Advances in Animal Science

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ADVANCES IN ANIMAL SCIENCE AND ZOOLOGY
ADVANCES IN ANIMAL SCIENCE

AND ZOOLOGY
VOLUME 11
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ADVANCES IN ANIMAL SCIENCE AND ZOOLOGY

AND ZOOLOGY
VOLUME 11
OWEN P. JENKINS
EDITOR

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CONTENTS
Preface vii
Chapter 1 Oropharyngeal Trichomonads in Wild Birds 1
María Teresa Gómez-Muñoz,
María del Carmen Martínez-Herrero,
José Sansano-Maestre
and María Magdalena Garijo-Toledo
Chapter 2 New Breeding Strategies
in Organic Dairy Farming 43
R. Rodríguez-Bermúdez, M. Miranda,
M. López-Alonso, J. Baudracco, R. Fouz,
I. Orjales and M. Garcia-Vaquero
Chapter 3 The Ecology and Conservation
of the West African Lion 73
Michael Campbell
Chapter 4 Girls of a Feather Flock Together:
Spatial Proximity in Adult Kin
in Greylag Geese (Anser anser) 111
Isabella B. R. Scheiber and Brigitte M. Weiß

vi Contents
Chapter 5 Estimating Associations between Amblyomma

Ticks and Wild Birds in a Neotropic Forest
from Survey Data 149
Ralph Maturano, Ronaldo R. Bastos,
Erik Daemon and João L. H. Faccini
Chapter 6 Cryptosporidiosis in Non-Human Primates 171
Sérgio Diniz Garcia,
Bruno Criado de Araújo Mendes,
Débora Barbosa Bruno,
Jaqueline da Silva Borégio, Marcela Dastre,
Vinícius Camarena Borges
and Katia Denise Saraiva Bresciani
Chapter 7 The Effect of Blueberries and Raspberries
on the Reproductive Behavior and Fecundity
of Drosophila Suzukii Females 189
Elizabeth Krohn, Jonathan Fingerut
and Scott McRobert
Chapter 8 Pheromone Communication Systems
in the Nasonia Species Complex 205
Jan Buellesbach
Chapter 9 Giardiasis in Birds 219
Elis Domingos Ferrari,
Bruno César Miranda Oliveira,
Mariele Fernanda da Cruz Panegossi,
Gisele Moraes dos Santos Reginaldo,
Walter Bertequini Nagata
and Katia Denise Saraiva Bresciani
Index 227

PREFACE
In this collection, the authors discuss oropharyngeal trichomonosis, a

disease that affects wild and domestic birds, summarizing the most
significant aspects of the disease: the biology, diagnosis and treatment
options. Recent scientific advances in the pathology, epidemiology and
control of the disease are also discussed. Following this, current animal
breeding strategies in organic farms are analyzed to determine the animal
breeds that could potentially benefit from different organic dairy farming
systems. In a subsequent review, a status assessment (based on a
comprehensive literature review) of the conservation issues of the West
African lion is conducted. The authors conclude that without an intensive
effort to rectify the factors for the extinction of the West African lion
(human population growth and urbanization, desertification, decline of
food sources, cattle herding, hunting and farming) and increased public
acknowledgement of this comparatively neglected lion subspecies, the
West African lion has a bleak future. This book includes a review of the
knowledge of extended family bonds between females in mammals and
birds, focusing on how waterfowl are particularly predisposed to female-
centered kin clans. A study is presented which investigates whether lineal
kin, i.e., mothers and their adult daughters, form long-term bonds in a well-
studied model organism within waterfowl, the greylag goose. The Atlantic
Forest biome is exhibited as one of the most biodiverse and threatened
biomes in the world. The authors conduct a quantitative analysis on the

viii Owen P. Jenkins
published data about parasitism by Amblyomma ticks on birds of the

Atlantic Forest, testing whether ecological and morphological variables of
the host are associated with parasitism by ticks. This compilation also
includes a review of Cryptosporidium, describing the history, taxonomy,
epidemiology cycle, clinical signs, diagnostic, treatment and prevention.
Next, exposure of Drosophila suzukii, a fruit fly species native to
Southeast Asia, to two fruits, blueberries and raspberries, is analyzed for
possible effects on sexual behavior and fecundity. The parasitoid jewel
wasp genus Nasonia is offered as an excellent model system to investigate
genetic, behavioral and ecological aspects of chemical communication.
Three main pheromone communication systems are discussed in light of
their respective chemical basis, hierarchical interplay, species-specificity
and potential contribution to prezygotic reproductive isolation. In closing,
the clinical signs of Giardia infection are discussed, including delayed
growth, dehydration, weight loss, and recurrent diarrhea. The occurrence
of Giardia spp. in birds from a particular site is studied to demonstrate the
extent of the disease, its specificity to its hosts, and its risk and
transmission potential.
Chapter 1 - Oropharyngeal trichomonosis is a disease that affects wild
and domestic birds. Some studies carried out in wildlife recovery centers
pointed it out as the main cause of entrance for birds of prey due to
infectious diseases. It causes small nodules and ulcers in the crop and other
locations of the upper digestive system of the animals. These small lesions
can coalesce and form large granulomas, which can provoke the death of
the animal by starvation. Trichomonas gallinae is considered the
etiological agent of the disease, a flagellated protozoon that is frequently
found in the oral cavity of columbiformes, which are considered the main
reservoir of the parasite. However, in the last decade, a great progress in
the molecular characterization of this and other protozoa has been reached,
and the number of genetic variants and even new species within the
trichomonads that inhabit the avian oropharynx has expanded. In this
review, the authors outline the latest descriptions of these parasites and
their host spectrum; more than 10 genetic variants or new species are
included. Although trichomonosis has been described in several groups of

Preface ix
birds, the higher impact is usually found on Accipitriformes,

Falconiformes and Strigiformes due to their predation or scavenging
habits. Psittaciformes and Passeriformes also show clinical signs of the
disease, and recently, several epidemic episodes of trichomonosis in
fringilids were described across Europe and North America. In addition,
chicks of endangered species like the Bonelli´s eagle (Aquila fasciata) are
frequently affected by the parasite, as several studies carried out in Spain
and nearby countries have proved. In this chapter, the authors summarize
the most important features of the disease, including the biology, the
diagnosis and treatment options. Additionally, the authors will describe the
recent scientific advances in the pathology, epidemiology and control of
the disease.
Chapter 2 - The selection of an appropriate breed in dairy farms will
have a huge influence on the animals’ welfare and production. This is
especially noticeable in animal production systems that aim to maximize
the use of on-farm resources (low-input production), i.e., organic farms.
The animal production in organic farming systems focuses on maximizing
the utilization of forage and improving the animals’ health and welfare,
while reducing the application of drugs routinely applied in the treatment
and prevention of diseases in the conventional farms. Thus, the selection of
animals adapted to this harsh farming conditions is essential for the success
of an organic farm. However, the current animal breeding strategies
adopted by the farmers do not differ between the intensive and the organic
animal production systems; i.e., the Holstein-Friesian is the most
commonly used breed in the organic and intensive dairy farms, despite the
poor production and adaptability of these animals to the organic production
systems. Recent studies showed that animals bred to produce high milk
yields in the conventional systems are not capable to adapt to pasture-
based systems. Cattle breeding strategies based on the selection of the
animals for functional traits, or the efficiency of the cows to use scarce
resources (inputs), could be a good strategy when selecting animals for
organic farms. This chapter analyzes the animal breeding strategies
currently performed in the organic farms, and discusses the novel strategies

x Owen P. Jenkins
and animal breeds that could potentially benefit different organic dairy
farming systems, including multifunctional farms.
Chapter 3 - The conservation of the lion (Panthera leo), a dominant
apex predator, indispensable ecological mainstay and popular wildlife
symbol, is one of the most important issues in current conservation biology
research and indeed is a relevant topic for other disciplines such as applied
ecology, biogeography and political ecology. The conservation issues for
the West African lion subspecies or variant (Panthera leo senegalensis or
Panthera leo leo) are particularly important, because the ecology and
status of this subspecies are less researched than those of the Southern,
Eastern, Asiatic and even extinct North African lions. The West African
lion is considered endangered and its changing populations, history, human
relations and current prospects are little known beyond presence data. This
chapter conducts a status assessment, based on a comprehensive literature
review, of the conservation issues of the West African lion. A key issue is
the lack of analysis of the human dimensions of lion presence in the
increasingly human dominated land cover of West Africa, and lack of
applications of relevant knowledge from studies of lions and other large
carnivores in other contexts and the multidisciplinary theoretical analyses
that could be applied to this issue. It is concluded that without an intensive
effort to rectify the factors for the rarity or extirpation of the West African
lion (extremely high human population growth and urbanization,
desertification, decline of food sources, cattle herding, hunting and
farming) and increased public acknowledgement of this comparatively
neglected lion subspecies, the West African lion has a bleak future. A
common, cross-border conservation policy among the West African
nations will be required, not only for lions but for prey species and human
land users.
Chapter 4 - In birds, the formation of stable social bonds beyond a
mated pair has only been reported in a few monogamous species with a
complex social system, such as in corvids. Also, in various species of
waterfowl, like swans, ducks and geese, such bonds are established not
only between pairs, but also within families, which may last far beyond
independence of offspring. Surprisingly, the story does not end here: Adult

