)
priodonta Fecundity ⬇ ⇧
A. silvestrii Life span/fecundity ⬇⬇/⬇ ⇧/⇧
Brachionus Life span/fecundity ⬇⬇/⬇ ⇧/⇧
calyciflorus
B. caudatus Life span/fecundity ⬇/⬇ ⇧/⇧
B. havanaensis Life span/fecundity ⬇⬇/⬇ ⇧/⇧
Keratella cochlearis Life span/fecundity ⬇⬇/⬇⬇ ⇧/⇧
Platyias Life span/fecundity ↔/⬇⬇ ↔/⇧
quadricornis
Synchaeta pectinate Life span/fecundity ⬇/⇧ ⇧/⬇
Synchaeta spec Life span/fecundity ↔/⇧ ⇧/⬇
Brachionus plicatilis Life span/fecundity ⇧/⬇ Yoshinaga et al.
(2000, 2001,
2003)
Protein content ⬇ Makridis and
Olsen (1999)
Mollusks
Babylonia formosae Larval growth ⇧ Zheng et al.
habei Metamorphosis ⬇ (2005)
(continued)
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Food availability
Low Intermit. High
Crassostrea gigas Growth rate, ⇧ Moran and
metabolic rate Delayed feeding Manahan (2004)
Growth of velar ⇧ Strathmann et al.
lobes (1993)
Illex illecebrosus Muscle protein ⬇ reviewed in Wells
and Clarke (1996)
Doryteuthis Survival ⬇ Vidal et al. (2006)
opalescens Muscle protein ⬇ reviewed in Wells
and Clarke (1996)
Meretrix meretrix Larval survival (⇧) Tang et al. (2006)
Octopus maya Juvenile growth ⬇ George-Zamora
Utilization of specific ⇧ et al. (2011)
AAs
Octopus spp. Muscle protein ⬇ reviewed in Wells
and Clarke (1996)
Ommastrephes Growth of statoliths ⬇ Yatsu and Mori
bartramii (2000)
Ruditapes Larval survival ⬇ Matias et al.
decussatus (2011)
R. philippinarum Larval survival after ⇧ Yang et al. (2010)
medium-term
starvationa
Echinoderms
Asterina miniata Cost of protein ⇧ ⬇ Pace and
synthesis Manahan (2007,
Lytechinus pictus Cost of protein ↔ ↔ 2006)
synthesis
Arbacia punctulata Energy consumption ⬇ Hill and
Lytechinus Energy consumption ⬇⬇ Lawrence (2006)
variegatus
Acanthaster planci Larval survival and ⬇ Lucas (1982)
development
Apostichopus Larval survival ⬇ Sun and Li (2012)
japonicus
Odontaster validus Time to select food ⬇ Kidawa (2009)
items
Pseudechinus Egg size ↔ ⇧ Poorbagher et al.
huttoni Larval morphol. ↔ (2010)
↔
plasticity
Larval mortality rate ⇧
Sterechinus 4-arm → 6-arm ↔ ↔ Marsh et al.
neumayeri larvae (1999)
(continued)
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Food availability
Low Intermit. High
Strongylocentrotus FoxOb activity ⇧ Carrier et al.
droebachiensis (2015)
Changes of fatty acid ⇧ Wessels et al.
patterns (2012)
S. intermedius Intestinal mypc ⬇ Yanjie et al.
transcription (2014),
S. purpuratus Utilization of specific ⇧ Meyer et al.
AAs (2007)
Tripneustes gratilla Growth ↔ Byrne et al.
Larval composition ↔ (2008)
Polychaetes
Various species Juvenile growth ⬇ Paterson et al.
With high proportions of (2006)
detritus
Hydroides elegans Larval ⬇ McEdward and
metamorphosis Discontinuous feeding Qian (2001)
Survivorship, ⬇ Qiu and Qian
settlement (1997)
Trochophore → ⇧
juvenile
Juvenile growth ⬇ Qian and
Pechenik (1998)
Capitella sp Larval mortality ⇧ Qian and Chia
Larval growth ⬇ (1993)
Settling rates ⬇
Larval span ⇧
Organic C content ⇧
Polydora ligni Larval mortality ⇧
Larval growth ⬇
Settling rates ⬇
Larval span ⇧
Organic C content ↔
Ophryotrocha Juvenile upper jaw ↔ Berruti (1980)
puerilis → definitive upper
jaw
Chaetognaths
Sagitta enflata Phosphorus excretion ⬇ Szyper (1981)
Nitrogen excretion ⬇
Crustaceans: Ostracods
Cyprideis torosa Anoxia tolerance ⬇ Gamenick et al.
(1996)
(continued)
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Food availability
Low Intermit. High
Cyprinotus Biomass ⬇ Grant et al. (1983)
carolinensis
Darwinula Stress tolerance ⇧ Rossi et al. (2002)
stevensoni
homozygous ♀ Starvation tolerance Low Rossi et al. (2004)
heterozygous ♀ Starvation tolerance High
Eucypris virens Clonal richness ⬇ Martins et al.
(2010)
Crustaceans: Cladocerans
Alona rectangula Population growth ⬇ ↔ ⇧ Nandini and
Sarma (2003)
Bosmina longirostris Survival/brood size ⬇/⬇ ⇧/⇧ ↔/⇧ Urabe (1991)
Ceriodaphnia dubia Growth rate ↔ ⇧ ⇧ Gama-Flores
Increasing with temperature et al. (2011)
Population growth ⬇ ↔ ⇧ Nandini and
Sarma (2003)
C. cf. dubia Life span ⇧ ⬇ ⬇ Rose et al. (2000)
Brood size ⬇ ⇧ ⇧
Growth rate ⬇ ⇧ ⇧
C. cornuta Mortality ⇧ Amarasinghe
Egg number ↔ et al. (1997)
Growth rate ↔
Life span ⬇ ⇧ ⬇ Nandini and
Fecundity ⬇ ⇧ ⬇ Sarma (2000)
D. hyalina + D. Offspring survival ⇧ ↔ ⬇ Gliwicz and
pulicaria Guisande (1992)
D. longispina Life span ⇧ ⇧ ⬇ Ingle et al. (1937)
D. hyalina × galeata Body length ⬇ ⇧ Doksæter and
Clutch size ⬇ ⇧ Vijverberg (2001)
D. laevis Population growth ⬇ ↔ ⇧ Nandini and
Sarma (2003)
(continued)
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Food availability
Low Intermit. High
D. magna Survival ⇧ Gliwicz (1990)
Life span ⇧ ↔ ⬇ Martínez-
Total offspring ↔ ⇧ ↔ Jerónimo et al.
(1994)
Brood size ⇧ Glazier (1992)
Juvenile growth rate ⬇ ⇧ ⬇ Giebelhausen and
Lampert (2001)
Offspring body size ⬇ Enserink et al.
Brood size ⇧ (1990)
Cd sensitivity ⬇
Offspring body size ⇧ ⬇ Gabsi et al.
(2014)
Predator avoidance ⇧ ⬇ Pauwels et al.
Reproduction ⬇ ⇧ (2010)
Sensitivity to ⬇ ⇧ Smolders et al.
osmotic stress (2005)
Offspring ⇧ Li and Jiang
Microcystis (2014)
resistance
Offspring disease ⇧ Mitchell and Read
resistance (2005)
D. parvula Survival ⇧ ⬇ ↔ Orcutt and Porter
Decreasing with temperature (1984)
D. pulex Growth rate ⇧ ⇧ ⇧ Gama-Flores
Decreasing with temperature et al. (2011)
Survival ⇧ ↔ ⬇ Latta et al. (2011)
D. pulicaria Survival ↔ ⇧ ↔
Survival ⇧ Gliwicz (1990)
D. reticulata Survival ⬇
D. cucullata Survival ⬇
Diaphanosoma Population growth ⬇ ↔ ⇧ Nandini and
brachyurum Sarma (2003)
D. excisum Mortality ⬇ Amarasinghe
Egg number ↔ et al. (1997)
Growth rate ⇧
Moina macrocopa Survival ↔ ⇧ ↔ Xi et al. (2005)
Decreasing with increasing
temperature
Life span/fecundity ⬇/⬇ Nandini and
Population growth ⬇ ↔ ⇧ Sarma (2000),
(2003)
(continued)
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Food availability
Low Intermit. High
M. micrura Mortality ↔ Amarasinghe
Egg number ⇧ et al. (1997)
Growth rate ⇧
Pleuroxus aduncus Life span/fecundity ⬇/⬇ Nandini and
Scapholeberis kingi Population growth ⬇ ↔ ⇧ Sarma (2000),
Simocephalus Life span/fecundity ⬇/⬇ (2003)
vetulus Population growth ⬇ ↔ ⇧
Crustaceans: Copepods
Heliodiaptomus Mortality ⬇ Amarasinghe
viduus et al. (1997)
Mesocyclops Mortality ⬇
thermocyclopoides
Paracartia grani Life span ⇧ Smith and Snell
Reproductive period ⇧ (2014)
Retarded ⇧ Calbet and
reproduction Alcaraz (1996)
Crustaceans: Cirripeds
Balanus amphitrite Larval survival ↔ Qiu et al. (1997)
Instar duration ⇧ Desai and Anil
(2004)
Crustaceans: Amphipods and Isopods
Abyssorchomene Glutamic acid ⇧ Janecki and
plebs content Rakusa-
Suszczewski
(2004)
Balloniscus sellowii Food retention ⇧ Wood et al.
Quality- (2012)
dependent
Diporeia spp. Lipid oxidation ⇧ Maity et al.
PUFA content ⬇ (2012a, b)
Phospholipids ⬇
Protein catabolism ⇧
Glutamine ⇧
Gammarellus Changes of fatty acid ↔ Wessels et al.
homari patterns (2012)
Gammarus fossarum Amylase activity ⬇ Charron et al.
(2014)
G. pulex Cd2+ susceptibility ⬇ ⇧ Alonso et al.
(2010)
(continued)
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Food availability
Low Intermit. High
Idotea baltica Population growth ⬇ Gutow et al.
rate (2007)
I. metallica Reproduction ⬇
survial ⇧
Marinogammarus Swimming activity ⬇ ↔ Mackintosh
obtusatus (1973)
Monoporeia affinis Survival ↔ Aljetlawi et al.
(2000)
Ammonia excretion ⬇ Lehtonen (1994)
Proasellus Ecdysteroid content ⬇ Mondy et al.
meridianus After (2014)
starvation
Themisto libellula Lipid content ⬇ Percy (1993)
Waldeckia obesa Arginine content ⇧ Janecki and
Rakusa-
Suszczewski
(2005)
Ammonia excretion ⇧ Chapelle et al.
(1994)
Crustaceans: Euphausiids
Euphausia hanseni Herbivory → ⇧ Huenerlage and
omnivory Buchholz (2013)
Metabolic ⬇
parameters
E. superba Metabolic activity ⬇Short- Auerswald et al.
Usage of body ⇧termd (2009)
reserves
Lipid reserves ⬇ Auerswald et al.
Long-term (2015)
Glucanase activity ⇧Short-term (McConville et al.
1986)
Herbivory → ⇧ Price et al. (1988)
omnivory
Polar lipids, sterols ⬇, ⬇ Virtue et al.
Short-term (1993)
Respiration ⬇ Atkinson et al.
Ammonium ⬇ (2002)
excretion Short-term
Larval lipid/protein ⬇/⬇ Meyer and Oettl
Short-term (2005)
(continued)
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Food availability
Low Intermit. High
Meganyctiphanes Haemocyanin levels ⬇ Spicer and
norvegica ATP levels ↔ Strömberg (2002),
Fructose-1,6- ⬇ Salomon and
bisphosphate levels Saborowski
(2006), Salomon
Pyruvate kinases ↔
et al. (2000)
Thysanoessa inermis Metabolic activity ↔ Huenerlage et al.
Lipid reserves ⬇ (2015)
Herbivory → ⇧?
omnivory
Crustaceans: Mysids
Gnathophausia Oxygen consumption ⬇ Hiller-Adams and
ingens Ammonia excretion ⬇ Childress (1983)
Leptomysis lingvura Respiration ⬇ Herrera et al.
Electron transport ↔ (2011)
system
Mysis mixta Isotopic composition ↔ Gorokhova and
Neomysis integer Isotopic composition ↔ Hansson (1997,
1999)
Sterol content ↔ Morris et al.
(1982)
Crustaceans: Decapods
Aegla platensis ♀♀ Gonadosomatic ↔ Silva-Castiglioni
index et al. (2015)
Hepatosomatic index ⬇
Carcinus aestuarii Immune competence ⬇ Matozzo et al.
Oxidative stress ↔ (2011)
C. maenas Larval body mass ⬇ Dawirs (1983,
Larval lipid reserves ⬇ 1984, 1987)
Zoea-I development ⬇
Zoea-II growth rate ⬇
Cherax Growth increments ↔ Calvo et al.
quadricarinatus (2012)
Crangon crangon Larval growth ⬇ Hufnagl et al.
Larval development ⬇ (2010)
Survival (↔)
Protein/carbohydrate/ ⬇⬇/⬇/ Moreira et al.
lipid reserves (⬇) (2015)
Fenneropenaeus Specific growth rate ⇧ Zhang et al.
chinensis Short-term (2010)
(continued)
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Food availability
Low Intermit. High
Homarus Molting interval ⇧ Abrunhosa and
americanus Kittaka (1997)
Myofibrillar proteins ⬇ D’Agaro et al.
Haemocytes ⬇ (2014)
Homarus gammarus Swimming activity ⬇ Schoo et al.
(2012)
Hyas araneus First zoea stage ⇧ Anger and Dawirs
Second zoea stage ⬇ (1981)
Jasus edwardsii Molting interval ⇧ Abrunhosa and
Kittaka (1997)
Glycogen reserves ⬇ (Simon and Jeffs
2013)
Lipid content ⬇ Smith et al.
Saturated FA ⇧ (2003)
Monounsaturated FA ⬇
J. verreauxi Molting interval ⇧ Abrunhosa and
Kittaka (1997)
Litopenaeus Body weight @ ↔ Walker et al.
vannamei 28 °C (2011)
Body weight @ ⬇
32 °C
Pathogen resistance ⬇ Lin et al. (2012)
Lysmata seticaudata Zoea → decapodid ⬇ Calado et al.
Decapodid → 1st juv ↔ (2010)
instar
Macrobrachium Survival of first ↔ Anger and Hayd
amazonicum larvae (2009)
M. equidens Larval survival ⬇ Gomes et al.
(2014)
Neocaridina davidi Juvenile stage I ⬇ Pantaleão et al.
survival (2015)
Juvenile stage II ⬇
survival
Juvenile stage III ↔
survival
Neohelice granulata Lipid reserves ⬇ Pellegrino et al.
(2013)
(continued)
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Food availability
Low Intermit. High
Nephrops norvegicus Adults ⬇ Mente (2010)
Hepatopancreas lipids ⬇
Survival
Adults ↔ Watts et al. (2014)
Protein from tail ⬇
muscle ⬇
Hepatopancreas size
& lipid reserves
Haemocyanin
Zoea ⇧ Pochelon et al.
Switching prey (2009)
preference
Palaemon elegans Zoea → decapodid ⬇ Calado et al.
Decapodid → 1st juv ↔ (2010)
instar
P. serratus Zoea → decapodid ⬇
Decapodid → 1st juv ↔
instar
Palaemon varians Zoea → decapodid ⬇
Decapodid → 1st juv ↔
instar
Larval development ⇧ Oliphant et al.
time (2014)
Larval growth rate ⬇
Panulirus cygnus 1st molting interval ⇧ Liddy et al.
(2003)
P. japonicus 1st molting interval ⇧ Mikami and
Takashima (1993)
Paralomis granulosa Proteins/lipids ⬇/⬇ Comoglio et al.
Oxygen consumption ⇧ (2005)
Procambarus clarkii Energy mobilization ⇧⇧ Powell and Watts
P. zonangulus Energy mobilization ⇧ (2010)
Insects
Chironomus tepperi Egg numbers ⬇ Colombo et al.
Offspring fecundity ⬇ (2014); Townsend
et al. (2012)
Chaoborus Feeding rates ⇧ Minocha and
Haney (1986)
(continued)
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Food availability
Low Intermit. High
Coenagrion puella Transient mass ⬇ Campero et al.
larvae Emergence Delayed (2008)
Immune response ⬇
Tunicates
Oikopleura dioica Respiration ↔ Lombard et al.
(2005)
Fishes
Acanthochromis Offspring size at ⬇/⬇ ⇧/⇧ Donelson et al.
polyacanthus hatching & survival (2009)
Acipenser High temperature ⇧/⇧ Verhille et al.
medirostris tolerance/HSP70 (2016)
Growth rate, lipid ⬇ ⇧ Haller et al.
content, protein ⬇ (2014)
content Triglycerides, ⬇
Salinity tolerance
⬇
A. transmontanus Salinity tolerance ⬇ Lee et al. (2015)
Heat shock protein ⬇ Han et al. (2012)
responses
Dicentrarchus Gonadal maturation Starvation In Kousoulaki
labrax ⬇ et al. (2015)
Protein oxidation in ⇧ Costantini et al.
the brain (2018)
Gadus morhua Fecundity ⬇ ⇧ Lambert and Dutil
(2000)
↔ ↔ Ouellet et al.
(2001)
G. morhua ♂♂ Sperm production ⬇ ⇧ Yoneda and
Wright (2005a)
G. morhua ♀♀ Potential fecundity ↔ ↔ Yoneda and
Somatic growth ⬇ ⇧ Wright (2005b)
C. morhua ♀♀ Egg survival ↔ ↔ Marteinsdottir
length, weight Larval survival ↔ ↔ and Steinarsson
(1998)
Gasterosteus Life span in natural ⇧ Gambling and
aculeatus habitats Reimchen (2012)
Life span ↔Dietary Inness and
restriction Metcalfe (2008)
⬇
Intermittent
feeding
(continued)
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Food availability
Low Intermit. High
Heterandria Number of offspring ↔ ⇧ ⇧⇧ Travis et al.
formosa ↔ ⇧ ⇧ (1987)
Number of broods
↔ ↔
Brood size ⬇ ⬇
Interbred intervals ↔ ↔
Offspring size
Offspring body size ⬇ Reznick et al.
(1996)
Hippocampus Survival after ⇧⇧ Sheng et al.
trimaculatus starvation for 24 h (2007)
Hippocampus kuda Survival after ⇧
starvation for 24 to
48 h
Nothobranchius Life span, laboratory ⇧ Terzibasi et al.
furzeri strain (2009)
Life span, wild ↔
straine
Female body mass ⇧ ⇧⇧ Blažek et al.
(2013)
Oryzias latipes Somatic growth ⬇ Davis et al.
Egg production ⬇ (2002)
Pleuronectes Fecundity ⬇ ⇧ Horwood et al.
platessa (1989)
Poecilia reticulata Neonates size ⇧ ⬇ Bashey (2006)
Offspring iridescent ⬇ ⇧ Evans et al.
area (2015)
Offspring sperm ⬇ ⇧
velocity
P. latipinna Matrotrophy ⬇ ↔ ⇧ Trexler (1997)
Pomoxis annularis Egg quality ⇧ Bunnell et al.
Ovary size ⇧ (2005, 2007)
Pseudopleuronectes Larval survival ⬇ ⇧ Buckley et al.
americanus Depending on ♀ size (1991)
Pungitius pungitius Maturation Delayed Ab Ghani and
Merilä (2015)
Salmo salar Egg size ⇧ ⬇ Jonsson et al.
(1996)
⇧ ⬇ Burton et al.
(2013)
Transcription of ⬇ Martin et al.
immune genes Starvation (2010)
(continued)
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Food availability
Low Intermit. High
S. trutta Egg size ⇧ ⬇ Einum and
Fleming (1999)
Salvelinus fontinalis Maternal fitness ⬇ ⇧ Hutchings (1991)
Simochromis Egg size ⇧ ⬇ Segers and
pleurospilus Offspring growth ⬇ ⇧ Taborsky (2011)
Offspring risky ⇧ ⬇
behavior
Maternal care ⬇ ⇧ Segers et al.
(2011)
Learning Superior on variable feeding Kotrschal and
performance Taborsky (2010)
Digestive efficiency ⇧ ⬇ Kotrschal et al.
Gut and liver weight ⇧ ⬇ (2014)
Sparus aurata Oxidative capacity in ⇧ Bermejo-Nogales
white skeletal muscle et al. (2011)
Growth ⬇ Metón et al.
(1999)
a
The point-of-no-return, when 50% of the larvae died off, was determined to be 18.7 d of starvation
b
Forkhead box protein
c
Major yolk protein gene
d
‘Short term’ and ‘long term’ = duration of starvation.
e
These results support the concerns of Ali et al. (2003) and Adler and Bonduriansky (2014) who
argue that the life span extension effect of DR is likely to be a laboratory artifact: in contrast with
captivity, most animals living in natural environments may fail to achieve life span extension under
DR. What, then, is the evolutionary significance of the suite of responses that extend life span in
the laboratory? The authors assume that these responses represent a highly conserved nutrient
recycling mechanism that enables organisms to maximize immediate reproductive output under
conditions of resource scarcity
4.2.1 Cnidarians
The trade-off between self-maintenance and reproduction has been addressed by

Tökölyi et al. (2016), who studied five freshwater Hydra spp. These are animals
with a simple body organization which occur in most freshwater habitats, where
they follow a predatory lifestyle. There was a significant interaction between food
level and species in determining budding rate. In H. vulgaris and H. circumcincta,
few buds were produced at the low food level, but the rate of asexual reproduction
increased steadily with food availability (Fig. 4.5a, c). In contrast, budding rate in
H. viridissima was high at low food levels, peaked at medium food supply, and
declined at the high level (Fig. 4.5b). The number of buds produced increased
sharply from low to medium food availability in H. oligactis, after which it leveled
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Fig. 4.5 Budding rate (a–c) of five species of Hydra on different food levels. Budding rate is the
total number of buds produced by three Hydra during a 6-day period (averaged for each strain
separately). (from Tökölyi et al. (2016), with permission from Springer; image ©CEW Steinberg)
off (Fig. 4.5c). Lastly, budding rate was very low in H. oxycnida (no buds were
produced with low food supply) and only increased slightly at higher levels
(Fig. 4.5c).
Budding rate and stress tolerance were negatively related to low and medium
level food availability (Fig. 4.6). In accordance with life history theory, Tökölyi
et al. (2016) found that stress tolerance and asexual reproduction were negatively
related across the strains investigated. Four of the five species can be positioned
along a fast–slow life history continuum, with H. vulgaris and H. circumcincta fol-
lowing relatively slow life-histories (high stress tolerance and low rates of asexual
reproduction), while H. viridissima and H. oligactis were at the opposite end.
4.2.2 Rotifers
Rotifers play an important role in many freshwater and brackish water plankton
communities. The populations are determined from bottom-up, depending on dif-
ferent food quantities and qualities. As threshold food levels for rotifers are higher
than for cladocerans they are often outcompeted when food concentrations are low-
ered by the effective clearance activity of cladocerans. Furthermore, food availabil-
ity has a significantly different impact on the various developmental phases
(embryonic, juvenile, adult). Walz (1995) summarized this variability in food sup-
ply effect for the model rotifer Brachionus angularis (Fig. 4.7). With increasing
food concentration, the duration of the juvenile phase (pre-reproductive period
between hatching and laying of the first egg) diminishes. The rate of juvenile
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Fig. 4.6 The relationship between budding rate and stress tolerance after 8 h of H2O2 treatment in
17 strains of Hydra belonging to five species. Budding rate and stress tolerance were quantified at
three different food levels: low (a), medium (b), and high (c). (from Tökölyi et al. (2016), with
permission from Springer)
1.5 embryonic
Rate of development, d-1
development
0.75
Spec. growth rate, d-1
1.0 juvenile development

Brachionus angularis
0.5
generation development
0.5 0.25
Keratella
cochlearis
0 5 10 15 20 0.0
0 5 10 20 30 40
Food concentration, mg L-1C
Food concentration, mg-1LC
Fig. 4.7 Left: The relationship between food concentration and rate of embryonic development,
rate of juvenile development, and rate of generation development in Brachionus angularis. For the
sake of clarity, the data points have been omitted. Right: Specific growth rates of the rotifers
Keratella cochlearis and Brachionus angularis, related to food concentration. For the sake of clar-
ity, the data points have been omitted. (Redrawn from Walz (1995), with permission from Springer;
images courtesy of the Encyclopedia of Life)
development also increased with food concentration (Fig. 4.7, left). This shortening
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of the pre-reproductive phase with higher food concentrations has been established
for many rotifers and planktonic crustaceans. For B. angularis, the rate of embry-
onic development was highest in medium-range food concentrations, where relative
egg size is smallest. An increase in juvenile development rate compensates for the
prolongation of the time of embryonic development, however. The rate of genera-
tion development, as an analogue to the population growth rate, therefore, reached
a plateau, according to a saturation function of the Monod model. The kinetics of
rates of development resembled those of the more general Monod-saturation func-
tions of population growth rates (Fig. 4.7, right). These relationships differ for roti-
fer species with different life-strategies: Keratella cochlearis reached lower
maximum growth rates at relatively lower food concentration than B. angularis
(Fig. 4.7, left).
Weithoff (2007) studied the effect of DR on life span and reproduction in the
rotifers Cephalodella sp. and Elosa worallii. The food concentration under DR con-
ditions was below the threshold for population growth. It was (1) tested whether the
rotifers start reproduction again after food replenishment, and (2) estimated whether
the duration of DR conditions is relevant to the persistence of a population in the
field. Only E. worallii responded to DR with an increase in life span at the expense
of reproduction. After replenishment of food, E. worallii started to reproduce again
within one day. With an increase in the duration of DR conditions of up to 15 days
(this was longer than the median life span of E. worallii under food saturation), the
life span increased and the lifetime reproduction decreased (Fig. 4.8). These results
show that in a variable environment, some rotifer populations can persist even dur-
ing long periods of severe food deprivation.
The papers referred to above showed that one isolate of a given species responds
to DR with more or less standardized reactions. However, does this assumption
really hold true and match reality, since natural populations consist of several
clones? Recently, Gribble et al. (2014) reported on patterns of intraspecific variabil-
ity in the response to DR by testing the effects of chronic DR (CDR) at multiple
food levels and of intermittent fasting (IF) in twelve isolates from the Brachionus
plicatilis species complex. While CDR generally increased or did not change life
span and total fecundity, IF caused increased, unchanged, or decreased life span,
depending upon the isolate, and decreased total fecundity in all but one isolate. Life
span under ad libitum (AL) feeding varied among isolates and predicted the life
span response to DR: longer-lived isolates under AL were less likely to have a sig-
nificant increase in life span under CDR and were more likely to have a significantly
shortened life span under IF (Fig. 4.9). The lack of trade-off between life span and
fecundity under CDR, and differences in life span and fecundity under CDR and IF,
even when average food intake was similar, shows that longevity changes are not
always directly determined by energy intake. It further indicates that CDR and IF
regimens extend life span through diverse mechanisms.
For multi-transgenerational heritage of effects after early-lifetime starvation in
Brachionus manjavacas, refer to Chap. 6 “Transgenerational Effects”.
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Fig. 4.8 Top: the survival of Cephalodella sp. (left) and Elosa worallii (right) fed Chlamydomonas
acidophila at saturated concentrations (full circles), dietary restricted (open circles) and starved
(no symbol, straight line). Error bars denote ±1 SE of the median calculated from survival analy-
ses. Bottom: age-specific reproduction (lxmx) of Cephalodella sp. (left) and E. worallii (right).
Note different x-axis scales. (from Weithoff (2007), with permission from Springer; images cour-
tesy of The Encyclopedia of Life)
4.2.3 Mollusks
In the wild, juvenile abalone feed on diatoms before transferring to a range of dif-
ferent species of macroalgae as they mature to adults. Buss et al. (2015) investigated
the feeding behavior of juvenile greenlip abalone (Haliotis laevigata) which were
fed live macroalgae and formulated diets at different rations. The authors observed
that DR had more effect than diet type on the feeding behavior of this mollusk. In
particular, DR induced greater movement, which has consequences for energy
expenditure. In the same abalone species, in fed and short-term starved individuals,
Sheedy et al. (2016) found that the starved animals had increased lipid, increased
arginine (Arg), and threonine (Thr) contents, but decreased leucine (Leu) and lysine
(Lys)] contents. Conservation of lipid combined with the loss of weight indicates
that during starvation, carbohydrate and protein, rather than lipid, were used as the
initial sources of energy to catabolize glycogen and phosphoarginine for ATP pro-
duction. This metabolic conservation appears to be characteristic of many marine
gastropods.
Like all cephalopods, Octopus maya, an endemic species from the Yucatan
Peninsula in Mexico, is a carnivorous species and, since protein is an important
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Fig. 4.9 Kaplan–Meier survival curves for 12 isolates of Brachionus spp. Isolates were fed at
100% and 10% of ad libitum food concentration or under intermittent fasting (IF). * denotes sta-
tistically significant difference between the treatment and the 100% food level (P < 0.05). (from
Gribble et al. (2014), with permission from Elsevier)
nutrient for tissue accretion and as an energy source, cephalopods require high
amounts of protein and AAs. Taking into consideration that octopuses have low
lipid reserves, it is very likely that fasting octopuses will mobilize AAs as the prin-
cipal source of energy in an attempt to maintain homeostasis. Therefore,
George-Zamora et al. (2011) evaluated the inanition effect of AA mobilization in
juvenile O. maya. Essential AAs were affected by fasting (Fig. 4.10).
O. maya used Thr, serine (Ser), and alanine (Ala) as metabolic fuel to face starva-
tion. Simultaneous increases in histidine (His), Arg, and Lys concentrations showed
possible accumulation of these AAs. It should be kept in mind that previous work
regarding nutritional requirements in other mollusks and fish species has shown that
high concentrations of certain AAs are not the result of de novo synthesis, but of
protein hydrolysis (Moran and Manahan (2004).
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Fig. 4.10 Essential amino acid (EAA, a) and nonessential amino acid (NEAA, b) content [g AA
(100 g protein) −1] of individual samples of arms of Octopus maya juveniles at 32 days post-
hatching during the acclimation period (control on day 12), and during the fasting period (on days
4 and 8 of fasting). Each AA was ordered from major to minor content with respect to the control.
(from George-Zamora et al. (2011), with permission from Elsevier)
4.2.4 Echinoderms
Marsh et al. (2001), Pace and Manahan (2006, 2007) studied the specific energy
requirement of protein synthesis under different food availability in three ecologi-
cally contrasting echinoderm species: the subtropical sea urchin Lytechinus pictus,
the Antarctic sea urchin Sterechinus neumayeri, and the asteroid Asterina miniata.
The painted urchin, L. pictus, is distributed in the Pacific off southern California.
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Rates of protein synthesis were measured in embryos and larvae using a protein
synthesis inhibitor. Pace and Manahan (2006) found that the energetic cost of pro-
tein synthesis in developing L. pictus was fixed at 8.4 J mg−1 protein synthesized
and was independent of large variations in protein synthesis rates during develop-
ment and growth. This finding for sea urchin development was in contrast to studies
with fish, where tenfold variable costs of synthesis within a species have been
reported (Pace and Manahan (2006) and references therein). In contrast to this sub-
tropical species, Marsh et al. (2001) reported that Antarctic sea urchin embryos and
larvae (S. neumayeri) have a very low cost of protein synthesis at 0.45 J mg−1 pro-
tein synthesized. Contrary to expectations of low synthesis with low metabolism at
low temperatures, protein and RNA synthesis rates exhibited temperature compen-
sation and were equivalent to rates in subtropical sea urchin embryos. Furthermore,
the transition rates from a four-arm to a six-arm larval stage were not affected by
food availability (Marsh et al. 1999).
The third echinoderm species studied, A. miniata, has a broad geographical habi-
tat range in the Pacific Ocean, from Alaska to Baja California. Its extensive spawn-
ing season is likely to result in developmental stages experiencing variable food
concentrations. In the larvae, different amounts of food obviously resulted in differ-
ent morphology (Fig. 4.11), organic biomass, and biochemical content (protein and
Fig. 4.11 Larvae of

Asterina miniata (29 d old)
grown under four different
feeding treatments with
equal amounts of
Dunaliella tertiolecta and
Rhodomonas sp. (a) Fed
40 algal cells μL−1, (b) 20
cells μL−1, (c) 4 cells μL−1,
(d) unfed. Arrows indicate
brachiolarial arm
development (ba) and
rudiment (r). Stomach
labeled as ‘st’. Scale bar
represents 300 μm. (from
Pace and Manahan (2007),
with permission from
Elsevier).
free AA pool). Surprisingly, faster growth rates were not just a function of more
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food, but also important alterations at the level of the whole organismal biochemical
processes. These alterations resulted in substantial differences in the energy effi-
ciency of growth.
Using the major yolk protein (myp) gene as a biomarker, Yanjie et al. (2014)
reported an interesting strategy in the sea urchin Strongylocentrotus intermedius
during fasting and refeeding periods. The authors found that the priority strategy
was to reduce myp transcription in the intestine if food is limited. Conversely, myp
gene transcription in the gonads did not decline, but increased continuously during
the fasting and refeeding periods. This is a smart strategy to protect reproductive
function and sustain the population.
4.2.5 Crustaceans
In crustaceans, one major pathway to cope with starvation is the increase in protein
catabolism. This pathway has been confirmed in many, but not all, crustaceans.
These include Neohelice granulata (Pellegrino et al. 2013), Litopenaeus vannamei
(Pascual et al. 2006), Abyssorchormene plebs (Janecki and Rakusa-Suszczewski
2004), Pontoporeia affinis (Lehtonen 1994), Waldeckia obesa (Chapelle et al. 1994),
Carcinus maenas (Dawirs 1987), and Homarus americanus (D’Agaro et al. 2014)
(Table 4.2). Some amphipods (Themisto libellula, Percy (1993)), krill (Euphausia
superba, Auerswald et al. (2015), Thysanoessa inermis, Huenerlage et al. (2015)),
and Nephrops norvegicus (Watts et al. 2014) prefer lipids as a major energy resource
under starvation, however. Yet other crustaceans favor lipids and glycogen, for
example, Jasus edwardsii (Smith et al. 2003), or first proteins and then lipids as in
Paralomis granulosa (Comoglio et al. 2005), or first proteins, then carbohydrates,
and eventually lipids (Crangon crangon (Moreira et al. 2015)).
4.2.5.1 Ostracods
Rossi et al. (2002) reported on an interesting study of Darwinula stevensoni under

multiple stresses with one being starvation and the other hypoxia. Individuals of this
small ostracod were kept under hypoxic conditions (< 2 mg L−1 O2) for 24 to 768 h
and remained unfed during the exposure. The controls were not fed during the
experiment either. The authors were surprised that a high proportion of animals
survived these harsh conditions. Even more strikingly, the treated animals were dis-
tinguished by having higher survival rates than untreated (control) ones during the
recovery period (Fig. 4.12). This result was more marked for individuals from a lake
than for those from a river. Obviously, the adverse effects of starvation were less-
ened under hypoxia, and vice versa. A low metabolic rate can be seen as an adapta-
tion to a decreased food supply as well as to low oxygen concentrations. Furthermore,
metabolic depression represents an advantage for survival: physiological processes
1.00
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0.90
0.80
0.70
Control L L
Treated L L
0.60
Control D L
Survival
Treated D L
0.50
Control LR
Treated L R
0.40
Control D R
Treated D R
0.30
0.20
0.10
0.00
0 200 400 600 800
Hours
Fig. 4.12 Survival of the ostracod Darwinula stevensoni during the recovery period. Circles indi-
cate control; triangles indicate results combined from different treatments. LL lake population,
12:12 L:D illumination rhythm; DL: lake population kept in darkness; LR: river population, illu-
minated; DR: river population, in darkness. (from Rossi et al. (2002), with permission from
Schweizerbart science publishers, www.schweizerbart.de)
run more slowly, substrates are conserved, the accumulation of metabolites is

limited (Rossi et al. 2002), and repair costs for damage from oxidative stress are
minimal.
4.2.5.2 C
ladocerans
Various cladoceran species have been the subject of life history studies: in fact,
these invertebrates are the best-studied group of aquatic animals and will be dis-
cussed in detail here. For instance, more than eight decades ago, classical studies on
the effects of food deprivation on longevity in cladocerans were published by Ingle
(1933) and Ingle et al. (1937). In the first paper, Ingle tested two feeding scenarios
with Daphnia longispina and Moina macrocopa, namely normal food ration and
food reduced by 1/3. With D. longispina, the author found life span extension by
Fig. 4.13 Survival curves 100

of Daphnia longispina
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under different starving

scenarios. ● average 50 Starved
longevity. (redrawn from until
Ingle et al. (1937), with 15th instar
permission from Wiley;
log10 of survivors (%)

image courtesy of the
Encyclopedia of Life)
10 Starved
thruought
life
5
Control,
well-fed
Daphnia
cf longispina
20 30 40 50 60
Age, days
12% to as high as 68%, however this was at the expense of reproduction. Starved
individuals reached sexual maturity later and had significantly fewer offspring than
“normal” ones. The same phenomenon was observed in the short-lived M. macro-
copa: animals living under poor food conditions had a 54% longer life span
(14 instead of 9 days), but reproduction reached only 70% of the “normal” levels.
In the later study, Ingle et al. (1937) answered the questions:
( 1) Would starvation throughout life produce the greatest prolongation of life? and
(2) Would D. longispina resume active rates of growth and reproduction if, after
various longer periods of starvation, they were cultured in normal medium for
the remainder of lifetime.
The answer to the first question is obvious: Daphnia starved until the fifteenth instar
lived 11 days (28%) longer than those starved throughout life (Fig. 4.13). Yet, even
Daphnia starved throughout life lived about 40% longer on the average than those
well fed throughout life. Any gain in longevity, however, took place at the expense
of reproduction: while being starved, Daphnia produced relatively few young.
When they were fed abundantly, even after being starved, they promptly produced
many more young in each brood, and broods were produced more frequently.
In starving D. magna, the greatest losses were those of dry weight, proteins, and
lipids (including those lost due to the release of young formed before starvation).
Furthermore, reductions in total carbohydrate and glycogen contents occurred dur-
ing the first day of starvation. The triglyceride:total lipid ratio decreased during
5–6 days of starvation from 0.52 to ca. 0.15. In D. magna starved for one to three
days, the respiration rate was low and the addition of food was followed by a
three- to four-fold increase in the respiration rate and increased swimming activity.
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In D. pulex, the respiratory quotient decreased from 1.13 to 0.71 over five days of
starvation. The decrease in the respiratory quotient demonstrated a change from
predominantly carbohydrate utilization to protein and fat metabolism (Smirnov
(2014) and references therein).
In starved D. magna females, the following events occur in midgut enterocytes:
depletion of lipid and glycogen reserves, swelling of mitochondria, reduction of the
endoplasmic reticulum and of dictyosomes, and a decrease in cell height. Hungry
Daphnia actively filter their food particles; it was noted that a period of about
30 min was sufficient to overcome the starvation effect. At starvation, growth con-
tinues for a time as a result of internal stores, and refeeding results in “catch-up”
growth (Smirnov (2014) and references therein).
It has been shown experimentally that under low-food conditions Daphnia spe-
cies develop larger filtering screens on their thoracic limbs (Fig. 4.14, right)
Fig. 4.14 Left: The elationship between measured parameters, setae thickness at base expressed
in setae length (RST1) in Daphnia magna or distal part of setae expressed in setae length (RST2)
and intrinsic rate of increase (r). Black triangles and line correspond to control, light gray rhom-
buses and line to Aphanizomenon treatment and dark gray circles and line to Cylindrospermopsis
treatment. (from Wejnerowski et al. (2017), courtesy of Springer); right: Larval filtering setae
(arrows) on the third thoracopod of D. magna. The arrowheads point to the setules, which termi-
nate in a hook-like structure. (from Klann and Stollewerk (2017), with permission from Elsevier)
(Lampert 1994). In addition, Repka et al. (1999) showed in D. galeata that the
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nutritional status of individuals was a more important cue for the phenotypic
response of the filter screen morphology than particle concentration. Ghadouani and
Pinel-Alloul (2002) found that daphnids used their phenotypic plasticity to respond
to changes in food quality and quantity. By using this strategy, daphnids can maxi-
mize their food uptake and thereby compensate for the scarcity of suitable food
encountered in oligotrophic conditions or even in eutrophic conditions, when phy-
toplankton communities are dominated by large inedible species.
The dispute about how far this phenotypic plasticity to food stress may be gener-
alized continues: for instance, Wejnerowski et al. (2017) reported that the thickness
and length of setae in D. magna increased in the presence of filamentous cyanobac-
teria (Fig. 4.14, left). Thickening of setae makes the filtering comb stronger, which
enables it to more effectively fulfill its function, especially if it lengthens under
low-food conditions. Moreover, cyanobacteria-induced setae thickening was posi-
tively correlated to the fitness of daphnids, which may indicate setae thickening as
a phenotypic adaptation to cope with food stress caused by filamentous
cyanobacteria.
Counter-intuitively, and unlike the effects typically observed in the transfer of
immunity in vertebrates, D. magna mothers under poor conditions produced off-
spring which were more resistant to bacterial infection than did mothers under
favorable conditions. This effect reflects adaptive optimal resource allocation,
where better quality offspring were produced in poor environments to enhance sur-
vival (Mitchell and Read 2005). Comparable effects were also observed in D. pulex:
Maternal food limitation triggered females to produce fewer, but larger neonates
that were more tolerant to toxic Microcystis aeruginosa (Li and Jiang 2014).
The life-extending effects of food quantities often follow a bimodal response.
One striking example was presented for D. magna (Pietrzak et al. 2010). Feeding
regime had a significant effect on survival, which was shortest under the highest
food concentration available (Fig. 4.15a). High investments in early fecundity were
associated with reduced life spans (Fig. 4.15b), emphasizing the trade-off between
these life history traits. This phenomenon is well expressed in the DST, which states
that the amount of energy available to an organism is divided between two func-
tions, namely, soma (body maintenance, growth, and life span) and reproduction. If
energy is mainly allocated to growth and body maintenance, this happens in disfa-
vor of reproduction, and vice versa. The correlation was most pronounced in the
pond clone B1 that markedly increased investments in the first clutch of offspring
under 1.5 and 4.5 mg L−1 C food regimes (Fig. 4.15b). Under these food regimes,
the shortest life spans were observed (Fig. 4.15a). Overall, an extension of life span
can be found under mild food stress conditions in D. magna, a hormetic response to
low doses inducing the opposite effect to that of large doses.
Latta et al. (2011) compared the short-lived D. pulex and the long-lived D. puli-
caria (Fig. 4.16) and found remarkable differences and deviations from the DST. The
short-lived D. pulex showed the classical relationship of enhanced life span in
response to reduced food quantity (Fig. 4.16, left), but not at the expense of repro-
duction (not shown). This is not in compliance with the DST. In contrast, the long-
a b
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1 120
100
0,8
Lifespan (days)
80
0,6
Survival
60
0,4
40
0,2
20
0
0 20 40 60 80 100 120 0
Age (days) 0 5 10 15 20 25
First clutch
Fig. 4.15 (a) Daphnia magna survival under five food regimes (from lightest to darkest line: 0.05,
0.15, 0.5, 1.5, and 4.5 mg C L−1); (b) Median life span plotted against median number of offspring
in the first clutch. Lake clone D1: white diamonds; pond clone B1: gray squares; lake clone D2:
black triangles. (from Pietrzak et al. (2010), with permission from Springer)
100
Daphnia pulex Daphnia pulicaria
80
9.1
Survivorship, %
60
16.9
2.6
40
28.6 9.1 28.6

20
16.9
2.6
0
0 40 80 0 40 80 120
Age, days Age, days
Fig. 4.16 Survival curves as a function of food concentration for Daphnia pulex and D. pulicaria.
Food concentrations in 104 cells mL−1. (redrawn from Latta et al. (2011), with permission from
Wiley; images courtesy of the Encyclopedia of Life and James F. Haney)
lived D. pulicaria did not gain any life-extending effects through diet restriction
(Fig. 4.16, right). Combined, the results provided evidence that the resource alloca-
tion model (DST) is not sufficient to explain the evolution of diet-mediated life span
plasticity; rather diet-mediated life span plasticity may vary both at the level of
populations and species.
In addition to the population scale, Gliwicz (1990) reported that diet-mediated
life history plasticity even affects the zooplankton community level. He provided
empirical evidence for the assumption that large-bodied cladoceran species were
superior competitors for resources, being able to grow and reproduce at lower food
concentrations. In particular, the threshold food concentration necessary to assure
15
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0.04
Survival, days
10
0.20
5
1.00
1 2 3 4 5 6 7 8
Brood number
Fig. 4.17 Survival of Daphnia hyalina neonates of different broods born to mothers grown at
three food levels (in mg C L−1). For the sake of clarity, the individual data points are omitted.
Similar differences were observed in Daphnia pulicaria. (redrawn from Gliwicz and Guisande
(1992), with permission from Springer; images courtesy of Mark Blaxter)
that assimilation equals respiration, was lower for large-bodied species (D. magna,
D. pulicaria) than it was for small-bodied species (D. cucullata, D. reticulata).
In a subsequent paper, Gliwicz and Guisande (1992) studied the maternal invest-
ment per offspring in clonal cultures of D. pulicaria and D. hyalina, two species
similar in body size. Mothers grown at high food levels produced large clutches of
smaller eggs, but their offspring could not survive long under starvation conditions
(Fig. 4.17). Genetically identical mothers grown at low food levels produced small
clutches of larger eggs, and their offspring, albeit low in number, were able to sur-
vive long periods of starvation. This shows that Daphnia mothers are capable of
assessing food level and use this information in adjusting their fractional per off-
spring allocation of reproductive resources. Furthermore, although no third and
later brood neonates were produced by D. pulicaria and D. hyalina grown at the
lowest food level, since most mothers died soon after producing the first brood, and
the remainder died after producing a second brood, it became obvious (Fig. 4.17)
that resistance to DR is passed on to the succeeding generation, probably even to the
generations succeeding them. (For more examples, refer to Chap. 6 “Trans
generational Effects”).
The small-bodied Daphnia parvula originates from North America and has
recently been introduced to other continents (Schrimpf and Steinberg 1982). It
experiences a wide range of temperatures (4–30 °C) and food conditions during its
annual population cycle. Orcutt and Porter (1984) determined the interacting effects
of temperature and food concentration on its life history traits. Animals were raised
at combinations of three naturally-experienced temperatures and food levels. D.
parvula showed an increase in survivorship with decreasing temperature at all food
levels. Number of broods/female, brood size, and the net reproductive rate increased
with increasing food at the two lower temperatures, but showed a mid-range food
optimum at the highest temperature (Fig. 4.18). The life history parameters, average
100
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105
50
10 °C
25 °C 20 °C
0
100 20 °C 104
Survival, %
50
10 °C
25 °C
0
100
103
20 °C
50
10 °C
25 °C
0
0 10 20 30 40 50 60 70 80 90
Days
Fig. 4.18 Survivorship curves for Daphnia parvula cohorts raised at all combinations of tempera-
tures (° C) and food concentrations of Chlamydomonas reinhardtii (cells mL−1). (redrawn from
Orcutt and Porter (1984), with permission from Springer; image A Schrimpf & CEW Steinberg)
life span, age at first reproduction, brood duration time, brood size and number of
young per reproductive female all showed a significant interaction between tem-
perature and food.
Callens et al. (2016) pointed out the significance of the symbiotic gut microbial
community in conditions of DR and starvation. Their study aimed to gain a better
understanding of host-microbiota interactions under different levels of food avail-
ability. The authors conducted experiments with D. magna and compared growth,
survival, and reproduction of conventional (symbiontic) with germ-free individuals
given varying quantities of food (see also Chap. 6 “Transgenerational Effects”). The
presence of the microbiota had strong positive effects on survival, when food was
sufficient, but had weaker effects under food limitation. There was also a significant
effect of both food availability and the microbiota on the age at first reproduction.
Symbiotic Daphnia receiving a high quantity of food reproduced significantly ear-
lier than those receiving an intermediate quantity. Furthermore, Daphnia receiving
the same amount of food always reproduced significantly earlier in the presence of
symbionts than when germ-free. To sustain populations of r-strategists like Daphnia
in the presence of predators, it is crucial to reproduce as early as possible and any
delay can be problematic.
The same combination of environmental factors was applied to Moina

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macrocopa. This microcrustacean inhabits small ponds and puddles worldwide and
possess a rather short life-cycle (Petrusek 2002; Elmoor-Loureiro et al. 2010). Xi
et al. (2005) studied the combined effects of four levels of temperatures and four
food densities on life history traits. Both food level and temperature, independently
and in concert, significantly influenced nearly all life history traits. The mean life
span was maximal at 18 °C at 1.0 × 106 cells mL−1 of Chlorella (Fig. 4.19). The
reproductive performance was a direct function of the food level, but the magnitude
of the food effect was temperature-dependent.
The survival and productivity of many cladocerans is significantly influenced by
food availability (Table 4.2). Food concentrations outside certain lower and upper
thresholds caused a decrease in reproduction. The study by Xi et al. (2005) showed
that the survival of M. macrocopa was markedly influenced by food level: exces-
sively high food concentrations caused decreased survival in M. macrocopa. This
decline in survivorship was most likely caused by an increased effort in food gather-
ing and reduced net energy assimilation. Other cladoceran species such as D. magna
have a constant ingestion rate at food concentrations from 0.2 to 20.0 mg mL−1 C
(Porter et al. 1982). Furthermore, Xi et al. (2005) found that higher temperature
(28 °C), low (0.5 × 106 cells mL−1) and high (4.0 × 106 cells mL−1) food concentra-
tions caused a decrease in fecundity of M. macrocopa. Both high temperatures
(33 °C) and high levels of food were detrimental to reproduction and survival. The
100
0.5 × 106 1.0 × 106
23 °C 28 °C 23 °C
33 °C
50 18 °C
28 °C 33 °C
Survivors, %
18 °C
0
100
2.0 × 106 4.0 × 106
18 °C
50 28 °C 23 °C
28 °C
33 °C 18 °C
33 °C
23 °C
0
0 5 10 15 20/0 5 10 15 20
Age, days
Fig. 4.19 Age-specific survivorship curves of Moina macrocopa grown at different levels of food
(Chlorella vulgaris cells mL−1) and temperature (°C). (redrawn from Xi et al. (2005), with permis-
sion from Wiley; image courtesy of Philipp Dachsel)
allocation of energy to metabolic processes at higher temperatures might leave less

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energy for reproduction than is possible at lower temperatures.

In another small planktonic cladoceran, Bosmina longirostris, growth, survivor-
ship and size and age at maturation, brood size, instar duration, and egg develop-
ment time were all strongly impacted by food quantity (Urabe 1991). Most notably,
the population growth rate decreased from 0.31 to 0.02 d−1, if the food concentration
dropped from 2.5 to 0.05 mg C L−1 (Fig. 4.20). This study also demonstrated that B.
longirostris ceased to grow after maturation at the lowest food concentration,
although it grew continuously at a high food concentration. This implies that B.
longirostris changed its pattern of resource allocation with changing food concen-
tration and invests its entire net intake in reproduction under poor food conditions,
thereby increasing its competitive capability.
With decreasing food concentration, this cladoceran species reduced its mature
size and brood size, and increased its maturation time. Similar trends have been
shown in various species of Daphnia. However, unlike Daphnia, B. longirostris did
not show any significant difference in the maturation instar among food
concentrations, most individuals maturing at the third instar (Urabe 1991) so it is
interesting to explore and simulate the competitive outcome of Daphnia and
Bosmina species at various levels of food availability.
Goulden et al. (1982) used three cladoceran species, D. magna, D. galeata, and
B. longirostris, to analyze the effect of low food levels on each population individu-
ally and in competition experiments. The demographic traits of all species were
affected by low food levels, but the smaller species, B. longirostris, suffered from
high death rates. In the competition experiments, D. magna was the numerical dom-
inant in high-food cultures with D. galeata, and D. galeata was the numerical domi-
nant in cultures with Bosmina. However, Bosmina coexisted with D. galeata, and in
medium-food competition experiments Bosmina became the numerical dominant
after the first oscillation cycle population. This finding appears inconsistent with the
hypothesis that competitive ability increases with body size because large animals
exploit low food densities better than small animals can. However, the results
12 1.0
A B
0.31 0.10
9
0.05
Brood size
Survival rate
0.25
6 0.20 0.5 2.50
3 0.11
0.02 0.0
0 0 5 10 15 20
Time, days
Fig. 4.20 Brood size (a) and survivorship (b) of Bosmina longirostris at four food concentration.
The figures in (a) denote population growth rates in d−1 and in (b) the food concentrations in mg C
L−1 (redrawn from Urabe (1991), with permission from Wiley, image courtesy of Michael Plewka,
plingfactory)
indicate that larger cladocerans do not always exclude smaller cladocerans in

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competition experiments.
One possible explanation comes from Goulden et al. (1982) who commented
that Bosmina coexists with Daphnia because Bosmina adults can survive the oscil-
lation cycles of the Daphnia populations, and when food was abundant, after the
decline of the Daphnia, the increase in resources enabled Bosmina to increase its
population size temporarily. This study reinforces the idea that different species of
the same community possess different diet-mediated life span plasticity. This state-
ment gets substantial support from two more studies (Kim et al. 2014a; Han et al.
2011). Kim et al. showed that reduced food levels did not extend the life span of a
D. pulex clone, which contrasts with previous studies indicating that variations in
the response to DR might be more common than previously thought. Kim et al.
showed that such responses are not even species-specific, they appear to be specific
to individual clones of a species instead. Consistent with this report, the life span of
the tropical Diaphanosoma dubium did not change if the food supply was modu-
lated (Han et al. 2011).
The cladocerans’ response in fecundity to food concentration and temperature,
and their interaction, differ with species (Table 4.2). The fecundity of M. micrura
was significantly influenced by temperature and food concentration. In contrast, the
fecundity of D. excisum, D. parvula, C. cornuta, D. hyalina × galeata, and M. mac-
rocopa was not significantly influenced by temperature, food concentration, or their
interaction. This was even true for net reproductive rates, in contrast to other cladoc-
erans in which the net reproductive rates depended on food concentration (Urabe
1991; Nandini and Sarma 2000; Rose et al. 2000).
The highly-diverse strategy of cladocerans to food availability alone and in com-
bination with temperature appears to be species-specific. The history of individuals
or populations, however, may have an additional impact via epigenetics. For
instance, different quality food algae and water matrices, such as autochthonous
humic-like substances, modulate the susceptibility of the individuals via epigenetics
and, thereby, favor specific responses to environmental challenges and suppress
others. This issue has not yet been addressed in detail, although there are several
strong indications (Bouchnak and Steinberg 2013, 2014; Menzel et al. 2011).
Recently, Steinberg et al. (2010) fed D. magna baker’s yeast as poor quality food
in comparison to a common green algal food (Raphidocelis subcapitata). Strong
oxidative stress became obvious in those Daphnia fed with yeast, however, mean
life span was extended by approximately 11 d (14%). This happened at the expense
of reproduction: the mean offspring numbers decreased to only 43% per female per
lifetime of the normally-fed individuals. One interesting feature in the yeast-fed
individuals was the upregulation of hsp60, coding the major heat shock protein 60.
The central role of the heat shock response in longevity has recently been confirmed
by Schumpert et al. (2014), who examined hsp70 expression in two Daphnia spp.
The long-lived D. pulicaria showed a more robust hsp70 induction than the short-
lived D. pulex.
These findings contradict one classical (but outdated) theory of aging, the free
radical theory (Harman 1956), which relates aging to increasing oxidative stress.
However, D. magna increased longevity because of, not despite, the oxidative stress
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caused by poor quality food (Steinberg et al. 2010). This contradiction can now be
explained by the theory of mitohormesis (Ristow 2014) (for details, refer to Vol. 2,
Plant Materials).
4.2.5.3 C
opepods
Herbivorous copepods, such as Calanus finmarchicus, C. glacialis, or A. tonsa, play

a key role in the marine pelagic food chain. Available studies point out the impor-
tance of clone-specificity (as with the rotifers, above) and feeding history in the
application of specific strategies to cope with food deprivation.
Hirche et al. (1997) used the egg production rate as a parameter of secondary
production. The authors exposed prefed and prestarved females of C. finmarchicus
to different concentrations of the diatom Thalassiosira antarctica. Egg production
increased with higher food concentrations, but much less after starvation. The
response to starvation was temperature dependent.
Later, Niehoff (2000) reported gonads in the same copepod remained immature
during starvation, or returned to an immature stage; no eggs were produced. After 2
weeks of starvation, reproductive activity resumed within a week of surplus feeding
(Fig. 4.21). However, egg production remained significantly lower than in females
fed continuously. When female C. finmarchicus were exposed to short starvation
intervals, reproduction decreased significantly owing to both a higher percent-
ages of non-spawning females and decreasing clutch sizes. These experiments
have shown that starvation periods have a strong influence on the reproduction of
Fig. 4.21 Rates of egg production and gonad development stages (GS) of (a) feeding and (b)
starving Calanus finmarchicus (image), 21 March to 14 April, 1997. Squares represent means of
three parallel experiments with groups of approximately 25 females, ■ fed, □ starving. Gonad
development is presented as the percentage of females in stages GS1 to GS4 and D (females with
degenerating oocytes). In all, 10–20 females were examined for each GS analysis. (from Niehoff
(2000), with permission from the Oxford University Press); (image courtesy of Russ Hopcroft,
C. finmarchicus, and this influence was dependent on the duration of starvation and
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the timing within the maturation cycle. Lifetime fecundity can be considerably
reduced, obviously during starvation, but also as a result of its long-term effects.
Overall, C. finmarchicus appears to be more sensitive to starvation than the freshwa-
ter cladocerans.
Under certain circumstances, energy requirements can be met by the catabolism
of endogenous proteins, but the non-selective use of endogenous protein for energy
will be detrimental to the animal in the long run. Mobilized proteins may include
enzymes and other vital organic molecules so their degradation will have far-
reaching effects on metabolic functions. Helland et al. (2003) examined protein
content in C. finmarchicus, in relation to diet. During the first 10 d of starvation,
protein content showed a moderate decline; however, during the following 21 d the
total content was drastically reduced. This reduction is equivalent to a protein loss
of 2.6 μg ind−1 d−1, corresponding to a reduction of 30.3 nmol N d−1. This finding
supports the notion of a sequential catabolism of endogenous nutrients during
starvation.
Madsen et al. (2008) compared the in situ egg production of C. finmarchicus and
C. glacialis in Disko Bay, western Greenland, under starvation and saturated food
conditions. Experiments with starved and AL fed females showed a significant dif-
ference in egg production rate between the two species, depending on sampling
time, i.e. gonad maturity and feeding history. The results showed varying use of
saturated food during the three phases of the bloom. For C. finmarchicus, no effect
of food level was observed during the first experiment in late April, whereas females
collected in early May, during the peak of the spring bloom, responded strongly to
changes in food concentration, with egg production which was 3-times higher than
that of the starved controls. In contrast, C. glacialis responded strongly to food
concentration in both late April and early May (Fig. 4.22). This study found that
Calanus females from the Disko Bay area continued to produce eggs without food
more than twice as long as those reported from other northern populations. This
observation indicated an adaptation to the Disko Bay environment, which has
unpredictable ice conditions and consequently large variations in the initiation of
the spring bloom.
4.2.5.4 A
mphipods and Isopods
The benthic amphipods Diporeia spp. (a conglomerate of several poorly defined

species, and glacial freshwater relict species) were once the predominant macroin-
vertebrates in deep, offshore regions of the Laurentian Great Lakes. However, since
the early 1990s, Diporeia populations have steadily declined across the area. It has
been hypothesized that this decline was due to starvation caused by increasing com-
petition for food diatoms with invasive dreissenid mussels (zebra and quagga). In
order to gain a better understanding of the changes in Diporeia physiology during
starvation, Maity et al. (2012b) starved Diporeia for 60 d and observed that
DR-stressed Diporeia responded with an increase in lipid oxidation and protein
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Fig. 4.22 Calanus finmarchicus and C. glacialis. (a, e) Spawning %; (b, f), clutch size; (c, g), egg
production (EP); and (d, h) cumulative EP for fed (●) and starved (○) females during 260 h incu-
bation. Values are mean of 10 females. Error bars indicate ±SE. (from Madsen et al. (2008), cour-
tesy of Inter-Research; © Inter-Research 2008)
catabolism; reductions in essential AAs (Pro, Thr, Phe); downregulation of

glycerophospholipid and sphingolipid metabolism; and a decrease in abundance in
polyunsaturated fatty acids (PUFAs) (Fig. 4.23). Abundance of 1-iodo-2-methylun-
decane, a metabolite closely related to insect pheromones, also decreased during
starvation. Decreases in these metabolites impaired reproductive function, growth,
and ultimately long-term survival. Overall, the hypothesis regarding the decline in
Diporeia gets some support and the next step should be to apply this new knowledge
to evaluating the nutritional status of feral Diporeia.
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Fig. 4.23 Metabolic pathways impacted during starvation in Diporeia based on KEGG and
including both polar and non-polar metabolites. (from Maity et al. (2012b), with permission from
Elsevier). DG diacylglycerols, DPA docosapentaenoic acid, EPA eicosapentaenoic acid, LsoPC
lysophosphatidylcholine, PC phosphatidylcholine, PG phosphatidylglycerol, PI phosphatidylino-
sitol, TG triacylglycerols, Thr threonine
To better understand the physiological responses of Diporeia to diverse stressors,

