Chapter 41
Invertebrates
Alisa L. Newton* and Roxanna Smolowitz**
*Wildlife Conservation Society, Bronx, NY, United States; **Roger Williams University, Bristol, RI, United States
INTRODUCTION
More than 99% of all animal species and nearly 1 million
named and an estimated 30 million unnamed species are
invertebrates (Ruppert et al., 2004). They suffer from a
variety of diseases related to their vertebrate counterparts.
By necessity this chapter will focus on a small number of
diseases in only a fraction of all invertebrates and aims
to provide an approach to assessing disease processes in
other invertebrates. The four major phyla (Cnidaria, Mol-
lusca, Arthropoda, Echinodermata), subphyla, and diseases
selected in this chapter were due to their ecological impor-
tance, commercial value, and importance to species in man-
aged care. These factors have driven the need for diagnostic
pathology and ancillary diagnostic testing in invertebrates
to advance veterinary care.
UNIQUE FEATURES
Clinical Pathology
The coelomic fluid and/or hemolymph serve as a transport
medium for nutrients, respiratory gases, and metabolites and
contain proteins and cells of innate defense. Invertebrates
inhabit a wide range of habitats and diverse physiological
mechanisms are involved in salt and water balance across
phyla. Normal clinical chemistries and electrolyte values
have been reported in some species and are reflective of this
balance (Gustafson et al., 2005; Punzo, 1983; Riddle, 1985;
Robertson, 1953, 1939; Schartau and Leidescher, 1983;
Zachariah et al., 2007). Many marine invertebrates are
osmotic conformers, maintaining the ionic concentration
and composition of extracellular fluids (hemolymph) simi-
lar to that of ambient sea water. All freshwater species are
osmotic hyperregulators and maintain an extracellular fluid
osmotic concentration higher than the environment. Brack-
ish water inhabitants show a combination of these factors,
Pathology of Wildlife and Zoo Animals. http://dx.doi.org/10.1016/B978-0-12-805306-5.00041-9
Copyright © 2018 Elsevier Inc. All rights reserved.
o­ smoconforming at high salinity and maintaining hyperos-
motic status at medium to lower salinities. Terrestrial inver-
tebrates maintain salt and water balance against evaporative
losses through drinking and food consumption (e.g., major
ions are obtained from the diet) (Larsen et al., 2014).
Invertebrates rely on innate immunity for their primary
protective response to infectious agents and lack the type
of adaptive immunity, challenge-specific long-term protec-
tion mediated by immunological receptors, present in ver-
tebrates. Whether invertebrates have an alternative adaptive
immunity, which allows for some degree of antigen specific
response and immunologic memory, remains a matter of
debate (­Arala-Chaves and Sequeira, 2000; Kurtz and Armit-
age, 2006). Invertebrate innate immunity relies on the inter-
actions between the cellular and protein components of the
hemolymph. The primary cellular components are generically
termed hemocytes, coelomocytes, or in some species amoe-
bocytes. They perform physiologic functions, such as nutri-
ent transport, digestion, shell mineralization, and excretion,
in addition to innate defense and wound healing. These cells
vary in differentiation depending on the species. A variety of
approaches have been used to characterize the cell types, which
include morphology, cytochemistry, function, flow cytometry,
density gradient configuration, and monoclonal antibodies to
surface proteins (Aladaileh et al., 2007). The simplest clas-
sification, which allows for comparison across species, sepa-
rates cells into three types based on the size and amount of
cytoplasmic granulation: hyaline (few to no granules), semi-
granular, and granular cells (Johansson et al., 2000). These
are also typically the only defining cellular features evident in
hematoxylin and eosin (HE) stained sections.
The humoral components of hemolymph serve an
important role in innate immune defense. They include
proteins involved in proteolytic cascades, such as coagula-
tion, melanization, and activation of Toll-like receptor and
complement-like reactions, as well as chemical m ­ ediators,
1019
1020 Pathology of Wildlife and Zoo Animals
such as cytokine-like factors. These proteolytic cascades
and chemical mediators are able to provide quick response
to pathogens and their components bear some resemblance
to those present in mammals. Hemolymph coagulation is
typically the first response of invertebrates to trauma or
pathogen invasion, particularly to bacterial pathogens.
This process has been best described in arthropods (fresh-
water crayfish and Atlantic horseshoe crabs) and less so
in insects and is discussed in more detail in the section
on non-infectious diseases. Different clotting processes
and proteins exist in different species (Jiravanichpaisal
et al., 2006; Ottaviani et al., 2004). Complement component
homologues identified in invertebrates include C3, Bf, and
mannan-binding lectin serine protease (MASP). Similar
to mammals, complement system opsonization enhances
phagocytosis, though alternative and lectin pathways are
believed to be less complex in invertebrates (Nonaka and
Yoshizaki, 2004). Cytokines identified in invertebrates
include interleukins (1α, 1β, 6), tumor necrosis factor α,
platelet derived growth factor (AB), transforming growth
factor (α, β, β1), epidermal growth factor, neurotrophic
factor, growth promoting factor, hemokinin, imaginal disc
growth factors, hemolymph trophic factor, and fibroblast
growth factor. Growth factors regulate cell proliferation,
differentiation, programmed cell death, stress response,
matrix production, and wound repair. Several cytokine-
like factors have also been identified in invertebrates.
These are important for immune responses because of
their effects on cell activation (PDGF-AB, TGF-B1, and
IL8) and chemotaxis (IL-1 α, TNF- α, IL8, PDGF-AB,
and TGF- β1). Other cytokine-like activities in inverte-
brates that are similar to those in vertebrates include IL-1
induction of TNF- α by hemocytes (Cerenius et al., 2010;
­Ottaviani et al., 2004).
Hemocytes provide defense through the process of
phagocytosis, nodulation and encapsulation, and pro-
duction of reactive oxygen intermediates. Phagocytosis
is initiated in an individual cell by recognition of a tar-
get agent (e.g., bacteria) either through receptor binding
or opsonization factors. Nodulation and encapsulation
occur in response to agents that are too large for phago-
cytosis. In this process, multicellular hemocyte aggre-
gates wall off pathogens in tissues. With chronicity,
these hemocyte foci demonstrate histomorphology not
dissimilar to granulomatous inflammation in lower ver-
tebrates (Jiravanichpaisal et al., 2006). A unique hemo-
cyte modulated response in invertebrates to infection or
focal tissue destruction is the formation of a tan to brown
pigmented, homogenous, often firm material referred as
melanization (Fig. 41.1). Prophenoloxidase is produced
and stored in hemocyte granules. When released and acti-
vated by microbial components like lipopolysaccharides
(LPS) and peptidoglycans from bacteria and β1–3 glu-
cans from fungi, the enzyme ­catalyzes the oxygenation
of monophenoles to quinones and ultimately the synthesis
FIGURE 41.1  Nodulation, encapsulation and melanization in the
digestive gland of a goliath bird eating spider. There is a focal area of
digestive gland necrosis surrounded by a rim of brown pigmented hyaline
material (melanization) and followed by a rim of hemocytes (nodulation
and encapsulation).
of melanin. P ­ henoloxidase is also important in pigmenta-
tion, ­sclerotization, wound healing, and encapsulation of
foreign materials (Cerenius and Söderhäll, 2004).
Anatomic Features
Prior to performing a necropsy examination on an inverte-
brate species, it is important to have an understanding of the
general body plan to assist in identification and determine
whether organs should be sampled or specimens should be
processed whole for histopathology. In addition to these
materials, texts of invertebrate zoology, invertebrate medi-
cine, and comparative histology as well as those that discuss
sampling and ancillary diagnostic testing serve as important
additional resources (Berzins et al., 2012; Williams, 1999).
Gross anatomy checklists are available in the Supplemen-
tal Materials of the necropsy chapter for the invertebrate
phyla presented here. There is considerable anatomic vari-
ety among invertebrates, and between them and their ver-
tebrate counterparts. Detailed description and images of
normal anatomy and histology are provided in the online
­Supplemental ­Materials.
Invertebrates are frequent asymptomatic hosts of many
viruses, protozoa, and metazoa, and can serve as reservoirs
for other species. When reviewing histopathology and ancil-
lary test results from new species, it is critical to assess for
associated tissue reaction to determine the significance of a
given result.
MULTIFACTORIAL CONDITIONS
Invertebrate diseases are frequently complex multifactorial pro-
cesses and the common terrestrial model of one-pathogen one-
disease simply does not apply in many cases. The ­fulfillment

of Koch’s postulates, where a single species of disease-causing
pathogen is successfully (1) isolated from an infected animal,
(2) grown in pure culture, (3) reinoculated into a healthy indi-
vidual, and (4) produces the same disease, is particularly chal-
lenging in invertebrates due to limited invertebrate cell lines
and the difficulty of culturing many invertebrate pathogens.
The marine environment is also particularly complex due to
the higher taxonomic diversity of hosts and parasites, differ-
ences in life histories, the more open nature of recruitment,
differences in anthropogenic impacts, and the potential for dif-
ferent modes of disease transmission (Mccallum et al., 2004).
It is important as we approach disease investigations in inver-
tebrates to consider the significant contribution of environ-
mental and other factors to susceptibility and pathogenesis and
to employ methods of investigation that include detailed ana-
tomic descriptions, ancillary diagnostic testing, and controlled
experimental design to arrive at the case description, etiology,
and pathogenesis of a disease (Work et al., 2008a).
The majority of coral diseases are complex processes
and a single primary etiologic agent has been confirmed
in only a handful of diseases to date. Even in those dis-
eases, other contributory factors are likely to be signifi-
cant. A thorough summary of the current understanding of
coral diseases was published by Woodley et al. (2016), and
exceeds what can be covered in this chapter. A few impor-
tant disease examples will be discussed later.
Bleaching in cnidaria, particularly scleractinian corals,
is the result of a breakdown of coral-algal symbiosis due to
loss of symbiotic photosynthetic algae (zooxanthella) or a
reduction in their per cell pigment concentrations. Temper-
ature extremes and solar radiation have received the most
focus, but other environmental factors, which include salin-
ity extremes, sediment, low temperature, desiccation, low
light, ultraviolet light, and reduced pH are considered as risk
factors. Contaminants, such as herbicides, copper, cyanide,
oil, and sunscreens may also serve as initiators. The primary
gross lesion associated with bleaching is patchy to diffuse
pallor of the affected colony (Baker and Cunning, 2016).
Three different tissue changes result in reduced zooxanthel-
lae densities in affected corals. These include degeneration
of the zooxanthellae within gastrodermal and epidermal
cells, release of zooxanthellae from the gastrodermis lin-
ing the mesenterial filaments, and sloughing of the gas-
trodermis and secondary loss of the zooxanthellae (Brown
et al., 1995). By light microscopy, intact zooxanthella should
be circular with a distinguishable nucleus, pyrenoids, and
assimilation bodies. Degeneration of zooxanthella is char-
acterized by a loss of circular profile, vacuolation, and in
some cases extreme enlargement. Zooxanthella loss from
gastrodermal cells is believed to be related to the changes
in coral metabolism associated with stress. Environmental
bleaching is typically due to a mixture of these three tis-
sue changes. Additional associated lesions seen with tem-
perature stress include epidermal and gastrodermal atrophy
and erosion, retractor muscle atrophy, and ultimately loss
Invertebrates Chapter | 41 1021
of tissue architecture, tissue necrosis to secondary zooxan-
thella necrosis (Glynn et al., 1985). If prolonged and severe,
bleaching results in coral mortality; milder bleaching events
can be reversible in weeks or months.
Colony collapse disorder (CCD) is a complex syndrome
affecting honey bee colonies, the hallmark of which is rapid
loss of adult worker bees. CCD is discussed here, prior to
other infectious diseases of bees, as some of the identified
contributory factors will be discussed in more detail later in
the text. In the fall of 2006, large scale losses of 30%–90%
of honey bee colonies were reported in the United States
and symptoms in affected colonies were not consistent
with known bee pathogens (Ellis et al., 2010). Character-
istic findings in affected colonies included (1) rapid loss of
adult worker bees; (2) brood populations in excess relative
to remaining adult bee populations; (3) lack of dead worker
bees within or surrounding affected hives; (4) delayed inva-
sion of common hive pests, such as small hive beetles and
wax moths; and (5) kleptoparasitism from neighboring honey
bee colonies (VanEngelsdorp et al., 2009). In colonies that
had not yet completely collapsed other findings included
(1) insufficient number of bees to maintain the number of
brood in the colony; (2) a workforce composed predomi-
nantly of younger adult bees; (3) persistence of the queen;
and (4) remaining bees that were reluctant to consume food
offered by bee keepers (Ellis et al., 2010). The three major
hypotheses regarding the cause of CCD include (1) novel or
resurgent pathogens; (2) poor nutrition and resultant devel-
opmental disorders; (3) environmental contaminants, which
include agricultural pesticides or chemicals used to control
common bee colony pests (Cornman et al., 2012). Other
possible factors suggested in the literature include man-
agement stress and poor genetic biodiversity in the queen
source (Ellis et al., 2010). Potential synergistic interactions
between two and more of the aforementioned hypotheses
(and potentially others) are likely.
To date, no single disease agent has been associated
with CCD, although infections are believed to play a critical
role. Epidemiologic studies of CCD have shown a higher
incidence and abundance of infectious agents in CCD col-
onies, which suggests they either are exposed to greater
numbers of pathogens, that coinfections can act synergisti-
cally, or that the colony is immune compromised ­(Cornman
et al., 2012; VanEngelsdorp et al., 2009). In particular
RNA viruses of the family Dicistroviridae [Israeli acute
paralysis virus (IAPV), acute bee paralysis virus (ABPV),
Kashmir bee virus (KBV), black queen cell virus (BQCV)]
and Iflaviridae [deformed wing virus (DWV)] and micro-
sporidia (Nosema spp.) have been implicated. Factors that
can affect colony immunocompetence include poor nutri-
tion and chronic sublethal exposure to agricultural or bee
keeping pesticides. Evidence for low-level exposure to
­environmental toxins is equivocal and neither individual
chemicals nor chemical load have been associated with
increased risk of CCD. Nutritional resources can affect
1022 Pathology of Wildlife and Zoo Animals
overwintering survival and bee longevity but analysis of
bee colonies for body weight, protein components, and lipid
stores has not shown a direct link between food resources
and collapse (VanEngelsdorp et al., 2009).
Epizootics of a process termed wasting disease have
been reported in the northeast Pacific across all sub-
phyla of echinoderms including sea stars, sea urchins,
brittle stars, and sea cucumber species since 1978 (Bates
et al., 2009; Dungan et al., 1982). Recently a large-scale
mortality event affecting at least 20 species of sea stars
devastated populations of sea stars along the Pacific coast.
During this outbreak sea star wasting disease (SSWD)
ultimately affected wild populations at more than 240 sub-
tidal and intertidal locations from southeastern Alaska to
southern California, and at least five Pacific coast public
aquaria that utilized “open” water systems. Pisaster, Pyc-
nopodia, and Evasterias were the most impacted genera
and mortality approached 100% in select species at many
locations. Clinical and gross findings associated with
wasting disease include (1) loss of body turgor (deflation)
and weakness; (2) foci of body wall pallor and tissue loss;
(3) sloughing of multiple rays and/or rupture of the body
wall with evisceration; and (4) death (Fig. 41.2). Consis-
tent histologic changes include epidermal degeneration,
necrosis, and ulceration with dermal separation, necrosis,
and inflammation. Community fingerprinting and bacte-
rial metagenomics performed during the recent mortality
event identified three candidate disease-associated bacte-
rial families including Bacteroidetes, Gammaproteobacte-
ria, and Spirochaetes. Viral metagenomes prepared from
symptomatic and asymptomatic sea stars identified several
candidate disease–associated metazoan viruses including
a sea star-associated densovirus (Parvoviridae) (Hewson
et al., 2014). Links between the histologic lesions and
FIGURE 41.2  Characteristic white lesion associated with sea star
wasting disease in an ochre sea star. Lesions begin on the ray near the
junction with the disc. White lesions may be areas of epidermal pallor or
areas of tissue loss with dermal and skeletal exposure. (Photo Courtesy of
M. Miner, University of California, Santa Cruz)
candidate disease-associated pathogens remain under
investigation; to date no infectious agents, including the
sea star-associated densovirus, have been demonstrated
by light or electron microscopy within affected sea star
tissues or lesions. Investigations of contributory envi-
ronmental factors to SSWD development and population
recovery are ongoing. Small changes in temperature, both
increased and decreased have been shown to influence the
progression and intensity of disease in different species
(Kohl et al., 2016; Menge et al., 2016). Seasonal changes
in reproductive potential, fluctuations in temperature (both
increase and decrease), host condition/acclimation, and
anthropogenic factors may further impact the dynamics of
the disease (Bates et al., 2009).
NON-INFECTIOUS DISEASES
Nutritional
Emaciation and dehydration in terrestrial invertebrates
are most apparent within the digestive organs, typically
the digestive gland. These organs often serve as sites
of energy/nutritional storage, similar to what is seen in
the liver and adipose stores of vertebrates. Gross lesions
are most apparent in arthropods and hexapods, and con-
sist of a pronounced decrease in the size of the abdomi-
nal segment or opisthosoma (Fig. 41.3A,B). Externally,
the reduced size of the digestive system will be grossly
apparent in some species. Histologically, the digestive
glands/organs are typically composed of pyramidal cells
with abundant cytoplasm that has variable vacuolation by
species. With emaciation, these cells are reduced in size,
vacuolation, and granulation. Etiologies are highly vari-
able and can be compounded by debilitation due to other
illnesses. Husbandry should always be investigated, par-
ticularly the lighting in photosynthetic species and prey,
water sources, lack of humidity or over ventilation in ter-
restrial invertebrates.
Molt death syndrome (MDS) of larval decapod
crustaceans is related to lipid nutrition, specifically low
dietary phospholipids, cholesterol, and n-3 highly unsat-
urated fatty acids. Gross findings are best described in
American lobster and include animals dying in a partially
molted condition or animals molting and having missing
or deformed appendages. Multifocal calcium deposits are
associated with the inner surface of the exuvial skeleton
and are visible grossly with the aid of a dissecting micro-
scope and scanning electron microscopy. These range in
size up to 0.2 mm diameter, are pink to colorless and are
primarily found on the inner surface of the claws and car-
apace. In severe cases, there is mottling of the carapace
and the cuticle of the thoracic limbs, tail segments, uro-
pods, and telson. Deposits are similar to calcite (CaCO3)
and may adhere to the new inner exoskeleton and cause
 Invertebrates Chapter | 41 1023

deformities that make shedding of the exuvium difficult

and may lead to death (Bowser and Rosemark, 1981;
Wang et al., 2016).