Preface xi
females maintain bonds with their collateral female kin, i.e., sisters,
throughout life [1]. Sisters benefit particularly during the breeding season,
if their long-term bonding partners help in raising offspring, thereby
reaping benefits via kin selection. However, this does not explain the
persistence of these bonds outside the reproductive season. Whereas
organization into matrilines is a common pattern in group-living mammals,
female-centered clan structures are quite unusual for birds. Yet, the
exceptional characteristic features of waterfowl breeding systems, such as
early pair formation, female-biased philopatry, long-term monogamy, and
prosocial development of offspring, may explain the formation of long-
term adult bonds among females. In this chapter the authors will first
review the knowledge of extended family bonds between females in
mammals and birds and will then dwell on why, based on their breeding
system, waterfowl are particularly predisposed to female-centered kin
clans. Second, the authors will present a hitherto unpublished experiment
investigating whether lineal kin, i.e., mothers and their adult daughters,
form long-term bonds in a well-studied model organism within waterfowl,
the greylag goose (Anser anser). The authors found that female and male
greylag geese form long-term social bonds with their parents, measured as
close proximity. Long-term social bonds are prevalent in waterfowl and
stand, hence, in stark contrast to other birds. The authors finish this chapter
by proposing mechanisms enabling the special social structure of
waterfowl, which includes long-term social bonds.
Chapter 5 - The Atlantic Forest biome is one of the most biodiverse,
and threatened in the world. Most studies on bird parasitism by ticks in
Brazil have been carried out in forest fragments of this biome, and
primarily focused on pathogens the ticks carry than the relationships
between ticks and birds. Accordingly, in this study the authors conducted a
quantitative analysis, by means of multiple correspondence analysis
(MCA), on the published data about parasitism by Amblyomma ticks on
birds of the Atlantic Forest. The hypothesis tested was that ecological and
morphological variables of the host (habitat occupation, eating habits, site
of occurrence, mass and length) are associated with parasitism by ticks.
Based on 403 tick/bird associations the authors found that the most

xii Owen P. Jenkins
prevalent species associated with Atlantic Forest birds are Amblyomma

longirostre, Amblyomma nodosum, Amblyomma calcaratum, Amblyomma
aureolatum and Amblyomma parkeri. All these species showed association
with birds inhabiting the forest understory and midstory. In addition, both
immature stages of A. aureolatum and nymphs of A. calcaratum were
associated with birds with terrestrial habits. Regarding diets, the most
common association was with insectivorous birds, followed by frugivorous
and nectarivorous birds. All tick species were associated with forest border
birds, while A. aureolatum was also associated with birds which forage in
open areas. As for bird length and mass, those with up to 40 cm and 40 g
are the most associated with tick larvae and nymphs. In terms of hosts, the
Thraupidae, Thamnophilidae and Furnariidae families were the most
parasitized.
Chapter 6 - The cryptosporidiosis is known since 1907, but there is no
much data compilation about it. 152 animal species have been described
with this parasite. With all the researchers found, it is necessary a review
about the disease, for a great understanding about the Cryptosporidium.
This research has the purpose to show all the information found in a
systematic review of articles about cryptosporidiosis in non-human
primates. For a better understanding, this article describe the historic,
taxonomy, epidemiology cycle, clinical signs, diagnostic, treatment and
prevention.
Chapter 7 - Drosophila suzukii, a fruit fly species native to Southeast
Asia, has become an invasive species and threat to agricultural production
in many parts of Europe and North America. Differing from other
Drosophilida, D. suzukii’s impact as a formidable agricultural pest, stems
from the ability of the females to use ripe fruit that is still on the vine as
sites for oviposition. Developing a greater understanding of the lifecycle
and behavior of D. suzukii may be key in controlling this pest. In this
study, exposure to two fruits, blueberries and raspberries, was analyzed for
possible effects on sexual behavior and fecundity of D. suzukii females.
Neither blueberries nor raspberries had significant effects on copulation
frequency, latency to copulation, nor copulation duration. However,
females exposed to whole blueberries or whole raspberries both prior to,

Preface xiii
and after, mating yielded significantly more offspring than females from
other housing conditions. These results add to the authors’ understanding
of the life history of a major agricultural pest.
Chapter 8 - The parasitoid jewel wasp genus Nasonia, encompassing
four species, has proven to be an excellent model system to investigate
genetic, behavioral and ecological aspects of chemical communication. In
recent years, it has become one of the best-investigated hymenopteran taxa
concerning pheromone-based sexual signaling, mate choice and assortative
mating behavior. A hierarchy of three main pheromone communication
systems can be clearly differentiated in Nasonia:
 A long-range pheromone produced in the male abdomen to attract

virgin females from longer distances.
 Female cuticular hydrocarbons that act as short-range contact
pheromones eliciting male arrestment, courtship and copulation
behavior.
 A male-produced mouthpart pheromone released during courtship,
hypothesized to induce female receptivity, but so far only
demonstrated to terminate the female response to the long-range
sex attractant.
These three different systems are discussed in light of their respective

chemical basis, hierarchical interplay, species-specificity and potential
contribution to prezygotic reproductive isolation.
Chapter 9 - The protozoan Giardia spp. is a flagellate intestinal
parasite and six species are recognized, being G. agilis that affects
amphibians, G. psittaci and G. ardeae that infect birds, G. microti and G.
muris that can be present in rodents and G. duodenalis that can cause
disease in humans and some domestic animals, according to their
assemblages, ranging from A to H. The individual can be infected by
consuming water and foods with the presence of cysts. The frequently
clinical signs seen in the Giardia infection are delayed growth,
dehydration, weight loss, and recurrent diarrhea. The occurrence of
Giardia spp. in birds from a particular site demonstrates the extent of the
disease, its specificity to its hosts, its risk and transmission potential.

In: Advances in Animal Science and Zoology ISBN: 978-1-53613-214-4
Editor: Owen P. Jenkins © 2018 Nova Science Publishers, Inc.
Chapter 1
OROPHARYNGEAL TRICHOMONADS
IN WILD BIRDS
María Teresa Gómez-Muñoz1,*, DVM, PhD,

María del Carmen Martínez-Herrero2,†, DVM, PhD,
José Sansano-Maestre3,‡, DVM, PhD
and María Magdalena Garijo-Toledo2,§, DVM, PhD
1
Universidad Complutense de Madrid. Facultad de Veterinaria,
Departamento de Sanidad Animal, Madrid, Spain
2
Universidad CEU Cardenal Herrera. Instituto de Ciencias Biomédicas,
Facultad de Veterinaria, Departamento de Producción y Sanidad Animal,
Salud Pública Veterinaria y Ciencia y Tecnología de los Alimentos,
Valencia, Spain
3
Universidad Católica de Valencia, Facultad de Veterinaria y Ciencias
Experimentales, Departamento de Producción Animal y Salud Pública,
Valencia, Spain
*
Email: mariateg@ucm.es.
†
Email: maria.martinez1@uchceu.es.
‡
Email: jose.sansano@ucv.es.
§
Email: marilena@uchceu.es.

2 M. T. Gómez-Muñoz, M. del C. Martínez-Herrero et al.
ABSTRACT
Oropharyngeal trichomonosis is a disease that affects wild and

domestic birds. Some studies carried out in wildlife recovery centers
pointed it out as the main cause of entrance for birds of prey due to
infectious diseases. It causes small nodules and ulcers in the crop and
other locations of the upper digestive system of the animals. These small
lesions can coalesce and form large granulomas, which can provoke the
death of the animal by starvation. Trichomonas gallinae is considered the
etiological agent of the disease, a flagellated protozoon that is frequently
found in the oral cavity of columbiformes, which are considered the main
reservoir of the parasite. However, in the last decade, a great progress in
the molecular characterization of this and other protozoa has been
reached, and the number of genetic variants and even new species within
the trichomonads that inhabit the avian oropharynx has expanded. In this
review, we outline the latest descriptions of these parasites and their host
spectrum; more than 10 genetic variants or new species are included.
Although trichomonosis has been described in several groups of birds, the
higher impact is usually found on Accipitriformes, Falconiformes and
Strigiformes due to their predation or scavenging habits. Psittaciformes
and Passeriformes also show clinical signs of the disease, and recently,
several epidemic episodes of trichomonosis in fringilids were described
across Europe and North America. In addition, chicks of endangered
species like the Bonelli´s eagle (Aquila fasciata) are frequently affected
by the parasite, as several studies carried out in Spain and nearby
countries have proved. In this chapter, we summarize the most important
features of the disease, including the biology, the diagnosis and treatment
options. Additionally, we will describe the recent scientific advances in
the pathology, epidemiology and control of the disease.
1. AVIAN OROPHARYNGEAL TRICHOMONOSIS
Avian oropharyngeal trichomonosis is a parasitic disease that has been

known for centuries. Although it has been described mainly in birds, there
are some references about the presence of the parasitic process affecting
even dinosaurs. First documented descriptions were found in falconry