Maity et al. (2013) conducted three 28-d experiments evaluating changes in the
metabolomes of Diporeia (1) fed diatoms (Cyclotella meneghiniana) versus starved,
(2) exposed (from Lake Michigan and Cayuga Lake) to quagga mussels (Dreissena
bugensis), and (3) exposed to contaminated sediments. Each stressor elicited a
unique metabolomic response. However, none of metabolomics patterns agreed
with that of the first starvation study; there appear to be yet more causes for the
Diporeia decline, to be discovered in studies to come.
Gutow et al. (2007) compared the life history responses to temporary DR of two
idoteid isopod species with contrasting life-styles. The coastally-distributed Idotea
baltica usually has unlimited access to food in benthic macroalgal belts. The oce-
anic I. metallica inhabits objects floating on the sea surface and frequently experi-
ences food limitation in the oligotrophic open oceans. I. baltica was severely
affected when food was temporarily limited, as indicated by survival rates
(Fig. 4.24). In female I. baltica, the mortality rate followed the same general pat-
tern, independent of feeding regime: the initial juvenile mortality rate was followed
by a fairly constant mortality rate in adults until the population became extinct.
Juvenile survivorship was clearly affected by food limitation: the longer the inter-
mittent periods of starvation, the higher the mortality. In female I. metallica, mortal-
ity followed the same pattern as in female I. baltica (Fig. 4.24). However, in contrast
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Fig. 4.24 Comparison of coastal Idotea baltica and oceanic I. metallica survival under different
quantitative feeding regimes (% temporal food availability): (●) 100%, (○) 50%, (▼) 33%, (▽)
25%, note different axes. (From Gutow et al. (2007), with permission from Inter-Research, ©Inter-
Research 2007; images courtesy of Mark Blaxter, Wikimedia, and Véronique Vion, doris.ffessm.fr)
Fig. 4.25 Left: Population growth rates (λ) of Idotea baltica and I. metallica under different feed-
ing regimes analyzed by Monod model. Right: Total lipid content of males of Idotea baltica and
I. metallica after different periods of starvation. *Statistically significantly higher lipid content of
unstarved male I. metallica. DW: dry weight. (from Gutow et al. (2007), with permission from
Inter-Research, © Inter-Research 2007)
to I. baltica, juvenile survival of I. metallica was not severely affected by food

limitation.
Predictably, DR led to longevity extension in the species which has an increased
plastic response: in both Idotea species, optimal food supply induced relatively
short life spans. However, whereas the reduction in temporal food availability did
not significantly extend the life span of I. baltica, this did happen in I. metallica,
with the lowest food supply resulting in the longest life span (Fig. 4.24).
Furthermore, the sensitivity of I. baltica to DR led to a significantly decreased
population growth rate (λ) (Fig. 4.25, left). I. metallica was less affected by tempo-
rary starvation. When food was limited, energy allocation was transferred in I.
metallica from development and reproduction to survival (indicative of increased
longevity, Fig. 4.24), enabling the isopods to outlast periods of unfavorable food

conditions. In this species, a reduced λ reflected slower gene propagation rather than
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reduced fitness. High lipid content allowed I. metallica to outlast periods of

starvation (Fig. 4.25, right). Freshly hatched I. metallica have better starvation
resistance than juvenile I. baltica, making the demographically-important juvenile
stage less vulnerable to unpredictable DR. The plastic responses of I. metallica to
variable food levels represent efficient adaptations to life under conditions of low
and unpredictable food availability and provide insight into the nutritional condi-
tions that organisms have to cope with during extended rafting voyages at the sur-
face of oligotrophic oceans.
4.2.5.5 E
uphausiids
Antarctic krill (Euphausia superba) survive winter with no, or little, feeding and,
consequently, its overwintering success is a key factor dictating population size.
However, it is not clear whether DR or an internal clock, set by the natural Antarctic
light regime, is responsible for non-feeding. Auerswald et al. (2015) addressed this
question and found that both metabolic activity and the activity of enzymes catabo-
lizing lipids decreased after the onset of starvation and remained low throughout
periods of food shortage, whereas lipid reserves declined and lipid composition
changed: phospholipids rose slightly, whereas triacylglycerol declined strongly. The
mass and size of krill decreased, while the inter-molt period increased. Depletion of
storage- and structural metabolites occurred in order of depot lipids then glycogen
reserves until proteins were used almost exclusively after 6–7 weeks of starvation.
Results confirmed the existence of various proposed overwintering mechanisms
such as metabolic slowdown, slow growth or shrinkage and use of lipid reserves.
These changes were set in motion by food shortage only, i.e. without the trigger of
a changing light regime.
Do all krill species respond in the same way to DR? Huenerlage et al. (2015)
carried out a comprehensive comparison. The arcto-boreal Thysanoessa inermis
appeared to cope with both successive and long periods of DR determined by strong
seasonality. This was in contrast to the subtropical upwelling krill species E.
hanseni, which followed a “hand-to-mouth” existence with only a limited capability
to survive long-term DR, due to very low lipid reserves (Fig. 4.26, left). In most
respects, the survival strategy of T. inermis was similar to that of the Antarctic E.
superba and the northern krill Meganyctiphanes norvegica (i.e. body shrinkage,
sexual regression, use of internal energy storage and opportunistic feeding)
(Table 4.2). However, differences were found in terms of the energy storage pattern
(long-term wax ester storage in T. inermis vs. short-term triacylglycerols, (Fig. 4.26,
right) and the outstanding characteristic of T. inermis which was not to reduce its
overall metabolism.
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Fig. 4.26 Left: Mean change in the lipid stages of Thysanoessa inermis over time of starvation
(T0 to T28). Error bars represent the standard errors of the means. * Significant difference to T0
(P < 0.0001). Right: Lipid class composition of female T. inermis over time of starvation (T0 to
T28) and after overwintering (OW; specimens from April 2013). Percentages are shown as
means ± SD. WE wax ester, TAG triacylglycerol, FFA free fatty acids, PE phosphatidylethanol-
amine, PC phosphatidylcholine. (from Huenerlage et al. (2015), courtesy of Inter-Research)
4.2.5.6 Decapods
In marine crustaceans, proteins are the primary energy source, because they have
limited lipid and carbohydrate storage capacity (Sánchez-Paz et al. 2007; Dall and
Smith 1986). However, what strategy do freshwater omnivorous crustaceans, such
as Aeglidae, apply to overcome food restriction? These animals are able to feed
directly on allochthonous plant matter and aquatic insect larvae. Silva-Castiglioni
et al. (2016) assessed the effects of different periods of starvation and refeeding on
macromolecule levels (glucose, proteins, triglycerides, and glycerol) in the hemo-
lymph, as well as circulating levels of cholesterol in Aegla platensis. During the
starvation period there were differences in cholesterol, glucose, and proteins
between the genders, but no differences in total lipid, triglycerides, or glycerol lev-
els were observed. The stomach was the most empty in the 30-d starvation group in
both females and males. Starvation appears to modulate gastric emptying, and pos-
sibly digestive processes, as well as induce preferential utilization of carbohydrates
and proteins. This enabled survival throughout the study period. The authors
observed maintenance of the gonadosomatic index and reduction of the hepatoso-
matic index in females during the study period with a return to baseline after refeed-
ing. Refeeding was insufficient to restore stomach fullness, but sufficient to restore
glucose and cholesterol levels.
Applying classical life history traits, Moreira et al. (2015) evaluated how the
brown shrimp (Crangon crangon) responded to prolonged DR in two seasons of the
year, and how this species mobilized its energetic reserves. Shrimps caught in June
(summer) and October (autumn) were placed in individual cages in experimental
aquaria and kept in starvation until the last shrimp died or was sacrificed. Summer
shrimp proved to be better prepared to endure starvation than those caught in
autumn: they survived longer, had a higher energy content, and higher lipid and
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protein content at the beginning of the experiments. The percentage of total body
protein decreased significantly in the first week, stabilized in the following week
and decreased again abruptly in the fifth week. The percentage of total lipids only
started to decrease after four weeks. This indicates that (1) C. crangon uses protein
as a first energetic resource, followed by carbohydrates and eventually lipids; and
(2) after 4 weeks of starvation, a critical point was reached when structural compo-
nents were mobilized to satisfy maintenance costs.
Importantly, the physiological condition of crustaceans is a central determinant
of their susceptibility to parasite or pathogen challenges. Susceptibility increases
with starvation. This has been demonstrated with young adults of L. vannamei chal-
lenged by Vibrio alginolyticus or a viral pathogen (white-spot syndrome virus) (Lin
et al. 2012) (Fig. 4.27). In the first stage of starvation (starved for 0.5–1 d), shrimps
exhibited a reduction in immune parameters including hemocyte count and down-
regulation of hsp70, but increased phenoloxidase activity. Oxidative stress coupled
with increased SOD activity per hemocyte is a common means of defense (Steinberg
2012). More specifically, compensatory upregulated ß-glucan-binding protein, per-
oxinectin, prophenoloxidase-activating enzyme, prophenoloxidase I and II, and
α2-macroglobulin expressions were observed.
Life history theory posits a fundamental trade-off between number and size of
offspring that structures the variability in parental investment across and within spe-
cies (see also Sect. 4.2.5.2. Cladocerans above). Higher per offspring investment
(POI) is associated with a greater degree of endotrophy and abbreviated larval
development. Often a higher POI is found in species inhabiting cooler, high latitude
regions. Oliphant et al. (2014) assessed the implications of between-brood-variation
in hatchling energy content (measured as carbon mass) on larval starvation resis-
tance and developmental plasticity within the caridean brackish-water shrimp
Palaemon varians. The authors tested the hypothesis that under food-limited condi-
tions, higher POI enables development through fewer larval instars. Under starva-
tion stress, larvae from broods of greater POI (measured as hatchling brood average
dry weight, DW) generally developed through fewer larval instars. As the starvation
period increased, larval development time increased, whilst larval growth rate, juve-
nile DW, juvenile carbon mass, and juvenile C:N ratio all decreased. Larval devel-
opment time generally decreased with increasing brood average dry weight. In
contrast, larval growth rate, juvenile DW, juvenile carbon mass, and juvenile C:N
ratio all increased with increasing larval brood average DW.
In particular, with greater periods of starvation, larvae increasingly developed
through longer developmental pathways, progressing through more larval instars
before reaching the juvenile stage. For example, the proportion of larvae developing
through six instars (the longest development pathway observed here) increased
from 0.01 under the 0S treatment to 0.12 under the 2S treatment and 0.50 under the
4S treatment (Fig. 4.28). Conversely, the proportion of larvae developing through
four instars (the shortest development pathway observed) decreased from 0.53
under the 0S treatment to 0.08 under the 2S treatment, and 0.03 under the 4S
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Fig. 4.27 Cumulative mortality rates of control Litopenaeus vannamei and 7-d starved shrimp,
challenged with Vibrio alginolyticus at 3.8 × 106 colony-forming units shrimp−1 and then released
into 35% seawater after 0.5–7 d (a), and cumulative mortality rates of control shrimp and 7-day
starved shrimp, challenged with white-spot syndrome virus (WSSV) at 1.5 × 103 copies shrimp−1
and then released into 35% seawater after 0.5–7 d (b). Data in the same time with different letters
differ significantly (P < 0.05) among different treatments. (from Lin et al. (2012), with permission
from Elsevier)
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Fig. 4.28 Proportions of Palaemon varians larvae developing through four, five, or six larval
instars under 0S, 2S, and 4S starvation treatments. 0S = 0 days starvation; 2S = 2 days starvation;
4S = 4 days starvation. (from Oliphant et al. (2014), with permission from Springer)
treatment (Fig. 4.28). The proportion of larvae developing through five instars was
highest under the 2S treatment (0.80) whilst it was 0.46 under the 0S treatment and
0.47 under the 4S treatment (Fig. 4.28).
Under both the 2S and 4S treatment, the number of larval instars during develop-
ment was influenced by brood average dry weight (Fig. 4.29). Under the 2S treat-
ment, the proportion of larvae developing through four instars increased with
increasing brood average dry weight, whilst the proportion developing through six
instars decreased (Fig. 4.29). Similarly, under the 4S treatment, the proportion of
larvae developing through five instars increased with increasing brood average dry
weight, whilst the proportion developing through six instars decreased (Fig. 4.29).
Therefore, under both 2S and 4S treatments, there was a tendency for larger larvae
at hatching to progress through relatively fewer larval instars than smaller larvae at
hatching (Fig. 4.29). The effects of POI on larval and juvenile traits depend on the
level of starvation stress; intermediate POI is advantageous under relatively benign
conditions, whilst higher POI becomes advantageous under the most unfavorable
conditions.
The ability to tolerate unfavorable conditions for growth and development, evi-
dent as larval development without the addition of extra larval instars and develop-
ment through fewer larval instars, would appear advantageous. In particular, P.
varians larvae with greater POI developed through fewer larval instars under starva-
tion conditions (Fig. 4.29). This likely resulted from greater internal reserves that
enabled development to continue via endotrophy in the absence of external food.
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Fig. 4.29 Logistic regressions of the relationships between the proportions of Palaemon varians
developing through (a) four instars, (b) five instars, or (c) six instars and brood average DW (μg)
for larvae under 0S, 2S, and 4S starvation treatments. Significant logistic regressions are indicated
by P values provided. (from Oliphant et al. (2014), with permission from Springer)
4.2.6 Fishes
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The growth hormone (GH) regulates numerous processes in vertebrates, including

growth promotion and lipid mobilization. During periods of DR, growth is arrested,
yet lipid depletion is promoted. This raises the question of how the growth promot-
ing actions of GH can be dissociated from its metabolic actions. Therefore, Norbeck
et al. (2007) used rainbow trout on different nutritional regimens to examine the
regulation of growth hormone (GH)–insulin-like growth factor-I (IGF-I) system
elements. Sensitivity to GH in the liver was reduced in fasting fish as evidenced by
reduced expression of GH receptor type 1 (ghr1) and ghr2 mRNAs and by reduced
125
I-GH binding capacity. Fasting-mediated growth retardation was accompanied by
reduced expression of total igf-I mRNA in the liver, adipose and gill, and by reduced
plasma levels of IGF-I. Sensitivity to IGF-I was reduced in the gill of fasting fish, as
indicated by reduced expression of type 1 IGF-I receptor (igrf1a and igfr1b)
mRNAs. By contrast, fasting did not affect expression of igfr1 mRNAs or 125I-IGF-I
binding in skeletal muscle and increased expression of igfr1 mRNAs and 125I-IGF-I
binding in cardiac muscle. These results indicate that the nutritional state differen-
tially regulates GH–IGF-I system components in a tissue-specific manner and that
such alterations both disable the growth-promoting actions of GH and promote the
lipid-mobilizing actions of the hormone.
We are now going to show that the predictions of evolutionary theory that DR
will increase survival probabilities also apply to fishes; however, there are numerous
relevant studies and the examples following will focus with particular emphasis on
livebearers, cavefishes, killifishes, and sticklebacks.
4.2.6.1 L
ivebearers
Guppies
In the Trinidadian guppy (Poecilia reticulata), Bashey (2006) tested the well-known
hypothesis that the larger offspring produced by food-limited mothers is an adaptive
response to low-food environments. Female guppies have been shown to grow less,
reproduce less, and bear fewer, larger offspring in response to food limitation
(Reznick and Yang 1993). Bashey (2006) examined the plasticity in offspring size
in response to maternal food level, and the consequences for offspring fitness, in
guppies from two natural populations in the Northern Range Mountains, Trinidad.
The goal was to identify whether plastic responses to food level varied between
populations and whether offspring size has consequences for offspring fitness. One
population was from a resource-limited, low-predation population, and the other
was from a high-resource, high-predation population.
Females from both populations produced larger, leaner offspring in response to
food limitation. However, the population that was thought to have a history of selec-
tion for larger offspring was less plastic in its investment per offspring in response
to maternal mass, maternal food level, and fecundity than the population under
selection for small offspring size. To test the consequences of maternal manipulation
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of offspring size on offspring fitness, Bashey (2006) raised the offspring of low- and
high-food mothers in either low- or high-food environments. No maternal effects
were detected at high-food levels, supporting the prediction that mothers should
increase fecundity rather than offspring size in noncompetitive environments. For
offspring raised under low-food levels, maternal effects on juvenile size and male
size at maturity varied significantly between populations, reflecting the initial dif-
ferences in the maternal manipulation of offspring size. Nevertheless, in both popu-
lations, increased investment per offspring increased offspring fitness. Under
low-food conditions, mothers from more plastic families invested more in future
reproduction and less in their own soma. Similarly, offspring from more plastic
families were smaller as juveniles and female offspring reproduced earlier. These
correlations show that a fixed, high level of investment per offspring can be favored
over a plastic response in a chronically low-resource environment. In addition, in
low-food environments, maternal environmental effects were found to influence off-
spring size throughout the juvenile period and the characteristics of offspring matu-
rity. Also, plasticity in investment per offspring was correlated with lower growth
and higher reproductive effort in both maternal and offspring generations.
Another interesting detail of food restriction effects in guppies was recently
reported by Evans et al. (2015). The authors showed that sexual trait expression was
susceptible to nutritional stress. The reliability of precopulatory male sexual signals
(notably iridescence) can be compromised by environmental stochasticity. Similarly,
the findings for post-copulatory sexual traits (notably sperm velocity) show that
genotypes coding for highly competitive sperm in one environment may perform
quite differently under different food conditions. In particular, a highly-significant
correlation between iridescent coloration and sperm viability in the high food treat-
ment broke down under DR (Fig. 4.30).
Mollies and Pygmy Perch
For another livebearer, the osmotolerant sailfin molly (Poecilia latipinna), Trexler
(1997) provided evidence of plasticity in the mode of embryo nourishment (matrot-
rophy) by females raised under contrasting environmental conditions. Female sail-
fin mollies raised at high- and low-food levels, produced neonates of similar mass
and percentage of fat by varying egg size and the amount of supplemental nourish-
ment provided to embryos as they developed. Female body size and brood size were
highly correlated with the low-food females producing smaller brood sizes. The
effect was greatest for the fish collected in Saint Marks, Florida (FL-SM) (Fig. 4.31).
In sailfin mollies, matrotrophy appears to be an adaptation that diminishes the
offspring size-offspring number trade-off by permitting a reduction in ovum size
and increase in fecundity without compromising neonate size. However, the matro-
trophic supplementation of yolk nourishment was greatest in relatively large females
raised under DR. Thus, matrotrophy may incur some energetic cost that renders it
inefficient for small females or for females with substantial or dependable energy
reserves available for reproduction.
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Fig. 4.30 Reduction of a precopulatory and a post-copulatory sexual trait in offspring from guppy
parents fed high and low food treatments. (from Evans et al. (2015), with permission from Wiley)
Why are female sailfin mollies not matrotrophic all of the time? Reznick et al.
(1996) hypothesized that obligate matrotrophy may yield suboptimal solutions to
the offspring size–number trade-off by fueling reproduction largely or solely by
recently acquired energy. Facultative matrotrophy permits maximum flexibility in
the use of stored and recently acquired energy, and may provide a solution to limita-
tions from strict matrotrophy. Also, matrotrophy may be more energetically expen-
sive per neonate than lecithotrophy, although the investment is spread over gestation.
Thus, facultative matrotrophy can be an adaptation to increase brood size while
diminishing the trade-off of offspring size and brood size seen in obligate leci-
thotrophic poeciliid species (Trexler 1997).
Evidence is growing that fishes do not always produce large eggs in hostile envi-
ronments (Morrongiello et al. 2012). Southern pygmy perch (Nannoperca australis)
inhabits a diversity of streams distributed throughout gradients of environmental
quality. Populations inhabiting increasingly harsh conditions produced more numer-
ous and smaller eggs. The within-female egg size variability increased as environ-
ments became more unpredictable. The authors argued that egg size had only a
minor influence on offspring fitness in harsh environments. Instead, maternal fitness
was maximized by producing many eggs. The increased variability in size may be
an example of bet hedging.1 Furthermore, there is support for the hypothesis that
1
Bet hedging occurs when organisms suffer decreased fitness in their typical conditions in
exchange for increased fitness in stressful conditions (Cohen 1966).
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Fig. 4.31 Fecundity of sailfin molly females (offspring number per unit body mass) raised on high
vs. low scope-for-reproduction treatments. The top panel illustrates the results for females raised
on the high-food level, and the bottom panel illustrates results for low-food females. Codes for
source populations are: SC, fish collected in South Carolina; FL or FL-SM, fish collected in Saint
Marks, Florida. (from Trexler (1997), with permission from the Ecological Society of America;
image of male sailfin molly courtesy of Tibias, Wikimedia)
egg size variability increased as the environmental predictability decreased. Other

authors, however, continue to claim that within-clutch variation, at least in salmon,
is more likely to be a reflection of the direct influences of the rearing environment
on a female’s ability to allocate resources evenly, and is not an evolutionary adapta-
tion as presented in the example above (Jonsson and Jonsson (2014) and references
therein).
4.2.6.2 C
avefish
The Mexican tetra (Astyanax fasciatus) includes surface dwellers with eyes and
eyeless forms (Fig. 4.32). The latter inhabit caves and are characterized by the
absence of eyes and melanin pigmentation. Cavefish also display behavior that
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Fig. 4.32 Two forms of the Mexican tetra (Astyanax fasciatus): left: surface dweller with eyes;
right: blind cave form. (Images by M. Kesl and H. Zell, courtesy of Encyclopeida of Life)
differs from that of surface forms: they show increased swimming/exploratory and
feeding behaviors; have lost sleep, perhaps to increase wakefulness and chances to
find food; do not present clear endogenous rhythmic activities, aggressive or school-
ing behaviors; and have a reduced alarm behavior (Volkoff 2016).
Due to the extreme and unproductive environment, cavefish can be expected to
suffer from DR or even starvation. However, they have adapted to become very
skilled at finding nourishment in an environment where food is often scarce: surface
fish placed in the dark are four times less efficient at finding food than cavefish and
rapidly show signs of starvation. The feeding success of cavefish can be attributed
to several characteristics that are worth considering in detail. First, cavefish display
increased food-searching behavior, in part due to constant locomotor activity that is
1.5-times higher than that of eyed fish. Specialized anatomical and behavioral fea-
tures allow the cavefish to maximize the detection of food. Cave and eyed Astyanax
have different brain structures, with cavefish having larger hypothalamic and olfac-
tory bulbs, larger hypothalamic serotonergic cells, and different brain transcription
levels of receptors for neurotransmitters, such as glutamate or cannabinoids.
Interestingly, even differences in energy homeostasis are seen between cave and
surface fish. In the fed state, cavefish exhibit lower standard metabolic rates, have
different triglyceride and glycogen body contents, and modified protein and lipid
metabolism in the pineal gland, compared with surface fish. Low metabolic rates
and high-energy reserves mean cavefish have higher survival rates than the surface
form during fasting periods (Volkoff (2016) and references therein).
Several peptides produced by the brain, and in the periphery, regulate feeding
and metabolism. These include hormones such as OX, CART, PYY, CCK, GHRL
(Fig. 4.1) and apelin, as well as metabolic enzymes and neurotransmitters, such as
tyrosine hydroxylase, serotonin, and target of rapamycin. A graphical summary of
known appetite-regulating peptides in blind Astyanax is presented in Fig. 4.33. For
instance, pyy brain mRNA levels increased after feeding, pointing at a role for PYY
as a short-term satiety factor, and a 10-d fasting period did not affect pyy brain
mRNA expression, showing that central PYY was not involved in long-term fasting
in this species (Wall and Volkoff 2013). This is in contrast to other fishes, where
fasting decreases pyy mRNA levels in the brain or gut (goldfish, yellowtail, sea
lamprey), so the response of PYY to feeding and fasting appear to be species-
Fig. 4.33 Summary of

known appetite-regulating
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peptides in blind Astyanax.

(a) Effects of injected
peptides on feeding
behavior in blind Astyanax.
The arrows with
arrowheads and bars
indicate stimulation and
inhibition, respectively. (b)
The effects of feeding
(meal) and fasting on the
expression of brain
peptides. Solid arrows
indicate an effect, whereas
dashed arrows indicate no
effect. (c) Known
interactions between brain
peptides. Double-ended
arrows with arrowheads
and bars indicate
stimulatory and inhibitory
interactions, respectively. ?
indicates that no
interactions have been
detected. CART cocaine-
and amphetamine-
regulated transcript, CCK
cholecystokinin, TOR
target of rapamycin, OX
orexin, PYY peptide YY,
TH tyrosine hydroxylase.
(from Volkoff (2016), with
specific and dependent upon the tissue examined (i.e., brain versus gut) and the
duration of fasting. Furthermore, there were no periprandial variations in brain cck
expression in either fed or unfed fish (Wall and Volkoff 2013), and that again con-
trasts with corresponding increases in cck expression in the brains and intestines of
other fish species, such as goldfish, channel catfish, zebrafish, or yellowtail (Volkoff
In cavefish, ghrelin injections increased food intake and induced an increase in
ox brain expression, indicating that the orexigenic actions of ghrelin was mediated
by ox in cavefish. In addition, the tor brain expression increased by both apelin and
ghrelin injections, but was not affected by either ox or cck injections (Penney and
Volkoff 2014), indicating that the actions of apelin and ghrelin involve the intracel-
lular PI3K/AKT/TOR signaling pathway.
Fasting induces increases in th brain mRNA levels, and brain th levels increased
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one hour after a scheduled feeding time in unfed fish (Wall and Volkoff 2013),
implying a role of th in regulating feeding. An overall increase in th mRNA in the
brain of fasted Astyanax could be due to an increase in locomotor activity and food
searching. Brain ox mRNA levels increased 1 h before and decreased 1 h after a
scheduled mealtime (Wall and Volkoff 2013), showing that ox acted as a short-term
hunger signal, as previously shown in several eyed fish species. The peak in ox
expression before mealtime can be linked to food anticipatory activity (FAA), an
increase in appetite and locomotor activity seen prior to mealtime in many other fish
(Volkoff (2016) and references therein).
Overall, blind Astyanax have evolved to maximize their energy intake and
metabolism in a subterranean habitat. Adaptations include: anatomical and behav-
ioral changes, as well as physiological changes. Also, blind cavefish appear to have
modified levels of certain neurotransmitters, such as dopaminergic and serotoner-
gic, pathways, and perhaps signaling cascades, for example, those involving
TOR. At the endocrine level, cavefish seem to possess typical vertebrate appetite-
regulating peptides that interact with each other, but their expression levels and
functions may have undergone modifications in order to confer the cavefish a more
efficient endocrine appetite-regulating system.
4.2.6.3 Killifishes
To examine the effect of food rations on allocation to growth and reproduction,

Vrtílek and Reichard (2015) analyzed responses to fluctuating resources in
Nothobranchius furzeri, a teleost fish with a rapid life history. The authors also
evaluated the resolution of the trade-off between egg size and number. As expected,
female N. furzeri responded strongly to ration manipulation, with a pronounced
decrease in fecundity associated with low rations (Fig. 4.34a). Due to the unpredict-
ability of the offspring environment, Vrtílek and Reichard (2015) expected no adap-
tive change in oocyte size. However, females responded to the quality of their
environment with an adaptive maternal effect, with females receiving a low ration
producing larger eggs (Fig. 4.34b). Further, a switch in ration size in either direction
was associated with a decrease in egg size. There was a trade-off between egg size
and number in half of the treatments, but high variability in egg size among females
made the relationship complex. Overall, N. furzeri females demonstrated high plas-
ticity in both growth rate and fecundity parameters. Females appear able to track
and respond adaptively to unpredictable changes in food availability in their
environment.
4.2.7 Summary of Starvation Effects
In addition to the detailed descriptions above, major responses of selected aquatic

animals to food availability are collected in Table 4.2.
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Fig. 4.34 (a) Comparison of female body mass across treatments. (b) Differences in mature
oocyte size among treatments. Different letters indicate significantly different groups. Error bars
denote 95% confidential intervals for treatment means. Grey points represent mean size of mature
oocyte for individual females. (from Vrtílek and Reichard (2015), with permission from Wiley). (c)
Red variant male of Nothobranchius furzeri. (from Blažek et al. (2013), courtesy of BioMed
Central). High-ration treatment (H) received food twice per day (08:00 and 16:00), while the low-
ration treatment (L) only received food at 08:00. Feeding consisted of an ad libitum amount of
frozen chironomid larvae. L-0, H-0 sexual maturation period; L-L, H-L, L-H, H-H sexual matura-
tion and adulthood. L-L low-low, H-H high-high, H-L switch from high to low, L-H switch from
low to high ration
4.2.8 Starvation: Point-of-no-Return
Starvation resistance can be described by critical periods in the larval development

termed as ‘point-of-no-return’ (PNR). PNR represents the average time, which the
larvae remain alive after exposure to starvation. In echinoderms, PNR is defined
more specifically as the threshold point during starvation after which larvae can no
longer settle even if food is provided. Subsequently, feeding cannot reverse the
nutritional stress imposed and further development is not possible. ‘Point-of-
reserve-saturation’ (PRS) is another, however, less frequently used, critical point for
larval development, which was defined as the time (in days) when the individuals at
a given stage of development are capable of moulting to the following stage without
additional food supply. Anger and Dawirs (1981) exemplified these terms by the
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PNR
50
2.5 PNR
100
PRS0
PRS
Delay of Z-1 development
50
2.0
a
b 10
1.5 8 )
ys
da
n(
6 tio
rva
sta
4 of
t ion
2
Dura
1.0
0 2 4 6 8 10
Commencement of starvation (day in Z1)
Fig. 4.35 Hyas araneus: Response pattern in the zoea-1 stage (Zl) to differential starvation. Delay
expressed as multiple of development duration in fed controls. Explanations in the text. (from
Anger and Dawirs (1981), with permission from Springer; drawing courtesy of hiero.ru)
response patterns of the first zoeal stage (Z1) of the great spider crab (Hyas araneus)
to starvation (Fig. 4.35): The delay in Z1 development shows differential effects of
starvation. The delay increases with duration of the starvation period, but even more
drastically with its advancing commencement within the stage. This pattern con-
firms the critical point concept. If starvation starts before the PRS502 is reached or if
it ends later than the PNR50 (shaded area, b in Fig. 4.35), then less than half of all
larvae have a chance to reach the second stage. The PNR100 and the PRS0 define the
ultimate limit for any further development (hatched area, a).
Studying two separate populations of the intertidal burrowing crab (Neohelice
granulata), Bas et al. (2008) showed that PNR values are no constants. Their study
revealed significant intraspecific variability of larvae to moult (after a limited initial
feeding period – even in the absence of food) (Fig. 4.36). These differences may, at
least partly, be correlated with differential productivity in the waters where larval
development occurs with site 1 being comparably low in plankton productivity.
Similar interspecific differences were found in three hatches of the mangrove marsh
crab (Sesarma curacaoense) (12.5–19.5 d) (Table 4.3). All three hatches showed a
2
Point-of-no-return, where 50% of the individuals have died.
4.3 Compensatory Growth 201
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Fig. 4.36 Neohelice granulata: Mortality and the Point-of-no-Return (PNR) at two sites. (from
Bas et al. (2008) with permission from Springer; image courtesy of Nortondefeis, Wikimedia)
significantly higher PNR than a later study by de Souza et al. (2017). Also in the
semiterrestrial N. granulata and the common shrimp (Crangon crangon), different
broods showed significantly different PNRs (Table 4.3). In C. crangon, larvae
hatching from winter eggs (winter larvae) were more resistant to temporary lack of
food or less dependent on initial feeding than summer larvae due to elevated quan-
tity of internally stored energy reserves in winter eggs.
Starvation resistance has been examined by PNR experiments in numerous
aquatic animals (Table 4.3). Larger larvae and those with more yolk, such as her-
ring, Japanese sand eel, plaice, and grunion, are more resistant to starvation, because
of their greater nutritional reserves than smaller larvae with poor reserves. In larvae
of the California grunion (Leuresthes tenuis), a point of irreversible starvation
appeared not to exist, as starvation could in fact be reversed at any point along a
survival curve of starved larvae (May 1971). The same seems to apply to the fresh-
water shrimp Macrobrachium jelskii endemic to South America, or the hard clam
Meretrix meretrix. Furthermore, is obvious that the PNR depends on the larval
stages as well as on the ambient temperature. The lower the temperature, the longer
the time to PNR.
4.3 Compensatory Growth
After food restriction combined with slowed development and restoration of favor-
able conditions, many, but not all (Table 4.1), animals are capable of compensatory
growth (CG), that is a period of accelerated growth. As is the case with any meta-
phor, CG means different things to scholars from different biological disciplines.
Evolution biologists and ecologists on the one hand and aquaculturists on the other
hand, consider DR, starvation, and compensatory and catch-up growth under clearly
different perspectives. Whereas the first ones are interested in mechanisms of
Table 4.3 Point-of-no-return (PNR) in the ontogenetic development of selected fishes and
invertebrates
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Species PNR50 in days Reference

Invertebrates
Apostichopus japonicus 4.6 Sun and Li (2014)
Babylonia formosae 12.5 Zheng et al. (2005)
habei
Carcinus maenas 6.9 at 12 °C; 3.8 at 18 °C Dawirs (1984)
Cherax quadricarinatus 8.7, stage III Calvo et al. (2012)
51, juveniles
Crangon crangon 3.5, summer larvae Paschke et al. (2004)
4.8, winter larvae
Crassostrea gigas 3–5 His and Seaman (1992)
Cyclina sinensis 12.5 Yang et al. (2008)
Euphausia superba 10–14, temperature-dependent Ross and Quetin (1989)
Exopalaemon 4.81 Zhang et al. (2015)
carinicauda
Fabia subquadrata 8 Harris and Sulkin
(2005)
Fenneropenaeus 7.86 Zhang et al. (2009b)
chinensis
Homarus americanus 6.9 Abrunhosa and Kittaka
(1997)
Hyas araneus 7.8 Anger and Dawirs
(1981)
Jasus edwardsii 4.2 Abrunhosa and Kittaka
J. verreauxi 6.5 (1997)
Loligo opalescens 3–4 Vidal et al. (2006)
Lophopanopeus bellus 9 Harris and Sulkin
(2005)
Macrobrachium jelskii Not reached within 5 d Rocha et al. (2016)
Maja brachydactyla 2.8 Guerao et al. (2012)
Meretrix meretrix Not reached within 9 d Tang et al. (2006)
Neocaridina davidi 16.15 for JI; 9.44 for JIII Pantaleão et al. (2015)
Neohelice granulata 1.6–3.3, different broods Giménez (2002)
Site 1: 2.5 at 18 °C; 3.5 at 27 °C Bas et al. (2008)
Site 2: 3.4 at 18 °C; 4.4 at 27 °C
Panulirus cygnus 4.6 d Liddy et al. (2003)
Petrolisthes laevigatus 7.2 August Gebauer et al. (2010)
3.7 November
Pleuroncodes monodon 3 Espinoza et al. (2016)
Ruditapes philippinarum 4.25, 17.54, and 22.17 for early, middle and Yan et al. (2009)
late umbo-veliger larvae
Sesarma cinereum 1.84 Staton and Sulkin
(1991)
(continued)
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S. curacaoense 12.5–19.5, 3 different hatches Anger (1995)
7.34 de Souza et al. (2017)
S. rectum 9.01
S. reticulatum 2.79 Staton and Sulkin
(1991)
Fishes
Achirus lineatus TtSa Houde (1974)
5.9 at 24 °C
4.09 at 32 °C
Acipenser schrenckii 16 Huang et al. (2007)
Ammodytes personatus 21 at 6.5 °C Yamashita and Aoyama
16 at 10.5 °C (1986)
11 at 15.5 °C
Ancherythroculter 11 Xiong et al. (2006)
nigrocauda
Anchoa mitchilli TtS Houde (1974)
5.24 at 24 °C
3.23 at 32 °C
Archosargus TtS
rhomboidalis 4.8 at 24 °C
3.23 at 32 °C
Clupea harengus 3–5 d for yolk-sac larvae Yin and Blaxter (1987)
6–7 for 36- and 60-d-old fish
8 in 30 d old fish In Gadomski and
15 in 88 d old fish Petersen (1988)
Coregonus lavaretus 11.1 Dabrowski et al. (1986)
C. peled 13.2
Engraulis japonicus 6 Wan et al. (2007)
E. mordax 15 Hunter (1976)
Epinephelus coioides 3 Primavera-Tirol et al.
(2014)
Gadus chalcogrammus 15 at 2 °C Yokota et al. (2016)
14 at 5 °C
10 at 8 °C
Gadus morhua 3–5 for yolk-sac larvae Yin and Blaxter (1987)
Heteropneustes fossilis 8 Mookerji and Rao
(1999)
Hippocampus kuda 4.82 Sheng et al. (2007)
H. trimaculatus 4.86
Hypsopsetta guttulata 9 Gadomski and Petersen
(1988)
Labeo rohita 14 Mookerji and Rao
(1999)
(continued)
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Leuresthes tenuis Not reached May (1971)
Misgurnus 9–10 Wang et al. (2010)
anguillicaudatus
Oreochromis 15–16, small to medium eggs Rana (1985)
mossambicus 21 large eggs
Paralichthys californicus 4 Gadomski and Petersen
(1988)
P. olivaceus 7.7 at 15 °C Dou et al. (2005)
5.2 at 18 °C
4.2 at 21 °C
Platichthys flesus 3–5 for yolk-sac larvae Yin and Blaxter (1987)
23 for 32-d-old fish
Pleuronectes platessa 23 Blaxter and Ehrlich
(1974)
Rhamdia voulezi 6 de Lima et al. (2017)
Salvelinus namaycush 52 at 7 °C Edsall et al. (2003)
24 at 12 °C
Sander lucioperca 15–16 Xu et al. (2017)
Scomber japonicus 3.33 Hunter and Kimbrell
(1980)
Scomberomorus 6.5 Shoji et al. (2002)
niphonius
Sebastiscus marmoratus 7–10 Iwamoto et al. (2016)
Siniperca scherzeri 5–6 at 23 °C Zhang et al. (2009a)
Trachurus 5–6 Yandi and Altinok
mediterraneus (2015)
T. symmetricus ca. 3 Hewitt et al. (1985)
a
Times to starvation: The time after hatching during which larvae must establish themselves as
active feeders, or risk starvation, this means that after their yolk supply is exhausted, larval fish
must begin feeding within some limited time span (Houde 1974)
survival of animal populations and the evolution of new species, the latter focus on
optimal production in their studies of aquatic farmed animals: if there was a delay
in growth due to low food availability, the question is put forward how the animals
in aquaculture overcome this productive drawback. Overall, all pros and cons can be
summarized as “there’s no gain without pain”, since compensatory and catch-up
growth result in decrease in other direct fitness components (Hector and Nakagawa
2012).
Compensatory growth reduces the variance in size by causing growth trajectories
to converge and is important to fishery management, aquaculture and life history
analysis because it can offset the effects of growth arrests (Ali et al. 2003). Even
more pronounced, if a farmed animal is known to possess the capability of over-
compensatory growth (Fig. 4.37), short-term starvation prior to refeeding is applied
to increase production.
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Fig. 4.37 Idealized patterns of growth compensation. (from Ali et al. (2003), with permission
from Wiley) and Lepomis auritus belonging to the classical model genus capable of overcompen-
sation. (image courtesy of the U.S. Fish and Wildlife Service, Duane Raver)
Several authors distinguish between CG and catch-up growth: CG refers to a

faster than usual growth rate, while catch-up growth implies the attainment of con-
trol size (Hector and Nakagawa 2012). Both forms of rapid growth may be an adap-
tive mechanism that buffers the growth trajectory of young organisms from
deviations caused by DR and depend on the experimental setup applied. Similarly,
recent research on the effect of DR on longevity found that whether animals were
multigenerational laboratory dwellers or not had a significant impact on their
response to dietary treatment (Nakagawa et al. 2012). The authors showed that the
reduction in both age-dependent and age-independent mortality rates determined
life-extension by DR among the well-studied laboratory model species (yeast, nem-
atode worms, fruit flies and rodents). The results indicate that convergent adaptation
to laboratory conditions better explains the observed DR–longevity relationship
than evolutionary conservation.
More practically, and in terms of a targeted growth trajectory, the consequence of
a CG response is the attainment of a size status relative to the size achieved by an
organism that has not experienced any phase of growth impairment. Implicitly, the
size of the latter organism is assumed to be optimum at every time, so the ratio
between the size of compensating and control animals when the compensatory
response has abated provides a measure of the effectiveness of the compensation. In
full compensation, the deprived animals eventually achieve the same size at the
same age as continuously fed contemporaries (Fig. 4.37). In partial compensation,
the deprived animals fail to achieve the same size at the same age as non-restricted
contemporaries, but do show relatively rapid growth rates, and may have better food
conversion ratios during the re-alimentation period. Overcompensation occurs
when the animals that had experienced a DR achieve a greater size at the same age
than non-restricted animals. The compensation is so strong that animals subject to
variable food supply exhibit a higher growth rate than those for whom food was
continuously available. This was observed in Lepomis hybrids (Fig. 4.37), but seems
to be a relatively rare outcome (Table 4.4). If the re-alimented fish resume growing

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at a rate characteristic of the size reached at the end of the deprivation period, no
compensation has occurred (Fig. 4.37) (Ali et al. (2003) and references therein).
Compensatory growth can be achieved through hyperphagia, or a combination of
hyperphagia and improved feed conversion efficiency (Ali et al. 2003; Qian et al.
2000). Furthermore, energy reallocation plays a major role in this process. Gurney
et al. (2003) studied in silico the phenomenon of overcompensatory growth and
hypothesized that hyperphagia acting alone cannot generate a sufficiently strong
compensatory response to allow the growth of a starved and refed individual to
equal, still less surpass, that of a continuously fed equivalent. However, when com-
bined with a mechanism that adjusts protein allocation between internal reserves
and structure in response to changes in assimilation rate in such a way as to increase
allocation to structure at high feeding rates, complete catch-up can occur over a
wide range. This in silico study gets substantial support from an experimental study:
Cui et al. (2006) reported that the deprived gibel carp showed a priority in recover-
ing their protein reserves in viscera, relative to carcass, during refeeding, and a pri-
ority in recovering their body protein reserves, relative to the lipid reserves.
Selected examples of invertebrates and fishes will be considered in detail. An
brief overview of whether or not aquatic animals are capable of CG is presented in
Table 4.4.
4.3.1 Invertebrates
4.3.1.1 Mollusks
The opalescent inshore squid (Doryteuthis opalescens) lives in the eastern Pacific
Ocean, from Mexico’s Baja California peninsula to Alaska. Vidal et al. (2006)
starved paralarvae of this squid for 2 and 3 days. Paralarvae did not survive 4 and
5 days of starvation. Larvae starved for less than 4 days showed CG that mitigated
the effects of starvation in that, at the end of the experiment, attained mean final
body weights were similar to the control treatment. Differences in the RNA:DNA
ratios between control and starved paralarvae were detected within 2 days of food
deprivation (Fig. 4.38). For paralarvae starved for 2 and 3 days, it took 1 day after
refeeding to attain RNA:DNA ratios not significantly different from the control
treatment. CG indicated that, for continuously fed paralarvae, growth was below the
maximum potential due to the survival of slower growing squid. Therefore, starva-
tion caused a strong selection for inherently fast-growing paralarvae. This study
also indicated a positive relationship between rapid growth and high survival.
The abalone Haliotis asinina is one major aquaculture species and possesses a
high phenotypic plasticity, as proven in an alternate starvation and refeeding 200-
day experiment in hatchery-bred individuals (Fermin 2002). When refed continu-
ously over 60 days, the starved groups showed a complete CG (Fig. 4.39) and
reached the same phenotypic features and survival as the control group. High
Table 4.4 Capability of selected invertebrate and fish species of compensatory growth (CG) after
dietary restriction
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No Partial
Species CG CG CG Over-CG References
Invertebrates
Acartia tonsa ✓ Malzahn and Boersma (2012)
Cherax quadricarinatus ✓* Stumpf et al. (2011)
Daphnia pulicaria ✓a Urabe et al. (2018)
Doryteuthis opalescens ✓ Vidal et al. (2006)
Exopalaemon carinicauda ✓ Zhang et al. (2015)
Fenneropenaeus chinensis ✓b Wu et al. (2000)
✓* Wu and Dong (2001)
✓c Wu and Dong (2002)
Haliotis asinina ✓* Fermin (2002)
H. discus hannai ✓ Cho et al. (2011)
Litopenaeus vannamei ✓* Zheng et al. (2008)
✓*d Zhu et al. (2016)
✓ Wasielesky Jr et al. (2013)
✓ Lara et al. (2017)
Lytechinus variegatus ✓*e Lawrence et al. (2003)
Macrobrachium nipponense ✓* Li et al. (2009)
M. rosenbergii ✓ de Almeida Marques and
Lombardi (2011)
✓ Singh and Balange (2007)
Penaeus monodon ✓*f Mohanty and Mohapatra
(2017)
Fishes
Acanthopagrus schlegelii ✓* Xiao et al. (2013)
Acipenser baerii ✓* Morshedi et al. (2013)
Brycon amazonicus ✓* Urbinati et al. (2014)
Carassius auratus gibelio ✓ Qian et al. (2000); Cui et al.
(2006)
✓* Xie et al. (2001)
✓* Zhu et al. (2004)
Catla catla ✓ Mohanta et al. (2017)
Centropomus parallelus ✓ Ribeiro and Tsuzuki (2010)
Cirrhinus mrigala ✓g Singh and Balange (2005)
✓ Mohanta et al. (2017)
Coregonus lavaretus ✓* Känkänen and Pirhonen (2009)
Cynoglossus semilaevis ✓ Tian et al. (2010); Fang et al.
(2017)
Cyprinus carpio ✓ Schwarz et al. (1985)
✓ Su et al. (2017)
Danio rerio ✓ ✓h Fuentes et al. (2013)
Dentex dentex ✓ Pérez-Jiménez et al. (2012)
(continued)
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No Partial
Dicentrarchus labrax ✓ Adaklı and Taşbozan (2015)
Gadus morhua ✓* Jobling et al. (1994)
✓ Bélanger et al. (2002)
✓* Hanssen et al. (2012)
Gasterosteus aculeatus ✓ Zhu et al. (2003); Lee et al.
(2013); Ali and Wootton (2001)
✓* Inness and Metcalfe (2008)
Hemibagrus nemurus ✓* Thongprajukaew and
Rodjaroen (2017)
Hippoglossus hippoglossus ✓ Heide et al. (2006)
Ictalurus punctatus ✓ Randolph and Clemens (1978)
✓* Chatakondi and Yant (2001)
✓* Gaylord and Gatlin III (2001)
✓ Kim and Lovell (1995)
✓* ✓* Li et al. (2005)
Large Small
fish fish
Labeo rohita ✓ Yengkokpam et al. (2014);
Mohanta et al. (2017)
✓ ✓ Srijila et al. (2014)
✓* Prabhakar et al. (2008)
Lates calcarifer ✓* ✓*i Tian and Qin (2004)
Leiocassis longirostris ✓* Zhu et al. (2005)
Lepomis ✓* Hayward et al. (1997)
cyanellus♀ × niacrochirus ♂
Megalobrama ✓ Zhu et al. (2014)
amblycephala
Morone chrysops×saxitilis ✓* Turano et al. (2007, 2008)
Picha et al. (2008a)
Nothobranchius furzeri ✓ Vrtílek and Reichard (2015)
Oncorhynchus mykiss ✓* Kindschi (1988)
✓* ✓*j Guzel and Arvas (2011)
✓* Quinton and Blake (1990);
Nikki et al. (2004); Blake et al.
(2006); Azodi et al. (2015)
✓ Dobson and Holmes (1984);
Sevgili et al. (2013a)
✓ ✓*k Taşbozan et al. (2016)
O. nerka ✓ Bilton and Robins (1973)
Oreochromis mossambicus ✓* Gabriel et al. (2018)
(continued)
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No Partial
O. mossambicus × O. ✓ ✓* Gabriel et al. (2017)
niloticus Wang et al. (2005)
Wang et al. (2000)
O. niloticus ✓* Gao et al. (2015)
✓ ✓l Ye et al. (2016)
Pagrus major ✓ Oh et al. (2007)
P. pagrus ✓ Caruso et al. (2012)
Pangasius bocourti ✓ Jiwyam (2010)
Paralichthys olivaceus ✓ (Huang et al. 2008); Cho et al.
(2012)
✓m Kim and Cho (2014)
Phoxinus phoxinus ✓ Russell and Wootton (1992)
Piaractus brachypomus ✓ Gómez-Peñaranda et al. (2016)
✓ Favero et al. (2018)
P. mesopotamicus ✓n Kojima et al. (2015)
Poecilia reticulata ✓ o
Auer et al. (2010)
Pungitius pungitius ✓ Ab Ghani and Merilä (2015)
Rutilus caspicus ✓ Abolfathi et al. (2012)
R. rutilus ✓ Van Dijk et al. (2005)
Salmo salar ✓ Mortensen and Damsgård
(1993); Nicieza and Metcalfe
(1997); Maclean and Metcalfe
(2001)
✓p Stefansson et al. (2009)
S. trutta ✓q Johnsson and Bohlin (2006)
✓ Pirhonen and Forsman (1998);
Sundström et al. (2013)
S. trutta labrax ✓* Kocabaş et al. (2013)
Salvelinus fontinalis ✓ Mlglavs and Jobling (1989);
Mortensen and Damsgård
(1993); Savoie et al. (2017)
Sander lucioperca ✓* ✓*r Mattila et al. (2009)
Sander vitreus ✓* Rosauer et al. (2009); Hayward
et al. (2015)
Scophthalmus maximus ✓ Sæther and Jobling (1999);
Blanquet and Oliva-Teles
(2010)
Sebastes inermis ✓ ✓s Oh et al. (2010)
Solea solea ✓ Piccinetti et al. (2015)
Sparidentex hasta ✓* Torfi Mozanzadeh et al. (2017)
Sparus aurata ✓ Peres et al. (2011)
✓* Eroldoğan et al. (2006)
Tinca tinca ✓ Myszkowski et al. (2010)
(continued)
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a
Compensatory feeding on P-restricted algae
b
Depending on the length of the starvation period
c
On protein restriction
d
On P and N restriction
e
Body maintenance at the expense of gonad development
f
Depending on the length of the starvation period
g
Protein content was significant lower
h
Due to biotechnological means, see below
i
Depending on duration of CR
j
Depending on the interval of feeding and fasting
k
Depending on dietary protein and lipid levels
l
Depending on duration of the starvation period
m
Restricted feed allowance
n
Dietary restriction <14 days
o
Transgenerational adverse effects, see below
p
Depending on refeeding rations
q
Depending on length of starvation period
r
Depending on frequencies of refeeding
s
Depending on refeeding rations
* = intermittent feeding
Fig. 4.38 Opalescent inshore squid (Doryteuthis opalescens). RNA:DNA ratios (mean±S.D.) of
paralarvae exposed to five different feeding treatments from 15 to 24 days old. (a) Control treat-
ment (constantly fed) and (b) 2, (c) 3, (d) 4 and (e) 5-day starved before refeeding. Filled squares
represent feeding days and empty squares represent days without food. Values within each day
with asterisks (*) are significantly different from the control. (from Vidal et al. (2006), with per-
mission from Wiley). Image: dorsal (top) and ventral views of adult squid (courtesy of Wikimedia)
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Fig. 4.39 Changes in mean daily growth rates in terms of shell length (a) and body weight (b) of
abalone, Haliotis asinina, reared under different feeding regimes. FR: full rations throughout (con-
trol); 5/5: starved for 5 days then refed for 5 days; 10/10: starved for 10 days then refed for 10 days.
(from Fermin (2002), with permission from Wiley)
phenotypic plasticity is obviously a common feature of abalones, since it also

applies to H. midae. Francis et al. (2008) showed that periodic bouts of starvation
were beneficial, allowing variable growth spurts when returned to full food rations.
4.3.1.2 Crustaceans
Ideally, the period of starvation does not impact the CG in individual groups. Li
et al. (2009) subjected young Oriental river prawn (Macrobrachium nipponense) to
increasing starvation periods (2–8 days). After refeeding, all starved groups reached
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Fig. 4.40 Specific growth rate (SGR) in Macrobrachium nipponense for individual groups during
compensatory growth. (from Li et al. (2009), with permission from Wiley; Image: coursety of
OpenCage, Wikimedia). Group C = control, no starvation: Groups S2 (starved for 2 days) to S8
(starved for 8 days)
at least the specific growth rate (SGR) of the control and declined afterwards, almost
parallel to the control (Fig. 4.40). After CG, the final weight of all groups did not
significantly differ from each other.
The red claw crayfish is an important species of freshwater crustacean for aqua-
culture. It has direct development and the first two lecithotrophic juvenile stages (JI
and JII) stay with their mothers for 10 to 15 days. After molting to stage juvenile III
(JIII), they become more independent from their mothers, beginning exogenous
feeding. The following stages (III, IV and V) last approximately 10 days each and,
according to this, after 30 days juveniles are found in stage juvenile V or VI. Juveniles
JIII can tolerate and compensate for short-term intermittent feeding. The time taken
when 50% of initially starved juveniles have lost the capability to molt obtained for
stage III juveniles was 4.28 days (Stumpf et al. 2010). Consequently, the question
arises of how long the intermittent feeding can be expanded and how this phase
impacts growth and survival at different stages of development of juveniles. Stumpf
et al. (2011) worked out that juvenile Cherax quadricarinatus can tolerate relatively
long periods of low food availability – an important adaptation for their survival in
changing environments and an attribute favorable for the production of the species.
In addition, the authors corroborated the ability to recover completely from short
periods of food deprivation in the early stage of development. Advanced juveniles
subjected to intermittent feeding regimes of 2–4 days have the same survival and
mass gain as daily fed individuals. The underlying mechanisms were higher feed
intake (hyperphagia) and increased food utilization efficiency (Stumpf and Greco
2015).
Currently, intermittent feeding practices are not utilized commercially in crusta-
cean farming of this species. In semi-intensive culture, where natural productivity in
the pond may partly satisfy the protein requirements of the red claw, the use of
intermittent feeding protocols under an adequate stocking density can be a good

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strategy to diminish costs due to the reduced use of formulated feeds (Stumpf et al.
2011).
Recently, Malzahn and Boersma (2012) reported of a lack of CG in the plank-
tonic herbivore A. tonsa. In order to investigate the response of herbivores’ growth
and nutritional condition to phosphorus-limited algal food, the authors fed this
copepod with the autotrophic flagellate Rhodomonas salina that was cultured
towards high and low phosphorus content. To test the effect of the duration of expo-
sure to low and high-P food on copepod growth, Malzahn and Boersma (2012) also
switched a subset of the copepods from high-P to low-P food for two to 12 days
before returning them to their original diet. P-limited prey clearly reduced the nutri-
tional condition of copepods, expressed as their RNA:DNA ratio and, consequently,
in their growth rates.
The significantly lower growth rate on P-restricted diets was mirrored when
compared to well-nourished copepods (Fig. 4.41). Very few of the animals on the
refed treatments were able to compensate for the initial feeding of poor quality
food. This study shows that P-limited food drastically reduces secondary production
and no CG occurs in the investigated copepod species. Hence, even short-term
exposure of herbivores to poor food has a lasting effect on secondary production.
In contrast to Acartia, the freshwater herbivore D. magna showed clear CG when
the starvation period was long enough (Bradley et al. 1991), while those daphnids
that were starved for shorter periods did not show signs of CG. Likewise, switching
Daphnia between high and low quality diets showed no CG after periods of poor
food. D. magna, when migrating from deep, nutrient-rich and cold layers to shallow,
Fig. 4.41 Deviation of growth rates of the marine herbivore Acartia tonsa between animals refed
on P diets and those reared exclusively on P diets in relation to the age of the animals. Dotted lines
denote the control values. (from Malzahn and Boersma (2012) with permission from Wiley)
warm and nutrient-depleted conditions, grew almost at maximum speed when when
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exposed to high-quality food for half or less of the day (Sterner and Schwalbach
2001), but did not exceed the growth of those fed a diet of constantly good quality
(Malzahn and Boersma 2012).
A major representative of East Asian aquaculture animals, the Chinese shrimp
(Fenneropenaeus chinensis), appears to have less phenotypic plasticity than, for
example, the abalone referred to above. Although shrimps subjected to periods of
starvation-and-refeeding showed a higher mean percentage weight gain than those
fed continuously AL, at the end of the experiment all treated shrimps failed to catch
up the controls in terms of body weight. This indicates an only partial
CG. Furthermore, feeding regimes significantly affected body lipid content but not
water, protein, ash, or energy content (Wu and Dong 2001). Only partial CG was
also reported for Macrobrachium rosenbergii after periods of DR (de Almeida
Marques and Lombardi 2011).
4.3.1.3 I nsects
De Block and Stoks (2008a) studied an interesting case, namely the cost of CG in
the willow emerald damselfly (Chalcolestes viridis). The authors tested the hypoth-
esis that an important cost of CG and, therefore, an evolutionary constraint on
growth rate in general is that growth leads to the accumulation of damage at the
cellular level, such as oxidative damage due to reactive oxygen species (ROS). The
authors assessed oxidative stress in a study where they generated CG in body mass
by exposing larvae of the damselfly C. viridis to a transient starvation period fol-
lowed by AL food. CG in the larval stage was associated with higher oxidative
stress in the adult stage. The results challenge a traditional view of life history the-
ory by supporting the notion that the costs of CG can be associated with ROS-
mediated trade-offs and not necessarily with resource-mediated trade-offs.
In addition to causing oxidative stress in the adult stage, CG in C. viridis also
impaired immune function. De Block and Stoks (2008b) determined the number of
hemocytes and the activity of prophenoloxidase (proPO) and phenoloxidase (PO) in
the experiment outlined above. Directly after starvation, immune variables were
reduced in starved larvae. Levels of proPO and PO remained low after starvation,
even after metamorphosis. In contrast, hemocyte numbers were fully compensated
by the end of the larval stage, yet were lower in previously starved animals after
metamorphosis. This study indicates that significant physiological traits can be
impaired by CG after starvation.
4.3.2 Fishes
The capability of fishes for CG is diverse as is the group of fishes itself. It ranges
from apparently lacking, as observed in common carp or gilthead seabream, to over-
compensation, as found in hybrid sunfish, chanel catfish, rainbow trout or hybrid
tilapia (Table 4.4). ‘Studies of identical species which have been conducted in the
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same laboratory and in different laboratories have produced contrasting results, for
instance with S. auratus, Oreochromis hybrids, P. brachypomus, L. rohita, I. pun-
catatus, Carassius auratus gibelio, Rutilus rutilus, G. morhua, or P. olivaceus.
There may be diverse reasons for the contrasting results, such as different ontogen-
tic and physiological stages, different strains of fish studied or different feeding
histories (Jobling et al. 1993, 1994; Sæther and Jobling 1999), or inconsistent and
variable feed qualities and non-standardized protocols for both dietary restriction
and refeeding (Zhu et al. 2003; Taşbozan et al. 2016).
Studies of juvenile and young R. rutilus demonstrate differences based on differ-
ent ontogenetic developmental stages. Méndez and Wieser (1993) proposed a gen-
eral model of metabolic responses of fishes to food deprivation and refeeding. The
model distinguishes between the following four phases: stress, transition, adapta-
tion and recovery. In contrast to this model, reporting a strong increase in glycogen
content during CG in individuals weighing 280–460 mg, Van Dijk et al. (2005)
showed that the body composition of the entire fish was not dramatically affected by
fasting and refeeding. Méndez and Wieser (1993) interpreted this phenomenon as a
tactic for rapid storage of food energy. This strategy, however, was apparently
restricted to small fishes, as this phenomenon was not observed in fish weighing
5–15 g; instead, juvenile roach had a higher conversion efficiency3 during CG than
during growth under control conditions (Van Dijk et al. 2002).
Another major reason for the discrepancies may be based on interindividual vari-
ations in response to CR. For instance, Jobling and Koskela (1996) found that a
feeding hierarchy was established in rainbow trout during restricted feeding.
However, when fish were switched from restricted to full rations and more food
became available, this hierarchy was relaxed and all the fish were given the oppor-
tunity to feed. Some of the fishes which had been suppressed during the period of
restricted feeding became hyperphagic and displayed high rates of growth
(Fig. 4.42). This results indicate strong interindividual variation in CR.
A similar phenomenon has been reported for Atlantic salmon (Maclean and
Metcalfe 2001). The authors found that social factors may prevent the individuals
from achieving the growth rates necessary for full compensation, since dominant
fish may be able to monopolize food supplies and thus show growth compensation,
while subordinates may not achieve the rates of food intake needed to compensate.
Zhu et al. (2001) compared CG in two species (the carnivorous stickleback G.
aculeatus and the omnivorous minnow P. phoxinus) following similar food depriva-
tion protocols after identical periods of starvation to determine inter-specific simi-
larities and differences. Both species experienced 1 or 2 weeks of starvation before
being AL-refed. The two species differed in their response to the starvation periods,
with minnows showing a lower weight-specific loss. Both species showed compen-
satory responses in appetite, growth and, to a lesser extent, growth efficiency.
Minnows wholly compensated for one and 2 weeks of starvation. At the end of the
experiment, sticklebacks starved for 2 weeks were still showing a compensatory
3
Specific growth rate/specific feeding rate.
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Fig. 4.42 Scatterplot showing growth rates (SGR) of individual rainbow trouts during the period
of restricted feeding (16 g food day−1) (period 1) and following the subsequent switch to full
rations (30 g food day−1) (period 2). Spearman rank correlations for individual growth rates during
periods 1 (restricted rations) and 2 (full rations) were not significant for any of the three tanks of
fish. The symbols represent different fish tanks. (from Jobling and Koskela (1996) with permission
from Wiley)
response and had not achieved full compensation. The compensatory responses of
the sticklebacks showed a lag of a week before developing during the refeeding
phase, whereas the response of the minnows was immediate. Analysis of lipid and
dry matter concentrations showed that the compensatory response restored reserve
lipids while also bringing the fish back to the growth trajectory of continuously fed
fish.
In a meta-analysis, Hector and Nakagawa (2012) added some general rules to the
above model considerations by showing that mortality was more strongly affected
by dietary treatment than reproduction, and negative fitness consequences were
more apparent after longer periods of realimentation. Males and females in single-
sex experiments had significantly higher fitness components than mixed-sex
experiments. Animals on an intermittent feeding regime were less likely to show
deficits in fitness components.
4.3.2.1 Overcompensatory Growth
Early attempts to use CG to increase fish growth rates in aquaculture have generally
failed because the rapid CG growth response elicited by food deprivation wanes as
soon as the refed fish approach the normal weight of control fish (Russell and
Wootton 1992). Applying fasting/refeeding cycles of 2 days/2 days to hybrid sun-
fish, Hayward et al. (1997) were the first to report overcompensatory growth by a
factor of two in a farmed fish. This successful short-term intermittent feeding

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prompted similar tests with other farmed invertebrates and fishes to increase pro-
duction. Positive reports are available for juveniles of L. vannamei, Chinese shrimp
(F. chinensis), black sea bream (Acanthopagrus schlegeli), zebrafish, channel cat-
fish, and rohu carp (L. rohita). However, intermittent feeding has been much more
often applied, as indicated in Table 4.4 by asterisks; several times with no or even
with adverse effects. As underlying mechanisms of overcompensatory growth,
hyperphagia and improved nutrient utilization and food conversion efficiency have
been discussed. In addition, and as mentioned above, energy reallocation may also
be fundamental to this process (Gurney et al. 2003).
Another mechanism has been responsible for the overcompensatory growth in
zebrafish reported by Fuentes et al. (2013) is inhibition of myostatin, the main nega-
tive regulator of muscle growth and development in vertebrates. For this purpose,
zebrafish underwent compensatory growth using fasting and refeeding trials.
Myostatin activity was blocked with dominant negative LAPD76A recombinant
proteins (applied six bathes for 2 h in 2 weeks) during the refeeding period, when a
rapid, compensatory muscle growth was observed. Treatment with LAPD76A
recombinant proteins triggered inactivation of the main signal transduction used by
myostatin to achieve its biological activity (SMAD) in skeletal muscles. Treated fish
displayed an overcompensation of growth characterized by greater muscle hyper-
trophy and growth performance than constantly fed, control fish (Fig. 4.43). This
study shows an attractive strategy for improving muscle growth by mixing a classi-
cal strategy (compensatory growth) with a biotechnological approach.
4.3.2.2 C
osts of Compensatory Growth
Life Span, Reproduction, or Stress Resistance?