Toxic
A wide array of organic and inorganic toxins is described
in invertebrates; many are intentionally used in pest
control. These agents are summarized in Invertebrate
Pathology: Noncommunicable Diseases (Sparks, 1972)
and Pathobiology of Marine and Estuarine Organisms
­(Gardner, 1993). One toxin of note in aquatic invertebrates
is copper. Copper is an essential micronutrient but toxic
concentrations of the bioavailable forms result in adverse
effects on aquatic and semiaquatic invertebrates. Environ-
mental factors, such as dissolved organic and inorganic
ligands, competition with dissolved cations (water hard-
ness), all significantly impact bioavailability and there-
fore total copper levels in water samples are insufficient
to predict toxicity. Sensitivity, morbidity, mortality, and
tissue response vary significantly by species and are inti-
mately related to species physiology and life stage (Arnold
et al., 2010; Grosell et al., 2007). For example, adult oys-
ters and clams are largely resistant to copper toxicity and
can sequester copper in hemocyte clusters in the gills and
mantle resulting in green discoloration in these areas,
while larval oysters, adult, and larval mussels (genus Myt-
ilus), and some echinoderms (sand dollar, sea urchin) are
considered some of the most copper sensitive taxa (Arnold
et al., 2010; Sparks, 1972).
The proposed pathogenesis of copper toxicity includes
oxidative stress, DNA damage, altered ion regulation, and
metabolic derangement due to impaired oxygen uptake.
In invertebrates, lesions described with copper toxic-
ity are similar to those described with other heavy met-
als and pollutants and are not specific to copper. Gross
lesions include “distress syndrome” in freshwater pulmo-
nate snails, which is characterized by immobility, muscu-
lar spasms, extension of the cephalopedal mass, inability
to attach the foot to substrate, swelling of the tentacles,
and epidermal sloughing. Gross findings in sea stars are
described as muscular weakness, loss of righting abil-
ity, lack of tube feet coordination, mouth relaxation, and
gastric eversion. Histologic lesions described in inver-
tebrates include zooxanthella expulsion in cnidarians;
branchial epithelial necrosis, amoebocytic inflammation
in the connective tissue, and necrosis and sloughing of the
mucosa in gastropods; nephridial necrosis in sea hares;
mucosal necrosis in the stomach and digestive gland atro-
phy in oysters; inflammation, necrosis, and vacuolation
in the digestive diverticula, kidney, and intestinal tract
of clams; branchial necrosis in crabs; and epidermal
FIGURE 41.3  The normal opisthosoma (A) compared to an opist-
hosoma in a goliath bird eating spider with dehydration (B). Note the
ulceration and irregular epidermal contour in numerous
overall decrease in size of the opisthosoma as well as the irregular contour aquatic invertebrate species. The latter is believed to be
(wrinkling) of the cuticle. the result of copper-induced peroxidation of membrane
1024 Pathology of Wildlife and Zoo Animals
lipid and impaired epidermal cellular membrane struc-
ture and function (Eisler, 2007; Gardner, 1993; LaDou-
ceur et al., 2016).
Age-Related/Degenerative
Senescence occurs at the end of a brief, single reproduc-
tive event in many invertebrate species (Moltschaniwskyj
et al., 2007). It is hypothesized due to the energetic costs
of gamete production and the act of mating (Franklin
et al., 2012). However, some species are able to mate over an
extended period of time and moderate the effects of reproduc-
tion, resulting in an uncoupling of reproduction and senes-
cence. Among invertebrates in managed care, senescence is
most often seen in cephalopods after spawning. Histologic
lesions associated with this process include nephritis, adenitis
of the accessory nidamental gland, generalized edema, and
frequently bacteremia. Bacteria can often be cultured from
the hemolymph or renal sac (Smolowitz, unpublished data).
Trauma and Environmental/Anthropogenic
The most common non-infectious disease processes encoun-
tered in invertebrates are direct trauma or organism stress.
The etiologies vary by situation but frequently overlap.
Environmental stress (e.g., temperature, pH, and anthropo-
genic), handling/husbandry, and social interactions are the
most frequent factors. Some examples of traumatic injuries
are discussed later under idiopathic diseases where environ-
mental factors and husbandry may be a significant compo-
nent. Gross lesions consistent with trauma vary from areas
of epidermal discoloration, tissue loss, and a loss of surface
features to exoskeletal fractures. Damage to the cuticle, epi-
dermis, or exoskeleton can result in loss of osmoregulatory
capacity, hemolymph loss and shock, opportunistic infec-
tion, and in many cases death.
The histologic response to injury has been described in
detail in many invertebrate species including soft and hard
corals (Meszaros and Bigger, 1999; Palmer et al., 2011),
black abalone (Armstrong et al., 1971), oyster (DesVoigne
and Sparks, 1968), freshwater mussel (Pauley and Hea-
ton, 1969; Pauley and Sparks, 1967), drosophila (Bidla
et al., 2005), wax moths (Rowley and Ratcliffe, 1978), horse-
shoe crabs (Bursey, 1977), sea cucumbers (Cowden, 1968;
Menton and Eisen, 1973), penaeid shrimp (Fontaine and
Lightner, 1973; Lightner and Redman, 1977), leeches
(Huguet and Molinas, 1992; Huguet et al., 1999; Tettamanti
et al., 2005), and octopus (Polglase et al., 1983; Feral,
1988). In most invertebrates, wound healing begins with
the formation of a hemolymph clot, which rapidly prevents
further hemolymph loss and entraps microbial pathogens
before they can enter and spread through the hemocoel
(Jiravanichpaisal et al., 2006). Clotting initiation is either
the result of a primary proteolytic cascade or is initiated by
degranulation of local hemocytes resulting in the formation
of a protein aggregate or “soft clot.” These proteins become
crosslinked in some species (the “hard clot”) and later
infiltrated by other types of hemocytes (scab formation;
Fig. 41.4) that seal the hemocoel from the outside environ-
ment and serve as the scaffolding over which the epidermis
can regenerate, cover the wound site, and replace the scab.
In cnidaria sedimentation and increased turbidity from
dredging, drilling, and agricultural runoff interferes with
coral autotrophy and heterotrophy by decreasing avail-
able light and photosynthetic activity, which causes polyp
retraction and interferes with prey capture. This is believed
to produce negative energy balance that may be further
complicated by increased energy demand to clear excess
sediment. Sediments can also cause local and systemic tis-
sue injury (Riegl and Branch, 1995). Gross lesions include
polyp swelling, tissue thinning, discoloration, pallor and
tight polyp retraction along with distention and discol-
oration of the oral disc (Vargas-Angel et al., 2006). Early
histologic changes include multifocal to diffuse changes in
the number and size of mucous secretory cells (mucocytes)
in the epidermis and gastrodermis. Mucocytes may show
variable uptake of pentachrome dyes with special staining,
suggesting a change in the composition of the mucus. At
3–4 weeks of exposure, colonies develop attenuation of the
epidermis and gastrodermis, atrophy of epidermal muco-
cytes, decreased mucus secretion, and eventually tissue
damage and necrosis.
Ulcerative lesions of the umbrella (bell rot) have been
reported in a number of jellyfish species in managed care
including moon jellies, Pacific sea nettles, Atlantic sea
nettles, lagoon jellies, blubber jellies, Sandaria jellies, and
white-spotted jellies (Fig. 41.5). Although a specific etiol-
ogy has not been identified, trauma and environmental stress
are considered significant contributing factors. Husbandry
FIGURE 41.4  Response to injury in a goliath bird eating spider. Clot
formation, hemocyte aggregation, and melanization at a site of focal limb
trauma.

practices resulting in irregular tank flow may allow for
collisions with tank walls, and increased tank density can
allow for conspecific collisions. Gross lesions range from
erosion to ulceration of the peripheral and dorsal aspects of
the bell ranging from 1 to 2 mm and up to 6 mm diameter.
Lesions can progress to full-thickness tears with secondary
herniation of the internal viscera. Histologic lesions include
epidermal necrosis (cell swelling, pyknosis, and lysis) of
the exumbrella and on occasion the subumbrella with ulcer-
ation and local amoebocyte infiltration. Cnidocytes are
spared. Secondary algal and bacterial invasion may be pres-
ent (LaDouceur et al., 2013; Steers et al., 2003).
The epidermis of cephalopod mollusks is very fragile
and heals poorly. The simple columnar epithelium is easily
damaged. Infection with opportunistic bacteria is common
FIGURE 41.5  Umbrella ulcers in a moon jelly. Along the aboral sur-
face of the bell there are numerous irregular areas of cavitation (dark ar-
eas) at sites of exumbrella ulceration. (Photo Courtesy of N. Mylniczenko,
Disney’s Animals, Science & Environment)
FIGURE 41.6  Traumatic lesions in cuttlefish. (A) Mantle trauma in cuttlefish (b) Cuttlebone fracture in a cuttlefish. (Part A: Photo Courtesy of
K. Conley, Disney’s Animals, Science & Environment; Part B: Photo Courtesy of C. Rodriguez, Disney’s Animals, Science & Environment)
Invertebrates Chapter | 41 1025
with secondary bacterial infections often resulting in septi-
cemia and death; bacteria of the genus Vibrio are the most
common opportunistic agents. Trauma to the mantle apex
(Fig. 41.6A) due to collision with tank walls after startling
or while trying to escape perceived threats is a frequent
occurrence in managed care (Seeley et al., 2016; Sherrill
et al., 2000). These lesions can be acute or chronic (follow-
ing multiple episodes of collision) and can result in deep
ulcerations that extend into the subjacent connective tissues
and ultimately in squid and cuttlefish into the pen or cuttle-
bone sac. In cuttlefish, mantle abrasions are reported due to
crowding and territorial competition between males. Cuttle-
fish have also been reported to develop a gas pocket beneath
the skin of the mantle secondary to hovering in the stream
of bubbles from an air stone. These gas pockets later rupture
resulting in a focal ulcer leading to similar complications as
blunt force trauma (Scimeca, 2012).
Cuttlebone fracture (Fig. 41.6B) is a frequent addi-
tional traumatic injury seen in cuttlefish. The cuttlebone
consists of lamellae of calcified chitin arranged in parallel
layers separated by pillars that are sealed along the lat-
eral and anterior margins by a thick calcified layer. These
layers are added sequentially, with new chambers added
ventrally. Ultimately what is created is a series of cham-
bers filled with either gas (ventrally) or fluid (dorsally)
that allow for near neutral buoyancy of cuttlefish. Fracture
is a common occurrence in both free ranging and managed
cuttlefish due to external trauma by predators or intraspe-
cies aggression, compression fracture due to excess hydro-
static pressure, or self-trauma (e.g., collisions with tank
walls) (Boletzky and Overath, 1991). Nutritional deficien-
cies and stressors, such as abnormal water temperatures
or elevated nitrogen cycle metabolites may impact nor-
mal cuttlebone mineral deposition in animals in managed
care leaving it brittle and more easily fractured (Sherrill
1026 Pathology of Wildlife and Zoo Animals
et al., 2000). Fractures can be either longitudinal or trans-
verse. Gross lesions are most often evident along the ante-
rior rim near the nuchal mantle edge. They consist of mild
thickening of the dorsal aspect of the bone and character-
istic dark lines along the ventral surface. Healing of lateral
shell fractures may result in an asymmetrical cuttlebone
shape, as recovery of normal shell growth is not always
possible after healing (Boletzky and Overath, 1991; Oest-
mann et al., 1997). Chronic low-impact trauma can cause
prolapse and proliferation of caudal cuttlebone membranes
and dermis reminiscent of a neoplasm. Occasionally the
stomach and cecum will herniate or the dorsal mantle will
detach from the cuttlebone. These complications often
lead to death (Oestmann et al., 1997). Healing is facili-
tated by secretions of the shell sac epithelium.
Inflammatory Non-infectious
Eversion syndrome is an idiopathic condition of captive
scyphomedusa jellyfish that results in bell eversion into a
concave position. This results in exposure of the manubrium
and gastric cavity, which often leads to death. The primary
histologic finding is striated muscle degeneration (loss of
striation and swelling) and necrosis and mesogleal degen-
eration characterized by thinning, fiber loss, and disarray.
Affected species include moon jellyfish, fried egg jellyfish,
Pacific sea nettle, Atlantic sea nettle, Northern sea nettle, and
purple striped jellyfish. Affected age groups are either young
growing animals (1–2 months old) or older animals (greater
than 6 months). A common etiology has not been identified
despite investigations into husbandry practices, environmen-
tal, and nutritional factors (Freeman et al., 2009).
­Idiopathic self-trauma (automutilation syndrome)
has been described in three species of octopus in man-
aged care: giant Pacific octopus, California two-spot octo-
pus, and Mexican four-eyed octopus (Dombrowski and De
Voe, 2007; Reimschuessel and Stoskopf, 1990). Animals
develop circular ulcers on the mantle, proximal arms, or
both. Mantle lesions begin as punctate ulcers that gradu-
ally enlarge over several weeks. They typically occur on
the anterior surface between the eyes or immediately above
the origin of the arms. Limb lesions include ulcers on the
dorsal surface, loss of suckers, or loss of the tips of mul-
tiple arms. These lesions are described as occurring acutely
(often overnight) with no history of exposure to live prey
or conspecifics. In many cases, the suckers and arm tips
are later recovered within the stomach of the animal, con-
firming self-mutilation. Histopathology has identified axial
nerve abnormalities including edema, neuritis, and necrosis
in many cases. Vascular lesions including brachial artery
thrombosis within the arms and mantle have also been noted
as well as myositis adjacent to areas of mantle damage. A
mixture of common opportunistic bacteria have been iso-
lated from these lesions including Aeromonas spp., Vibrio
spp., and Staphylococcus spp. The etiology of this syn-
drome is unknown, but hyperesthesia secondary to the neu-
ral and vascular lesions is suspected.
Neoplastic
Neoplasia is a documented occurrence across all inverte-
brate taxa, but challenges remain with regard to tumor diag-
nosis, identification, and treatment. Spontaneous, hereditary
or acquired (e.g., radiation, chemical, or viral) genetic alter-
ations, well described in vertebrates, also occur in inver-
tebrates (Robert, 2010a; Tascedda and Ottaviani, 2014).
Reports of neoplasia are frequent in phylum Mollusca and
subphylum Hexapoda (class Insecta), infrequent in phylum
Cnidaria and subphylum Crustacea , and are not yet docu-
mented in phyla Porifera or Echinodermata. This distribu-
tion is likely biased by the intensive monitoring that occurs
in invertebrates of economic importance.
There are many challenges to documenting neoplasms
in invertebrates. Images and descriptions of gross and
microscopic findings in the older literature, which would
allow for comparison or peer evaluation of the identifica-
tion, are inadequate (Scharrer and Szabo Lochhead, 1950).
The classifications that exist for mammalian tumors are dif-
ficult to apply to invertebrate neoplasms and may not be
appropriate. Ancillary testing to facilitate cell identifica-
tion, such as immunohistochemistry, is generally unavail-
able. Therefore, it is critical for pathologists to be familiar
with the identification of invertebrate tissues, normal tissue
reaction, inflammation, and wound repair in a given species
when considering a diagnosis of neoplasia. The character-
istics used to define neoplasms in vertebrates, such as self-
sufficiency in growth signals, insensitivity to antigrowth
signals, evasion of apoptosis, unlimited replicative poten-
tial, sustained angiogenesis, tissue invasion, and metasta-
sis, may be challenging to fulfill across invertebrate taxa.
Organs, organ systems, cell types, and metabolic reactions
are different and the anatomy of most invertebrate circula-
tory systems (partially open or lacking organization) makes
documenting angiogenesis extremely difficult (Hanahan
and Weinberg, 2000; Peters et al., 2012).
The fundamental characteristics used to differentiate
benign from malignant neoplasms include (1) degree of cel-
lular differentiation and anaplasia; (2) rate of growth; (3)
local invasion; and (4) evidence of local or distant metasta-
sis (Kumar et al., 2015). These features should be applied
with caution as invertebrate neoplasms have rarely been
studied over time, mitotic figures can be transient, cell divi-
sion may be under heavy hormonal control, and evidence
of local invasion and metastasis can be difficult to assess in
species with open circulatory systems (Peters et al., 2012).
The majority of neoplasms described in invertebrates have
been reported to be benign, largely due to a lack of variation
in cell size and shape (anaplasia), lack of mitotic figures,