Oropharyngeal Trichomonads in Wild Birds 3
books, where the disease is explained as the appearance of typical lesions

on birds of prey. For example, there is a book on falconry called The
Hunting of Birds, from the 14th century, and the London’s Turbervile book
of 1575 in which the typical lesions are described.
At first, the etiological agent was named Cercomonas gallinae
(Rivolta, 1878). Later, in the year 1938, its taxonomic classification was
reviewed by Dr. Robert M. Stabler, from the University of Colorado. He is
one of the authors who has contributed most to knowledge about avian
trichomonosis through numerous scientific publications.
Since then, other scientists, especially in the United States, have been
responsible for studying different aspects related with pathogen
mechanisms, morphology, immunology, biology, and protozoan treatment.
Some of the scientists to be highlighted are: Dr. Richard M. Kocan, from
the US Bureau of Sport Fisheries and Wildlife, Patuxent wildlife research
center, Laurel, Maryland; Dr. Bronislaw M. Honigberg, from the
University of Massachusetts; Dr. Norman D. Levine from the University of
Illinois and Dr. Lionel G. Warren, from Rice University, Houston, Texas,
among others.
Trichomonosis is one of the most relevant infectious diseases among
wild birds, both from urban (Boal et al., 1998; Stenkal et al., 2013) and
rural environments. Raptors are the most frequently reported with the
disease in the studies carried out. Wendell et al., in 2002, analyzed the
causes of admission to the Veterinary Hospital of Colorado University
(USA) in a retrospective study from 1995 to 1998. Trichomonosis
accounted for 44% among the infectious diseases in raptors and it was the
second cause of admission in importance after trauma. In a different study
carried out in Spain, the authors found the same percentage of
trichomonosis among the infectious diseases in raptors, being Strigiformes
and Falconiformes the most affected (Molina-López et al., 2011).
Remarkably, the disease has been considered emergent in passeriformes
since 2005 (Pennycot et al., 2005) and it has spread out through Europe
and America, being a cause of population decline.

2. THE AGENT: TAXONOMY, MORPHOLOGY,

LIFE CYCLE AND TRANSMISSION
Traditionally, the etiological agent of avian oropharyngeal

trichomonosis was considered T. gallinae. However, in the last decade
several genetic variants or new species have been described. In this
section, we describe T. gallinae, since other species and variants will be
discussed in a different section.
2.1. Taxonomy
Trichomonas gallinae (Rivolta, 1878) belongs to the family

Trichomonadidae, order Trichomonadida, class Trichomonadea, phylum
Parabasalia (Brugerolle and Lee, 2000; Cepicka et al., 2010):
Empire Biota
Domain Eukaryotes (Chatton, 1925)
Phylum Parabasalia (Honigberg, 1973)
Class Trichomonadea (Cepicka, 2010)
Order Trichomonadida (Kirby, 1947)
Family Trichomonadidae (Chalmers and Pekkola, 1918)
Subfamily Trichomonadinae (Chalmers and Pekkola, 1918)
Genus Trichomonas (Donné, 1836)
Species Trichomonas gallinae (Rivolta, 1878)
All trichomonads are unicellular flagellated protists classified in the

supergroup Excavata, which includes not only parasitic organisms, but also
free-living and commensal ones (Sleigh, 1991). The species grouped in the
order Trichomonadida are anaerobic and present a complex cytoskeleton,
three to five flagella at the apical pole, one recurrent flagellum, axostyle,
costa and pelta (Mehlhorn et al., 2009).

T. gallinae is classified in the subfamily Trichomonadinae, one of the

two that represent the family Trichomonadidae. Other parasites of human
and veterinary importance, like Trichomonas vaginalis or Tetratri-
chomonas gallinarum are also included in this subfamily. As these
protozoa lack mitochondria, they have an alternative route for ATP
synthesis that is fulfilled by hydrogenosomes, cytosolic organelles that do
not require an oxygen influx.
The genus name, Trichomonas, derives from the Greek word “thrix-”
meaning hair and “-monas” that indicates a single unit, regarding their one
cell structure.
2.2. Morphology
The parasitic stage of T. gallinae is called “trophozoite” (from the

Greek word “troph-” meaning feed and “–zoite”, from “–zoon”, animal). It
has an average cell size of 7 to 11 µm and its morphology can vary from
ovoid to pyriform or spherical. It has a great elasticity that allows frequent
transitions into these different forms (Borror, 1988; Mehlhorn et al., 2009;
Martínez-Díaz et al., 2015) (Figure 1).
The trophozoite presents five flagella arising from the periflagellar
canal, which is located at the apical pole of the cell. Four of them, the
anterior flagella, measure from 11 to 18 µm. The fifth one, called the
recurrent flagellum, is attached to the cell membrane in all its extension.
This flagellum accompanies the undulating membrane, which measures
between 2/3 and 3/4 of the cell’s length. Basal bodies are located at the
origin of each flagellum, and present a typical structure of a central
centriole and external microtubule triplets (Mehlhorn et al., 2009).
It presents an oval nucleus whose mean measures are 2.5-3.4 µm
length and 2.2 µm width and is found next to the basal bodies of the
flagella (Mehlhorn et al., 2009; Martínez-Díaz et al., 2015). Near the
proximal pole and close to the nucleus, a curved microtubule complex
called the pelta is located (Honigberg and Brugerolle, 1990; Cepicka et al.,
2010). The costa is another long dimension cytoskeleton structure, located

below the undulating membrane, with filaments arranged in a herringbone

pattern.
Figure 1. Schematic representation of a T. gallinae trophozoite. AF: anterior flagella,

RF: recurrent flagellum, UM: undulating membrane, N: nucleus, AX: axostyle.
Original drawing by Mónica Caridad Benito Torrecilla.
Finally, the axostyle is a characteristic support structure with its final

segment extending from the posterior pole of the cell. It is composed by a
single row of microtubules that extend throughout the central axis of the
trophozoite, starting from the nucleus up to the end of the cell and
extending beyond (Stabler, 1941).
For the species T. gallinae, the development of a cystic wall to
generate resistance forms has not been described. However, electron
microscopy studies have revealed a morphotype called pseudocyst. Its

morphology can be oval or spherical and has no flagella or undulating

membrane. This form appears in in vitro conditions, and is related to
stressful situations, such as an inadequate temperature. Pseudocysts have
been found in intestinal trichomonads of reptiles, rodents, birds and
amphibians (Mattern et al., 1973; Stachan et al., 1984; Friedhoff et al.,
1991). In addition, their formation is documented in parasitic species of the
urogenital tract of bovids, such as Tritrichomonas foetus and humans, such
as T. vaginalis. These resistant forms adhere to the host tissue at a higher
rate than the trophozoite stage, which indicates that they could have an
important effect on the transmission and the infective capacity of the
parasite (Mariante et al., 2004). In addition, their transition to the
trophozoite stage has been documented, being also competent to multiply
(Granger et al., 2000; Pereira-Neves et al., 2003; Castro et al., 2016).
Analysis through electronic microscopy has also allowed for a more
precise study of the morphology of the trophozoite (Figure 2). It has a complex
cytoskeleton composed of four types of kinetosome rootlets involved in the
trophozoite morphology, including the structures of the costa, pelta and
parabasal filaments. In addition, some filamentous structures have been
described in cytoplasmic granules associated with the axostyle and costa.
Figure 2. Trophozoites (left) and pseudocyst stage (right) of T. gallinae. AX: axostyle,
AF: anterior flagella, UM: undulating membrane. Original drawing by Mónica Caridad
Benito Torrecilla, adapted from Tasca and De Carli (2003).

2.3. Life Cycle and Transmission
T. gallinae has a direct life cycle, with trophozoites multiplying by

longitudinal binary fission, one cell will divide to form two new ones
(Stabler, 1941). Their preferred location is the mucosa of the upper
digestive tract, from the oropharyngeal cavity and crop to the proximal
esophagus. Nevertheless, affection of internal organs such as liver, lungs,
pericardium, air sacs and pancreas, has also been reported in certain strains
of the parasite. Indeed, some strains target particular tissues, namely,
Jones’ Barn and Eiberg strains are hepatotrophic, while Mirza strain is
cephalotrophic (head sinuses, orbital regions, brain and neck) (Stabler and
Engley, 1946; Jaquette, 1950; Pérez-Mesa et al., 1961; Kocan and Herman,
1971; Narcisi et al., 1991). However, the Jones’ Barn strain primary
showed tropism the lungs instead of the liver of mourning doves (Zenaida
macroura) and rock pigeons (Columba livia) in experimental infections.
Non pathogenic strains, like Lahore and Stabler-gallinae (SG), have also
been described (Honigberg et al., 1964; Kocan, 1969a; Nadler and
Honigberg, 1988).
The primary route of transmission is by direct contact with the saliva
of an infected bird, although contaminated food and water also act as
sources of infection (Kocan, 1969b; Kietzmann, 1990; Bunbury et al.,
2007). Indeed, experimental transmission of the parasite through the
ingestion of contaminated food has been proved in columbiformes. Recent
studies have showed that bird baths may act as potential vehicles with
viable trophozoites for at least 16 hours (Purple and Gerhold, 2015; Purple
et al., 2015).
Contaminated carcasses also remain infective for at least 48 hours after
death, acting as a reservoir for ornitophagous and scavenging birds (Erwin
et al., 2000). Moist grains are also able to maintain the parasite’s viability
for at least five hours (Kocan, 1969b).
Regarding the number of trophozoites needed to initiate the infection,
it seems that only one trophozoite of the Jones’ Barn strain is enough to
develop the disease and cause death in pigeons in less than 15 days post-
infection (Stabler and Kihara, 1954).