The single greatest expense in aquaculture is feed. To further optimize feed effi-
ciency, culturists are attempting to improve feeding regimens. One method being
intensively explored is restricting feeding for varying periods, which can induce
CG. The following question arises: is compensatory growth for free?
Inadequate nutrition, however, is known to affect immune function and disease
resistance and the effect of restricting feed on immunity is poorly understood.
Therefore, CG can be expected to be associated with costs, typically increased mor-
tality or reduced future reproduction. Interestingly, the results are rather diverse. For
instance, Johnsson and Bohlin (2005) did not observe reduced costs of the compen-
satory response in brown trout in a field trial within the timespan of the experiment.
Therefore, the authors hypothesized that wild brown trout have an adapted “buffer
capacity” to withstand fluctuations in food supply, allowing restoration of lost lipid
reserves when feeding conditions improve. However, CG increased mortality costs,
namely winter mortality (Johnsson and Bohlin 2006).
Inness and Metcalfe (2008) investigated the effects of CG on both mortality tra-
jectories and reproductive investment. Using experimental studies of the three-
spined stickleback, the authors showed that animals going through cycles of
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Fig. 4.43 Schematic diagram summarizing the effects of fasting, refeeding, and LAPD76A treat-
ment on SMAD signaling pathway activation, muscle cellularity, and growth performance in
zebrafish. The schematic diagram illustrates the events occurring in (a) Fasted zebrafish, (b) refed
zebrafish, and (c) refed zebrafish treated with LAPD76A for 2 weeks. (from Fuentes et al. (2013)
short-term deprivation followed by CG had a much shorter life span but the same
investment in reproduction as those able to feed each day on the same total amount
of food. By contrast, continuous DR has no effect on life span but caused a reduc-
tion in female reproductive effort.
Similarly, Lee et al. (2013) showed again in G. aculeatus that CG led to a reduc-
tion in median life span of approximately 15%; conversely, decelerated growth
extended life span. These life span effects were independent of eventual size attained
or reproductive investment in adult life.
Nothobranchius furzeri responded with full CG to improved food conditions,
and, unlike stickleback, obviously not at the expense of fecundity (Vrtílek and
Reichard 2015). Females undergoing full CG were able to invest in fecundity at the
same level as females kept on a high ration throughout the experiment. Since the
experiment was terminated after 8 weeks, other life history traits, particularly life
span, were not recorded. Therefore, the question of whether or not CR took place at
the expense of longevity, remained unanswered. A reduced life span would inevita-
bly lead to decreased lifetime reproduction. The brown trout example above points
out the significance of long-term studies.
Organisms must carefully regulate energy intake and expenditure to balance
growth and trade-offs with other physiological processes, such as reproduction,
Fig. 4.44 Regulatory roles

of hypothalamic NPY in
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increasing appetite and

concomitantly suppressing
reproduction and sexual
behavior in response to
negative energy balance.
(from Kalra and Kalra
(2004), with permission
from Elsevier)
because nutritional infertility may result (Fig. 4.44). Consequently, under limited

food conditions, impairment of important life history traits may occur if the energy
homeostasis is imbalanced in favor of body growth. The unambiguous experimental
evidence that high abundance of neuropeptide Y (NPY) promoted hunger and
simultaneously shut off sexual drive and the reproductive process was critical in
enunciating the concept that reciprocal control of appetite and reproduction was
inherent in the NPY molecule itself. Since NPY is universally present in vertebrates
and its orexigenic effects have been preserved through invertebrate and vertebrate
evolution, its function is considered to be of fundamental importance (Kalra and
Kalra 2004).
This argument applies particularly to the following study. Auer et al. (2010) used
a food manipulation experiment to examine the reproductive consequences of CG in
P. reticulata. CG did not affect adult growth rates, litter production rates or invest-
ment in offspring size. However, CG had negative effects on litter size, independent
of the effects on female body length, resulting in a 20% decline in offspring produc-
tion (Fig. 4.45). These costs were delayed until later litters. Overall, reproductive
costs to accelerated juvenile growth may play an important role in the evolution of
CG and other rapid juvenile growth responses.
In terms of CR impact on immunity parameters, the results are diverse and even
contrasting. For instance, Corrales and Noga (2011) found adverse effects, Caruso
et al. (2012) none, whereas Mohapatra et al. (2015, 2017) showed beneficially influ-
ences of starvation on pathogen defense. Table 4.5 presents an overview of mixed
results in fish, and two contrasting studies show the underlying details.
Antimicrobial polypeptides (AMPP) belong to the most potent traits of the innate
immune defense system, comprising host-produced peptides and small proteins.
Among the most prevalent and potent AMPPs in fish are histone-like proteins (HLP)
and piscidins. Corrales and Noga (2011) collected gill, skin, blood and spleen sam-
ples from fish fed either at a high, moderate or low feed rate or fasting rate. Tissue
antibacterial activity, HLP and piscidin levels were determined between days 0 and
Fig. 4.45 Trajectories of

litter size for experimental
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(grey) and control (black) 70

Poecilia reticulata. Dotted
lines represent observed 60
individual trajectories;
solid lines represent 50
Litter size
predicted trajectories for
each treatment. (from Auer 40
et al. (2010) with
permission from Wiley) 30
20
10
0
18 21 24 27 30 33 36
Female length (mm)
Table 4.5 Effects of dietary restriction and compensatory growth on immunity traits in selected
fish species
Beneficial No effect Adverse
Species Immunity trait effect observed effect References
Barbonymus Non-specific × Eslamloo et al.
schwanenfeldii imunity (2017)
Megalobrama Hypoxia resistance × Li et al. (2016)
amblycephala
Mesopotamichthys Red blood cell × × Najafi et al.
sharpeyi number × (2015)
Hematocrit
Immunity
Morone saxatilis×M. Antimicrobial × Corrales and
chrysops polypeptides Noga (2011)
Oreochromis Resistance to toxic × Abdel-Tawwab
niloticus metals (2016)
Pagrus major Pathogen × Mohapatra et al.
resistance (2015, 2017)
P. pagrus Hemolytic activity × Caruso et al.
(2012)
Paralichthys Hemoglobin, × Kim et al.
olivaceus hematocrit, total (2014b)
cholesterol
Piaractus Lysozyme, × Gimbo et al.
mesopotamicus cholesterol (2015)
Sparidentex hasta Lysozyme × Torfi
Mozanzadeh
et al. (2017)
80. On day 80, the fasting group had significantly less antibacterial activity and
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piscidin 4 concentration in the gills. Fasted fish also had the most rapid mortality
rate after Ichthyophthirius multifiliis challenge. This study indicates that prolonged
fasting results in increased pathogen/parasite susceptibility. In contrast to this study,
Mohapatra et al. (2017) reported that short-term starvation of red sea bream before
a bacterial infection resulted in increased oxidative stress response, along with
altered energy metabolism. In addition to altered transcription of several iron
homeostasis and lipid metabolism-related genes, pre-starvation also influenced the
programmed cell death mechanism in the body, consequently triggering the defense
mechanism in the fish to fight against Edwardsiella tarda infection. Thus, an alter-
nating starvation and refeeding regime at the appearance of an infection can be
adopted in fish at the farm level as a counter measure against bacterial invasion – at
least in this species.
Can Compensatory Growth Be Induced?
If CG is to be initiated on refeeding, changes in the physiological state of fish during
the deprivation period must signal a discrepancy between the actual state and the
state of fish that have not experienced that deprivation. It is unclear which, if any, of
the changes observed during deprivation provide the error signals for CG when food
becomes freely available (Ali et al. 2003). How do specific food components regu-
late CG or which specific food compounds does CG require?
Fish, whose metabolism is largely based on lipids and proteins, store lipids in the
liver, viscera, and muscle. Lipids are broken down early in starvation in, for exam-
ple, European eel, European sea bass, pike, rainbow trout, and Nile tilapia.
Consequently, changes in lipid levels (and composition as shown in Clarias gariepi-
nus) take place before and during CG and, during refeeding, the lipid reservoirs
have to be replenished first, followed by protein reservoirs (Ali et al. 2003).
Therefore, it can by hypothesized that lipids act as elicitor of CG.
Silverstein and Plisetskaya (2000) illustrated the importance of dietary fat with
an impressing test. Channel catfish was manipulated by diet composition to reach
different levels of fat content and then fed the same diet. In the first 2 weeks follow-
ing a switch to the same diet, the low-fat catfish ate more than the high-fat fish.
These results are in accord with the expectations of a theory on appetite regulation
based on maintenance of energy reserves within narrowly defined limits. Arctic
charr showed an inverse relationship between food intake (% body mass day−1) and
carcase lipid (% wet mass) and visceral lipid (% wet mass). For juvenile Piaractus
brachypomus, a correlation between food intake and visceral fat content was
reported. A comparison between temporal changes in food intake and body compo-
sition indicated that the level of food intake in re-alimented fish decreased, as vis-
ceral fat content approached the level in controls fed at satiation level (Ali et al.
(2003) with references therein).4 Consistent with this, Zhu et al. (2005) studied the
CG responses of the carnivorous Chinese longsnout catfish (L. longirostris) in a
4
Excerpt taken with permission from Wiley.
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Fig. 4.46 Survival percentage of Labeo rohita fingerlings after challenge with Aeromonas
hydrophila. Different letters indicate significant differences (P < 0.05) (mean ± S.E.) (from
Yengkokpam et al. (2016) with permission from Elsevier). Fish in the control group (C) were fed
to satiation level twice daily with the diet containing 30% crude protein (CP) throughout the exper-
imental period. Fish in the other four treatment groups were deprived of feed for 3 weeks and then
refed to satiation for 5 weeks with a diet containing 25 (25P), 30 (30P), 35 (35P) or 40 (40P) % CP.
7-week study and observed that CG restored the fat to lean body mass ratio in the
deprived fish.
Reyes and Baker (2017) added significant details to the CG issue. They manipu-
lated early-life feeding regimens in three-spined stickleback (G. aculeatus) to study
compensatory responses in growth rate and lipid storage during the first growing
season. Early in life, the stickleback populations undergo a crucial survival period,
during which nutrient fluctuations may result in a biological trade-off where grow-
ing large is more important than lipid storage for competition and ultimately sur-
vival. The results demonstrated that CG responses in juvenile stickleback depend on
the timing of deprivation during the first growing season and, further, that responses
to late-season deprivation have favoured development of a larger body frame enter-
ing the overwintering season over lipid regeneration.
One result not compatible with this model was the observation that higher pro-
tein, rather than lipid, diet was essential while refeeding food-deprived rohu carp in
order to restore the growth, immunity and anti-oxidant capability of the fish. The
higher protein diet also imparted enhanced immune capability that was even higher
than daily-fed fish as revealed by an A. hydrophila challenge (Fig. 4.46) (Yengkokpam
et al. 2016).
Together, these studies indicate that the compensatory response is induced by
internal physiological factors rather than dietary inducers. However, the question
remains whether transgenerational effects of CG are even feasible. Do the offspring
of fasted and refed parents successfully compete with offspring of permantly well-
fed parents?
Box: Growth Factor as Biomarker for Growth

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Several growth factors control growth of animals. The insulin-like growth fac-
tor (IGF) system plays a central role in neuroendocrine regulation, and ele-
ments of IGF signaling have been well conserved among the vertebrates. A
brief description of the primary components of this system as found in mam-
mals is presented in Box Figure 1. IGF ligands signal through the IGF1 recep-
tor. The IGF2 receptor targets the ligand to degradation. The IGF binding
proteins (IGFBPs) modulate the bioavailability of IGFs in the extracellular
environment, thereby influencing ligand–receptor interactions. IGFBP3 and
-5 can form ternary complexes with IGFs and the acid-labile subunit (ALS);
most serum IGFs are in such a complex with IGFBP3 (Wood et al. 2005).
Box Fig. 1 The mammalian insulin-like growth factor (IGF) system. (from Wood et al.
(2005), with permission from Elsevier)
Studies of IGF-mediated growth in fish clearly show that the somatomedin

hypothesis (Le Roith et al. 2001) can explain the neuroendocrine regulation of
growth (Box Fig. 2) (Wood et al. 2005). Somatomedins are hormones that
promote cell growth and division in response to stimulation by the growth
hormone (GH). To demonstrate the function of GH, Devlin et al. (2001)
treated rainbow trout eggs from a very slow-growing wild strain with exoge-
nous growth hormone protein. The treated trout grew much faster than non-
transgenic sibling controls (Box Fig. 3).
(continued)
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Box Fig. 2 Schematic representation of the modern somatomedin hypothesis. Originally

developed through studies in mammals, the hypothesis is now well supported by research
on the neuroendocrine regulation of postnatal growth in fish. Solid lines indicate positive
stimulation; dashed lines indicate negative stimulation. (from Wood et al. (2005), with
Box Fig. 3 Effect of

exogenous growth
hormone in rainbow
trout. Phenotype of
growth hormone-
treated domestic and
wild strains. (from
Devlin et al. (2001),
the Nature Publishing
Group)
(continued)
Environmental factors regulate hypothalamus-mediated GH secretions

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from the pituitary gland; circulating GH stimulates the secretion of IGFs and
IGFBP3 from the liver, and possibly from nonhepatic tissues. Hepatic and
nonhepatic IGFs stimulate tissue growth under the modulatory influence of
locally synthesized IGFBPs.
Studies in fish have revealed an important additional role for IGFs in the
maintenance of osmoregulatory homeostasis, in part through direct stimula-
tion of ion transporter enzymes in osmoregulatory tissues (e.g., gills).
Although the effects of the IGF system on growth, osmoregulation, and repro-
duction have been reasonably well described in fish, new lines of evidence
indicate that IGF signaling may also be essential as a central regulator of
embryonic and larval development in fish (Box Fig. 4) (Wood et al. 2005).
Box Fig. 4 Insulin-like growth factor (IGF) signaling (indicated by arrows), in association
with hormones and IGF binding proteins (IGFBPs), affects multiple aspects of fish develop-
ment and physiology. Its importance for growth, osmoregulation, and reproduction has
been relatively well defined; more recent studies indicate a central role for IGF signaling in
embryonic growth and development. IGF signaling may also serve to facilitate the switch
between reproductive and somatic development (indicated by the dashed, bidirectional
arrow) (from Wood et al. (2005), with permission from Elsevier). Abbreviations: CORT
cortisol, GH growth hormone, GTHs gonadotropins, Ins insulin, T3 triodothyronine
How can this basic knowledge be translated into fish breeding and aqua-
culture practice? It is now well documented that IGF1 is nutritionally regu-
lated. Dyer et al. (2004) studied the correlation between growth rate and
circulating IGF1 concentrations of barramundi (L. calcarifer), Atlantic
salmon, and southern bluefin tuna (Thunnus maccoyii). The authors found
that varying dietary compositions resulted in detectable growth rate differ-
ences which could also be identified by changes in the IGF1 levels. Measuring
(continued)
IGF1 can therefore be a useful tool to detect, and predict, subtle changes in
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growth and provide an alternative method to traditional techniques for opti-

mizing diet formulation.
Reinecke et al. (2005) extended this aspect by reporting that in bony fish,
not only igf1 mRNA, but also igf2I mRNA have been detected both in liver
and in numerous other organs, such as brain, eye, gills, heart, gastrointestinal
tract, pancreatic islets, kidney, skeletal muscle, spleen, and male and female
gonads. Furthermore, there is evidence that in bony fish both the igf1 and igf2I
are controlled by GH in all organs.
One striking example of IGF1 as biomarker for fish growth has been pre-
sented by Brown et al. (2012) in Nile tilapia (Box Fig. 5). A positive correla-
tion was found between diet as an independent variable and resultant changes
in measurable hepatic igf1 mRNA. The igf1 expression did not have a linear
correlation with the dietary parameters; instead, the relationship of growth to
the gene expression appears to be a sigmoid curve. One important source of
variance in these data was the social status of the fish; even under identical
experimental conditions within the same treatment groups, subordinate male
fish had consistently lower growth rates and igf1 expression values compared
to dominant male fish (Brown et al. (2012) and references therein).
14
12
10
Weight gain (g)
8
y = 0.931 + 1.880 (x)
6 r 2 = 72.9
4
2
0
0 100 200 300 400 500 600
Relative IGF-1 mRNA
Box Fig. 5 Correlative relationship of weight gain resulting from various feeding regimens
with relative hepatic concentrations of igf1 mRNA detected in Nile tilapia. The positive
correlation between weight gain and igf1 mRNA indicates the value of this molecular
method as an indicator of growth. (from Brown et al. (2012), with permission from Wiley)
Significant correlations between plasma IGF1 and specific growth rate in

fish induced through manipulations in the feeding regime or dietary composi-
tion have been reported for Mozabique tilapia, Nile tilapia, hybrid striped
(continued)
bass, coho salmon, Atlantic salmon, and barramundi (Picha et al. (2008a) and
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references therein). Interestingly, even the feed efficiency is correlated to the

IGF1 system (Box Fig. 6). Supporting results have recently been reported for
juvenile mirror carp fed graded protein diets (Huang et al. 2016).
Box Fig. 6 Relationship between hepatic igf1 mRNA level and feed efficiency of Nile
tilapia. (from Qiang et al. (2012) with permission from Elsevier)
Studies with results that contradict these findings also exist. In his review,
Beckman (2011) summarized that potential sources for this variation include
both biological and methodological issues and include differences in how
growth is defined (changes in length or weight), the duration of growth
assessed (weeks to months) and how growth is calculated (total change, rate,
percent change). Yet these methodological differences cannot account for all
the variation found. Instead, a number of physiological conditions and envi-
ronmental factors might influence IGF1 level and the subsequent relation of
that IGF1 level to growth rate.
As shown, IGF1 and growth relations generally remain concordant after
changes in nutrition (consumption rate or diet). Differences in IGF1 level of
juvenile, maturing male and maturing female fish are common and IGF1–
growth relations appear discordant between these groups. Acute changes in
temperature and salinity induce discordant relations between IGF1 and
growth, but acclimation to persistent differences in environmental conditions
generally result in concordant relations. Overall, by discriminating between
fish of differing physiological status and discerning and categorizing differ-
ences among environments, one may effectively use IGF1 as a growth index
for fishes (Beckman 2011).
4.4 Compensatory Growth in Populations

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In terms of population maintenance, it can be expected that natural selection favors

the evolution of mechanisms mitigating negative fitness consequences of early-life
growth limitation, whether resulting from food restriction or some other unfavor-
able environmental condition. However, only a few studies have investigated the
genetic basis of CG potential and existence of genetically-based population differ-
entiation in CG potential. Significant population differences in CG responses were
observed in Atlantic silversides (Menidia menidia) (Schultz et al. 2002) and in the
Atlantic salmon (Fraser et al. 2007), but not in the Atlantic cod (Purchase and Brown
2001). Fraser et al. (2007) found that individuals from the long-distance migrating
salmon population exhibited stronger CG response to food deprivation than those
from the short-distance migrating population. It is likely that this difference reflects
the need for long-distance migrants to reach a large size to offset the energetic costs
of long migration and to compensate for the shorter time they spend on feeding
grounds.
To disentangle the interplay between genetic and nutritional factors, Ab Ghani
and Merilä (2015) studied population differentiation, genetic basis, and costs of CG
potential in the euryhaline nine-spined sticklebacks (P. pungitius) differing in their
normal growth patterns. As selection favors large body size in pond and small body
size in marine populations, the authors expected CG to occur in the pond but not in
the marine population. By manipulating feeding conditions (high, low, and recovery
feeding treatments), the authors found clear evidence for CG in the pond but not in
the marine population.
In the marine population, overcompensation occurred in individuals from the
recovery treatment and eventually they grew larger than those from higher feeding
treatment. In both populations, the recovery feeding treatment reduced maturation
probability. The recovery feeding treatment also reduced the survival probability in
the marine, but not the pond, population (Fig. 4.47). Analysis of interpopulation
hybrids further showed that both genetic and maternal effects contributed to the
population differences in CG. Hence, apart from demonstrating intrinsic costs for
recovery growth, both genetic and maternal effects were identified to be important
modulators of CG responses. Overall, this study demonstrated the occurrence of CG
in response to early-life food restriction in a pond population of nine-spined stick-
lebacks, as well as evidence for significant catch-up (but not for compensatory)
growth in a marine population. In other words, although marine fish were not
observed to accelerate their growth in response to removal of food restriction above
routine levels, as was the case of the pond fish, fish from both populations compen-
sated for early growth restriction by reaching similar (pond) or larger (marine) sizes
than their conspecifics grown with unlimited food rations. Experiments with pure
and hybrid crosses further indicated that the observed population differences in
growth responses had a partially genetic basis (Ab Ghani and Merilä 2015).
4.4 Compensatory Growth in Populations 229
a MM high feeding
MP high feeding
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100
PM high feeding
PP high feeding
MM low feeding
MP low feeding
90 PM low feeding
PP low feeding
80
70
The probability of survival (%)
60
0 20 40 60 80 100
b
MM high feeding
100 MP high feeding
PM high feeding
PP high feeding
80 MM low feeding
MP low feeding
PM low feeding
PP low feeding
60 MM recovery feeding
MP recovery feeding
PM recovery feeding
40 PP recovery feeding
20
100 200 300 400 500 600

Time (DAH)
Fig. 4.47 The probability of survival among four cross-types of nine-spined sticklebacks (a)
before and (b) after initiation of the recovery feeding treatment. M marine, P pond. For the hybrids,
the first abbreviation denotes origin of father, the second origin of mother. (from Ab Ghani and
Merilä (2015), with permission from Wiley), DAH days after hatch
4.5 Regulation of Compensatory Growth

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Growth is under endocrine control, and the role of hormones in CG is increasingly

well understood (Volkoff 2006). Our understanding of the endocrine mechanisms
during this phenomenon is commonly limited to observations in a few model spe-
cies or extrapolations from studies in higher vertebrates. Won and Borski (2013)
reviewed the state-of-the-art of endocrine regulation; in order to elicit a CG response,
endogenous energy reserves must first be moderately depleted to alter endocrine
profiles that, in turn, enhance appetite and growth potential. During this catabolic
phase, elevated ghrelin and GH production increase appetite and protein-sparing
lipolysis, while IGFs are suppressed. During refeeding, temporal hyperphagia pro-
vides an influx of energy and metabolic substrates that are then allocated to somatic
growth by IGF signaling. Under appropriate conditions, refeeding results in hyper-
anabolism and a steepened growth trajectory relative to constantly fed control
fishes. The response wanes as energy reserves are re-accumulated and homeostasis
is restored.
During the preceding catabolic phase (fasting, Fig. 4.48b), orexigens stimulate
appetite as endogenous energy reserves are depleted. Ghrelin and cortisol stimulate
GH production and elevate circulating GH levels in order to free energy-storing
lipids. However, hepatic GH resistance and the growth-inhibitory effects of cortisol
and somatostatins suppress IGF production under the pretext that conditions are
unfavorable for growth. Reduced negative feedback from IGF1 further permits GH
levels to rise. A critical period of catabolism is therefore required to induce hor-
monal changes that prime the fish for hyperphagia and super-potentiate the growth
axis. During refeeding (Fig. 4.48c), CG is fueled by the hyperphagic influx of exog-
enous energy and substrates.5 The food is assimilated with heightened efficiency as
the result of modifications to metabolic substrate absorption, in part attributable to
residual elevated GH levels. Hepatic GR receptors (GHRs) are reinstated and GH
sensitivity returns, followed by a steep rise or even overcompensation in IGF1 pro-
duction. Production of IGFs during refeeding is also influenced by a decline in
growth-inhibitors. Substrate and energy availability, enhanced assimilation effi-
ciency, and augmentation of the growth axis culminate in a hyperanabolic, rapid
growth phase until a lipostat-like mechanism initiates the return to basal appetite
and a growth axis profile representative of a normal growth trajectory (Fig. 4.48a).
Some empirical studies illustrate this regulation.
In hybrid striped bass, Picha et al. (2008b) showed that during the phase of
refeeding and the ensuing CG response, elevations in growth rates are not only
driven by a combination of hyperphagia and improved feed conversion, but also
heightened levels of IGF1, IGFBPs, and IGF2. This can be identified in elevated
hepatic transcripts (e.g. igf1, igf2, ghr1, and ghr2) to levels exceeding those in
5
Although rather comprehensive, the model description by Won and Borski (2013) is still simpli-
fied. For more details, refer to the in silico study by Gurney et al. (2003) and the empirical work by
Cui et al. (2006) discussed above.
4.5 Regulation of Compensatory Growth 231
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Fig. 4.48 Endocrine regulation of growth and appetite during normal anabolism, catabolism, and
hyperanabolism resulting from feeding status. Growth is regulated by the GH/IGF axis; GH
secreted into circulation by the pituitary binds its receptor (GHR) to stimulate hepatic IGF1 pro-
duction that systemically drives somatic growth and exerts negative feedback on GH secretion.
Lipolysis is an alternate function of GH during catabolism. Peripheral signals from a lipostatic
mechanism (anorexigenic), possibly leptin, and ghrelin (orexigenic) regulate energy intake by
modulating NPY and other neuropeptides in the central feeding center. Ghrelin also functions as a
GH secretagogue. Arrows show the direction of regulatory pathways; widening/narrowing of
arrows represents a dynamic increase/decrease in a component over the duration of a particular
metabolic state. (a) During regular feeding, energy homeostasis is maintained by matching energy
intake and expenditure. Peripheral signals counter-regulate appetite centrally. Growth is regulated
by nominal levels of circulating GH that stimulates IGF1 production via hepatic GHRs. (b) Fasting
necessitates catabolic processes to provide energy for basal metabolism. Rising ghrelin production
stimulates both appetite and circulating GH levels. Elevated lipolytic GH levels exploit stored
energy reserves, decreasing lipostatic signaling. Reduced hepatic GHR expression desensitizes the
liver to GH-induced IGF1 production. (c) Refeeding signifies the switch from catabolic to anabolic
processes. Temporally elevated orexigens carried over from fasting drive hyperphagia. The return
to the positive energy status is characterized by the resumption of hepatic GH sensitivity and a
steep rise in circulating IGF1 levels that promotes accelerated growth. Eventually, the repletion of
energy reserves and negative feedback from IGF1 returns GH and appetite to nominal levels, mark-
ing the return to normal growth rates. (PIT pituitary, HYP hypothalamus, NPY neuropeptide Y, GH
growth hormone, GHR growth hormone receptor, IGF1 insulin-like growth factor I). (from Won
and Borski (2013), courtesy of Frontiers in Endocrinology)
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Fig. 4.49 Hepatic mRNA levels in control hybrid striped bass fed to satiation twice daily and
treatment fish subjected to 3 weeks feed restriction (grey bar) followed by 6 weeks of refeeding
twice daily to satiation. Hepatic mRNA levels were expressed as (a) total hepatic igf1, (b) igf2, (c)
the Type I Ghr (ghr1), and (d) Type II Ghr (ghr2) copy number/body weight. Asterisks represent
significant differences between groups at each time point (*P < 0.05; **P < 0.01;***P < 0.001).
(from Picha et al. (2008b), with permission from the BioScientifica Ltd.) (Image courtesy of the
International Game Fish Association. Artist: Duane Raver)
normally fed fish which is likely to be a contributing factor to the accelerated growth
rates characteristic of CG responses (Fig. 4.49). In addition to dietary restriction and
CG, IGF1 is responsive to increased ration size, increased crude protein shares and
increased plant protein supplementation (Picha et al. 2008a). Reports showing the
involvement of IGF1 in the CG process are available for gilthead sea bream and
rainbow trout (Montserrat et al. 2007a, b) or juvenile Atlantic halibut (H. hippoglos-
sus) (Hagen et al. 2009).
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Fig. 4.50 Transcription of myog in white muscle of Megalobrama amblycephala at different time
points during starvation and refeeding. Significant differences at P < 0.05 are labeled with different
letters; mean ± SEM of each mRNA quantity is shown for each stage tested. (from Zhu et al.
(2014) with permission from Elsevier) (Image courtesy FAO)
Now the question arises of how muscle growth is regulated. Myogenin (myog) is
a muscle-specific basic transcription factor that plays an essential role in regulating
skeletal muscle development and growth. Therefore, it is not surprising that, for the
recovery of the muscle mass after refeeding, myog plays a central role during CR,
as shown in Wuchang bream (M. amblycephala) (Fig. 4.50) (Zhu et al. 2014).
A more comprehensive picture of CG has recently been drawn by Rescan et al.
(2017) from a study of muscles from fasted/refed trout, where genes promoting
myofibre growth, but not myofibre formation, are upregulated. This indicates that a
compensatory muscle growth response, resulting from the stimulation of hypertrophy
but not the stimulation of hyperplasia, occurred after refeeding (Fig. 4.51). It is
interesting to know whether or not this mechanism also applies to the muscle growth
in biotechnologically treated zebrafish (Fig. 4.43).
4.5.1 Appetite-Regulating Hormones
Leptin and ghrelin are two appetite-regulating hormones that have been recognized
to have a major influence on energy balance. Leptin is a mediator of long-term regu-
lation of energy balance, suppressing food intake and thereby inducing weight loss
(anorexic effect). Ghrelin (acronym: growth hormone release inducing = ‘hunger
hormone’), on the other hand, is a fast-acting hormone and plays a role in meal
initiation and energy usage. Both hormones are agonists and regulate appetite to
achieve energy homeostasis.
In Arctic charr (Salvelinus alpinus), Frøiland et al. (2010) showed the antago-
nisms of ghrelin and leptin. There was a significant seasonal effect of stomach ghrl
and liver lep transcription; ghrl transcription decreased 3-fold from March to late
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Fig. 4.51 The compensatory muscle growth response involves only a subpart of the molecular
signature of the hyperplastic growth zone. Venn diagram representing the distribution of genes of
the compensatory muscle growth signature and genes upregulated in the superficial hyperplastic
growth zone of the late trout embryo. Functional categories inferred from genes common to the
compensatory muscle growth and the hyperplastic growth zone signatures are detailed and major
functional categories specific to hyperplastic growth zones are mentioned. The 312 genes specific
of the compensatory muscle growth response were mostly related to translation, protein folding,
RNA processing and ribosome biogenesis. (from Rescan et al. (2017), courtesy of BioMed Central)
June, and subsequently increased to the spring level by the end of August and
remained high (Fig. 4.52a). Liver lep transcription was low in spring, followed by a
gradual increase (Fig. 4.52b).
Several studies address the activity of leptin in fish and aquatic invertebrates and
showed that leptin is not involved in CG. The studies were conducted in rainbow
trout (Murashita et al. 2008b), Arctic charr (Frøiland et al. 2010), Atlantic salmon
(Johnsen et al. 2011; Trombley et al. 2012; Rønnestad et al. 2010; Murashita et al.
2011), European sea bass (Gambardella et al. 2012), striped bass (Morone saxatilis)
(Won et al. 2012), medaka (Chisada et al. 2014), Schizothorax prenanti (Yuan et al.
2014), Mandarin fish (Siniperca chuatsi) (Yuan et al. 2016), and Chinese mitten
crabs (Jiang et al. 2009).
In contrast with studies confirming leptin as adiposity signal, no significant dif-
ferences in the leptin expression were found in control, overfed and fasting goldfish,
pointing out the lack of a relationship between nutritional status and leptin (Tinoco
et al. 2012). Similar phenomena have also been reported from common carp
(Huising et al. 2006). Also for Atlantic salmon, the role of leptin is less explicit than
a b
1.00 0.10
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Relative ghrelin mRNA expression
Relative leptin mRNA expression

a
a
0.80 0.08 b
ac
ab
0.60 0.06
bc
0.40 b 0.04
0.20 0.02 a a
a
0.00 0.00
Mar Apr May Jun Jul Aug Sep Oct Nov Mar Apr May Jun Jul Aug Sep Oct Nov
Fig. 4.52 Above: seasonal changes in mean (± SEM) stomach ghrelin (a) and liver leptin (b)
transcription levels in Arctic charr. The results are expressed as target copy to β-actin copy ratio.
Different letters denote significant differences between dates (P < 0.05) (from Frøiland et al.
(2010), with permission from Elsevier). Below: image of Salvelinus alpinus. (from Bloch (1782–
1784), courtesy of the Biodiversity Heritage Library)
expected, since Moen and Finn (2013) showed that short-term, but not long-term,
food restriction causes differential expression of leptin.
Recently, Jönsson (2013) reviewed the orexigenic function of ghrelin in various
fish species and demonstrated the functional differences between stomachless and
stomach species (Fig. 4.53). Most ghrelin is produced and secreted in the stomach
(rainbow trout) or intestine (goldfish, a stomachless species), but there is also evi-
dence of local ghrelin production in the hypothalamus. In goldfish, vagus nerve
afferents mediate the signal from gut-derived ghrelin to the brain. In the hypothala-
mus, ghrelin stimulates orexigenic populations of NPY neurons and orexin neu-
rons. Orexin may also stimulate ghrelin-containing neurons. This mode of action
of ghrelin in the hypothamalus of goldfish leads to increased food intake. In rain-
bow trout, ghrelin suppresses food intake by acting on the anorexigenic corticotro-
pin-releasing hormone (CRH) neurons. This pathway is hypothesized to involve
proopiomelanocortin/cocaine and amphetamine regulated transcript (POMC/

CART), but the potential direct action of ghrelin on these neurons has not yet been
investigated. The regulatory mechanisms by which ghrelin alters the activity of
hypothalamic neurons containing orexigenic and anorexigenic neuropeptides are
unknown. How and to what extent ghrelin in the blood crosses the blood-brain bar-
rier to access the growth hormone secretagogue receptor (GHS-R) in the hypo-
thalamus has not yet been demonstrated and there is still a question whether ghrelin
communicates via the vagus nerve in rainbow trout.
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Fig. 4.53 Schematic model of action of ghrelin (Ghr) on food intake in goldfish (left-hand side of
figure) and rainbow trout (right-hand side of figure). Details in the text. (from Jönsson (2013), with
4.5.2 Neuropeptides
Orexigenic as well as anorexigenic hormones act through a network of peptides,

such as neuropeptide Y (NPY), hypocretin (orexin) neuropeptide (HCRT), melanin-
concentrating hormone (MCH), agouti-related proteins (AgRPs), cholecystokinin
(CCK), proopiomelanocortin (POMC), prepro-orexin (pOX), or cocaine and
amphetamine regulated transcript (CART) (Valen et al. 2011; Le et al. 2016; Gomes
et al. 2015). Here, we shall focus on NPY, a well understood neuropeptide, and
CCK and CART, newly identified neuropeptides, with increasing recognition and
significance in appetite regulation in fish.
All fishes produce two neuropeptide Y (NPY)-related peptides, namely NPY and
peptide YY (PYY) (Volkoff et al. 2005). In their pioneering paper, López-Patiño
et al. (1999) demonstrated the significance of NPY in the feeding behavior of gold-
fish (Fig. 4.54). This graph shows food intake during discrete and cumulative inter-
vals after intracerebroventricular injection of NPY. Food intake was significantly
increased by 1 μg of NPY at two hours post injection. During the later discrete
intervals, none of the doses tested significantly modified food intake.
To date, many papers on appetite-regulating peptides are inventory studies in
various aquatic invertebrate and fish species applying different methods. In inverte-
brates, neuropeptides F and, particularly, short neuropeptides F are viewed as homo-
logs of NPY (Nässel and Wegener 2011; Mercier et al. 2007). Neuropeptides Y and
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Fig. 4.54 Food intake (mg) of goldfish after intracerebroventricular administration of 1 μl saline
alone or containing increasing doses of neuropeptide Y (NPY) at 0–2 (top), 2–8 (middle) and 0–8
(bottom) h postinjection, in goldfish. The x-axis represents absolute doses of neuropeptide Y (μg)
in all graphs. *P < 0.05. (from López-Patiño et al. (1999), with permission from Elsevier)
F are potent stimulators controlling feeding behavior, energy homeostasis,

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reproduction, and tissue/organ regeneration (Suwansa-Ard et al. 2015; Kreshchenko

et al. 2008).
Such peptides have been identified in:
• Annelida: polychaete worms (Capitella teleta) and leeches (Helobdella robusta)
(Veenstra 2011);
• Mollusks: great pond snails (Lymnaea stagnalis) (de Jong-Brink et al. 2001),
abalones Haliotis asinina (Nuurai et al. 2010), anemone cones (Conus anemone)
(Le et al. 2003), sea hares (Aplysia californica) (Rajpara et al. 1992), giant owl
limpets (Lottia gigantean) (Veenstra 2010), octopuses (Octopus vulgaris)
(Suzuki et al. 2003), Akoya pearl oyster (Pinctata fucata) (Stewart et al. 2014),
Pacific oyster (Crassostrea gigas) (Bigot et al. 2014; Stewart et al. 2014), sunray
Venus clam (Macrocallista nimbosa) (Zatylny-Gaudin and Favrel 2014);
• Crustaceans: American lobster (Homarus americanus) (Jiang et al. 2012), ice
krill (Euphausia crystallorophias) (Toullec et al. 2013), water fleas Daphnia
pulex (Christie et al. 2011b; Dircksen et al. 2011) and D. magna (Christie et al.
2008; Christie and McCoole 2012), Eucyclops serrulatus (Christie 2015),
amphipods Echinogammarus veneris, Hyalella azteca, and Melita plumulosa
(Christie 2014b), penaeid shrimps Marsupenaeus japonicus, P. semisulcatus6
(Kiris et al. 2004), P. monodon (Sithigorngul et al. 2002), P. marginatus, L. van-
namei (Christie 2014a; Christie et al. 2011a), and Fennropenaeus chinensis
(Wang and Xiang 2003), spiny lobster (Panulirus interruptus) (Ye et al. 2015),
ghost crab (Ocypode ceratophthalma) (Hui et al. 2013), shore crab (Carcinus
maenas) (Ma et al. 2009), mudflat crab (Chiromantes haematocheir) (Honma
et al. 1996), mud crab (Scylla paramamosain) (Bao et al. 2015);
• Lampreys: sea lamprey (Petromyzon marinus) (Pérez-Fernández et al. 2014);
• Fishes: and countless more fish species (for a review, see Volkoff et al. (2005)).
In fishes, as in mammals, NPY interacts with a number of appetite regulators.
NPY is a neurotransmitter in the brain and in the autonomic nervous system and
causes growth of fat tissue (Kuo et al. 2007). Within the central nervous system,
production of NPY is responsive to food restriction, insulin, dietary lipid, carbohy-
drate, and protein. NPY, in turn, regulates pituitary hormone secretion in the GH,
thyroid, and reproductive axes. Identification of the receptors which mediate the
actions of NPY in fish is still being studied (Volkoff 2006).
Fasting induced an increase in npy hypothalamic expression in goldfish
(Narnaware and Peter 2001) (Fig. 4.55), salmon (Silverstein et al. (1998), or blunt
snout bream (M. amblycephala) (Ji et al. 2015). In all fish species, refeeding reversed
these effects.
6
It is noteworthy that the administration of vertebrate NPY dramatically increased food intake in
both penaeid shrimps, pointing at a conserved role for NPY and possibly a degree of sequence
homology in crustacean species, most likely in the part of the hormone that interacts with its
receptor.
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Fig. 4.55 Relative neuropeptide Y (npy) mRNA levels of goldfish in various brain regions of the
goldfish: telencephalon-preoptic (a) and hypothalamus (b) subjected to feeding, food deprivation
for 72–75 h or refeeding for 1–3 h after 72-h food deprivation. Significant differences at P < 0.05;
letters not joined by underlining indicate groups that differ significantly. (from Narnaware and
Peter (2001), with permission from Elsevier). Image of Carassius auratus (from Bloch (1782–
1784), courtesy of the Biodiversity Heritage Library)
Nutrient composition and feeding patter influence npy expression. Narnaware

and Peter (2002) showed that goldfish had a preference for high carbohydrate and
high fat diets, with no preference shown for high protein diets. The findings in gold-
fish are supported by studies in the Mozambique tilapia. Glucose treatment signifi-
cantly increased the npy mRNA levels, indicating that NPY-containing neurons may
be a “glucosensor” as reported in mammals (Riley Jr et al. 2009), facilitating a
macronutrient selection, at least in these two fish species. Whether this phenomenon
applies, not only to these two, but also to other fish species remains unknown, as
well as the physiological mechanism involved in diet selection in fish (Kulczykowska
and Sánchez Vázquez 2010).
Evidence is increasing that NPY and no other appetite regulators are central to
the food intake mechanism. Recently, Hosomi et al. (2014) showed that npy but not
cck (cholecystokinin, another appetite modulator in many vertebrates) is signifi-
cantly transcribed in yellowtail (Seriola quinqueradiata) during fasting.
The CART produces similar behavior in animals in response to cocaine and
amphetamine, but conversely blocks the effects of cocaine when they are
co-administered. The mRNA of the CART peptide is extensively distributed in the
brain of sharks and teleosts, such as goldfish, catfish, zebrafish, medaka, Senegal
sole, and Atlantic cod (Subhedar et al. (2014); Bonacic et al. (2015) and references
therein). In zebrafish, starvation resulted in a significant decrease in CART-positive
cells in the nucleus recessus lateralis and nucleus lateralis tuberis hypothalamic
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regions, indicating a function in energy homeostasis. (Akash et al. 2014).

Cholecystokinin (CCK) is a peptide hormone of the gastrointestinal system
responsible for stimulating the digestion of fat and protein and plays an antagonistic
role to that of peptide Y (PY), a NPY-related peptide; CCK stimulates, whereas PY
represses enzyme secretion. Inventory studies identified and located the release of
the peptide hormones in different tissues. For instance, van Wormhoudt et al. (1989)
identified gastrin/CCK-like peptides from the hemolymph of two penaeid shrimps
(M. japonicus, L. stylirostris). Later, sulfakinins were found in P. monodon (Johnsen
et al. 2000), L. vannamei (Torfs et al. 2002), and, in silico, in D. pulex (Christie and
McCoole 2012), Eucyclops serrulatus (Christie 2015), and M. rosenbergii
(Suwansa-Ard et al. 2015). CCK and sulfakinins originate from a common ancestral
gastrin/CCK-like peptide and have identical functions in arthropods (Staljanssens
et al. 2011).
In rainbow trout, Jensen et al. (2001) documented the presence of CCK-like pre-
prohormones in the brain. Kamisaka et al. (2005, 2013) reported that CCK-
producing cells in the gut developed “meal-responsiveness” later in post-hatch
development in Atlantic herring. In winter skate (Raja ocellata), CCK, as well as
NPY and CART, peptides were present in brain, gut, and gonads. Two weeks of
fasting induced a NPY increase in telencephalon and a CCK increase in the gut, but
had no effects on hypothalamic NPY, CART and CCK, or on telencephalon CART
(MacDonald and Volkoff 2009). In Atlantic herring larvae, CCK participates in the
regulation of digestive processes (Rojas-García et al. 2011).
Rainbow trout challenged by reduced food intake had higher cart mRNA in the
hypothalamus, whereas npy mRNA did not follow the macronutrient-induced
changes (Figueiredo-Silva et al. 2012). This supports the role of CART in mediating
satiation in fish, whereas the physiological role of NPY in regulating food intake
under normal physiological and feeding conditions requires further study. Nguyen
et al. (2013) showed that, at least in juvenile cobia (Rachycentron canadum), NPY
serves as orexigenic factor.
For the channel catfish, Peterson et al. (2012) described the interplay of GRLN,
CART, NPY, and CCK as CART, NPY, and the two CCKs (CCKa and CCKb) in the
regulation of feeding. In the brain, but not in the hypothalamus, the antagonistic
interplay between CCK and NPY is clearly visible (Fig. 4.56). The function of
GRLN during feeding is not certain as both plasma and mRNA concentrations were
relatively unchanged during the peri-prandial experiment.
Recently, Ji et al. (2015) examined the transcription of ghrelin, npy, and cck in
blunt snout bream. The three genes were expressed in a wide range of adult tissues,
with the highest expression levels of ghrelin in the hindgut, npy in the hypothala-
mus, and cck in the pituitary. Starvation increased the expression levels of ghrelin
and npy in the brain and intestine (Fig. 4.57, left), and decreased the the t ranscription
of cck (Fig. 4.57, right). Refeeding brought the expression levels of the three genes
back to control levels. These results indicated that the feeding behavior of blunt
snout bream was regulated by the concerted action of ghrelin, npy, and cck.
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Fig. 4.56 Hypothesized scheme for control of CCK release of pancreatic enzymes and the
nutrient-specific involvement of CCK and PY regulatory loops in fish. (from Murashita et al.
(2008a), with permission from Elsevier)
In order to determine how different nutrients stimulate the synthesis of CCK and
PY in S. quinqueradiata, Murashita et al. (2008a) measured cck and py mRNA lev-
els in the digestive tract after oral administration of a single bolus of either
phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein),
oleic acid (fatty acid) or tri-olein (triglyceride). Casein, oleic acid, and tri-olein
increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not
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Fig. 4.57 Changes in expression of NPY (left) and CCK (right) in the brain (a) and intestine (b)
of blunt snout bream responding to fasting and refeeding. Different italic lowercase letters above
the columns indicate significant differences in the control group and bold lowercase letters for
experimental group (P < 0.05). * indicates a significance difference between experimental and
control groups on the given day (P < 0.05). (from Ji et al. (2015) with permission from Elsevier)
affect the activity of any of these enzymes. cck mRNA levels rose, while py mRNA
levels were reduced in fish administered casein, oleic acid, and tri-olein. These
results confirm that CCK and PY maintain antagonistic control of pancreatic
enzyme secretion in yellowtail after intake of protein and/or fat. The supposed
modes of action of macronutrients are summarized (Fig. 4.56).
Dietary fat and protein are strong stimulators of CCK release in yellowtail, while
carbohydrates are weak stimulants. The increased synthesis of CCK and trypsin in
casein-administered yellowtail compared well with results reported in larval Atlantic
herring, where synthesis of CCK and trypsin was observed after tube-feeding bovine
serum albumin as intact protein (Murashita et al. (2008a) and references therein).
Cahu et al. (2004) reported that CCK levels of low-hydrolysate protein-fed sea bass
larvae were higher than in fish fed high-hydrolysate protein indicating that intact
protein stimulates CCK release more than hydrolyzed protein. The higher cck
mRNA level in casein-administered yellowtail was therefore probably mediated by
the CCK-releasing factors. In tri-olein fed yellowtail, the cck mRNA increase was
delayed and it seems that the level of cck mRNA depended on the quantity of oleic
acid in the intestinal lumen. It is therefore possible that tri-olein stimulated the
secretion of pancreatic digestive enzymes through other pathways than CCK. Overall,
the inverse expression patterns of cck and py observed in the study of Murashita
et al. (2008a) point out that the control of pancreatic enzyme secretion by CCK and
NPY-related peptides differ in fish and mammals.
4.5.3 Transcription of Growth Regulators

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A few studies have focused on the importance of apetite-regulating hormones and

neuropeptides during early teleost development. Gomes et al. (2015) analyzed the
expression patterns of the appetite-controlling factors ghrelin, npy, peptide yy (pyy),
pro-opiomelanocortin (pomc-c), and cart. Transcription was studied in response to
feeding in developing Atlantic halibut larvae, before (pre-metamorphic stage 5) and
during metamorphosis (stages 8 and 9B), and also in response to a fast–refeed chal-
lenge. The authors showed that ghrelin transcription increased in synchrony with
stomach development, while cart was significantly reduced during larval develop-
ment; pyy was upregulated 1 and 3 h after feeding in stage 5 (Fig. 4.58). Transcription
of other appetite-controlling factors did not change in response to feeding. Fasting–
refeeding trials (majority of larvae in metamorphosing stage 7) revealed a down-
regulation of pomc-c 30 min after refeeding, while ghrelin, pyy, and npy transcription
increased 2, 4 and 5 h after refeeding, respectively. This paper shows that transcrip-
tion of key appetite-controlling factors started early during development in Atlantic
halibut and was not correlated with metamorphosis, with the exception of ghrelin.
This also indicates that pyy may mediate satiety early in larval development. The
differing response times of pomc-c, ghrelin, pyy, and npy to food are intriguing and
require further exploration to understand the role of each player in appetite
control.
Which role do peptide transporters play in CG? Oligopeptide transporter 1
(pept1), for instance, is an integral plasma membrane protein responsible for the
uptake of dietary di- and tri-peptides in cells. It transports the peptides against a
concentration gradient by coupling the movement of substrate across the membrane
with the movement of protons down an inwardly directed electrochemical proton
gradient. A unique feature of PepT1 is the capability to transport nearly all possible
di- and tripeptides, including differently charged species.
Terova et al. (2009) showed that pept1 was highly expressed in the proximal
intestine (Fig. 4.59) with lower levels of expression in the gill, brain, heart, liver and
spleen of European sea bass. Nutritional status significantly influenced pept1 tran-
scription in the proximal intestine, inducing a downregulation during prolonged
fasting (35 days) and an upregulation during the recovery from fasting, the inverse
of that observed so far in mammals and birds (Verri et al. 2011). These findings
document a direct correlation between the transcription of an intestinal transporter
primarily involved in the uptake of dietary protein degradation products and body
growth.
Furthermore, the Δ6 desaturase transcription was enhanced (Terova et al. 2012).
The absolute mRNA levels of Δ6 desaturase in the proximal intestine in response to
the feeding trial are presented in Fig. 4.60. The analysis showed significant changes
in Δ6 desaturase expression levels during the experiment. Prolonged fasting caused
a significant decrease in Δ6 desaturase transcripts. The recovery from fasting was
associated with a significant increase in mRNA transcript levels until the end of day
4 of refeeding, consistend with CG. Subsequently, 10 days after refeeding, the Δ6
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Fig. 4.58 Ontogenetic and periprandial mRNA expression of the selected genes in Atlantic halibut
larvae at stages 5, 8 and 9B. Ghrelin was analyzed in the GI tract and npy, pyy, pomc-c and cart
were analyzed in the brain. The mRNA expression of the selected genes was analyzed before feed-
ing, and 1 h and 3 h after feeding. Results are shown as mean ± SEM of the normalized expression
(MNE), using the reference gene eef1a1. * indicates statistically significant difference (P < 0.05).
(from Gomes et al. (2015), with permission from Elsevier)
desaturase mRNA copy number levels decreased compared to the previous time
point tested, and then returned to control levels at the end of 21 days of refeed-
ing. In addition to other genes to be identified, pept1 and Δ6 desaturase are central
to CG.
Calpains, a superfamily of intracellular calcium-dependent cysteine proteases,
are involved in the cytoskeletal remodeling and wasting of skeletal muscle. They are
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Fig. 4.59 Transcription levels of oligopeptide transporter 1 (pept1) in Dicentrarchus labrax prox-
imal intestine in the course of the experiment. Fish were sampled before fasting (day 0), 4 days
after fasting (4 days fasted), at the end of fasting (35 days fasted), and then sequentially at 4, 14
and 21 days following refeeding. The means of five animals in each group are shown. * indicates
significantly different means from controls, for each time point tested (P < 0.05). (from Terova
et al. (2009), with permission from Elsevier)
Fig. 4.60 Expression levels of Δ6 desaturase RNA in proximal intestine of the European sea bass.
The mRNA copy number was normalized to 100 ng total RNA. The mean values of five animals in
each group are shown. Bars indicate standard error of the mean. * indicates significantly differ-
ences from controls for each time point tested (P < 0.05). (from Terova et al. (2012), with permis-
sion from Wiley)
generated as inactive proenzymes which are activated by N-terminal autolysis

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induced by calcium-ions. Preziosa et al. (2013) assessed the effect of nutrient
restriction and subsequent refeeding on the expression of clpn1, clpn2, and clpn3 in
skeletal muscle in channel catfish. Starvation for 35 days influenced the expression
of all three genes, whereas the subsequent refeeding did not change the expression
of these genes. This means that, after a starvation and refeeding period, the expres-
sion patterns of calpain genes and the catalytic activity of the protein indicated
either further degradation of myofibrillar proteins for AA supply or the qualitative
remodeling of the muscle. Moreover, under wasting conditions, clpn3 was immedi-
ately downregulated, showing that its activity is different to that of the ubiquitous
calpains required for skeletal muscle homeostasis.
Lipids are the most important energy store and their replenishment is central. In
a comprehensive study, Rimoldi et al. (2015) evaluated how the transcription of
genes controlling lipid metabolism in European sea bass was modulated in a tissue-
specific manner in response to fasting and refeeding. The approach focused on 29
genes in which desaturases, elongases, triacylglycerol lipases, fatty acid-binding
proteins, β-oxidation and oxidative phosphorylation enzymes, phospholipid-related
enzymes, and lipid homeostasis-regulating transcription factors were represented.
This study clearly indicated tissue-specific molecular signatures that were regulated
to a large extent by nutrient supply. Fasting activated the lipolytic machinery in
adipose tissue, liver, and muscle, whereas lipogenesis genes were downregulated in
liver and adipose tissue. Genes involved in phospholipid and oxidative metabolism
were differentially regulated in liver and skeletal muscle of fasted individuals.
However, 12 days of refeeding were sufficient to reverse the transcription of key
genes.
This study showed a tissue-specific regulation of lipid-related genes according to
the different metabolic capabilities of each tissue, the brain being the most resistant
organ to changes in nutrient and energy availability and the liver the most respon-
sive one. It is noteworthy that stearoyl-CoA desaturase 1b (scd1b) proved to be one
of the most informative markers of lipogenesis in liver and adipose tissues. In paral-
lel, lipoprotein lipase (lpl) and lipoprotein lipase-like (lpl-like) transcription pro-
vided an accurate indication of the lipid flux between adipose tissue and liver.
Conversely, upregulation of group XIIB secretory phospholipase A2 (pla2g12b),
adipose triglyceride lipase (atgl), and peroxisome proliferator-activated receptor α
(pparα) are good indicators of the activation of the hepatic lipolytic machinery
(Fig. 4.61). In muscle, however, lpl, lpl-like and endothelial lipase (el), together
with carnitine palmitoyltransferase 1A (cpt1a) and succinate dehydrogenase cyto-
chrome b560 subunit (sdhc), are excellent markers of the nutritional status.
The innovative information presented in this paper provides valuable informa-
tion on fish lipid metabolism that could be successfully applied in the aquaculture
industry to monitor the metabolic status of farmed fish in order to optimize feeding
protocols and new diet formulations.
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Fig. 4.61 Graphical representation of fold-changes of differentially expressed genes (fasted vs

control and refeeding vs control) for mesenteric adipose tissue (a, b) and liver (c, d) of European
sea bass (Dicentrarchus labrax). Gene names grouped by function: long-chain PUFA metabolism:
elovl1, elovl4, elovl5, elovl6, fads2, scd1b; phospholipid metabolism: lpcat1, lpcat2, pemt,
pla2g12b; lipoprotein and triacylglycerol metabolism: lpl, lpl-like, hl, el, atgl, cel, hsl, lipa; fatty
acid β-oxidation and oxidative phosphorylation: cpt1a, hadh, cs, nd5, sdhc, cyb, cox1; cholesterol
metabolism: cyp7a1; transcriptional regulation: pparα, pparβ, pparγ. (from Rimoldi et al. (2015),
with permission from Springer)
4.5.3.1 Information from Transgenic Animals
Using GH transgenic coho salmon (O. kisutch), Kim et al. (2015) drew a fairly
complete picture of appetite-regulating brain gene expression. They compared the
expression of multiple genes important in appetite regulation within brain regions
and the pituitary gland (PIT) of GH transgenic (fed fully to satiation or restricted to
a wild-type ration throughout their lifetime) and wildtype individuals. It became
obvious that differences in both genotype and ration levels resulted in differentially
expressed genes associated with appetite regulation in transgenic fish, including
elevated agrp1 in the hypothalamus (HYP) and reduced mch in PIT. Transcription
of agrp1, gh, and ghr were higher in transgenic than wild-type fish in HYP and in
the preoptic area (POA). These data are consistent with the known roles of orexigenic
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factors in foraging behavior, acting via GH and through MC4R receptor-mediated

signaling. igf1 mRNA was elevated in fully-fed transgenic fish in HYP and POA,
but not in ration-restricted fish. However, both of these types of transgenic animals
have very pronounced feeding behavior relative to wild-type fish, showing that
IGF1 does not play a direct role in appetite stimulation acting via paracrine or auto-
crine mechanisms. The present findings provide new insights on mechanisms
involved in altered appetite regulation in response to chronically elevated GH, and
on the potential pathways by which elevated feeding response is controlled, inde-
pendently of food availability and growth.
Multiple and complex responses in expression of appetite-regulating genes
(including agrp1, gh, igf1, ghr, mch, and cart) have been observed in transgenic fish
(Fig. 4.62). Appetite regulation is likely maintained by a balance between orexi-
genic and anorexigenic factors, including those influencing the melanocortin sys-
tem in neurons located in the POA/HYP/PIT axis. Overexpression of GH stimulates
agrp1 expression that, in turn, stimulates feeding behavior, possibly by antagoniz-
ing the anorexigenic actions of α-msh at MC4R. In addition, the decreased expres-
sion of the anorexigenic factor cart observed in transgenic fish may also further
increase feeding motivation. These actions persist even when growth rate is restricted
in transgenic fish by ration limitation. Many genes known to be involved in appetite
regulation (i.e. agrp2, bbs, cck, gal, ghrh, glp, gnrh, hcrt, lep, mc4r, trh, and tsh) did
not differ in brain expression between transgenic and wild-type fish, indicating an
increased appetite and foraging behavior in transgenic fish may be controlled by a
limited number of appetite-related genes and pathways.
In fact, evidence of impairment of major life history traits is accumulating. Since
from mammals, it is well understood that GH excess causes an increment in the
metabolic rate and in ROS generation that accelerate the ageing process, Rosa et al.
(2010) expanded this approach to zebrafish. They analyzed the phenotype of spinal
curvature and expression of genes related to the anti-oxidant defense system and
myogenesis in muscle of 8- and 30-month-old GH-transgenic males. Analyses of
gene transcription revealed that both SOD isoforms were downregulated only in
30 month-old animals (Fig. 4.63), while glutamate cysteine ligase was downregu-
lated in GH-transgenic zebrafish. Acceleration of the spinal curvature and a reduc-
tion in the expression of myogenin at both ages and MyoD in the old fish were also
observed. Although neurolipofuscin accumulation (indicative of aging) was not sig-
nificant in GH-transgenic zebrafish, the estimated maximum longevity was signifi-
cantly lower in this group. The results indicate that GH overexpression reduced the
Fig. 4.62 (continued) those that are unaffected by transgenesis are shown in grey. A yellow back-
ground within tissues indicates effects of transgenesis regardless of ration level (i.e. TF and TR
differ from NT fish), while green background indicates effects of transgenesis are present only
when ration is unrestricted (i.e. only TF fish differ from NT fish). Red arrows indicate stimulation,
blue blunt-end lines indicate suppression, and green arrows indicate effect of increased nutrients.
? Indicates pathways for which effects are not fully defined in fish. (from Kim et al. (2015), with
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Fig. 4.62 Endocrine regulation of appetite-related genes mediated by overexpressed growth hor-
mone. (a) Study regions in fish brain; POA Preoptic area of the hypothalamus, HYP Remainder of
the hypothalamus, PIT pituitary, OB olfactory bulb, T telencephalon, ON optic nerve, OT optic
tectum, MB midbrain, C cerebellum, HB hindbrain, SC spinal cord. Shaded areas indicate tissues
examined in the current study. (b) The influence of GH transgenesis and nutrient levels on endo-
crine and genetic regulation of appetite and growth in salmon. Genes and proteins/peptides that are
increased in GH transgenic fish are shown in red, those that are decreased are shown in blue, and
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Fig. 4.63 Antioxidant defense system gene expression profiles in zebrafish. The graphs represent
the relative gene expression in the 8- (a) and 30-month-old (b) GH-transgenic animals compared
to non-transgenic siblings at the same age. The dashed-line represents the relative expression of
NT animals. The gene expression was normalized by the expression of ef1a. Statistically signifi-
cant differences (P < 0.05) in the gene expression levels between NT and GH-transgenic animals
are denoted by *. cat catalase, gpx-se glutathione peroxidase selenium dependent, gclc glutamate
cysteine ligase catalytic subunit, Cu, Zn-sod superoxide anion dismutase isoform, Mn-sod super-
oxide anion dismutase cytosolic isoform. (from Rosa et al. (2010), with permission from Elsevier)
transcription of the anti-oxidant defense system and myogenesis-related genes that

accelerated senescence in the transgenic zebrafish. In other words, accelerated
growth causes aging.
In addition to accelerated senescence, transgenic fish showed increased oxygen
demand and this impaired the ability of juvenile zebrafish to sustain an aerobic
metabolism and induced anaerobic metabolism when the fish were challenged with
low oxygen levels (Almeida et al. 2013). In short, transgenic fish were less stress-
resistant than wild-type individuals. Adverse effects of transgenesis persist even in
adult fish. For instance, Batista et al. (2014) discovered that the excess of GH
impaired the immune functions in GH transgenic zebrafish. Furthermore, Figueiredo
et al. (2013) reported that in normal mature gonads in 2-year-old male transgenic
zebrafish, a significant general decrease was observed in all spermatic and repro-
ductive parameters analyzed: motility, motility period, membrane integrity, mito-
chondrial functionality, DNA integrity, fertility, and hatching rate (Fig. 4.64).
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Fig. 4.64 Comparison of spermatic parameters between GH-transgenic (T) and non-transgenic
(NT) male zebrafish. (a) % of motile cells; (b) motility period (s); (c) % of functional mitochon-
dria; (d) % membrane integrity; (e) % DNA integrity; (f) % fertilization and hatching. * statisti-
cally significant differences (P < 0.05). (from Figueiredo et al. (2013), with permission from
Elsevier)
Taken together, the findings in transgenic zebrafish provide a clear corroboration

of the nutritional infertility hypothesis and show that, not only fertility, but several
major life history traits are impacted. Even on the ecosystem scale, GH transgenic
fish have an adverse impact, since they can behave cannibalistically (Fig. 4.65) and
derange population and food web structures, as shown in coho salmon populations.
Biomass in the low-ration environments, mimicking natural food availability
conditions, showed declines in all populations containing transgenic fish, whereas
populations containing only non-transgenic animals continued to gain biomass
throughout the experiment, albeit slowly because of limited food availability
(Fig. 4.66) (Devlin et al. 2004).
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Fig. 4.65 Cannibalistic activity by transgenic salmon. Note the caudal fin of a prey fish protruding
from the mouth of the transgenic salmon predator. (from Devlin et al. (2004), courtesy of the
National Academy of Sciences of the USA)
30.0
Low Ration Level
Average population biomass (g)
25.0 a
20.0
15.0
10.0
(NN) - Low
(TN) - Low
5.0 b
(TT) - Low
b
0.0
0 2 4 6 8 10 12 14
Week
Fig. 4.66 Biomass of populations reared under low rations. NN pure non-transgenic (wild-type)
population, TN mixed population, TT pure transgenic population. (from Devlin et al. (2004), cour-
tesy of the National Academy of Sciences of the USA)
4.6 Concluding remarks
Despite the heterogeneity of questions addressed and methods applied, several key
issues of DR become obvious, particularly from the comparison of invertebrate
response and fish response:
• Low food availability strongly modulates longevity, however, not in a unidirec-
tional manner. In many invertebrates, such as rotifers and cladocerans, life span
is extended. Contrary to expectations based on evolutionary theory, DR can even
reduce life span, particularly in fishes.
• In addition to life span extension, further survival probabilities can increase, such
as offspring tolerance to pathogens and toxic food, as well as predator
avoidance.
4.6 Concluding remarks 253
• Low food availability reduces fecundity in many animals, but leads to larger eggs
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and embryos than under conditions of high food supply.