and a lack of organ invasion or metastatic spread (­Robert,
2010). The establishment of the Registry of Tumors in
Lower Animals (RTLA), a cooperative project between
the National Institutes of Health’s (NIH), National Cancer
Institute (NCI), and the Smithsonian Institution’s National
Museum of Natural History in 1965 greatly advanced our
understanding of the occurrence, gross and microscopic
pathology, and etiology of invertebrate neoplasms. It is
notable that out of the 1550 invertebrate submissions made
to the RTLA database and evaluated by the RTLA patholo-
gists between 1965 and its closure in 2007, only 30% were
ultimately designated to be neoplasms (Peters et al., 2012).
Disseminated neoplasia of presumed hematogenous
(hemic) origin (also termed hemic neoplasia, hemic sar-
coma, hematopoietic neoplasia, and hemocytic leukemia) is
one of the most commonly described neoplastic conditions
of invertebrates. This condition is described in 15 species of
bivalve mollusks as well as white shrimp, and mutant strains
of Drosophila (Farley, 1969a,b; Lightener and Brock, 1987;
Shrestha and Gateff, 1986). The cytologic and histologic
features are similar across species and include the presence
of large, round, anaplastic cells in connective tissues, blood
vessels, sinuses of the visceral mass, muscle, and mantle
tissue (Fig. 41.7). These cells are 2–4 times the diameter of
a normal hemocyte, with a nuclear to cytoplasmic volume
ratio of 1:1. They contain one or more prominent nucleoli
and one to several vesicles containing neutral triglycerides
and lipids. Mitotic figures are common. Infiltrating cells
cause displacement, compression, and necrosis of the gill,
gonad, and connective tissue, and arrest gametogenesis by
causing gonad atrophy and degeneration. Death is the ulti-
mate outcome of this tumor due to the effects of organ dis-
ruption and dysfunction of the immune system. Gross findings
in affected individuals are nonspecific and considered general
FIGURE 41.7  Disseminated hemic neoplasia in the gill of a softshell
clam. Neoplastic hemocytes fill and expand the vascular spaces of the gill
resulting in distortion of the tissue architecture.
Invertebrates Chapter | 41 1027
signs of poor doing in bivalves including emaciation, pallor
of the digestive gland and mantle recession (Barber, 2004;
Elston et al., 1992).
An etiology for disseminated neoplasia has not been
identified in bivalves or crustaceans. A clear consensus has
not been reached regarding the exact cell of origin in most
tumors. Most authors favor a hemic origin due to similari-
ties between the neoplastic cells and granular or hyaline
hemocytes and detection of neoplastic cells first in the cir-
culatory system; a gonadal origin has also been proposed
(Barber, 2004; Farley, 1969b). A relationship between prev-
alence and environmental carcinogens, biotoxins, stress, and
genetics has been suggested but not proven (Barber, 2004;
Böttger et al., 2013; Walker et al., 2011). There is a varia-
tion in the occurrence of disseminated neoplasia between
species, locations, and time of year suggesting that tumor
development is likely a complex, multifactorial process
(Walker et al., 2011). In a single case report of disseminated
neoplasia of shrimp, lesions consistent with penaeid shrimp
virus (IHHN) were present in the neoplastic hematopoietic
tissue suggesting a possible viral infection (Lightener and
Brock, 1987). Metzger et al. (2016) has recently shown
that these tumors in bivalves are most likely the result of a
transmissible neoplastic cell, and in most cases these cells
are species specific (or at least only infect closely related
species). A high number of Steamers, a retrotransposon, are
expressed only in neoplastic cells.
Tumor-like lesions of the carapace have been described
in crustaceans and classified as papillomas or hamarto-
mas, a proliferation of disorganized tissue indigenous to the
examined site (Peters et al., 2012; Shields and Small, 2013).
Carapace tumors arising from the abdominal somites occur
in brown shrimp, California brown shrimp, American lob-
ster, and other lobster species (Peters et al., 2012; Shields
and Small, 2013; Sparks and Lightener, 1973). Affected
crustaceans are often adults at an intermolt stage. Carapace
tumors are broad based, firm exophytic proliferations with a
papilliform to rugose surface and increased apical pigmenta-
tion (Fig. 41.8). On cut section, tumors are fibrous with a
superficial convoluted cuticle of variable thickness covering
the surface. Histologically, the epidermis and subepidermis
are continuous with the adjacent unaffected areas, but epi-
thelial cells within the mass demonstrate altered histomor-
phology described as hypertrophy and hyperchromasia in
shrimp and columnar with globose apical vacuoles in lobster.
Adnexa (tegmental glands, setal inserts, and pigment cells)
are locally to multifocally absent. When present in shrimp,
adnexa are increased in number and in an abnormally deep
location relative to the epidermis. Mitotic figures are not
observed but the strong hormonal control of cell division in
crustaceans may limit the ability for mitoses to occur outside
times of eccdysis even in neoplastic lesions. Tumor stroma
is composed of fibrous connective tissue with scattered mus-
cle fibers, few arterioles, and small numbers of infiltrating
1028 Pathology of Wildlife and Zoo Animals
FIGURE 41.8  Hamartoma of the carapace of an American lobster. A
bright blue broad based exophytic papilliform mass protrudes from the ar-
throdial membrane between the thorax and the abdominal somites. (photo
Courtesy of J. Shields, Virginia Institute of Marine Science)
hemocytes. The etiology of these carapace lesions is cur-
rently unknown. Infectious agents have not been identified.
In lobster, local trauma, bleeding, and wound repair are a
suggested etiology, but the lack of adnexa suggests epider-
mal disruption is more complex than simply failing to unfold
during eccdysis, and is more consistent with a neoplasm. It
is not clear if animals affected with these lesions can survive
molting due to the lack of tegmental glands, which provide
lubrication during eccdysis, and the size of the lesions, which
would likely adhere to the instar during molting (Shields and
Small, 2013; Sparks and Lightener, 1973).
Growth anomalies in cnidaria are reported in more than
40 species of scleractinian coral, 5 gorgonians, and 1 hydro-
zoan (Domart-Coulon et al., 2006). Despite having a simi-
lar external appearance and gross features, the histologic
morphology of these lesions have significant interspecies
differences and not all growth anomalies have been clas-
sified as neoplasms (Burns and Takabayashi, 2011; Loya
et al., 1984; Peters et al., 1986; Work et al., 2008b). Growth
anomalies present as discrete, nodular protrusions from the
colony and share the gross characteristics of fewer polyps
per surface area, decreased pigmentation (fewer zooxan-
thella per polyp) and finer (more porous) skeletal structures
than normal. These foci also grow more rapidly than the
adjacent normal appearing tissues.
Calicoblastic epitheliomas, tumors resulting from
abnormal proliferation of the ectodermal cells that produce
the aragonite skeleton, are described in both Acropora spp.
and Montipora spp. corals (Peters et al., 2012). Histologi-
cally, there is proliferation of gastrovascular canals and the
associated calicoblastic epidermis (calicodermis) with a
loss of normal polyp structures and decreased numbers
of ­gastrodermal zooxanthellae, mesenteries, and gonads.
Affected calicoblasts, instead of having a squamous mor-
phology, are cuboidal to columnar and resemble those
found near the rapidly growing regions in corals (Peters
et al., 1986). The designation of these lesions as neoplastic
and specifically malignant is controversial but is based on the
histologic features that suggest rapid, uncoordinated growth
of the tissue and skeleton, and destruction of adjacent normal
tissue (Domart-Coulon et al., 2006; Peters et al., 1986). Lack
of anaplasia and mitotic figures are cited as the reason for
considering these growth anomalies or hyperplasias rather
than true neoplasms (Robert, 2010; Work et al., 2016). In
areas of anomalous growth, the loss of polyps and mucus
secretory cells makes affected corals susceptible to sediment
accumulation, which causes tissue necrosis and filamen-
tous algae overgrowth in the coral skeleton and can cause
regional polyp or entire colony death (Peters et al., 2012).
Although the normal argonite crystalline structure is retained
in areas of growth anomalies, the skeletal density is reduced,
further predisposing the affected area to erosion and preda-
tion. In Montipora spp., similar anomalies reduce fecundity
of affected colonies and in some cases nearly 70% of the
colony is replaced by growth anomalies (Burns and Taka-
bayashi, 2011). The etiology of these lesions is not known.
Viral infections and UV light exposure have been proposed
but remain to be proven (Work et al., 2008b).
A number of different benign external tumors, papil-
lomas, polypoid tumors, or polypoid growths, affect the
body wall in gastropod and bivalve mollusks. The locations
and species affected include the foot of freshwater mussels,
oysters, and a butter clam and the base of the siphon of soft
shell clams and a horse clam (DesVoigne et al., 1970; Pau-
ley, 1966; Pauley and Sparks, 1967; Pauley et al., 1968).
These tumors are focal, exophytic, and firm with a tex-
ture and color similar to the adjacent tissue of the foot and
siphon; some cases have a firm but flexible stalk (Pau-
ley, 1966). Histologically, the tumors are covered by normal
tall columnar epithelium that is often thrown into convo-
luted folds. Multifocal hyperplasia of the basophilic glan-
dular cell layer may occur. The neoplastic portion of these
growths consists of a compact, disorganized proliferation of
smooth muscle cells originating from the muscle beneath
the epithelium of the foot or siphon. Mitotic figures are not
a feature of any of the described tumors and all are classi-
fied as benign. The etiology of these neoplasms is unknown
although some authors have suggested environmental con-
taminants while others have described them as spontaneous
(Odintsova et al., 2011; Pauley and Sparks, 1967).
Exophytic lesions grossly similar to those described
earlier in bivalves have been described in apple snail and
giant African snail, Pacific oyster, Sydney rock oyster, and
multiple planaria species. Histologically these tumors are
composed of large altered epithelial cells forming convo-
luted acini and covering a fibrous connective tissue stalk
containing bands of muscle. The altered epithelial cells
indicate a different cell of origin and tumor type and the
term adenopapilloma, epithelioma, or epidermal papil-
loma is applied to these lesions (Mitchelson, 1972; Peters
et al., 2012; Tascedda and Ottaviani, 2014). All are similarly
described as benign and of unknown etiology.

Gonadal tumors are described in 14 species of marine
bivalve mollusks from 3 continents. Species include ocean
quahog, bay scallop, cockle, Pacific oyster, Eastern oyster,
chalky macoma, Northern quahog, hybrid quahog, soft shell
clam, blue mussel, bluff oyster, and razor clam (­Darriba
et al., 2006; Hesselman et al., 1988; Peters et al., 1994).
Three histologic patterns are documented in these neo-
plasms: germinomas, tumors of germ cell origin; fibroma/
myofibroma, tumors of stromal/connective tissue origin;
and gonadoblastoma, mixed tumors containing both com-
ponents (Peters et al., 1994). Germinomas are composed
of undifferentiated germ cells that fill individual follicles,
efface the gonadal area or spread to other adjacent tissues.
Stromal tumors are composed of neoplastic cells that are
myxoid to vesicular to spindle shaped and efface normal
reproductive tissue as they grow. In mixed tumors, there is
a central region that consists of dense fibrous tissue and the
surrounding margins are neoplastic germ cells with either
ovarian or spermatogenic differentiation. The latter group
of tumors is reported infrequently but has an aggressive
pattern of growth with evidence of extension through the
follicular basement membrane, invasion of the adjacent
connective tissue of the gill, and mass formation along the
wall of the branchial vein (Peters et al., 1994). Small foci
of isolated neoplastic cells in the gill microvasculature in
a soft shell clam were consistent with metastasis. Gonadal
neoplasia has also been described in a male limpet (Carella
et al., 2009). The etiology of gonadal neoplasms is currently
undetermined. Environmental pollutants and/or viruses are
proposed but causal relationships remain to be confirmed
(Peters et al., 1994, 2012).
INFECTIOUS DISEASES
Viral-like particles (VLPs) have been reported in the plum-
rose anemone, a cnidarian that lacks symbiotic zooxanthella,
and the temperate sea anemone, which do not. In corals VLPs
are reported in Pavona danai, Acropora formosa, Acropora
muricata, Zoanthus spp., and Montastraea faviolata, and
their zooxanthella. It remains undetermined if this asso-
ciation is mutualistic, opportunistic, or pathogenic. Viruses
have also been reported but evidence is not definitive.
DNA Viruses
Large DNA viruses of the families Poxviridae (Entomo-
poxvirinae), Iridoviridae, Ascoviridae, Baculoviridae,
Nudiviridae, Hytrosaviridae, Nimaviridae (Whispovirus),
and Malacoherpesviridae, and small DNA viruses includ-
ing family Parvoviridae (Densoviriae), Bidnaviridae, and
Circoviridae are important pathogens of aquatic and terres-
trial invertebrates.
Herpes-like viral infections of larval and juvenile
Pacific oyster caused by oyster herpes virus 1 microvar-
iant (OsHV-1 µv), have resulted in disease and high
Invertebrates Chapter | 41 1029
mortality in aquacultured animals, first in France, then in
other locations on the Atlantic coast of Europe and in Italy
(Renault et al., 2014). Disease outbreaks are associated with
warmer summer temperatures (>16ºC). Infected animals
do not show disease at lower temperatures (13ºC) (Pernet
et al., 2015). Another OsHV, a different strain from the one
previously mentioned, has also been implicated as the cause
of mortality in aquacultured juvenile Pacific oysters in Cali-
fornia and other locations along the west coast of the United
States. Eosinophilic inclusion bodies are noted in the hyper-
trophic nuclei of hemocytes, connective tissue, and epithe-
lial cells in oysters from Europe but not in oysters from the
United States. Despite these differences, OsHV associated
mortality has been confirmed in Europe and the United
States using molecular methods (Friedman et al., 2005).
Abalone viral ganglioneuritis due to abalone her-
pesvirus (AbHV) (OIE and USDA reportable disease) is
reported in farmed and wild greenlip, blacklip, and hybrid
abalone in Australia and cultured diversicolor abalone
cohoused with black abalone in Taiwan. Mortality rates
across all age groups of up to 90% and 70%–80% have
been reported in Australian outbreaks and in adult and
juvenile abalone in epizootic and experimental infections
of diversicolor abalone, respectively. Horizontal transmis-
sion has been confirmed experimentally. Affected animals
demonstrate curling of the foot, excessive mucus produc-
tion, swelling and protrusion of the mouth parts, eversion of
the radula, and abnormal spawning or bloating. Foot abnor-
malities may include irregular peripheral concave elevation
of the foot, lack of foot movement, poor righting reflex, and
reduced pedal adhesion to substrates. Death occurs within
1–2 days. Histologically, there is individual cell necrosis
of the nervous tissue including cerebral, pleuropedal, and
buccal ganglia, branches of the pedal nerve and peripheral
nerves. Margination of the chromatin and central pallor
suggestive of intranuclear inclusion bodies may be seen in
neurons (Hooper et al., 2007). Conventional and real-time
PCR testing and in situ hybridization are available for con-
firmation (Crane and Corbeil, 2016).
At least 20 viral diseases have been identified in shrimp
and prawns (Ganjoor, 2015). Only a few will be discussed
here. For information on all of the viruses on the WOAH
list, please see (http://www.oie.int/en/international-stan-
dard-setting/aquatic-manual/access-online/).
The agent of white spot disease (WSD) also termed
white spot syndrome virus (WSSV) is a species of Whis-
povirus. The virus, a DNA virus and the only member of
the Nimaviridae family, can be found in all decapod crus-
taceans in many locations throughout the world, but is
best known for causing disease with significant morbidity
and mortality in several species of aquacultured penaeid
shrimp. Characteristic gross lesions are white spots in the
carapace. Depending on the severity, the spots can coalesce
to form extensive areas of white discoloration across the
carapace. Microscopically, the virus infects ectodermal and
1030 Pathology of Wildlife and Zoo Animals

­ esodermal tissues and forms large, eosinophilic to baso-

m epithelium, hematopoietic tissues, and connective tissue;
philic inclusions in hypertrophic nuclei resulting in necro- but not tissues of endoderm-derived organs (hepatopan-
sis of infected cells (Fig. 41.9). White spots in the cuticle creas and gut epithelium, etc.). Histologically it causes
are due to infection and necrosis of the underlying cuticular multifocal acute necrosis of the cuticular epithelium and
epithelium. The virus is very stable in dead tissues and sur- dermis (Lightener and Brock, 1987) (Fig. 41.11). The
vives freezing (Reville, 2005). Heavy infections have been disease first occurred in the 1992 in Ecuador and is now
reported in other decapods without marked disease. Gross found in shrimp farms throughout the world however, evi-
and microscopic findings combined with disease outbreaks dence indicates some strains of the virus were acquired
and molecular identification of the virus are used in diag- from wild broodstock in waters not contiguous with the
nosis. In addition to cultured shrimp, the virus has been Americas (Tang et al., 2012).
responsible for disease outbreaks in semicultured crayfish Monodon baculovirus (MBV) disease (also Penaeus
in Louisiana (Baumgartner et al., 2005). Numerous other monodon-type baculovirus), (USDA reportable disease),
crustaceans and aquatic insects are thought to be carriers/ occurs in various species of cultured penaeid shrimp and
reservoirs of the virus. is found in global shrimp culture (Bower et al., 1994). The
Infectious hypodermal and haematopoietic necrosis virus can be horizontally transmitted in water contaminated
(IHHN) (OIE reportable disease) is a densovirus (brevi- with broodstock feces and also appears to be vertically
densovirus) in the Parvoviridae family. IHHN causes
acute disease with high mortality in juvenile and subadult
aquacultured blue shrimp. Infected animals exhibit abnor-
mal behavior in the water column and irregular white to
buff colored spots are seen through the carapace. Chronic
infections resulting in poor growth and deformities, such
as bent bodies, deformed rostrums, and wrinkled antennae
cause runt-deformity syndrome (RDS) in white shrimp
(Ganjoor, 2015; Lightner, 2006). Histologically, eosino-
philic intranuclear inclusions are noted in the cuticular
epithelium and other ectodermal and mesodermal origin
tissues in acute outbreaks (Fig. 41.10). Inclusions are not
identified in chronic disease and diagnosis in these cases is
usually accomplished using molecular methods.
Taura syndrome virus (TSV; Discistroviridae family
Aparavirus genus (OIE reportable disease) infects sev-
eral shrimp species. It causes disease primarily in newly
metamorphosed, postlarval (PL) aquacultured blue and FIGURE 41.10  Infectious hypodermal and hepatopancreatic necro-
white shrimp; however, larger shrimp may develop disease sis virus in shrimp. Throughout the cuticular epithelium there are numer-
if not previously exposed. The virus infects the c­ uticular ous eosinophilic intranuclear inclusions.