3. NEW SPECIES AND VARIANTS OF

OROPHARYNGEAL TRICHOMONADS IN BIRDS
3.1. New Species: Trichomonas stableri
3.1.1. Introduction
In 2011-2012 a new species was described in USA: Trichomonas
stableri (Girard et al., 2014a). Named in honor of Professor Robert M.
Stabler (1904-1985), who was one of the great pioneers of avian
trichomonosis research. This new species was isolated from adults of
Pacific Coast band-tailed pigeons (Patagioenas fasciata monilis) in
California. According to the authors, the pathogenic character of the
protozoan was equal to T. gallinae, as lesions were located in the
oropharyngeal cavity, esophagus and lungs, with healthy carriers found as
well. Coinfections with T. gallinae were also detected.
Figure 3. From left to right: T. gallinae, slender form of T. stableri and rounded form
of T. stableri. Original drawing by Mónica Caridad Benito Torrecilla, adapted from
Girard et al., 2014a.

Biological factors related to the life cycle of the birds, increased their
susceptibility to disease. The population studied in California performs a
migration into Alaska and was subjected to game pressure, being
recognized in decline since 1966 (Keppie and Braun, 2000). T. stableri was
identified during mortality events that occurred in winter and early spring
migration and in one breeding bird without clinical signs.
3.1.2. Host Spectrum

Up to date, this species has only been reported on Pacific Coast band-
tailed pigeons.
3.1.3. Morphology
T. stableri has four anterior flagella with the common external
structures that characterize T. gallinae: axostyle projection in the caudal
pole of the cell and a recurrent flagellum that forms the undulating
membrane. Two morphotypes were described: the slender and the rounded
form (Figure 3). Although T. gallinae trophozoites have an inconstant
shape, with a particular grade of pleomorphism, are generally defined with
a pyriform, ovoid or spherical form (Stabler 1941; Tasca and De Carli,
2003; Mehlhorn et al., 2009; Girard et al., 2014a; Martínez-Díaz et al.,
2015).
3.1.4. Phylogenetic Analysis

Authors reported that T. stableri isolates had 92.1-93.4% nucleotide
similarity with T. gallinae strains for the ITS1/5.8S/ITS2 region, while
99.6% was obtained in comparison with T. vaginalis strains (Girard et al.,
2014a).
3.1.5. Co-Existence with T. gallinae

The band-tailed pigeon population of California, USA, was carefully
studied to unravel the epidemic pattern of avian trichomonosis. Findings
indicated that during non-epidemic periods, birds harbored mainly T.
gallinae Fe-hydrogenase subtype A2, whereas T. gallinae Fe-hydrogenase

subtype A1, T. stableri and Tritrichomonas blagburni n. sp.-like were

found in lower abundance. In contrast, during the winter or early spring
migration, periods where birds suffered from higher stress levels and
outbreaks occurred, T. gallinae Fe-hydrogenase subtype A2 and T. stableri
were isolated (Girard et al., 2014b). Infection was detected in 96% of the
birds affected during mortality events of trichomonosis. The epidemiology
of the disease was similar to the outbreak of a wintering woodpigeon
population reported from Spain (Villanúa et al., 2006).
This parasitic disease was pointed out as another key factor to monitor
during the study of the population dynamics, due to their steadily decline.
Additionally, the detection of the causative agent of the European finch
trichomonosis epidemic (T. gallinae Fe-hydrogenase subtype A1) in
healthy carriers also raised the concerns about the potential transmission to
other bird species.
3.2. New Species: Trichomonas gypaetinii
3.2.1. Introduction
As a consequence of an epidemiological study of oropharyngeal
trichomonads carried out on wild birds admitted to wildlife recovery
centers from Spain, a new species of trichomonad was described from
cinereous (Aegypius monachus) and Egyptian vultures (Neophron
percnopterus, Martínez-Díaz et al., 2015). Its name was assigned as T.
gypaetinii, due to its presence in birds from the subfamily Gypaetinae (Old
World vultures).
3.2.2. Host Spectrum

T. gypaetinii has been reported from cinereous vultures, Egyptian
vultures, bearded vultures (Gypaetus barbatus; Grabensteiner et al., 2010)
and one American bald eagle (Haliaeetus leucocephalus; Kelly-Clark et
al., 2013).

Figure 4. T. gypaetinii and T. gallinae trophozoites. Left: T. gypaetiniii. Right: T.

gallinae. Scale bars of 10 µm. Reproduced with permission from Martínez-Díaz et al.,
2015.
3.2.3. Morphology
Like T. gallinae, with an ovoid to pyriform cell body shape, smaller
size (mean measures 8x6 µm for T. gypaetinii vs. 11x7 µm for T. gallinae),
and a longer and prominent axostyle projection (Martínez-Díaz et al.,
2015; Mehlhorn et al., 2009) (Figure 4).
3.2.4. Phylogenetic Analysis

T. gypaetinii shared 90-91% nucleotide homology with the
ITS1/5.8S/ITS2 regions of T. gallinae isolates. This new organism was
more similar to T. stableri and T. vaginalis strains (up to 97% for the
previously cited genetic region) than to T. gallinae (Martínez-Díaz et al.,
2015).
3.3. New Variants
Since 2001, new variants of oropharyngeal trichomonads from wild

birds with more genetic homology to other trichomonads than to T.
gallinae had been characterized (Table 1). The unraveling of the etiologic
complex of avian trichomonosis helps to understand the epidemiology of
the disease. These latest investigations denote that the boundaries between
new species or subspecies are difficult to establish, with multiple genetic

and morphological variables that need to be considered. In any case,

further research will determine the extent and host distribution of these
new variants or organisms.
Table 1. New species and variants of oropharyngeal trichomonads

detected in birds
Trichomonad new Host Country Reference

species/variants
T. vaginalis-like Common ground-doves USA Gerhold et al.,
(Columba passerina) 2008
Trichomonas sp. Mockingbirds (Mimus Anderson et al.,
polyglottos) 2009
T. vaginalis-like Bearded vultures Czech Grabensteiner et
(later T. gypaetinii) Republic al., 2010
T. tenax-like Racing pigeons Austria
T. vaginalis-like Striped owls (Asio clamator) Brazil Ecco et al., 2012
Simplicomonas sp. Green-winged saltators
(Saltator similis)
T. vaginalis-like Bald eagles (Haliaeetus Canada Kelly-Clark et al.,
(later T. gypaetinii) leucocephalus) 2013
T. tenax-like European turtle doves, UK Lennon et al.,
woodpigeons and stock doves 2013
(Columba oenas)
T. stableri Pacific Coast band-tailed USA Girard et al.,
pigeons 2014a
T. blagburni-like Pacific Coast band-tailed Girard et al.,
pigeons 2014b
T. vaginalis-like Egyptian vultures Spain Martínez-Herrero
(later T. gypaetinii) et al., 2014
T. canistomae-like European turtle doves
(Streptopelia turtur) and
goshawks (Accipiter gentilis)
T. gypaetinii Cinereous and Egyptian Martínez-Díaz
vultures et al., 2015
Trichomonas sp. European turtle doves UK Stockdale et al.,
2015

4. PRESENCE IN DIFFERENT GROUPS OF BIRDS
4.1. Order Accipitriformes
Diurnal raptors constitute one of the most affected groups of birds, due
to their contact with columbiformes, the reservoir hosts of the protozoan,
either as a food resource or at water stations (Purple et al., 2015). Avian
trichomonosis would probably be a major infectious disease for this group
of birds since, probably, prehistoric times (Wolff et al., 2009).
Ornithophagous birds of prey, especially those that include
columbiformes on their diets, are at a higher risk of exposure and infection.
For instance, Wieliczko et al., (2003) found a 100% infection rate in
goshawk nestlings of Poland, and Krone et al., (2005) found a 65.1%
prevalence in Berlin’s nests with a low rate of lesions. Also, 85% of
Cooper’s hawk (Accipiter cooperii) nestlings were infected in Arizona
(Urban et al., 2014). Later, Martínez-Herrero et al., (2014) reported that
ornithophagous raptors were at a lower risk of suffering from avian
trichomonosis in comparison with non-strict ornithophagous ones,
probably due to a host-parasite adaptation.
Studies on wild, free-living raptors are still scarce and surveillance
efforts are restricted mostly to threatened species. For example, nestlings
of Bonelli’s eagle (Aquila fasciata), an endangered species of the western
Mediterranean basin, had 36% prevalence in one study of Spain (Real et
al., 2000) and 42.9% in Southern Portugal (Höfle et al., 2000). Two
publications highlighted that avian trichomonosis was the primary cause of
admission for birds of prey due to infectious diseases in wildlife recovery
centers in retrospective studies (Wendell et al., 2002; Molina-López et al.,
2011) while Martínez-Herrero et al., (2014) found that 11.4% of predator
birds admitted in several wildlife hospitals were infected.
Koyama et al., (1971) published the findings of T. gallinae in secretary
birds (Sagittarius serpentarius), the only report on the species up to the
current authors’ knowledge.