• Upon food scarcity, larvae of several mollusks and sea urchins, as well as cladoc-
erans, develop larger capacity for clearing food particles from suspension. This
indicates that functionally similar examples of developmental plasticity have
evolved more than once and may be widespread (Strathmann 1978).
• Based on energy mobilization during long-term starvation in two sympatric
Procambarus species, high availability of nutrients favors the survival of P.
clarkii whereas P. zonangulus may have an advantage over P. clarkii when envi-
ronmental conditions and food availability delay emergence from the burrows in
the early fall. This indicates that even relatively small shifts in energy allocation
in response to different life history traits define separate ecological niches.
• In the damselfish A. polyacanthus, the inverse applies: juveniles from parents in
good condition were longer and heavier at hatching than juveniles from parents
in poor condition.
• In two Hippocampus species tested, short- to medium-term starvation signifi-
cantly increased the survival rate of juveniles.
• Even strains and clones of the same species may respond differently to DR as
shown with Brachionus sp., P. americanus, and N. furzeri, pointing at the, at least
partly, genetic basis of the response to DR and of the CG.
• Whereas intermittent feeding improves learning behavior in S. pleurospilus, it
reduces the life span in the stickleback G. aculeatus.
• The results, whether or not the female constitution of Atlantic cod positively
influences egg quality and embryo survival, are inconsistent. This indicates a
limited impact of the egg characteristics on their viability and hatching success.
Particularly, studies with invertebrates (e.g., Brachionus, Ceriodaphnia,
Daphnia, Moina) indicate that diet-dependant life history traits vary if challenged
by environmental stress. This indicates that the trade-off between different life his-
tory traits is not fixed and depends on specific environments which fluctuate sto-
chastically. Although temperature is a major challenge for poikilotherms, it is not
the only one; predation risk or exposure to natural and synthetic xenobiotic chemi-
cals can be as significant as temperature (for a review, see Steinberg (2012)). These
challenges may work individually or in concert, and thus change the energy alloca-
tion between body maintenance (growth, repair, or longevity) and reproduction.
Some issues are not yet sufficiently understood and deserve empirical studies to
come. This applies to the costs of DR and CG, as well the role of ghrelin and leptin.
In the case of the costs of CG, only a few and even contradictory reports exist.
Whereas most studies show adverse effects of CR and CG on immunity traits in
fishes, a few did not find any and one laboratory continues to present evidence that
fasting increases the resistance to pathogens. In total, the cost issue is not adequately
covered, probably because the productivity aspect dominates most of the aquacul-
ture studies.
Many, but not all, aquatic animals demonstrate CG. In a few cases, overcompen-
satory growth (Lepomis hybrids, Haliotis midae) or absence of CG (A. tonsa,
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Fig. 4.67 Hypothetic transgenerational inheritance due to dietary restrictions via epigenetics.
(modified from Shaw et al. (2017), with permission from the American Chemical Society)
common carp, gilthead sea bream) have been observed. Partial and full CG were the
most frequent response to DR in fishes and aquatic invertebrates. Furthermore,
intermittent feeding resulted in overcompensation in only a few cases. Together,
these studies indicate that the compensatory response is induced by internal physi-
ological factors, rather than dietary inducers. However, the question remains
whether transgenerational effects of CG are even feasible. Do the offspring of fasted
and refed parents successfully compete with offspring of permantly well-fed par-
ents? The seahorse example points out a transgenerational effect by which the sur-
vival rate of juveniles increased if the parental generation had been diet-restricted.
Can acquired resistance to starvation and more general stress be passed to subse-
quent generations, most likely by epigenetic mechanisms as hypothesized in
Fig. 4.67? If so, the broodstock can intentionally be improved, instead of using the
more stochastic genetic approach.
In addition to the seahorse example, a few more encouraging studies do exist
with results that directly or indirectly support the assumption, such as heat stress-
resistance in Artemia (Norouzitallab et al. 2014), increased progeny resistance to
heat and starvation stresses from starved parental C. elegans (Jobson et al. 2015), or
fitness advantage when mothers and their progeny experience DR also in C. elegans
(Hibshman et al. 2016)
Aquaculture should be prepared to be fertilized by these intriguing studies.
References 255
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Ye JD, Chen JC, Wang K (2016) Growth performance and body composition in response to dietary
protein and lipid levels in Nile tilapia (Oreochromis niloticus Linnaeus, 1758) subjected to
normal and temporally restricted feeding regimes. J Appl Ichthyol 32(2):332–338. https://doi.
org/10.1111/jai.13004
Yebra L, Berdalet E, Almeda R, Pérez V, Calbet A, Saiz E (2011) Protein and nucleic acid
metabolism as proxies for growth and fitness of Oithona davisae (Copepoda, Cyclopoida)
early developmental stages. J Exp Mar Biol Ecol 406(1–2):87–94. https://doi.org/10.1016/j.
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Yengkokpam S, Sahu NP, Pal AK, Debnath D, Kumar S, Jain KK (2014) Compensatory growth,
feed intake and body composition of Labeo rohita fingerlings following feed deprivation.
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Yengkokpam S, Debnath D, Sahu NP, Pal AK, Jain KK, Baruah K (2016) Dietary protein enhances
non-specific immunity, anti-oxidative capability and resistance to Aeromonas hydrophila in
Labeo rohita fingerlings pre-exposed to short feed deprivation stress. Fish Shellfish Immunol
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Yokota T, Nakagawa T, Murakami N, Chimura M, Tanaka H, Yamashita Y, Funamoto T (2016)
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ment of first-time spawning male Atlantic cod, Gadus morhua. ICES J Mar Sci 62(7):1387–
1393. https://doi.org/10.1016/j.icesjms.2005.04.018
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Yoshinaga T, Hagiwara A, Tsukamoto K (2001) Effect of periodical starvation on the sur-
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271. https://doi.org/10.1016/S0022-0981(02)00574-9
Yuan D, Wang T, Zhou C, Lin F, Chen H, Wu H, Wei R, Xin Z, Li Z (2014) Leptin and cholecys-
tokinin in Schizothorax prenanti: molecular cloning, tissue expression, and mRNA expression
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timing of initial feeding on growth and survival of spotted mandarin fish Siniperca scherzeri
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Zhang C, Li Z, Li F, Xiang J (2015) Effects of starvation on survival, growth and development
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https://doi.org/10.1111/are.12490
Chapter 5
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Chrononutrition – ‘The Clock Makes Good

Food’
Abstract As all organisms on Earth, also fishes and aquatic invertebrates are sub-
ject to circadian rhythms triggered by external zeitgebers and controlled by gene
transcription; all life history traits change in a circadian manner. This rhythmicity
applies to digestive enzyme activity, regulated by endogenous systems, and which
can be measured even under fasting conditions. Contrary to mammals, teleost fishes
appear not to have a master clock. In aquatic invertebrates, the circadian control is
less well understood than in fish. In both animal groups, transcription of metabolic
and, particularly, also biotransformation genes show clear circadian rhythmicity.
The latter determine the toxicity of natural and synthetic xenobiotic chemicals.
Therefore, one can predict that several of the so-called antinutrional dietary com-
pounds may lose their ‘anti’-character, if the farmed animals are fed during the
acrophase of biotransformation genes transcription.
Organisms that live on the Earth are subject to environmental variables that display
cyclic variations, such as light, temperature, and tides. Since these cyclic changes in
the environment are constant and predictable, they have affected biological evolu-
tion through selecting the occurrence of biological rhythms in the physiology of all
living organisms, from prokaryotes to mammals. Biological clocks confer on organ-
isms an adaptive advantage, as they can synchronize their behavioral and physiolog-
ical processes to occur at a given moment in time, when the effectiveness and
success would be greater and the cost and risk for the organisms would be lower.
Among environmental synchronizers, light has been the mostly widely studied to
date. However, also other environmental signals, such as temperature, tides, oxygen
content, food availability, or predation, play an important role in biological rhythms,
especially in aquatic animals (López-Olmeda 2017). Anticipating these environ-
mental changes allows organisms to adjust all of their metabolic and behavioral
processes in advance and to do everything ‘on time’.
Hence, biological clocks have evolved, and even in the absence of any environ-
mental clue, they autonomously oscillate with a circadian (circa = about, dia = day)
period. When food delivery is restricted to the same time every day, fish, like other
animals, display food-anticipatory activity under a light–dark cycle. Even when
maintained under constant light conditions, the animals can rapidly synchronize

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290 5 Chrononutrition – ‘The Clock Makes Good Food’
their activity pattern to the restricted food availability (Zhdanova and Reebs 2005;
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Naruse and Oishi 1994). For instance, when Pacific white shrimps (L. vannamei)
operated a self-feeding system, they displayed nocturnal feeding and locomotor
rhythms, with activity peaking after lights off and decreasing at the end of the dark
phase. These rhythms persisted under constant dark conditions and were driven by
an endogenous clock (Santos et al. 2016).
With respect to fish nutrition, already more than two decades ago, Sugita et al.
(1990) had presented clear evidence of daily, and most likely circadian, fluctuations
of the fecal flora of the common carp. Genus Aeromonas was detected predomi-
nantly in all the fecal samples of five fish specimens, and Enterobacteriaceae,
Pseudomonas, Bacteroides type A and other Bacteroidaceae occurred abundantly in
88–96% of fecal samples. These results indicate that those five bacteria were indig-
enous organisms in the carp’s intestines. However, those bacteria fluctuated daily in
the same fish specimen, although no special tendencies were recognized (Fig. 5.1).
Body maintenance in general, and growth, body repair, reproduction, or death, in
particular: all life history traits are depending on time. In these processes, biological
clocks are central to a proper functioning of organisms, and in synchronizing them
with their environment. Also, food uptake is subject to the internal clock. The syn-
chronization of biological processes with the environment takes place on various
time scales, from seconds to years and even decades. Referring to food and feeding
behavior, the most interesting and best-studied rhythm is the circadian rhythm. In
this respect, teleosts represent one of the most successful groups of vertebrates, with
a wide range of adaptations to a great variety of even contrasting habitats. For this
reason, fishes provide insight into the circadian clocks and how they adapt to vari-
ous environmental conditions during evolution.
Aeromonas
Enterobactericeae
Pseudomonas
Bacteroides type A
Bacteroidaceae
Others
2 4 6 8 10
Bacterial no. (Log CFU g-1)
Fig. 5.1 Daily variation of the predominant fecal flora in the intestinal tract of common carp. Each
pack of bars shows from above to below day 1 (white) to day 5 (black); CFU colony forming units.
(From Sugita et al. 1990, with permission from Wiley)
5 Chrononutrition – ‘The Clock Makes Good Food’ 291
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Fig. 5.2 Diurnal activity of digestive enzymes in Nile tilapia: pepsin-like, alkaline proteases, tryp-
sin, chymotrypsin, amylases, and lipases. Black bars represent basal conditions (fasting fish) with
solid line (P < 0.05); marbled bars with dashed lines represent ad libitum feeding conditions
(P < 0.05); Black sections in the bar at the bottom of the figure represent night time; Values are
mean ± SE. (From Montoya-Mejía et al. 2016, courtesy of the West Pomeranian University of
Technology in Szczecin)
The circadian rhythm of the digestion will be exemplified in a fish (Nile tilapia)
and an invertebrate (Macrobrachium tenellum) species. Montoya-Mejía et al. (2016)
showed that these fishes have circadian natural rhythms for digestive enzyme activ-
ity, regulated by endogenous systems, and which can be measured even under fast-
ing conditions (Fig. 5.2). In particular, in juvenile Nile tilapia the circadian cycle of
concentrations of total soluble protein, protease, pepsin-like, trypsin, chymotrypsin,
amylase, and lipase were determined. The baseline (fasting) and feeding conditions
(ad libitum, AL) were sampled every hour for 24 h. The basal peak of enzyme activ-
ity in the intestine occurred at 18:44 h for amylase, at 19:57 h for proteases, and
20:29 for trypsin. The minimal activity for most enzymes, appeared between 4:51 h
(amylase) and 10:13 h (lipases). In the AL feeding treatment, stomach activity

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(pepsin-like) had maximal activity at 20:06 h and minimal activity 05:46 h. Intestinal
amylase activity covered an extended period of low enzymatic activity beginning at
05:46 h and ending at 12:59 h. The peak activity of the digestive enzymes occurred
within 18:44–20:29 h. In general, secretion of digestive enzymes was positively
stimulated by food, for all enzymes assayed with Nile tilapia having a higher diges-
tive enzyme activity at night than during the day. Doubtless, knowledge of the cir-
cadian cycle of digestive enzymes, and modifications initiated by food, is useful to
establish feeding times. If feeding schedules are adjusted to coincide with maxi-
mum natural peaks, food efficiency will increase, which will be reflected in weight
gain of the fish and provide more profitable yields for aquaculture.
The circadian cycle of digestive enzymatic activity of the general proteases,
lipases, and amylases in the juvenile prawn Macrobrachium tenellum is biphasic at
08:00 and 20:00 h, with a fluctuation of less activity throughout the day (Fig. 5.3),
Fig. 5.3 Left: Basal specific digestive enzymatic activity of lipases, amylases, general proteases,
chymotrypsin (×10−2), and trypsin (×10−2) in Macrobrachium tenellum. The vertical lines at each
point show the standard deviation. The vertical lines on the chart indicate schedules 08:00, 12:00,
and 20:00 h used as references. (From Espinosa-Chaurand et al. 2017, with permission from
Elsevier). Right: Male M. tenellum. (From Espinosa-Chaurand et al. 2011, courtesy of the
Universidad Autónoma Metropolitana, Unidad Iztapalapa)
5.1 How Does a Biological Clock Work? 293
which can be affected by the alteration of the natural photoperiod, modifying

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enzyme concentrations and basal peaks (Espinosa-Chaurand et al. 2017). The
biphasic behavior of digestive enzymatic activity, at 08:00 and 20:00 h, relates to
the ecological activity of this species with feeding habits during the early hours of
the day and night.
Biphasic circadian cycles have been described in several crustacean species:
Palaemon serratus, Penaeus notalis, P. californiensis, Callinectes arcuatus,
Litopenaeus schmitti (Espinosa-Chaurand et al. (2017) and references therein) and
appear to be the rule.
In contrast to mammals, the circadian system of fish shows impressive flexibility,
as the same fish species can exhibit diurnal or nocturnal behavior, and shift from one
type of phasing behavior to another depending on the season or ontogenetic stage
(Idda et al. 2012). In addition, Herrero and Lepesant (2014) reported that, in the
European sea bass (D. labrax), even temperature profoundly impacts the circadian
gene transcription pattern. The authors showed that the winter/spring destabiliza-
tion of daily rhythmic patterns can be linked to cold water temperatures. Such find-
ings may have a potential application to the control of reproduction in aquaculture.
Melatonin may function as a mediator of temporal cues in the pituitary acting on the
expression of the genes of the cryptochrome family (cry) (explanation below). Thus,
rhythms in clock genes in the pituitary of sea bass seem to be the result of a multi-
component system involving several elements, including temperature, photoperiod
and signaling molecules such as melatonin.
The hormone melatonin is also a key regulatory molecule in invertebrates, such
as Daphnia. The rate-limiting enzyme in melatonin synthesis is the arylalkylamine
N-transferase (AANAT). Schwarzenberger and Wacker (2014) identified three
genes coding for insect-like AANATs in Daphnia, of which the authors measured
the gene expression in an ecologically relevant light–dark cycle. They demonstrated
that Daphnia’s insect-like aanat transcription oscillated in a daily manner, and that
the highest peak of expression after the onset of darkness was followed by a peak of
melatonin production at midnight. In most organisms, melatonin synthesis is due to
rhythmic expression of genes of the circadian clock, since transcription of aanats is
directly linked to a circadian transcription factor. In Daphnia, melatonin synthesis
is obviously coupled to the expression of clock genes, and that insect-like aanats of
crustaceans have a similar function as aanats of vertebrates: the initiation of mela-
tonin synthesis.
5.1 How Does a Biological Clock Work?
Conceivably, every cell in the body can contain an intrinsic clock mechanism.
Although some cells may initiate oscillations only in response to specific internal or
external factors, others constantly express this function. The continuously oscillat-
ing cells and structures specializing in providing circadian signals to the entire
organism are called central oscillators. The most renowned of them is the
suprachiasmatic nucleus (SCN) of the hypothalamus in mammals, a neuronal struc-

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ture defining the majority (if not all) of the circadian rhythms in this group. The
autonomous oscillations displayed by peripheral cells and tissues may remain inde-
pendent, or can synchronize with each other and with the central oscillators, orga-
nizing complex networks and affecting multiple physiological functions in a
species-specific manner (Zhdanova and Reebs 2005).
5.1.1 Fishes
Contrary to mammals, the existence of a central oscillator (master clock) in teleosts

has not been demonstrated to date, and therefore, the fish circadian system is con-
sidered to be less hierarchical than the mammalian one (Isorna et al. 2017); instead,
the fish circadian system is a net of circadian oscillators (Fig. 5.4).
The presence of clock genes in a variety of tissues from different species sup-
ports the existence of an extended net of oscillators in teleost central and peripheral
locations. Clock genes have been found in zebrafish and goldfish and in a variety of
tissues of other fish, including the retina, brain, hypothalamus, pituitary, liver, head
kidney, skin, gut and gonads (Sánchez-Bretaño et al. 2015). In their comprehensive
review, Zhdanova and Reebs (2005) listed many markers of self-sustained circadian
rhythms in a wide variety of fish species, from food uptake, body color changes,
over electric discharge, locomotion, and melatonin production, to courting and
parental behavior. In fact, all life history traits change in a circadian manner.
The molecular core of these biological clocks is based on interlocked transcrip-
tional and post-translational auto-regulatory feedback loops of a set of genes called
clock genes. In general, the clock regulation of transcription relies on the rhythmic
regulation of chromatin accessibility (Menet et al. 2014). In mammals, this tran-
scription regulation leads to rhythmic expression of approximately 10% of all genes.
In addition to such transcriptional regulation of the circadian clock, posttranscrip-
tional and translational regulations have been reported to serve important roles in
maintaining circadian rhythms (Tahara and Shibata 2013).
Most studied are the biological clocks in zebrafish and to a lesser degree also
goldfish and cave fishes (Cavallari et al. 2011; Beale et al. 2013) (Fig. 5.7). Since
circadian clocks influence most aspects of physiology and behavior, it is not surpris-
ing that circadian oscillators exist in nearly all fish cells. In zebrafish, peripheral
clocks are directly triggered by light – similar to the situation in Drosophila (Idda
et al. 2012). However, central to the translation of light into biomolecular, biochem-
ical, physiological, and behavioral reactions is the pineal organ (also known as the
pineal body, conarium, or epiphysis cerebri) with the induction of certain clock
genes (cryptochrome and period elements). The two genes cry1 and cry2 code for
the cryptochrome proteins CRY1 and CRY2, two flavoproteins sensitive to blue
light. Period genes per1 and per2 (both with several homologs) code for the period
proteins PER1 and PER2. Oscillations in transcript levels and corresponding pro-
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Fig. 5.4 The fish circadian system: a net of circadian oscillators. The fish circadian system is
composed of a net of oscillators that are widely distributed throughout the entire organism. These
oscillators are entrained by external inputs, such as the light–darkness and feeding–fasting cycles
and should be linked to generate outputs (such as locomotor activity and metabolic rhythms) in a
coordinated manner. The retina, pineal gland and probably some deep brain photoreceptors are
directly targeted by light, which then entrains the endogenous clocks in such structures (shown
in blue). Other organs that contain circadian clocks, such as the gut and liver, are probably targeted
by any feeding- or metabolic-related signals, which mainly synchronise these oscillators to the
energetic status of the animal (shown in green). The head kidney is probably entrained by both
external signals (shown in purple). These endocrine organs (pineal gland, pituitary gland, gut, liver
and head kidney) release hormones (melatonin, pituitary hormones, ghrelin, leptin and cortisol) in
a time-dependent pattern, which may provide a temporal message to specific-hormone receptors.
This diagram only shows the most studied endocrine organs that are functionally related to the
circadian system; however, other oscillators also probably exist. The continuous lines indicate the
connections that are currently known to exist in fishes, whereas the dashed lines illustrate hypo-
thetical connections that have not yet been reported. ENC, other encephalic nuclei; HT, hypothala-
mus; PIT, pituitary gland. (From Isorna et al. (2017), with permission from the Society for
Endocrinology)
teins have a period of approximately 24 h (= circadian). Cryptochromes act as light-

independent and period proteins as light-dependent inhibitors of CLOCK-BMAL1
proteins (transcription factors), two master genes/proteins of the circadian clock.
CLOCK-BMAL1 acts only in its heterodimeric form and rhythmically activates the
expression of their transcriptional repressors PER and CRY through a specific pro-
moter sequence (‘e-box’). Rhythmic CLOCK:BMAL1 binding to the DNA pro-
motes rhythmic chromatin opening (Fig. 5.5) (Menet et al. 2014).
In the European sea bass, del Pozo et al. (2012b) found that the daily expression
of cry1 was rhythmic in brain, heart and liver with the acrophase around zeitgeber
time (ZT) 03:15 h (after the onset of light). Similarly, the daily expression of cry2
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Fig. 5.5 Illustration of the mechanism by which CLOCK:BMAL1 regulates the expression of its
target genes. E-box (enhancer box) is a DNA sequence found in some promoter regions in eukary-
otes that acts as a protein-binding site. CLOCK:BMAL1 functions in a similar way to pioneer
transcription factors and regulates the DNA accessibility of other transcription factors (Y, Z).
Contrary to the permanent chromatin opening associated with lineage commitment however,
CLOCK:BMAL1-mediated chromatin opening is dynamic and occurs every day. (From Menet
et al. 2014, courtesy of the Cold Spring Harbor Laboratory Press)
Cry1 Cry2
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3 2
Log (fold change)
Log (fold change)

2.5 1.8
1.6
2 1.4
1.2
BRAIN 1.5 BRAIN 1
1 0.8
0.6
0.5 0.4
0.2
0 0
3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0
Zeitgeber Time Zeitgeber Time
2.5 1.6
Log (fold change)
Log (fold change)

1.4
2 1.2
1.5 1
HEART HEART 0.8
1 0.6
0.5 0.4
0.2
0 0
3 6 9 12 15 18 21 0 3 6 9 12 15 18 21 0
3 1.6
Log (fold change)
2.5 Log (fold change) 1.4

1.2
2 1
LIVER 1.5 LIVER 0.8
1 0.6
0.4
0.5 0.2
0 0
3 6 9 12 15 18 21 0 1 3 6 9 12 15 18 21 0
Fig. 5.6 Relative expression of cry1 and cry2 in sea bass brain, heart, and liver. Every point rep-
resents the mean cry expression of 3–4 fish (except at ZT 00:00 h and 09:00 h in liver of one
sample) and the error bars show the standard error about the mean (SEM). The black and white
bars above the graphs show the dark and light phases, respectively. The zeitgeber time (ZT, in
hours) is represented on the horizontal axis, while the relative expression as fold change (log10) is
plotted on the vertical axis. * indicates the time point which differed by ANOVA (P < 0.01). The
dotted line denotes the Cosinor(Cosinor analysis is used in the analysis of biologic time series that
demonstrate predictible rhythms. It uses the least squares method to fit a sine wave to a time series
and method is applicable to non-equidistant data (Cornelissen 2014)) adjustment (P < 0.01). (From
del Pozo et al. 2012b, with permission from Elsevier)
was rhythmic in the liver, peaking at ZT 03:28 h, whereas in brain, the acrophase
was at ZT 11:08 h (shortly prior to the extinguishing of light) (Fig. 5.6). The maxi-
mum expression peak of period 1 gene (per1) in several tissues (brain, retina, liver
and gut) has been observed close to light onset in several species (data not shown).
Overall, sea bass per1, cry1, and cry2 expressions in all tissues, where their rhyth-
micity has been reported (except cry2 in the brain), were in phase, and so the pro-
teins encoded by these genes (PER1, CRY1 and CRY2) could join together and
form the Per-Cry complex, which, in turn, would inhibit the cry transcription, clos-
ing the negative loop of the molecular mechanism that directs the circadian clock.
Besides the clock genes, a systematic microarray analysis showed that approxi-
mately 100 genes were significantly upregulated upon light exposure. These genes
belong to diverse functional groups (Idda et al. 2012). A study of the blind cavefish,
Phreatichthys andruzzii (Fig. 5.7), revealed that even fish excluded from the light
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Fig. 5.7 Fish species subject to intensive clock gene studies. Phreatichthys andruzzii, Danio rerio,
and cultivated goldfish breed, Carassius auratus. (Courtesy of the Public Library of Science;
©CEW Steinberg)
trigger for millions of years do retain a clock, albeit an abnormal one that is no lon-
ger entrained by light but can still be triggered by the feeding rhythm (Cavallari
et al. 2011). When studying the blind form (cavefish) of the Mexican tetra (Astyanax
mexicanus), Beale et al. (2013) showed that two major triggers can entrain the
biological clock: light plus food availability. As we shall see below, these two trig-
gers act in concert and even mutually reinforce each other.
In goldfish, the daily light–dark cycle and feeding schedule are able to synchro-
nize two classic outputs (overt rhythms) of the circadian system: the daily locomo-
tor activity and food anticipatory activity (FAA) rhythms. These two synchronizers
also drive clock gene rhythms in the liver of this teleost. When only one signal (LD
cycle or feeding time) is present, per1a rhythms in the liver keep the circadian pat-
tern, but their amplitudes decrease (Fig. 5.8), indicating that both environmental
signals work together in sustaining the molecular clockwork in the liver. The ampli-
tude of the per1a rhythm is higher in the hepatic oscillator than in the brain, indicat-
ing that the liver is highly sensitive to the feeding schedule in goldfish
(Sánchez-Bretaño et al. 2015) (Fig. 5.9).
A more detailed picture of appetite regulation is displayed in Fig. 5.10, showing
the central appetite regulators in fish and interrelations between them. The influence
of environment and peripheral regulators is only cursorily sketched. Kulczykowska
and Sánchez Vázquez (2010) summarized that regulation of food intake involves
interaction among the circadian and homeostatic control systems in the central ner-
vous system, the gastrointestinal tract, and the environment. The hypothalamus that
receives, integrates, and transmits relevant internal and external signals, is recog-
nized as the primary center of regulation of food intake. The neuroendocrine factors
that originate from the hypothalamus either stimulate or inhibit food intake, so that
nutritional demands of the organism can be fulfilled and energy balance can be
achieved. Appetite regulation is a physiological mechanism in which a variety of
neurohormones and neuropeptides interact (Fig. 5.10). This complex system is very
sensitive to any disturbance. Fish in farms and fish in a natural environment are
equipped with the same combination of neurohormones to regulate food intake, but
Fig. 5.8 Daily rhythms of

gper1a relative expression
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in the liver of goldfish

under different
environmental conditions.
(a) Goldfish maintained
under 12L:12D and
scheduled-fed at ZT 2. (b)
Goldfish maintained under
12L:12D and randomly fed
(RF). (c) Goldfish
maintained under LL and
scheduled-fed at ZT 2; ZT
Zeitgeber time. (From
Sánchez-Bretaño et al.
2015, with permission
from Elsevier)
they meet different challenges, particularly with regard to the type of food and the
feeding schedule.
The circadian system of fish is composed of a central pacemaker within the brain
and at least two peripheral oscillators located in the retina and the pineal organ. The
pineal organ in fish can act as one of the several circadian pacemakers in a circadian
system. In rainbow trout, for instance, the pineal organ does not seem to be a central
pacemaker that controls the feeding rhythm, because the removal of the pineal organ
does not disrupt the daily feeding rhythm (Kulczykowska and Sánchez Vázquez
In the Senegalese sole (Solea senegalensis), Navarro-Guillén et al. (2017) pre-
sented further details of this regulation. Their study supports the existence of a regu-
latory loop between cholecystokinin (CCK) and trypsic activity in pre- and
post-metamorphic Senegal sole larvae. This assumption is based in the simultane-
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Fig. 5.9 Regulation of the goldfish hepatic oscillator by photic and non-photic cues. As an ele-
ment of a complex circadian net, the liver of the goldfish receives inputs, possesses the molecular
clockwork machinery and probably drives outputs or overt rhythms. The daily rhythmic expression
of gPer1a in this organ is driven by both environmental zeitgebers, light–dark cycle and feeding
time. Abundance of per genes in the liver can be also modulated by ghrelin and glucocorticoids,
showing that these hormones could be involved in the cross talking among different oscillators.
Orexin seems to induce per expression in the brain and in the gut, but not in the liver. Continuous
black lines indicate the hormones that modify clock gene expression (from per family) in the cor-
responding tissue. (From Sánchez-Bretaño et al. 2015, with permission from Elsevier)
ous opposite trends (increasing vs. decreasing as a function of the postprandial time)
of these two digestive products, rather than on their absolute values. Additionally,
CCK level was also modulated by ingestion activity, tending to be lower when lar-
vae were being fed and higher when food was not available. Furthermore, larvae
were able to synchronize digestive functions to very different feeding regimes,
although it seems to be important to have a diurnal feeding phase during pre-
metamorphic stages for proper development.
Vera et al. (2007) presented evidence of feeding entrainment of locomotor activ-
ity rhythms, digestive enzymes and neuroendocrine factors in the goldfish. The
authors observed that periodically fed goldfish showed FAA in locomotor activity
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Fig. 5.10 Appetite regulators in fish brain. Orexigenic factors orexigenic (= appetite stimulating:
black letters on white): NE (norepinephrine), NPY (neuropeptide Y), orexins, galanin, AgRP
(agouti-related protein) and ghrelin. Anorexogenic factors (= appetite suppressing: white letters on
black): CCK/gastrin (cholecystokinin–gastrin), CART (cocaine and amphetamine-regulated tran-
script), MCH (melanin-concentrating hormone), MSH (melanocyte-stimulating hormone), tachy-
kinins, melatonin (Mel), DA (dopamine) and 5-HT (serotonin). Arrows: interactions between
regulators and the influence of the environment and peripheral factors. Dotted lines: interactions
via the circulatory system. (From Kulczykowska and Sánchez Vázquez 2010, with permission
from Wiley)
as well as in amylase and NPY: amylase secretion was synchronized by periodic

food delivery and hypothalamic NPY production anticipated the feeding time.
Alkaline protease and melatonin in the gastrointestinal tract were higher after feed-
ing, whereas plasma cortisol levels were reduced. Plasma melatonin remained
unmodified before and after mealtime. These results show that scheduled feeding
entrained both behavioral and certain physiological patterns in goldfish, FAA being
of adaptive value to anticipate a meal and to prepare the digestive physiology of fish.
The anticipation is controlled endogenously because it still persists after 2 days of
fasting. In goldfish maintained on a daily scheduled feeding regime, an increase in
the npy mRNA level in the telencephalon-preoptic region and in the hypothalamus
shortly before feeding has been demonstrated (Narnaware et al. 2000).
Later, Vera et al. (2013) refined their previous findings. Regardless of the meal-
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time, the daily rhythm of clock gene expression in the brain peaked close to the
light–dark transition in the case of bmal1 and clock (both form the positive loop of
the molecular clock), and at the beginning of the light phase in the case of per2 and
cry1 (both form the negative loop of the molecular clock), showing the existence of
phase delay between the positive and negative elements of the molecular clock. In
the liver, however, the acrophases of the daily rhythms differed depending on the
feeding regime. This study shows that the sea bream clock gene expression is
endogenously controlled and in the liver it is strongly entrained by food signals,
rather than by the light–dark cycle, and that scheduled feeding can shift the phase of
the daily rhythm of clock gene expression in a peripheral organ (liver) without
changing the phase of these rhythms in a central oscillator (brain), indicating uncou-
pling of the light-entrainable oscillator from the food-entrainable oscillator.
With variations, the sea bream example appears to be valid for the majority of
finfishes.
5.1.2 Invertebrates
Circadian rhythms in physiology and behavior have also been documented in

numerous crustacean species. Little, however, was known about the underlying
molecular or cellular machineries. Tilden et al. (2011) presented the first in silico
genomic identification of a putative circadian system in the cladoceran crustacean
Daphnia pulex. Bernatowicz et al. (2016) published an analysis of the transcription
of selected putative clock and clock-associated genes in D. pulex under experimen-
tal conditions. They analyzed the abundance of 20 gene transcripts throughout the
day in the whole bodies of D. pulex and found that 15 of these genes were transcrip-
tionally active, and most had daily expression level changes (Fig. 5.11 and Table 5.1).
According to the functional classification of their homologs in insects, these genes
may represent elements of the Daphnia molecular oscillator core and its input and
output pathways. Studies of PERIOD (PER) protein, one of the main clock compo-
nents, revealed its rhythmic expression pattern in the epidermis, gut, and ovaries.
Finally, the cycling levels of many of these clock components observed in animals
Fig. 5.11 (continued) ferred for 6 days to permanent darkness (DD) (black lines marked with cir-
cles) or continous light (LL) (light-gray lines marked with squares). qRT-PCR was performed
using clock-gene-specific primers, and expression was normalized to gapdh and Ef-1α as reference
genes. Samples from 10 to 12 individuals were collected every 4 h over the course of 24 h (from
Zt/Ct 0 to Zt/Ct 0 of the next day). Graphs represent efficiency-corrected, normalized, relative gene
expression values (±SEM) from three independent replicates for each time point (all values are
compared to the highest value received for the given gene, which is set to 100%). Points marked
on graphs by different letters denote values that differ significantly (P < 0.05), as calculated by
one-way ANOVA followed by Bonferroni’s post hoc test. The horizontal bars indicate the day
(white), night (black), and subjective day (shaded) phases, with times expressed as Zeitgeber time
(Zt) and circadian time (Ct). (From Bernatowicz et al. 2016, with permission from Wiley)
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Fig. 5.11 Temporal changes in transcript levels of the period (per) (a), timeless (tim (a, b and h)
(b–d), clock (clk) (e), cycle (cyc) (f), and doubletime (dbt) (g) genes in Daphnia pulex. Graphs
represent the expression levels determined by qRT-PCR using total RNA extracted from the whole
bodies of females held in light–dark cycle (LD) (dark gray lines marked with diamonds) or trans
Table 5.1 Clock genes in Daphnia pulex studied by Bernatowicz et al. (2016)
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Abbreviation Protein Putative function

clk Clock Transcription factor involved in the circadian rhythm (clock gene);
together with bmal(1,2…) the positive loop of the molecular clock
cyc Cycle Involved circadian regulation of gene expression by promoting
transcription in a negative feedback mechanism
dbt Doubletime Kinase that phosphorylates PER protein that regulates the
molecularly-driven, biological clock controlling circadian rhythm
per Period Period circadian regulators; with cry1 and tim the negative loop of
the molecular clock
tim a, b, h Timeless a, Essential proteins that regulate the circadian rhythm; part of a
b, h transcription-translation negative feedback loop involving the
period (per) and cry genes and their proteins
reared in continuous light led to the conclusion that the Daphnia oscillator, even if
it is structurally similar to the oscillators of other arthropods, can be considered a
particularly important adaptive mechanism for living in environments with extreme
photoperiods.
The experimental design involved keeping animals under three different light
regimens, thus allowing the identification of genes that are likely candidates for
constituting the endogenous (circadian) oscillator mechanism of Daphnia, as their
expression oscillates under constant darkness. Interestingly, Bernatowicz et al.
found that the expression of many of these genes varies over the day in animals liv-
ing under continuous light. This finding is consistent with known data on the cycli-
cal behavior of Daphnia (such as diel vertical migration (DVM), Fig. 5.12) under
continuous light. Many marine and freshwater zooplankter carry out DVM which
appears to be triggered by endogenous rhythm machines (Cohen and Forward Jr.
2009; Brierley 2014; Gaten et al. 2008).
In contrast to the study of Bernatowicz et al. (2016), Rund et al. (2016) did not
detect 24 h sinusoidal rhythmic expressions of any of the putative canonical clock
genes [clk, cyc, per, par domain protein 1ε, vrille, four of the eight tim paralogs (a
− h), cry1, cry2, or pigment dispersing hormone (a neuronal clock output gene)]
(Fig. 5.13). However, genes in many functional groupings exhibited 24 h rhythms in
their expression patterns under diel conditions and highlighted the rhythmic expres-
sion of immunity, oxidative detoxification, and sensory process genes. This appar-
ent contradiction may be due to the use of different clones of D. pulex. It is also
feasible that D. pulex has an alternative, noncanonical, core molecular clock that
operates differently from well-characterized insect clocks. In fact, under constant
light conditions, D. magna has been reported to have an unusually long 28 h free-
running period (Ringelberg and Servaas 1971; Harris 1963). Alternative clock
mechanisms have been proposed in a number of invertebrates including the nema-
tode, C. elegans (van der Linden et al. 2010), the sea squirt (Ciona intestinalis)
(Minamoto et al. 2010), and in other crustaceans such as the speckled sea louse
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Fig. 5.12 Twenty-four hour rhythmic changes in Daphnia’s environment. Daphnia are exposed to
different environmental conditions and stressors as the 24 h day progresses. This variation is a
consequence of the daily rising and setting of the sun, the chronobiology of other organisms in the
environment, and Daphnia’s pattern of diel vertical migration (DVM) through the water column.
The sun brings changes in temperature, ambient light, UV radiation, and increased risk of fish
predation (which locate Daphnia visually) in populations found in large bodies of water. Similarly,
as Daphnids move down the water column they are exposed to decreasing ambient light of chang-
ing wavelengths, and reduced UV radiation, temperature, and oxygen levels. Some parasites of
Daphnia live in the sediment at the bottom of water bodies, so risk of being parasitized is increased
at times of day that the Daphnia are lower in the water column (daytime in populations from large
water bodies). (Rund et al. 2016, courtesy of BioMed Central Ltd.)
(Eurydice pulchra) (Zhang et al. 2013), and the giant river prawn (Macrobrachium
rosenbergii) (Yang et al. 2006).1
In addition to Daphnia, Christie et al. (2013) documented clock elements in
Calanus finmarchicus, and Nesbit and Christie (2014) in copepods of the genus
Tigriopus. Teschke et al. (2011) proved the existence of a circadian clock in the
Antarctic krill (Euphausia superba). More importantly, these authors showed that
the expression of the canonical clock gene cry2 was highly rhythmic both in a light–
dark cycle and in constant darkness, indicating that it is an endogenous circadian
timing system (Fig. 5.14).
Subsequently, De Pittà et al. (2013) showed that the Antarctic krill has evolved
rhythmic physiological and behavioral mechanisms to adapt not only to daily, but
also to seasonal, changes. Furthermore, the authors presented the first insight into
the genetic regulation of physiological changes that occur around the clock during
an Antarctic summer day under natural conditions (Fig. 5.15).
The Norway lobster (Nephrops norvegicus), is a burrowing decapod with a rhyth-
mic burrow emergence (24 h) governed by the circadian system. The current knowl-
edge of Nephrops circadian biology is phenomenological, as is the case for almost
all crustaceans. In an attempt to elucidate the putative molecular mechanisms under-
lying circadian gene regulation in Nephrops, Sbragaglia et al. (2015) used a tran-
scriptomics approach on cDNA extracted from the eyestalk, a structure playing a
1
References taken from Rund et al. (2016)
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Fig. 5.13 Clock gene expression in Daphnia pulex. Day and night are indicated by the horizontal
white/black bars. Expression is presented as raw fluorescence value. Error bars represent SEM of
technical replicates. (From Rund et al. 2016, courtesy of BioMed Central Ltd.)
Fig. 5.14 Transcript levels of cry2 in heads of the Antarctic krill (Euphausia superba) were mea-
sured by quantitative PCR from two independent time course experiments, in 2008: full 24 h cycle
under (a) light–dark (LD) conditions and (c) at the third consecutive day in constant darkness
(DD), and 2010: full 48 h cycle at the first and second consecutive day in DD (b). (From Teschke
et al. 2011, courtesy of the Public Library of Science)
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Fig. 5.15 Oscillatory patterns of differentially expressed genes in Antarctic krill involved in ener-
getic and metabolic processes. (a) Schematic representation of the daily distribution of metabolic
processes resulting from the transcriptional signature of several differentially expressed genes
throughout the 24-hour cycle. The different metabolic processes are indicated by gradiently colored
arrows showing the time of the day corresponding to the higher expression levels of gene groups.
The lengths of the arrows and darker colors indicate intervals and peaks of expression, respectively.
Local times are indicated at the bottom of the figure, where an indicative representation of light
intensity is also shown. The breakdown of energy-yielding nutrients (glycolysis, the Krebs cycle
and the electron-transport chain) and energy storage pathways (glycogen synthesis and fatty-acid
synthesis) are specifically activated in the early morning, while glycogen mobilization, gluconeo-
genesis and fatty-acid catabolism are used as stored energy sources in the evening and throughout
the night. (b) Gene transcription involved in energetic and metabolic processes are represented. The
color of each gene corresponds to the metabolic process in which it is involved. Dashed lines indi-
cate differentially expressed genes identified by a one-way ANOVA test, while the solid lines rep-
resent differentially expressed genes characterized by a sinusoidal expression pattern detected by
CircWaveBatch analysis. Microarray expression values are represented as log2 mean ± standard
deviation. Genes are represented with the following abbreviations: fructose 1,6-bisphosphatase
(FB), glyceraldehyde-3-phosphate dehydrogenase (GPD), pyruvate kinase (PK), 6-phosphofructo-
kinase (6P), citrate synthase (CS), ATP synthase b (ATP), succinate dehydrogenase type C (SD),
isocitrate dehydrogenase [NAD] subunit beta mitochondrial (ID), acetyl-CoA acetyltransferase
(ACA), short-chain specific acyl-CoA dehydrogenase (ACD), acetyl-CoA carboxylase (ACC), ATP
citrate lyase subunit beta (ACL), glycogen debranching enzyme-like (GDE), glycogen synthase
(GS), and glycogenin-1 (G). (From De Pittà et al. 2013, courtesy of the Public Library of Science)
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Fig. 5.16 Canonical clock gene expressions in Nephrops eyestalk. Measurements were normal-
ized to α-act and 18S and expressed as fold change with respect to a control time point (7,30).
Vertical bars represent the confidence limits. Black and white bars represent dark and light hours,
respectively. Timeless shows a significant expression and period tends to have expression
(P < 0.05). Letters indicate the output of the Tukey’s post hoc test (a > b). (From Sbragaglia et al.
2015, courtesy of the Public Library of Science)
crucial role in controlling behavior of decapods. They found a list of candidate clock
genes and focused on canonical ones: timeless, period, clock, and bmal1. The puta-
tive Nephrops clock genes showed high levels of identity with known crustacean
clock gene homologs. The authors also found a vertebrate-like cryptochrome 2
(cry2). Only timeless had a robust and period tended to have a diel pattern of expres-
sion, indicating that the molecular clockwork of crustaceans shows some differ-
ences from the established model in Drosophila melanogaster (Fig. 5.16).
5.2 Food and Circadian Gene Transcription
Besides major nutrients, all feeds contain less well digestible, or even so-called
antinutritional, compounds, which induce digestive or biotransformation gene
transcription.
5.2.1 Major Nutrients
Almost all papers describing the circadian regulation of food digestion in aquatic
animals study degrading enzymes of the macronutrients: proteins, carbohydrates,
and lipids. For instance, Felip et al. (2015) showed in a fishmeal replacement trial
5.2 Food and Circadian Gene Transcription 309
that carbohydrates supplied to sea bream in the afternoon were not used as effi-
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ciently as those supplied in the morning. Nevertheless, they demonstrate a clear

circadian rhythmicity of food digestion.
It is well understood that seasonal and circadian changes in the environment can
influence not only feeding behavior, but also the dietary selection in fish. In goldfish,
in which feeding behavior is not confined to the light or the dark phase, there are
strong timing preferences for macronutrients: carbohydrate during the light phase,
protein during the dark phase and fat in the transition phase, which shows that light
is a crucial external regulator of feeding preferences in fish (Kulczykowska and
Sánchez Vázquez (2010) and references therein).
In his review, Soengas (2014) pointed out major diet constituents in the regula-
tion of food intake in fish. In recent years, evidence has been obtained in several
fishes, mainly in rainbow trout, regarding the presence and functioning in brain
areas of metabolic sensors informing about changes in the levels of nutrients such
as glucose and fatty acids. The activity of these sensors relate to the control of food
intake, through changes in the expression of anorexigenic and orexigenic neuropep-
tides. In contrast to lipid or carbohydrate metabolism, very few studies have inves-
tigated the regulation of target gene expression or activity of corresponding enzymes
in relation to essential micronutrients such as vitamins, minerals or trace elements
(Panserat and Kaushik 2010). Actually, almost no paper has considered, or even
mentioned, natural xenobiotics and the corresponding biotransformation enzymes;
however, the increased longevity of Nothobranchius treated with resveratrol clearly
demonstrates the beneficial effect of natural xenobiotics.
In zebrafish, light-induced gene transcription identified 117 light-regulated
genes, with the majority being induced, but some repressed, by light (Weger et al.
2011). Cluster analysis grouped the genes into five major classes that showed regu-
lation at all levels of the organization. The regulated genes covered a variety of
functions, and the analysis of gene ontology categories revealed an enrichment of
genes involved in circadian rhythms, stress response, and DNA repair (Fig. 5.17).
The emphasis in this paper, however, was put on the genes involved in the circadian
pattern, rather than on the specific involvement of biotransformation genes and
enzymes. Nevertheless, a number of genes appeared in various metabolic pathways
(Fig. 5.17, left part). Several of these genes functioned in the mitochondria involved,
for instance, in the electron transport chain, reflecting the potential multiple role
that mitochondria play in Reactive Oxygen Species (ROS) metabolism. Overall, the
question of whether or not metabolic and, particularly, biotransformation enzymes
are subject to the circadian pattern remained open for a long time; for biotransfor-
mation enzymes, it has only very recently been answered (Vera et al. 2018; Carmona-
Antoñanzas et al. 2017) (see below).
Li et al. (2015) developed a computational method to integrate both circadian
gene expression and metabolic network. Applying this method to the zebrafish cir-
cadian transcriptome, the authors have identified large clusters of metabolic genes
containing mostly genes encoding proteins involved in purine and pyrimidine
metabolism in the metabolic network showing similar circadian phases. The de
novo purine synthesis supplies crucial building blocks for DNA replication.
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Fig. 5.17 Gene ontology (GO) hierarchy and enrichment statistics for biological processes in the
zebrafish. GO terms within the biological process ontology that are significantly enriched in the
light-induced gene set are indicated in color, with the color shade corresponding to the enrichment
P-value. (From Weger et al. (2011), courtesy of the Public Library of Science)
In the sea bass, a fish species with dual feeding behavior (switching from diurnal
to nocturnal in winter, and returning to diurnal feeding in spring), the feeding
rhythm (diurnal vs. nocturnal) strongly influenced the daily patterns of digestive
function and clock gene expression in the liver, but not in the brain. As a conse-
quence, daily blood glucose variations observed in both groups showed higher
glucose levels occurring at night in nocturnal, as well as in diurnal, fish, although
only diurnal sea bass displayed a significant daily rhythm. Furthermore, the highest
values of amylase activity coincided with the feeding phase of fish; that is, in noc-
turnal sea bass, the maximum was reached in the late afternoon (ZT 18:00 h),
whereas in diurnal sea bass it was during the night (ZT 03:39 h) (del Pozo et al.
2012a). Again, this study comprised basic metabolic, namely catabolic, enzymes
and did not consider biotransformation pathways of natural xenobiotics.
Yúfera et al. (2014) started an inventory of the transcription rhythmicity of two
digestive genes in their dependence on the feeding protocol in the gilthead sea
bream Sparus auratus. The authors investigated daily rhythms in stomach fullness,
gastric and intestinal pH, as well as pepsin activity and expression of pepsinogen
and proton pump in juvenile fish under three different feeding protocols: one daily
meal, two daily meals, and continuous feeding during the daytime. The feeding
protocol affected significantly the rhythm of gastric pH and pepsin activity pattern.
In contrast to these biochemical variables, the transcription of both genes remained
practically constant, with only slight fluctuations. Although the food was not char-
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acterized, and the transcription of only two genes was evaluated, this study is a
promising step indicating that the combination of chronobiology and nutrition has
entered the aquatic phase as chrononutrition.
Currently, evidence is accumulating that the introductory question, of whether or
not the clock makes good food, has to be answered with a ‘yes’. For instance,
Paredes et al. (2014) investigated the circadian expression of key genes involved in
lipid metabolism in the liver of Sparus aurata and their synchronization to light–
dark and feeding cycles. All lipid-related genes investigated exhibited well-defined
daily rhythms with distinct phases in the liver, indicating a clear time gap between
the catabolic and anabolic processes. The lipolysis-related genes: hsl (hormone-
sensitive lipase), pparα (peroxisome proliferator-activated receptor-α), and lpl
(lipoprotein lipase) showed a nocturnal maximum expression, while lipogenesis-
related (fatty acid synthase, fas) and fatty acid turnover (cyclooxygenase, cox-2)
genes showed a mostly diurnal rhythm, and pparα was nocturnal. Most strikingly,
the main zeitgeber driving these rhythms appears to be the light–dark cycle and not
the feeding time. Furthermore, even under constant dark conditions, several genes
showed circadian rhythmicity. During constant dark conditions, these genes were
especially pparα and hsl; during constant light, these were the clock genes bmal1
and cry1. However, the transcription was much lower than in the light/dark scenario
(Mata-Sotres et al. 2015). These findings show that lipid utilization in the liver is
rhythmic and strongly, but not completely, synchronized with the light/dark cycle,
regardless of feeding time.
Comparable studies were conducted, for instance, with European sea bass
(Azzaydi et al. 2000), European eel (López-Olmeda et al. 2012), beluga (Sudagar
et al. 2012), Senegalese sole (Marinho et al. 2014), rainbow trout (Hernández-Pérez
et al. 2015), and zebrafish (Paredes et al. 2015). In the Senegalese sole, López-
Olmeda et al. (2016) provided the first evidence of daily rhythms and differential
day/night effects in growth factors. In addition, the authors showed that physiologi-
cal effects on that axis induced by growth hormone (GH) are dependent on the time
of day of its administration. Therefore, exogenous GH was more effective when it
was administered during the night (the active phase of this fish species), especially
as regards the stimulatory effect on hepatic igf1 expression levels. Interestingly,
Hernández-Pérez et al. (2015) showed that the circadian rhythm of cortisol and all
analyzed genes of the glucose and lipid metabolism in the liver of rainbow trout
exhibited well-defined daily rhythms that persisted even in the absence of light and/
or food, indicating the endogenous nature of such rhythms. Subsequently, a circa-
dian rhythm of expression of genes from the somatotropic axis was also shown in
Nile tilapia (Costa et al. 2016a, b).
Furthermore, Mata-Sotres et al. (2016) confirmed corresponding results in devel-
oping gilthead sea bream larvae. The authors measured changes in the enzyme
activity (trypsin, lipases and α-amylase) and gene expression (trypsinogen-try,
chymotrypsinogen-ctrb, bile salt-activated lipase-cel1b, phospholipase A2-pla2,
and α-amylase-amy2a) during a 24 h cycle in larvae reared under a 12/12 h light/
dark photoperiod. The enzyme activity and gene expression exhibited behaviors
markedly dependent on the light/dark cycle in all tested ages. The patterns of activ-
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ity and expression of all tested enzymes were compared to the feeding pattern found
in the same larvae that showed a rhythmic feeding pattern with a strong light syn-
chronization. In the four tested ages, the activities of trypsin, and to a lesser extent
lipases and amylase, were related to feeding activity. The changes in the expression
of protease precursors (try and ctrb) are shown in Fig. 5.18. Both try and ctrb
showed apparently different daily profiles at different ages. However, in both cases
a decreasing trend during the light period and/or an increasing trend during the dark
period can be observed, probably as an anticipation of the forthcoming ingestion of
food that will take place during the next light period. It follows that the enzymatic
activities are being regulated at translational and/or post-translational level. The
potential variability of enzyme secretion during the whole day is an important factor
to take into account in future studies. A particularly striking consequence of the
present results is the reliability of studies based on only one sample per day taken at
the same hour of the day, as those focused on assessing the ontogeny of digestive
enzymes.
In contrast to the studies above indicating an endogenous rhythmicity of clock
genes, Feliciano et al. (2011) reported that there is not such an obvious expression
in the goldfish, since the feeding time synchronized the rhythmic clock gene expres-
sion in its brain and liver. The authors showed that in the absence of any light/dark
cycle, a feeding schedule regime is sufficient to maintain both the rhythmic expres-
sion of several clock genes in the optic tectum and gPer1a in the hypothalamus.
However, in the liver, most of the clock genes studied presented significant daily
rhythms in phase with the last food supply, even in randomly fed fish.
Interestingly, García-Meilán et al. (2014) provided evidence that meal timing
affects the protein-sparing effect of carbohydrates in sea bream. In particular, the
authors applied a commercial diet (C) (48% protein and 20% lipids) and a highly
digestible carbohydrate diet (CH) (37% protein, 12.5% lipids, and 40% high-
digestible carbohydrates) to feed sea bream juveniles for an 8-week period. In the
commercial diet, more than 60% of the ingredients originated from various plants,
whereas the only component of plant origin in the CH diet was wheat. To determine
the best time to administer carbohydrates, and the possible protein-sparing effect,
diet was provided in the morning and in the afternoon. Interestingly, the measured
acid protease activity was anticipated, and was higher when the next meal would
have more protein (Fig. 5.19). The smaller ration given to sea bream in the after-
noon led to a lower pancreatic release of alkaline protease and α-amylase (Fig. 5.20)
and an upregulation of d-Glc and l-Ala absorption capacity. A higher transit rate
was measured when sea bream were fed the CH diet. When high-digestible carbo-
hydrates were administered in the morning, and the commercial diet in the after-
noon, the authors observed a better assimilation of both diets due to compensatory
mechanisms, such as an increase in l-Lys, d-Glc and l-Ala absorption capacity
after the morning food, and a higher pancreatic release of alkaline protease and
amylase after the afternoon food. In contrast, when high-digestible carbohydrates
were given in the afternoon, only a significant upregulation of the capacity to absorb
l-Lys was detected. Thus, the inclusion of highly digestible carbohydrates in the
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Fig. 5.18 Relative expression of proteases (try and ctrb) (solid line) compared with the gut full-
ness (dashed line) of Sparus aurata larvae at 10, 18, 30 and 60 dph. Different letters represent
significantly different values (P < 0.05) within the same age (mean ± SEM). The white and gray
parts of the graph symbolize the light and dark periods. (From Mata-Sotres et al. 2016, with per-
mission from Elsevier)
diet improved digestion and absorption processes when administered in the morn-
ing, leading to a protein-sparing effect that yielded growth comparable to that of fish
fed an exclusively commercial diet.
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Fig. 5.19 Stomach acid protease activity in sea bream in the morning (white bars) and in the
afternoon (black bars). Values are expressed as the mean ± SEM of eight fish. Significant differ-
ences (P < 0.05) between diets are shown by different letters and significant differences between
meal times are shown by an asterisk. C commercial diet, CH highly digestible carbohydrate diet.
(From García-Meilán et al. 2014, with permission from Elsevier)
Fig. 5.20 Sea bream: (a) Total protease, (b) α-amylase and (c) lipase activity in pyloric ceca
(white bars) and proximal intestine (gray bars). Values are expressed as the mean ± SEM of eight
fish. Significant differences (P < 0.05) between diets are shown by different letters; significant dif-
ferences between intestinal segments (P < 0.05) are shown by different numbers, and significant
differences between meal times are shown by *. (From García-Meilán et al. 2014, with permission
from Elsevier)
One can simply hypothesize that combined with biotransformation, the under-
standing of aquatic animal feeding can be significantly improved, and several of the
so-called antinutrional compounds may lose their ‘anti’-character.
5.2.2 Xenobiotic or Antinutritional Compounds

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Any feed, and particularly feed of herbal origin contains a variety of natural xeno-
biotic chemicals, often termed antinutritional factors (Castillo and Gatlin III 2015;
Goopy and Murray 2003; Krogdahl et al. 2010; Krogdahl et al. 2015). This termina-
tion and the underlying mechanistic perspective, however, do not adequately cover
the biological action of natural xenobiotics. Therefore, it is not surprising that only
a few papers consider natural or synthetic xenobiotics in feed for aquatic animals.
In a recent paper, Søfteland et al. (2014) studied synthetic xenobiotics, such as pes-
ticides and polycyclic aromatic hydrocarbons, as contaminants in plant ingredients
of fish feed, and the biochemical and biomolecular response of the challenged
fishes; this is a long-overdue step to include xenobiotics as food ingredients, since
the majority can be of natural origin and both classes of xenobiotics activate identi-
cal metabolic pathways (Steinberg 2012). Consequently, the question has to be
addressed: Are the corresponding genes and enzymes transcribed and active all day
and night or do they have a certain circadian rhythm?
One the best-studied examples of food and circadian gene transcription is the
history of alcohol. Ethanol is a natural xenobiotic and has entered the socio-cultural
sphere of human beings. The diurnal fluctuations in susceptibility towards con-
sumption of ethanol are well known, not only in science but even in public. It is
controlled by one per gene (Brager et al. 2011; Rosenwasser 2015). This example
of ‘chrononutrition’ (or better in its inverted version ‘chrono-toxicology’) illustrates
that even animals more complex than C. elegans or Drosophila, namely mammals,
exhibit clear circadian metabolic patterns and that comparable phenomena must,
therefore, be anticipated also in fishes.
The greatest moiety of natural xenobiotics is comprised of plant secondary metab-
olites (PSMs). The primary cause for the production of PSMs is their shielding effects
against adverse environmental triggers, particularly excess light energy, ozone, or
UV irradiation – where applicable (Close and McArthur 2002). Flavonoids facilitate
this task; this is a chemically diverse group of PSMs and comprises anthocyanidins,
flavonols, flavones, flavanols, flavanones, chalcones, dihydrochalcones, dihydrofla-
vonols, isoflavonoids, and pterocarpanes. PSMs also function as feed allelochemi-
cals, most often as repellents; the ultimate goal is that the herbivores avoid this food
source. In a so-called co-evolutionary arms race, consumers have generated offensive
tools, the major being detoxification abilities. Even more, based on the long-term
coexistence of PSMs, which can be considered natural xenobiotics and the biotrans-
formation system in consumers, an originally adverse chemical stress was reversed to
an eventually overall beneficial process, as we shall examine soon.
Biotransformation is the chemical modification made by an organism to a chemi-
cal compound, and is known to metabolize a wide variety of chemical compounds,
facilitated by the rather low specificity of many enzymes involved; one major pri-
mary function is to maintain biochemical homeostasis. As far as animals in general,
and herbivores in particular, are concerned, homeostasis is challenged by
biochemical compounds taken up via food: the PSMs. Complete avoidance is typi-
cally not possible due to the ubiquity and diversity of PSMs. Yet, there are physio-
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logical mechanisms that animals can use to detect the consequences of ingested
PSMs. Central is the biotransformation system, which comprises four distinct
phases (Steinberg 2012):
• Phase 0: Excretion of parent compounds by transporter proteins, with permeabil-
ity glycoproteins being major and well-studied representatives.
• Phase I: Oxidative, reductive, or hydrolytic transformation of a chemical com-
pound by enzymes. Hydrophilicity of the xenobiotic compound increases, mainly
through the activity of cytochrome P450 (CYP) enzymes.
• Phase II: Conjugative transformation of a chemical compound by a variety of
transferase enzymes, such as glutathione transferases (GSTs) or glycosyltrans-
ferases (UGTs); hydrophilicity of the xenobiotic compound increases further.
• Phase III: Absorption, distribution, and excretion of metabolites by transporter
proteins.
The low substrate specificity of the biotransformation enzymes allows that,
besides the natural xenobiotics, other synthetic xenobiotics introduced into the envi-
ronment by man, such as, for instance, pesticides or dioxins, can be more or less
metabolized; there is no fundamental difference. However, there is a great differ-
ence between metabolites and synthetic xenobiotics: the latter being generally
highly toxic (Steinberg 2012). Therefore, studies on the circadian transcription of
biotransformation genes of animals exposed to synthetic xenobiotics explain the
underlying biomolecular mechanism also of PSM compounds. One intriguing
example study of chronotoxicology has been published on Drosophila melanogas-
ter (Hooven et al. 2009). The authors compiled literature data on Drosophila
exposed to synthetic pesticides. It became obvious that the genes implicated in pes-
ticide metabolism and resistance exhibited peaks in expression clusters in the day-
time, particularly late afternoon, and clusters within several hours of each other.
This is particularly striking in the body: The expression of two GSTs and three
P450s oscillate in phase, peaking at ZT6-8, i.e., late afternoon. When all P450s and
redox partners, esterases, GSTs, and UGTs are included, in addition to those impli-
cated in pesticide metabolism, the second group of genes peaked between late night
and early morning (Fig. 5.21). Later, Erion et al. (2016) showed that the rhythmic
expression of the cytochrome P450 transcripts depended upon clocks in neurons
expressing neuropeptide F.
The findings in Drosophila clearly show that the capability of metabolizing
‘problematic’ substrates is not evenly distributed; instead, it peaks a few times dur-
ing short periods of time over the day, and the corresponding lethality of exposed
fruit was significantly reduced. In the meantime, supporting reports of the rhythmic-
ity of biotransformation gene expression come, for instance, from another
Drosophila species, D. suzukii (Hamby et al. 2013), the brown planthopper
(Nilaparvata lugens) (Kang et al. 2017), and the mosquitoes Anopheles gambiae
(Balmert et al. 2014; Rund et al. 2011) and Aedes aegypti (Yang et al. 2010).
Similarly, immune-related genes are also subject to circadian rhythmicity, as shown
in the mosquito A. stephensi (Murdock et al. 2013).
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Fig. 5.21 Example of chronotoxicology: Peak expression times of rhythmically expressed genes
(y-axis) [cytochrome P450s (cyps), P450 redox partners, esterases (ests), glutathione transferases
(gsts), and glycosyltransferases (ugts)] implicated in (natural and synthetic) xenobiotic metabo-
lism and resistance in Drosophila melanogaster. The peak time of expression (x-axis: zeitgeber
time, ZT) is plotted against frequency of rhythmic genes reported (y-axis). Solid colums and regu-
lar text are from studies of fly head. Blue colums and bold text are from fly body. (From Hooven
et al. 2009, courtesy of the Public Library of Science)
In other words: the clock makes the poison. Consequently, we can also hypoth-
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esize that the clock facilitates good, or even excellent, food; implying that optimal
metabolism of natural xenobiotics in the food is limited to a few short periods of the
day if chrononutrition applies. We can also hypothesize that this assumption applies
also to aquatic animals and their nutrition.
The latter assumption gets some support from a circadian study of the impact of
synthetic xenobiotic chemical compounds in zebrafish, sea bream, and Atlantic
salmon (Sánchez-Vázquez et al. 2011; Vera et al. 2010, 2018; Vera and Migaud
2016). Sánchez-Vázquez et al. showed that the toxicity and effectiveness of two
anesthetic substances were highest during daytime, the active phase of fish, thus
indicating a link between the daily rhythms of behavior and toxicity. However, since
the authors did not look for the periodicity of detoxifying enzymes, the underlying
mechanism remained obscure.
Only a few papers deal with the diurnal transcription rhythm of biotransforma-
tion enzymes in aquatic animals. For instance, one paper focused specifically on the
effects of ethanol on fishes. For fishes, this compound is a xenobiotic, rather than a
recreational drug as for some mammals. Vera et al. (2018) showed that ethanol tox-
icity exhibits daily rhythmicity in zebrafish larvae and adults. The ethanol effect was
more detrimental when fish were exposed in the morning, whereas the toxicological
and neurobehavioral responses were attenuated when exposure was carried out at
night. In addition, the paper revealed that the expression of genes involved in etha-
nol detoxification is under circadian regulation in zebrafish liver (Fig. 5.22). Cosinor
analysis in complete darkness (DD) revealed that the expression of all genes (adh5,
adh8a, aldh2.1, and aldh2.2) showed circadian rhythmicity, with their acrophases in
phase and located between CT = 20:36 h and CT = 21:38 h, at the end of the subjec-
tive night. The expression of adh5, aldh2.1, and aldh2.2 also displayed significant
statistical differences between time points, peaking at CT22 in all cases and show-
ing the lowest levels in the period CT10–14. However, the genes involved in ethanol
detoxification failed to show significant daily rhythms during the light–dark cycle
(LD) (not shown).
Circadian regulation of hepatic detoxification seems to be among the key roles of
the biological clock. The liver is the major site for biotransformation, and in mam-
mals it contains several clock-controlled transcription factors that act as circadian
regulators of detoxification genes. Therefore, Carmona-Antoñanzas et al. (2017)
explored the existence of daily and circadian expression of transcription factors
involved in detoxification, as well as the temporal profile of a set of their target
genes in zebrafish liver. Zebrafish were able to synchronize to an LD cycle and dis-
played a diurnal pattern of activity. In addition, the expression of clock genes pre-
sented daily and circadian rhythmicity in the liver. Regarding the detoxification
genes, the major target gene of aryl-hydrocarbon receptor (AhR), cyp1a, showed
daily and circadian expression, with an acrophase 2 h after ahr2. Under LD, abcb4
also showed daily rhythmicity. However, the expression of six detoxification genes
showed circadian rhythmicity under DD, including cyp1a and abcb4, as well as
gstr1, mgst3a, abcg2, and sult2_st2 (Table 5.2). In all cases, the acrophases of these
genes were found during the second half of the subjective night. This indicates that
1000 1000
adh5 adh8a
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a
Normalized Relative Units
800 ab 800
ab
600 ab ab 600
b
400 400
200 200
0 0
CT2 CT6 CT10 CT14 CT18 CT22 CT2 CT6 CT10 CT14 CT18 CT22
1200 1200
aldh2.1 aldh2.2 a

1000 1000
a ab
ab
800 800
ab ab
ab ab ab
600 ab 600
b b
400 400
200 200
0 0
CT2 CT6 CT10 CT14 CT18 CT22 CT2 CT6 CT10 CT14 CT18 CT22
CT (h) CT (h)
Fig. 5.22 Relative expression of ethanol metabolizing genes (alcohol dehydrogenases) in the liver
of zebrafish kept in constant darkness (DD). The gray and black bars at the top of the graph indi-
cate the subjective photophase and darkness phase, respectively. Data are shown as the mean rela-
tive units (RU) ± SE. Superscript letters indicate statistically significant differences (P < 0.05). The
dotted black line represents the sinusoidal function determined by Cosinor analysis. CT circadian
time. (From Vera et al. 2018, courtesy of the Public Libray of Science)
their expression is clock-controlled, either directly by core clock genes or through

transcription factors. This study presents new data demonstrating that the process of
detoxification in fishes is also under circadian control. The results showed further
that time of day should be considered when designing toxicological studies or when
administering drugs to fish – or when to administer feed with so-called antinutri-
tional factors.
Why do we focus on natural xenobiotics such as PSMs, termed in aquaculture
antinutritional factors, and their potential timely biotransformation or even degrada-
tion in fishes? This is much more than a personal bias. There are at least two good
reasons to consider PSMs in depth. In ‘Transgenerational Effects’, we have, in the
case of a specific anthraquinone, learned that PSMs have a strong potential to act as
epigenetic agents interfering within the translation of mRNAs. Furthermore, based on
the long-term coexistence of PSMs that can be considered natural xenobiotics and the
biotransformation system in consumers, an originally adverse chemical stress was
reversed to an eventually overall beneficial process. Due to its low substrate specific-
ity, the biotransformation system metabolizes a wide variety of natural xenobiotic
Table 5.2 Biotransformation genes in fishes studied by Carmona-Antoñanzas et al. (2017)