FIGURE 41.9  White spot disease in shrimp. Within the cuticular epitheli- FIGURE 41.11  Taura syndrome virus in shrimp. The cuticular epithe-
um, hypertrophied nuclei contain large eosinophilic to basophilic inclusions. lium (right) demonstrates characteristic acute necrosis.

transmitted (Kanjanasopa et al., 2015). The disease causes
slow growth, decreased feeding, lethargy, and gill fouling.
Mortality can be high in late larval to postmetamorphic
juvenile shrimp (Bower et al., 1994). Adults generally do
not express disease. Heavily infected larvae and early juve-
niles exhibit a white midgut that is visible through the thin
abdominal carapace and have an enlarged hepatopancreas.
Histologically, the enlarged hepatopancreatic or midgut
epithelial cell nuclei contain eosinophilic triangular inclu-
sions (paracrystalline occlusion bodies) with marginated
chromatin (Fig. 41.12). Necrosis and sloughing of the epi-
thelium follows. These paracrystalline inclusions may also
be detected in squash preparations of the dissected hepa-
topancreas or in the feces stained with malachite green
­(Kanjanasopa et al., 2015).
RNA Viruses
Yellow head disease of shrimp (YHD) (OIE reportable
disease) is caused by yellow head virus (YHV), gill-
associated virus (GAD) and six other closely related viral
strains. YHV is related to nidoviruses in the Coronaviri-
dae and Arteriviridae families. GAD is considered a new
member of the genus Okavirus (Lightner et al., 2012). Both
are ssRNA viruses in the family Roniviridae. YHV is pri-
marily seen in Asia while GAD is reported in Australian
shrimp farming facilities. There are no confirmed cases of
YHV in the Americas. YHV is potentially lethal to most
cultured penaeid shrimp strains. Mass mortality occurs in
the early to late juvenile stages. Gross lesions are character-
ized by bleaching of the carapace and yellow discoloration
of the cephalothorax due to the abnormally yellow hepato-
pancreas. The hepatopancreas is frequently soft in affected
animals when compared to healthy shrimp. YHV targets
the lymphoid organ, hemocytes, hematopoietic tissue, gill
FIGURE 41.12  Monodon baculovirus disease in shrimp. Within the
hepatopancreatic tubules are characteristic eosinophilic triangular intra-
nuclear inclusions.
Invertebrates Chapter | 41 1031
lamellae, subcutaneous connective tissue, gut, antennal
gland, gonads, and nervous system (nerve tracts and gan-
glia). Histologic lesions include characteristic deeply baso-
philic cytoplasmic inclusions approximately 2 µm in size
or smaller. The lymphoid organ, stomach, and gills are the
best locations for detection of inclusions. Lymphoid organ
necrosis may also be seen. Confirmation is through in situ
hybridization along with RT-PCR and sequencing.
Infectious myonecrosis of shrimp (OIE reportable dis-
ease), is caused by a double stranded RNA totivirus and
causes significant mortalities in juvenile and adult farmed
Pacific white shrimp. Pacific blue shrimp and black tiger
shrimp have been infected experimentally. Disease out-
breaks may follow stressful events, such as feeding, capture,
or sudden environmental changes (salinity, temperature).
Gross and histologic findings are similar to those seen with
white tail disease of shrimp. Animals present acutely with
focal to locally extensive white areas in the striated muscles
that correspond to areas of necrosis. The distal abdominal
segments and tail fan are the most severely affected and can
become necrotic and red in some shrimp. The primary tar-
get organs are striated muscle (skeletal and less often car-
diac), connective tissue, hemocytes, and lymphoid organ
parenchyma. Histologic lesions include coagulation to lytic
necrosis frequently accompanied by edema and hemocyte
infiltration in affected striated muscle. With chronicity,
inflamed muscle fibers are replaced by fibrocytes and con-
nective tissue interspersed with hemocytes and regenerat-
ing muscle fibrils. A consistent finding is hypertrophy of
the lymphoid organ caused by accumulation of lymphoid
organ spheroids and spheroids in ectopic locations (gills,
heart, antennal gland, and ventral nerve cord). Transmis-
sion can occur horizontally through cannibalism. Vertical
transmission is suspected in carriers. Confirmation and dif-
ferentiation from white tail disease requires a combination
of nested PCR or ISH and histopathology (Nibert, 2007;
OIE, 2013; Poulos et al., 2006).
Two agents, Macrobranchium rosenbergii nodavirus
(MrNV) and extra small virus (XSV) cause white tail
disease of shrimp, an OIE reportable disease that causes
severe mortalities in larval and postlarval giant freshwater
prawn. Early juveniles are also susceptible while adults
are resistant and can act as carriers. Target organs include
gill, muscles of the head, heart and abdomen, ovaries, pleo-
pod and tail muscle. Transmission via horizontal routes is
reported in hatcheries and a vertical route of infection is
suspected from carrier animals. Hosts include artemia and
aquatic insects. Clinical signs include lethargy, opaque
abdominal musculature, degeneration of the uropods, and
telson with up to 100% mortality in 4 days. Histologically,
striated muscle of the cephalothorax, abdomen, and tail
demonstrate acute necrosis characterized by hyaline degen-
eration, necrosis and lysis, and intramuscular edema. Large
oval or irregular basophilic cytoplasmic inclusion bodies,
1032 Pathology of Wildlife and Zoo Animals
1–4 µm diameter, are present in the muscles of the abdo-
men, cephalothorax, and interstitium of the hepatopancreas.
Diagnostic confirmation includes RT-PCR, dot-blot hybrid-
ization, in situ hybridization, and ELISA testing (Hameed
and Bonami, 2012; Widada et al., 2004).
Caribbean spiny lobsters, an important commercially
fished species, are endemically infected with Panulirus
argus virus 1 (PaV1). Infection occurs by water exposure
and contact is potentiated by the behavior of these gregari-
ous lobsters. High mortality (>40%) can develop in infected
juveniles. Uninfected lobsters can detect infected juveniles
and behaviorally segregate themselves from infected ani-
mals. Adults appear to be resistant to the ­disease but that
may be partially behaviorally based. Grossly, juvenile
lobsters are lethargic before death and have milky hemo-
lymph that does not clot (Fig. 41.13A). Microscopically
large eosinophilic intranuclear inclusions with marginated
chromatin can be found in the fixed hemocytes of the hepa-
topancreas (Fig. 41.13B) and connective tissues through-
out the body as well as in circulating hemocytes. Infection
causes destruction and hemocytopenia as the disease pro-
gresses (Behringer et al., 2011).
The most significant viral pathogens of honey bees are
chronic bee paralysis virus (CBPV), deformed wing virus
(DWV), and viruses of the family Dicistroviridae: acute
bee paralysis virus (ABPV), Kashmir bee virus (KBV), and
Israeli acute paralysis virus (IAPV). The honey bee mite
Varroa destructor is an important vector of these viruses.
Chronic bee paralysis (CBP) is caused by a unique virus
with a fragmented positive-stranded RNA genome com-
posed of two major RNAs. It shares some characteristics
with Nodaviridae and Tombusviridae family viruses, but
is considered as the type species of a new group of posi-
tive-stranded RNA viruses (Ribière et al., 2010). Infection
FIGURE 41.13  Panulirus argus virus 1 in Caribbean spiny lobster. (A) Milky white discoloration of the hemolymph characteristic of affected
animals. (B) Histologically large eosinophilic intranuclear inclusion bodies are present throughout the fixed hemocytes of the hepatopancreas. (Photos
Courtesy of J. Shields, Virginia Institute of Marine Science)
results in two distinct syndromes, both of which can be pres-
ent in affected colonies to varying degrees. Bees with type 1
syndrome are unable to fly, often crawl on the ground or up
plant stems, and may form masses of thousands of affected
animals that huddle together on top of the cluster in the
hive. Affected animals develop severe abdominal swelling
secondary to distention of the honey sac with fluid. Symp-
tomatic animals die within a few days. In severely affected
colonies there can be sudden colony collapse at the height of
summer leaving only the queen and a few workers remain-
ing. Type 2 syndrome has several names (black robbers,
little blacks, and hairless black syndrome) that all reflect
the characteristic loss of hairs on the body, which causes the
animals to appear dark to almost black and shiny or greasy.
They are smaller than healthy animals, have a broad abdo-
men and can fly initially but are attacked by healthy bees
in the colony and become flightless, develop body tremors,
and die. The virus is neurotropic and in situ hybridization
has demonstrated infection and replication within neurons
of somata and neuropile regions of the protocerebron and
deuterocerebron. Targeted neurons in portions of the brain
include the mushroom bodies and central complex, which
are involved in sensory processing, learning, memory stor-
age, control of motor patterns, locomotor control and orien-
tation behavior, and the antennal lobes which have primary
sensory function (Olivier et al., 2008; Ribière et al., 2010).
Histopathologic lesions other than localization of viral
replication in these regions are not described. A two step
RT-PCR test with a fluorescent probe to quantify viral load
has been described (Blanchard et al., 2008, 2007). Viral
spread is horizontal and associated with host density within
the hive. Confinement and crowding of bees due to poor
weather conditions, or increased bee colony density within
areas of limited foraging promote viral spread. Increased

bee contact due to crowding results in cuticle abrasion and
transmission (Genersch, 2010).
Until recently there were no described viruses of echi-
noderms. Metagenomic studies in three species of healthy
urchins from Hawaii and five species of sea stars recovered
viral sequences matching single stranded DNA densovi-
ruses (family Parvoviridae). Densoviruses have received
a great deal of attention due to their association with sea
star wasting disease (discussed previously) (Gudenkauf
et al., 2014; Hewson et al., 2014). However, to date, no
studies have confirmed a location of viral replication or the
presence of densoviral particles in association with lesions
of wasting disease.
Bacteria
Bacterial infections in invertebrates are common particu-
larly in situations where other environmental factors (abnor-
mal temperature, biofouling, and toxins) contribute to
debilitation. The respiratory apparatus and digestive system
are primary targets in hexapods and arthropods. The epider-
mis and body wall are most frequently affected in echino-
derms. Gram-negative bacteria, often mixed populations,
are frequently identified. However, filter feeding bivalves
can accumulate large numbers of bacteria from surround-
ing sea water and can act as passive carriers, therefore it is
important to consider that a positive culture does not neces-
sarily correlate with disease (Lauckner, 1983). Gross lesions
are often not apparent in arthropods and mollusks. In echi-
noderms, particularly sea urchin, multifocal loss of spines,
pedicellaria, and tube feet, epidermal ulceration with swell-
ing of the epidermis at the tissue margin, and black discol-
oration of the exposed test are evident, a condition known
as bald urchin disease (Becker et al., 2008; Maes and Jan-
goux, 1984). Histopathology associated with invertebrate
bacterial infections may include multifocal lamellar necrosis
in the gills or book lungs, multifocal necrosis and hemo-
cyte infiltration in the digestive gland, multifocal necrosis
and hemocyte infiltration in the connective tissue, and in
cases of sepsis bacteria present within circulating hemocytes
(Fig. 41.14). Histologic lesions associated with bald urchin
disease include multifocal epidermal necrosis, disorganiza-
tion of the skeletal muscle, coelomocyte infiltration, and the
presence of mats of bacteria with frequent secondary invad-
ers (i.e., ciliates, algae, and fungi) (Gilles and Pearse, 1985).
Vibrios are Gram-negative, motile bacilli that are wide-
spread in coastal and estuarine environments. Some spe-
cies comprise serious pathogens of aquatic invertebrates.
­Vibrio-induced bleaching is one of the few coral diseases for
which Koch’s postulates have been fulfilled. Two examples
include bacterial bleaching of Oculina patagonica due to
Vibrio shiloi and coral bleaching and lysis in Pocillopora
damicornis and Paramuricea clavata due to Vibrio coral-
liilyticus. Environmental temperature and nutrients are risk
Invertebrates Chapter | 41 1033
FIGURE 41.14  Bacterial sepsis in a greenbottle blue tarantula. He-
mocytes within the pericardium (bottom) and ventricular lumen (top left)
contain cytoplasmic aggregates of small bacilli consistent with circulating
bacteremia.
factors to the development and progression of disease. Vib-
rio shiloi causes the death of endosymbiotic zooxanthella
within the gastrodermis. Vibrio coralliilyticus causes tissue
lysis due to the synthesis of a potent metalloproteinase at
temperatures above 26ºC. Gross lesions in V. shiloi infection
include multifocal areas of loss of pigmentation particularly
in colonies in shallow water at depths of 1–6 m. Histologic
lesions confirm large aggregates of bacteria at the border
between affected and unaffected tissue and surrounding
released zooxanthellae on the tissue surface. Transmission
electron microscope (TEM) images demonstrate intracel-
lular bacteria within the ectoderm, vacuolar change in the
gastrodermis, and degeneration of zooxanthellae. Gross
lesions in V. coralliilyticus infection include white patches,
typically starting on the outgrowth/polyp extensions of the
branch and later become confluent. Tissue lysis results in
exposure of the underlying skeleton. Scanning electron
microscopy (SEM) confirms high numbers of rod-shaped
bacteria on the surface, below the necrotic tissue and on the
zooxanthellae. (Woodley et al., 2016).
Acute hepatopancreatic necrosis disease (also called
early mortality syndrome) of penaeid shrimp is caused by
a unique strain of Vibrio parahaemolyticus that produces
a plasmid-encoded toxin. Grossly, the hepatopancreas,
which usually appears brown, appears white when exam-
ined through the carapace (http://www.enaca.org/publica-
tions/health/disease-cards/ahpnd-disease-card-2014.pdf).
Microscopically, infection results in acute necrosis of the
hepatopancreatic tubular epithelium, sloughing of degen-
erate cells into the lumen of the tubules, marked intratu-
bular hemocytic inflammation, and late development of a
secondary massive bacterial infection (Tran et al., 2013).
In addition to gross and microscopic findings, molecular
methods to detect the gene responsible for toxin production
1034 Pathology of Wildlife and Zoo Animals
have been developed. (http://www.oie.int/fileadmin/Home/
eng/Internationa_Standard_Setting/docs/pdf/Aquatic_
Commission/A_AAC_March_2015.pdf).
Brown ring disease caused by Vibrio tapetis predomi-
nant 1 (Vibrio P1) causes severe disease and mortality in
aquacultured Manila clams in Europe. Cockles and other
bivalves in affected areas are infected but do not show dis-
ease. Grossly, brown rings of conchiolin are observed at the
edges of the pallial line and the edge of the shell. Micro-
scopically, the brown rings are composed of a mixture of
necrotic periostracum and inner poorly formed layers of
the prismatic and nacreous shell along with hemocytes
and other cell debris from the mantle. It appears that the
­bacteria attach at the palladial line—an area where the shell
epithelium of the outer mantle lobe detaches from the shell
(Paillard and Maes, 1995). Disease is associated with colder
temperatures (4ºC) and is not noted at temperatures above
22ºC. In animals that recover, nacre covers the inner surface
of the shell lesion forming an irregularity in the inner shell
surface, a process termed nacrezation, and circumferential
growth can then continue. While soft tissue lesions are not
noted with this disease, a decrease in glycogen occurs and
may be the cause of mass mortalities in the stressful winter
period. Disease caused by V. tapetis has been noted in fish
in areas where infected clams are found (Paillard, 2004).
Shell disease is a general term used in reference to any
crustacean with carapace erosions. In the American lobster,
epizootic shell disease (ESD) has recently been identi-
fied (Fig. 41.15) and is common in wild caught American
lobsters along the northeast US coast (specifically Massa-
chusetts and Rhode Island) (Smolowitz et al., 2005). The
disease begins as deep erosions on the dorsal cephalotho-
racic carapace in late summer (a few months after molting).
Lesions progress both laterally across the carapace surface
and into deeper layers of the carapace, and can result in
ulcerations into the underlying carapace epithelium and
dermis with significant dermal inflammation. Ulceration
FIGURE 41.15  Epizootic shell disease in an American lobster.
Throughout the dorsal cephalothoracic carapace and extending along the so-
mites are extensive, frequently coalescent areas of severe cuticular erosion.
may result in adhesions of the old shell to the new, inability
to completely molt and mortality. If an animal can molt suc-
cessfully, there are generally no indications it was affected
before molting. No significant internal tissue changes are
noted in animals without ulcerations. Affected berried
females, who usually carry eggs (berries) on the ventral
abdomen for 9–12 months, will molt early; early molt and
egg loss are partly responsible for decreased lobster pro-
duction in diseased areas. The cause of ESD has not been
determined but evidence suggests that elevated water tem-
peratures at the time of molting (Glenn and Pugh, 2006)
and the occurrence of specific types of bacteria (Aquima-
rina homaria and others) in the lesions are important in the
development of the disease (Chistoserdov et al., 2012).
Other forms of shell disease described in American lob-
sters are enzootic shell disease (EnSD), which is similar
to ESD, but is rarely found in fished populations and is of
unknown cause, black spot disease which presents as focal
to focally extensive deep black erosions that develop sec-
ondary to carapace trauma, and impoundment shell dis-
ease (ISD). ESD can be grossly distinguished in early stages
from ISD when ISD has not yet spread across the surface of
the shell (Smolowitz et al., 1992). Early ISD begins around
the bilaterally symmetrically positioned tegmental gland
ducts and sensory hairs and therefore lesions are bilaterally
symmetrical; ESD lesions are not. In all three of these dis-
eases bacteria are frequently cultured from lesions, but are
considered secondary invaders not primary pathogens.
Gaffkemia (red tail) in lobster is caused by the bac-
terium, Aerococcus viridans. This Gram positive polysac-
charide capsule producing coccus forms tetrads and infects
American and European lobsters. Most infections occur
through breaks in the carapace during molting. The bac-
teria proliferate in the open vascular system of the lobster
(Fig. 41.16). Grossly, infected animals are lethargic and the
arthrodial membranes on the ventrum of the tail as well as
hemolymph samples are pink to red. Hemocytopenia even-
tually occurs and the vascular system is filled with tetrads
of bacteria. The bacteria do not cause direct organ necro-
sis in chronic disease, rather tissues atrophy. The increased
density of captured lobsters maintained in impoundment/
holding areas at >16°C is considered a contributory factor
to disease development. The disease is not seen in recently
caught wild animals. Gram stains of hemolymph and his-
tologic examination are commonly used diagnostic meth-
ods as is culture of the bacteria. Oxytetracycline has been
approved by the United States Food and Drug Administra-
tion (FDA) for use in treatment of this disease.
Necrotizing hepatopancreatitis (NHP) is an OIE
reportable disease that causes high mortalities in penaeid
shrimp. It is caused by a Gram-negative, dimorphic
intracellular rickettsia-like organism (RLO; unclassi-
fied alphaproteobacterium) that targets the tubular cells
of the hepatopancreas. Based on genome sequencing
information, the name Hepatobacterium penaei has been