4.2. Order Anseriformes
Descriptions of affected birds of this order are minimal. There is one

report from farmed ducks that suffered from respiratory and intestinal
involvement, with inflammation of the infraorbital sinuses, nasal cavity
and trachea, with mucofibrino-purulent discharge that resembled the
disease (Tsai et al., 1997). However, Tetratrichomonas anatis was
determined as the etiological agent with no further genetic characterization.
4.3. Order Columbiformes
Different levels of prevalence are found in the oldest reservoir host of

T. gallinae: birds from the Order Columbiformes. For instance, McKeon et
al., (1997) described 46% of infection in Senegal doves (Spilopelia
senegalensis) and Lennon et al., (2013) obtained 86% prevalence in
European turtle doves (Streptopelia turtur) and Eurasian collared doves
(Streptopelia decaocto). In contrast, 5.6% of infected mourning doves
(Zenaida macroura) were reported by Schulz et al., (2005). Obviously,
local and seasonal variations occur, but infection in columbiformes is
described as endemic. Indeed, this group of birds is thought to be
responsible of the worldwide distribution of the protozoan (revised in
Forrester and Foster, 2008).
Epidemics with high mortality and morbidity that occur at periodic
time intervals have been described in some species. This is the case of
Pacific Coast band-tailed pigeons in USA (Rogers et al., 2016a). The
species was monitored in 1951 (Stabler) and lately, mortality events have
been increasing. The negative effects of avian trichomonosis on the
population dynamics have raised the concerns about the transmission of
these new species or variants of oropharyngeal trichomonads among wild
birds. Seasonality is also another factor that is frequently present in avian
trichomonosis episodes of wild columbiformes. Some examples are: the
massive mortality reported by Höfle et al., (2004) in a wintering
population of woodpigeons (Columba palumbus) in Spain, the highest

prevalence during winter and summer in Spanish rock pigeons (Columba

livia) by Sansano-Maestre et al., (2009) or with mild temperatures and low
rainfall in the case of Mauritian columbiformes (Bunbury et al., 2007). In
this last case, the endemic species was threatened by this infectious disease
that increased their risk of extinction.
4.4. Order Falconiformes
This group of birds is commonly affected of trichomonosis due to their

predatory habits. The ancient falconry technique implied that the contact
with the protozoan might occur with high frequency. Indeed, Samour and
Naldo (2003) diagnosed 393 of 7,085 cases, being 5.5% of the patients
from the Falcon Specialist Hospital of Saudi Arabia.
In Spain, there are two studies from admissions to wildlife recovery
centers. Sansano-Maestre et al., (2009) determined 12.5% prevalence in
peregrine falcons (Falco peregrinus) and 10.3% in common kestrels
(Falco tinnunculus). Later, Martínez-Herrero et al., (2014) reported 4.2%
and 14.2% infection rates in the same hosts.
Besides, captive raptor breeding centers would be at risk of infection if
a proper preventive management is not implemented (Forbes, 2016).
Several measures, like control of food and water sources and avoidance of
contact with wild birds, in addition to an individual proper diagnosis, are
required (Kubiak and Forbes, 2016). In fact, Martínez-Herrero et al.,
(2014) found 4.4% prevalence in a lesser kestrel (Falco naumanni) captive
breeding center, with animals sub-clinically infected, without lesions and
normal feeding behavior.
4.5. Order Galliformes
The name of T. gallinae derives from this group of birds, as the first
description of this parasitic infection was made on them (Rivolta, 1878)
although it is not commonly found in this type of birds. Poultry and turkeys

could also suffer the disease, with studies reported from industrial
production (Davidson et al., 1985; Willoughby et al., 1995; Mirzaei et al.,
2016). It seems a rare affection of wild gallinaceous birds.
4.6. Order Gruiformes
Bailey et al., (1996) reported avian trichomonosis in captive rufous-

crested bustards (Eupodotis ruficrista). Columbiformes were the likely
source of the infection, due to the uncontrolled access to the food and
water farm supplies. The same etiology was determined in houbara
bustards (Chlamydotis undulata) and Kori bustards (Ardeotis kori) with
caseous inflammatory lesions at the oropharynx (Silvanose et al., 1998).
4.7. Order Passeriformes
A notable importance and attention has been driven during recent years
to avian trichomonosis in this group of birds. The disease was normally
rare in wild passeriformes, although in 2002 an outbreak in house finches
(Carpodacus mexicanus) and house sparrows (Passer domesticus) was
reported in the USA (revised in Forrester and Foster, 2008). In 2009,
Anderson et al., informed about positive cases in house finches, several
corvids species (scrub jays-Aphelocoma californica, crows-Corvus
brachyrhynchos and ravens-Corvus corax) and mockingbirds attended in a
wildlife recovery center of California. Moreover, in 2010 Anderson et al.,
published that avian trichomonosis reached the level of emerging disease
for house finches. Other reports were informing about the disease in
Canadian finches: American goldfinches (Carduelis tristis) and purple
finches (Carpodacus purpureus; Forzán et al., 2010).
In the UK this group of birds suffered a massive epizootic of the
disease (Pennycott et al., 2005; Lawson et al., 2006; Duff et al., 2007;
Robinson et al., 2010). Carcasses were recovered from gardens and
submitted for examination. A 35% and 21% decrease was observed in the

population of greenfinches (Chloris chloris) and chaffinches (Fringilla

coelebs), respectively, during next years. The first cases occurred in 2005-
06 and extended to European countries in subsequent years: Sweden,
Norway, Finland, France, Austria and Slovenia (Neimanis et al., 2010;
Gourlay et al., 2011; Ganas et al., 2014). Avian trichomonosis was
determined as a novel emergent disease for wild birds, with unknown
consequences in the population dynamics of raptor and other sympatric
bird species (Robinson et al., 2010; Chi et al., 2013; Zu Ermgassen et al.,
2016).
The likely route of infection for passeriformes was determined to be,
mainly, through the ingestion of contaminated food or water in areas
shared with columbiformes (Robinson et al., 2010).
4.8. Order Psittaciformes
Mostly psittaciformes kept as pets are reported with cases of avian

trichomonosis. Lumeij (1994) informed about cases in Amazon parakeets
(Amazona spp.) and conure species such as Aratinga parakeets (Aratinga
spp.). The popular budgerigars (Melopsittacus undulatus) were the subject
of an investigation at their native country, Australia. Researchers stated
11.4% prevalence from captive flocks (McKeon et al., 1997). Later, an
exhaustive revision of clinical cases was done by Park (2011). This author
described the disease from 146 psittaciformes, including budgerigars,
rainbow lorikeets (Trichoglossus haematodus), purple-crowned lorikeets
(Glossopsitta porphyrocephala) and cockatiels (Nymphicus hollandicus).
4.9. Order Strigiformes
Nocturnal birds of prey are another frequently affected group of birds.