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Abbreviation Protein Function

abcb4 Multidrug resistance protein 3 Transporter through membranes
abcg2 ATP-binding cassette Transporter through membranes
subfamily G member 2
ahr2 Aryl-hydrocarbon receptor 2 Regulation of biological responses to planar
found in zebrafish aromatic (aryl) hydrocarbons
cyp1a Cytochrome P450, family 1, Phase I xenobiotic and drug metabolism
subfamily A
gstr1 Glutathione transferase Rho1 Phase II xenobiotic and drug metabolism
mgst3a Microsomal glutathione Phase II xenobiotic and drug metabolism
transferase 3a (in zebrafish),
sult2_st2 Sulfotransferase Phase II enzyme; transfer of a sulfo group from
a donor molecule to an acceptor alcohol or
amine
compounds and may thereby produce stimulating metabolites (Steinberg 2012). This
can easily be demonstrated with plant polyphenols such as quercetin, resveratrol,2
rosmarinic acid, caffeic acid or blueberry extracts (Wilson et al. 2006; Pietsch et al.
2010, 2011) which are thought, and sometimes even proven, to be beneficial for
health, longevity, or reproduction. For instance, polyphenols can, but may not neces-
sarily, extend life span and increase tolerance towards environmental stresses of
exposed animals (Saul et al. 2011). The classical assumption that the underlying
mechanism is based on antioxidative properties of the phenolic compounds does not
hold true, since evidence is emerging that some polyphenols do not have this property
(Saul et al. 2011); rather they sometimes tend to have prooxidative properties (Menzel
et al. 2011). Furthermore, many polyphenols do not follow a simple linear dose–
effect relationship; instead, they show hormetic behavior (Saul et al. 2013) character-
ized by a low dose stimulation and high dose inhibition. The resulting dose–effect
curve is J-shaped or inverted U-shaped (Calabrese and Baldwin 2003).
Applying transcriptomics to polyphenol-exposed C. elegans, individual tran-
scription profiles and Gene Ontology Trees have been revealed (Pietsch et al. 2010,
2012). This indicates that, with certain polyphenols, the antioxidant property may
play a minor role in the beneficial effects on life traits, but the major regulation is
genetically controlled and specific for the applied polyphenols. Genes coding
2
Bass et al. (2007) reported an interesting inter-laboratory comparison of expected and actually
occurring longevity effects of resveratrol in C. elegans: The results were variable, with resveratrol
treatment, resulting in slight increases in life span in some trials but not in others. This statement
agrees well with our own unpublished findings showing that resveratrol impacts could not be
reproduced in various consecutive trials (Saul, Pietsch, Menzel, Steinberg, unpublished). To date,
the most plausible explanation is that all trials disregarded internal diurnal rhythms mediated by
the timing protein LIN-42 (Jeon et al. 1999). Very recently, Hendriks et al. (2014) showed that gene
transcription may change by a factor > 10. In particular, nearly one fifth of detectably expressed
transcripts oscillate with an 8 h period, and hundreds change that much. Consequently, it is not a
surprise that life extension effects cannot be reproduced unless individuals of exactly the same
period within the biological rhythm are exposed.
enzymes of biotransformation Phase I and II – in charge of xenobiotic metabolism

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equivalent to stress defense – and antioxidant enzymes were overrepresented in

polyphenol-exposed C. elegans individuals (Pietsch et al. 2012). Overall, life exten-
sion is combined with an initial oxidative stress followed by the specific interaction
of the polyphenol with the genome. This finding contradicts classical assumptions
of aging mechanisms, such as the free oxygen radical theory by Harman (1956) that
assumes that the development of ROS is the major cause of aging. However, the
findings are well in compliance with the newly emerging concept about the inevita-
bility of oxidative stress in longevity and health, e.g. the mitochondrial hormesis
hypothesis (Schulz et al. 2007; Ristow et al. 2009). This issue will be revisited when
displaying and discussing nutritional effects of plant extracts in aquatic animals.
The polyphenol-mediated life span extension has been demonstrated with inver-
tebrates such as C. elegans or M. macrocopa (Pietsch et al. 2010, 2011) but appears
to be questionable for mammals such as mice (Strong et al. 2013). How do fish
behave? Do they differ from other animals? Since fish are lower vertebrates, but not
mammals, they may respond completely differently to plant polyphenols. And in
fact, they do. This should not be tested with long-lived fish, such as some koi carps
or sturgeons, but with short-lived species, namely killifish that inhabit ephemeral
savanna water bodies that are only filled with water during the rainy season. Fish
occur only during the rainy season when these savanna depressions are filled with
water, and survive the dry season as diapaused eggs or embryos buried in the soil.
Annual desiccation limits their life span to several weeks to months. Therefore,
Nothobranchius furzeri and N. guentheri have entered the research on aging – for
several years (Genade et al. 2005; Genade and Lang 2013).
Feed supplementation of resveratrol resulted in a statistically significant increase
in longevity that was dose-dependent (Fig. 5.23a). Life extension was observed both
in males and females (Fig. 5.23b). Furthermore, longevity was not linked to loss of
fertility. Also, resveratrol slowed down the age-dependent increase in death rate and
raised the mortality rates of treated fishes over those of control-fed fishes during the
first weeks after administration (Fig. 5.23c) (Valenzano et al. 2006). This fact indi-
cates that resveratrol exposure started as a chemical stress with adverse outcome
before it eventually prolonged life span.
In a crossing experiment, the recent mapping of life span quantitative trait locus
(QTL) revealed that life span determination in N. furzeri appears to be polygenic
(Kirschner et al. 2012). The authors detected four QTLs affecting life span. Taken
together, the QTLs explained 27.4% of the total life span variance in the F2 popula-
tion; life span was moderately heritable and did not show sex-specificity. Due to the
focus on potential longevity genes, this study did not take into consideration the
transcription of stress-related (biotransformation, oxidative stress) genes. Therefore,
the genes responsible for increased lethality during the early exposure phase remain
undiscovered to date.
The N. furzeri case is not singular; rather its sister species N. guentheri (Fig. 5.24
right) behaved comparably upon resveratrol exposure (Fig. 5.24 left). This began at
12 weeks of age and continued through the life of the fish. Resveratrol extended
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Fig. 5.23 Survivorship in resveratrol-exposed Nothobranchius furzeri. (a) Survival curves of five sepa-
rate experiments and of a reference set. Two trials of control-fed fishes (solid and empty red triangles);
reference survival of untreated fishes (black dotted); 24 mg/g resveratrol-treated fishes; two trials of
120 mg resveratrol-treated fishes (solid and empty black circles); 600 mg resveratrol-treated fishes
(blue). (b) Comparison between the age-dependent survival of males and females in controls and
120 mg/g resveratrol-treated fishes. Control males (black line) and females (dotted black line); 120 mg/g
resveratrol-treated males (blue line) and females (dash and dot line). (c) Death trajectories in controls
(black) and 120 mg g−1 resveratrol-treated fishes (red). (From Valenzano et al. 2006, courtesy of Elsevier)
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Fig. 5.24 Resveratrol treatment (RT) extends the life span of Nothobranchius guentheri. Hazard
functions are suppressed by RT through much of the life span, but increase to the value of normally
aging fish later on (from Genade and Lang (2013), with permission from Elsevier. Image: © Hristo
Hristov – AQUASAUR, courtesy of the U.S. Geological Survey. [2018]. Nonindigenous Aquatic
Species Database. Gainesville, Florida. [Accessed 2/15/2018])
median survival by 42.9%. Resveratrol restrained the mortality rate up to 52 weeks

of age, whereafter hazard functions3 increased (Genade and Lang 2013).
Not only does resveratrol extend the life span of N. guentheri, but it also improves
its cognitive performance. Yu and Li (2012) showed that the resveratrol-treated fish
exhibited a higher rate of performance than the control fish. Furthermore, the data
indicated that resveratrol had the property of protecting N. guentheri from neurode-
generation, as well as retarding the aging-related histological markers in lipofuscin
formation and in the expression of senescence-associated β-galactosidase activity.
In conclusion: the natural xenobiotic example may be considered an extreme.
Nevertheless, we hypothesize that, based on improved knowledge of the circadian
pattern of digestive and biotransforming enzymes, the effectiveness of feed contain-
ing PSMs, in terms of health, reproduction, and longevity, may be significantly
improved, which, in turn, may reduce the production costs and environmental
impact of aquaculture. With this in mind, we will check the effects of various food
components of aquatic animal nutrition from the mechanistic perspective from indi-
viduals to generations (Volume II of Aquatic Animal Nutrition).
Although the understanding even of mammal chrononutrition is still in its infancy,

and does not yet include interesting substrates such as polyphenols, it is far ahead of
that of fishes and aquatic invertebrates, and may serve as a lighthouse for future
3
The hazard function is defined as the event rate at time t conditional on surviving up to or beyond
time t.
studies of aquatic animal nutrition. Recent findings indicate that meal timing is
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crucial, with both intermittent fasting and feeding adjusted to the diurnal rhythm
improving health and function, independent of overall intake (Fontana and Partridge
2015). Figure 5.25 illustrates that the circadian system in mammals tightly regulates
energy metabolism, and feeding has a feedback to clock genes. Nevertheless, the
evidence is growing that food intake late at night, irrespective of its chemical com-
position, leads to obesity in mammals (Fig. 5.26) (Tahara and Shibata 2013).
Overall, we can expect the optimal timing of food intake, by understanding the cir-
cadian changes in the transcription of corresponding genes of digestion and bio-
transformation, or the connected metabolic activity, to maintain or improve animal
health, reproduction, or longevity.
Fig. 5.25 Circadian changes affect the functions of food metabolism, including digestion and
absorption of food, and energy metabolism. Considering these factors when determining the tim-
ing, amount, and composition of food intake can benefit health and body-weight gain. (From
Tahara and Shibata 2013, with permission from Elsevier)
Fig. 5.26 Late night food intake (ex. 23:00) had a greater impact on the changes in the peripheral
clock phase, and promoted irregular feeding times. This could be because late night dinner comes
after a lengthy fasting period, compared to after breakfast. (From Tahara and Shibata 2013, with
References 325
We shall eventually be able to replace the mouse by a fish species in the clock
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graph (Fig. 5.26), probably by a goldfish (Vivas et al. 2011), a gilthead sea bream
(Vera et al. 2014), a zebrafish (Villamizar et al. 2014), an Atlantic salmon (Valen
et al. 2011), or a rainbow trout (Soengas 2014). Significant progress has been made
in identifying the neurohormonal regulation of food intake and response to nutrients
in fish. Furthermore, many studies of the circadian transcription of major digestion-
as well as growth-related genes in fish are driven by the light/dark rhythm, regard-
less of feeding time, and make it very likely that this rhythmicity may apply also to
the biotransformation pathway.
Nevertheless, there remains something to be discovered, and intriguing results
can be anticipated. If the feeding schedules are adjusted to coincide with maximum
natural peaks of digestive and biotransformation enzymes, food efficiency will
increase, which will be reflected in weight gain of the fish, and provide more profit-
able yields for aquaculture.
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Chapter 6
VetBooks.ir
Transgenerational Effects – ‘Your

Offspring Will Become What You Eat’
Abstract Parental effects, which are well documented for aquatic wildlife and
farmed species, are a major source of phenotypic plasticity of the succeeding gen-
erations and determine their persistence in the ecosystem. Theory predicts that,
under low food availability, parents, mainly mothers, may decrease their fecundity
and increase their investment per offspring to have greater reproductive success.
These predictions come true in most, but not all, studies, if dietary gross quality or
reduced quantity of appropriate diets are considered. Under these circumstances,
the offspring can be more resistant to environmental stresses and starvation, pos-
sesses an improved innate immunity, shows courageous behavior and improved
learning. However, if individual dietary compounds (amino acid patterns, methio-
nine content, polyunsaturated fatty acids, vitamin E content) are unbalanced or
insufficiently supplied in the parental diets, reduced hatching successs as well as
elevated embryonic mortality and malformation can be observed. Very recently, it
became obvious in rotifers that parental dietary signals can be passed to tens of suc-
ceeding generations. DNA-dependent and DNA-independent inheritance (e.g. pri-
ons, protein feed back loops, hormones, metabolites) and epigenetics are discussed
as underlying transgenerational mechanisms. Less well understood, than maternal
effects, are paternal effects; however, increasing evidence supports the notion that at
least some epigenetic marks acquired during spermatogenesis can be sustained
through embryonic development as reported for a tubeworm and Atlantic cod.
The second part of this chapter comprises brief descriptions of major epigenetic
mechanisms, their time scale, and persistence, followed by the question of how
environmental signals, in general, and diet-mediated signals, in particular, translate
into detectable epigenetic markers. The body of evidence of environmental markers
affecting the DNA methylations status, is rapidly increasing, wherease diet-mediated
studies are still in their infancy. For instance, in zebrafish the lack of dietary vitamin
E reduced the availability of methyl donors and caused lasting metabolic impair-
ments in the embryos. Also only a few studies report on food-mediated hsp90
expression; but the link to HSP90 as a capacitor of phenotypic traits is still com-
pletely missing. In contrast to this multifunctional chaperon, knowledge about small

https://doi.org/10.1007/978-3-319-91767-2_6
334 6 Transgenerational Effects – ‘Your Offspring Will Become What You Eat’
non-coding RNAs (microRNAs) and their distribution in fasted and well-fed ani-
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mals is rapidly developing and clearly exceeds the state of stocktaking.
The transfer to offspring of phenotypic traits acquired by adult parents, as espoused

by Lamarck (1809), is an old notion in biology. In particular, significantly before the
exchange of key thoughts on evolution in letters between the almost forgotten Welsh
scholar Alfred Russel Wallace and Charles Darwin (Knapp 2013) and half a century
before Darwin published On the Origin of Species, the French naturalist Jean-
Baptiste Lamarck outlined his own theory of evolution. A cornerstone of this was
the idea that characteristics acquired during an individual’s lifetime can be passed
on to its offspring.
In its day, Lamarck’s theory was generally ignored. Now the situation is changing.
Over the past decade, it has become clear that environmental factors, such as diet or
biotic as well as abiotic stress, can have biological consequences that can be trans-
mitted to succeeding generations (Koonin 2014). Doubtless, inherited phenotypic
traits tune the variability of a given phenotype to match the variability of the acting
selective pressure. Various forms of epigenetic inheritance are now firmly established
as an important complement to the vertical transmission of genetic information in
diverse organisms. Lamarckian evolution is now reality rather than myth (Young
2008); however, the study of its impact and generality is in its infancy for many
groups of animals. Therefore, this chapter will gather several illustrative examples,
discuss the mechanisms, and try to figure out the state of the art in aquatic animals.
A few intriguing examples will be introduced regarding the Lamarckian ideas
and their updating. The first example is a transgenerational ecotoxicological one
rather than a diet-based one. Chamorro-García et al. (2013) reported that the expo-
sure of mice to tributyltin (TBT) modulates critical steps of adipogenesis through
retinoid X receptors (RXRs) and that prenatal TBT exposure predisposes multipo-
tent mesenchymal stem cells to become adipocytes by epigenetic imprinting into the
memory of the mesenchymal stem cell compartment. Via drinking water, female
mice (P0) were exposed to TBT throughout pregnancy. F1 offspring were bred to
yield F2, and F2 mice were bred to produce F3. F1 animals were exposed in utero and
F2 mice were potentially exposed as germ cells in F1 mice, but F3 animals were never
exposed to the chemical. Prenatal TBT exposure increased most white adipose tis-
sue depot weights, the adipocyte size, and the adipocyte number and reprogrammed
multipotent mesenchymal stem cells toward the adipocyte lineage at the expense of
bone in all three generations. The results showed that early life obesogenic exposure
can have lasting effects. Very recently, this obesity pathway after TBT exposure has
also been discovered in zebrafish (Ouadah-Boussouf and Babin 2016).
With the mycoestrogen zearalenone, Schwartz et al. (2013) documented trans-
generational effects of P0 short-term exposure on F1 generation in zebrafish. The
growth of F1 males increased if the P0 generation had been exposed to 1.0 μg L─1
zearalenone; however, the improved growth was combined with reduced fecundity
of the F1 generation.
Studies of transgenerational effects often focus on single environmental vari-
ables. However, in nature, it is unlikely for one factor to vary independently from
6.1 Parental Effects 335
others, and there are likely to be trade-offs between different stressors. In fact, the
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next example is diet based. It concerns a member of the moth family Noctuidea,
since corresponding works on aquatic wildlife are not yet available. In the cab-
bage looper (Trichoplusia ni), Shikano et al. (2015) examined how nutritional
stress influences the transgenerational immune priming with a sublethal challenge
by the bacterial pathogen Bacillus thuringiensis. There was a significant trade-off
between transgenerational immune priming and the transfer of nutritional stress
tolerance: parents transferred resistance to pathogens but not nutritional stress
tolerance (Fig. 6.1).
The study highlights the trade-offs that can modulate the occurrence and magni-
tude of transgenerational effects and illustrates the importance of assessing interac-
tions between multiple environmental variables. This argument must be kept in
mind when we report and discuss transgenerational effects in aquatic wildlife, as
most of these studies are in their infancy and focus on diets only.
Except for a few comprehensive and convincing field studies, such as the research
in the Experimental Lakes Area on fathead minnow (Pimephales promelas) exposed
to low concentrations of the potent 17α-ethynylestradiol (Kidd et al. 2007) or a
multigenerational laboratory study with Poecilia reticulata1 (Volkova et al. 2015), a
clear demonstration of heritable transgenerational effects in aquatic wildlife is lack-
ing. However, the studies have not been extended to the F4 generation, which is
considered to be essential in species reproducing by recombination (Rosenfeld
2014). Therefore, these studies cannot rule out other stimulating factors for the gen-
erational effects, such as phenotypic plasticity or developmental plasticity.
Therefore, it is not known whether the altered phenotypes pass to subsequent gen-
erations. One can easily extend this statement: even less is known about the food-
mediated transgenerational modification of aquatic wildlife. We try to collect bits
and pieces and complete the emerging mosaic to form a discernible picture by add-
ing information from terrestrial animals. We aim to demonstrate the fascinating
variety of transgenerational effects and start with a seemingly easy-to-understand
issue, diet-mediated parental effects.
6.1 Parental Effects
Parental effects are the causal influence of the parental genotypes or phenotypes on
the offspring phenotype (Wolf and Wade 2009). Early life history stages are most
sensitive to environmental stress, so transgenerational phenotypic plasticity,
whereby the parental environment and offspring environment interact to alter the
phenotype of the offspring, is viewed as the key to promoting persistence in the face
of environmental changes. The non-genomic transmission of maternal life history
traits from one generation to the next illustrates the pervasive influence of maternal
1
Only P0 individuals were exposed to 17α-ethynylestradiol, and the study was extended to the F2
generation, which still showed disturbed behavior.
Fig. 6.1 Effects of

parental diet quality,
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parental Bacillus
thuringiensis (Bt)
challenge, and offspring
diet quality on cabbage
looper (Trichoplusia ni)
offspring’s resistance to:
(a) Bt, measured as the
lethal concentration of Bt
in an artificial diet that
kills 50% of offspring
(LC50; IU mL−1 diet) and
(b) the baculovirus
TnSNPV, measured as the
lethal dose of virus that
kills 50% of offspring
(LD50; OB larva−1). The
error bars represent 95%
confidence intervals. (From
Shikano et al. 2015, with
permission from Wiley)
care on offspring’s development and the high degree of phenotypic plasticity.

Theory predicts that the optimal solution to the quality–number trade-off depends
on environmental conditions, whereby parents who decrease their fecundity and
increase their investment per offspring will have greater reproductive success when
the environmental quality is low. Therefore, the decision to produce many poorly
provisioned offspring or a few well-provisioned offspring can have a profound
effect on parental reproductive success and the maintenance of populations in fluc-
tuating environments (Stahlschmidt and Adamo 2015).
Parental effects are a major source of phenotypic plasticity and may influence
offspring phenotypes and, in concert with environmental demands, even their per-
sistence and survival in the ecosystem. Specifically, parents have to divide the
energy available for reproduction between investment per offspring and fecundity.
Subsequently, both the parental legacy and the current environmental conditions can
affect offspring life histories; however, their relative importance and the potential
relationship between these two influences have rarely been investigated in aquatic
animals. Parental effects themselves have often been described, and they have been
reviewed excellently by Green (2008). However, the question regarding whether
these effects last longer than one generation remains unanswered. Nevertheless, if
one expands the view to short-lived terrestrial animals, it soon becomes obvious that
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parental effects are long-lasting, most probably via epigenetic heritage. Before we
touch on the intriguing mechanisms, we shall highlight some recent studies on
parental, maternal, and paternal effects. While there has been long-standing interest
in the role of transgenerational plasticity via the maternal line (maternal effects), the
evidence is increasing that paternal effects can also play a role.
Studying the springtail Orchesella cincta, which is found in the litter layer of
temperate forests, Zizzari et al. (2016) measured developmental plasticity in
response to paternal or maternal food availability over two generations in the perfor-
mance of offspring. The authors found adverse effects of food limitation on several
life history traits and the reproductive performance of both parental sexes. The food
conditions of both parents contributed to the offspring phenotypic variation, provid-
ing evidence for dietary transgenerational effects. Parental diet influenced sons’ age
at maturity and daughters’ weight at maturity. Specifically, being born to food-
restricted parents allowed offspring to alleviate the adverse effects of food limita-
tion without reducing their performance under well-fed conditions. Therefore,
parents raised on a poor diet primed their offspring for more efficient resource use.
However, a mismatch between maternal and offspring food environments generated
sex-specific adverse effects: female offspring born to well-fed mothers showed
decreased flexibility in dealing with low-food conditions. Notably, these maternal
effects of food availability were not observed in sons. Finally, Zizzari et al. (2016)
found that the relationship between age and size at maturity differed between males
and females and showed that offspring life history strategies in O. cincta are primed
differently by the parents. Parents with a poor diet acted in concert in improving
sons’ age at maturity but not their weight at maturity, because male mating success
in Orchesella is not size dependent. Compared with males, Orchesella females are
subject to more severe developmental constraints. This might explain the positive
transgenerational effects of diet on daughters’ weight at maturity.
Although mothers with a poor diet improved the performance of their daughters,
poorly fed daughters showed the highest delay of maturation time if their mothers
were well fed. Conversely, sons born to well-fed mothers had a similar maturation
time irrespective of their own diet. These results indicate that a rich maternal diet
induced low resource-use efficiency in the daughters that was detrimental only
under poor nutritional conditions. Thus, the environmental mismatching between
maternal and offspring diet generated adverse effects in female offspring only.
Low food availability often leads to larger progeny than high food availability.
Beneficial parental influences are expected to be the most important when offspring
experience poor environmental conditions. Because for most species, however, the
potentially complex relationships between the parental effects and the juvenile envi-
ronment were unknown, Donelson et al. (2009) experimentally tested the interact-
ing effects of differences in food supply during both the parental and the juvenile
stage on the growth and survival of a tropical damselfish, Acanthochromis poly-
acanthus. Using a design in which breeding pairs were fed either a low- or a high-
quantity diet and their offspring were reared on either a low- or a high-quantity diet,
the relative importance of parental effects on the growth and survival of offspring
under different environmental conditions was assessed. Juveniles from parents in

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good condition were longer and heavier at hatching than juveniles from parents in
poor condition. Parental effects on juvenile size were evident up to 4 weeks post-
hatching but disappeared by 7 weeks. The offspring of good-condition parents had
a higher early survival rate than the offspring of poor-condition parents when reared
in a low-food environment. By contrast, the parental condition did not affect juve-
nile survival in the high-food environment. These results show that parental effects
on offspring performance are the most important when poor environmental condi-
tions are encountered by juveniles. Furthermore, parental effects observed at hatch-
ing may often be moderated by compensatory mechanisms when the environmental
conditions are good. Furthermore, parental effects can influence the mortality
schedules of juveniles with a lasting effect on population replenishment and popula-
tion genetic structure, but these effects are likely to be more pronounced in low-
quality habitats or during periods of increased environmental stress.
An intriguing phenotypic example of diet-based parental transgenerational
effects was recently reported for brown trout (Salmo trutta, Fig. 6.2, below) (Van
Leeuwen et al. 2016). Brown trout exhibit partial migration, with some individuals
remaining in freshwater (freshwater-resident) while others undertake anadromous
migration, gain most of their adult size at sea, and then return to fresh water to
spawn. The option adopted by an individual trout is thought to be determined partly
by its growth performance in early life, which, in the stochastic and dynamic envi-
ronment of freshwater streams, may be dependent on its flexibility. To examine the
potential effects of parent type on phenotypic flexibility, Van Leeuwen et al. (2016)
measured the metabolism, growth, and morphology of full-sibling groups of off-
spring from freshwater-resident and anadromous parents both before and after a
switch in diet. The authors found that fry had a higher growth rate and a more
rounded head and body shape when reared on Chironomid larvae than when they
were reared on Daphnia, but diet had no effect on the standard metabolic rate.
Interestingly, the offspring of anadromous parents were less able to maintain their
growth rate when fed on Daphnia than those of freshwater residents and showed a
correspondingly greater increase in growth following a switch from Daphnia to
Chironomid larvae. The offspring of anadromous parents also showed less morpho-
logical flexibility in response to diet than the offspring of freshwater residents. The
results of this study indicate that offspring from freshwater-resident and anadro-
mous parental life history strategies show some differences in phenotypic flexibility
that may be consistent with the future habitats that individuals may encounter. The
offspring of migratory fish appear to be morphologically less flexible and less able
to maintain growth on a poor-quality diet than the offspring of
freshwater-residents.
Fontagné-Dicharry et al. (2017) and Seiliez et al. (2017) described the effect of
parental dietary methionine (Met) on the life history traits of rainbow trout o ffspring.
Three plant-based diets differing in their Met level were fed to the broodstock (male
and female) for 6 months prior to spawning. The offspring survival and growth were
the highest in fry from broodstock fed excess Met. Furthermore, the diet deficien-
cies in Met affected the activation and expression of several key metabolic factors
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Fig. 6.2 Above: Schematic diagram of the morphological shape response for offspring from anad-
romous (Anad) and freshwater-resident (Res) brown trout parents during time periods when fish
were reared on Daphnia (gray arrows) and Chironomid larvae (black arrows). The diets were
switched at the start of interval 2. Note the equivalent morphological responses to diet of the two
offspring types during intervals 1 and 2 (i.e., a similar degree of initial morphological divergence
between fry on the Daphnia and on the Chironomid larvae diets and a similar effect of a diet
switch, as measured by the Mahalanobis distance, D). However, by interval 3 the offspring of
freshwater-resident parents showed greater dietary-induced morphological divergence (D = 3.67)
than the offspring of anadromous parents (D = 2.54). (From Van Leeuwen et al. 2016, with permis-
sion from the NRC Research Press); below: image of Salmo trutta. (From Bloch 1782–1784,
courtesy of the Biodiversity Heritage Library) (The Mahalanobis distance is a measure of the dis-
tance between point P and distribution D. It is a multidimensional generalization of the idea of
measuring how many standard deviations away P is from the mean of D. This distance is zero if P
is at the mean of D and grows as P moves away from the mean: along each principal component
axis, it measures the number of standard deviations from P to the mean of D)
in the offspring, indicating nutritional programming in fish by means of parental

nutrition. In particular, the expression of genes involved in appetite regulation and
muscle growth was affected and assessed at the swim-up stage (which corresponds
to the beginning of exogenous feeding) and at 21 days post-first feeding (Fig. 6.3).
The swim-up fry from the Met-deficient broodstock group (BD) had a higher
expression of pomc along with a lower expression of the npy feeding peptide
(Fig. 6.3a). This parental effect remained significant in the 21-day fed fry for pomc
but disappeared for npy (Fig. 6.3b). The highest expression of the anorexigenic
feeding peptide POMC in fry fed the Met-deficient diet indicated reduced appetite
in that group. Dietary Met deficiency in the broodstock enhanced the expression of
myog and decreased the expression of fmhc in fry at the swim-up stage (Fig. 6.3a).
The parental effects on myog were no longer significant 21 days later (Fig. 6.3b). A
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potential mechanism behind this expression pattern is not yet obvious.

The expression of gdh was upregulated in BD offspring in both the swim-up
stage and 21 days post-first feeding (Fig. 6.3a, b). This increased gdh mRNA level
indicates that the process of AA deamination is very sensitive to dietary Met imbal-
ance at this developmental stage (Skiba-Cassy et al. 2016). The expression of sod2
was lower in BE offspring in both the swim-up and the 21-day post-first-feeding fry
(Fig. 6.3a, b). Broodstock Met deficiency led to upregulation of the expression of
lecithotroph at both the swim-up stage and 21 days post-first feeding (Fig. 6.3).
Further studies need to evaluate the long-term persistence of the parental effects
over time and generations and to elucidate whether the mechanisms are epigenetic.
Fig. 6.3 Effects of rainbow trout broodstock Met nutrition on offspring whole-body gene expres-
sion related to the appetite, growth, and quality of offspring at the swim-up stage (a) and fed dif-
ferent Met levels at a constant water temperature of 17 ± 1 °C for 3 weeks (b). The columns
represent means ± SEM and are normalized to ef1α mRNA and expressed as fold changes of
mRNA abundance compared with the control group. Within each stage and each diet-related effect
(broodstock or fry diet), means not sharing a common superscript letter are significantly different
(P < 0.05) (from Fontagné-Dicharry et al. (2017), with permission from Elsevier). BD, BA, and
BE indicate broodstock diets that are deficient, adequate, or in excess of the established require-
ment; genes: poma, proopiomelanocortin A; npy, neuropeptide Y; myog, myogenin; fmhc, fast myo-
sin heavy chain; gdh, glutamate dehydrogenase; sod2, superoxide dismutase 2; and ldlr, low-density
lipoprotein receptor
In terms of phenotypic traits, parental Met deficiency reduced the egg size and
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decreased the later growth performance of the offspring. This decreased growth of
offspring from broodstock fed the Met-deficient diet was in line with data obtained
in rainbow trout fed high levels of plant protein ingredients. The parental Met excess
also decreased the growth performance of offspring compared with the adequate
parental Met level, despite its beneficial effect on the offspring’s survival.
In a series of papers, Maret G. Traber and her coworkers studied comprehen-
sively how vitamin E (VE) deficiency in zebrafish translates into embryos and life
history traits. α-Tocopherol is probably required during embryonic development to
protect PUFAs that are crucial to development, specifically arachidonic (ARA) and
docosahexaenoic (DHA) acids. Additionally, ARA and DHA are metabolized to
bioactive lipid mediators via lipoxygenase enzymes, and α-tocopherol may directly
protect, or it may mediate the production and action of, these lipid mediators
(Lebold and Traber 2014). Miller et al. (2012) identified that such diets produced
embryos with increased morphologic abnormalities and mortality (Fig. 6.4). When
VE-depleted adults were spawned, they produced viable embryos with depleted
α-tocopherol concentrations. The VE− embryos exhibited a higher mortality at 24 h
post-fertillization and a higher combination of malformations and mortality at 120 h
post-fertilization than embryos from parents fed VE+ or lab diets (Fig. 6.4, right).
This study pioneered in documenting that VE is essential for normal zebrafish
embryonic development. This study points also out that the dietary range of VE sup-
ply appears to be rather small, since it cannot be warranted that the malformed or
dead embryos on parental VE+ diet (0.5 g kg−1 α-tocopherol) indicated the begin-
ning of adverse hypervitaminosis effects.
On the biomolecular level, Miller et al. (2014) refined that parental diet impacts
the basal embryonic transcriptome. The VE− embryos had significant differential
expression of 2656 transcripts compared to VE+ diet embryos. Of these transcripts,
1661 were repressed and 995 were elevated in the VE+ diet when compared to the
VE− diet embryo transcript levels (Fig. 6.5c). The elevated transcripts were impli-
cated in gene expression, AA metabolism, organ morphology and nervous system
development (clusters #2 and #3, Fig. 6.5c). The repressed transcripts were associ-
ated with pathways involved in cell-to-cell signaling and interaction, embryonic and
tissue development (cluster #1, Fig. 6.5c). Notably across all three clusters many
differentially expressed transcripts were linked. These changes in gene expression
can lead to long-term physiological or functional effects, and could also alter sus-
ceptibility to other stressors. In the absence of VE, lipid peroxideation would over-
whelm the antioxidant capacity of the embryo through rampant lipid oxidation
(primarily in the highly-oxidizable PUFAs), perturbing the ‘quiet’ metabolic state
in the embryo. Metabolic regulation would switch to an ‘active’ state in response to
the increased stress from the lack of VE. This is supported by the significantly
increased expression of both peroxisome proliferation-activated receptor γ, co-
activator 1-α and co-activator 1-β mRNA in the VE− embryos. Both are nuclear
receptor cofactors with global biologic functions, chiefly serving as mediators of
energy metabolism (Miller et al. (2014) with references therein).
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Fig. 6.4 Left: Typical zebrafish morphology at 120 hpf. Representative pictures from the three
diet groups are shown after 5 days (120 h). The eye and otic vesicle are indicated on all fry; mal-
formations are illustrated on the image of the deficient fish. CF, cranial–facial malformation; BA,
bent anterior–posterior axis; PE, pericardial edema; SB, swim bladder malformation; YSE, yolk-
sac edema. Right: Malformations and mortality of zebrafish embryos. (a) Increased mortality was
observed in the (V)E− embryos at 24 and 120 hpf compared with the other diet groups, but at
24 hpf, the differences between diet groups did not reach statistical significance. Mortality
increased from 24 to 120 hpf in the VE− (squares) and the VE+ embryos (triangles), but not in the
lab diet embryos (circles). At 120 hpf, the VE− embryos displayed significantly higher levels of
mortality compared with the E+ and lab diet embryos (diet effect). (b) Higher levels of both mal-
formations and mortality were observed at 120 hpf in the VE− embryos compared with VE+ or lab
diet embryos; VE+ had greater malformations than did lab diet embryos. Embryos were analyzed
in 96-well plates, one embryo per well with 48–120 embryos per group per spawn. Results are
expressed as percentages affected per total number of embryos (n = 6 spawns per group). (From
Miller et al. 2012, with permission from Elsevier)
McDougall et al. (2016) found that α-tocopherol deficiency in zebrafish embryo

caused the specific depletion and increased turnover of DHA-containing phospho-
lipid and lyso-phospholipids,which may compromise DHA delivery to the brain
and thereby contribute to the functional impairments observed in VE− embryos.
Furthermore, VE deficiency was associated with mitochondrial dysfunction with
concomitant impairment of energy homeostasis. The observed morbidity and mor-
tality outcomes could be attenuated, but not fully reversed, by glucose injection
into VE-deficient embryos at developmental day one. Thus, embryonic VE defi-
ciency in vertebrates leads to a metabolic reprogramming that adversely affects
methyl donor status and cellular energy homeostasis with lethal outcomes
(McDougall et al. 2017a). Taken together, embryonic VE deficiency causes lasting
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Fig. 6.5 Global effects of parental diet on embryonic transcription in zebrafish. (a). Venn diagram
displaying differentially represented transcripts. Both Lab and (V)E− groups were first compared
to the VE+ defined diet control group; significant transcripts from each set were then compared as
represented in the figure b and c. Transcripts from Lab (b) and VE− (c) that were significantly
different from the VE+ controls were grouped using bi-hierarchical clustering as displayed in the
heatmap. General annotations for each cluster are noted on the right side of the heat maps. (From
Miller et al. 2014 with permission from Elsevier)
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Fig. 6.6 (VE–) E– larvae have sustained metabolic perturbations to the cellular antioxidant net-
work despite consuming adequate diets for 7-days. (a) Antioxidant network scheme showing inter-
action of antioxidants with lipid radicals and consumption or NAD(P)H. (b) VE– and VE+ larvae
relative response data was normalized against QC sample intensities for each individual metabo-
lite. Boxes shown in Red (increased in VE– or Blue (increased in VE+) represent metabolites that
were higher in VE– or VE+ larvae, respectively. Black boxes indicate relative levels of a given
metabolite are not shown in the figure. Statistical significance (P < 0.05) was determined using the
Holm-Sidak method for multiple comparisons of normalized intensity values. Shown are
means±SEM, p-values are indicated as **** < 0.0001. Abbreviations: LOO lipid radical, LOOH
oxidized lipid, α-TOC-OH, α-tocopherol; α-TOC-O·, α-tocopheroxyl radical; AA ascorbic acid;
A● ascorbate radical, DH-A dehydroascorbate, GSH glutathione, GSSG glutathione disulfide,
PUFAs polyunsaturated fatty acids. (From McDougall et al. 2017c, with permission from Elsevier)
behavioral impairments due to persistent lipid peroxidation and metabolic pertur-

bations (Fig. 6.6) that are not resolved via later dietary VE supplementation
(McDougall et al. 2017c).
6.1.1 Maternal Effects
Maternal effects are the causal influence of the maternal genotype or phenotype on
the offspring phenotype (Wolf and Wade 2009), in other words the environment that
mothers provide to their offspring, their provision of nutrients, and the environment
that offspring of the same clutch share. These effects have come to be recognized as
an important influence on offspring fitness (Carter et al. 2004). The impact on the
offspring can last for only one up to several generations; this means that simple
effects, whereby eggs or embryos receive direct stress response information (pro-
teins, mRNAs) from their mothers or are directly challenged by environmental
stress during their development, clearly differ from true increases in phenotypic
diversity by epigenetic mechanisms. Several reviews have emphasized the need for
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transgenerational studies to be carried out through at least the F3 or F4 generations

(Rosenfeld 2014). This is the correct scholarly definition of animals with recombi-
nation as the major mode of reproduction; and, in fact, studies with mammalian
models have documented that global dietary restriction (DR) during pregnancy
results in a specific phenotype that can be passed transgenerationally to the second
and third generations (Ponzio et al. 2012). Occasionally, second-generation off-
spring of food-restricted grandmothers were heavier but shorter-lived (Araminaite
et al. 2014). However, even a one-generation effect can be regulated epigenetically.
Furthermore, comparatively short-term, one-generational acclimation to fluctuating
environments is central to sustaining the population. In addition to this, many inver-
tebrate animals reproduce mainly asexually. Therefore, the terms “epigenetics” and
“epigenetic mechanism” will be used in a more general, broader sense. Some exam-
ples may highlight food-based inherited life history traits and trigger more detailed
future studies in non-mammalian animals in general and aquatic animals in
particular.
6.1.1.1 I nvertebrates
The high degree of variability in shellfish reproductive success has been shown to
be attributable partly to gamete quality, sperm–egg interaction, and differential via-
bility of genotypes. To identify oocyte quality, a global transcriptional approach
assists in understanding the complex molecular mechanisms underneath oocyte
maturation and quality. de Sousa et al. (2015) proved the feasibility of this approach
by identifying indicative individual genes in the European clam, Ruditapes decus-
satus. Apoptosis appeared as one important pathway that was found to be more
abundantly represented in poor-quality oocytes and negatively correlated with the
veliger yield. Among the candidate genes, dnaj (hsp40) homolog, tumor necrosis
factor receptor, and caspase 8 appeared to be candidates of particular interest for
more specific and precise individual functional investigation in relation to the repro-
ductive success of R. decussatus.
Pauletto et al. (2017) improved this approach in king scallop (Pecten maximus)
by combining the identified genes into biological processes. They showed that
genes coding proteins involved in cytoskeletal dynamics, the serine/threonine
kinases’ signaling pathway, mRNA processing, the response to DNA damage, apop-
tosis, and the cell cycle are crucial for both oocyte maturation and developmental
competence (Fig. 6.7). This study resulted in a species-specific microarray platform
for the king scallop to identify differentially expressed genes in relation to oocyte
quality and to investigate the transcriptional features of both stripped and spawned
scallop oocytes.
Transgenerational effects are often assumed to have adaptive value as a driver of
variation in offspring and parental fitness and have only occasionally been reported
for aquatic wildlife. Recently, García-Roger and Ortells (2018) showed that the
survival of a population of the well-known euryhaline rotifer Brachionus plicatilis
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Fig. 6.7 Enrichment analysis of transcripts significantly correlated with the veliger rates of the
king scallop (Pecten maximus). Significant enriched (biological process) BP_direct terms obtained
through the enrichment analysis performed on the transcripts significantly correlated with the veli-
ger rates. The green bars identify the number of correlated genes belonging to the annotation term.
Only terms with minimum gene counts of five are reported. The numbers in the bars correspond to
the fold enrichment reported for each term. (From Pauletto et al. 2017, courtesy of the Public
Library of Science; image courtesy of the FAO)
is a function of the resource allocation of individuals. In particular, the production

of diapausing eggs is an ecological strategy commonly used by zooplanktonic
organisms to cope with adverse conditions in periods of varying habitats. Traits
related to diapause (e.g., the time spent in diapause, the hatching fraction of dia-
pausing eggs, or the amount of reserves allocated to them) are combined by natural
selection to favor adaptation to particular habitat conditions. García-Roger and
Ortells studied population differentiation in survival, hatching propensity, and the
amount of lipid reserves stored in diapausing eggs and found a negative relationship
between lipid content and survival (Fig. 6.8).
Non-mammal studies on nutritional deprivation are more frequent than those on
nutritional obesity. For instance, Frost et al. (2010) examined whether phosphorus
(P) limitation of Daphnia magna altered the responses of its offspring to inade-
quate P nutrition. Mother Daphnia consuming P-poor algal food produced smaller
neonates, which had lower body P content than control (P-rich) mothers. These
offspring from P-stressed mothers, when fed P-rich food, grew faster and repro-
duced on the same schedule as those from P-sufficient mothers. In contrast, the
offspring from P-stressed mothers, when fed P-poor food, grew more slowly and
had delayed reproduction than their sisters born to control mothers and fed P-poor
diets. The authors also presented some evidence that daughters from P-stressed
mothers were more susceptible to infection by a virulent bacterium. This study
showed not only that P-stress is transferred across generations but also that its
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Fig. 6.8 Relationships between diapause-related traits in Brachionus plicatilis diapausing eggs.
(a): survival and estimated lipid content—measured as pixel intensity after oil red O staining. (b):
survival and hatching fraction. The dots represent population averages of diapause-related traits,
and the error bars are ±1 SE. For each plot, linear regression is represented by a solid line (—) and
95% confidence intervals (upper and lower bounds) are represented by short dashed lines (--).
(From García-Roger and Ortells 2018, with permission from Springer)
effect on offspring varies depending on the quality of their own environment, such
as the presence of pathogens.
With two clones of D. magna, Lyu et al. (2016) showed that maternal consump-
tion of non-toxic Microcystis induced tolerance to toxic Microcystis in offspring.
The authors fed neonates from mothers fed toxic and non-toxic Microcystis. The
neonates demonstrated enhanced growth and reproduction compared with neonates
produced from mothers fed only a green algal diet. This means that Daphnia neo-
nates can tolerate toxic cyanobacteria when their mothers were fed diets containing
non-toxic or toxic strains of cyanobacteria. Furthermore, elevated RNA–DNA ratios
and SOD and CAT activities in neonates fed diets containing Microcystis demon-
strated that the underlying mechanisms also involved processes associated with
metabolism and antioxidative status.
Daphnia magna mothers experiencing poor nutritional conditions produced off-
spring that were more resistant to bacterial infection than mothers in favorable con-
ditions (Chapter 4 ‘Dietary Restriction, Starvation, Compensatory Growth’). This
effect occurred when mothers who were well provisioned during their own develop-
ment then found themselves reproducing in poor conditions. These effects are likely
to reflect adaptive optimal resource allocation whereby better-quality offspring are
produced in poor environments to enhance survival (Mitchell and Read 2005).
Comparable effects were also observed in D. pulex: maternal food limitation trig-
gered females to produce fewer but larger neonates that were more tolerant of toxic
Microcystis aeruginosa (Li and Jiang 2014).
Given that the size of offspring individuals is indicative of their energy content,
Gabsi et al. (2014) demonstrated that offspring size (OS) is significantly related to
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Fig. 6.9 Dependence of the mean offspring size on the maternal body size (a) and age (b) in
Daphnia magna in relation to the food level. (From Gabsi et al. 2014, with permission from EDP
Sciences)
maternal body size and age at all food concentrations (Fig. 6.9a, b). The slopes of
the different regression equations linking OS to these two variables were generally
steeper at low (0.05 and 0.075 mg C) than at higher food concentrations (0.1–1 mg
C), indicating that well-fed mothers invest less in offspring quality than low-fed
mothers. This could be confirmed on the basis of a daily amount of ingested carbon:
independent of the maternal body size, there was a significant negative relationship
between OS and the ingested carbon, since the largest neonates were born to moth-
ers with the smallest amount of ingested carbon and the smallest neonates to moth-
ers with the largest amount of ingested carbon.
Furthermore, large females (body size≥3.5 mm) reared at high food concentra-
tions produced smaller offspring than low-fed ones of the same size (Fig. 6.9a).
However, for smaller mothers (body size≤3 mm), OS had a non-linear response to
feeds: it increased with decreasing food concentration from 1 to 0.1 mg C and then
decreased when food decreased down to 0.05 mg C. In comparison, females of the
same age produced smaller offspring with increasing food concentration (Fig. 6.9b).
This example confirms that the adaptive benefits of mothers’ larger per offspring
investment are greater under stressful than under benign conditions.
In Daphnia, the most commonly described maternal effect is when a mother
adjusts the reaction norm of her descendant to the conditions anticipated in the
future. It is a so-called “anticipatory maternal effect” (AME; for a review, see
Marshall and Uller (2007)). Recently, Mikulski and Pijanowska (2017) investigated
the relative importance of maternal and own risk perception in the multi-trait
response of D. magna to the presence of fish. It appears that a maternal effect is
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Fig. 6.10 Survival of experimental Daphnia magna offspring; the treatment code indicates mater-
nal (first letter) and own (second letter) experience (F) or lack of experience (C) of fish presence.
(Mikulski and Pijanowska 2017, courtesy of Springer)
involved in shaping some key traits of adaptive changes in life history of the off-
spring: the duration time of egg holding in the brood chambers, age and size at first
reproduction (primiparia), and number of first-clutch neonates. Other traits, such as
the duration time of the release of the first clutch of neonates from the brood cham-
ber and the size of neonates, are determined by the environmental risk. The relative
contributions of maternal and own perception of risk in shaping individual pheno-
types depend on the time needed to express a particular trait (Fig. 6.10). In particu-
lar, adaptive changes that are expressed shortly after the exposure to a predation
threat can be determined upon direct perception of environmental risk. However,
developmental changes requiring a longer time to be expressed in offspring depend
primarily on the maternal perception of the risk. Similarly, the maternal effect domi-
nates the expression of traits that are primarily or solely determined in the early
stages of ontogenesis.
Maternal effects are widely observed, but their adaptive nature remains difficult
to describe and interpret. Coakley et al. (2018) investigated adaptive maternal
effects in a clone of D. magna, experimentally varying both maternal age and mater-
nal food and subsequently varying food available to offspring. The authors had two
main predictions: that offspring in a food environment matched to their mothers
should fare better than offspring in unmatched environments, and that offspring of
older mothers would fare better in low food environments. Coakley et al. (2018)
detected numerous maternal effects, for example offspring of poorly fed mothers
were large (Fig. 6.11), whereas offspring of older mothers were both large and
showed an earlier age at first reproduction. However, these maternal effects did not
clearly translate into the predicted differences in reproduction. Thus, the predictions
about adaptive maternal effects in response to food variation were not yet met in this
genotype of D. magna and remain to proven in studies to come.
In the Australian midge Chironomus tepperi, Townsend et al. (2012) showed
that, under DR, adverse effects occur in filial generations. First instar larvae were
subjected to food treatments with larval density controlled and offspring were raised
either under the same nutritional conditions as their parents or under standard con-
ditions. In DR treatments, adults in the P0 generation experienced delayed emer-
gence and females produced fewer egg masses. The P0 diet affected the performance
Fig. 6.11 The effect of

mothers (G0) food and age
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(defined by clutch) on
offspring body size (G1
generation). Error bars
represent one standard
error around the mean. LF
indicates low maternal
food, and HF indicates
high maternal food. (From
Coakley et al. 2018,
courtesy of Wiley)
of F1 with continued exposure, and the quality of the offspring was compromised.
Although sex ratios were not skewed, males and females responded differently to
DR, especially in the F1 generation, in which female development was more delayed
than in the P0 generation. This indicates that females are more sensitive to DR than
males. The adverse impact even translates into further life history traits: the off-
spring of parents reared under DR also had a shorter development time and lower
reproductive output than the offspring of parents raised under excess food but did
not change their susceptibility to chemicals, shown by exposure to Zn (Colombo
et al. 2014).
Studying the marine copepods Calanus finmarchicus and Euterpina acutifrons,
Guisande et al. (1999, 2000) put forward an interesting hypothesis: the amino acid
(AA) composition and hence the concentrations of essential AAs (EAAs) in the diet
determine their potential use for growth and reproduction. If the available pool of
free AAs (FAAs) is unbalanced relative to the demands for protein synthesis, deami-
nation of the excess AAs will occur and part of the ration will be lost through catab-
olism and ammonium excretion. In particular, the fecundity was mainly determined
by food concentration, while hatching success was correlated with food quality.
Elevated hatching success was obtained when the similarity between the AA com-
position of eggs and females and between the AA composition of females and food
algae was high (Fig. 6.12). However, reduced hatching success was observed when
the proportion of diatoms in the phytoplankton exceeded a certain threshold (>70%).
This study confirmed earlier observations that diatoms are non-suitable food for
copepods; for instance, the diatom Thalassiosira rotula adversely affects reproduc-
tive success in the copepod Acartia clausi (Ianora et al. 1996). Later, it was demon-
strated that two classes of oxylipins, polyunsaturated aldehydes and non-volatile
Fig. 6.12 Relationship
between hatching success
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in Euterpina acutifrons and

similarity between the
amino acid composition of
females and the seston
3–20 μm size fraction.
Percentage of diatoms in
the phytoplankton
community lower (□) and
higher (■) than 70%.
(From Guisande et al.
2000, courtesy of
Inter-Research; ©Inter-
Research 2000)
oxylipins, in the diatoms are responsible for the apparently toxic effect on copepod
reproduction (Ianora et al. 2015).
In a laboratory study with C. finmarchicus, Helland et al. (2003) detected that the
highest cumulative egg production was correlated with a high degree of similarity
in the free pool of EAA in the food suspension and the female copepods, thus sup-
porting the above hypothesis of Guisande et al. Studying A. tonsa, Broglio et al.
(2003) tried to expand this hypothesis to the FA patterns in the food source. The
diets, offered in monoculture, were the heterotrophic ciliates Strombidium sulcatum
or Mesodinium pulex, the heterotrophic dinoflagellate Gymnodinium dominans, the
autotrophic cryptophyte Rhodomonas salina, and the autotrophic dinoflagellate
Gymnodinium sanguineum. The diets were related to egg production efficiency and
reproductive success. Clear differences were found in the FA contents and the com-
position of the different diets offered, but these differences did not correspond to the
variations in egg production efficiency. However, egg viability was again correlated
with the ingestion of certain prey essential FAs (Fig. 6.13). In Acartia erythraea,
Chen et al. (2012) demonstrated that food quality (ingested PUFAs) was important
for egg production in addition to food quantity. Among PUFAs, DHA was the most
important specific fatty acid based on its high partial correlation coefficient for the
egg production rate. Egg hatching success was significantly correlated with the per-
centage of the three major ω3 unsaturated fatty acids (α-linolenic acid ALA, eicosa-
pentaenoenic acid EPA, and DHA) in diets. In Temora longicornis, particularly
DHA and the ratio of DHA/EPA have the strongest steering effect on the hatching
effect (Arendt et al. 2005). Overall, these studies of marine copepods clearly show
that the maternal food composition, the AA and FA patterns, modulates the hatching
success of F1 offspring.
Continuing the studies with A. tonsa, Acheampong et al. (2011) demonstrated
that food limitation did not affect the biochemical composition of adult copepods
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Fig. 6.13 Acartia tonsa. Relationship between hatching success and ingestion rates of fatty acids.
(a) Polyunsaturated fatty acids (PUFAs); (b) essential fatty acids (EFAs) (18:
3ω3 + 20:5ω3 + 22:6ω3). Asymptotic curves are shown: Y hatching success, X ingestion of fatty
acids. (From Broglio et al. 2003, courtesy of Inter-Research; ©Inter-Research 2003)
but did affect the production rate and biochemical composition of the eggs.
Food-limited females moderated the cost of reproduction by producing eggs with-
out much modification to the substrates that they ingested.
It is easy to predict that maternal influences are also closely related to offspring
survival. The study by Sato and Suzuki (2010) examined the effects of female body
size on larval body size, weight, and survival period in the coconut crab (Birgus
latro). Larval size increased significantly with increasing female size. Larvae
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Fig. 6.14 Rotifer Brachionus plicatilis: life span, fecundity, and oxidative-stress resistance of
mothers cultured under different feeding regimes. (a) Rotifers fed ad libitum (AL) or under calorie
restriction (CR), providing food on every other day. Boxes indicate 24 h. (b) The life span (solid
line) of the CR group was about 50% longer than that of its AL counterpart. The number of daugh-
ters (broken line) of the CR group was significantly lower than that of its AL counterpart. (From
Kaneko et al. 2011, with permission from Wiley)
hatched from larger females showed significantly longer survival periods under
non-fed conditions.
Increasing evidence shows that food quantity matters for determining the mater-
nal effects that are forwarded to offspring. For instance, Kaneko et al. (2011) inves-
tigated DR—here termed as calorie restrictions (CR)—effects on the life span,
oxidative stress resistance, and the expression levels of two antioxidant enzymes
[CAT and SOD1 (=Mn-SOD)] in the parthenogenetic euryhaline rotifer Brachionus
plicatilis during two consecutive generations. Rotifers under CR lived 50% longer
than those fed AL (Fig. 6.14) in association with the enhancement of oxidative
stress resistance and increased mRNA levels of cat and sod1. The daughters of the
CR-treated mothers lived 20% longer than those of the mothers fed AL regardless
of food-rich and CR conditions for the daughters (Fig. 6.15). Furthermore, the
daughters from CR-treated mothers were endowed at birth with a greater ability to
resist oxidative stress and increased mRNA levels for cat but not for sod1. In agree-
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Fig. 6.15 Brachionus plicatilis: life span and oxidative stress resistance in daughters from ad
libitum (AL) and calorie-restricted (CR) mothers. The mothers were transferred daily to freshly
prepared media, with AL feeding or CR feeding, and their daughters were subjected to the experi-
ment. The different superscripts indicate significant differences. (a) The neonates produced by the
AL and CR mothers at the age of 5 days were cultured under AL or CR feeding conditions. (b) The
neonates produced by the AL and CR mothers at the age of 5 and 7 days and each cultured in the
medium containing 50 mM paraquat (a model chemical provoking oxidative stress in exposed
organisms) under AL feeding. (From Kaneko et al. 2011, with permission from Wiley)
ment with the mRNA expression patterns, CR increased the protein levels of CAT
and SOD1 in eggs and the whole body of mothers, respectively.
In B. calyciflorus, DR-mediated life span extension was combined with a tran-
scription increase of four hsp genes: hsp40, hsp60, hsp70, and hsp90 (Yang et al.
2014a). Because under non-DR conditions the transcription of hsp70 decreased
with age, which indicated waning of the heat shock response with aging, the authors
concluded that the DR-mediated upregulation of the four hsp genes is a major rea-
son for longevity in this rotifer. Furthermore, DR significantly increased the tran-
scription level of Mn-SOD, CuZn-SOD, and CAT, and the improved antioxidant
protection of aging individuals can be another reason (Yang et al. 2013). Overall, it
would be challenging to figure out whether HSP90 in Brachionus also has the capa-
bility to act as a capacitor of phenotypic traits (see Sect. 6.2.2.3).
After collecting evidence for transgenerational effects of maternal nutrition, the
question arises of how decreased fecundity and increased investment per offspring
translate into more than one succeeding generation. Does only the first filial genera-
tion benefit from this maternal decision or does it propagate to further generations?
Do the individual’s actions affect not only its own survivorship and reproductive
performance but also those of its offspring and later descendants? Does increased
investment per offspring translate into offspring fitness? From a population dynamic
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perspective, these effects are important, because they mean that a population’s
response to environmental change may be time lagged to some degree, with inter-
generational effects operating as a source of intrinsic delayed density dependence
(Plaistow et al. (2006) and references therein).
Since extracting detailed information on the strength and duration of maternal
effects from field data is fraught with difficulty, as one has to disentangle the effects
of changes in juvenile density from changes in juvenile quality and from cohort
effects created by other aspects of the immediate neonatal environment, correspond-
ing studies are rather scarce. Plaistow et al. (2006) therefore undertook an experi-
mental study to measure the strength of intergenerational effects on a number of life
history traits in a number of different environments over three generations, using the
soil mite Sancassania berlesei in a crossed factorial design (studies with aquatic
animals obviously do not exist). Their study design enabled them to separate the
relative influence of current and past environments on multiple key life history traits
and measure the nature, strength, and duration of intergenerational effects across a
range of environmental backgrounds.
Plaistow et al. (2006) showed that manipulating the parental food environments
of soil mites produced intergenerational effects that were still detectable in the life
histories of descendants three generations later. Intergenerational effects vary in dif-
ferent environments and from one generation to the next. In low-feed environments,
variation in the egg size altered the trade-off between age and size at maturity and
had little effect on the egg size produced in subsequent generations. Consequently,
intergenerational effects decreased over time. In contrast, in high-feed environ-
ments, variation in the egg size predominantly influenced the trade-off between
fecundity and adult survival and generated increasing variation in the egg size
(Fig. 6.16). As a result, the persistence and significance of intergenerational effects
varied between high- and low-feed environments. Context-dependent intergenera-
tional effects can therefore have complex but important effects on population
dynamics.
In a subsequent paper, Plaistow and Benton (2009) showed that maternal effects
are complex. At the individual level, maternal effects influence different traits in
context-dependent ways. There is no simple correlate of any univariate measure of
maternal performance and offspring performance. Large females can lay small eggs
(when young or if starved) or large eggs (when they age). Large eggs can hatch into
offspring who grow fast or slowly. If the offspring grow slowly, they can increase
their survival or later fecundity depending on their food levels. This complexity
implies that there is no single hypothesis regarding how maternal effects may act at
the population level. Instead, the paper shows that perturbing the population age
structure (i.e. the initial conditions) may dampen or accentuate the population-level
manifestation of maternal effects.
Plaistow and Benton (2009) concluded that interactions between intergenera-
tional effects in different environments, and the influence that they have on the
strength and persistence of maternal effects, have generally not been measured.
Therefore, it can be argued that maternal effects will have a greater influence on the
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Fig. 6.16 Effect of high and low original parental environments on the reaction norm of age and
size at maturity of their offspring, grandoffspring, and great-grandoffspring in the soil mite
Sancassania berlesei. The 95% range lines of age and size are shown for high (solid lines) and low
(dashed lines) parental environments. Offspring from a high original parental environment start off
smaller and older at maturity than offspring from a low original parental environment in the P0 (F1)
generation (the maternal effect). In the F2 generation (the grandmaternal effect), grandoffspring
transient dynamics of populations that initially experience benign conditions for

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one or more generations. However, studies of interactions between intergenerational

effects in other species are required before their significance can generally be
understood.
Recently, a particularly intriguing transgenerational experiment of starvation
was reported by Kamizono et al. (2017) for Brachionus manjavacas. Starvation
affects the sexual reproduction of this cyclic parthenogenetic rotifer. Short-term
starvation of rotifers that experienced starvation immediately after hatching from
resting eggs can cause high induction of sexual reproduction (mixis). Kamizono
et al. (2017) detected that the signal is epigenetically inherited up to the fortieth
generation (Fig. 6.17), since the mixis induction in offspring from starved stem
females was significantly greater than in those from non-starved stem females. This
mechanism can facilitate colonization by favoring population growth via female
parthenogenesis and by decreasing the food requirements for survival and
reproduction.
6.1.1.2 F
ishes
Usually it is assumed that most of the parentally induced variation in larval traits is
attributable to maternal effects propagated through the egg characteristics and gen-
erally paternal effects have not been considered. As a consequence, much of the
previous research has focused on detecting maternal effects in the phenotypic traits
of eggs, and evidence for significant maternal effects on the egg size is now well
established for many species (Chambers and Leggett 1996). Paternal or male effects,
on the other hand, are almost always considered as synonymous with genetic effects,
since the only significant contribution of the sperm is DNA. The ability to observe
potential paternal and hence genetic effects on early life history has, however, been
impaired by empirical weaknesses (milt from multiple males when fertilizing eggs
or eggs from a single spawning event often being fertilized by multiple males)
(Bang et al. 2006). The variable results concerning the degree to which males and
females affect larval traits are probably dependent on the difference in the egg size
between females. If the egg sizes are similar, it may allow male-induced effects to
be observed, but these effects may be masked if the egg sizes are very different
(Bang et al. 2006 and references therein).
Bang et al. (2006) used a factorial mating design to analyze the maternal and
paternal effects on the early life history traits of Atlantic herring (Clupea harengus)
Fig. 6.16 (continued) from low original parental environments are larger under high-feed condi-
tions but take longer to mature under low-feed conditions, resulting in an expanded reaction norm
compared with that of high-parental-environment grandoffspring. Finally, in the F3 generation, the
maximum and minimum ranges of size and age at maturity are similar for the two treatments,
although there is clearly still a difference in the distribution of the points for individuals from high
and low original parental environments. The effect of a medium original parental environment was
similar to that of the low parental environment and was therefore omitted to improve the clarity of
the plots. (From Plaistow et al. 2006, with permission from the University of Chicago Press)
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Fig. 6.17 Changes in the mixis induction with every tenth generation starting from the starved or
non-starved rotifer mothers. An open circle represents the mixis induction (%) of 8 clones into
each generation from non-starved mothers (control), while the filled circle represents those from
stem female starved for 12 h. The open squares and filled squares represent the average mixis ratio
in each group. The upper part of different letters on the x-axis represents significant differences
among the combination of experimental groups and generations (2-way ANOVA, experimental
group*generation, P = 0.000; a < b < c < d, P < 0.05). (From Kamizono et al. 2017, with permis-
sion from Springer)
larvae and partition the observed phenotypic variance into its underlying genetic
and non-genetic components. A summary of the recorded early life history traits of
all larvae at the hatch is depicted as box plots in Fig. 6.18. There were parental
effects on all of the examined traits. A sire effect was evident on the standard
length, yolk sac volume, and RNA:DNA ratio. A dam effect was evident on the
standard length, yolk sac volume, and dry weight, with females explaining a par-
ticularly large part of the variation in yolk sac volume and dry weight. Overall,
maternal effects were detected in the larval weight and yolk sac volume, while
paternal and hence genetic effects appeared in the larval length, yolk sac volume,
and RNA:DNA ratio. The findings also showed that increased emphasis should be
placed on the importance of male influence on the success of early larval fishes
(Refer to Sect. 6.1.2).
A fatty-acid-based maternal effect was detected by Patterson and Green (2014)
in Gulf killifish (Fundulus grandis), a euryhaline cyprinodont. The authors utilized
experimental variations in exogenous FAs available to spawning F. grandis to con-
struct a multitissue evaluation of FA allocation and quantify effects on reproductive
Fig. 6.18 Box plots of an