FIGURE 41.16  Gaffkemia in an American lobster. The vascular spac-
es throughout the hepatopancreas are distended and filled with bacteria
(Aerococcus viridans).
proposed. Early in infection small, pleomorphic, intra-
cytoplasmic bacteria are present in the apical cytoplasm
or fill hepatopancreatic tubular epithelial cells resulting
in cellular hypertrophy of individual to small patches of
cells. Later in infection, there is more prominent tubular
epithelial cell hypertrophy or epithelial attenuation with
tubular dilation. Hemocytic infiltrates are present and
tubular necrosis is mild and typically affects distal tubular
segments. At the end stage of disease, there are linear areas
of tubular epithelial necrosis that extend to the medial and
proximal segments. Necrotic tubules are collapsed, contain
luminal hemocytes, and have a hyalinized and fragmented
basement membrane. Melanization and lamellar peritu-
bular interstitial fibrosis is present and remaining tubules
demonstrate moderate to severe dilation and epithelial
attenuation (Fig. 41.17). The RLO are Gram-negative but
discrete organisms are best seen by Steiners staining (Fre-
lier et al., 1992). Elevated temperature (near 30ºC) and
elevated salinity [30 ppt (part per thousand)] contribute to
disease occurrence. Diagnosis is confirmed by PCR (Loy
et al., 1996; Nunan et al., 2008).
Two of the most economically important bacterial dis-
eases in honeybees are American and European foulbrood,
both of which are reportable to OIE (Forsgren, 2010).
American foulbrood is a bacterial infection of the larval
stages of the honey bee (Apis mellifera and other Apis spe-
cies) by the Gram-positive, spore forming bacillus Paeni-
bacillus larvae. It occurs worldwide. Gross lesions consist
of a mottled appearance of the colony due to a mixture of
healthy capped, unhealthy uncapped, and empty brood cells
(OIE, 2008). Capped cells containing infected larvae are
moist and dark colored and become concave and puncture
as infection progresses. Affected larvae change color from
creamy white to brown, and develop a semifluid, glue-like
consistency (ropy stage) that can be drawn out as a thread
with a probe or matchstick, and can serve as a method of
Invertebrates Chapter | 41 1035
FIGURE 41.17  Necrotizing hepatopancreatitis in shrimp. Within the
hepatopancreatic interstitium are numerous hemocytic infiltrates that sur-
round aggregates of the rickettsia-like organism. Adjacent tubules are di-
lated and contain luminal hemocytes.
field diagnosis. A month after the ropy phase, the larval
material dries to a hard, dark scale (foulbrood scale) tightly
adhered to the lower cell wall (De Graaf et al., 2006). The
scale contains millions of spores that are a source of disease
transmission between colonies and can remain infectious for
more than 35 years and withstand heat, cold, drought, and
humidity. Bacterial spores are transmitted orally by nurse
bees or via spore contamination at the base of a brood cell
(OIE, 2008). Transmission via hive contamination neces-
sitates hive destruction by burning to prevent subsequent
transmission from colony to colony. Spores are infectious
only to larvae, which are most susceptible 12–36 h after
hatch. These spores pass through the foregut and germinate
in the midgut where they colonize the epithelium and mas-
sively proliferate, eventually filling the lumen, penetrating
the peritrophic membrane, and passing through the epi-
thelium to the hemocoel. Histologic lesions include initial
vacuolation and eventual lytic necrosis of the midgut epi-
thelium. Diagnosis is based on confirmation of the bacte-
ria and clinical signs either through direct testing of colony
material or following specialized culture with Paenibacillus
larvae agar (PLA), MYPGP agar, BHIT agar, and Columbia
sheep agar. A conventional PCR test for rapid confirmation
and identification after culture and a nested PCR that allows
for direct analysis of spore solutions are also useful in con-
firming infection. Colony samples should consist either of
a section of comb (20 cm2) containing affected brood cells
or collection of affected larval remains with a sterile swab.
Smears made at the apiary can be used to confirm diagnosis
through the presence of characteristic small Gram-positive
bacilli both singly and in chains, or with carbol fuchin stain-
ing of larval smears which highlights the spores. Appropri-
ate sampling techniques, storage, shipping, and testing are
described in the OIE Manual of Diagnostic Tests and Vac-
cines for Terrestrial Mammals, 2017.
1036 Pathology of Wildlife and Zoo Animals
European foulbrood of honey bees caused by (Melis-
sococcus plutonius) is the most widespread brood disease
in England and has broad distribution that includes most
locations where apiculture is practiced except New Zealand
(Forsgren, 2010). The name is derived from the foul odor
associated with infected colonies. This disease results in the
death of larvae at 4–5 days of age predominantly in unsealed
broods. Gross diagnostic features during colony assessment
include dead larvae that are not in their normal coiled posi-
tion but are instead stretched lengthwise within the cell or
twisted along the walls, and a distinct larval color change
from normal white-tan to yellow, progressing to brown,
and finally black-gray as larvae decompose. Some larvae
die in sealed cells resulting in sinking of the cap, similar to
American foul brood. M. plutonius is a microaerophilic to
anaerobic, Gram-positive, lanceolate coccus found singly,
in pairs and in chains. Secondary/concurrent bacterial infec-
tion is common and can lead to misdiagnosis of other bacte-
rial etiologic agents. Pathogenesis includes ingestion of the
bacterium with contaminated food followed by rapid prolif-
eration within the larval midgut. Although not all infected
larvae may be killed, those that survive infection are stunted
compared to uninfected animals. Confirmation of infection
can be made through smear preparations of larvae showing
gross lesions or with a rapid detection kit.
Withering syndrome (WS) is an OIE reportable, fatal
disease of abalone caused by the rickettsiales-like pro-
karyote (WS-RLP) Candidatus xenohaliotis californiensis
(Friedman et al., 2000; Gardner et al., 1995). All members
of the genus Haliotus in free ranging, cultured and labora-
tory settings can develop infection although susceptibil-
ity varies by species. Black and white abalone populations
suffer severe mortality, with only moderate mortality seen
in red abalone and little to no mortality seen in free rang-
ing pink and green abalone (Friedman et al., 2007; Moore
et al., 2009, 2001). The disease is most prominent in free
ranging and cultured populations along the Pacific coast
from central California through Baja California, Mexico, but
the geographical range is broad due to global culture of red
abalone and secondary exposure of native species. Infected
abalone are known to have been transported to Chile, China,
Taiwan, Iceland, Ireland, Israel, Spain, Thailand, and Japan.
External gross lesions include poor body condition and foot
muscle atrophy following a clinical period of anorexia and
lethargy. Internal gross lesions include retraction of the vis-
ceral tissues and digestive gland atrophy. On ­histopathology,
the target organ is the digestive tract. Organisms invade the
epithelium of the posterior esophagus and digestive gland
causing epithelial hypertrophy, necrosis, and low cuboidal
to squamous epithelial metaplasia. Deeply basophilic, api-
cal cytoplasmic bacterial colonies form inclusions that range
from approximately 10–20 µm diameter and are evident in
affected digestive tissue on routinely stained sections. WS-
RLP inclusions are also detectable on squash preparations of
digestive tissues stained with the nucleic acid-specific fluo-
rochrome Hoechst 33258. Pedal atrophy is characterized by a
diffuse decrease in the size of skeletal muscle fibrils with sec-
ondary wide separation of the fibrils by extracellular matrix
material. Transmission is likely fecal-oral and early infection
is located in the posterior esophagus and to a lesser extent in
the intestine. The pathophysiology of the disease is physio-
logic starvation secondary to the digestive gland changes that
result in depletion of glycogen reserves, catabolic changes in
the pedal musculature, muscular and digestive gland atrophy,
and ultimately death (Friedman et al., 2000).
Diagnosis of WS requires consistent gross lesions with
confirmation of RLP infection either by histopathology
or in situ hybridization (method of choice) coupled with
PCR (conventional and quantitative) and sequence analy-
sis (Antonio et al., 2000; Crosson et al., 2014; Friedman
et al., 2000). Definitive diagnosis of WS must be conducted
according to World Organization of Animal Health (OIE)
standards in the Manual of Diagnostic Tests for Aquatic
Animals (OIE, 2013).
A second RLP, the stippled RLP (ST-RLP) has been
identified in California abalone but at this time it is consid-
ered nonpathogenic and is found in tissues only at low lev-
els. A phage hyperparasite of the WS-RLP has been detected
that may reduce the pathogenicity of the bacterium. WS-
RLP and ST-RLP the phage variant can be differentiated by
light microscopy based on their differential staining proper-
ties on routine HE staining and the cytoplasmic location of
the inclusion (Crosson et al., 2014; Friedman et al., 2014).
Roseovarious oyster disease (ROD, also known as
juvenile oyster disease, JOD) is caused by an alpha-pro-
teobacteria, Roseovarius crassosstreae. The bacterium
infects juvenile eastern oysters and causes significant accu-
mulation of flaky/mucoid conchiolin between the membra-
nous mantle and the nacre of the shell. Affected animals
show cupping of the left valve and shortening of the right
valve with death of affected animals (often >90%) within
6 weeks of initial signs of infection. The disease was first
identified in 2002 and is noted sporadically in aquacultured
juvenile oysters in the northeastern United States from
Maine to New York. Histologically, mantle epithelium pro-
gresses from a hyperplastic/hypertrophic condition to ulcer-
ation of the shell epithelium of the mantle with buildup of
debris (conchiolin) between the ulcerated mantle and the
shell. Increases in hemocyte numbers are noted throughout
all tissues in early to mid-disease stages. This agent prolif-
erates in juvenile oysters less than 25 mm in shell height and
only in seawater greater than 21ºC and salinity between 25
and 32 ppt. Larger juveniles show signs of the infection but
survive the disease. Diagnostics include characteristic gross
lesions, histological evaluation of tissues, and PCR identi-
fication of the bacteria from infected oyster tissues. Resis-
tance to the disease appears to develop in the F1 generation
by using survivors as broodstock (Boettcher et al., 2005).

Fungi and Algae
Fungal infections, similar to bacterial infections, are com-
mon in situations where other environmental disruption
(abnormal temperature, biofouling, toxins) also contributes
to debilitation. Microsporidia are important pathogens of
invertebrates, infecting host groups from all major taxa in
all environments. Of the genera identified to date, nearly
half infect aquatic organisms including 50 genera in aquatic
arthropods and 21 genera in aquatic, nonarthropod inver-
tebrates (Dubuffet et al., 2013; Stentiford et al., 2013).
They cause significant morbidity and mortality in beneficial
insects (pollinators), farmed Crustacea and in some cases
are used as biological control for insect pests. Horizontal
and vertical transmissions are both possible. Though most
major organ and tissue types can be affected, infection is
typically restricted to one organ, tissue, or cell type in an
individual. Regardless of tissue type, histologically micro-
sporidia are evident in large numbers in the host cell cyto-
plasm. Some form sporophorous vesicles, a barrier between
the parasite and the host cell cytoplasm. Host cells are fre-
quently hypertrophied. Xenoma formation occurs in numer-
ous hosts and tissue types.
Crayfish plaque (also Crayfish aphanomyciasis, La
Peste etc.) (Edgerton et al., 2002; Longshaw, 2011) is a
fungal disease primarily of the carapace (cuticle) of many
species of crayfish. It is caused by the Oomycetes (water
mold), Aphanomyces astaci. The fungus infects through
small breaks in the arthrodial membranes primarily at
molting and proliferates within the carapace tissues. Gross
lesions include dark brown-black (melanized) or white to
brown spots and patches on the arthrodial membranes of the
ventral cephalothorax and abdomen. The fungus is highly
branched, approximately 7 µm to 9 µm in width, and non-
septate. Spore formation is absent until late stages of infec-
tion. Examination of wet mounts with and without Giemsa
FIGURE 41.18  Fungal infection of the ocellus and gill in an Atlantic horseshoe crab. (A) The ocellus and surrounding carapace are multifocally
eroded and an irregular white-tan plaque covers the local surface. (B) Within the gill lamellae there are multifocal areas of white discoloration, regional
lamellar thickening and necrosis.
Invertebrates Chapter | 41 1037
staining helps with identification. Histological sections are
also diagnostic. Molecular methods are available but due to
the many strains of fungus, may not be accurate.
Fungal, algal, and cyanobacterial diseases are common
causes of exoskeletal and respiratory disease in Atlantic
horseshoe crabs. Green algal (chlorophycophytal) infec-
tion of the carapace is one of the most common diseases in
wild and captive animals. It causes severe pitting and defor-
mity of the dorsal exoskeleton, eyes (ocelli), arthrodial mem-
branes, and the base of the telson resulting in shell deformity,
abnormal molting, end stage eye disease, and full thickness
perforation of the carapace. Lesions have a characteristic
dark green color. Histologically, there is multifocal erosion
of the cuticle with rhizoidal processes of algal zygotes seen
extending between the chitin laminae of the carapace. These
lesions can be full thickness, penetrating the carapace, and
infiltrating the internal viscera and creating portals for sec-
ondary bacterial infections. Histomorphology in one study
identified the algae as being from the family Ulvaceae
(Braverman et al., 2012). Fungal infections are an issue seen
primarily in managed care when animals are housed without
sand substrate and can have similar lesions to those previ-
ously described with algal infection (Fig. 41.18A,B). Infec-
tion primarily targets the book gills and operculum resulting
in thickening and yellow discoloration of the cuticle and
book gill leaflets. Ultimately there is extensive necrosis of
the book gill lamellae with extensive tissue loss. Cyano-
bacterial gill disease due to filamentous blue-green algae
(Oscillatoria spp.) can also colonize the thin chitinous book
gill lamellae. Gross lesions include swelling and rupture of
gill leaflets and extensive tissue necrosis and loss (Smith and
Berkson, 2005) (Fig. 41.19A). Lesions in fungal and cya-
nobacterial gill disease are histologically similar. Gill leaf-
lets are expanded by increased numbers of hemocytes that
frequently form large aggregates obstructing the lumen of
the leaflet (Fig. 41.19B). Distal to these aggregates ­leaflets
1038 Pathology of Wildlife and Zoo Animals
FIGURE 41.19  Cyanobacterial gill disease in an Atlantic horseshoe crab. (A) Gross lesions include black discoloration of the gill lamellae, lamellar
swelling, and multifocal lamellar necrosis. (B) Histologically a thick mat of cyanobacteria covers the lamellar surfaces and the lamellar vascular spaces
are infiltrated and obstructed by hemocyte aggregates.
often show signs of regional necrosis similar to embolic dis-
ease. With fungal disease, hyphae are present both within
the sinuses as well as penetrating through the gill leaflets.
With cyanobacterial gill disease, thick mats of organisms fre-
quently coat both surfaces of individual leaflets.
Aspergillosis of sea fan corals (Gorgonia spp.) due to
infection with the Aspergillus sydowii causes mild morbid-
ity to severe localized mortality events in the Caribbean
(Alker et al., 2001; Kim et al., 2006). Gross lesions affect
primarily the fan portion of the colony and include multifo-
cal tissue loss, distinct purple discoloration of the viable tis-
sue at lesion margins, and large nodular growths (galls) on
the fan of otherwise normally pigmented and intact colonies.
Lesions characteristically occur away from the colony edge
along major veins. The purple discoloration is the result of
increased production of purple sclerites (skeletal elements),
a nonspecific response to exposure to biotic agents. Histo-
logic lesions include fungal infiltration, tissue necrosis, and
extension of fungal elements into the axial skeleton. Granu-
lar amoebocyte infiltration and melanization often surround
fungal hyphae. The margin between infected and healthy
tissue is devoid of coral polyps and has a lower density of
zooxanthella (Smith et al., 1998). Stressors, such as ele-
vated temperature and nutrient pollution likely contribute to
disease development (Woodley et al., 2016).
Algal infections are reported in echinoderms. Cocco-
myxa ophiura infects two species of brittle stars (ophiu-
rids) causing multifocal patches of subepidermal green
discoloration along the aboral surface of the disc and rays.
Histologically, the algae infiltrate the dermal and mutable
connective tissues of the body wall in the space between
the dermal ossicles. Lesions progress and coalesce resulting
in epidermal necrosis, dissolution of the dermal ossicles,
infiltration of the connective tissue, and ossicles with algal
cells and eventually ray autotomy. A similar disease process
caused by C. astericola occurs in sea stars (Jangoux, 1986).
Chalk brood of honey bees due to the fungus Asco-
sphaera apis typically affects colonies in the spring during
cool and humid weather conditions and affects larval stages
exclusively. Larvae typically die before pupation, which
affects colony populations. Infection is spread through the
colony by food contaminated with fungal spores (asco-
spores). Ascospores germinate in the midgut of infected
larvae and the fungus invades the viscera. White masses
of mycelia are visible through the skin of infected larvae
and eventually break through the posterior end of the lar-
vae after death. Following death, hyphae cover the entire
cadaver growing from the posterior end toward the head
(Aronstein and Holloway, 2013).
Metazoa
Invertebrates serve as intermediate hosts for a wide variety
of metazoan parasites. Pleurocercoids (intermediate forms
of tapeworms) of Callibothrium verticallis are found in the
anterior midgut ceca of Pagurus spp. The final host is the
dogshark. Pleurocercoids cause significant damage to the
blind ended tubules of the glands and cause severe necro-
sis, rupture, and secondary bacterial infections that lead
to death (Smolowitz et al., 1993). Numerous pleuroceroid
larvae can also be found in the cecal lumen of squid and
other cephalopods. Typhlitis secondary to pleuroceroid lar-
vae has not been reported. Licnophora sp. (Phylum Cili-
ophora, Class Spirotichea) infest several genera of animals
including gastropods with and without shells (limpets and
sea hares) as well as seahorses, corals, clams, and others.
Their importance as a disease causing agent varies by host
species and environmental conditions, which may promote
their sometimes opportunistic, nature.
Nematode infections of arachnids are restricted to the
Mermithidae family. At least 51 species of spiders and
harvestmen have been recorded as mermithid nematode