Obviously, the diet of the species will determine the likelihood of the
infection. Several studies have described the lesions and pathology of fatal
cases (Tanabe, 1925; Jessup, 1980; Schulz, 1986; Pokras et al., 1993;

Sansano-Maestre et al., 2009; Ecco et al., 2012; Martínez-Herrero et al.,

2014; Rogers et al., 2016b).
In USA, Schulz (1986) investigated the occurrence of the parasite
among barn owls (Tyto alba) and stated that trichomonosis was the most
prevalent infectious disorder. In Spain, 14% prevalence was reported from
a wildlife recovery center (Sansano-Maestre et al., 2009). The parasite has
been recovered from even Eurasian scops-owls (Otus scops), a 16-20 cm
body-length species of 60-135 weight grams, with a mainly insectivorous
diet (Martínez-Herrero et al., 2014).
4.10. Other Orders with Sporadic Reports
In 1938, Hees described a case in a gull (Order Charadriiformes) in

northern UK (revised in Forrester and Foster, 2008).
5. CLINICAL SIGNS AND LESIONS
Clinical presentation of avian trichomonosis usually consists in a

nonspecific symptomatology. Birds appear in a low body condition, with
ruffled feathers and lethargic behavior, anorexia, reluctance to fly and
depression (Figure 5). Feathers around oral cavity and nostrils may appear
stuck to the beak as result of regurgitation. Diarrhea, when present, is often
characterized by yellow pasty stools. Some animals may display
respiratory signs such as labored breathing, sinusitis and nasal discharge
(Krone et al., 2005;Anderson et al., 2010; Neimanis et al., 2010; Madani et
al., 2015; Forzán et al., 2016).
Lesions of avian trichomonosis are usually restricted to upper digestive
system, including oropharyngeal cavity, esophagus and crop. The absence
of cystic forms of the trophozoite prevents the possibility of resistance to
the acidic medium of the proventriculus and thus, the digestive tract below
is rarely affected. However, infection of upper and low respiratory airways

is possible, and could represent a diagnostic challenge for the veterinarian

(Samour and Naldo, 2001).
The formation of yellow-white nodules and plaques in the oral cavity
are characteristics of avian trichomonosis but are not pathognomonic
lesions. Vitamin A deficiency, candidiosis, poxvirus infection,
aspergillosis, capillariosis and bacterial stomatitis might display similar
gross appearance.
Figure 5. Clinical signs of avian oral trichomonosis. Left: stuck feathers around nostrils
and oral cavity in a common kestrel (Falco tinnunculus). Right: reluctance to fly and
apathy in a Eurasian collared dove (Streptopelia decaocto).
Lesions grow in a few days since the infection is established. At first,

trophozoites attach to the epithelial cells inducing a damage, which leads to
a separation and removal of the cells. The disruption of the epithelial layer
favors the entrance of trichomonads and opportunistic bacteria to deeper
epithelial layers. The cellular reaction of the immune system of the bird
aids in the formation of an early stage granuloma.
Initial oral lesions are white, ulcerative and are located at the
oropharynx (Krone et al., 2005). As the disease progresses, multiple new
ulcerated areas may appear and coalesce. The infiltration of inflammatory
cells (plasma cells and macrophages) surrounding the affected area causes
the formation of caseo-necrotic granulomas. Chronic granulomas usually
are larger than the tracheal opening, show a characteristic yellow
coloration and may spread to the esophagus and crop (Figure 6) (Krone et
al., 2005; Anderson et al., 2010; Borji et al., 2011; Ecco et al., 2012;
Rogers et al., 2016). In rare cases, granulomas may extend to the choanae,

sinuses, and inner ear as well as the bone at the base of the skull (Ecco et
al., 2012; Kunca et al., 2015; Rogers et al., 2016). Some of these
granulomas can eventually block the oral opening and prevent the intake of
food and water or even cause the death by asphyxia or suffocation (Real et
al., 2000; Samour and Naldo, 2001). These gross findings appear
frequently in raptors and pigeons with chronic trichomonosis. Passerines
rarely present these macroscopic lesions in the lumen of the oropharynx,
which difficult the presumptive diagnosis. Domestic pigeons with
macroscopic oral granulomas frequently sustain lesions in other organs
such as trachea, lungs, liver and kidney (Borji et al., 2011; El-Khatam et
al., 2016).
Histologically there is a generalized congestion and edema of affected
tissues. Yellow granulomas correspond with caseous necrosis areas and
surrounding tissues may develop coagulative necrosis. Passeriformes
frequently develop an epithelial hyperplasia of the esophagus and crop
with the consequent thickening of the mucosa (Anderson et al., 2010). The
epithelium has multifocal areas of lymphoplasmacytic, heterophilic,
granulomatous or necrotizing esophagitis associated with bacteria (Ecco et
al., 2012; Girard et al., 2014a). Similar lesions are observed in the
epithelium of the trachea (Borji et al., 2011; Madani et al., 2015; El-
Khatam et al., 2016).
Figure 6. Yellow-white nodules and plaques in the oral cavity of a common kestrel
(Falco tinnunculus) and a Bonelli’s eagle (Aquila fasciata).
When present, lesions in the lungs are characterized by congestion and

inflammatory cell infiltration in the bronchial wall and in the peri-

bronchial tissue. The liver shows focal areas of necrosis with bile duct
epithelial hyperplasia and vacuolar degeneration of hepatocytes. Renal
tubular necrosis and multiple foci of mononuclear inflammatory cells are
the most consistent pathologic findings in renal tissues.
6. DIAGNOSIS
Although clinical signs and pathological findings are very suggestive

of avian trichomonosis, the confirmation and identification of the species
should be performed in the laboratory.
The classical method to diagnose avian oropharyngeal trichomonosis
consists in wet-mount cytology. This is a non-expensive test in which the
sample, taken from the oropharyngeal cavity and crop with a swab
moistened in physiological saline solution, is extended on a microscopic
slide. The characteristic movement of trophozoites facilitates their
detection. The inconvenience of this method is a low sensitivity (more than
two times lower) when compared with the culture (Bunbury et al., 2005)
and the examination of the slide should be performed immediately after
obtaining the sample, which makes difficult its application in the field.
Cytologies stained with a metachromatic method, such as Diff-Quick or
Giemsa, has been used. However, the risk of false negative diagnosis is
high, especially early at the infection when low trophozoite numbers may
be present.
Cultures of oropharyngeal trichomonads in specific media is regarded
as the most sensitive method for the diagnosis of the infection (Wieliczko
et al., 2003; Krone et al., 2005; Gerhold et al., 2008; Sansano-Maestre et
al., 2009; Grabensteiner et al., 2010; Zimre-Grabensteiner et al., 2011; Chi
et al., 2013; Girard et al., 2014a and b; Martínez-Herrero et al., 2014;
Kunca et al., 2015; Martínez-Díaz et al., 2015; McBurney et al., 2015).
The main advantage of parasite culture is that it can be used as a transport
medium from the field to the laboratory, facilitating the detection of the
trophozoites. A commercial kit for the diagnosis of Tritrichomonas foetus
from cattle (InPouch® TF, Biomed Diagnostics, San Jose California

EEUU) is available and can be used for the diagnosis of avian

trichomonosis.
Polymerase chain reactions (PCR) of different loci (ITS1/5.8S/ITS2
region, α-tubulin gene, small rRNA subunit, Fe-hydrogenase gene) have
been used in order to confirm the presence of parasite DNA (in carcass
samples and cultures). PCR analysis is mandatory for the identification of
the species taking into consideration the complex etiology of
oropharyngeal bird trichomonosis (Höfle et al., 2004; Villanúa et al., 2006;
Gaspar da Silva et al., 2007; Gerhold et al., 2008; Grabensteiner et al.,
2010; Lawson et al., 2011; Chi et al., 2013; Ganas et al., 2014; Girard et
al., 2014a; Martínez-Herrero et al., 2014; Martínez-Díaz et al., 2015).
Recently, FTA cards were used to preserve DNA from DNases and other
degrading processes. In that way, subsequent molecular analysis would be
easier to perform from samples taken in the field, which would be a great
advantage (Holt et al., 2015). Unfortunately, there are no studies
comparing PCR results from oropharyngeal swabs with cultures and its
validation as a diagnostic method.
7. TREATMENT AND PREVENTION
Different drugs have been proved effective for the treatment of avian
trichomonosis. For decades, nitroimidazole drugs were the treatment of
choice in affected birds. Metronidazole, dimetridazole, ipronidazole,
ronidazole and carnidazole have been successfully used in different species
of birds, directly or mixed with food or drinking water (Lumeij and
Zwijnenberg, 1990). Among them, dimetridazole is a drug with a lower
therapeutic index in birds, inducing hepatoxicity in passerines, psittacines
and fledglings. The indiscriminate use of sub-therapeutic doses in racing
pigeons has leaded to the development of resistant strains to these drugs.
Special care should be taken when administering medications in food and
drinking water, as sick birds have reduced or altered intake, with a
consequent sub-medication and treatment failure, which can trigger the

development of resistance (Lumeij and Zwijnenberg, 1990; Muñoz et al.,

1998; Zimre-Grabensteiner et al., 2011).
Since sick animals have poor body condition, it is mandatory to correct
the electrolytic balance and the catabolic state with supplementary energy
sources. Wide-spectrum antibiotic drugs should be used in order to prevent
secondary bacterial infections. Oral and nasal flushing with topical
disinfectants, such as 0.5% chlorexidin, could reduce the microbial charge.
In some cases surgical scission of the granuloma may be necessary.
Recently, extracts of Artemisia sieberi and garlic plants (Allium spp.)
have proved their efficacy against T. gallinae, which promises a new
source of anti-trichomonad agents that could be used as preventive and
therapeutic drugs (Seddiek et al., 2014; Yussefi et al., 2017).
In the absence of vaccines and drugs as effective preventive
treatments, the infection can be controlled by avoidance of elevated
concentrations of birds (especially columbiformes) at food stations and
drinkers, implementing disinfection protocols and replacing food
frequently at these points.
8. FACTORS THAT TRIGGER PARASITE PATHOGENICITY
As we have mentioned before, infections by T. gallinae are frequent in