Atlantic herring (Clupea
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harengus) study. (a) Yolk

sac volume, (b) larval
standard length, and (c)
RNA:DNA ratio. The top
and bottom of the box
represent the upper and
lower quartiles,
respectively. The solid and
dashed horizontal lines
within the box represent
the median and the mean
value of the data,
respectively. The upper and
lower limits of the
whiskers represent the
largest and smallest values
within 1.5 interquartile
ranges of the top and the
bottom of the boxes,
respectively. Data outside
the whiskers are
considered to be outliers
and represented by solid
circles. Parental
combinations: 1–5: male 1
with females 1–5; 6–10:
male 2 with females 1–5;
11–15: male 3 with
females 1–5. (Modified
from Bang et al. 2006,
Elsevier)
output and offspring fitness. No significant decrease in fecundity occurred in ani-

mals consuming low levels of long-chain poly-unsaturated FAs (LC-PUFAs),
although the embryo viability rates were affected. Furthermore, maternal dietary
FA variation produced differences in starvation tolerance, hypo-osmoregulatory
ability, and acute thermal stress tolerance for larvae. The FA composition of eggs
and tissues from spawning females point at the potential biosynthesis of LC-PUFAs
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from shorter-chain (C18) precursors in F. grandis and indicate that this species pos-
sesses physiological mechanisms allowing the maintenance of the reproductive
function when subjected to dietary deficiencies in FAs that are generally considered
to be essential for marine fishes.
In two papers, Houde et al. (2015, 2011) tried to unravel the genetic, maternal,
paternal, and environmental factors influencing the phenotypic variance of survival
and fitness-related traits in juvenile Atlantic salmon from three allopatric popula-
tions. There was a shift from maternal environmental to genetic effects during
development in two of the populations. That is, the maternal environmental effects
were larger at early (egg and alevin) life stages, whereas the non-additive effects
were larger at the latter (fry) life stage. The amount of genetic effects was small,
showing that the traits respond slowly to selection.
Little is known about how maternal effects vary throughout ontogeny. Therefore,
Lindholm et al. (2006) estimated the contribution of maternal effects to offspring
body size from birth until 1 year of age in the live-bearing fish Poecilia parae. In
both sexes, the maternal effects on body size were initially high in juveniles (i.e., the
timing of sexual maturity) and then declined to zero at sexual maturity. Furthermore,
there was no relationship between early life maternal effects and adult longevity,
showing that maternal effects, although important early in life, may not always
influence late life history traits. This indicates that these effects may not persist to
later life history stages (i.e., adult longevity).
Egg and Embryo Quality
Also in fish, AME are widespread and non-genetic in nature and comprise a class of
phenotypic effects that parents have on phenotypes of their offspring that are unre-
lated to the offspring’s own genotype. The effects may take one or more of the fol-
lowing routes: (1) Epigenetic inheritance; (2) cytoplasmic inheritance; (3) maternal
nutrition either via the egg or via pre- and post-natal supplies of food; (4) transmis-
sion of pathogens and antibodies through the pre-natal blood supply or by post-natal
feeding; (5) imitative behavior; (6) interaction between sibs either directly with one
another or through the mother. Maternal effects can be interpreted as an important
mechanism that allows a (presumably adaptive) phenotypic response in offspring to
an environmental cue perceived by the parents. In other words, mothers might adjust
the phenotypes of their offspring in response to cues, perceived by the mother, about
the environment her offspring will encounter, in a way that enhances offspring fit-
ness (Bernardo (1996) and references therein).
Taborsky and her coworkers exemplified such non-genetic effects in the African
maternally mouth brooding cichlid Simochromis pleurospilus; in particular, they
showed, how maternal food abundance determines juvenile growth and behavior
(Segers et al. 2011; Segers and Taborsky 2011; Taborsky 2006).
Females of this cichlid adjust their egg size in response to their own juvenile
growth conditions (Taborsky 2006). Mothers that were raised on a low food regime
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Fig. 6.19 Simochromis pleurospilus: Growth trajectories of the four treatment groups for standard
length. Means and standard errors of the mean are shown for each measurement. The lines connect
the means. (From Segers and Taborsky 2011, picture of a juvenile by Karin Schneeberger, with
produced on average larger eggs than conspecifics raised on high food levels irre-
spective of prevailing food conditions. Segers and Taborsky (2011) examined the
potential survival benefits of hatching from a larger egg when facing food scarcity
in S. pleurospilus. Egg size may determine initial hatchling size and it may directly
affect growth trajectories. The authors hypothesized that in addition egg size may
affect growth and survival prospects indirectly via altering risk-taking behavior.
Therefore, they hand-raised individuals from a broad range of egg sizes, and fol-
lowed their growth trajectories and their behavioral development individually. They
experimentally removed maternal care as potential determinant of offspring size,
and disentangled the effects of egg size and resource availability on offspring growth
trajectories and behavior in the absence of additional variance generated by com-
petitive interactions among siblings.
Segers and Taborsky (2011) expected that under high-food conditions, pheno-
types will converge quickly, both with regard to size and behavior, whereas under
low-food conditions initial size differences are retained for longer with large-egg
fish behaving more cautiously than small-egg ones. Food level affected the growth
of the fish, as the high-food individuals grew faster than the low food ones (Fig. 6.19)
and were larger at the start of the experimental period; body mass increase followed
the same patterns as standard length. Furthermore, larger eggs gave rise to larger
young that had a higher burst swimming speed. Food ration greatly influenced long-
term growth, while egg size, predominantly affected fish size during the first 2 weeks
of life. However, the large egg size caused a size advantage of juveniles persisting
throughout the experimental period.
Fig. 6.20 The total time

the fish (Simochromis)
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spent hiding during 10 min

observation time during the
three trials for each
treatment group. S-L
small-egg/low-food, L-L
large-egg/low-food, S-H
small-egg/high-food, L-H
large-egg/high-food. The
boxplots show medians,
quartiles and fifth and
ninetyfifth percentiles.
(From Segers and Taborsky
2011, with permission
from Wiley)
Additionally, Segers and Taborsky (2011) examined whether a lack of body

reserves drives juvenile behavior: fish from small eggs hid longer, whereas food
treatment as main effect did not affect the time spent in shelter by a fish. Egg size
and food ration interactively affected the hiding and foraging behavior of young
(Fig. 6.20). In the low-food treatment, individuals from small eggs spent less time
in shelter and showed a higher commitment to foraging than individuals from large
eggs. In a natural setting, this should markedly increase predation risk of young
originating from small eggs, particularly in poor environments. In contrast, when
the food was plentiful juveniles behaved similarly, irrespective of egg size.
Overall, egg size strongly affects body size in S. pleurospilus, and fish hatched
from larger eggs, swim faster. When food is scarce, juveniles originating from
smaller eggs show more risk-prone behavior that might make them more suscepti-
ble to predation. Thus, the more cautious behavior of individuals originating from
larger eggs may increase their survival chances when growing up under harsh con-
ditions. The observed effects of egg size on juvenile growth and behavior were most
pronounced in the first weeks of life, but weakened with time (Segers and Taborsky
2011).
One additional highly influential transgenerational mechanism by which parents
can affect the phenotype and survival of their offspring is the quality of brood care.
The results of this trait can best seen in mouthbreeders as shown again with the
African cichlid S. pleurospilus. Segers et al. (2011) found that food-deprived females
produced smaller young and engaged less in brood care behavior than well-nourished
females. Final brood size and, related to this, female protective behavior were inter-
actively determined by nutritional state and predator exposure: well-nourished
females without a predator encounter had smaller broods than all other females and
at the same time were least likely to take up their young after a simulated predator
attack. In sum, food treatment influenced brood care patterns most strongly.
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Fig. 6.21 Fish on a switched diet have a superior learning performance. Relationship between
early nutrition and associative learning performance in juvenile (left panel) and adult (right panel)
Simochromis pleurospilus. In both tests neither early nor late resource availability influenced later
learning performance. The interaction between treatments was significant, however, indicating that
animals which experienced a switch between treatments during their upbringing outperformed
those fed on constant rations. Left panel: mean residuals (±SE) of correct choices after accounting
for the number of tests fish participated in; right panel: mean residuals (±SE) of correct choices
after accounting for participation in tests and for previous testing experience. Experimental food
treatments: NHH (high food treatment), NLL (low food treatment), SHL (switched from high to low
food), and SLH (switched from low to high food). (From Kotrschal and Taborsky 2010, courtesy of
the Public Library of Science)
In natural habitats, food supply is strongly fluctuating. Flexible or innovative

behavior is advantageous, especially when animals are exposed to frequent and
unpredictable environmental perturbations. Improved cognitive abilities can help
animals to respond quickly and adequately to environmental dynamics, and there-
fore changing environments may select for higher cognitive abilities. Increased cog-
nitive abilities can be attained, for instance, if environmental change during
ontogeny triggers plastic adaptive responses improving the learning capacity of
exposed individuals. Kotrschal and Taborsky (2010) reported an interesting case by
testing the learning abilities of S. pleurospilus in response to experimental variation
of environmental quality during ontogeny. Individuals of this cichlid that experi-
enced a change in food ration early in life outperformed fish kept on constant rations
in a learning task later in life (Fig. 6.21), irrespective of the direction of the imple-
mented change and the mean rations received. This difference in learning abilities
between individuals remained constant between juvenile and adult stages of the
same fish tested 1 year apart. The results indicate a pathway by which a single
change in resource availability early in life permanently enhances the learning abili-
ties of animals. Early perturbations of environmental quality may signal the devel-
oping individual that it lives in a changing world, requiring increased cognitive
abilities to construct adequate behavioral responses.
To recall, the environmental quality influences the relationship between invest-
ment per offspring and offspring fitness. The optimal investment per offspring will
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Fig. 6.22 Left: Image of Salmo salar. (From Bloch 1782–1784, courtesy of Biodiversity Heritage
Library). Right: Relationships between optimal egg size of S. salar and environmental measures
of eight streams, including 95% confidence intervals. (Rollinson and Hutchings 2013,with permis-
sion from The University of Chicago Press)
increase as environmental quality decreases. To test this prediction, Rollinson and

Hutchings (2013) released juvenile Atlantic salmon (Fig. 6.22, left) into eight wild
stream environments, and monitored subsequent growth and survival of juveniles.
Rollinson and Hutchings (2013) described that optimal egg size was greater
when the quality of the stream environment was lower as the estimated habitat qual-
ity. Across streams, the mean size of stream gravel and the mean amount of incident
sunlight were the most important individual predictors of optimal egg size (Fig. 6.22,
right). Within streams, juveniles recaptured in stream subsections that featured
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Fig. 6.23 Images of Atlantic halibut (Hippoglossus hippoglossus) and turbot (Scophthalmus max-
imus). (From Bloch 1782–1784, courtesy of the Biodiversity Heritage Library)
larger gravels and greater levels of sunlight also grew relatively quickly. The size of
stream substrates is a driver of population regulation in Atlantic salmon, because
offspring production eclipses the amount of juvenile habitat that is available, such
that the acquisition of a fixed, food-based territory centered within river gravels is
paramount to juvenile survival. The size of stream gravel reflects the availability and
quality of juvenile territories for simple geometric reasons, given that an amalgam
of large gravel will have relatively more and larger inter-gravel spaces, and that
juveniles use these gravel interstices for shelter. Canopy closure was negatively
related to juvenile growth. An increase in incident sunlight stimulates the growth of
periphyton, and this has the direct result of increasing local abundance of aquatic
invertebrates, particularly grazers. Therefore, juvenile salmon occupying territories
in sunlit areas have greater access to food.
Numerous studies have highlighted the maternal contributions to the develop-
ment of embryos, including transcripts that regulate cell division and determine
oocyte polarity, pattern development during early and late embryonic stages, and
the transition from maternal to zygotic gene expression and translation. Since most
fish embryos develop independently within an enclosed egg envelope, they rely on
compounds deposited within the oocytes during their various stages of develop-
ment. In addition to regulatory nucleic acids (maternal DNA and RNA), these
include proteins and other compounds that contribute to the structure and function
of the egg envelope and the bulk molecular cargo that will be used as a source of
cellular energy and structural components for the formation of embryos and larvae.
These latter components notably include yolk lipids and proteins deposited during
oocyte growth and water acquired at the same time and during cytoplasmic matura-
tion (Lubzens et al. 2017). Two examples may illustrate this topic.
In Atlantic halibut (Fig. 6.23), Mommens et al. (2014) identified the maternal
transfer of innate and adaptive immune system transcripts to embryos and their rela-
tion to future embryo developmental potential. Especially, irf7 (which encodes the
interferon regulatory factor 7, a transcription factor central to the transcriptional
activation of virus-inducible cellular genes) and mhc2a (which encodes major his-
tocompatibility complex IIα, molecules found in antigen-presenting cells) tran-
scripts can serve as molecular markers of embryo quality. Furthermore, in the
European sea bass, irf7 can serve as a biomarker for egg quality, besides rnf213
(which encodes E3 ubiquitin-protein ligase RNF213, involved in protein ubiquitina-

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tion) (Żarski et al. 2017). The authors also found new genes (namely usp5, plec, and
cenpf),2 the expression patterns of which have not yet been reported to be quality
dependent in any fish species. These results stress the importance of genes, or
groups of genes, being involved in protein ubiquitination, translation, DNA repair,
and cell structure and architecture, probably the mechanisms that contribute to egg
developmental competence, at least in the European sea bass.
Transgenerational Metabolic Programming
Fuiman and Perez (2015) reported metabolic programming and its functional con-
sequences in a marine fish, the red drum (Sciaenops ocellatus). The authors demon-
strated that maternal provisioning of eggs with the essential FA, DHA, varies with
the DHA content of the maternal diet. During a brief developmental window, DHA
plays a role in establishing the metabolic capacity of the offspring for its own uptake
or storage (for details, refer to the Volume II Chapter ‘Lipids and Fatty Acids’). This
has protracted and possibly permanent effects on the ecologically important sur-
vival skills of individuals and important implications for the dynamics of popula-
tions and food webs. Particularly, larvae with high PUFA (e.g., ARA and DHA)
content had good escape performance if challenged by a predator (Fuiman and
Ojanguren 2011). The transgenerational link that metabolic programming estab-
lishes between the ingestion of EFAs by adults and the fitness of offspring repre-
sents one specific pathway through which variations in EFA synthesis by primary
producers might transmit through food webs to alter and stabilize animal popula-
tions and communities (Perez and Fuiman 2015).
Another of the very few examples of metabolic and developmental programming
was reported by Chi et al. (2015). The authors studied the expression patterns and
activity of trypsinogen/trypsin of turbot (Fig. 6.23) and showed that trypsin/tryp-
sinogen in embryos was deposited in the oocytes before the gastrula stage and then
synthesized by the embryo (Fig. 6.24). The trypsin in turbot embryos not only is
involved in egg yolk degradation but also serves some other purposes, such as
embryo development and the hatching process.
Without doubt, diet-mediated transgenerational metabolic programming and its
underlying mechanisms—probably epigenetic ones—deserve much more attention
in future studies.
2
USP5 = ubiquitin carboxyl-terminal hydrolase 5, a deubiquitinating enzyme; PLEC = plectin-like
isoform, a giant protein acting as a link between the three main components of the cytoskeleton;
and CENPF = centromere protein F, involved in physical cell division.
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Fig. 6.24 Expression of trypsinogen transcripts during the different stages of development in
turbot (Scopthalmus maximus). The relative gene expression of trypsinogen was quantitated from
unfertilized eggs to 30 days using qPCR. un unfertilized eggs, 2c 2 cells, 8c 8 cells, bla blastula
stages, gas gastrula, kv Kupffer’s vesicle, hea: heart. (From Chi et al. 2015, with permission from
Elsevier)
Offspring Immunity and Fecundity
The maternal control directing the very first hours of life is of pivotal importance for
ensuring proper development of the growing embryo. Thanks to the finely regulated
inheritance of maternal factors, including mRNAs and proteins produced during
oogenesis and stored in the mature oocyte, the embryo is sustained throughout the
so-called maternal-to-zygotic transition, a period of development characterized by a
species-specific length of time, during which critical biological changes regarding
cell cycle and zygotic transcriptional activation occur (Miccoli et al. 2017).
After dietary administration of probiotics and prebiotics, beneficial parental
responses can be transferred to the succeeding generation. These dietary supple-
mentations have been recognized as playing important roles in many biological
systems, including immune response, growth, development, and reproduction (see
also Volume 2 ‘Functional Aquafeed’). However, to date, only a few studies have
focused either on the relation among probiotics and maternal factors or on probiot-
ics’ ability to modulate maternal transcripts qualitatively and/or quantitatively. For
instance, Miccoli et al. (2015) studied the effects of Lactobacillus rhamnosus
administered to parental zebrafish on the control of maternal factors involved in
autophagic, apoptotic, and dorsalizing processes during zebrafish embryonic devel-
opment. Dietary probiotics induced significant changes in both maternal and zygotic
mRNA levels involved in embryonic development. The maternal autophagy-
regulating genes investigated—ambra1a, ambra1b, beclin, and lc3—as well as
those involved in apoptosis—caspase3, bcl2, and bax—were modulated in disfavor
and favor of the treated group, respectively. The key transcripts ruling the dorsaliz-
ing process—goosecoid and chordin—were also subject to significant regulation of
their gene expression.
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Fig. 6.25 q-PCR graphs reporting the temporal gene expression of ambra 1a1, ambra 1b, beclin
1, caspase 3, bax, bcl2, goosecoid, and chordin in Danio rerio eggs and embryos. mRNA levels
normalized against 18S for the control (CTRL) and treated (PROBIO) groups. The error bars indi-
cate mean ± SD. The confidence interval was set at 95% (P < 0.05). (From Miccoli et al. 2015,
courtesy of the S. Karger AG, Basel).
A typical pattern of the maternally inherited transcripts exists throughout the first
8 h of development (Fig. 6.25). The gradual exploitation of mRNAs, and therefore
the constant decrease in their expression levels, were clearly deduced in all four
cases (Fig. 6.25a–c). Evidently, in the majority of the developmental stages ranging
from 0 hpf to 4 hpf, except for beclin1 and ambra1a levels, embryos descending
from probiotic-treated fish have a statistically significant lower availability of
autophagy-related transcripts (Fig. 6.25a–c). An opposing scenario was detected at
8 hpf, a stage at which treated embryos showed a greater quantity of ambra1a and
beclin1 with respect to controls.
A situation similar to those uncovered for the first 4 h of development (i.e. higher
levels of autophagy-related transcripts in control embryos than in treated ones) was
identified in the remaining developmental stages (12, 24, and 48 hpf) for ambra and
beclin 1 genes (Fig. 6.25a). The hatching rates increased in the probiotic-fed P0
fishes (Fig. 6.26). Overall, the study by Miccoli et al. (2015) indicated that the sup-
plementation of Lactobacillus rhamnosus induced remarkable changes in the
Fig. 6.26 Hatching rates

of the two experimental
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groups of Danio rerio. The

percentage values were
transformed with the
arcsen function and
submitted to Student’s
t-test statistical analyses
(P < 0.05). (From Miccoli
et al. (2015), courtesy of
the S. Karger AG, Basel)
maternal and zygotic control of F1 fish, enabling them to undergo faster and more
successful embryonic development.
Concerning the significance of autophagy, Benato et al. (2013) reported that in
zebrafish the expression of the corresponding genes is driven by maternally con-
trolled transcripts stored in the oocytes during the oogenesis. Such transcripts fol-
low the typical maternal tendency, being firstly very abundant then progressively
exploited and eventually replaced by corresponding mRNAs by the zygotic machin-
ery itself. Autophagy—the process of self-eating—leads to the degradation of cyto-
plasmic components for the dynamic remodeling of subcellular compartments,
turnover and recycling of macromolecules, and regulation of cellular activity
through the control of specific intracellular signaling pathways. This fundamental
process is also implicated in the systemic response to starvation and immune chal-
lenges as well as antitumorigenesis and anti-senescence. Recent studies have also
highlighted an important role for autophagy in embryonic development (Wada et al.
2014), with cardiac morphogenesis being one major process (Lee et al. 2014).
In addition to dietary probiotics, prebiotics, such as β-glucans, are well-
documented “conductors of immune symphonies” (Dalmo and Bøgwald 2008) and
can be used safely to promote the non-specific immunity in offspring of fishes. For
instance, Jiang et al. (2016) reported that dietary β-glucan enhances the contents of
complement component 3 and factor B in eggs of zebrafish (Fig. 6.27). Component
3 is a protein of the immune system, central to the complement system, thereby
contributing to innate immunity, and complement factor B is an acute-phase protein
that increases during inflammation.
To test whether the embryos derived from β-1,3 glucan-fed females were more
resistant to bacterial challenge, the embryos at the 4- to 8-cell stage from control
and experimental groups were microinjected with phosphate-buffered saline (PBS)
or PBS plus live A. hydrophila. The mortality of the embryos from glucan-fed D.
rerio was significantly lower than that of the embryos from control fish (Fig. 6.28a).
To prove further that the embryos of glucan-fed females have stronger antibacterial
Fig. 6.27 Contents of

complement compound 3
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(C3, a) and factor B (Bf, b)

in serum and zebrafish egg
cytosol. Data are expressed
as mean ± SD from three
experiments. * and **
indicate significant
differences at P < 0.05 and
P < 0.01. (From Jiang et al.
(2016), with permission
from Elsevier)
activities than those of control fish, a specific region of the A. hydrophila 16S rRNA
gene was amplified. Little difference was observed in the expression of the 16S
rRNA gene of A. hydrophila in the embryos from glucan-fed and control zebrafish
soon after the bacterial injection (Fig. 6.28), but its expression in the embryos from
glucan-fed zebrafish was markedly lower than that in the embryos from control fish
6 and 12 h after injection. Overall, feeding female zebrafish with β-glucan had few
detrimental effects on the number of spawned eggs and their embryonic develop-
ment. Therefore, brood fish can be fed with β-glucan as an alternative strategy to
improve the immune status and disease resistance of fish embryos.
In fishes, the maturation of lymphoid organs and immunocompetence is delayed,
with very limited synthesis of specific antibodies during embryonic development
and early larval stages; therefore, maternally derived immunity is essential in these
early phases. Qin et al. (2014) compared the effects of two probiotic strains,
Lactobacillus rhamnosus CICC 6141 (a highly adhesive strain) and L. casei BL23
(a weakly adhesive strain), on zebrafish reproduction and their offspring’s innate
level of immunity to waterborne pathogens. During probiotic treatments from day
7 to day 28, both Lactobacillus strains, and especially L. casei, significantly
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Fig. 6.28 Increase in antibacterial activity of glucan-treated zebrafish embryos. (a) The embryos
from non-treated and glucan-treated females were injected with PBS alone or with PBS plus live
A. hydrophila. The cumulative mortality 24 h after injection was calculated. (b) The embryos from
non-treated and glucan-treated females were injected with PBS alone or with PBS plus live A.
hydrophila. Five embryos were sampled each time 0, 6, and 12 h after injection and used to isolate
the DNAs for amplification of the specific region of the A. hydrophila 16S rRNA gene by qRT-
PCR. Data are expressed as mean ± SD from three experiments. * and ** indicate significant dif-
ferences at P < 0.05 and P < 0.01. (From Jiang et al. 2016, with permission from Elsevier)
increased the fecundity in zebrafish: higher rates of egg ovulation, fertilization, and
hatching were observed (Fig. 6.29). Increased densities of both small and large
vitellogenic follicles, seen in specimens fed either Lactobacillus strain, demon-
strated accelerated oocyte maturation. Feeding either strain of Lactobacillus upreg-
ulated the gene expression of leptin (a hormone that regulates appetite), kiss2 (a
peptide that stimulates gonadotropin release), gnrh3 (gonadotropin-releasing hor-
mone), fsh (a follicle-stimulating hormone), lh (a luteinizing hormone), lhcgr (a
luteinizing hormone/choriogonadotropin receptor), and paqr8 (a progestin and adi-
poQ receptor), which are regarded as enhancing fecundity and encouraging oocyte
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Fig. 6.29 The effects of the probiotics on the fecundity of brood fish and larval quality in zebraf-
ish. The number of ovulated eggs per week by females (a), the fertilization rate of eggs (b), the
hatching rate of fertilized eggs (c), and alkaline phosphatase (AP) activity in the whole-body sam-
ples (d) of offspring in a temporal series. The results are shown as mean ± SEM for each treatment.
A significant difference is denoted by a single asterisk (P < 0.05). Labels: 6141 = L. rhamnosus
CICC 6141 treatment group; BL23 = L. casei BL23 treatment group. (From Qin et al. 2014, with
permission from the Society for Reproduction and Fertility)
maturation. Concomitantly, the gene expression of bmp15 (bone morphogenetic

factor 15) and tgfb1 (transforming growth factor β) was inhibited. Both genes code
for factors that prevent oocyte maturation. The beneficial effects of the Lactobacillus
strains on fecundity diminished after the feeding of the probiotics was discontin-
ued, even for the highly adhesive gut Lactobacillus strain. Administering L. rham-
nosus for 28 days was found to affect the innate immunity of offspring derived from
their parents, as evidenced by a lower level of alkaline phosphatase activity in early
larval stages. In summary, using either L. rhamnosus or (especially) L. casei as a
food additive was an important avenue through which to stimulate follicle matura-
tion, enhance fecundity, and improve egg quality in zebrafish. Furthermore, admin-
istering L. rhamnosus can affect the fitness level of offspring during early stages of
larval development. In particular, after probiotic treatment, the quality of the
embryos improved, as evinced by higher fertilization and hatching rates (Fig. 6.29a–
c) and by lower alkaline phosphatase (AP) activity in the offspring’s early stages of
development (Fig. 6.29d). The underlying mechanism remained obscure; neverthe-
less, this study indicated the potential for using these two probiotics strains (either
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Fig. 6.30 The sperm epigenome: a messenger of ancestral exposures. Schematic overview of
environmentally acquired epigenetic changes and disorders in the offspring through the paternal
germ line. Examples of studies on transgenerational inheritance include exposures to malnutrition
(such as famine) or over-nutrition, a low-protein diet, vitamin or micronutrient deficiencies, a high
fat diet, plastic-derived toxins, obesity, smoking, distress, paint and solvents, pesticides, ionizing
radiation, and war. Although the molecular components are largely known, it is unclear how they
are interlinked and how or when the environment interferes in these processes. (From Soubry
2015, and references therein and courtesy of Elsevier)
separately or combined) for the beneficial manipulation of stock reproductive capa-

bilities in aquaculture.
6.1.2 Paternal Effects
Paternal effects are the causal influence of the paternal genotype or phenotype on
the offspring phenotype (from Wolf and Wade 2009). Increasing evidence supports
the idea that at least some epigenetic marks acquired during spermatogenesis can be
sustained through embryonic development. A cartoon (Fig. 6.30) links the effects of
several environmental exposures with potential molecular changes during male
gametogenesis in mammals, causing persistent epigenetic alterations and pheno-
typic consequences in the next generation(s). The sperm epigenetic machinery
includes DNA methylation, histone modifications, and transcription of non-coding
RNAs [such as microRNAs (Fullston et al. 2013), see below]. Male germ cells
develop from primordial germ cells (PGCs) into spermatogonia (SG) before puberty.
They further differentiate into spermatocytes (SC) and finally spermatozoa (SZ)
during each reproductive cycle. Enzymes, such as DNA methyltransferases
(DNMTs) and histone deacetylases (HDACs), often form a link between these com-
ponents; they are important for fine-tuning the intermolecular effects. Unbalanced
reactive oxygen species (ROS) generation may also trigger this fine-tuning.
Environmental messages are able to alter the epigenetic machinery in male germ
cells. If the effects persist, these alterations may be beneficial (green), they may
disturb homeostasis or metabolism (orange), or they may be harmful (red) to the

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next generations (Soubry 2015).

In fishes, several studies designed explicitly to identify paternal effects have
shown that paternity can account for a significant portion of variation in phenotypic
expression and survival during early life history in Atlantic herring (Høie et al.
1999), brown trout (Vøllestad and Lillehammer 2000), and haddock (Probst et al.
2006; Rideout et al. 2004). Furthermore, previous studies on winter flounder have
reported paternal effects on fertilization success as well as on the survival and devel-
opment of embryos and larvae (Butts and Litvak 2007a, b). In contrast to these spe-
cies, studies performed on Atlantic cod in the Baltic Sea and Atlantic herring have
found no paternal effects on early life history traits or if so only via maternal–pater-
nal interactions (Bang et al. 2006; Chambers and Leggett 1996; Chambers et al.
1989; Trippel et al. 2005).
Recently, Kroll et al. (2013) found contrasting results when evaluating the poten-
tial influence of paternity on rates of mortality and development in eggs and larvae
of Atlantic cod. Paternity had a strong influence on the fertilization success, hatch-
ing success, cumulative embryonic mortality, larval standard length, eye diameter,
yolk sac area, and cumulative larval mortality. Paternal contributions to embryonic
and larval development were still evident despite differences in female quality,
showing that sire effects on offspring are undeniable and can serve as important
sources of variation during early life stages in fishes.
To date, key knowledge gaps remain:
1. Whether paternal effects act to increase or decrease offspring performance
remains largely unexplored;
2. The relative roles of maternal and paternal effects have rarely been disentangled;
and
3. The role of environmental variation, a key determinant of the benefits of trans-
generational plasticity, has not been explored with regard to paternal effects.
In ectothermic organisms, such as fish and invertebrates, reproductive success
and population abundance will decline under unfavorable thermal conditions if vital
life history traits are rigid in response to changing temperatures. Therefore,
Guillaume et al. (2015) explored all three issues using the marine tubeworm
Galeolaria caespitosa, an important habitat-forming species in southern Australia,
and checked the stress tolerance of the progeny. Although the authors did not explic-
itly check the nutritional impact, this study pointed out important transgenerational
modification of the phenotypic plasticity. The authors found that both paternal and
maternal experiences affected key stages of offspring performance (fertilization and
larval development) and, surprisingly, paternal effects were often stronger than
maternal effects. Furthermore, they found that paternal effects often reduced off-
spring performance, especially in fluctuating environments compared with stable
environments. The strong paternal impact can clearly be seen in fertilization success
(Fig. 6.31): this depended on a combination of the thermal histories of both mothers
and fathers. Maternal experience had no effect on fertilization success when fathers
had experienced a warmer environment, but, when fathers had experienced a cooler
environment, fertilization success was higher when mothers had experienced a
Fig. 6.31 Fertilization
curves showing the
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combined effects of male

and female acclimation
temperatures on
fertilization success in the
tubeworm Galeolaria
caespitosa. Adults were
acclimated to either cooler
(15.5 °C) or warmer
(21.5 °C) water
temperatures for 14 days
before use in fertilization
assays. Fertilization
success is pooled across
assay temperatures. The
error bars are ±SE. (From
Guillaume et al. 2015, with
warmer environment. This paper shows that, while transgenerational plasticity may
play an important role in modifying the impacts of internal and environmental
stresses, these effects are not uniformly positive. Importantly, paternal effects can
be as strong as, or stronger than, maternal effects and environmental variability
strongly modifies the impacts of paternal effects.
A comparable paternal effect has recently been shown in Atlantic cod (Dahlke
et al. 2016): fathers modified thermal reaction norms for hatching success, and
paternally mediated differences in offspring viability coincided with vital morpho-
genetic processes during early embryogenesis, showing that genetic processes were
primarily effective during this period. The study used two replicated trials with indi-
viduals from the Northwest Atlantic and the Baltic Sea. Each trial included five
temperature treatments encompassing optimum conditions as well as the amount of
ocean warming projected for the year 2100. In both trials, the mean hatching suc-
cess significantly decreased towards thermal extremes. However, half-sibling fami-
lies varied in their response to different incubation temperatures, as indicated by
significant paternity × temperature interactions and the crossing of reaction norms.
The influence of paternity itself was highly significant. High variation in daily
embryo survival among the half-sibling families and temperature treatments was
observed during the blastula and gastrulation stages (until 100% epiboly), while
almost no mortality occurred during the subsequent development and throughout
the hatching period. Paternally mediated differences in daily embryo survival arose
during specific developmental stages, irrespective of the incubation temperature
(Fig. 6.32b–c). Compared with either earlier or later time points (days post-
fertilization), the coefficient of variation among half-sibling groups was signifi-
cantly higher on days coinciding with stages I and II (late blastula until 100%
epiboly, Fig. 6.32a). The relationships between the succession of embryonic devel-
opment and the temperature were curvilinear and best described by a two-parameter
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Fig. 6.32 Paternally mediated variation in the daily survival rates of cod (Gadus morhua) embryos
in relation to the developmental age and temperature. (a) The pictures show successive develop-
mental stages during embryogenesis. Scale bar = 1 mm. (b and c) The bubble plots display pater-
nally mediated differences in daily survival rates until hatching among (b) Atlantic and (c) Baltic
cod half-sibling families in relation to developmental age (days post-fertilization) and temperature.
The bubble size indicates the magnitude of among-family variation expressed as the coefficient of
variation (% CV). The gray shaded bubbles coincide with the developmental stages during the
critical period (highlighted in a) and denote days with significantly higher CVs than others (black
bubbles). (c and d) Relationships between development and temperature of (d) Atlantic and (e)
Baltic cod embryos. The developmental times (days) until the completion of successive develop-
mental stages (Ia, Ib, II, III, IV, and hatch) are plotted versus the incubation temperature. Different
symbols indicate the hatching times of half-sibling families (±SEM), while gray symbols represent
samples obtained from mixed-family incubations. Temperature-dependent development rates were
fitted by two-parameter exponential decay models. (From Dahlke et al. 2016, with permission
from Elsevier)
exponential decay model (Fig. 6.32d–e). Overall, the paternal contribution repre-

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sents a relevant resource for adaptation to ocean warming in cod.

Paternity even has the potential to modulate the starvation resistance. However,
this impact varies with the fish species studied. Whereas in Baltic cod paternal
effects were only revealed through interaction with respective females and did not
contribute solely to explaining variability in early life history (Trippel et al. 2005),
in haddock males had a strong impact on these traits (Rideout et al. 2004).
Specifically, the hatching success, larval standard length, myotome height, jaw
length, and yolk size were affected by paternity. This paper pointed out that the
importance of males in the early life history success of marine fishes has long been
underrated.
In contrast to the gadid species, in Atlantic salmon there seems to be no obvious
paternal effect. Einum (2003) showed that the effect of the egg size on the offspring
performance, and hence the optimal egg size, was independent of the paternal body
mass. The egg size, in turn, had a strong effect on the body mass at yolk absorption,
causing juveniles originating from large eggs to outgrow their siblings from small
eggs. In addition, in juveniles of winter flounder (P. americanus), a paternal contri-
bution to their growth strategy could not be manifested (Fraboulet et al. 2010).
6.1.2.1 M
ale Pregnancy
Male pregnancy is a unique form of male parental care, exclusively found among
pipefishes, seahorses, and seadragons. In many of these species, males possess a
specialized epithelial structure known as a brood pouch, and mating involves the
transfer of eggs to the male’s pouch, after which the male carries the developing
embryos until they emerge as independent juveniles. Paczolt and Jones (2015)
reported details of a nutritional effect on this paternal care. They assumed that males
invested differently in broods depending on the availability of food and used the
Gulf pipefish (Syngnathus scovelli) to test this prediction by monitoring the growth
rate and offspring survivorship during the pregnancies of males under low- or high-
feed conditions. The study showed that pregnant males grew less rapidly on average
than non-pregnant males and that pregnant males under low-feed conditions grew
less than pregnant males under high-feed conditions (Fig. 6.33). Offspring survivor-
ship, on the other hand, did not differ between feed treatments, indicating that male
Gulf pipefish sacrificed investment in somatic growth and thus indirectly sacrificed
future reproduction in favor of current reproduction. However, a positive relation-
ship between the number of failed eggs and the male growth rate in the low-feed
treatments indicated that undeveloped eggs reduce the pregnancy’s overall cost to
the male compared with broods containing only viable offspring. In other words, in
resource-limited environments males maintain their investment in their current
brood, despite the costs associated with pregnancy, and instead sacrifice somatic
growth. Furthermore, males appear to be capable of redirecting resources from
failed eggs in their broods to somatic growth.
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Fig. 6.33 The effects on growth of pregnancy and food availability in Gulf pipefish. Growth was
measured before and during pregnancy and standardized to correct for the effect of male body
length. Positive values indicate that pregnant males grew more than non-pregnant males of the
same size; negative values indicate that pregnant males grew less than non-pregnant males of the
same size. (a) Males grow less during pregnancy than in the time period before pregnancy; (b)
pregnant males on high food grow more than males on low food, regardless of mate size. (From
Paczolt and Jones 2015, courtesy of the Public Library of Science; image © Joseph Tomelleri,
courtesy of the University of Texas at Austin)
As we shall see, several recent studies have shown that nutrition in general and
DR in particular activate most of the currently known epigenetic mechanisms,
although only in the parental generation itself and without checking the diet-induced
effects on succeeding progeny. It is only a matter of time until this gap will be
bridged; therefore, we have to define and explain these mechanisms.
6.2 What Is Epigenetics?
Interactions between heredity and environment can engage distinctly different epi-
genetic mechanisms. Such induced epigenetic state(s) can be reversed by the
removal or alteration of the factor, the addition of a different environmental factor,
6.2 What Is Epigenetics? 379
or emigration from the specific environment. This mitotic transgenerational effect is

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termed “context-dependent” epigenetic change (Walker and Gore 2011), because it

requires continued exposure to the environmental insult. Alternatively, the epigen-
etic modification can be incorporated into the germline, a process termed “germline-
dependent” epigenetic change. In this manner, the effect manifests in each generation
even in the absence of the causative agent. Context-dependent epigenetic modifica-
tion is fundamentally different from germline-dependent epigenetic modification.
Although both have been attributed “transgenerational” properties, only in the latter
(germline) instance will the trait be passed to the next generation even in the absence
of any continued exposures or stimuli (Crews and Gore 2011).
Tollefsbol (2014) summarized the development of the definition of “epigenetics”
as follows. It is now apparent that the influence of epigenetic information carried
out by processes such as DNA methylation, histone modifications, and non-coding
RNA is not limited to mitotic cell-to-cell inheritance but also extends to meiotic
generational inheritance. This needs to be distinguished in the definition of epi-
genetics. Therefore, a more modern definition of epigenetics is the modification of
phenotypic traits that can be inherited mitotically during cell division and meioti-
cally during transgenerational reproduction without changes in the DNA sequence.
Based on the definition above, transgenerational inheritance should always be cou-
pled with DNA recombination. If an observed phenomenon is not connected to the
germline, Tollefsbol (2014) recommended the term “transplacental or parent-to-
offspring epigenetic effects.”
From an ecological point of view, the definition above is recombination centered,
since it is based solely on sexual reproduction. However, in many non-vertebrate
animal species, although sexual reproduction does occur, various forms of asexual
reproduction are the preferred way to maintain a population for many generations,
as long as the environmental conditions are relatively stable. Relatively stable means
that the abiotic environmental variables do not reach the catastrophic state but stay
in the tolerable range and that biotic interactions, such as pests, diseases, or patho-
gen infections, do not happen. In natural pristine ecosystems, the supply of nutrients
is never in excess; rather, the opposite applies and oligotrophy remains combined
with dietary restriction at higher trophic levels. Consequently, energy saving within
the process of energy allocation is a central issue. Animals that engage in recombi-
nation every generation face considerable costs, such as the production and mainte-
nance of two mature bodies, one female and one male. Animals with the potential
for asexual reproduction save energy for the production of male bodies during peri-
ods of asexuality and can allocate this energy to offspring production. For instance,
many insects, crustaceans, rotifers, nematodes, and protozoans and some tardi-
grades can switch between asexual and sexual reproduction, and in most protozoans
asexual reproduction is dominant.
Even during relatively stable environmental conditions, it is advantageous for
asexually reproducing invertebrates to be prepared for environmental stresses to
occur and pass the acquired stress resistance to subsequent generations, most likely
by epigenetic mechanisms. For this reason, we will continue to use the term “trans-
generational effects” in the broader sense even for parthenogenetic populations,
particularly for such effects as those described by Beisson and Sonneborn (1965) or
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later by Csaba (2014), Menzel et al. (2011), or Suhett et al. (2011). Therefore, we
adopt the simplified definition and refer to intergenerational, transgenerational, and
multigenerational effects interchangeably.
6.2.1 Time Scales of Epigenetic Inheritance
Inherited epigenetic changes can last for only one but also up to thousands of gen-
erations. Because of the inherent instability of epigenetic inheritance, fixation of an
epigenetically determined phenotype is likely to be less stable than fixation through
a genetic selection mechanism.
Compromising HSP90 in D. melanogaster by geldanamycin, a potent and spe-
cific inhibitor of this stress protein/capacitor, Sollars et al. (2003) reported that
reduced activity of HSP90 induced a heritably altered chromatin state. The altered
chromatin state was evidenced by expression of the morphogen wingless in eye
imaginal discs and a corresponding abnormal eye phenotype, both of which were
epigenetically heritable in at least 13 subsequent generations (Fig. 6.34), even when
the function of HSP90 was restored.
The following example of epigenetic inheritance covers not only another mecha-
nism but also even more generations than the Drosophila example. Studying the
unicellular ciliate Tetrahymena pyriformis, Csaba (2014) reported an impressive
case of transgenerational epigenetic inheritance. Hormonal (chemical) imprinting is
already present at the unicellular level, for instance in Tetrahymena. The imprinting
was epigenetically caused, fixed, and transmitted to an indefinite number of genera-
tions executed through changes in the methylation pattern and probably also through
Fig. 6.34 Geldanamycin selection experiments (black columns) showed higher frequency of out-
growth in later generations. vtd3 is a stable mutation in the eyes. (From Sollars et al. 2003, with
permission from the Nature Publishing Group)
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Fig. 6.35 Time and concentration course study of the growth and viability of Tetrahymena pyri-
formis (insert) imprinted by insulin in generations 500 (a) and 1000 (b). The data represent
means ± SD of three parallel measurements. The significance is related to the control at a given
time point. * P < 0.05. (From Köhidai et al. 2012, with permission from Elsevier; image courtesy
of Dave Roberts, myspecies)
other epigenetic mechanisms. The strength of imprinting can decrease but was still
observed months after treatment, that is, after thousands of generations.
Taking the neurotransmitter 5-hydroxytryptamine (5-HT, serotonin) as a metabo-
lite of diet-born tryptophan (Trp), Köhidai et al. (2012) published one of the very
few studies of aquatic animals and diet-mediated epigenetically transmitted proper-
ties. The authors imprinted Tetrahymena cells with 5-HT and traced inherited life
history traits even in generations 500 and 1000 (Fig. 6.35). The underlying mecha-
nism, however, remained obscure.
Future studies will show that diet-mediated epigenetically acquired phenotypes
apply to aquatic animals in general; the first indications are emerging, since several
biomolecular phenomena, fundamental to epigenetics, have recently been detected
in fishes and aquatic animals fed various diets.
6.2.2 Epigenetic Mechanisms
The epigenome consists of chromatin and its modifications as well as a covalent

modification by methylation of cytosine rings found in the dinucleotide sequence
CpG. The epigenome determines the accessibility of the transcription machinery
Fig. 6.36 Impacts of Parental malnutrition

parental malnutrition on
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offspring programming via

epigenetic modifications. Epigenetics
These modifications are DNA methylation
transmitted to the progeny, Histone modification
setting up a metabolic state HSP90
that is best adapted to the
parent environment. If the
environment of the Genome activity and gene transcription
progeny deviates from that
of the parents, their
metabolic state is no longer Post-transcriptional modifications
adapted to their microRNAs; mRNA turnover
environment, resulting in alternative splicing
lifelong adverse
consequences. (Strongly
modified after Wang et al. Protein expression and physiological activity
2012). For more
mechanistic details, see Offspring programming
Box Methylation
Maldevelopment of offspring by impairment of

physiological functions
retarded growth and development,
reduced immune function, impaired health
that transcribes the genes into messenger RNAs (mRNAs). Inaccessible genes are
therefore silent, whereas accessible genes are transcribed (Szyf et al. 2008).
Research over the past decade has focused first on two major molecular mecha-
nisms that mediate epigenetic phenomena—DNA methylation and histone modifi-
cation—and more recently HSP90 as a capacitor and the action of microRNAs
(miRNAs) and other post-transcriptional modifications as indicated for parental
malnutrition (Fig. 6.36). For the sake of simplicity in the following sections, the
post-transcriptional modifications are subsumed under epigenetics (sensu latu).
To demonstrate that progeny metabolism is adapted to the parental environment,
we refer to Buescher et al. (2013), who studied Drosophila. The authors focused on
responses to a high sugar diet fed to adult females and recorded slight increases in
circulating sugars and decreased glycogen storage in larvae from exposed mothers.
A different response, however, was observed when these progeny were followed to
adulthood, with decreased glucose and increased glycogen stores in adult males, the
opposite of the larval phenotype (Fig. 6.37). A challenge of adult progeny with the
high sugar diet exacerbated these phenotypes and led to more dramatic increases in
triglyceride and trehalose levels than those seen in the control progeny, indicating a
propensity in these progeny for diet-induced obesity. Interestingly, increased circu-
lating sugars persisted in larval stages through the F2 generation. A number of lipid
and carbohydrate metabolic genes also changed their expression in the F1 progeny.
Unlike metabolite levels, however, some of these changes were not exacerbated
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Fig. 6.37 Drosophila display transgenerational inheritance of metabolic state. Female flies were
fed a high-sugar diet and their offspring were raised on a normal diet (Buescher et al. 2013). F1
larvae had increased glucose and trehalose levels and decreased glycogen levels, while F1 adults
showed the opposite effect. Adults also displayed increased triglycerides compared with controls.
Larval changes in glucose and trehalose can be transmitted to the F2 generation. (From Somer and
Thummel 2014, with permission from Elsevier)
when the progeny were challenged with the high-sugar diet. On the contrary, for
some genes, a high-sugar challenge in the progeny of high-sugar-fed mothers equil-
ibrated expression to control levels of low-sugar-fed progeny from low-sugar-fed
parents. Although not precisely correlated, these “rescued” changes in gene expres-
sion support the model that progeny metabolism is adapted to the parental environ-
ment (Somer and Thummel 2014).3
6.2.2.1 DNA Methylation, Histone Methylation, and Acetylation
DNA methylation involves the addition of a methyl group to the five position of
cytosine (one of the four bases of DNA), which occurs in animals in the context of
CpG (cytosine followed by guanine) dinucleotides, whereas in plants the position is
somewhat more flexible. This modification can be inherited through cell division.
DNA methylation is typically removed during zygote formation and reestab-
lished through successive cell divisions during development. DNA methylation is a
crucial part of normal organismal development and cellular differentiation in higher
organisms. DNA methylation stably alters the gene expression pattern in cells.
Common wisdom was that once DNA methylation patterns were formed during
development, they remained stable thereafter (Razin and Riggs 1980). This classical
model predicted that any epigenetic variations would form exclusively during gesta-
3
Excerpt taken with permission from Elsevier.
Folic acid
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DHF
B6 THF Methionine SAM Unmethylated DNA
5.10-MTHF B12 DNMTs
B2 5-MTHF Homocysteine SAH Methylated DNA
Fig. 6.38 Simplified schematic of the folic acid metabolic pathway resulting in DNA methylation.
(From Vanhees et al. 2014, with permission from Springer)
tion but not later in life. Recent data imply that environmental exposures can alter
the epigenome after birth, supporting the hypothesis that DNA methylation and
chromatin modification machineries remain active and dynamic throughout life
(Szyf et al. 2008).
Methyl donors are supplied by food (Vanhees et al. 2014) or even by ubiquitous
environmental matrices, such as humic substances (Menzel et al. 2011), the largest
reservoir of organic carbon in all ecosystems (Steinberg 2003). For instance, diet-
based methylation of DNA occurs via the folic acid metabolic pathway (Fig. 6.38).
In this pathway, the micronutrient folic acid (vitamin B9) is first reduced to dihydro-
folate (DHF), which is then reduced to tetrahydrofolate (THF). 5,10-methylene-
THF (5,10-MTHF) is formed by adding a methylene group to THF. In this step of
the pathway, vitamin B6 (B6) serves as an essential co-enzyme. Next, 5,10-MTHF
is reduced to 5-methyl THF (5-MTHF) with the aid of the essential co-enzyme vita-
min B2 (B2). 5-MTHF then donates, with the co-enzyme vitamin B12 (B12), its
methyl group to homocysteine, resulting in the formation of methionine (Met).
Subsequently, Met donates its methyl group to DNA via S-adenosylmethionine
(SAM) and becomes S-adenosylhomocysteine (SAH). Besides Met, the AAs gly-
cine (Gly), histidine (His), and serine (Ser), as well as choline, betaine, and vitamin
B12, can serve as methyl donors.
Contrary to methyl donors, dietary polyphenols may adversely affect DNA
methylation (Fang et al. 2007). Certain dietary polyphenols, such as (2)-epigallocat-
echin 3-gallate (EGCG), the active compound from green tea and genistein from
soybean, have been demonstrated to inhibit DNA methyltransferases (DNMT) in
vitro in human cancer cell lines. This inhibitory activity is associated with the
demethylation of the CpG islands in the promoters and the reactivation of
methylation-silenced genes. Many other polyphenolic compounds have lower activ-
ity levels in inhibiting DNMT (Fig. 6.39). Since a number of genes become abnor-
mally methylated during tumorigenesis, the reversal of hypermethylation-induced
inactivation of key tumor suppression genes by dietary DNMT inhibitors appears to
be an effective approach to reducing the health burden of cancer (Li and Tollefsbol
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Fig. 6.39 Inhibition of 5-cytosine DNA methyltransferase activity in an esophageal cancer cell
line by different polyphenols. (From Fang et al. 2007, with permission from the American Society
for Nutrition)
2010). Although corresponding papers are not yet available, this statement can eas-
ily be expanded to aquatic animals.
Histone proteins are the basic building blocks of chromatin. In contrast to DNA
that is modified only by methylation, histones can be modified by methylation, acet-
ylation, phosphorylation, biotinylation, ubiquitination, sumoylation, and ADP-
ribosylation (Fig. 6.40). The location of the histone modifications is at the histone
tails. Lysine residues in the histone tails can be either methylated or acetylated, and
Arg residues can be methylated (Choi and Friso 2010).
DNA methylation, histone acetylation and deacetylation, and histone methyla-
tion all work together to build up chromatin structures, which in coordination may
shift from a transcriptional permissive state to a transcriptional inactive state and
vice versa (Fig. 6.41) (Strietholt et al. 2008). Genes are inactivated when the chro-
matin is condensed, and they can be transcribed when the chromatin is opened
(relaxed). Methylation, demethylation, acetylation, and deacetylation of histone
proteins are performed by histone methyltransferase, histone demethylase, histone
acetyltransferase, and histone diacetyltransferase, respectively. Histone activities
are also influenced by phosphorylation, ubiquitination, ADP-ribosylation,
sumoylation, and glycosylation. In sum, the metabolism of AAs and vitamins (B6,
B12, and folate) plays a key role in the provision of methyl donors for DNA and
protein methylation. In addition to these mechanisms, siRNAs target several mole-
cules, for instance DNA methyltransferase itself, prevent cytosine methylation, and
maintain the accessibility of the gene for transcription factors.
Among epigenetic changes, DNA methylation and histone modification are asso-
ciated with severe diseases, particularly various types of cancers, in a number of
ways, many of which are regulated by dietary components mostly found in plants.
From human nutrition, it can be deduced that genistein, resveratrol, curcumin,
EGCG, 3,3′-diindolylmethane (DIM), diallyl disulfide, garcinol, procyanidin B3,
quercetin, sulforaphane, and other isothiocyanates have the capacity to affect his-
tone modifications (Gao and Tollefsbol 2015) and are most likely to improve the
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Fig. 6.40 Nucleosome and histone modifications. Each nucleosome comprises an octamer of his-
tone molecules and double-stranded DNA. The amino termini of histones, which are called histone
tails, can be modified post-translationally and function as signal integration platforms. The main
epigenetic modifications at histone tail sites are: lysine and arginine methylation (MetK and MetR,
respectively), phosphorylation (P), acetylation (Ac), and ubiquitination (Ub). (From Choi and
Friso 2010, with permission from the American Society for Nutrition)
health status and promote the longevity of aquatic animals fed these compounds as
well (Pietsch et al. 2010, 2011, 2012; Saul et al. 2010, 2011).
Lai et al. (2016) found an epigenetic mechanism responsible for the response of
Daphnia magna to hypoxia. Hypoxia occurs when the amount of dissolved oxygen
falls below 2.8 mg L−1 in aquatic environments and can cause transgenerational
effects not only in fish but also in invertebrates. The authors collected embryos (F1)
from adults (P0) that were previously exposed to hypoxia/normoxia for their whole
life. Comparative transcriptome analysis showed 124 differentially expressed genes,
including 70 up- and 54 downregulated genes under hypoxia. Gene ontology analy-
sis highlighted three clusters of genes that revealed acclimatory changes of hemo-
globin, suppression in the vitellogenin gene family, and histone modifications.
Specifically, the expressions of histone h2b, h3, and h4 (Fig. 6.42) and histone
deacetylase 4 (hdac4) were deregulated.
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Fig. 6.41 Close interactions between DNA methylation and histone modifications. (a) Relaxed
chromatin is accessible to transcription factors (TFs). Chemical modifications (green) of the core
histones (yellow) result in a relaxed chromatin structure. (b) DNA methyltransferases (DNMTs)
add methyl groups (gray triangles) to CpG dinucleotides, resulting in gene silencing that can affect
the former modification of the histones. (c) The chemical modification (red) to the core histone
results in a condensed and inactive chromatin structure. TFs are sterically hindered and cannot
bind to their recognition sequence on the DNA. (From Strietholt et al. 2008, courtesy of BioMed
Central)
Fig. 6.42 Quantitative PCR results of histone h3, h4, and h2b and expression in F1 embryos of
Daphnia magna. The embryos were collected from adults that were previously exposed to hypoxia
or normoxia for their whole life. Data are presented as the means±SEM; * P < 0.05. (From Lai
et al. 2016, with permission from Elsevier)
6.2.2.2 Diet-Mediated DNA Methylation

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The above description of this major epigenetic mechanism and its inheritance,
mainly derived from mammals, demonstrates the general significance of the dietary
impact on this pathway, which will be unraveled in aquatic animals too.
A major growth regulation pathway is governed by the retinoid X receptors,
RXRs. RXRs have been found in many aquatic animals (Box RXR). However,
nutrition-mediated methylation and its transgenerational implication have so far
only been studied in mammals. Furthermore, interesting information also exists on
transgenerational inheritance from toxicology.4 For instance, Volate et al. (2009)
showed in mice that green tea reduced intestinal cancer by upregulating the retinoic
X receptor alpha (rxrα). The underlying mechanism was a significant reduction of
the methylation in the promoter region.
Complex regulation of obesity was proposed by Mischke and Plösch (2013) in
their perspective paper. Substantial evidence has linked early postnatal nutrition to
the development of obesity later in life. Epigenetic mechanisms have been indicated
to be involved in this process (Lillycrop and Burdge 2015). Therefore, Mischke and
Plösch (2013) proposed that early postnatal nutrition (breast and formula feeding)
epigenetically programs the developing organs via modulation of the gut microbi-
ome and influences the body weight phenotype, including the predisposition to obe-
sity. Specifically, the early-age feed patterns are known to determine the gross
composition of the early gut microbiota. In turn, the microbiota produce large quan-
tities of epigenetically active metabolites, such as folate and short chain fatty acids
(butyrate and acetate). The spectrum of these produced metabolites depends on the
composition of the gut microbiota. Hence, it is likely that changes in gut microbiota
that result in an altered metabolite composition might influence the epigenome of
directly adjacent intestinal cells as well as other major target cell populations, such
as hepatocytes and adipocytes. Nuclear receptors and other transcription factors
(RXR and others) could be physiologically relevant targets of this metabolite-
induced epigenetic regulation.
Recently, McDougall et al. (2017a, b, c) published an interesting study on
adverse dietary impact of VE deficiency on the methylation pathway in zebrafish.
The authors fed spawing parents VE-sufficient (E+) and VE-deficient diets, ana-
lyzed the VE and VC contents as well as metabolites of the methylation pathway,
and observed depletion of both choline, choline-derived and other methyl donors
(e.g. betaine, S-adenosylmethionine, Met) in VE− embryos, which is consistent
with the decreased global DNA methylation status from 1 to 4 dpf in E− vs. E+
embryos. This reduced availability of methyl donors increased with time and is
most expressed in the 120 hpf embryos (Fig. 6.43). Overall, this study indicates that
VE deficiency causes lasting metabolic impairments in zebrafish embryos.
4
Some of the earliest studies of changes in DNA methylation associated with environmental fac-
tors in fishes examined the effect of xenobiotic chemicals, and (eco)toxicology continues to be an
important focus of DNA methylation studies in fishes (refer to Metzger and Schulte 2016).
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Fig. 6.43 Relative response intensities of choline and methylation pathway intermediates. (V)E–
and E+ embryo. Shown are means ± SEM; P-values are for VE×Age interactions, unless indicated
otherwise. Paired comparison, P-values are indicated as * < 0.05, ** < 0.005,*** < 0.001,****
<0.0001. (From McDougall et al. 2017b, courtesy of Elsevier)
Box RXR in Aquatic Animals

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Retinoid receptors (RXRs) are members of the nuclear receptor super family
and are hypothesized to play key roles in hormonal regulation and lipid
metabolism of both vertebrates (Volate et al. 2009) and invertebrates, particu-
larly insects (Hult et al. 2015; 2011). RXR can form both homodimers or
heterodimers with many other nuclear receptors, including peroxisome
proliferator-activated receptors (PPARs), and therefore bind to DNA response
elements inducing the transcription of genes involved in xenoprotection, lipid
homeostasis, and development.
There are, however, clear differences between different vertebrate groups,
for instance fishes and mammals. Different types of vertebrate RXRs (αRXR,
ßRXR, and γRXR) have arisen from multiple duplication events. The adaptive
evolution mechanism has preserved duplicate RXR paralogs. The study of
Philip et al. (2012) sheds some light on their role in development and adapta-
tion by indicating that the evolution of RXR could be linked to an increase in
complexity of the organism (Box Fig. 6.1), where different types of cells that
require the same basic biochemical process are triggered by different agents
or with different intensities.
Rxr genes have been identified in many fish species, e.g., zebrafish (White
et al. 1994; Zheng et al. 2015), rainbow trout (Cleveland and Manor 2015),
rockfish (Sebastiscus marmoratus) (He et al. 2009; Zhang et al. 2013),
Japanese flounder (Haga et al. 2003), thicklip grey mullet (Chelon labrosus)
(Raingeard et al. 2009), Nile tilapia (He et al. 2015), yellow catfish
(Pelteobagrus fulvidraco) (Pan et al. 2016), medaka (Oryzias latipes)
(Hayashida et al. 2004), golden pompano (Trachinotus ovatus) (Yang et al.
2015), European sea bass (Villeneuve et al. 2005), Atlantic bluefin tuna
(Thunnus thynnus) (Maisano et al. 2016), common sole (Solea solea) (Ribecco
et al. 2012), Senegalese sole (S. senegalensis) (Darias et al. 2012), sea bream
(Ribecco et al. 2011), goldfish (Bremer et al. 2012), burbot (Lota lota) (Olsvik
et al. 2013), and Atlantic salmon (Carmona-Antoñanzas et al. 2016).
Rxr genes and their activity have been proven also in the ascidian
Halocynthia roretzi (Maeng et al. 2012), in crustaceans, such as brachyuran
crabs (Paratelphusa sp., Uca pugilator, Gecarcinus lateralis, Carcinus mae-
nas) (Sarika and Anilkumar (2014) and references therein), mud crab (Scylla
paramamosain) (Girish et al. 2017), common shrimp (Crangon crangon)
(Verhaegen et al. 2011), waterflea (Daphnia magna) (Jordão et al. 2015),
freshwater prawn (Macrobrachium nipponense) (Shen et al. 2013), amphipod
(Gammarus fossarum) (Gouveia et al. 2018), in midge larvae (Chironomus
riparius) (Morales et al. 2013), in mollusks, such as great pond snail (Lymnaea
stagnalis) (Carter et al. 2010), European physa (Physa acuta) (Morales et al.
2018), limpet (Patella vulgate) (Gesto et al. 2016), Zhikong scallop (Chlamys
farreri) (Lv et al. 2013), and sea urchin (Strongylocentrotus nudus) (Kim and
Sohn 2017).
(continued)
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Box Fig. 6.1 Comparison of the rxr gene expression patterns during the embryonic devel-
opment stages of Mus musculus and Danio rerio: Comparison of the rxr gene expression
patterns during the embryonic development stages of Mus and Danio for multiple homolo-
gous anatomical structures, showing subfunctionalization and neofunctionalization of rxr
genes in zebrafish following duplication events. (From Philip et al. 2012 with permission
from Elsevier)
Invertebrates
Compared with vertebrates, invertebrates are characterized by relatively low global

cytosine methylation levels [5–10% vs. 0.17–0.90% (Gavery and Roberts 2010;
Menzel et al. 2011; Sarda et al. 2012)]. Despite the low methylation levels, inverte-
brates experience changes in cytosine methylation as a result of environmental
changes (Asselman et al. 2015). With D. magna, the latter authors tested the hypoth-
esis that changes in global cytosine methylation depend, among other autochtho-
nous and environmental factors, on food quality. Therefore, two clones, a Finnish
one (Xinb3) and a German one (Iinb1), were exposed to four types of food that
varied widely in quality: normal-quality food (a mixture of the green algae
1.5
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[5mdC]/[dG]% (treatment/control)
1.0
0.5
0.0
Raphidocelis
Control
Chlamydomonas
Cryptomonas
Microcystis
non-toxic
Microcystis
Fig. 6.44 Daphnia magna: mean global DNA methylation level in all treatments for the two geno- toxic
types Iinb1 (gray columns) and Xinb3 (hatched columns) normalized to the mean corresponding
control methylation (i.e. control for all treatments). The error bars represent the standard deviation.
The black stars denote exposures that are significantly different from the corresponding control
treatment (P < 0.05). The gray stars denote exposures for which Iinb3 and Xinb3 differ signifi-
cantly from one another (P < 0.05). (From Asselman et al. 2015, modified, with permission from
Wiley)
Raphidocelis subcapitata and Chlamydomonas reinhardtii), excellent food quality

(the unicellular alga Cryptomonas), non-toxic low-quality food (non-microcystin-
producing Microcystis), and toxic low-quality food (microcystin-producing
Microcystis) (Fig. 6.44). Two striking outcomes were obvious: first, a significant
decrease in global DNA methylation was observed for the Iinb1 genotype exposed
to non-microcystin-producing Microcystis; second, global DNA methylation was
significantly lower in the Iinb1 genotype than in the Xinb3 genotype if fed
Cryptomonas and non-toxic Microcystis. Evidently, different genotypes respond
differently to nutritional changes in terms of their global DNA methylation pattern.
These differences do not depend on the food quality, as the same pattern was
observed for both food types. The authors quantified changes in the DNA methyla-
tion level only in the generation that was exposed to the stressor; the response to
some stressors might be greater in the offspring generation. This effect was observed
by Vandegehuchte et al. (2009) after exposing D. magna to zinc concentrations.
Significant changes in the global DNA methylation level occurred only in the F1
generation and not in the exposed P0 generation itself.
Fig. 6.45 Image of the sea

lamprey (Petromyzon
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marinus). (From Bloch