hosts across various habitats. Poinar provides a detailed
summary of recorded instances of mermithid infection in
arachnids (Poinar, 1985). Symptoms include abnormalities
in body size and shape including swelling of the opistho-
soma, altered epigynum, malformed palpi, legs that are
shorter and thicker than normal, and poorly developed
male secondary sexual characteristics. Coils of the para-
site may be visible through the integument as the nematode
eventually occupies the entire abdomen and occasion-
ally the cephalothorax. This results in the digestive gland
reduction or absence, and loss of the prosomatic muscles,
the entire digestive system, or the reproductive system
(Poinar, 1985).
Nematodes are also common endoparasites of milli-
pedes and are found across a similarly wide range of species
(Hopkin and Read, 1992). Rhabditiform and rhigonema-
tid nematodes have been recovered from fecal samples of
myriapods (Chitty, 2012). Although previously not thought
to be pathogenic, nematodes with morphology consistent
with rhigonematids have been documented to cause midgut
ulceration and transmural hemocytic inflammation in Puerto
Rican white and giant African millipedes (Fig. 41.20). At
necropsy, affected animals are also in thin body condition
with evidence of digestive gland atrophy.
Shells of various invertebrates are often infested by
various shell boring organisms. Some types of sponges
(Cliona spp.) infest the shells of bivalves, dissolving the
shell and establishing colonies on the shell surface. Colo-
nization results in brittle, easily broken shells or pinpoint
round holes in the shell when the sponge is no longer
present. The shells of most bivalves and some other inver-
tebrates, such as abalone, are infested by Polychaeta (Poly-
dora spp. and Boccardia spp.) worms that borrow into the
matrix of the shells and form tunnels. Sand and mud is often
found in the tunnels along with the worms and forms “mud
blisters” when the tunnel is viewed from the inner surface of
the bivalve shell. These tunnels occasionally serve as homes
for other invertebrates.
Dicyemida (Rhombozoa) are a phylum of unusual
organisms that may be related to round worms, although
their life cycle is not similar. All life stages, vermiform
embryos, nematogens, rhombogens, sexually produced lar-
vae, and infusoriform embryos are found in the renal sacs
surrounding the appendages (kidney) of cephalopods. The
number and maturity of the organisms parallels the age/
growth of the cephalopod. Dicyemids are host-specific but
more than one species of dicyemid may be found in the
same host. The method of infection is not known although
it is suspected that the infusoriform larvae may both exit
and enter via the renal pore that empties into the mantle cav-
ity. Dicyemids attach to the renal tubule/crypts of the renal
appendages that fill the lumen of the renal sac into which
urine is deposited. Some species have also evolved to only
live in the sac surrounding the branchial heart appendage.
Invertebrates Chapter | 41 1039
FIGURE 41.20  Rhabditiform nematode infection in the midgut of
a millipede. Extending from the mucosa deep into the submucosa is a
dense hemocytic infiltrate surrounding innumerable cross sections of adult
rhabditiform nematodes.
Dicyemids tend to infect cephalopod species that spend
time in the benthic layer (primarily octopi and some spe-
cies of cuttlefish). An inflammatory response is usually not
associated with infection, but kidney necrosis and increased
debris in the renal sac may be seen in heavy infections
(Furuya and Tsuneki, 2003).
Protozoa
Coccidia are global pathogens that affect a wide range of
organs and species of invertebrates. In gastropod mollusks,
the renal coccidian Merocystis kathae has been reported in
dog whelks. Margolisiella haliotis is also often seen in the
renal epithelium (nephridium) of black, red, pink, green,
flat, and pinto abalone concurrently affected with wasting
syndrome (González et al., 2014). These coccidia do not
typically result in gross lesions or morbidity. All develop-
mental stages include oocysts, macrogametes, microgameto-
cytes, and meronts are evident on histopathology with no to
moderate hemocytic nephridial infiltrates present ­(Friedman
et al., 1995; González et al., 2014). All life stages exist
within a parasitophorous vacuole. Transmission is direct and
horizontal; the life cycle is completed within a single host.
Many species of decopod crustaceans can be infected
with histophagous, holotrich ciliates causing bumper
car disease, so called due to the shape of the organ-
ism. The most commonly diagnosed infection occurs in
American lobster and is caused by Mugardia sp., but
other genera (Paranophrys sp., Anophrys sp., etc.) o­ ccur
in other ­decopods i­ncluding several crabs (Hibbits and
Sparks, 1983). The disease can be found in low prevalence
in several types of decopods and only rarely causes severe
disease in wild animals, typically in late winter/early spring.
1040 Pathology of Wildlife and Zoo Animals
The method of entry appears to be through cuticular breaks
and may occur most commonly in the gills (Cawthorn
et al., 1996). Clinically, animals are lethargic. Histologi-
cally, when present in low abundance, the parasite is pri-
marily located in the sinusoids around the hepatopancreas.
As the disease progresses, small granulomas/encapsu-
lations occur in several areas (including the gills) and in
late stages, hemocytopenia occurs and abundant ciliates
may be found in the vascular system throughout the ani-
mal but especially in areas underlying the carapace. Direct
or Giemsa-stained examination of the hemolymph for the
characteristic, active, ciliated, bumper car-shaped protozoa,
or histologic examination are used for the diagnosis. Direct
or stained imprints of the hepatopancreas may detect early/
light infections in lobsters.
Paramoebiasis in decopod crustaceans has been respon-
sible for both endemic and epizootic disease and mortality in
blue crabs and American lobsters. Paramoeba perniciosia
infection occurs at molting. Lobsters may sustain a back-
ground infection without progression to mortality. Infec-
tions of blue crabs lead to death and most mortality is noted
in high density holding tanks in the fall and winter (John-
son, 1977; Newman and Ward, 1973). Grossly, crabs show
gray and lobsters red discoloration along the ventrum when
the disease is most severe, and hemolymph is white (crabs)
or pink (lobsters). Early in disease development, P. perni-
ciosa are histologically found in the connective and neural
tissues and are associated with hemocyte micronodulation
and encapsulation. In late stages of the disease, hemocyto-
penia and lack of response to the proliferation of organisms
in the vascular system is noted. In lobsters with chronic low
grade infections, fewer and melanized (chronic), micronod-
ular foci containing paramoeba are found in neural and
other tissues (Mullen et al., 2004). Since the range of these
two crustaceans overlap, it is possible that lobsters may be
a reservoir host.
Hematodiniosis in several species of crustaceans
including copepods and amphipods is caused by parasitic
dinoflagellates in the family Syndiniceae (Stentiford and
Shields, 2005). Infection by the genus Hematodinium sp.
produces the most economically important diseases in deca-
pod crustaceans. These include gray crab disease of the blue
crab, bitter crab disease of snow crabs, and postmolt syn-
drome in Norway lobsters. Ingestion and infection of the
hemolymph through the gastrointestinal tract is the primary
method of infection (Walker et al., 2009). Severely infected
animals take on a cooked appearance and the hemolymph
becomes milky (Small, 2012). Histologically, as the disease
progresses abundant mononucleated or binucleated amoe-
boid trophonts can be seen in the hemolymph along with
occasional vermiform plasmodia. Small encapsulations in
several organs are seen in early infections. In late infections
(1–2 weeks after infection), there is wasting of the tissues
and hemocytopenia with abundant dinoflagellate forms
filling the vascular spaces. Single nucleated amoeboid tro-
phonts can be easily mistaken for hemocytes histologically
and on examination of hemolymph samples. Diagnosis is
by gross and histologic evaluation and molecular methods.
H. perezi can be diagnosed by staining a sample of hemo-
lymph with oil red O, which results in lysosome staining
in the parasite but not in the hemocytes (Stentiford and
Shields, 2005).
Marteilia refringens belongs to the protozoan parasite
phylum Paramyxea and causes Marteiliosis, an OIE report-
able disease. It causes significant disease in aquacultured
and wild populations of European flat oysters on the west
coast of Europe. However, other species of bivalve, includ-
ing clams, scallops, and mussels can be infected with M.
refringens, or a closely related species, in various parts of
the world. Associated morbidity and mortality vary greatly
and some bivalve species may be carriers. Tissues of mor-
bid animals are thin and edematous. Infection occurs via
ingestion when a primary/stem cell enters the digestive sys-
tem and infects digestive gland epithelial cells forming a
“nurse cell.” Nurse cells undergo consecutive internal cleav-
age to produce 8–16 intracellular sporonts, each containing
4 spores and a few refringent bodies that can be seen histo-
logically (thus its species name) (Fig. 41.21). Histology is
used to diagnose the infection but smears/digestive gland
imprints on Wrights Giemsa stained slides can be used in
moderate to severe infections. Molecular diagnostic meth-
ods are also available.
Perkinsiosis caused by the dinoflagellate Perkinsus
marinus (also termed Dermo) is an important, OIE report-
able disease in bivalves. P. marinus is one of the two dis-
eases that have caused great reductions in the population of
eastern oysters on the east coast of the United States. The
infectious form, a trophozoite, enters primarily though the
FIGURE 41.21  Digestive gland of a European oyster infected with
Marteilia refringens. Digestive gland epithelial cells form a “nurse cell”
containing 8–16 sporonts per cell. Each contains four spores and a few
refringent bodies.

FIGURE 41.22  Perkinsus sp. infection in the gill of a soft-shelled clam.
Hemocytes are distended and contain sporangia filled with trophozoites.
gastric epithelium and digestive gland tubules and can also
enter through the mantle and gill epithelium. The parasite
is most active in oysters in seawater at a salinity of 12–28
ppt and at temperature greater than 18ºC. The trophozo-
ites are engulfed by the hemocytes in which they prolifer-
ate and form signet ring profiles consisting of a hemocyte
containing a 2–4 µm round parasitic vacuole. Occasionally,
a refringent vaculoplast can be indentified in the vacuole.
The mature trophozoite undergoes karyokinesis, then cyto-
kinesis to form a rosette within the cells. Mature rosettes,
called sporangia, are filled with trophozoites causing swell-
ing of the hemocyte (Fig. 41.22). Evidence strongly sug-
gests that the agent can inhibit apoptosis of the hemocyte
until it has finished its replication cycle, at which time
the host cell lyses and the newly formed trophozoites are
released to infect naïve hemocytes. Infected hemocytes cir-
culate throughout the open vascular system of the oyster. In
early infections, moderate mixed hemocytic (granulocytic/
agranunlocytic) inflammation is noted, especially in and
adjacent to the gastric epithelium or digestive gland tubules.
In late infections, few normal hemocytes remain in the tis-
sues and tissues appear thinned and edematous. The agent
is directly infective and trophozoites are released from
dead, decaying animals as well as with the feces from live
animals. The slowly progressive disease has spread and is
now found in aquacultured and natural populations of east-
ern oysters in the United States from Maine to Louisiana
where it usually causes death in the fall. P. marinus has also
recently been identified in other oyster species in the Gulf
of Mexico, and the Pacific oyster, which is cultured on the
west coast of the United States, can become infected but
does not exhibit disease. Histopathological examination is
not as sensitive or specific a diagnosis as is PCR/qPCR, and
is not as sensitive as the Ray’s fluid thioglycollate media
(RFTM) method (Smolowitz, 2013).
Invertebrates Chapter | 41 1041
Other Perkinsus species have been isolated from vari-
ous bivalve species around the world. The most important
of these is P. olseni, which is an OIE reportable disease.
Grossly, P. olseni and other Perkinsus sp. infections result
in small to large, white to brown nodules in the tissues of
the body, gill and mantle. Microscopically, foci of infection
are characterized by hemocytic inflammation that contain
and surround or attempt to surround signet ring and rosette
forms of the parasite. Trophozoites of P. olseni can be up
to 40 µm in diameter, much larger than those of P. mari-
nus. P. olseni has been identified in Tridacnid clams (dis-
played in aquariums) and other cultured bivalves, such as
Palourde clams, and infection has caused major mortality in
these and other bivalves. It is also a directly infective agent.
Perkinsus species can be cultured in RFTM, but culture is
nonspecific if more than one Perkinsus species is present
(Sheppard and Phillips, 2008).
Haplosporidium nelsoni, also named multinucleated
sphere unknown (MSX), is in the Class Ascetosporea,
Phylum Cercozoa. MSX produces spores but they are con-
sidered more primitive than other Cercozoa. It was first
identified in eastern oysters in late 1950 to early 1960 in cul-
tured animals from New Jersey to Virginia (United States).
It is now found in eastern oysters throughout the northeast
United States and into Canada and along with P. marinus,
and is responsible for decimating populations of aquacul-
tured and wild eastern oysters along the eastern United
States. Its most common form in tissues is a multinucleated
plasmodia. The infectious form is a spore that is first found
between epithelial cells of the gill, where it develops into a
multinucleated plasmodia (Fig. 41.23). The plasmodia later
breaks through the basement membrane of the branchial
epithelium and enters the open vascular system, where it
continues to proliferate through multiple serial divisions
that produce more multinucleated plasmodia. Inflamma-
tion at sites of infection in the gills is intense and local-
ized to areas of infection. The inflammatory response is
ineffective in destroying or isolating the plasmodia, which
proliferate in the open vascular system. As the disease pro-
gresses, the inflammatory response is greatly muted. Ter-
minally, connective tissue and other cells are shrunken and
animals develop generalized edema with increased “brown
cells” (spent tissue hemocytes) in the tissues. Mortality
is most severe in the late summer/early fall. The parasite
grows best in oysters in salinities greater than 15 ppt and
in warm water up to 20ºC; H. nelsoni is reported to die in
oyster tissues at temperatures greater than this. Spores are
formed in the digestive tubules of juvenile oysters in some
circumstances, but the reason/conditions for this are not
understood. While sporulation kills the juvenile oysters, the
spores are not infective to other adults or juveniles indicat-
ing that this is an indirect infection that requires another
host in the parasite’s life cycle. To date, other hosts have not
been identified but movement of infected animals to areas
1042 Pathology of Wildlife and Zoo Animals

FIGURE 41.23  Multinucleated sphere unknown (MSX) in the digestive gland of an eastern oyster. (A) Multinucleated plasmodia in the sinusoids
and (B) sporulated plasmodia of Haplosporidium nelsoni in the digestive gland epithelium.