some avian species or in certain populations living under circumstances
that favor the spread of the disease. In columbiformes, most of the infected
animals show no apparent clinical signs of disease, or might suffer
subclinical infections (<1% in Sansano-Maestre et al., 2009; 1.9% in
Bunbury et al., 2007 as examples). Only a small percentage of them
develop macroscopic lesions or die. This situation has led to some
skeptical parasitologists to doubt about the pathogenic character of this
protozoon. Still, we have to think that a similar situation occurs in many of
the pathogens infecting animals. We do not doubt on the pathogenic
character of Salmonella and, if we isolate it from an apparently healthy
animal, we call it “healthy carrier”. A similar situation occurs with
trichomonads in general. As far as we know, some species of trichomonads

can be infecting the host during several months: T. vaginalis and T. foetus
(revised in Hirt et al., 2002; Tolbert and Gookin, 2016). The same situation
has been described in T. gallinae infected birds, in which the parasite can
persist in a host for at least 20 months (Bunbury et al., 2007).
Although the idea of “healthy carrier” for T. gallinae is being
increasingly accepted nowadays among protozoologists, we are giving
some hints trying to explain the situation in which the presence of T.
gallinae is not accompanied by the appearance of lesions. Risk factors for
the development of the disease associated with the host (host susceptibility,
immune response, stress situations) or associated with the parasite
(genotype associated virulence) are analyzed.
Little has been studied on the sub-clinical effects of the infection and
some authors support the idea that a reduced survival rate in
columbiformes is a consequence of T. gallinae infection (Bunbury et al.,
2007). A different situation has been described in other avian groups,
mainly raptors, but specially during the last decade also fringilids, in which
the infection is highly associated with the presence of macroscopic lesions
and even with mortality events in bird populations (Sansano-Maestre et al.,
2009; Lawson et al., 2011; revised in Amin et al., 2012; Martínez-Herrero
et al., 2014).
Since many of the surveys investigating the presence of T. gallinae in
birds are cross-sectional studies, it is difficult to have a picture on the
progression of the infection. It is possible that the infection occurred
recently and there are no lesions present, but it could also be possible that
the infection (and disease) was old and the parasite still persists even when
evident signs of the disease are not present any longer. Bunbury et al.,
(2007) screened a population of the endangered Mauritian pink pigeon
(Nesoenas mayeri) for two years and found a high prevalence rate with a
low percentage of animals with active signs of trichomonosis, but
suggested that a prolonged infection with the parasite, even without
evident clinical signs, might affect the oncoming survival rate of the birds.
Prevalence was higher with age, a fact that supports the hypothesis of long
carrier status of previously infected animals. Lennon et al., (2014)
observed that although the percentage of animals hosting T. gallinae was

higher in adult columbiformes, the percentage of animals with lesions was

higher in young individuals.
The high rate of infection accompanied by a low percentage of animals
with clinical signs, and the relationship between prevalence rate and host
age is a regular observation under a stable endemic situation. However,
when an outbreak occurs, there is a high percentage of birds with clinical
signs among the infected individuals in a short period of time. This
situation can appear in naïve animals or in previously exposed animals
under circumstances that favor the spread of the disease by reduction of the
immune responses. Migration, food stress, climatic conditions, breeding
season, sharing of water or food sources with columbiformes, are some
examples (Keppie and Braun, 2000; Höfle et al., 2004; Robinson et al.,
2010; Lawson et al., 2011; Lennon et al., 2014; McBurney et al., 2015;
Stockdale et al., 2015).
The parasite can be pathogenic under certain circumstances, and risk
factors are needed for the development of the disease. If those factors are
not present, the disease will not develop. In this sense, a cross sectional
study was published by Martínez-Herrero et al., in 2014 pointing out some
of the factors than can influence the development of lesions in infected
animals. Among them, the diet type in raptors, and the parasite genotype
were more relevant than other factors, as Sansano-Maestre et al., (2009)
previously suggested. When raptors feed exclusively on birds, lesions due
to trichomonosis were less frequent. This situation can be explained by an
efficient response of the immune system, and if the animal has frequent
contact with the parasite, it gets refractory to the development of disease,
even if the animal maintained the infection for a long time. In this sense,
several authors have observed an inverse relationship between prevalence
rate and percentage of animals showing gross lesions (Bunbury et al.,
2007; Martínez-Herrero et al., 2014; Kunca et al., 2015).
Risk factors associated with the parasite, such as genotype, have been
analyzed, since many genetic variants have been described. Some of them
can be pathogenic while others are not, as it has been described for other
protozoa (Eimeria spp., Cryptosporidium spp. as examples). Many of the
genetic variants have never been associated with disease. Others, however,

have been found when lesions are present and they might appear as single
or mixed infections (Sansano-Maestre et al., 2009; Grabensteiner et al.,
2010; Girard et al., 2014b; Martínez-Herrero et al., 2014). Since there are
many variants without enough epidemiological and clinical data, we have
focused in the two most frequent genetic groups of T. gallinae: ITS-OBT-
Tg-1 (firstly described by Gerhold et al., in 2008 as genotype A), and ITS-
OBT-Tg-2 (firstly described by Gerhold et al., in 2008 as genotype C).
Both have been detected in animals with lesions, which means that both
have the potential to induce disease. This pathogenicity has been
demonstrated by in vitro virulence assays in cell cultures by Amin et al.,
(2012). In the study, the authors showed how different genotypes of T.
gallinae induce cytopathogenic effect on the cells while Tetratrichomonas
gallinarum did not provoke the same effect. More in vitro studies are
required to clarify this issue. Perhaps the point is to focus on the virulence
degree of the different genotypes instead of the strict categorization of
pathogenic/non-pathogenic.
An association between genotype ITS-OBT-Tg-1 and the presence of
gross lesions (including fatal cases) have been observed for the first time
by Sansano-Maestre et al., in 2009, and later on by other authors including
more avian species (Chi et al., 2013). In 2015, McBurney et al., found that
finches and rock pigeons with clinical signs of trichomonosis carried the
same genotype (ITS-OBT-Tg-1), while healthy animals carried the other
genotype (ITS-OBT-Tg-2). Stockdale et al., in 2015 detected four genetic
variants of T. gallinae in European turtle doves, but all birds with clinical
signs carried genotype ITS-OBT-Tg-1. Although this genotype has also
been detected in animals without clinical signs, it might be possible that
they had suffered the disease in the past (Zu Ermgassen et al., 2016).
Also, the number of fatal cases or cases with gross lesions in which
only genotype ITS-OBT-Tg-2 have been detected is much lower. The
genotype of T. gallinae is associated with the bird species, although the
most frequent genotypes have been detected in a wide variety of them. In
Europe, genotype ITS-OBT-Tg-2 is highly associated with columbiformes
and in some studies up to 100% of the animals carry the parasite (Lennon
et al., 2013, 100% incidence in adult European turtle doves). Mixed

infections (including more than one genotype) have been reported by

different authors and it could be possible that when a mixed infection is
present only one genotype is detected by culture or PCR-sequencing. To
solve that problem, a direct amplification and sequencing of the swabs
obtained from lesions is recommended. The only study involving genotype
ITS-OBT-Tg-2 with the development of lesions was done in
columbiformes in Egypt (El-Khatam et al., 2016), in which genotype ITS-
OBT-Tg-2 seems to appear more frequently, especially in pigeon farms. In
this study, only one genotype of the parasite was found, and it seemed that
a small percentage of animals developed lesions. Whereas genotype ITS-
OBT-Tg-2 could also produce some degree of lesions, a control group of
animals infected with genotype ITS-OBT-Tg-1 is lacking and for that
reason it is difficult to obtain a clear conclusion from the study.
A question that has still not been investigated in depth is the
association of genotype with a categorization of the disease; that is, cases
of lethality, internal organs affected, severity of oropharyngeal lesions, and
so on.
9. RECENT ADVANCES AND FUTURE STUDIES
We have seen during the past years how the diversity of avian
oropharyngeal trichomonads has increased. The description of new species,
probably circumscribed to certain hosts, is one of the issues that will be
investigated and enrich our knowledge of avian trichomonosis in the near
future.
Another fact that needs more clarification is which characteristics, if
any, are associated with the genotype of the parasite. For example, is there
a similar growth rate between both genotypes? Do they need the same
nutrients to grow in vitro? This is an important issue, since the necessity of
nutrients can explain differences in isolation success, or even in the
virulence of the isolates. In this regard, the effect of certain nutrients on the
growth of different genotypes of the parasite needs further investigation.
The isolation and maintenance of certain genetic variants in the laboratory,