1782–1784, courtesy of the
Biodiversity Heritage
Library)
Fishes
In animals, growth is regulated by the growth hormone (GH). In fish, GH partici-

pates in almost all major physiological processes in the body, including the regula-
tion of ionic and osmotic balance, lipid, protein, and carbohydrate metabolism,
skeletal and soft tissue growth, reproduction, and immune function. Studies have
indicated that GH affects several aspects of behavior, including appetite, foraging
behavior, aggression, and predator avoidance (Reinecke et al. 2005). GH binds to its
receptors, GHR, in the target organs mainly in the liver and stimulates the synthesis
and release of insulin-like growth factor-I (IGF-I). IGF-I is involved in the regulation
of protein, lipid, carbohydrate, and mineral metabolism in the cells, differentiation
and proliferation of the cells, and ultimately body growth (Moriyama et al. 2000).
In the sea lamprey (Petromyzon marinus, Fig. 6.45), development and maturation
are combined with a dramatic ontogenetic dietary shift: larval lampreys are freshwa-
ter filter feeders that burrow into streambeds. During the larval stage, lampreys go
through a series of metamorphoses until the final transformation into a parasitic tis-
sue-feeding adult that migrates to the marine environment. Metamorphosis entails an
intricate process that implies a complex gene network. The well-known hox gene
family endorses an important role with regard to the structural organization along the
major body axis throughout the entire vertebrate development. hox genes encode
axial patterning transcriptional regulatory proteins in all bilaterians. Recently,
Covelo-Soto et al. (2015) explored the epigenetic regulation of metamorphosis in the
sea lamprey. They found that particularly hox genes involved in morphogenesis were
differently methylated, along with genes related to the water balance and to the
osmotic homeostasis, all associated with a marine environment adaptation. The
utmost adult and larvae difference was encountered for the unmethylated state, being
significantly higher in the larvae. It can be hypothesized that the ontogenetic diet
shift could be one causative agent for the observed difference in the methylation pat-
tern. Furthermore, changes in DNA methylation also play a role in establishing varia-
tions in life history traits, as in the rainbow trout between smolt and resident juveniles
(Baerwald et al. 2015), and may again be connected to the corresponding diets.
Since the transmembrane growth hormone receptor GHR is central to growth
regulation, it can be expected that both quantitative and qualitative modulation of
the food supply affect this receptor epigenetically. In fact, Zhao et al. (2015) showed
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Fig. 6.46 Total number of methylated cytosines of gh (A) in the pituitary as well as growth rates,
growth hormone (gh), growth hormone receptor 1 (ghr1), and growth receptor 2 (ghr2) mRNA
expression in male and female Nile tilapia. Methylation is inversely related to the growth rate and
gene transcriptions. Different superscripts indicate significant differences. (From Zhong et al.
2014, with permission from Elsevier)
starvation-induced changes in the methylation status of ghr. Furthermore, Fukada

et al. (2004) found that ghr expression is reduced following three-week fasting of
masu salmon (Oncorhynchus masou). The binding rate of GH to GHR decreased,
and the growth-promoting function of GH was weakened and then slowed down the
fish growth. However, in Mozambique tilapia, ghr expression in muscle increased
during fasting and then declined below the control levels upon refeeding (Fox et al.
2010). These results indicated that GHR is sensitive to the nutritional status and
then affects the growth condition. Consequently, ghr and other growth-promoting
genes can be considered as potential and functional candidate genes for selecting
high-growth fish.
In Japanese flounders, high methylation of the CpG site caused a lower gene
expression level of cyp17-II (Ding et al. 2012), and, in Nile tilapia, the methylation
of the gh promoter was negatively correlated with gh expression in the pituitary and
the growth rate (Fig. 6.46) (Zhong et al. 2014). In the halfsmooth tongue sole, the
promoter methylation regulated the ghr1 expression (Fig. 6.47).
Overall, these papers described the impact of methylation on the transcription of
growth-related genes and on growth and energy conversion. It is very likely that the
methylation status depends not only on the food quantity (starvation vs. feeding) but
also on the food quality, namely the presence and quantity of methyl donors. In
rainbow trout, Craig and Moon (2013) observed that a carbohydrate load induced
hypomethylation of genomic DNA (Fig. 6.48). The methyl pool necessary for DNA
methylation is derived from S-adenosylmethionine, which is originates from Met
adenylation (see Proteinaceous Nutrients, Volume II). It was hypothesized that, if
the dietary Met was restricted, then a decrease in the degree of total DNA methyla-
Average DNA methylation level of exon 8

Gene expression DNA methylation
1.1 100.0%
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Relative expression level of GHR

1
0.9 80.0%
0.8
0.7
60.0%
0.6
0.5
40.0%
0.4
0.3
0.2 20.0%
0.1
0 0.0%
AA AG GG
Fig. 6.47 The correlations between the gene expression and the average CpG methylation level of
the ghr1 gene of different genotypes of half-smooth tongue sole (Cynoglossus semilaevis) (from
Zhao et al. (2015), with permission from Springer). Individuals were divided into three genotypes,
AA, AG, and GG according to the L1 locus, the GG genotype having one more CpG site because
of the mutation, and into two genotypes, AA and GG, based on the L2 locus
Fig. 6.48 Effects of dietary manipulation of carbohydrate load and methionine restriction on the
percentage of methylation (5-methylcytosine; 5-mC) of genomic DNA in both liver (black col-
umns) and muscle (gray columns) tissues harvested from Oncorhynchus mykiss 6 h post feeding.
a, b
Mean values for a tissue (liver or muscle) with unlike letters were significantly different
(P < 0.05). * The mean value was significantly different from that for liver (P < 0.05). 12% +: 12%
carbohydrate load with 1.5% methionine content; 12% ─: 12% carbohydrate load with 0% methio-
nine; 22% +: 22% carbohydrate load with 1.5% methionine content; 22% ─: 22% carbohydrate
load with 0% methionine. (From Craig and Moon 2013, with permission from the Cambridge
University Press)
tion would be observed. However, Met restriction did not significantly affect the
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total DNA methylation (Fig. 6.48). This indicates that Met restriction may not limit
the methyl pool available for DNA methylation and that other methyl sources or
methylgenic pathways compensate for the decrease in the methyl pool, such as beta-
ine, choline, and homocysteine remethylation.
Although Met restriction did not affect the total DNA methylation, trout fed with
the 22% carbohydrate diet had significantly reduced total DNA methylation in both
liver and muscle (Fig. 6.48). A reduction in DNA methylation in trout fed the
high-carbohydrate diet raises concerns related to gene expression, genomic stabili-
zation, and the possible development of secondary complications related to Met
restriction, such as cancer (Craig and Moon 2013). In addition to these unanswered
questions, the problem of whether this hypomethylation status can be transferred to
succeeding generations also merits further evaluation.
In common garden experiments with parthenogenic Artemia, Norouzitallab et al.
(2014) showed that an environmental stress activitated the complex epigenetics
machinery. On exposure to nonlethal heat shocks, Artemia experienced an increase
in levels of HSP70 production and developed tolerance toward lethal heat stress and
resistance against pathogenic Vibrio campbellii. Interestingly, these acquired phe-
notypic traits were transmitted to three successive generations, none of which were
exposed to the parental stressor. This transgenerational inheritance of the acquired
traits was associated with altered levels of global DNA methylation and acetylated
histones H3 and H4 in the heat-shocked group compared to the control group, where
both the parental and successive generations were reared at standard temperature.
These results indicated that epigenetic mechanisms, such as global DNA methyla-
tion and histones H3 and H4 acetylation, have particular dynamics that are crucial in
the heritability of the acquired adaptive phenotypic traits across generations.
Therefore, it can predicted that feeding dietary methyl donators bears a high poten-
tial for DNA methylation and, thereby, initiating epigenetically inheritable traits.
Box Methylation
Highlights of Gene Methylation in Fish Mediated by Exogenous Triggers
Global methylation patterns in zebrafish embryos have been described
(Ribas and Piferrer 2014), and recently an epigenetic mechanism involving
DNA methylation of the gonadal aromatase promoter was proposed to explain
the effects of temperature on the sex ratio in European sea bass (Navarro-
Martín et al. 2011). Furthermore, methylation contributes significantly to the
antiviral innate immune defense in grass carp (Ctenopharyngodon idella)
challenged by the grass carp reovirus (Shang et al. 2015, 2016). The authors
showed that the promoters of one specific retinoic acid-inducible gene I (rig-
I), the cirig-I (Ctenopharyngodon idella rig-I), and one specific melanoma
differentiation associated factor 5 (cimda5), were methylated. Both genes are
crucial members of virus recognition receptors.
(continued)
Previously, in the Japanese flounder (Paralichthys olivaceus), MDA5 has

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been testified to induce an antiviral response to the viral hemorrhagic septice-

mia virus; MDA5 has been found to serve as a positive regulator in antiviral
immune responses in channel catfish (Ictalurus punctatus) and rainbow trout
(O. mykiss). Nevertheless, to date the regulatory mechanism of MDA5 antivi-
ral transcription has only been deciphered in the grass carp (Shang et al.
(2015) with reference therein).
Another process in which methylation of genes plays a central role is sex-
ual maturation, with aromatase cytochrome P450 (P450arom) as the key
player. P450arom, the product of cyp19, is a sex steroid hormone and a key
component of the aromatase complex converting androgens into estrogens.
These estrogens play a vital role in sex differentiation and gonad develop-
ment. In Nile tilapia, the expression of cyp19a1a at all ovarian stages points
to an important role for estrogen in female sex determination and the mainte-
nance of phenotypic sex. Furthermore, as a sexual dimorphic marker, the fork
head transcription factor gene 2 (foxl2) is expressed in the ovaries of medaka
(Oryzias latipes), rainbow trout, Nile tilapia, halfsmooth tongue sole, brown
spotted reef cod (Epinephelus merra), and Japanese flounder (Si et al. (2016)
and references therein). Recently, Si et al. (2016) reported that foxl2 is
involved in the regulation of cyp19a1a transcription. In addition, the whole
methylation levels of CpG rich regions were inversely correlated with the
foxl2 expression during ovary development, indicating that the DNA methyla-
tion pattern in the coding region influences the foxl2 mRNA expression.
Consequently, an epigenetic mechanism of foxl2 regulated the cyp19a1a tran-
scription: (1) foxl2 expression was regulated by the DNA methylation level of
the CpG sites in its coding region; and (2) the binding efficiency of the tran-
scription factors in the promoter and coding region of cyp19a1a was influ-
enced by DNA methylation.
In brown trout, migratory trout (sea trout) and sedentary trout (freshwater
trout) coexist and interbreed. Morán et al. (2013) identified dramatic differ-
ences in genome-wide methylation patterns between hatchery-reared and sea-
water brown trout. Furthermore, they demonstrated that salt-enriched diets
can trigger short-term genome-wide methylation changes in hatchery-reared
trout. However, these changes only lasted for a short period of time.
Determining the duration of this effect will result in increased survival of
hatchery-reared trout in seawater when fed on salt-enriched diets. Altogether,
these results indicate that salt-induced alterations to DNA methylation pat-
terns play an important role in enabling fish acclimation to seawater condi-
tions, with important economic consequences for fish farming.
The modulation of gene methylation has not yet entered nutritional stud-
ies; instead, it plays a central role in aquatic ecotoxicology and stress ecology.
Hypoxia is amongst the most widespread and pressing problems in aquatic
environments and is able to affect adversely not only the exposed individuals
(continued)
but also succeeding generations. For instance, Wang et al. (2016) demon-
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strated that fish (Oryzias melastigma) exposed to hypoxia showed reproduc-

tive impairments (retarded gonad development and a decrease in sperm count
and sperm motility) in the F1 and F2 generations despite these progeny (and
their germ cells) having never been exposed to hypoxia. The authors further
showed that the observed transgenerational reproductive impairments were
associated with a differential methylation pattern of specific genes in sperm of
both P0 and F2 coupled with relevant transcriptomic and proteomic alterations
that impair spermatogenesis. The discovered transgenerational and epigenetic
effects indicate that hypoxia poses a dramatic and long-lasting threat to fish
populations (Box Fig. 6.2). Because different dietary substances also possess
high potential for methylating genes, these results also shed light on the
potential dietary transgenerational effects.
Some examples from aquatic ecotoxicology demonstrate the significance
of gene and histone methylation under chemical challenge. For instance,
Pierron et al. (2014) investigated whether chronic exposure to contaminants
experienced by wild female fish (Anguilla anguilla) throughout their juve-
nile phase can affect the DNA methylation status of their oocytes during
gonad maturation. Fish were sampled at two locations, a low and a highly
contaminated one. The DNA methylation levels of the genes encoding for
Box Fig. 6.2 Schematic diagram of the hypoxia-induced epigenetic effects in Oryzias
melastigma. (From Wang et al. 2016, courtesy of Nature Communications)
the aromatase and the receptor of the follicle-stimulating hormone were

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higher in contaminated fish than in fish from the clean site. The hypermeth-
ylation was associated with a decrease in the transcriptions. Finally, greater
gonad growth was observed in fish from the reference site in comparison
with contaminated fish.
Corrales et al. (2014) exposed zebrafish to benzo[a]pyrene (BaP). BaP is
an established co-carcinogen and reproductive and developmental toxicant
(Steinberg 2012). BaP exposure in animals has been linked to infertility and
multigenerational health consequences. The authors investigated aberrant
changes in promoter DNA methylation in zebrafish embryos and larvae fol-
lowing parental and continued embryonic waterborne BaP exposure. They
detected that parental exposure followed by embryonic BaP exposure signifi-
cantly reduced egg production and offspring survival in zebrafish. BaP caused
global DNA hypomethylation in larvae 96 h past fertilization (hpf). An
increase in CG methylation and a decrease in gene expression were observed
in dazl (a gene involved in gametogenesis). In contrast, decreased CG meth-
ylation and increased gene expression were found in c-fos (a proto-oncogene).
BaP altered CG, CHH, and CHG methylation in many of the target cancer and
developmental genes. The changes were greater at 96 hpf than at 3.3 hpf,
showing that DNA methylation is more susceptible to BaP modification dur-
ing late embryogenesis or organogenesis than during gametogenesis and early
embryogenesis. In sum, BaP exposure in early life alters promoter CG meth-
ylation and gene expression in cancer and development-related genes, possi-
bly leading to higher risk of adult diseases in later life.
Very recently, Metzger and Schulte (2017) demonstrated that even a com-
parably weak environmental trigger, such as temperature, influences the DNA
methylation pattern. They showed that altered temperature during develop-
ment had prolonged effects on DNA methylation levels in three-spined stick-
leback (Gasterosteus aculeatus) and that the modifications were also
associated with the plastic adult acclimation response to environmental tem-
peratures. In addition, Metzger and Schulte demonstrated that the persistent
effects of developmental plasticity on DNA methylation patterns affected
regions of the genome where the DNA methylation patterns were also modi-
fied during adult acclimation. Consequently, one can hypothesize that triggers
that provide methyl donors themselves, such as certain dietary compounds,
will have at least a similar, if not an even stronger, impact on the methylation
pattern. Verification of this hypothesis is long overdue.
6.2.2.3 HSP90 as a Capacitor of Phenotypic Traits

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Besides DNA methylation and chromatin methylation and acetylation, further

mechanisms of chromatin modification take place, such as chromatin regulation
(Tirosh et al. 2010) or heat shock proteins as “capacitors” of morphological change.
HSP90 appears to be the best understood capacitor.5 The HSP90 chaperone assists
in the folding of proteins that are metastable signal transducers, such as kinases,
transcription factors, and ubiquitin ligases. HSP90 is normally present at much
higher concentrations than needed to maintain these proteins, allowing it to act as a
buffer, protecting organisms from phenotypic consequences that would otherwise
be caused by genetic variants of these proteins. Because protein folding is so sensi-
tive to environmental stress, changes in the environment can exhaust the chaperone
buffer, unmasking vulnerable polymorphisms (Rohner et al. 2013). While most of
the novel phenotypes are likely to be maladaptive, their frequency and variety may
boost the chances of natural selection to find a new route through the current adap-
tive landscape (Pigliucci 2003).
The recognition of the HSP90 as a capacitor was based on laboratory studies by
Rutherford and Lindquist (1998). Since then, it has been debated whether capaci-
tance is a laboratory curiosity or a major force in evolution (Siegal 2013). However,
the body of evidence showing that HSP90 can act as a capacitor even in the wild
population is growing. For instance, Chen and Wagner (2012) showed that HSP90
helps to buffer deleterious variation in fruit flies derived from a wild population,
particularly under heat stress. Furthermore, Rohner et al. (2013) provided the first
evidence for HSP90 as a capacitor for morphological evolution in a natural setting
with fishes by discovering its role in the evolution of eye loss in the cavefish
Astyanax mexicanus. This species exists in eyed surface and eye-less cave forms.
Rohner et al. (2013) applied environmental stress in the form of a change in water
conductivity, typical of cave natural habitats. Under these conditions, fish embryos
upregulated HSP90. Since changes in water conductivity are a relative soft environ-
mental trigger, it can therefore be expected that stress connected with food quality
and quantity and feeding regimes will affect the HSP90 machinery similarly and
release cryptic variations.
Food-Mediated hsp90 Expression
Environmental challenges, including food deprivation, are driving forces for many
adaptations (Steinberg 2012). For example, simple starvation affects HSP expres-
sion in larval gilthead sea bream, rainbow trout (Cara et al. 2005), and European sea
bass (Antonopoulou et al. 2013). The latter authors detected changes in cells’ pro-
tective mechanisms, such as the expression of HSPs and the activation of MAPKs,
and the antioxidant defenses in the present investigation revealed a tissue distinct
5
Studying cryptic genetic variations in D. melanogaster, Takahashi (2013, 2015) reported that
there are many more capacitors than only HSP90.
Table 6.1 Changes in the induction levels of HSPs, the phosphorylation of MAPKs, and the
enzyme activity levels of European sea bass (Dicentrarchus labrax) between 2S–2F (2 months of
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starvation followed by 2 months of feeding) and 1F–3S (1 month of feeding followed by 3 months
of starvation) compared with the control (4 months of feeding, no starvation) diet treatment (dark
gray indicates an increase, light gray indicates a decrease, and white indicates no change)
Intestine Liver Red muscle White muscle

2S–2F 1F–3S 2S–2F 1F–3S 2S–2F 1F–3S 2S–2F 1F–3S
HSP70
HSP90
p38
MAPK
ERKs
GPx
CAT
SOD
From Antonopoulou et al. (2013), with permission from Elsevier
and differential cellular stress response and antioxidant capacity (Table 6.1), which
can be related to stress induced both by food deprivation and by refeeding. The
induction of these mechanisms seems to be a biochemical adaptation strategy that is
closely related to the fact that many fish experience periods of starvation as part of
their natural life cycle.
This study confirms the essentials of a former study by Cara et al. (2005). These
authors examined the HSP70 and HSP90 protein expression in early life stages of
the gilthead sea bream after 12-hour food deprivation and in early life stages of
rainbow trout after 7-day food deprivation. The results clearly demonstrated that
food deprivation enhanced HSP70 and HSP90 protein expression in the larvae of
both species. The most interesting result, however, was that food deprivation obvi-
ously induced cross-tolerance, because the food-deprived trout larvae that had
higher HSP70 and HSP90 protein content survived even heat shocks. This result
contrasts those obtained with juveniles of Senegalese sole that did not show differ-
ences in HSP90 expression if the food rations were modulated (Salas-Leiton et al.
2010). Obviously, this reaction is species specific.
Feeding Siberian sturgeon (Acipenser baerii) a β-glucan-enriched diet, Simide
et al. (2016) found that the level of hsp90 expression was lower than that in fish fed
with a control diet (Fig. 6.49). This could be explained by a lower metabolic rate;
the low basal expression level had no impact on the fishes’ capacity to induce an
efficient HSP response under conditions of stress. Whether this hsp90 expression
pattern translates into the next generation is still an open question.
Batista et al. (2016) examined the changes in innate immune parameters and the
gut microbiota fed with graded plant ingredients with or without probiotic or yeast
supplementation to Senegalese sole. The hsp90b mRNA levels in the distal intestine
were strongly downregulated with the use of high content of plant ingredients in the
diet containing the multispecies probiotic and autolyzed yeast, respectively. Moreover,
probiotic supplementation resulted in the upregulation of the hsp90b transcript levels
in the distal intestine. Whether or not these changes in the transcript levels have the
potential to contribute to transgenerational effects merits further evaluation.
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Fig. 6.49 Boxplots of heat shock protein 90 (hsp90) expression in Siberian sturgeon fed a control
(D1) and β-glucan-enriched diet (D2) under environmental control conditions. The crosses indi-
cate means, while the boxes indicate the seventy-fifth percentile (top line), median (middle line),
and twenty-fifth percentile (bottom line) and the error bars indicate extreme values of the data
range. Different superscripts indicate significant differences among conditions. (From Simide
et al. 2016, courtesy of Springer)
Although the above papers leave unanswered the question of how these findings
translate into life history traits of the affected and succeeding generations, they
clearly describe alterations in the HSP90 content in fishes challenged by the rela-
tively mild stress of starvation, and an impact on filial generations can be expected
and should be taken into account when culturing fish and aquatic invertebrates with
reference to optimal performance and production.
6.2.2.4 MicroRNAs in Action
Action of microRNAs
MicroRNAs (miRNAs) are a new large class of non-coding, endogenous, small

RNA that regulates gene expression by translational repression. miRNAs are
encoded by the genome, contain about 22 nucleotides, and are found in plants, ani-
mals, and some viruses with functions such as RNA silencing and post-transcriptional
regulation of gene expression. They play roles in controlling DNA methylation and
histone modifications, creating a highly controlled feedback mechanism. Vice versa,
epigenetic mechanisms, such as promoter methylation or histone acetylation, can
modulate miRNA expression (Choi and Friso 2010). For the sake of easy readabil-
ity, we will apply the term “miRNAs” in the more general sense for almost all small
non-coding RNAs.
García-Segura et al. (2013) summarized the way in which macro- and micronu-
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trients affect miRNA transcription in mammals:

• Amino acid-deficient diets can lead to changes in miRNA expression, with miR-
182, miR-183, miR-199a, miR-705, and miR-1224 being involved;
• The decreased or augmented availability of glucose has been recognized as a
factor that modulates miRNA expression, particularly miR-466 h-5p;
• Some fatty acids can alter the expression of, for instance, miR-10a, miR-24, miR-
122, miR-192, and miR-375;
• Vitamins also have the potential to modulate the expression of miRNAs. For
instance, diets deficient in methyl donors (folate, methionine, and choline) lead
to hepatocellular carcinoma accompanied by increased expression of let-7a,
miR-17-92, miR-21, miR-23, miR-130, and miR-190 and decreased expression of
miR-122. Vitamin D appears to be connected to miR-125b, and VE deficiency has
been shown to result in a decrease in miR-122a.
Recently, miR-10a and miR-10b have been identified as repressing granulosa cell
development in ovaries during folliculogenesis and inducing apoptosis (Tu et al.
2017). These anticancer effects exerted by the miR-10 family on granulosa cells are
conserved among different species; for instance, miR-10 has been found in zebrafish
(Woltering and Durston 2008) and L. vannamei shrimps (Huang et al. 2017). In the
shrimp, the host miR-10a plays an important, positive role in the replication of the
white spot syndrome virus (WSSV). Huang et al. (2017) concluded that shrimp
miR-10a is co-opted by these and other WSSV genes to enhance WSSV replication
(Fig. 6.50).
In fishes, many miRNAs are central to growth, development, metamorphosis,
and stress response (Bizuayehu and Babiak 2014; Campos et al. 2014; Flynt et al.
2009; Fu et al. 2015; Wan et al. 2015). A significant induction of miR-210 was found
in primary ovarian follicular cells of the marine medaka (Oryzias melastigma)
exposed to hypoxia, and gene ontology analysis further highlighted the potential
roles of miR-210 in cell proliferation, cell differentiation, and cell apoptosis: in sum,
miR-210 plays an anti-apoptotic role in ovarian cells (Tse et al. 2015).
Furthermore, evidence is significantly accumulating that miRNAs play a crucial
role in pathogen resistance, that is, endogenous vs. exogenous RNA. miRNA-
mediated resistance has been described in zebrafish (Ordas et al. 2013), channel
catfish (Xu et al. 2013), rainbow trout (Bela-ong et al. 2015), large yellow croaker
(Larimichthys crocea) (Qi et al. 2014), Asian seabass (Lates calcarifer) (Xia et al.
2011), a snakehead (Channa sp.) cell line (Liu et al. 2016), grass carp (Xu et al.
2015, 2016), blunt snout bream (Megalobrama amblycephala) (Yuhong et al. 2016),
tongue sole (Cynoglossus semilaevis) (Yan et al. 2016), and three-lined tongue sole
(C. abbreviatus) (Sha et al. 2014).
Most information on the action and regulatory pathways of miRNAs in aquatic
invertebrates stems from the model genus Daphnia and shows that miRNA are cen-
tral to the development (Chen et al. 2014; Ünlü et al. 2015; Wheeler et al. 2009) and
stress response (Chen et al. 2015) of this invertebrate species, many of which appear
to be crustacean- and arthropod-specific miRNAs (Blythe et al. 2012). The pathogen
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Fig. 6.50 Schematic representation of how shrimp miR-10a is co-opted by the white spot syn-
drome virus (WSSV) to enhance viral replication. Pv-miR-10a upregulates the expression of at
least three WSSV genes (vp26, vp28, and wssv102) by targeting their 5′ untranslated region.
Alignment followed by RNAhybrid prediction indicates that several other important WSSV genes
are also likely to be upregulated by host miR-10a. (From Huang et al. 2017, courtesy of Frontiers
in Immunology)
responsiveness of miRNAs has also recently been documented for a huge variety of
invertebrates, such as Pacific whiteleg shrimp (Litopenaeus vannamei) (Zeng et al.
2015), Japanese tiger prawn (Marsupenaeus japonicus) (Gong et al. 2015; Yang
et al. 2014b; Zhu et al. 2016), black tiger shrimp (Penaeus monodon) (Li et al.
2013b), Chinese mitten crab (Eriocheir sinensis) (Ou et al. 2012), red swamp cray-
fish (Procambarus clarkii) (Ou et al. 2013; Xu et al. 2014), mud crab (Scylla
paramamosain) (Li et al. 2013a), flat oyster (Ostrea edulis) (Martín-Gómez et al.
2014), and sea cucumber (Apostichopus japonicus) (Zhang et al. 2014).
MicroRNAs in Action
Recently, Seemann et al. (2017) provided novel histopathological evidence that BaP
is a transgenerational skeletal toxicant in medaka. Bone impairments persist to F3
adult fish descended from P0 great grandparents that were exposed to 1 μg L─1 BaP
for 21 days. The screening of conserved bone miRNAs and the identification of
potential changes in osteogenic miRNA/mRNA expression (including miR-214,
osx, opn) associated with osteoblast activity in F3 male fish shed light on the miRNA-
epigenetic circuit underlying BaP-induced transgenerational skeletal impairments.
It is well understood that hypoxia leads to reproductive impairments in males of
the marine medaka via DNA methylation (Box Methylation). In human cell cultures,
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Fig. 6.51 All expressed miRNAs in the Normoxia 24 h and Hypoxia 24 h groups of Macrobrachium
nipponense. The Venn diagram displays the distribution of 882 co-expressed miRNAs between the
Normoxia 24 (left, green circle) and Hypoxia 24 (right, blue circle) groups. Pink circles indicate
the differentially expressed unique miRNAs (P < 0.001). (From Sun et al. 2016, with permission
from Elsevier)
Yu et al. (2015) found that under hypoxia the increased expression of miR-98 leads
to the downregulation of cyp19A1 mRNA and protein expression and contributes to
a reduction in estradiol production. In addition, Tse et al. (2016) showed that hypoxia
also alters the testicular functions in male individuals through miRNA regulation. In
the testis, nine miRNAs were significantly upregulated and five were downregulated
by hypoxia. Bioinformatic analysis revealed that, among the genes targeted by the
hypoxia-responsive miRNAs, many are closely related to the stress response, cell
cycle, epigenetic modification, sugar metabolism, and cell motion. Particularly,
miR-125-5p impaired the euchromatic histone-lysine N-methyltransferase 2, the
epigenetic regulator of transgenerational reproduction. In zebrafish, He et al. (2017)
detected that miR-125c plays a pivotal role in cellular adaptations to hypoxic stress
and has a great impact on the early embryonic development of zebrafish.
The oriental river prawn (Macrobrachium nipponense) is an important commer-
cial aquaculture species, and is sensitive to hypoxia. By deep sequencing of four M.
nipponense tissues (gill, hepatopancreas, muscle and hemocytes), Sun et al. (2016)
identified 267 unique miRNAs, including 203 conserved and 64 prawn-specific
miRNAs. Annotation of targets revealed a broad range of biological processes and
signal transduction pathways regulated by M. nipponense miRNAs. In addition, 882
co-expressed and 39 specific (25 normoxia-specific and 14 hypoxia-specific) miR-
NAs (Fig. 6.51) that may be involved in the response to hypoxia were confirmed
using miRNA microarray analysis from the four prawn tissues combined.
Recently, Andreassen and Høyheim (2017) pointed out the importance of miR-
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NAs as important regulators of a number of key genes in immune system gene net-
works. Challenge studies conducted in several species have identified differently
expressed miRNAs associated with viral or bacterial infection. The results from
these studies identified several miRNAs that are likely to have evolutionary con-
served functions that are related to immune response in teleost fish. Changed expres-
sion levels of mature miRNAs from the five miRNA genes miRNA-462, miRNA-731,
miRNA-146, miRNA-181, and miRNA-223 were observed following viral as well as
bacterial infection in several teleost fish. Furthermore, significant changes in expres-
sion of mature miRNAs from the five genes miRNA-21, miRNA-155, miRNA-1388,
miRNA-99, and miRNA-100 were observed in multiple studies of virus infected fish
while changes in expression of mature miRNA from the three genes miRNA-122,
miRNA-192, and miRNA-451 were observed in several studies of fish with bacterial
infections. The function of the evolutionary conserved miRNAs responding to
infection depended on the target gene(s) they regulate. A few target genes have been
identified while a large number of target genes have been predicted by in silico
analysis. The results indicated that many of the targets were genes from the host’s
immune response gene networks.
The emerging evidence has pointed out another field of action of miRNAs: eco-
logical speciation. In the following Chap. 7 ‘Trophic Diversification and Speciation’,
we shall learn that dietary resource competition has the potential to induce ecologi-
cal branching and speciation.
6.2.2.5 Small RNAs and Dietary Restriction (DR)
One specific mechanism of DR-mediated longevity deserves mentioning. Evidence

from animal studies and human famines indicates that starvation can affect the
health of the progeny of famished individuals (Fig. 6.52). However, it is not clear
whether starvation affects only immediate offspring or has longer lasting effects; it
is also unclear how such epigenetic information is inherited. To date, it is well
understood that double-stranded (ds)RNA-mediated gene silencing in the nematode
C. elegans can be maintained over dozens of generations via transgenerational
inheritance of distinct pools of small interfering (si)RNA (Rechavi et al. 2014).
These authors showed that the affected target genes were involved in nutrition,
affirming the biological relevance of the discovered phenomenon. Furthermore, the
heritability of the dsRNA-based starvation response depended on a dsRNA-binding
protein and a germline-expressed argonaute protein, indicating that a specific RNAi6
pathway was involved. The transgenerational transmission of the miRNA-based
starvation response clearly comes across as inheritance of an acquired trait.
Supportingly, Kogure et al. (2017) found that components of the miRNA machinery
6
A biological process in which RNA molecules inhibit gene expression or translation by neutral-
izing targeted mRNA molecules.
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Fig. 6.52 Caenorhabditis individuals with a starvation history show an extended life span. A
comparison of the life span of N2 (wildtype) worms whose great-grandparents had experienced
starvation at the first larval stage with animals whose great-grandparents were continuously fed.
The x axis shows the life span in days of adulthood. The y axis shows the fraction of worms alive.
N2 derived from starved great-grandparents show a life span extended by 37% in comparison with
N2 that were continuously fed over generations. (From Rechavi et al. 2014, with permission from
Elsevier)
play an important role in mediating intermittent fasting-induced longevity via the

regulation of fasting-induced changes in gene expression.
The studies by Rechavi et al. (2014) and Kogure et al. (2017) presented the dra-
matic possibility that, at least in multicellular eukaryotes, epigenetic adaptation is a
general strategy to cope with all types of environmental challenges. The heritability
of the epigenetic adaptations defies the common assumption that “evolution has no
forecast.” Indeed, the transgenerational persistence of epigenetic changes would be
selected precisely because lineages that are capable of predicting that stress condi-
tions typically last longer than a single generation span and maintaining the adapta-
tion across generations would have an advantage over clueless rivals. Still, the
limitation remains that, at least at face value, this highly efficient Lamarckian adap-
tation strategy does not lead to long-term evolutionary change (Koonin 2014).
The central question “can epigenetics translate into genetics?” can now be
answered “by epigenetic mechanisms that ‘flirt’ (interact) with the DNA, such as
non coding RNAs-mediated gene regulation, and DNA-independent inheritance
mechanisms (e.g. prions, protein feed back loops, hormones, metabolites)” (Pilpel
and Rechavi (2015) and references therein). Following the reasoning of Houri-
Ze’evi et al. (2016), the different epigenetic mechanisms for the long-term inheri-
tance of acquired properties are not equivalent. Instead, the authors showed that
exposure to dsRNA activates a feedback loop whereby gene-specific RNAi
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Fig. 6.53 The duration of epigenetic responses underpinning transgenerational inheritance is

determined by an active mechanism relying on the production of small RNAs and modulation of
RNAi factors, dictating whether ancestral RNAi responses would be memorized or forgotten.
(From Houri-Ze’evi et al. 2016, with permission from Elsevier). dsRNA double-stranded RNA,
siRNA small interfering RNA
responses dictate the transgenerational duration of RNAi responses mounted against

unrelated genes, elicited separately in previous generations (Fig. 6.53). RNA-
sequencing analysis revealed that, aside from the silencing of genes with comple-
mentary sequences, dsRNA-induced RNAi affected the production of heritable
endogenous small RNAs, which regulate the expression of RNAi factors.
Manipulating genes in this feedback pathway changes the duration of heritable
silencing. Such active control of transgenerational effects could be adaptive, since
ancestral responses would be detrimental if the environments of the progeny and the
ancestors were different.
Diet-Mediated microRNA Effects

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There has been and continues to be a debate regarding whether dietary miRNA
maintain its regulatory ability in consumers—whether there is a so-called cross-
kingdom effect (see, for instance, Witwer and Zhang (2017)). Similarly, Zhou et al.
(2017) stated that, in plants, nematodes, and microbes, small RNAs (sRNAs) can
mediate inter-kingdom communication, environmental sensing, gene expression
regulation, host parasite defense, and many other biological functions. Strikingly,
the study by Zhou et al. showed that ingested plant miRNAs are transferred to blood,
accumulate in tissues, and regulate transcripts in consuming animals. While Zhou’s
research and subsequent studies by other independent groups has explored the
emerging field of miRNA-mediated crosstalk between species further,7 some groups
have reported negative results and questioned its general applicability (Kang et al.
2017). Therefore, further studies carefully evaluating the horizontal transfer of
exogenous sRNAs and their potential biological functions are urgently required.
Since none of the studies to date have been carried out with aquatic animals, the
potential cross-kingdom of dietary miRNAs will not be followed any further in this
chapter; instead, it will focus on dietary effects on the expression and action of
endogenous miRNAs in aquatic consumers.
Invertebrates
The anthraquinone emodin8 is often applied in aquaculture as an immunopotentiator

in fish and crayfish (also see, Volume II, Plant Materials). Feeding the red swamp
crayfish emodin-containing diets, Xu et al. (2014) identified 106 mature miRNAs
(belonging to 68 miRNA gene families) and 5 novel miRNAs from hepatopancreas
of crayfish fed emodin diets. Six miRNAs were significantly upregulated and 29
miRNAs were significantly downregulated. Function annotation of the predicted
target genes of the novel miRNAs indicated that they are likely to be involved in the
innate immune response, growth, metabolism, cellular process, biological regula-
tion, and stimulus response (Fig. 6.54).
7
From mammalian studies; however, evidence is growing that such an effect is likely to exist:
Zempleni et al. (2015) reported that the body of evidence is sufficient to consider milk cow miRNA
bioactive compounds in human foods. Furthermore, Lukasik et al. (2018) found all the studied
plant food-derived miRNAs (miR166a, miR156a, miR157a, miR172a, and miR168a) in breast milk
from healthy volunteers.
8
= 6-methyl-1,3,8-trihydroxyanthraquinone. The term ‘emodin’ is derived from the Himalayan
rhubarb, Rheum emodi. Emodin is present in many plants, such as rhubarb (Rheum officinale), aloe
(Aloe barbadensis), senna (Cassia angustifolia), Thunberg (Polygonum multiflorum), and Japanese
knotweed (Fallopia japonica). Emodin has been widely used in traditional medicine in Eastern
Asia, especially in China.
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Fig. 6.54 Red swamp crayfish (Procambarus clarkii): gene ontology (GO) analysis of novel
miRNA target genes between control and feeding trial libraries (blue: control, red: feeding trial).
(From Xu et al. 2014, with permission from Elsevier)
Fishes
Growth factors, regulatory proteins, and transcription factors have been identified as
participating in the regulation of myogenesis in fish. MicroRNAs have emerged as
a new class of key regulators of myogenesis. Among the 21 miRNAs identified with
high abundance in the fast muscle of adult Mandarin fish or Chinese perch (Siniperca
chuatsi), 19 miRNAs were expressed differentially in adults and juveniles (Zhu
et al. 2015). The postprandial changes in the transcript abundance were determined
for the 21 miRNAs following a single satiating meal in the juveniles after fasting for
1 week. The results showed that the 7 miRNAs (miR-10c, miR-107a, miR-133a-3p,
miR-140-3p, miR-181a-5p, miR-206, and miR-214) were sharply upregulated or
downregulated within 1 h after refeeding. These miRNAs may be the most promis-
ing candidate miRNAs involved in a fast-response signaling system that regulates
fish skeletal muscle growth. Target prediction and expression analysis showed that
4 miRNAs (miR-10c, miR-107a, miR-140-3p, and miR-181a-5p) can play a role in
regulating the translation of target gene transcripts, such as myostatin, following
acute anabolic stimuli.
A heat map summary and hierarchical clustering analysis were performed
according to the similarity in their expression across different postprandial times
(0–96 h). Two clades were shown by hierarchical clustering of the miRNAs through-
out the trial (Fig. 6.55). The first clade showed six pairs of closely linked miRNAs
(miR-103 and miR-107a; miR-21 and miR-1a; miR-181a-5p and miR-10c; miR-
133a-3p and miR-22a; miR-26a and miR-199-3p; and miR-199-5p and miR-152a).
The second clade showed a close relation and covariation of miR-140-3p and miR-
0h 1h 3h 6h 12h 24h 48h 96h

miR-499
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miR-21 2.08
miR-1a
miR-133a-3p
miR-22a 1.39
miR-23a
miR-143
miR-133b-3p 0.69
miR-107a
miR-103
miR-181a-5p 0.00
miR-10c
miR-206
miR-26a -0.69
miR-199-3p
miR-199-5p
miR-152a -1.39
miR-214
miR-101a
miR-140-3p -2.08
miR-146a
Fig. 6.55 Heat map summary of hierarchical clustering of miRNAs in skeletal muscle during fast-
ing–refeeding periods in Chinese perch. In the heat map, the green and red colors, respectively,
indicate a decrease and an increase. The absolute signal intensity ranges from −2.08 to +2.08, with
corresponding color changes from green to red. (From Zhu et al. 2015, with permission from
Springer)
0h 1h 3h 6h 12h 24h 48h 96h

miR-133b-3p 1.82
miR-133a-3p
1.21
miR-206
0.61
miR-181a-5p
0.00
miR-214
miR-26a -0.61
miR-146 -1.21
miR-1a -1.82
Fig. 6.56 Heat map summary of hierarchical clustering of miRNAs in skeletal muscle during fast-
ing–refeeding periods in grass carp. Green and red respectively denote a decrease and an increase.
The absolute signal intensity ranged from −1.82 to +1.82, with corresponding color changes from
green to red. (From Zhu et al. 2014, courtesy of Science Press)
146a, which was subsequently clustered with miR-101a and miR-214 (Fig. 6.55).
The transcription of several miRNAs in the fast skeletal muscle of Chinese perch
responded quickly to a single meal after 7 days of fasting. This paper indicates that
refeeding induces a coordinated expression of several miRNAs involved in strong
resumption of myogenesis with feeding. These miRNAs are involved in regulating
fish myotomal muscle growth.
Do carnivorous and herbivorous fish species differ in their miRNA pattern
responsible for muscle growth? Following a comparable experimental approach but
with less miRNA, Zhu et al. (2014) studied the grass carp (C. idella). miR-1a, miR-
133a-3p, miR-133b-3p, miR-146, miR-181a-5p, miR-206, and miR-214 were sig-
nificantly elevated 1 or 3 h after refeeding in the fast muscle of grass carp. The
clustering of the miRNAs throughout the trial showed three clades (Fig. 6.56). The
first clade included two pairs of closely linked miRNAs (miR-133b-3p and miR-
133a-3p; miR-206 and miR-181a-5p) that clustered together. The fact that miR-
181a-5p and miR-206 clustered together indicates a close relation and coordination
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regulation of these miRNAs towards the resumption of myogenesis following

refeeding. The second clade clustered miR-26a with miR-146 expression. MiR-1a
was isolated. This pattern is in fairly good agreement with the carnivorous Chinese
perch (Fig. 6.55).
Recently, Mennigen (2016) emphasized the significance of miRNAs in develop-
ment and growth, sexual maturation, and endocrine regulation in rainbow trout.
Several exogenous and endogenous stimuli are crucially involved in regulating
energy metabolism in teleost fish (Fig. 6.57). Exogenous stimuli include several
abiotic factors, such as temperature, photoperiod, salinity, and hypoxia, as well as
biotic factors, such as con- and interspecific interaction and nutrition. These exog-
enous factors are integrated by endogenous factors, including endocrine and cyto-
kine signaling molecules.
In teleost fish, as in other vertebrate classes, the regulation of metabolic miRNAs
is still only poorly understood. Evidence for a role of exogenous metabolic stimuli
in regulating metabolic miRNAs stems from studies investigating acute nutritional
challenges following a short-term fast. In rainbow trout, for example, the abundance
of miR-122, a hepatic regulator of glucose and lipid homeostasis, is postprandially
regulated following the ingestion of a single meal after a short-term fast. A one-
week fast in zebrafish has been shown to increase the abundance of let-7d and miR-
140-5p, predicted to target hepatic mRNAs coding for subunits of 5′adenosin
monophosphate-activated kinase (AMPK), an important metabolic sensor. These
findings show acute and prolonged responses of metabolic miRNAs to nutrient
availability in several teleost species. In addition to nutrient quantity, studies inves-
tigating the effect of nutritional quality have found that the macronutrient composi-
tion also alters metabolic miRNAs: a single high-fat meal acutely suppressed the
lipogenic miR-122 abundance in rainbow trout, and several hepatic miRNAs altered
the expression profiles in response to a prolonged high-fat diet in the Blunt snout
bream (Mennigen (2016) and references therein).
With regard to endogenous metabolic stimuli, the evidence indicates a prominent
role for endocrine regulation of hepatic miR-122 expression in rainbow trout and
zebrafish. In rainbow trout, acute insulin administration in fasted rainbow trout and
in primary hepatocytes acutely increased miR-122 abundance, showing that insulin
may at least partially mediate the observed postprandial effects following the inges-
tion of a single meal after a one-day fast in the same species. The role of several
other important metabolic stimuli has not yet been addressed, and there is an acute
need to determine how metabolic miRNAs can integrate metabolic stimuli to regu-
late teleost energy metabolism (Mennigen (2016) and references therein).
Finally, light is appearing at the end of the fish tunnel: in a series of papers, the
laboratory of Jun Qiang (Chinese Academy of Fishery Sciences, Wuxi) identified
the involvement of various miRNAs of a fast-growing tilapia strain (GIFT, geneti-
cally improved farmed tilapia) in nutrition and stress response. Specifically, Qiang
et al. (2017) reported that miR-29a post-transcriptionally downregulated stearoyl-
CoA desaturase (scd) by binding to its 3′ UTR. miR-29a expression was negatively
correlated with scd expression in the liver. The inhibition of miR-29a led to a
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6.2 What Is Epigenetics?
Fig. 6.57 Proposed scheme of the metabolic function of miRNAs at different levels of biological organization in rainbow trout. (From Mennigen 2016, with
413

significant increase in scd expression on day 60 induced by a saturated FA diet,

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thereby increasing the conversion of 16:0 and 18:0 to 16:1 and 18:1, respectively.
Overall, miR-29a is critical in regulating lipid metabolism homeostasis in the liver,
and this may provide a basis for understanding the biological processes and thera-
peutic intervention encountered in fatty liver.
In sum, the post-transcriptional action of miRNAs as an epigenetic mechanism
appears to be of paramount importance in many pathways and to be affected strongly
by nutrition. It can be expected that future studies will show that starvation and
refeeding (the quantitative side of nutrition) of fish significantly influence not only
the expression of miRNAs but also the various food ingredients—the qualitative
side of nutrition. Furthermore, it will be challenging to figure out how the action of
miRNAs translates into the life history traits of the progeny. Since transgenerational
plasticity across multiple generations has recently been discovered in marine stick-
lebacks in response to changing thermal environments (Shama and Wegner 2014),
the corresponding step specifically concerning the nutrition of fishes and aquatic
invertebrates is overdue and appears to be very promising.
Transgenerational effects of parental diets occur on various levels and through vari-
ous mechanisms, whereby both diet quantity and diet quality are effective. Parental,
especially maternal, effects are well understood. It becomes obvious that there is no
uniform response to dietary challenges—the responses appear to be species specific
and reflect the different strategies of the species to sustain their populations.
These effects can, for instance:
• Increase resistance against pathogens and natural xenobitotics, such as cyanotox-
ins. For instance, Daphnia offspring possessed increased resistances if they were
produced by mothers under poor nutritional conditions. In general, the per off-
spring investment of Daphnia under stressful conditions is greater than that
under benign conditions. Also in fishes, improved immunity acquired via pre-
and probiotic food can be transferred to the offspring, as reported for several
species;
• Produce contrasting results from coconut crabs. Larvae hatched from larger
(well-fed) females showed significantly longer survival periods under non-fed
conditions;
• Show contrasting behavior for the midge Chironomus tepperi under a reduced
food supply: the offspring developed less well and were less reproductive;
• Increase hatching success if the AA patterns of food algae match those of the
invertebrate mothers, as shown in marine copepods. Similarly, Met-deficient
diets in rainbow trout led to short-term reduced appetite in the fry. Parental Met
deficiency reduced the egg size and decreased the later growth performance of
the offspring;
• Determine the metabolic capacity and even the escape performance of the off-
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spring via metabolic programming, as shown in the red drum. PUFAs play a
crucial role in this process;
• Increase innate and adaptive immunity by maternal transfer of corresponding
transcripts to embryos;
• Probably determine whether or not brown trout fry become freshwater residents
or anadromous wanderers. Interestingly, the offspring of migratory fish appear to
be less able to maintain growth on a poor-quality diet than those of freshwater
residents;
• Modulate starvation (dietary or calorie restriction) resistance. Dietary restriction
can extend the life span and can be passed to the offspring, as exemplified by the
euryhaline rotifer B. calyciflorus;
• Induce sexual reproduction, as found in the rotifer Brachionus manjavacas; the
corresponding signal can be inherited epigenetically up to the fortieth
generation.
It is obvious that many of the described transgenerational effects were reported
from studies of invertebrates. These studies can serve as a role model for future
projects with fishes. For instance, does the parental diet quality have an influence on
the sexual determination of the offspring?
Until recently, paternal effects were thought to have only a genetic background.
However, evidence from invertebrates and fishes, indicating that epigenetic marks
acquired during spermatogenesis can be sustained through embryonic development,
is increasing. In addition, evidence is currently growing that epigenetic inheritance
can last for many generations, as shown not only in ciliates but also in insects.
Several epigenetic mechanisms appear to be feasible: methylation of the DNA and
the histones, HSP90 acting as a capacitor for morphological evolution, or the action
of miRNAs. To date, it is hard to judge which mechanism may be the most plausible
one, particularly because convincing field evidence for HSP90 as a capacitor has
been presented recently, eye-less and eyed cavefish with HSP90 being strongly
involved. On the other hand, methylation of DNA and histones is the classical
mechanism, for which many examples are available. As the most recently discov-
ered mechanism, the action of non-coding small RNAs (miRNAs) bears consider-
able potential to cause mutation-like changes in the epigenome, particularly because
not only endogenous but also exogenous (dietary) miRNAs9 are central. However,
the proof for aquatic animals that diet-mediated epigenetic changes in life history
traits can be transmitted to succeeding generations is awaiting substantiation.
9
Here, I take it for granted that exogenous miRNAs possess regulating capacities in the hosts, that
cross-kingdom effects do occur.
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Zhang J, Zuo Z, Zhu W, Sun P, Wang C (2013) Sex-different effects of tributyltin on brain aro-
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marmoratus). Mar Environ Res 90:113–118. https://doi.org/10.1016/j.marenvres.2013.06.004
Zhang P, Li C, Shao Y, Chen X, Li Y, Su X, Li T (2014) Identification and characterization of
miR-92a and its targets modulating Vibrio splendidus challenged Apostichopus japonicus. Fish
Zhao JL et al (2015) Polymorphisms and DNA methylation level in the CpG site of the GHR1 gene
associated with mRNA expression, growth traits and hormone level of half-smooth tongue
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sole (Cynoglossus semilaevis). Fish Physiol Biochem 41:853–865. https://doi.org/10.1007/

s10695-015-0052-y
Zheng L, Xu T, Li D, Zhou J (2015) A representative retinoid X receptor antagonist UVI3003
induced teratogenesis in zebrafish embryos. J Appl Toxicol 35:280–286. https://doi.
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Zhong H et al (2014) DNA methylation of pituitary growth hormone is involved in male growth
superiority of Nile tilapia (Oreochromis niloticus). Comp Biochem Phys B 171:42–48. https://
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Chapter 7
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Trophic Diversification and Speciation –

‘Your Eating Fuels Evolution’
Abstract This concluding chapter presents evidence that the dietary basis of fishes
and aquatic invertebrates has, due to initial individual specialization, the potential to
cause trophic diversification and induce parapatric or even sympatric speciation.
Well known examples comprise not only fish and, to a lesser degree, invertebrate
species flocks in ancient lakes, but also rapid speciating fish taxa, such as Lepomis
gibbosus, Cyprinodon spp., Salvelinus alpinus, Gasterosteus aculeatus, and
Amphilophus spp. These fish species can radiate within a few decades or even years.
Underlying mechanisms are genetics and epigenetics, e.g. the action of microR-
NAs. From ciliates, which pass acquired properties to hundreds of generations, also
methylation of DNA and histones has been reported. Therefore, genetics and epi-
genetics are not mutually exclusive; rather, they can enforce each other, and non-
genetic mechanisms should, therefore, be in the focus of studies to come.
Evolutionary branching can be found in many diverse ecological systems and inter-
actions, including resource competition, interference competition, predator–prey
systems, spatially structured populations and metapopulations, host–parasite sys-
tems, mutualistic interactions, and mating systems (Geritz et al. 2004). With respect
to nutrition, species diverge morphologically and ecologically, when they compete
for limited food resources. As they diverge, the likelihood of interbreeding declines
(Grant and Grant 2017).
Resource-use polymorphisms are associations between heritable phenotypic
variation and habitat use within populations. These polymorphisms can offer
insights into the mechanisms by which adaptation can occur despite gene flow, and
into the importance of these mechanisms for the formation of new species.
Ecological speciation is therefore the process by which barriers to gene flow evolve
between populations as a result of ecologically-based divergent selection (Nosil
2015). In other words, sympatric speciation is a likely outcome of competition for
resources (Dieckmann and Doebeli 1999). Central to the environmental resources is
food; the competition for food can lead to trophic diversification and eventually to
parapatric or even sympatric speciation. It can at least promote ecological and phe-
notypic variation. This is because rare phenotypes of one species may have access

https://doi.org/10.1007/978-3-319-91767-2_7
432 7 Trophic Diversification and Speciation – ‘Your Eating Fuels Evolution’
to alternative resources, thereby escaping competition with more common pheno-

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types. However, intraspecific competition is also thought to maintain intraspecific

variation, trophic polymorphism, or even drive speciation (Svanbäck and Bolnick
2007 and references therein). In fact, this is evolution in action!
The life history and ecological traits contributing to ecological speciation in
freshwater fishes appear to be controlled by a few loci with rather large phenotypic
effects, or may be superimposed by epigenetics (see below). This phenotypic vari-
ability is indicated by prey foraging and capturing traits, such as jaw morphology,
body shape, tooth shape and number (Puebla 2009), topographic distributions and
the composition of photoreceptors (found in sting rays (Garza-Gisholt et al. 2015)),
or electric organ discharges (Tiedemann et al. 2010).
Until recently, genetic considerations have predominated in the discussion of
evolution and selection. Compared to the enormous progress made in genetics, there
has been relatively little systematic effort to analyze the environmental effects on the
phenotype, and their evolutionary consequences. The plastic phenotype, stigmatized
by poorly understood environmental influences, has sometimes been lost from view
as the focus of selection (West-Eberhard 1989); furthermore, this concept has been
espoused most infamously by Lysenko (1948). However, already three decades ago,
West-Eberhard claimed that the “phenotype” includes all aspects of an organism
other than the genotype, from the enzyme products of the genes to learned behaviors
and the effects of disease. If one considers that the “environment” includes both the
external surroundings of an organism and the internal conditions affecting gene
expression, “phenotypic plasticity” is seen to “encompass an enormous diversity of
kinds of variability” and one can add eventual “speciation”. In short, phenotypic
plasticity is an environmentally-based change in the phenotype (Via et al. 1995).
In the same period of time, Meyer (1987) experimentally demonstrated a similar
phenomenon without genetic change and used the term “epigenetic”: persistent
behavioral differences associated with different diets during ontogeny caused
marked differences in the trophic morphology of the Jaguar cichlid (Parachromis
managuensis, Fig. 7.1). Young fish of the same lab-spawned brood were assigned
randomly to one of two groups: group I was fed nauplii of Artemia salina, and
group II was fed commercially available flake food and lab-reared nematode worms
for 8 months. Thereafter, both groups were fed a common diet. Phenotypes that dif-
fered significantly at 8.5 months converged almost completely at 16.5 months
(Fig. 7.1). If feeding on two different diets was continued after 8.5 months, the
phenotypes remained distinct. Differences in diet and possibly in feeding mode
appeared to have caused these phenotypic changes. Overall, Meyer showed that
heterochrony1 is involved in diet-induced morphological differences between indi-
vidual cichlid fish.
Further examples of diet-induced phenotypic plasticity have since been reported
in a variety of fish species (Table 7.1); the fish literature is particularly rich with
examples of resource-based or trophic polymorphisms. Discrete resource polymor-
1
In evolutionary developmental biology, heterochrony is a change in the timing or rate of events,
leading to changes in size and shape
7 Trophic Diversification and Speciation – ‘Your Eating Fuels Evolution’ 433
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change of diet
group I
group Il
acutorostral phenotypic
acceleration plasticity
shape
control group
obtusorostral
retardation
age
Fig. 7.1 Jaguar cichlid. Left: X-rays images of (a) group I fish and (b) group II fish, after 8 months
of feeding on different diets. Group I fish show the acutorostral morphology, and group II fish
exhibit a more obtusorostral morphology. Right: Qualitative model showing the ontogenetic tra-
jectories and the assumed heterochronic processes responsible for the observed developmental
patterns (applying heterochronic terminology to intraspecific ontogenetic events). At 8.5 months of
age, before the diet change, the fish in group II show an obtusorostral morphology, which retards
the shape growth-rate relative to the “normal” (acutorostral) developmental trajectory of group
I. The acceleration in shape growth rate after the diet was changed is relative to group I, which
remained acutorostral in shape, and to the control group, which continued to feed on flake food and
remained obtusorostral in shape. (From Meyer 1987, with permission from Wiley). Inserted image
of Jaguar cichlid (courtesy of George Chernilevsky, Wikimedia)
phisms probably occur more frequently than is generally appreciated. Furthermore,

this phenomenon may be underestimated as a diversifying force and potentially
plays important roles in population divergence and the initial steps in speciation. In
an ecological context, it is important in resource partitioning and reducing intraspe-
cific competition (Skulason and Smith 1995).
Phenotypic differences between morphs vary widely across taxa and depend on
whether they are based on morphology, life history traits, or behavior. They are
highly correlated with habitat use in general, and diet in particular. Fishes typically
differ in jaw morphology (Fig. 7.2, size and jaw shape), and size, shape and number
of gill rakers. Other external and internal structures may also be involved, including
the size and shape of the fins, body and head depth, and structure of the stomach and
gut. Benthivorus morphs generally have a blunt snout, the lower jaw is shorter than
the upper jaw, and they have a less streamlined body than planktivorous and piscivo-
rous morphs, which have a pointed snout and jaws that are similar in length.
Differences in life history, such as growth pattern, age at maturity, reproductive
investment, fecundity and egg size are especially common in morphs of salmonids.
Differences are seen in foraging behavior, techniques, migratory behavior and social
behavior (Skulason and Smith (1995) and references therein).
Furthermore, the notion that cultured fishes develop a morph that differs from
their wild conspecifics has become nearly axiomatic. A commonly supervened
Table 7.1 Resource-induced phenotypic plasticity in selected fish species

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Species Affected trait/ecological

Trivial name Scientific name niche References
Alewive Alosa Foraging morphology Post et al. (2008)
pseudoharengus
Midas cichlid Amphilophus cf. Pharyngeal jaw apparatus/ Elmer et al. (2010)
citrinellus lip morphology
Midas cichlid A. labiatus Lip morphology Colombo et al. (2013)
Midas cichlid A. tolteca Lip and jaw morphology Kusche et al. (2014)
Mediterranean Aphanius fasciatus Morphology of the Tigano et al. (2001)
killifish pharyngeal and oral jaws
Alluaud’s haplo Astatoreochromis Lower pharyngeal jaws Young (2013) and
alluaudi (Fig. 7.2) Gunter and Meyer
(2014)
African cyprinid Barbus neumayeri Trophic morphology, Schaack and Chapman
feeding performance (2004)
Barbs Barbus spp. Head, fork length Nagelkerke et al. (1994)
Cichlid Cichlasoma minckleyi Feeding on snails and In Skulason and Smith
plant material (1995)
Dwarf cisco Coregonus artedii Size, body shape, life Shields and Underhill
history traits (1993)
Lake whitefish C. clupeaformis Benthivory, planktivory, In Skulason and Smith
piscivory, migration (1995)
European C. lavaretus Skull & gill raker Østbye et al. (2005)
whitefish morphology, intestine
length
Scandinavian Coregonus spp. Benthivory, planktivory, In Skulason and Smith
whitefish piscivory (1995)
Zebrafish Danio rerio Head, body, and fin Shukla and Bhat (2017)
morphology
Western Gambusia affinis Jaw morphology, feeding Ruehl and DeWitt
mosquitofish performance (2005)
Three-spined Gasterosteus Benthivory, planktivory, In Skulason and Smith
stickleback aculeatus piscivory, migration (1995)
Three-spined G. aculeatus Brain morphology? Park et al. (2012)
stickleback telencephalon
Eartheaters Geophagus spp. Pharyngeal jaw apparatus Wimberger (1992)
Haplochromine Haplochromis spp. Body shape Machado-Schiaffino
cichlids et al. (2015)
Texas cichlid Herichthys Head morphology Stauffer and Gray
cyanoguttatus (2004)
Goodeid fish Ilyodon spp. Strong indication of In Skulason and Smith
differences in food (1995)
Pumpkinseed Lepomis gibbosus Body form, gill raker In Skulason and Smith
sunfish architecture/benthivory, (1995)
planktivory
(continued)
7 Trophic Diversification and Speciation – ‘Your Eating Fuels Evolution’ 435
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Orangespotted L. humilis Gill rakers, pharyngeal Hegrenes (2001)
sunfish jaws
Bluegill sunfish L. macrochirus Benthivory, planktivory Ehlinger (1990)
Tanganyika Lobochilotes labiatus Lip morphology Colombo et al. (2013)
cichlid
Florida Micropterus Skull morphology Wintzer and Motta
largemouth bass salmoides floridanus (2005)
Lake Victoria Neochromis spp. Jaw morphology Bouton et al. (1999)
chichlids
Round goby Neogobius Pharyngeal morphology Andraso et al. (2017)
melanostomus
Sockeye salmon Oncorhynchus nerka Benthivory, planktivory, Skulason and Smith
migration (1995)
Rainbow smelt Osmerus mordax Benthivory, planktivory, Skulason and Smith
Spotted tilapia Pelmatolapia mariae Head morphology Stauffer and Gray
(2004)
Eurasian perch Perca fluviatilis Size-specific growth and Hjelm et al. (2000)
body morphology
South American Percichthys trucha Gill raker length Ruzzante et al. (1998)
perch Yolk-sac size, upper jaw Crichigno et al. (2014)
length
Cichlids Perissodus spp. Eating scales from left vs. Skulason and Smith
right side of a live fish (1995)
Aufwuchs-feeding Petrochromis Morphology, physiology, Sturmbauer et al. (1992)
cichlid orthognathus foraging behavior
Trinidadian guppy Poecilia reticulata Body, scull, paired fin Robinson and Wilson
morphology (1995)
Mountain Prosopium Snout size and shape Whiteley (2007)
whitefish williamsoni
Neotropical fish Saccodon spp. Different techniques in Skulason and Smith
eating algae (1995)
Brown trout Salmo trutta Benthivory, planktivory, Skulason and Smith
Arctic charr Salvelinus alpinus Benthivory, planktivory, Snorrason et al. (1994);
piscivory, migration Adams and Huntingford
(2004)
Brook charr S. fontinalis Foraging tactics McLaughlin et al.
(1999)
Lake charr S. namaycush (figure Eye position, gape size, Muir et al. (2016)
footnote) gill raker length and
spacing
Red drum Sciaenops ocellatus Jaw morphology, feeding Ruehl and DeWitt
performance (2007)
(continued)
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Turbot Scophthalmus Growth performance Wang and Ma (2015)
maximus
Aufwuchs-feeding Tropheus moorii Morphology, physiology, Sturmbauer et al. (1992)
cichlid foraging behavior
Minnows
Striped shiner Luxilus Snout, head, fin Jacquemin and Pyron
chrysocephalus morphologies (2016)
Redfin shiner Lythrurus umbratilis
Emerald shiner Notropis atherinoides
Sand shiner N. stramineus
Bluntnose Pimephales notatus
minnow
Fig. 7.2 Phenotypic plasticity in the lower pharyngeal jaws of the cichlid fish Astatoreochromis
alluaudi. A mechanically stimulating diet, such as hard-shelled snails induces a molariform phe-
notype with larger molar-like teeth set in a larger, denser jaw, in comparison with the papilliform
phenotype, which develops in response to a soft, nutritionally equivalent diet of pulverized snails.
Photographs were kindly provided by Helen Gunter. (From Young 2013, courtesy of Wiley)
c orollary is that exposure to culture causes predictable and consistent morphologi-

cal changes that together form a common “cultured phenotype”. From their meta-
analysis, Wringe et al. (2016) confirmed that aspects of a general “cultured
phenotype” exist. The meta-analysis analysis revealed that cultured fish had shorter
heads and upper jaws and fins. The diet of cultured fish likely promotes the develop-
ment of smaller heads, and jaws, because fish which are fed non-elusive, prepared
diets (Meyer 1987), as well as fish fed a greater ration, have been shown to develop
smaller heads and jaws (Wringe et al. (2016) and references therein).
7.1 Individual Specialization 437
7.1 Individual Specialization