without disease results in ­transmission indicating that the

other hosts are commonly found in many areas. The agent
and a mild form of the disease in low prevalence have been
identified in Pacific oyster. Transport of Pacific oyster to the
east coast of the United States is believed to be responsible
for the introduction of MSX into eastern oyster popula-
tions. Histopathological examination of tissues (especially
the gills in early infections) is the most common method of
diagnosis. Molecular methods have been published but are
not commonly used.
Haplosporidium costale, also called seaside organism
(SSO), is a close relative of MSX and also infects eastern
oysters. The disease was first identified in 1962 in oysters
grown in high salinity waters on the Atlantic coast of Vir-
ginia (United States). Recently the disease has been found
in aquacultured oysters in more northern states. The portal FIGURE 41.24  Seaside organism (SSO) in the digestive gland of an
of entry is primarily through the digestive tract epithelium, eastern oyster. Multinucleate plasmodia of Haplosporidium costale with-
unlike MSX. Early SSO infections are identified when in the connective tissue surrounding the digestive tubules.
multinucleated plasmodia are present in the sinusoids in
connective tissues around the stomach and digestive gland no intermediate host has been identified. The parasite grows
(Fig. 41.24). As in MSX, plasmodia proliferate over time best in oysters held in salinities 25 ppt or above. Diagnosis
and fill the vascular system of the oyster. Inflammation is of SSO is hampered by the similar appearance of the plas-
moderate in early infections but as the disease progresses, modia to that of MSX. MSX plasmodia are usually slightly
the number of viable hemocytes decreases and inflamma- bigger and more eosinophilic, but that is not consistent in all
tion is not seen late in the disease. Unlike MSX, plasmodia cases. Molecular confirmation is very helpful in these cases.
of SSO synchronously sporulate throughout the sinusoids in Bonamia spp. parasites are considered members of
late May to early June in adult oysters. If abundant plasmo- Haplosporidia although no spore form has been identified.
dia are present in the sinusoids, the animals will die during The disease, also termed microcell disease and hemocytic
sporulation. If few plasmodia are present, the animals will disease, was first reported in the United States during the
survive the event. Those that survive often appear thin and mid-1960s, but disease caused by Bonamia spp. occurs
edematous. Microscopically, the only remaining sign that an worldwide. Several species of bivalve can be infected by
animal was infected are numerous “brown cells” in circula- the two most important species, which produce varying
tion. It only rarely causes severe disease in wild animals, morbidity and mortality especially during warmer months.
typically in late winter/early spring. Transmission using B. ostreae is most problematic in European flat oyster. B.
spores from infected oysters does not cause infection in exitiosa, and the genetically similar B. roughleyi, are found
naïve oysters so indirect infection methods are suspected but primarily in European flat oyster in New Zealand, Spain,

and Italy. Importantly, some species of oysters, including
Pacific oyster, a commonly aquacultured oyster in Europe
and the United States and Suminoe oyster, which was con-
sidered as a replacement species in aquaculture for Eastern
oyster, become infected but do not develop disease, and
are considered carriers. Bonamia spp. infect the hemo-
cytes of oysters (primarily adults) and form few to many
2–5 µm round cells within the cytoplasm of the hemocyte.
It is directly infective. Grossly, yellow discoloration of
the gills may be noted. Histologically, in early stages of
the disease, an increase in circulating hemocytes is noted
in affected animals and multifocal hemocyte aggregates/
small encapsulations contain few to many Bonamia sp.
organisms within the cytoplasm of some of the hemocytes
(Fig. 41.25). Interestingly, da Silva (2008) found a cor-
relation between high percentage of agranulocytes in the
hemolymph and increased probability of infection of that
strain of oyster. Histology and molecular methods can be
used to diagnose the disease. Additionally, imprints of the
heart on coated slides stained with Giemsa stain can be
diagnostic. B. ostreae and exitiosa are both OIE reportable.
Quahog parasite unknown (QPX) is a protozoan
belonging to the phylum Labrinthomorpha and class Thraus-
tochytrid. It is an opportunistic pathogen that causes disease
in hard clams (Mercenaria mercenaria) on the east coast of
the United States. It was first identified in clams in Canada
in the late 1940s then in aquacultured clams in the United
States in 1996. It has caused disease in New York and Vir-
ginia, however, the prevalence and severity of the disease
were highest in areas of Massachusetts. The protozoa infect
the mantle of clams close to the base of the siphon; almost
all infected animals show nodules and swellings in this area.
This is the area where pseudofeces are deposited and it is
hypothesized that the increased contact time between the
parasite, pseudofeces, and the mantle allows for infection
into the tissues of that area. More advanced infections show
spread of the organisms throughout the clam vascular sys-
tem. The inflammatory reaction to the invading parasite is
seasonally dependent. In the early spring and late fall, the
inflammatory response is poor to moderate and character-
ized by numerous proliferating organisms within an often
thick coating of tenacious mucus (produced by the para-
site) surrounded by only a few layers of responding hemo-
cytes. In the summer (when metabolism of the clam peaks),
the inflammatory response is intense with many layers of
hemocytes ­forming hemocytic nodules with the occasional
occurrence of hemocyte derived multinucleated giant cells
and histologic evidence of destruction and phagocytosis of
the invading organism. Diagnosis can be accomplished with
stained or unstained squash preparations of the nodules in
the mantle or with histological examination. Molecular
diagnostic tools are also available (Smolowitz, 1998).
Many benthic oriented cephalopods, including most
octopi and several species of cuttlefish, are infected by
Aggregata sp., highly host specific coccidians that develop
Invertebrates Chapter | 41 1043
FIGURE 41.25  Bonamia spp. in a Suminoe oyster. Within the diges-
tive gland connective tissues, hemocytes contain numerous 2–5 µm round
parasites within the cytoplasm.
unusual forms in the gastrointestinal tract of the host
(Estévez et al., 1996). Both gametogony and sporogony
occur in the submucosa of all parts of the tract, but are usu-
ally most notable in the cecum. Grossly, infection is char-
acterized by white, sometimes confluent, nodules (1 mm
and less) throughout the gastrointestinal tract. Microscopi-
cally, in severe infections, numerous parasites in various
life-stages are seen (Fig. 41.26). Severely affected animals
show a loss in body condition (Gestal et al., 2002). A sec-
ond less well known, ciliated parasite, Chromidina sp., is
sometimes found in the renal sacs of epi- and mesopelagic
squids and octopi (Furuya et al., 2004).
Bodonid flagellates of the genus Ichthyobodo have
been intermittently reported in cephalopods, particularly
octopus, but their host-parasite interaction has not been
studied in detail. The parasite is free-swimming in the water
column and can attach to epithelial surfaces, particularly
targeting the gills in octopus. There are no gross lesions and
diagnosis is primarily made by histopathology. Histologi-
cally there is swelling, degeneration, necrosis, and erosion
of the branchial epithelium with hemocytic infiltration of
the submucosa. Ichthyobodo spp. are pyriform, 6–10 µm
long, 3–6 µm wide and are attached to the apical epithelium
frequently in large numbers that are densely packed (Seeley
et al., 2016).
Ectoparasites
Tubellarids (flatworms) of many different genera infest and
infect several types of invertebrates. A commonly encoun-
tered example is the Triclad tubellarid (Bdelloura spp.)
found on the gills and body of Atlantic horseshoe crabs.
Large white flat worms are visible on the gills, ventrum, and
arthrodial membranes. Adults are not believed to cause dis-
ease, but the egg cases in the gills can cause pressure necro-
sis of the lamellae causing inflammation and opportunistic
1044 Pathology of Wildlife and Zoo Animals
infections (Fig. 41.27A,B) Tubellarid (Rhabdocoela) infec-
tions of the opalescent nudibranch used in research, involve
a flat worm that invades the coelom through the digestive
tract prior to collection. The turbellarid develops there, and
eventually lyses the body wall of the host causing death. It
forms cocoons and the resulting embryo invades a new slug
host. Tubellarians also infest the gills and digestive tracts
and potentially cause disease in various bivalves (Brun
et al., 1999; Uddin et al., 2011).
Amphipods, small copepods, flatworms, and nudi-
branchs are important ectoparasites of cnidaria. All cause
tissue damage and loss to their respective hosts resulting
in morbidity and mortality. Hyperiid amphipods are obli-
gate parasites of gelatinous zooplankton that cause ecto-
parasitism in jellyfish and comb jellies in managed care.
FIGURE 41.26  Agreggata spp. in the esophagus of a cephalopod. Ex-
panding the submucosa are nodular aggregates of coccidia demonstrating
both gametogeny and sporogony.
FIGURE 41.27  Bdelloura infection in the gill of an Atlantic horseshoe crab. (A) Cytology of a gill leaflet contains a focal egg casing from a Triclad
turbellarid. (B) Histologically, adult Bdelloura are embedded within the gill lamella causing focal distortion.
During larval development gravid females select host
medusa, bite holes into the tissue, and place larvae into
the openings near the gonadal and gastric tissues. Devel-
oped females feed on host tissues and on prey captured by
the medusa (Boonstra et al., 2015; Crossley et al., 2009).
Dugesia, Waminoa, and Convolutriloba sp. flatworms can
cause significant ectoparasitism in coral reef tanks. Flat-
worms of the order Polycladida are of particular concern
and affect acroporid corals (Acropora eating flatworms).
Gross lesions in affected Acropora consist of white bands
of tissue loss which begin at the base and move dorsally
along the branches. Diagnosis can be made through scrap-
ings at the margins of tissue loss or through identification
with a dissecting microscope (Fig. 41.28A,B). The cope-
pod Tegastes acroporanus is an ectoparasite of acroporid
corals and actively feeds on coral tissues causing reduced
polyp extension, loss of coral tissue, and zooxanthellae pig-
ments, reduced growth and death (Carl, 2008). The com-
mon name for this disease is “red bug” and the copepods
are visible to the naked eye and with a dissecting micro-
scope (Fig. 41.29A–C). Nudibranchs can be ectoparasites
in coral reef aquaria, feeding on Montipora plate corals
and earning the moniker Montipora-eating nudibranchs
(Fig. 41.30). These organisms also cause tissue loss in
affected coral colonies and are visible with the naked eye
or by dissecting microscope though they can be difficult to
detect during daylight hours (Carl, 2008).
Acarapisosis (also acariosis or acarine disease) of
honey bees is an OIE reportable disease caused by the Tar-
sonemid mite Acarapis woodii, an internal pathogen of the
respiratory system of the adult honey bee Apis mellifera
and other Apis species. Disease has been documented in
North and South America, Europe, and the Middle East.
These mites reproduce within the respiratory tract and feed
upon the hemolymph of host bees. They are approximately
 Invertebrates Chapter | 41 1045

FIGURE 41.28  Acropora eating flatworm infection in Acropora spp. coral. (A) A single branch of an affected coral demonstrating tissue loss begin-
ning at the base of the branch. (B) Cytology of the margin of the viable tissue recovered characteristic planaria.

FIGURE 41.29  Tegastes acroporanus (red bug) infection of an Acropora spp. coral. (A) The affected branch demonstrates regionally extensive pallor
and multifocal tissue loss, (B) Superficial copepod parasites with a yellow body and prominent red focus on the caudal ramus consistent with Tegastes
visible on the surface through a dissecting microscope and (C) recovered in a mucus sample.

the airways, physical obstruction of the respiratory tract by

FIGURE 41.30  Montipora eating nudibranchs on a Montipora spp.
coral. A representative branch affected coral demonstrating tissue pallor
and loss with visible ectoparasitism. Innumerable nudibranch parasites are
present at the border between viable tissue and tissue loss.
150 µm in diameter and are found predominantly in the pro-
thoracic trachea but can be present in the head, thoracic, and
abdominal air sacs. Infection causes mechanical d­ amage to
mites or lesions, and hemolymph loss. With heavy parasit-
ism gross lesions, specifically opaque brown-black foci in
the tracheal walls develop due to inflammation and mela-
nosis. Mortality from this infection is moderate to high and
can be a cofactor in colony losses (Cepero et al., 2014).
Diagnosis is by microscopy, enzyme-linked immunosorbent
assay (ELISA) or the observation of guanine, a mite waste
product. Microscopic confirmation requires identification of
the mites first within the trachea and then after removal for
positive identification. Spread is through direct contact with
newly hatched (less than 10 days old) bees who are the most
susceptible to novel infection.
Small hive beetle infestation is caused by Aethinia
tumida, a beetle native to Africa that was introduced to the
United States, Egypt, Canada, and Australia by commercial
bee movement. Adult and larval beetles consume larvae, pol-
len, honey, and bee brood. The adult females lay eggs in the
hive and the beetle larvae feed within the hive, then leave
to pupate in the soil where the adults hatch and can fly to
new hives. Spread can be rapid and over a range of several
1046 Pathology of Wildlife and Zoo Animals
kilometers. If infestation is severe, bees will abandon the hive.
Diagnosis is made by positive identification of beetles within
the hive (World Organisation for Animal Health, 2016a).
Tropilaelaps of honey bees is an OIE reportable disease
due to infection with a few different species of Tropilaelaps
mites, most frequently Tropilaelaps clareae and T. koeni-
gerum. The mites feed on the brood (larvae and pupae).
Gross lesions include an irregular pattern of sealed and
unsealed brood and deformities in adult bees. Spread is by
direct contact, either bee to bee or by movement of brood
frames. Diagnosis is by positive morphologic identification,
although molecular testing is available (World Organisation
for Animal Health, 2016b).
Varroosis of honey bees, an OIE reportable disease, is
due to infection with an ectoparasitic mite that affects both
adult bees and the brood. Of the four species of mite, Varroa
destructor is the most important. These mites have nearly
a worldwide distribution with the exclusion of ­Australia
and the southern island of New Zealand. Gross findings
include shrunken abdomens in adult bees and visible exter-
nal mites. Spread is by direct contact between adult bees
and ­movement of infested bees or bee brood. In addition to
direct effects, this mite is also an important vector for viral
diseases of honey bees, including transmission of a virus
that causes wing deformity (World Organisation for Animal
Health, 2016c).
 Invertebrates Chapter | 41 1046.e1

E-SLIDES
41.e1 Haplosporidium nelsoni (MSX) infection, eastern oyster, whole body. These two sections are taken through
the bodies of two eastern oysters. The sections show tissue changes and hemocytic inflammation associated with
infection by parasite, Haplosporidium nelsoni (MSX). (see Fig. 41.23). eSlide: VM05211
41.e2 Perkinsus infection, eastern oyster, whole body. The bottom section shows a severe infection with the parasite,
Perkinsus marinus (Dermo) associated with a diffuse hemocytic inflammation. (see Fig. 41.22). eSlide: VM05212
1046.e2 Pathology of Wildlife and Zoo Animals
E-ONLY CONTENTS
ADDITIONAL UNIQUE FEATURES
The four major phyla (Cnidaria, Mollusca, Arthropoda,
Echinodermata) and specific subphyla (Table e1) and dis-
eases covered in this chapter were selected due to their
ecological importance, commercial value, and importance
to species in managed care. The majority of invertebrates
discussed in the chapter “ Invertebrates” are Eumetazoa
and have true tissues that are divided into germ layers and
dedicated to one or few functions. Functional counterparts
of familiar vertebrate tissues are frequently present and
include integumentary systems, skeletal systems, circula-
tory systems, digestive systems, respiratory systems, ner-
vous systems, sensory systems, and excretory systems. A
pseudocoelom exists in Nematodea, Acathocephala, and
five other minor phyla. Most other invertebrates have a true
coelom, a body cavity formed within the mesoderm that
contains coelomic fluid and multiple organ systems often
suspended by mesenteries. The coelom provides ample
space for gonadal development and expansion of gut to
accommodate large prey and is the basis of the hydrostatic
skeleton in some species (discussed later). In more complex
TABLE e1 Phyla and Subphyla of Ecological,
Commercial, and Captive Importance
1. Cnidaria
a. Anthozoa—anemones, soft and stony corals, sea fans
b. Scyphozoa—sea jellies
c. Hydrozoa—Hydra, Portuguese man-of-war
2. Mollusca
a. Gastropoda—sea snails, sea slugs, freshwater snails, abalone
b. Cephalopoda—octopus, squid, cuttlefish, nautilus
c. Bivalvia—clams, mussels, oysters
3. Arthropoda
a. Chelicerata
i. Arachnida—spiders, scorpions
ii. Xiphosura—horseshoe crabs
b. Crustacea
i. Malacostraca—crabs, lobsters, crayfish, shrimp
c. Myriapoda
i. Chilopoda—centipedes
ii. Myriapoda—millipedes
d. Hexapoda
i. Insecta
1. Hymenoptera—bees
2. Lepidoptera—butterflies
3. Coleoptera—beetles
4. Echinodermata
a. Asterozoa
i. Asteroidea—sea stars
ii. Ophiuroidea—basket stars
b. Echinozoa
i. Echinoidea—urchin
ii. Holothuroidea—sea cucumbers
invertebrates, the coelom is partly or completely divided
into a connected series of compartments, such as the peri-
cardium, renocoel and gonocoel of molluscs, or the tubular
systems in echinoderms.
Most invertebrates have a complex body wall formed of an
external epidermis, a medial connective tissue ­compartment
and an internal epithelial barrier that lines either a simple body
cavity (pseudocoelom or coelom) or a gut cavity (gastroder-
mis). The epidermis forms a critical barrier to the external
environment and allows for internal regulation and homeo-
stasis. In some species, it is also important for nutritional and
respiratory transport functions. As in vertebrates, the epider-
mis is composed of a single layer of epithelial cells bound
together by intercellular junctions composed of cell adhesion
proteins (cadherins). The cells are often ciliated (one or many
cilia per cell) and can further differentiate into structural and
secretory cell types with intervening pigment cells in some
species. Epidermal cells secrete and lie upon a thin layer of
dense, fibrous extracellular matrix (ECM), the basal lamina,
which is composed of collagen type IV; hemidesmosomes
composed of integrins anchor epithelial cells to the basal
lamina. The epidermis is covered by a secreted glycoprotein
extracellular matrix (ECM), the cuticle, which varies in com-
plexity by species (Fig. e1A,B).
Invertebrate connective tissue compartments consist of
a minimum of proteoglycan ECM and embedded protein
fibers; most species also have an additional cellular compo-
nent. The thickness varies as does the composition. In some
species, the connective tissue compartment consists only
of the epidermal and gastrodermal basal laminae. In oth-
ers, a loose ECM, the reticular lamina, and additional cells
(fibroblasts, amoebocytes) are present. Similar to mamma-
lian tissues, collagen is a common connective tissue fiber in
invertebrates. The extracellular matrix provides attachment
points for cells, pathways for cellular movement, and an
environment for cellular differentiation.
The skeletal systems in invertebrates can be fluid or
solid and develops from modifications of the internal or
external ECMs (connective tissue compartment or cuticle).
Skeletons form a structure that supports and transmits the
force of muscular contractions and provides protection
from predation, injury, infection, and the environment.
Fluid or hydrostatic skeletons are a feature of cnidaria and
many annelids. Their bodies are supported by the balance
between ­pressurized water within their gastrovascular cav-
ity or coelom and the body wall which is often reinforced
with a mesh of inelastic fibers arranged in crossed-helical
pattern. These fibers are composed of proteins, typically col-
lagens, which are either embedded in the cuticle (annelids)
or in the reticular lamina beneath the epidermis (cnidarian).
Solid skeletons may be either endo or exoskeletons and are
composed of solid materials that can be either pliant, able
to be deformed but return to their original shape, or rigid,

FIGURE e1  Examples of normal epidermis, adnexa, and surface structures. (A) Epidermis from a mottled sea star demonstrating the complex na-
ture of the epidermis and cellular differentiation. (B) Normal epidermis, adnexa, and cuticle of the opisthosoma (abdomen) of a greenbottle blue tarantula
demonstrating the exo and endocuticle and epidermis with basement membrane. (C) American lobster epidermis demonstrating similar layers.
resistant to any change in shape (Chapman, 1958). Endo-
skeletons originate from the ECM of a connective tissue
compartment that is stiffened either by protein crosslink-
ing or secretions from extracellular matrix cells. Exoskel-
etons originate as a ­thickened form of the cuticle that is
made more rigid either by chemically cross-linking the
ECM proteins (insects), or by adding mineral to the ECM
(crustaceans). Pliant skeletons are composed of a compos-
ite of proteins, polysaccharides and water, and they range
in c­onsistency from watery gels, like in the mesohyl of
sponges and mesoglea of comb jellies, to the stiff or springy
mesoglea of many jellyfish or the skeletal material in the
hinge of clams or supportive structures of sea fans (gorgo-
nin). Pliant skeletons can be made stiffer by the addition of
rigid pieces, mineral spicules, sand grains, or organic fibers
that include collagen (spongin), chitin, or cellulose. Rigid
skeletons are composed of a composite of materials that
are frequently mineralized. The cuticle is composed of lay-
ers of chitin and other proteins primarily produced by the
­underlying c­ olumnar epithelium. In many arthropods the
exoskeleton contains both rigid and pliant skeletal compo-
nents, with the rigid skeleton being the “hard” carapace and
a more pliable exoskeleton made of flexible chitin, termed
arthrodial membranes, act as a joint and allow bending and
movement of the sections of the hard carapace.
There are three different types of muscle cells in inverte-
brates that further define the type and structure of ­muscle tis-
sue present. Epitheliomuscular cells have actin and myosin
filaments in a fixed arrangement at the basal part of epithelial
cells. Myoepithelial cells are similar to epitheliomuscular
cells but their apex is not exposed to the epithelial surface.
Myocytes, which are true muscle cells that have lost their
epithelial characteristics, are specialized solely for contrac-
tion. In addition to three muscle cells, there are three types
of muscles in invertebrates. These include smooth muscle,
which is slow contracting with tension possible over a wide
Invertebrates Chapter | 41 1046.e3
range of stretch lengths and, often seen in animals with
extensible bodies or similar appendages (e.g., tentacles);
cross striated muscle that demonstrates rapid contraction but
develops tension with limited excursion and is often associ-
ated with bodies or appendages that travel fixed distances
(e.g., claws, limbs, jaws); and obliquely striated muscle with
intermediate performance between smooth and cross stri-
ated muscle that contracts more quickly but over a wider
excursion and is most often associated with soft-bodied and
extensible animals, such as Annelids and Cephalopods.
The nervous and sensory systems in invertebrates per-
mit adaptive responses to environmental stimuli. Movement
toward more favorable environmental conditions occurs
by sensing these factors and conveying information to the
musculoskeletal system. In the simple invertebrates, there
are single cells (independent effectors) that have a sensory
surface and a motile portion. In slightly advanced inverte-
brates, sensory and motor functions are separated and in the
most advanced invertebrates there is a clear system of con-
ducting cells (interneurones). Interneurons undergo further
organization in more advanced species into subepithelial
nerve nets, differentiation of nerve nets into functionally
distinct parts (e.g., echinoderms); or aggregation of nerve
cell bodies into network of functionally distinct ganglia
(e.g., mollusks, arthropods) (Fig. e2 A,B,C). In cephalopod
mollusks, the central nervous system consists of multiple
lobes connected to each other to from a ring around the
esophagus at the level of the eyes.
The sensory system in invertebrates detects environmen-
tal conditions including electromagnetic (light, infrared),
mechanical (sound, vibrations, touch, pressure, gravity)
and chemical (taste, smell) stimuli. The receptor neurons
evolved from ciliated epithelial cells and their sensory sur-
faces are typically modifications of cilia or microvilli. In
some cases, groups of photoreceptors, chemoreceptors, and
mechanoreceptors are locally concentrated in the epithelium
1046.e4 Pathology of Wildlife and Zoo Animals
FIGURE e2  Examples of normal nervous system structures. (A) The
radial nerve cord of echinoderms is a V-shaped structure running the length
of each ray in the ambulacral groove and consists of an outer ectoneural
(sensory) nerve and an inner hyponeural (motor) nerve. The example here
is from a mottled sea star.(B) The nerve cord of myriapods consist of giant
nerve fibers that connect with pairs of ganglia present for each of the first
four trunk segments and two pairs for each diplosegment to provide motor
control. The example here is in a giant African millipede. (C) The cephalo-
thoracic nerve mass in arachnids is composed of multiple ganglia that pro-
vide motor (dorsal pathways in the ganglion) and sensory (ventral pathways
in the ganglion) function. The example here is in a goliath bird eating spider.
and form discrete sensory organs. Statocysts (gravity recep-
tors) and photoreceptors/eyes are examples of such special-
ized sensory structures. Statocysts consist of a capsule lined
by mechanoreceptive cells that contain a central, dense
particle, the statolith. As an animal changes orientation,
gravitational forces pull on the statolith, which changes and
stimulates different areas of the statolith lining and conveys
a sense of orientation with regard to up and down. Statoliths
are notably present in bivalve mollusk larvae and juveniles
but are usually not seen in adults. Photoreceptors can be
dispersed as individual cells over the integument or into a
single location forming an eye. The simplest form of an eye
is an ocellus of which there are two types. A pigment spot
ocellus consists of a patch of photoreceptors interspersed
with pigment cells. Pigment cup ocellus forms a cup into
which the photoreceptor elements project. These can be
either everted (photoreceptor elements project between the
pigment cells into the lumen of the cup) or inverted (pho-
toreceptors project into the cup lumen not through the pig-
ment cells. Pigment cells in all eyes serve the function of
shading the photoreceptors, which enables the animal to
determine the direction a light source. Some invertebrates
have comparatively advanced eyes that share a “camera
type” structure that is similar to vertebrate eyes and likely
represents convergent or parallel tissue evolution. These
include cephalopod mollusks and arachnids which have a
complex eye connected to the central nervous system by a
short optic nerve (Fig. e3 A,B).
The cardiovascular system in most invertebrates is open
although the extent to which the vascular system is open
varies greatly. In open vascular systems, the hemolymph
is pumped by a heart into the body cavities and hemocytes
diffuse back to the circulatory system through tissue cells
rather than having a direct return through closed vascular
structures. A heart-like structure exists in many species and
can consist of a thin, relatively indistinct muscular vessel
(e.g., myriapods) or muscular tube with arterial branches
that sits in a pericardial sinus that is open to venous return
(e.g., arachnids), or more complex hearts like what is pres-
ent in mollusks and that consist of a ventricle and two atria
separated by atrioventricular valves and lies within a peri-
cardial sac. Mollusks also have a vascular system, with an
aorta that exits the ventricle and forms an aortic bulb close
to the ventricle. Arteries and arterioles branch from the aorta
and carry hemolymph to various parts of the body. The
circulatory system of cephalopod mollusks is considered
the most developed of any invertebrate. The sinusoids are
reduced in size and are connected by a set of well-developed
arteries and veins that differ from other invertebrates by hav-
ing thicker walls with multiple layers of tissues. Each cepha-
lopod has three hearts: a systemic heart located subcentrally
to the mantle midpoint that receives oxygenated hemolymph

FIGURE e3  Normal invertebrate eye structures. (A) The median eye
of a greenbottle blue tarantula demonstrating from dorsal to ventral the
cornea, lens, vitreous cells, visual receptor cells, pigment cells, and the
start of the optic nerve. (B) The eye from a mantis shrimp demonstrating
(dorsal to ventral) corneal lens, crystalline cone, retinal cells, and optic
nerve fibers.
from the gills; and two branchial hearts that receive hemo-
lymph from the body and pump it into the gills for oxygen-
ation (Fig. e4A,B).
The excretory organs in invertebrates are highly
variable and waste exchange is often paired with gas
exchange. In coelenterates, ammonia simply diffuses
across the body wall and disperses in the water (Ruppert
et al., 2004). In other species, filtration of the circulatory
system fluid (hemolymph) results in urine production. In
mollusks, this occurs through the heart—pericardial organ
complex and the hydrostatic pressure that dictates filtra-
tion is generated by cardiac contraction which pushes
hemolymph into the pericardial cavity after which it
flows through the renopericardial canal to the kidney or
Invertebrates Chapter | 41 1046.e5
FIGURE e4  Examples of normal invertebrate hearts. (A) The sac-like
heart composed of cross striated muscle fibers of an arachnid. The heart
lies within a pericardial sac. Blood flows from the pericardium into the
heart via dorsal and lateral ostia. This example is from a goliath bird eating
spider. (B) One of the branchial hearts in a cephalopod mollusk demon-
strating the thick muscular wall which pumps hemolyph from the body to
the gills for oxygenation. The example is from an octopus.
nephridium (Fig. e5A, B). Podocytes that form a selective
filtration barrier are described associated with the basal
lamina of the auricle, pericardial gland, and kidney in
many species. In crustaceans, the gills are the most impor-
tant organs of ­osmoregulation with c­ ontribution from the
antennal glands. The excretory system of insects is com-
posed of the Malpighian tubules and the gut, particularly
the hindgut. Malpighian tubules consist of a single layer
of squamous epithelial cells forming a blind-ended tube
1046.e6 Pathology of Wildlife and Zoo Animals
FIGURE e5  Examples of normal excretory organs (nephridia). (A)
Nephridia of clams, is a long tube intimately associated with the heart
(heart–kidney complex). Hemolymph is passed into the nephridia where
selective secretion and absorption of ions is performed by the nephridial
epithelium. (B) The nephridia (antennal gland) of an American lobster,
consists of a proximal labyrinth and distal bladder. The glandular spongy
labyrinth contains numerous folds and the walls secrete and absorb ions
selectively to maintain balance in the hemolymph.
that range from 2 to 70 mm length and 2–250 in number.
Urine formation involves active transepithelial secretion
of ions (K, Na, and Cl) into the lumen of the Malpighian
tubule and passive flow of water due to the created gra-
dient. The primary urine is then modified by the midgut
through reabsorption or secretion. Water, ion, and metab-
olite reabsorption occurs in the rectum and produces
strongly hyperosmotic or hypoosmotic excretia. In many
terrestrial insects, the hindgut can recover nearly all of the
water from gut contents and fluid secreted into the gut by
Malpighian tubules (Larsen et al., 2014).
Invertebrates have developed a wide range of respi-
ratory systems to facilitate oxygen exchange with their
environment. In many species, oxygen uptake is improved
through countercurrent exchange where hemolymph flow
is in the reverse direction to air or water. Respiratory sur-
faces have thin highly vascularized walls to minimize dis-
tance for oxygen exchange between ­respiratory medium
and hemolymph (Leake, 1975) (Fig. e6 A,B). Gills are
present in many invertebrate species and may be exter-
nal or internal, and singular or multiple and segmentally
arranged. In echinoderms, there are three types of struc-
tures that can serve respiratory functions—the papulae
(external coelomic extensions), tube feet, and peristomal
gills (Fig. e6 C). The respiratory system of most insects
consists of an open tracheal system, a series of airways
that end internally in fine branches in tissues (Mill, 1997).
Most arachnids have two different types of respiratory
systems, one pair of localized book lungs, and one to two
pairs of tubular trachea ­(Foelix, 1996) (Fig. e6 D). Gas
exchange along respiratory surfaces may be facilitated
by the environment (natural water movement) or through
circulation using cilia, body movements, or respiratory
pumps.
Invertebrate digestive systems grow increasingly more
complex with animal size. The simplest forms, seen in
cnidaria, are simple sacs with a single opening for intake
of food and waste discharge. They are lined by a single
layer of epithelium, the gastrodermis, which is secretory
and absorptive; digestion is intracellular. In other inver-
tebrates, the digestive system is more complex, with a
single opening for intake of food and a separate opening
for discharge. In many invertebrates, the gut has special-
ized areas for grinding, storage, digestion, and absorp-
tion. Specialized structures are also present for capturing
food, such as ciliary tracts in bivalve molluscs, tentacles
in cnidaria, suckers in cephalopod molluscs, and radulae
in gastropod molluscs. Digestion is partly extracellular
and the gut lining is functionally separated into regions
that secrete different enzymes (Leake, 1975). Many inver-
tebrates have accessory digestive glands with functions
similar to those of the liver and pancreas in vertebrates
(Fig. e7 A–C).
Invertebrates employ a wide variety asexual (budding
and fission) and sexual (production of two types of ­gametes)
reproductive strategies. Male and female gametes may be
produced by one individual (hermaphrodite) or by two indi-
viduals of opposite sexes (dioecious). In aquatic animals
fertilization is often external, though internal f­ertilization
occurs in some species that live in fast moving water
(Fig. e8 A–C) (Leake, 1975).
 Invertebrates Chapter | 41 1046.e7

FIGURE e6  Examples of normal respiratory organs. (A) The normal book gill of an Atlantic horseshoe crab demonstrating the fine chitinous lamel-
lae, which increase gill surface area and permit oxygen exchange with the environment. (B) A nautilus gill demonstrating the folded lamellae on the gill
leaflets which increase surface area. (C) The papulae of a mottled sea star which are hollow, thin evaginations of the coelom that project from the body
wall surface. Gas exchange with the coelomic fluid occurs directly with the environment over the epidermis and internal coelomic epithelium. (D) The
normal book lung of a brown bark scorpion demonstrating the fine layers of chitin that increase surface area and permit oxygen exchange between the
hemolymph and the environment.
1046.e8 Pathology of Wildlife and Zoo Animals

FIGURE e8  Examples of normal reproductive organs. (A) The normal

FIGURE e7  Examples of normal digestive organs. (A) The glandular
pyloric stomach of a mottled sea star. (B) The hepatopancreas (digestive
gland) of an American lobster. (C) The digestive gland of an octopus.
ovary of a tarantula demonstrating folliculogenesis and vitellogenesis. (B)
The normal testis of a Puerto Rican white millipede demonstrating sperm
and spermatophore production. (C) The normal hermaphroditic gonadal
tubules in a bay sea scallop.

REFERENCES Leake, L.D., 1975. Comparative Histology: An Introduction to the Micro-

scopic Anatomy of Animals. Academic Press, New York.
Chapman, G., 1958. The hydrostatic skeletonin the invertebrates. Biol.
Mill, P.J., 1997. Invertebrate respiratory systems. Handbook of Physiology
Rev. 33, 338–371.
Comparative Physiology. Wiley-Blackwell, 1009-1096.
Foelix, R.F., 1996. Biology of Spiders. Oxford University Press, New York.
Ruppert, E.E., Fox, R.S., Barnes, R.D., 2004. Invertebrate
Larsen, E.H., Deaton, L.E., Onken, H., O’Donnell, M., Grosell, M.,
Zoology : A Functional Evolutionary Approach. Thomson-Brooks/
­Dantzler, W.H., Weihrauch, D., 2014. Osmoregulation and excretion.
Cole, Belment, CA.
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