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Following the trophic diversification, Bolnick et al. (2003) and Snowberg et al.
(2015) stressed the ecology of individuals rather than idealized populations of aver-
aged individuals. However, many ecologically generalized populations are com-
posed of relatively specialized individuals that selectively consume a subset of their
population’s diet, in a phenomenon known as “individual specialization”. These
specialized individuals can serve as a basis for radiation. Consequently, the Niche
Variation Hypothesis2 posits that this individual specialization can arise during eco-
logical release if niche expansion occurs mainly through diet divergence among
individuals, leading to greater morphological variation (Fig. 7.1).
To test this hypothesis, Snowberg et al. (2015) checked whether intra-population
diet variation is correlated with intra-population morphological variation, across 12
lacustrine populations of the three-spined stickleback. First, they used behavioral
observations, isotopes, and gut contents to show that, within populations, individu-
als differ in microhabitat use and diet. Second, the authors showed that some popu-
lations exhibit more diet variation than others, as evidenced by differences in both
isotopic and gut content variations among individuals. Finally, the authors con-
firmed that populations with greater dietary variation are more morphologically
variable. However, this relationship was only significant when the total morphologi-
cal variance was examined, not for individual morphological traits – in contrast to
Meyer’s example. This discordance may reflect among-population differences in
the relationship between individual morphology and diet. Because morphology–
diet relationships can differ between populations, morphological variance may be a
poor predictor of actual diet variation when diverse populations are being
compared.
In terms of habitat use, individual stickleback typically foraged in a mixture of
microhabitats both within and across days (Fig. 7.3). Snowberg et al. (2015)
observed a fair repeatability of individuals’ microhabitat use across days. A few
individuals were observed using microhabitats more evenly, or switching habitats
across days. Despite some day-to-day variation in individual diets, stickleback
exhibited substantial among-individual variation in microhabitat use (Fig. 7.3). On
average, pairs of individuals shared only 44% of their microhabitat use. Furthermore,
the authors found significant inter-individual variation in foraging behavior. There
was also an observation-day effect indicating that all fish shifted towards a particu-
lar microhabitat on certain days, although the intra-individual variation effect size
was much greater.
Overall, very little is known about how the degree of individual specialization
might alter the dynamics of ecological communities. Theoretical models assume
that intraspecific diet variation may profoundly alter the dynamics of single popula-
tions or predator–prey interactions (Snowberg et al. (2015) and references therein).
The Niche-Variation Hypothesis postulates a positive relationship between ecological niche

2
width and morphologic (or genetic) variability and has been put forward by Van Valen (1965).
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Fig. 7.3 Variability of the foraging behavior among individual stickleback. Each individual is
represented by a column, subdivided to represent the proportion of feeding strikes that the indi-
vidual directed against various microhabitats (designated by colored shading). Numbering below
each column: total number of feeding strikes (# Strikes) observed for the individual (top row), and
the number of times the individual was observed (# Obs., bottom row). Only those individuals
observed on at least four separate occasions are presented (n = 17). Column on the right: mean
habitat use by the individuals represented in this figure, which was very similar to the overall mean
habitat use by all individuals. (From Snowberg et al. 2015, with permission from Springer)
Documenting empirical patterns of individual specialization, as the authors have

done, is a necessary prerequisite towards addressing broader questions about the
consequences of niche variation within natural populations. Ecologically variable
populations of stickleback may provide a good model for understanding the effects
of variation in specialization on population, community, and ecosystem dynamics.
However, the authors note that direct measures of individual and population niches
are likely to be preferable to the use of morphology (see below) as a proxy for ecol-
ogy across populations or species.
7.2 Underlying Mechanisms of Speciation
With a few exceptions, the underlying biomolecular, mechanistic basis of ecological

speciation is still considered purely genetic. Coding mutations, affecting the genome
architecture, are generally assumed to be involved in morphological change
(reviewed in Küpper et al. (2015) and Martin and Orgogozo (2013)) and regulatory
variation, for example via transcription, is another major source of phenotypic vari-
ation that can enhance the adaptive potential of protein-coding genes through a
variety of mechanisms (Singh et al. 2017).
For instance, Gunter et al. (2013) presented the transcriptional basis of diet-
induced phenotypic plasticity in the East African cichlid fish, Astatoreochromis
alluaudi. This species displays adaptive phenotypic plasticity in its pharyngeal jaw
apparatus. In response to different diets, the pharyngeal jaws change their size,
7.2 Underlying Mechanisms of Speciation 439
shape, and dentition: hard diets induce an adaptive robust molariform tooth
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phenotype with short jaws and strong internal bone structures, while soft diets
induce a gracile papilliform tooth phenotype with elongated jaws and slender inter-
nal bone structures (Fig. 7.2). To gain insight into the molecular underpinnings of
these adaptations, the transcriptomes of the two divergent jaw phenotypes were
examined. The study identified a total of 187 genes whose expression differed in
response to hard and soft diets, including immediate early genes, extracellular
matrix genes and inflammatory factors. Furthermore, these genes included various
mechano-responsive genes identified so far in the bones and teeth of mammals.
Much is known about the genes involved in cichlid jaw and craniofacial develop-
ment. However, it is still unclear what salient sources of variation gave rise to
trophic-niche specialization, which is facilitating adaptive radiation. Recently, alter-
native splicing and differential gene expression have been documented as mecha-
nisms in cichlid adaptive radiation (Singh et al. 2017). Alternative splicing, or
differential splicing, is a regulated process during gene expression that results in a
single gene coding for multiple proteins. The proteins translated from alternatively
spliced mRNAs will contain differences in their AA sequence and, often, in their
biological functions. The connection of genotype, gene regulation, and environmen-
tal input in shaping adaptive morphological structures is not fully understood
(Pigliucci 2001; Pfennig et al. 2010), but it has become clear that adaptive mor-
phologies are not only shaped in a complex developmental pathway, but during the
entire life span of an individual and in continuous feedback with the environment,
with food being central (Singh et al. (2017) and references therein).
Singh et al. (2017) explored two sources of transcriptional variation that may
underlie species-specific disparities in jaw morphology. Using whole transcriptome
RNA-sequencing, the authors analyzed differences in gene expression and alterna-
tive splicing, at the end of post-larval development, in the fully functional jaws of
six species of cichlids from the Lake Tanganyika tribe Tropheini. The data revealed
a surprisingly high degree of alternative splicing events compared with gene expres-
sion differences among species and trophic types. This indicates that differential
trophic adaptation of the jaw apparatus may have been shaped by transcriptional
rewiring of splicing as well as gene expression variation during the rapid radiation
of the Tropheini. Specifically, genes undergoing splicing across most species were
found to be enriched in pharyngeal jaw gene ontology terms. Overall, jaw transcrip-
tional patterns on the post-larval developmental stage were highly dynamic and
species-specific. This work indicates that shifts in alternative splicing can have
played a more important role in cichlid adaptive radiation, and possibly adaptive
radiation in general, than currently recognized.
Doubtless, genetics are the central mechanism of rapid speciation, but likely not
the only one. During the past genetic period, non-genetic inheritance of phenotypic
traits was often classified as parascientific: “Modern synthesis has deemed it impos-
sible to deal with the long known facts on the role of epigenetic inheritance. More
than adequate evidence on epigenetic hereditary changes in animals and plants has
existed for a long time; Beisson and Sonneborn (1965)3 and others produced
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experimental evidence on the transmission of “acquired characters” in Protozoa;

abundant evidence on induction and transmission at a cellular level of epigenetic
marks (DNA methylation and histone modification) and of epigenetically induced
changes is accumulating at an accelerated pace’ (Cabej 2013). In Chap. 6,
‘Transgenerational Effects’, we referred to the intriguing paper by Csaba (2014),
who showed that a hormonal epigenetic imprinting in the protist Tetrahymena was
fixed and transmitted to an indefinite number of generations – comparable to muta-
tions. “No attempts are made to deal with the epigenetic control of gene expression
and the sudden heritable changes in cases of transgenerational developmental plas-
ticity that affect not one individual but whole populations simultaneously. Ignoring
this body of evidence is unexplainable at best” (Cabej 2013). However, to date,
changes are emerging slowly, but nevertheless clearly (also, see footnote to “Lake
Victoria”).
Even in alternative splicing, miRNAs can be involved, since miRNA-based gene
silencing and alternative splicing have emerged as two key post-transcriptional
mechanisms that function on a genome-wide scale to regulate tissue development
and differentiation. The interplay between miRNA and splicing regulation has been
demonstrated in cell culture models, and Kalsotra et al. (2010) identified a hierarchy
in which rapid postnatal up-regulation of specific miRNAs controls the expression
of alternative splicing regulators (in mice) and the subsequent splicing transitions of
their downstream targets. The authors demonstrated a direct role for miR-23a/b,
microRNAs which are also renowned for their action in FA and AA rapid metabo-
lism and, eventually, skeletal muscle growth (Zhu et al. (2014) and references
therein) (also, see Chap. 6 ‘Transgenerational Effects’). Further miRNAs identified
so far which interfere with alternative splicing are miR-124, miR-10a/10b, miR-28,
and miR-505 (Kelemen et al. 2013). Also, miR-10a is involved in fatty acid metabo-
lism in mammals (García-Segura et al. 2013) and has been found in zebrafish and
whiteleg shrimps (Huang et al. 2017; Woltering and Durston 2008).
These findings indicate that genetics and epigenetics are not mutually exclusive;
rather, they can enforce each other. Furthermore, there is no reason to assume that
this concerted mechanism is restricted to mammals and does not likewise apply to
lower vertebrates and even to invertebrates. In support of this, Kelemen et al. (2013)
stated that an emerging theme in protein functions is the regulation of mRNA stabil-
ity through miRNAs, which can be affected by alternative splicing of the miRNA-
binding sites.
The following paragraphs will highlight intriguing examples of speciation likely
based on resource competition and will try to answer the question of how long such
speciation may take to happen. We shall focus on such communities which have
proven monophyletic, and on species flocks that have developed in situ. This is the
case almost exclusively for fish communities.
3
For the sake of completeness, full references were added to the reference list below.
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7.3.1 Ancient Lakes
The oldest lakes in the world (e.g., the African Rift Lakes, Lake Baikal, Lake Ohrid,
Lake Biwa, Lake Titicaca, Caspian Lake, and the Malili Lakes of Sulawesi) shelter
endemic faunas that provide the most celebrated examples of ecological diversifica-
tion and speciation (Fig. 7.4). They are the laboratories of evolution. The fact that
the faunas of these lakes originated largely by intra-lacustrine speciation, and not by
multiple invasion, is now well established and accepted.
The process of trophic diversification and ecological speciation is documented in
cichlid fishes inhabiting lentic ecosystems throughout the African Rift Valley
(Fig. 7.5). Kocher et al. (1993) stated that convergent morphologies are associated
with adaptations to specific habitats and resources and that the cichlid communities
in Lakes Tanganyika and Malawi are characterized by the sympatric occurrence of
convergent forms.
Furthermore, studies of sculpin species flocks in Lake Baikal, Siberia, and of a
cyprinid species flock in Lake Biwa, Japan strongly support this statement.
Fig. 7.4 Endemic diversity and physical features of major ancient lakes of the world. (From
Cristescu et al. 2010, with permission from Wiley)
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Fig. 7.5 The differing feeding habits of cichlid fish in the three African lakes with the highest
cichlid diversity – Lake Malawi, Lake Tanganyika, and Lake Victoria – provide a sample of the
diversity of ecological niches they occupy. Brawand et al. (2014) present the genome sequences of
one species from each of these lakes and two river-dwelling cichlids (indicated by red stars). (From
Jiggins 2014, courtesy of the Nature Publishing Group)
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There are more than 2000 species of cichlid fish, the majority of which are found in
three large African lakes. This species radiation is the result of somewhere between
20 million and 45 million years of evolution, although, remarkably, around 500 spe-
cies found in one of these lakes, Lake Victoria, arose in only the past 100,000 years
(Jiggins 2014).
The species diversity of African cichlids is matched by their diversity in both
ecology and morphology (Fig. 7.5). The fishes occupy a huge range of ecological
niches – ranging from some fairly standard fishy activities, such as eating algae or
mollusks, through to the more bizarre, such as scale eaters, which use their asym-
metric jaws to nibble scales from the sides of other fishes. Many of these forms have
evolved, apparently independently, in each of the different lineages, which have
undergone varying degrees of species radiation. The genomes reported by Brawand
et al. (2014) provide evidence for a burst of gene duplications associated with spe-
cies radiation. This implies that natural selection has favored the retention of dupli-
cate genes in African cichlids, perhaps in part owing to their role in adapting to new
environments (Jiggins 2014). Furthermore, transposable elements obviously play an
important role in generating variation among species in ecologically important mor-
phological traits (Brawand et al. 2014).
Divergence in gene function can also occur through changes in gene regulation,
without a change in the protein-coding sequence; Brawand et al. (2014) also
addressed this. The cichlid genomes show evidence for enhanced rates of evolution
in putative regulatory elements, and high evolutionary turnover in miRNAs, known
for their regulation of gene expression. Furthermore, the genomes reveal 40 new
microRNA-encoding genes that, intriguingly, show complementary patterns of
expression relative to the genes they are hypothesized to regulate. This indicates that
they are involved in suppressing gene expression, perhaps to stabilize and refine
expression patterns that have been acquired during radiation.
Selected examples will illustrate the shift of trophic evolutionary niches.
Lake Victoria
The African Great Lakes contain as many or even more fish species than all of the
rivers and lakes of Europe combined. About 90% of the fish species in each lake
belong to a single family, the cichlids (Cichlidae; Teleostei), and are endemic to that
lake. Estimates of the phylogenies of these species flocks show that the species of
Lakes Victoria, Malawi, and Tanganyika have evolved in situ. Even more remark-
ably, for Lakes Malawi and Victoria, the species flocks are derived from one or only
a few closely related ancestral species and are all haplochromines (Alphen et al.
2004 and references therein).
Over 500 haplochromine species have been described from Lake Victoria alone
(Brawand et al. 2014). Previously, Nagl et al. (2000) proposed that the ancestors of
the Lake Victoria flock were trophic generalists, which lived in the East African
river systems in reproductive isolation from other haplochromine populations at

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least 1.4 million years ago (Mya). From this population, a series of subpopulations
separated in close succession 0.1–0.2 Mya. The founders of the Lake Victoria flock
entered the forming lake ~12,400 years ago and began to radiate by adapting to the
various ecological niches emerging in the lake. The rapid radiation was possible
because most of the mutations necessary for the morphological and behavioral
adaptations to these niches were already present as polymorphisms in the gene pool
of the large founding population. The mutations were sorted out into distinct com-
binations and fixed by natural selection.4
Later, Bouton et al. (2002) were able to experimentally demonstrate adaptive
phenotypic plasticity in Neochromis greenwoodi (Fig. 7.6), a rock-dwelling haplo-
chromine cichlid from Lake Victoria. For these cichlids, it may contribute to the
success of the settlement after migration between rocky outcrops or islands, where
newly colonized patches of rock offer different food regimes and different competi-
tors. In their experiments, Bouton et al. (2002) simulated such situations by raising
fry on different diets and with different competitors in such a way that they could
predict the direction of anatomical changes from functional demands. The authors
expected an indirect effect of interspecific competition: selectively removing one
4
In detail, the authors argued: “It is inconceivable, however, that all of the mutations responsible
for the morphological adaptations differentiating the flock’s individual species arose within a mere
12,400 years. Three important conclusions must, therefore, be drawn. First, the mutations must
have already been present in the founding population as polymorphisms, but in combinations
which allowed the maintenance of the generalist (non-specialist) phenotype characteristic of river-
ine haplochromines. Only in the lake, under the selection pressure to produce phenotypes adapted
to specific ecological niches, were the mutations sorted into new combinations, allowing the
appearance of various specialized phenotypes in the different niches. Second, to furnish all of the
necessary polymorphisms (it can reasonably be assumed that most of the morphological characters
differentiating the flock’s species are polygenetically controlled), the flock’s founding population
must have been large. These two conclusions are supported by the demonstration that nuclear gene
polymorphisms are widely shared not only among the various species of the Lake Victoria flock
but also between the Lake Victoria flock and the riverine species. Third, because of the shortness
of the time interval since the beginning of the radiation, the species of the Lake Victoria flock can-
not be expected to be fixed for any mutations apart from those responsible for the morphological
differences between them. Unless the latter are identified, it may therefore be futile to search for
molecular markers that could decipher the phylogeny of the flock (Nagl et al. (2000) and refer-
ences therein).
These arguments are plausible in the pure genetic view of increasing the diversity of morphot-
ypes, because genetic drift is unlikely to produce changes in predictable periods of time, correlated
with the environment. However, the view disregards corresponding epigenetic possibilities that do
not happen incidentally in unpredictable periods of time, but happen directed by environmental
stresses/challenge in the exposed generations themselves and can be inherited to succeeding gen-
erations. The epigenetic modification will last, at least, as long as the environmental stress/chal-
lenge persists. In terms of food sources, this can be considerably long periods.
The in depth evaluation of this hypothesis is a request to research approaches to come and starts
being set into practice. For instance, Brawand et al. (2014) discussed the involvement of miRNAs
in the species explosion of African cichlids (see above). Kapralova et al. (2014) and Gudbrandsson
et al. (2016) identified differences in the miRNA expression in two contrasting ecomorphs of the
Arctic charr (see below).
Fig. 7.6 Image of a male

Neochromis greenwoodi
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from Lake Victoria.

(Courtesy of Kevin
Bauman)
prey-type by the competing species would leave N. greenwoodi with the other.
Bouton et al. (2002) found the predicted phenotypic responses to food in trophic
traits of N. greenwoodi. Long-term selection and possibly genetic assimilation of
plastic traits may drive the populations further apart and may ultimately result in
allopatric speciation. The chance of subsequent speciation depends on the size of
the genetic neighborhood (Kawata 2002). Since haplochromine cichlids lack pre-
mating migration and larval dispersal, they typically have a small genetic neighbor-
hood. This enhances the chance of sympatric speciation (Bouton et al. 2002).
Lake Tanganyika
The cichlid Astatotilapia burtoni occurs in both Lake Tanganyika and inflowing riv-
ers. Theis et al. (2014) focused on the early phases of adaptive divergence driven by
variations in the food source. Generally, lake fish feed more on softer and smaller
food particles, whereas stream populations feed more on hard-shelled and larger
food items. Lake fishes have higher bodies, a more superior mouth position, longer
gill rakers, and more slender pharyngeal jaws than stream fishes, and show a plant/
algae and zooplankton-based diet, whereas stream fishes prefer snails, insects, and
plant seeds. The test for reproductive isolation between closely related lake and
stream populations did not detect population-assortative mating. Analyses of F1 off-
spring reared under common garden conditions indicate that the detected differ-
ences in body shape and gill raker length do not constitute pure plastic responses to
different environmental conditions, but also have a genetic basis.
Adaptive radiation can well take place even in a single guild, for instance the
herbivores. In Lake Tanganyika, five cichlid tribes have acquired herbivory: grazers,
browsers, scrapers, biters, and scoopers. These tribes have no sexual dichromatism,
and therefore, one can focus on the effect of ecological opportunity in the adaptive
radiation of these herbivorous cichlids. Sixteen species of the herbivorous cichlids
coexist on a rocky littoral slope in the lake. Seven of them individually defend feed-
ing territories against intruding herbivores to establish algal farms. Hata et al. (2014)
collected epiphyton from these territories at various depths and also gathered fish
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Fig. 7.7 Schematic diagram of hypothesis for adaptive radiation in Baikal sculpins. The origin of
Baikal sculpins is inferred as an ancestral benthic species of the genus Cottus. (From Goto et al.
2015, with permission from Springer)
specimens. Algal farms differed significantly in their composition among cichlid

species, even in the same ecomorph, due in part to their habitat-depth segregation.
The algal species composition of the stomach contents and algal farms of each spe-
cies differed, indicating that cichlids selectively harvest their farms. This study
revealed food niche separation based on habitat-depth segregation among coexist-
ing herbivorous cichlids in the same ecomorphs on a rocky shore.
7.3.1.2 L
ake Baikal
Within the Baikalian sculpins, many taxa are specialists. The most speciose group
of freshwater sculpins belongs to the genus Cottus with 64 species. The extraordi-
nary radiation of cottoid fishes from a single common ancestor (Fig. 7.7), with spe-
cies differentially adapted to the deeper benthic and open pelagic habitats, might
represent an irrefutable example of divergent natural selection as a major cause of
their reproductive isolation (Goto et al. 2015).
Sherbakov (1999) nicely summarized the molecular analyses of several species
flocks of Baikalian endemic organisms to provide support for the two contrasting
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Fig. 7.8 (a) Summary of evolutionary histories of three Baikalian invertebrate and one Baikalian
vertebrate species flocks: amphipods (Gammaridae, Crustacea), sub-endemic Baikalian genus
Choanomphalus (Pulmonata, Mollusca), endemic family Baicaliidae (Prosobranchia, Mollusca),
and Cottoidei (sculpin fishes) inferred from molecular phylogenetic studies. The width of the black
areas corresponds to the diversity of the species flocks at a given period; the grey areas represent
hypothetical lineages that have no known descendants. In the case of Baicaliidae, ancient fossils
are known, but their taxonomic relationship to contemporary species is doubtful; thus, the area is
shaded a lighter grey. The question mark indicates that, owing to a lack of fossils, it is unknown
whether sculpins existed in Lake Baikal before approximately 2.5 million years ago. (b) Trends in
annual temperature (unbroken line) and humidity (broken line) for the past 5 million years with
which to compare the critical periods in the development of the lake’s biodiversity. (From
Sherbakov 1999, with permission from Elsevier)
evolutionary scenarios (Fig. 7.8): there are both very young species assemblages
(sculpins and Baicaliidae) and very ancient and polyphyletic groups (gammarids
(Fig. 7.9) and Choanomphalus). It is most likely that the general cooling at the
beginning of the Pleistocene caused major environmental transitions and these chal-
lenges led to a general rearrangement of the Baikalian fauna. Only some of the
recent species assemblages contain survivor lineages. The speciose groups of
Baikalian animals mentioned in Sherbakov’s review by no means exhaust the vari-
ety of species flocks in Lake Baikal. The unexplored or almost unexplored groups
comprise, for instance, ostracods, nematodes, and infusorians. Some estimated
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Fig. 7.9 Selected gammarid species from the eulittoral of Lake Baikal. Above: Eulimnogammarus
maackii, Eu. cruentus, Eu. verrucosus; below: Eu. cyaneus, Pallasea cancelloides; Dorogostaiskia
(formerly Brandtia) parasitica. (Photographs courtesy of V.V. Pavlichenko, Irkutsk)
times of presumably sympatric or parapatric branching likely due to resource com-

petition in Baikalian taxa are listed in Table 7.2.
Most species of amphipods are euryphagic and opportunistic generalists. However,
many taxa have become specialists, such as necrophages which evolved morphologi-
cal adaptations to search actively for the bodies of dead animals. To mention only a
few more, phytophile (Pallasea) and spongiophile (Dorogostaiskia, formerly
Brandtia) taxa have evolved in which differentiation by substrate has been combined
with trophic differentiation. Pallasea spp. feed on the algae that they inhabit, and the
spongiophile Dorogostaiskia parasitica eats, but not exclusively, the tissue of the
Lubomirskiid sponge on which it lives (Takhteev 2000; Mekhanikova 2010).
7.3.1.3 L
ake Biwa
From ancient Lake Biwa, Japan, the ecological speciation of the Japanese gudgeon
Sarcocheilichthys (Cyprinidae, Fig. 7.10, right) has been documented (Komiya
et al. 2011). In this ancient lake, different bottom environments create habitats with
different benthos communities, which cause selection in benthivorous fishes. The
authors described the nature of the eco-morphological and genetic divergence
among local populations of the Japanese gudgeon, which inhabit contrasting habi-
tats in the littoral zones, namely rocky and pebbly habitats. Eco-morphological
analyses revealed that Sarcocheilichthys variegatus microoculus from rocky and
pebbly zones differed in morphology and diet (Fig. 7.10, left), and that populations
from rocky environments had long heads and deep bodies, which are expected to be
advantageous for capturing cryptic or attached prey in structurally complex, rocky
habitats.
Table 7.2 Estimated times (millions of years ago, Mya) of presumably parapatric and sympatric
branching likely due to resource competition
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Estimated time of
Species flock Habitat branching, Mya References
Fishes
Amphilophusa cf. citrinellus Lake Apoyeque, ~0.0001 Elmer et al. (2010)
Nicaragua
Gasterosteus aculeatus Lake Constance, <0.00015 Marques et al. (2016)
adjacent streams
Cyprinodon Laguna <0.008 Horstkotte and Plath
Chichancanab, (2008)
Mexico
Labeobarbus spp Lake Tana, Ethopia 0.01–0.025 de Graaf et al. (2007)
Oncorhynchus nerka: North Pacific <0.010 Taylor et al. (1996)
sockeye–kokanee forms Multiple times
Gasterosteus aculeatus Holoarctic lakes <0.010 Rundle et al. (2000)
Multiple times and Bell et al. (2016)
Haplochromini cichlids Lake Victoria 0.1–0.2 (0.0124) Nagl et al. (2000)
Oryzias Lake Poso, Sulawesi 0.84 Mokodongan and
Yamahira (2015)
Nothobranchius furzeri Southern 0.875 Dorn et al. (2011)
Incomati vs. Limpopo clades Mozambique
Altolamprologus Lake Tanganyika >1 Koblmüller et al.
compressiceps–A. calvus (2017)
Eviota spp. Coral reefs, eastern ~1 Tornabene et al.
Indonesia/Palau (2015)
Sailfin silverside fish Lake Matano, 1–2 Herder et al. (2008)
Sulawesi
Australoheros forquilha–A. Rivers in upper and 2.3–3.3 Piálek et al. (2012)
ykeregua middle Uruguay
Australoheros angiru–A. 4.2–6.0
minuano
Sculpins Lake Baikal 3.3–8.0 Goto et al. (2015)
Crenicichla lacustris– C. Rivers in upper and 6–8 Piálek et al. (2012)
missioneira middle Uruguay
Invertebrates
Coral-reef shrimp Synalpheus Coral reefs, 0.007–0.009 Duffy (1996)
brooksi Caribbean Panama
Amphipods Lake Baikal 0.01–0.015 Naumenko et al.
(2017)
Gastropods Lanistes spp. Lake Malawi 0.6 Schultheiß et al.
(2009)
Gastropods Tegula spp. Baja California 1 Krug (2011)
Gastropods Ancylus Lake Ohrid 1.4 Albrecht et al. (2006)
scalariformis, A. lapicidus, A.
tapirulus
(continued)
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Estimated time of
Species flock Habitat branching, Mya References
Hermit crabs Calcinus spp. Coral reefs >2 Malay and Paulay
worldwide (2010)
Gastropods Acroloxus spp. Lake Ohrid 1.3–1.4 Stelbrink et al. (2016)
Sea urchins Echinometra Atlantic and eastern 1.27–1.62 Alvarado and
lucunter–E. viridis Pacific Solís-Marín (2014)
Gastropods Tylomelania spp. Ancient lakes, 1–2 von Rintelen and
Sulawesi Glaubrecht (2005)
Asexual Ostracods Lake Baikal 5–8 Schön et al. (2012)
The character of the speciation in Amphilophus spp. is still debated: Barluenga et al. (2006a, b) vs.
a
Schliewen et al. (2006)
Fig. 7.10 Stomach contents of Sarcocheilichthys variegatus microoculus captured in rocky and
pebbly zones. (From Komiya et al. 2011, courtesy of Public Library of Science; photograph by
Hiroshi Senou, Fish Collection of Kanagawa Prefectural Museum of Natural History,
KPM-NR0092023)
Sarcocheilichthys biwaensis, a rock-dwelling specialist, exhibited similar mor-

phologies to the sympatric congener, S. v. microoculus, except for body/fin color-
ation. Genetic analyses based on mitochondrial and nuclear microsatellite DNA
data revealed no clear genetic differentiation among local populations between the
species. Although the morphogenetic factors that contribute to morphological diver-
gence remain unclear, the paper by Komiya et al. indicates that the gudgeon popula-
tions in Lake Biwa show a state of resource polymorphism due to differences in the
bottom environment. This example of resource polymorphism emphasizes the
importance and generality of feeding adaptation as an evolutionary mechanism that
generates morphological diversification and leads to speciation.
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Fig. 7.11 Ecomorphological and dietary comparison of the Uruguay River Crenicichla species
flock. Live representatives of C. minuano (1), C. tendybaguassu (2), C. missioneira (3), and C.
celidochilus (4) are laterally associated with their whole body warp transformation grids (a), sum-
maries of their stomach contents (b), lower pharyngeal jaw warp transformation grids (c) and
representative lower pharyngeal jaw (d). (From Burress et al. 2013, courtesy of the Public Library
of Science)
7.3.2 Rivers
Most studies so far have focused on lentic habitats. In addition, Burress et al. (2013)
showed that the same mechanisms are valid in lotic environments. The authors inves-
tigated the ecological and morphological diversification of a recently discovered
lotic predatory Neotropical cichlid species (Crenicichla) flock in subtropical South
America. They documented morphological and functional diversification using geo-
metric morphometrics, stable C and N isotopes, stomach contents and character evo-
lution. This species flock displays species-specific diets and skull and pharyngeal
jaw morphology (Fig. 7.11). Moreover, this lineage appears to have independently
evolved away from piscivory multiple times and derived forms are highly specialized
morphologically and functionally relative to ancestral states. Ecological, trophic-
based speciation played a fundamental role in this radiation. This paper revealed
novel conditions of ecological speciation including a species flock that evolved: (1)
in a piscivorous lineage, (2) under lotic conditions and (3) with pronounced morpho-
logical novelties, including hypertrophied lips that appear to have evolved rapidly.
However, no concrete time span for this development was indicated.
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Fig. 7.12 Individual net diversification rate (rG) of subclade vs. subclade ages. Black circles rep-
resent high-quality feeders; green circles represent low-quality feeders. Dashed line represents the
rG value over which all clades showed significant higher than expected species richness. Crown
ages are according to mean node ages in BEAST analysis. Circle sizes are proportional to the
number of species in each clade. (From Lobato et al. 2014, courtesy of the Public Library of
Science)
7.3.3 Coral Reefs
Coral reefs are diverse underwater ecosystems held together by calcium carbonate
structures secreted by corals. Shallow coral reefs form some of the most diverse
ecosystems on Earth: covering <0.1% of the ocean’s surface, coral reefs harbor
about one-third of all marine fishes or ~5000 species.
Lobato et al. (2014) tested the hypothesis that the diversification of reef fish lin-
eages is shaped by ecological opportunities. In fact, the authors presented a new
perspective in which the emergence of low-quality feeding modes may underpin the
diversification in a broad range of reef fish families (Fig. 7.12). This complies well
with the hypothesis that fishes in nutrient-poor, humic-rich, and base cations-poor
freshwater environments, such as the Rio Negro system in South America, had to
develop a plethora of mechanisms to cope with this situation – resulting in the most
diverse freshwater fish fauna worldwide (Steinberg et al. 2007). We can predict that
in coral reefs, the Rio Negro and geochemically comparable systems, evolution is
still in action and will allow many more intriguing species to develop.
In damselfishes (Pomacentridae), multiple transitions appear to have taken place
in several ways. When examining patterns for diet and feeding mode, Floeter et al.
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Fig. 7.13 Diet and feeding mode reconstruction mapped on a time-calibrated phylogenetic tree
for 303 (of ∼630) species of wrasses and parrotfishes (family Labridae). Color-coding depicts dif-
ferent feeding modes of adults. The timescale is dated in million years ago (Mya). Pie charts rep-
resent the probability of the ancestral state in each node. Clade abbreviations: Hyp Hypsigenyines;
Lb Labrines; Chl Cheilines; Scr Scarines; Cirr Cirrhilabrus; Lbr Labrichthyines; Mcr
Macropharyngodon. (From Floeter et al. 2017, with permission from Wiley)
(2017)5 summarized that such transitions and associated morphological alterations

represent cases in which ecological opportunity for the exploitation of different
resources drives speciation and adaptation. Similar trophic strategies (i.e. pelagic,
intermediate and benthic feeding) and morphologies (oral jaw shape and body size)
evolved repeatedly over the last 20 Mya. The diversity of trophic strategies
(Fig. 7.13) and ecomorphological traits within this family is attributable to conver-
gent radiations throughout its phylogenetic history. The development of different
morphs has been so successful that, in many cases, planktivorous species were
described as separate genera due to different morphologies. We shall come across a
comparable issue with the Arctic charrs below.
Despite the outstanding diversity of trophic groups found in labrids, certain
feeding modes are highly conserved within lineages (Fig. 7.13). For instance, the
variety of modes of herbivory/detritivory (browsing, scraping and excavating) is
5
Excerpts taken with permission from Wiley.
mostly restricted to the parrotfish clade (Scarini). Macroalgae browsing is probably

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the ancestral mode of herbivory within parrotfish, followed by the origin of “scrap-
ing” in the Atlantic restricted Sparisoma genus (∼18 Mya) and in Scarus/
Hipposcarus lineage (∼12 Mya), and finally “excavating” in Bolbometopon/
Cetoscarus (∼9 Mya) and Chlorurus genera (∼7 Mya). Foraminifera feeding
(15 Mya, Macropharyngodon), coral feeding (∼20 Mya; Larabicus,
Diproctacanthus, Labropsis, Labrichthys), and fish cleaning (<10 Mya) are the
most recent feeding strategies in the Labridae family and arose within the crown
group julidines. In the Labroides lineage, fish cleaning as adults evolved only once
and is derived from a coral-feeding lineage in Labrichthyines (∼9 Mya; Fig. 7.13).
Corallivory appeared earlier in the Labridae family (∼29 Mya, Fig. 7.13). This
revolution in the reef functional system is concordant with the expansion of
Acropora and Pocillopora corals. In contrast, corallivory evolved very recently and
independently across the Chaetodontidae. Overall, from reconstructions across
several groups, examples of independent transitions to planktivory become obvi-
ous: ecological opportunity for the exploitation of different resources drives specia-
tion and adaptation (Floeter et al. 2017).
7.4 Convergent Evolution
The food-based bottom-up control, described so far at the individual and population
level, also takes place on a global scale. It is the convergent evolution, the indepen-
dent evolution of similar features in species of different lineages. It creates analo-
gous structures that have a similar form or function, but were not present in the last
common ancestor of those groups. The different populations occupy similar eco-
logical niches. For instance, despite divergent evolutionary histories, Neotropical
cichlids (Cichlidae) and Nearctic sunfishes (Centrarchidae) appear to have similar
functional morphotypes and, therefore, occupy similar ecological niches. This is
evident from morphology, diet, and stable isotope analysis (Montaña and Winemiller
2013). Concordant patterns of interspecific similarity in morphology and trophic
ecology in these distantly-related groups support the hypothesis of convergent adap-
tive evolution.
A similar striking case of evolutionary convergence has been proposed for the
weakly electric South American gymnotiform and African mormyriform fishes.
Despite being distantly related and occurring in different zoogeographic regions,
these fishes are phenotypically and ecologically similar in many important respects,
including body form, swimming behavior, feeding behavior, reproductive behavior,
nocturnal activity, and the generation and reception of electric impulses (Montaña
and Winemiller (2013) and references therein).
From marine fishes, Friedman et al. (2016) reported an example of ecomorpho-
logical convergence in planktivorous fishes, namely in surgeonfishes. Often induced
7.4 Convergent Evolution 455
by shared ecological specialization, homoplasy6 hints at underlying selective pres-

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sures and adaptive constraints that deterministically shape the diversification of life.
Friedman et al. (2016) examined the feeding morphology in 30 acanthurid species
and, combined with a pre-existing phylogenetic tree, compared the fit of evolution-
ary models across two diet regimes: zooplanktivores and non-zooplanktivorous
grazers (Fig. 7.14). The findings indicate that zooplanktivorous species are converg-
ing on a separate adaptive peak from their grazing relatives. Zooplanktivorous acan-
thurids tend to develop a slender body, reduced facial features, smaller teeth and
weakened jaw adductor muscles than their grazing relatives. However, despite these
phenotypic changes, lineages appear to have not yet reached the adaptive peak asso-
ciated with plankton feeding even though some transitions seem to be over 10 mil-
lion years old. These findings demonstrate that the selective demands of pelagic
feeding promote repeated ecomorphological convergence within the surgeonfishes.
While much progress has been made in understanding the role of convergence in
driving aquatic (e.g., cichlids) radiations, little is known about its macroevolution-
ary effects across environmental gradients. Davis and Betancur (2017) used a suite
of recently developed comparative approaches integrating diverse aspects of dietary
data, habitat affiliation, morphology, and phylogeny to assess convergence across
several well-known tropical–temperate fish families in the percomorph suborder
Terapontoidei (Fig. 7.15), a clade with considerable phenotypic and ecological
diversity radiating in both marine and freshwater environments. The authors dem-
onstrate significant widespread convergence across many lineages occupying equiv-
alent trophic niches, particularly feeding habits such as herbivory and biting of
attached prey off hard substrates. These include several examples of convergent
morphotypes evolving independently in marine and freshwater clades, separated by
deep evolutionary divergences (tens of millions of years).
Soares et al. (2013) based their study on the identification of the ecomorphologi-
cal patterns that characterize the fish species found in tide pools in the Amazonian
Coastal Zone in the Pará State, Brazil. Representatives of 19 species were collected
during two field campaigns in 2011. The trophic guild, dominance, and residence
status of each species were established, and morphometric data were obtained for
each species. Principal Component Analysis (PCA) was utilized for the evaluation
of ecomorphological attributes and grouped species with pelagic habits with benthic
ones. With regard to feeding, the PCA formed two statistically significant axes
(Fig. 7.16). The species with the highest scores on the first axis are characterized by
relatively high mouths and relative head length, both of which are related to poten-
tial prey size. In the case of the second axis, the species with the lowest scores pres-
ent relatively long digestive tracts and large eyes, with the former variable being
related to feeding habits, and the latter to visual foraging behavior. It is interesting
to note that, in this analysis, the engraulid species remains isolated from all other
6
Homoplasy is a character shared by a set of species but not present in their common ancestor. A
good example is the evolution of the eye which has originated independently in many different
species. Further good examples are the development of succulence in cacti and euphorbias in dry
environments or the insect-feeding aerial birds swallows (Hirundinidae) and swifts (Apodidae).
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Fig. 7.14 Phylomorphospace projection of acanthurid species on the first two principal compo-
nents. Blue and green circles denote zooplanktivorous and non-zooplanktivorous species, respec-
tively. Fish silhouettes illustrate the outlying species on each axis, whereas superimposed red lines
designate the traits loading high (>0.82) on that Principal component (PC) and, therefore, the
major sources of variation along that axis. PC1 scores are negatively correlated with adductor
mandibulae mass, distances between eye and pectoral fin, and distances between the eye and ante-
rior tip of the premaxilla. PC2 scores load negatively with gill raker length. (From Friedman et al.
2016, with permission from Wiley)
Fig. 7.15 One specimen

of the Terapontoidei, the
Bermuda Chub (Kyphosus
sectatrix) (courtesy of
Kevin Bryant,
7.4 Convergent Evolution 457
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Fig. 7.16 Principal Components Analysis (PCA) based on the seven ecomorphological indices
related to the feeding of 19 fish species collected from tide pools on the Amazonian Coastal Zone
in 2011. Hypotheses concerning the interpretation of the ecomorphological indices highly corre-
lated with the principal axes. Codes: Atherinella cf. brasiliensis (ABR); Amphichthys cryptocen-
trus (ACR); Amphiarius phrygiatus (APH); Bathygobius soporator (BSO); Batrachoides
surinamensis (BSU); Butis koilomatodon (BKO); Colomesus psittacus (CPS); Epinephelus itajara
(EIT); Engraulidae gen. (ENG); Gobiesox barbatulus (GBA); Gymnothorax aff. funebris (GFU);
Lutjanus jocu (LJO); Mugil aff. curema (MCU); Mugil aff. hospes (MHO); Mugil sp. (MSP);
Omobranchus punctatus (OPU); Rypticus randalli (RRA); Sphoeroides greeleyi (SGR);
Thalassophryne nattereri (TNA). (From Soares et al. 2013, courtesy of the Sociedade Brasileira de
Ictiologia)
species (Fig. 7.16) due to the high relatively long digestive tracts and large eye val-
ues, which indicate that the species forages visually and feeds from vegetal sources
(planktonic herbivore). In summary, the grouping together of herbivores, carnivores,
and omnivores indicates a weak relationship between morphology and diet. The
relatively long digestive tract observed in the engraulid species correctly diagnosed
the species as herbivore. While the morphological features related to foraging
behavior overlapped between the trophic guilds, competition among the different
groups may be minimized by the variation in prey size.
In the study of Soares et al. (2013), the groups were segregated by the potential
prey size, independent of the taxonomic relationships between the different species.
This shows that the trophic structure of the community is organized in terms of the
ecomorphological characteristics of the species rather than along phylogeny dis-
tance or evolutionary lineages.
7.5 Rapid Speciating Taxa

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Several fish taxa are excellent witnesses for sympatric and parapatric speciation:
pumpkinseed sunfish, Cyprinodon, grunters, three-spined stickleback, and Arctic
charr.
7.5.1 Pumpkinseed Sunfish
Foraging-related diversification has been described in many taxa, but many ques-
tions remain about the contribution of such diversification to reproductive isolation
and potentially sympatric speciation. Colborne et al. (2016) used stable isotope
analysis of diet and morphological analysis of body shape to examine phenotypic
divergence between littoral and pelagic foraging ecomorphs in a population of
pumpkinseed sunfish (Lepomis gibbosus, Fig. 7.17).
The authors examined the reproductive isolation between ecomorphs by compar-
ing the isotopic compositions of nesting males to eggs from their nests (a proxy for
maternal diet) and used nine microsatellite loci to examine genetic divergence
between ecomorphs. The results supported the presence of distinct foraging eco-
morphs in this population (Fig. 7.17) and indicated that there was significant posi-
tive assortative mating based on diet. Interestingly, Colborne et al. (2016) did not
find evidence of genetic divergence between ecomorphs, however, indicating that
isolation was either relatively recent or not strong enough to result in genetic diver-
Fig. 7.17 Box plot estimates of the littoral prey resource contribution to the diets of male and
female pumpkinseed (Lepomis gibbosus) collected from the littoral and pelagic habitats. Stable
isotopic compositions of liver tissue samples were used in independent mixing models for each sex
and sampling period. The box plots represent the inner 50% of observations, with the mean value
indicated by the line within each box. The whiskers represent the 90th and 10th percentiles and
dots are the 95th and 5th percentiles. (From Colborne et al. 2016, with permission from Wiley).
(Image of Lepomis gibbosus courtesy of the U.S. Fish and Wildlife Service and Duane Raver)
7.5 Rapid Speciating Taxa 459
gence at the microsatellite loci. This paper indicates that the studied pumpkinseed
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fishes have not yet become separate species, but rather represent a population in the
early stages of phenotypic diversification along the speciation continuum.
7.5.2 Cyprinodon
Cyprinodon is a genus of small pupfishes found in fresh, brackish, and salt water.
While most species have separate distributions, six (C. beltrani, C. labiosus, C.
maya (all imaged in Fig. 7.18), C. simus, C. suavium, and C. verecundus) are
endemic in Laguna Chichancanab, Mexico (Horstkotte and Plath 2008). The authors
tried to answer the fundamental question in sympatric speciation of how trophic
divergence is achieved by three of the six species. They examined divergent evolu-
tion of preferences for different feeding substrates. In a test aquarium, the authors
presented four feeding substrates (sand, gravel, a plastic plant, and blank bottom),
but no actual food was offered. The four feeding substrates were chosen to mirror
the most common substrate types in Laguna Chichancanab. Previous studies dem-
onstrated that benthic food items prevail in the diet of most Cyprinodon species. C.
beltrani preferred sand, whereas C. labiosus preferred gravel. F1 hybrids of both
species showed intermediate preferences (Fig. 7.18).
Cyprinodon maya searched for food equally at all substrates. As the test fish were
reared under identical laboratory conditions (i.e., in the absence of feeding sub-
strates), the species-specific preferences appear to be genetically fixed, indicating the
rapid divergent evolution of feeding behaviors. Therefore, the rapid evolution of mor-
phological structures and feeding substrate preferences appear to have interacted in
facilitating niche partitioning and trophic segregation in sympatric speciation events.
Fig. 7.18 The mean (±SD) numbers of feedings attempts at the different substrate types (gravel,
sand, the blank bottom, or the plastic plant) in Cyprinodon beltrani, C. labiosus, the F1 hybrids (C.
beltrani × C. labiosus), and C. maya. Inserted figure: experimental setup. (From Horstkotte and
Plath 2008, with permission from Springer)
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Fig. 7.19 Images of selected Terapontidae: yellowtail trumpeter (Amniataba caudavittata),

Crescent grunter (Terapon jarbua); silver grunter (Mesopristes argenteus) (courtesy of Carpenter,
Wikimedia; Thomas Hardwicke (1755–1835) Illustrations of Indian Zoology; F. G. Levrault
(1828–1849) Histoire naturelle des poisons, Biodiversity Heritage Library)
7.5.3 Terapontidae (Grunters)
Davis et al. (2012) reported that the marine-freshwater transitions of the Australian
continent were associated with the evolution of dietary diversification in the tera-
pontid grunters (Teleostei: Terapontidae, Fig. 7.19). The authors examined the role
of these major habitat transitions and trophic diversification in a radiation of
Australasian fishes using a new molecular phylogeny incorporating 37 Terapontidae
species.
A combined mitochondrial and nuclear gene analysis showed that ancestral tera-
pontids appear to have been euryhaline in habitat affiliation, with a single transition
to freshwater environments producing all Australasian freshwater species. The map-
ping of terapontid feeding modes onto the molecular phylogeny–predicted carnivo-
rous dietary habits was displayed by ancestral terapontids, which subsequently
diversified into a range of additional carnivorous, omnivorous, herbivorous, and
detritivorous dietary modes upon transition to freshwater habitats. Comparative
analyses indicate that following the freshwater invasion, the single freshwater clade
has exhibited an increased rate of diversification at almost three times the back-
ground rate evident across the rest of the family.
7.5.4 Three-Spined Stickleback
Previous Canadian studies identified two distinct trophic “morphotypes” in the

three-spined stickleback (Gasterosteus aculeatus), each associated with a specific
lake environment. Populations inhabiting benthic-dominated environments were
found to possess reduced gill raker number and reduced gill raker length but
increased upper jaw length relative to populations from limnetic environments. It
has been proposed that the inter-population variability in trophic morphology is a
response to trophic resource differences between lakes and between habitats within
one lake (Larson 1976; Lavin and McPhail 1985; Bell 1976).
Later, Rundle et al. (2000) presented empirical evidence from the wild by using
the post-Pleistocene radiation of three-spined sticklebacks to infer natural selection
in the origin of species. The authors tested sympatric species of three-spined stick-
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Fig. 7.20 Sampling sites in the Lake Constance area and lake and stream ecotypes of three-spined
stickleback. (a) Map of Lake Constance. In stream 1, both ecotypes breed in sympatry and thus
opportunity for gene flow between ecotypes is geographically unconstrained, while in stream 2,
ecotypes breed in distant parapatry or effective allopatry, and geographical opportunity for gene
flow is therefore strongly restricted. The authors sampled stickleback early in the breeding season,
during the migration of the lake ecotype into streams, before site S1 in stream 1 was reached by
lake stickleback, but when both migrant lake and resident stream stickleback were present at inter-
mediate sites S1a and S1b along stream 1. (b) Pictures show representative males of both lake (L)
and stream (S) ecotypes in full breeding colors, and alizarin-red stained to highlight skeletal fea-
tures. (From Marques et al. 2016, courtesy of the Public Library of Science)
lebacks inhabiting small, low-elevation lakes in coastal British Columbia, Canada.

These populations derived recently from the marine three-spined stickleback that
colonized freshwater after the retreat of the glaciers at the end of the Pleistocene.
One species of each sympatric pair is a large-bodied Benthic that feeds on inverte-
brates in the littoral zone; the other species is a smaller, more slender Limnetic that
feeds primarily on plankton in open water. The Benthic and Limnetic from a given
lake constitute biological species: they are reproductively isolated by strong assorta-
tive mating, ecologically based post-mating isolation, and probably sexual selection
against hybrid males. Phenotypic differences between sympatric species have a
genetic basis and persist over multiple generations in a common laboratory environ-
ment. Both comparative and direct experimental evidence indicate that divergent
selection caused by competition for resources has contributed to the evolution of
these phenotypic differences (Rundle et al. 2000 and references therein).
Actually, the three-spined stickleback is a super-fast evolving fish split into two
species in same lake system. Marques et al. (2016) examined genomic differentia-
tion between migratory lake and resident stream ecotypes (Lake Constance,
Fig. 7.20) of the three-spined stickleback reproducing in sympatry in one stream,
and in parapatry in another. A similar differentiation has also been reported for the
parapatric lake-stream system of Lake Geneva, Switzerland (Lucek et al. 2013).
Importantly, these ecotypes started diverging less than 150 years ago. Consistent
with incipient ecological speciation, the authors found significant genomic
d ifferentiation between ecotypes both in sympatry and parapatry. Of the 19 islands

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of differentiation resisting gene flow in sympatry, all were also differentiated in

parapatry and were thus likely driven by divergent selection between habitats. These
islands clustered in quantitative trait loci controlling divergent traits among the eco-
types, many of them concentrated in one region with low to intermediate recombi-
nation. These findings show that adaptive genomic differentiation at many genetic
loci can arise and persist in sympatry at the very early stage of ecotype divergence,
and that the genomic architecture of adaptation may facilitate this.
7.5.5 Arctic Charr
Another excellent witness of fast radiation is the Arctic charr (Salvelinus alpinus).7
The Arctic charr is native to alpine lakes and arctic and subarctic coastal waters. Its
distribution is circumpolar. Comparable to the three-spined stickleback, the Arctic
7
Muir et al. (2016) question whether Arctic charr is really “the most diverse vertebrate”; instead,
they present convincing evidence that Lake charr (Salvelinus namaycush) is more diverse (Figure
footnote) than its relative; however, it is much less well studied with respect to, e.g., trophic
speciation.
Figure footnote Lake charr morphs from Great Bear Lake (from top to bottom: half-breed;
piscivore, invertivore, butterfly [common names used by local fishing guides]); Great Slave Lake
(lean, two deep-water siscowet-like morphs); Lake Mistassini (lean, humper-like morph); Rush
Lake [humper-like morph (Salvelinus namaycush huronicus; Hubbs 1929), lean]; and Lake
Superior (siscowet, humper, redfin and lean). Illustrations by Paul Vecsei (from Muir et al. (2016),
with permission from Wiley).
charr easily splits into various morphs. For instance, Iceland is noted for the evolu-
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tion of four morphs of the Arctic charr: small benthic, large benthic, small limnetic
and large limnetic (Malmquist et al. 1992). The morphs exhibited dietary specializa-
tions, strongly correlated with distinct differences in trophic morphology. Benthic
morphs foraged on zoobenthos, especially the mollusk Lymnaea peregra. Small
Limnetics fed extensively on open water foods, such as zooplankton, whereas large
Limnetics preyed mainly upon three-spined stickleback less on Arctic charr. Diet of
Limnetics differed slightly between habitats. The food segregation between Benthics
and Limnetics was established from age 1.
Likewise, Knudsen et al. (2006) described two reproductive isolated morphs of
Arctic charr, termed profundal and littoral charr, according to their different spawn-
ing habitats8; they co-occur in the postglacial Lake Fjellfrøsvatn in North Norway.
The profundal charr lives in deep water their entire life and have a maximum size of
14 cm, while the littoral charr grow up to 40 cm. The two morphs have different
dietary niches in the profundal zone: the profundal charr ate typical soft-bottom
prey (Chironomid larvae, pea mussels, and benthic copepods (Harpacticoida)),
while the young littoral chars mainly consumed crustacean zooplankton. The
profundal morph of Fjellfrøsvatn therefore utilizes a food resource niche that the
littoral morph did not exploit. This indicates that intraspecific resource competition
has driven an incipient ecological speciation. The sympatric ecological divergence
within the profundal habitat is possible because unexploited food resources (soft
bottom profundal prey) are available. Apparently, this represents a case of incipient
segregation by expanding to new resource types (niche invasion), and not by subdi-
vision of one broad ancestral niche.
Later, Knudsen et al. (2007) compared the Arctic charr populations from
Fjellfrøsvatn and a neighboring sub-arctic lake (Lille Rostavatn). They found pro-
found differences in the development of trophic polymorphisms in the two lakes. In
the species-poor fish community in Fjellfrøsvatn, individual charr showed differ-
ences that linked habitat, diet, and morphology to polymorphic ecotypes. In the
more species-rich Lille Rostavatn, with strong interspecific resource competition,
no such differences were present; in this lake, only pelagic food resources were
utilized. Also the contrasting results from these neighboring lakes support the gen-
eral hypothesis that access to multiple resource types and subsequent niche special-
ization are central for the development of repetitive radiation of fishes in young
post-glacial lakes.
Intriguingly, Kapralova et al. (2014) were the first (to the best of my knowledge)
to consider epigenetics as one of the major underlying mechanisms of sympatric
radiation. The authors proved that miRNAs − a major class of developmental regu-
lators − are central regulators in the process of adaptive regulation. Sequences of
many miRNAs are highly conserved, yet they often exhibit temporal and spatial
heterogeneity in expression among species and have been proposed as an important
reservoir for adaptive evolution and divergence. With this in mind, Kapralova et al.
(2014) studied the miRNA expression during embryonic development of offspring
8
Distinct profundal charr morphs are often recognized as separate species.
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Fig. 7.21 Two contrasting Arctic charr morphs differing in size, coloration and head morphology.
Top: Arctic charr from aquaculture stock is large, silvery and has a pointed snout and long lower
jaw, Bottom: small benthic charr from Thingvallavatn is small, dark and has a sub-terminal mouth
and rounded snout and is sexually ripe. (From Kapralova et al. 2014, courtesy of the Public Library
of Science)
from two contrasting morphs of the highly polymorphic Arctic charr, namely a
small benthic morph from Lake Thingvallavatn and an aquaculture stock. These
morphs differ extensively in morphology and adult body size (Fig. 7.21). The
authors established offspring groups of the two morphs and sampled at several time
points during development. Four time points (3 embryos and one just before first
feeding) were selected for high-throughput small-RNA sequencing. The authors
identified a total of 72 conserved and novel miRNAs that showed significantly dif-
ferent levels of expression in the two contrasting morphs. Hierarchical clustering of
the 53 conserved miRNAs revealed that the expression differences are confined to
the embryonic stages, where miRNAs such as sal-miR-130, sal-miR-30, sal-
miR-451, sal-miR-133, sal-miR-26, and sal-miR-199a were highly expressed in the
aquaculture specimens, whereas sal-miR-146, sal-miR-183, sal-miR-206, and sal-
miR-196a were highly expressed in the embryos of the small benthic morph. The
majority of these miRNAs have previously been found to be involved in key devel-
opmental processes in other species such as development of the brain and sensory
epithelia, skeletogenesis, and myogenesis.
Subsequently, Gudbrandsson et al. (2016) looked for the putative targets of the
interesting miRNA candidates and found a significant difference between the two
morphs in genes involved in energy metabolism and blood coagulation, as well as
in the differential expression of 7 genes in the developing head that are associated
consistently with benthic vs. limnetic morphology. This study implicates multiple
genes and molecular pathways in the divergence of the two charr morphs, and, most
importantly, epigenetics is central to this regulation.
7.6 Time Span of Trophic Speciation 465
7.6 Time Span of Trophic Speciation

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Finally, the question arises how long does it take one population to progress toward
different full, reproductively isolated species? The recorded estimates of fish spe-
cies differ considerably from a few tens to millions of years ago (Mya) (refer to
7.3.3 and Table 7.2). This is not surprising, however, because the underlying biomo-
lecular mechanisms appear not to be only pure stochastic, genetics (longer-term
speciation), but may be epigenetically superimposed (short-term initiation), particu-
larly in the “super-fast” evolving communities, as recently demonstrated; neverthe-
less, this issue deserves extensive future considerations.
Recently, Elmer et al. (2010) reported on an astonishingly rapid sympatric eco-
logical differentiation of crater lake cichlid fishes within historic times. The authors
used genetic and coalescence approaches to infer the colonization history of Midas
cichlid fishes (Amphilophus cf. citrinellus) that inhabit a very young crater lake in
Nicaragua: the ~1800 year-old Lake Apoyeque (Fig. 7.23). This lake holds two
sympatric, endemic morphs of Midas cichlid: one with large, hypertrophied lips and
another with thin lips (Fig. 7.22). The authors tested the associated ecological, mor-
phological and genetic diversification of these two morphs and their potential to
represent incipient speciation.
The genetic analysis revealed that crater Lake Apoyeque was colonized in a sin-
gle event from the large neighboring great lake Managua only about 100 years ago.
This founding in historic times is also reflected in the extremely low nuclear and
mitochondrial genetic diversity in Apoyeque. Elmer et al. (2010) found that sympat-
ric adult thin- and thick-lipped fishes occupy distinct ecological trophic niches.
Diet, body shape, head width, pharyngeal jaw size and shape and stable isotope
values all differ significantly between the two lip-morphs.
This study provides empirical evidence of eco-morphological differentiation
occurring very quickly after the colonization of a new and vacant habitat.
Exceptionally low levels of neutral genetic diversity and inference from coalescence
indicates that the Midas cichlid population in Apoyeque is much younger (~100 years
or generations old) than the crater itself (~1800 years old). This indicates either that
the crater remained empty for many hundreds of years after its formation or that
Fig. 7.22 Two morphs of Midas cichlid (Amphilophus cf. citrinellus) are found in Lake Apoyeque:
one with fleshy lips (left individual; “thick-lipped”) and the other with thin, normal A. citrinellus
lips (right individual; “thin-lipped”). (From Elmer et al. 2010, courtesy of BioMed Central Ltd.)
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Fig. 7.23 Divergence along the benthic–limnetic axis in Nicaraguan crater lakes. In western
Nicaragua (Central America), several crater lakes have been colonized independently by Midas
cichlids from the great lakes (Lake Managua and Lake Nicaragua). Midas cichlids in crater lakes
Apoyo and Xiloá have speciated along the benthic-limnetic axis. The schematic drawings indicate
high-bodied “benthic” specimens that rather live and forage in the littoral zone, while the slender-
bodied “limnetic” individuals explore the open water column. This study focuses on Amphilophus
tolteca from the small and young crater lake Asososca Managua. (From Kusche et al. 2014, cour-
tesy of Wiley)
remnant volcanic activity prevented the establishment of a stable fish population

during the early life of the crater lake. Based on the findings of eco-morphological
variation in the Apoyeque Midas cichlids, and known patterns of adaptation in
Midas cichlids in general, the authors hypothesize that this population is in a very
early stage of speciation (incipient species), promoted by disruptive selection and
ecological diversification.
In a neighboring crater Lake Asososca (Fig. 7.23), which harbors the sister spe-
cies Amphilophus tolteca, Kusche et al. (2014) observed a similar adaptive specia-
tion. The authors investigated the variation in morphology and diet as well as their
correlations along the benthic-limnetic axis in this extremely young Midas cichlid
species. They found that A. tolteca varied continuously in ecologically relevant
traits such as body shape and lower pharyngeal jaw morphology. The correlation of
these phenotypes with niche suggested that individuals are specialized along the
benthic-limnetic axis. No genetic differentiation within the crater lake was detected
based on genotypes from 13 microsatellite loci. Overall, Kusche et al. (2014) found
that individual specialization in this young crater lake species encompasses the lim-
netic as well as the benthic macro-habitat. Yet there is no evidence for any diversifi-
cation within the species, making this a candidate system for studying what might
be the early stages preceding sympatric divergence.
In support of this, Malinsky et al. (2015) discovered and characterized an early-
stage adaptive divergence of two cichlid fish ecomorphs in a small (700 meters in
diameter) isolated crater lake in Tanzania, Lake Massoko. The ecomorphs show clear
differences in traits normally associated with adaptive radiation in cichlid fishes,
including body shape, pharyngeal jaw morphology, diet, or microhabitat preference.
References 467
These Central American and African cichlid examples obtain further strong sup-
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port by the above-mentioned studies of Lake Constance and Lake Geneva stickle-
backs, which reported that these lake and stream populations (ecotypes) started
diverging less than 150 years ago. Similarly striking results were recently obtained
with three-spined sticklebacks. In 2009 and 2011, Bell et al. (2016) released about
3000 reproductively mature anadromous three-spined stickleback into two Cook
Inlet lakes. The authors sampled the source population and made annual samples
from the two introduced populations. Anadromous stickleback released into the
lakes produced abundant progeny, many of which survived and became reproduc-
tively mature the following spring. Both populations experienced demographic
bottlenecks in 2013 and 2014 and began to recover in 2015. Observations indicate
that sticklebacks in both lakes resemble anadromous stickleback, but by 2015 about
20% of the specimens in one population had a highly heritable, Limnetic pheno-
type. These observations indicate that substantial evolution occurred during the first
few generations.
Food fuels even evolution!
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VetBooks.

Aquatic Animal Nutrition

Humboldt University at Berlin

ISBN 978-3-319-91766-5 ISBN 978-3-319-91767-2 (eBook)

Library of Congress Control Number: 2018953153

© Springer Nature Switzerland AG 2018

Berlin, Germany Christian E. W. Steinberg

1 Introduction – ‘You Are What You Eat’................................................... 1

3.2 Fishes................................................................................................ 80

3.2.1 Microbiome Ontogenesis...................................................... 81

6 Transgenerational Effects – ‘Your Offspring Will Become

6.1 Parental Effects................................................................................. 335

AAs amino acids

Autocrine (signaling) a form of cell signaling in which a cell secretes a signaling

chemical that binds to receptors on that same cell

Chaperon protein stabilizing new proteins to ensure correct folding

or helping to refold proteins that were damaged by cell

dbt encodes DOUBLETIME protein; a kinase that phosphory-

lates PER protein that regulates the molecularly driven,

fMHC fast myosin heavy chain, ATP-dependent motor protein,

involved in muscle contraction and other motility pro-

GRP gastrin-releasing peptide: a neuropeptide, stimulating the

Lecithotrophy nourishment and development of the embryo only via the

yolk originally contained within its egg; opposite:

Myogenin (= myog, myogenic factor 4) transcription factor involved

in the coordination of skeletal muscle development or

tim the negative loop of the molecular clock

RNAi RNA interference or RNA silencing: biological process in

which RNA molecules inhibit gene expression or transla-

tim a, b, h encode timeless proteins; essential in regulating the circa-

dian rhythm; part of a transcription-translation negative

Introduction – ‘You Are What You Eat’

‘You are what you eat!’

© Springer Nature Switzerland AG 2018 1

(Fig. 1.1) (Jordan 1905).

Fig. 1.2 A fly-eating frog

even spanning through evolutionary time.

hypothesis will surely challenge the paradigm of the so-called anti-nutritional

factors in animal feed in general, and for aquafeed in particular;

reducing potential constraints of production, and keeping it as productive, efficient,

of speciation. Alternatively, the action of microRNAs and other epigenetic

processes are beginning to be taken into consideration. In short-lived animals, such

Diets and Digestive Tracts – ‘Your Food

Abstract This chapter is an inventory of basic digestive mechanisms and of how

2.1 Digestive Tract

To understand the short-term, as well as the long-term and even transgenerational

© Springer Nature Switzerland AG 2018 9

incorporated numerous adaptations for the efficient breakdown and absorption of

Functional trophic group

by consuming a greater proportion of autochthonous material (algae and aquatic

macrophytes) instead of allochthonous material (terrestrial plant fragments and ter-

2.2.1 Protein Digestion

2.2.2 Lipid Digestion

Efficient lipid digestion requires emulsifiers in the mixture of food—mainly pro-

2.2.3 Carbohydrate Digestion

involved in carbohydrate digestion: pancreatic α-amylase and disaccharidases in the

brush border membrane of the intestinal epithelial cells (Fig. 2.5).

2.3 Ontogenesis and the Intestine

Berlin, Germany Christian E. W. Steinberg

1 Introduction – ‘You Are What You Eat’................................................... 1

3.2 Fishes................................................................................................ 80

3.2.1 Microbiome Ontogenesis...................................................... 81

6 Transgenerational Effects – ‘Your Offspring Will Become

6.1 Parental Effects................................................................................. 335

2.1 Digestive Tract

2.2.1 Protein Digestion

2.2.2 Lipid Digestion

2.2.3 Carbohydrate Digestion

2.3 Ontogenesis and the Intestine

2.4 Herbivory, a Disadvantageous Acquization Strategy?

2.5 Starvation and Gut Morphology

2.6 Trophic Positions: An Omnivores’ Dilemma?

2.7 Concluding Remarks

3.2.1 Microbiome Ontogenesis

3.2.2 Does a Core Microbiome Exist?