under our experience, is not easy. If we rely solely on culture for

epidemiological studies, then we may exclude some genetic variants which
grow slower or do not grow at all in the most frequent employed culture
media.
In order to optimize the results obtained in epidemiological studies, a
consensus diagnostic technique among laboratories, sensitive enough to
detect distinct species and low number of protozoa will be desirable. In this
sense, it can be possible that PCR carried out on DNA isolated directly
from oral swabs is a good option if targets with tandem sequence repeats
are used (ITS1/5.8S/ITS2 region and small rRNA subunit). The possibility
of sequencing and BLAST analysis of positive results is also an advantage
if we want to obtain a detailed picture of the epidemiological situation.
The virulence of the isolates needs more in vitro and in vivo evidences.
Although several studies have focused on the higher virulence of genotype
ITS-OBT-Tg-1, a small percentage of animals infected with genotype ITS-
OBT-Tg-2 and other less frequent species (such as T. stableri) develop
lesions as well. An important fact that has to be present to validate
epidemiological studies is that the wide spread genotypes are isolated from
the same bird species. If only one of them is isolated (genotype-host
association) it will be difficult to extract any conclusion. In vitro virulence
assays using both genotypes are necessary to clarify the pathogenicity
associated with the genotype of the isolate.
The virulence mechanisms of the parasite have not been elucidated yet.
Although a couple of studies identified several proteins present in T.
gallinae, which are similar to some virulence factors previously described
in T. vaginalis, more studies focusing on the proteomic composition of the
parasite are needed. The presence of cystein peptidases or GADPH are
some of the above mentioned factors (González-Díaz et al., 2011; Amin et
al., 2012), but there are, still, many proteins to be described in this
protozoan.
The mechanisms of transmission of the parasite can be direct, by
feeding on infected preys or by feeding the young or during courtship, but
they can also be indirect, when sharing of contaminated food or water is a
common practice among birds. The role of pseudocysts in disease

transmission has been poorly investigated (Tasca and De Carli, 2003),

although several studies focused on the persistence of the parasite on food
or water have been carried out (Kocan, 1969b; Kietzmann, 1990; Erwin et
al., 2000; Bunbury et al., 2007; Purple and Gerhold, 2015; Purple et al.,
2015). Description of survival and mechanisms of development of
pseudocysts will be a valuable contribution to the epidemiology of the
disease.
Another factor that has not been investigated in depth so far is the
presence of concomitant flora that can exacerbate the disease. The presence
of Pseudomonas aeruginosa has been pointed out as a factor that
complicates the lesions (Samour, 2000). However, more studies including
a general view of the flora present in healthy and infected populations of
birds (metagenomics) are needed in order to clarify the influence of the
microbiota on the progression of the disease. Also, the possibility of gene
transferring among microorganisms present in the mucosae could be
investigated. This idea has been suggested by some authors who found
similar surface protein families in individuals colonizing mucosal surfaces
(Hirt et al., 2002).
In order to control the disease, a mechanism of immunization of naïve
birds under management or the development of a vaccine will be a great
contribution, especially for endangered species in which oropharyngeal
trichomonads constitutes a menace for them.
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Chapter 2
NEW BREEDING STRATEGIES

IN ORGANIC DAIRY FARMING
R. Rodríguez-Bermúdez1, M. Miranda2,
M. López-Alonso1, J. Baudracco3, R. Fouz4,
I. Orjales2 and M. Garcia-Vaquero5
1
Department of Animal Pathology, Veterinary Faculty,
Universidade de Santiago de Compostela, Lugo, Spain
2
Department of Anatomy,
Animal Production and Clinical Veterinarian Sciences,
Veterinary Faculty,
Universidade de Santiago de Compostela, Lugo, Spain
3
Agricultural Cience Faculty,
Universidad Nacional del Litoral, Esperanza, Santa Fe, Argentina
4
Africor Lugo, Ronda de Fingoi, Lugo, Spain
5
School of Veterinary Medicine, University College Dublin,
Belfield, Dublin, Ireland

44 R. Rodríguez-Bermúdez, M. Miranda, M. López-Alonso et al.
ABSTRACT
The selection of an appropriate breed in dairy farms will have a huge

influence on the animals’ welfare and production. This is especially
noticeable in animal production systems that aim to maximize the use of
on-farm resources (low-input production), i.e., organic farms. The animal
production in organic farming systems focuses on maximizing the
utilization of forage and improving the animals’ health and welfare, while
reducing the application of drugs routinely applied in the treatment and
prevention of diseases in the conventional farms. Thus, the selection of
animals adapted to this harsh farming conditions is essential for the
success of an organic farm. However, the current animal breeding
strategies adopted by the farmers do not differ between the intensive and
the organic animal production systems; i.e., the Holstein-Friesian is the
most commonly used breed in the organic and intensive dairy farms,
despite the poor production and adaptability of these animals to the
organic production systems. Recent studies showed that animals bred to
produce high milk yields in the conventional systems are not capable to
adapt to pasture-based systems. Cattle breeding strategies based on the
selection of the animals for functional traits, or the efficiency of the cows
to use scarce resources (inputs), could be a good strategy when selecting
animals for organic farms. This chapter analyzes the animal breeding
strategies currently performed in the organic farms, and discusses the
novel strategies and animal breeds that could potentially benefit different
organic dairy farming systems, including multifunctional farms.
Keywords: local breed, cattle, animal breeding, sustainability, pasture-

based system, multifunctional farm, organic, intensive, production
ORGANIC FARMING - HISTORICAL BACKGROUND
The organic farming practices emerged in the 20th century as a global

reaction and willingness to change the existing farming sector. The organic
production is a holistic system designed to optimize the productivity and
the lifestyle of communities, including the management of multiple
resources such as soil, crops, livestock and people (OMAFRA, 2009). The
creation in 1972, of the International Federation of Organic Agriculture
Movements (IFOAM), was a huge milestone towards the development of

New Breeding Strategies in Organic Dairy Farming 45
the current organic farming practices. IFOAM, together with multiple

stakeholders around the World, worked on the development of ecological,
social and economic management systems aligned with their standards.
The principles of organic livestock production included the preservation of
the biodiversity, the development of husbandry practices adapted to the
natural behaviors of the animals and the sustainable management of the
farms (IFOAM, 2005).
Despite the spread and acceptance of the principles of organic farming
in Europe in the early 1960’s (Padel et al., 2004), the biggest increase of
the organic farming practices was appreciated in the late 1990’s, with over
120,000 organic farms registered in 1999 (Padel, 2000). The number of
animals raised in organic farms increased exponentially since then, with
several countries leading the change from the intensive to the organic
production systems, including Austria, France, United Kingdom, Sweden,
Italy and Spain (EC, 2013). In fact, the organic farming practices are
currently growing at a range of 10 to 20% per year in Europe, while the
conventional farming systems are declining due to the economic global
crisis (Díez et al., 2012). The number of dairy cows raised in organic farms
reached 0.7 million animals, representing approximately 3% of the total
dairy cattle registered in the European census in 2011 (EC, 2013).
Despite this growth, the organic farms are still limited in number and
small in size when compared to the intensive farms. As an example, 85%
of the organic farms are managed by a sole proprietorship or by a family
partnership (Organic Farming Research Foundation, 2003). Furthermore,
the organic farms in Europe invest less in technology than the conventional
farms. Recent studies showed that the organic dairy farms were 4.5% less
technically efficient than the intensive ones (Sipilainen & Oude-Lansink,
2005). The use of the technological advances designed for the intensive
farms could increase the productivity of the organic systems up to 5.3%
(Kumbhakar et al., 2009). However, the technological investments in the
organic farming practices seemed to be related to the farmers’ personal
philosophy, concerning animal welfare and production, rather than to
economic reasons (Escalante et al., 2013). The diversification of the
production in organic farms, including the production of cheese (Nauta

et al., 2006c), yogurt (Van Loo et al., 2013) or the use of the farm to attract
tourists (Villarino Pérez et al., 2009), could increase the profits of these
small and non-technological organic farms (San Segundo-Barahona, 2008;
Pouliquen, 2014).
The organic farming sector has recently attracted the attention of the
scientific community, evidenced by the scientific studies on the
management practices in organic farming systems (Organic Research,
2015) and the creation of the Network for Animal Health and Welfare in
Organic Agriculture (NAHWOA) (4th NAHWOA, 2001). The current
research in the organic farming sector focuses on the evaluation of the
animals’ health and the maximization of the use of on-farm inputs.
However, little information is available on the breeds, and the genetic
profile of the cattle, that could produce more efficiently in organic farming
systems (Ahlman, 2010).
BREED DIVERSITY - CURRENT SCENARIO

IN CONVENTIONAL AND ORGANIC DAIRY FARMS
The American Holstein-Friesian breed largely dominates the dairy

production Worldwide (Zenger et al., 2007; Oltenacu & Broom, 2010;
Felius et al., 2015), representing approximately 95% of the total number of
animals raised in the dairy sector (Oltenacu & Algers, 2005; Oltenacu &
Broom, 2010). This breed, originated in the United States using animals
imported from Northern Europe, was hugely exported globally in the early
1970s (Oltenacu & Broom, 2010; i.e., the North American Holstein-
Friesian semen exports reached 8 million doses in 1997 (Oltenacu &
Broom, 2010). The huge expansion of this breed could be attributed to the
high milk yields of the Holstein-Friesian cows and the improvement of the
reproductive technology, needed to export semen and embryos
(Brotherstone & Goddard, 2005; Oltenacu & Broom, 2010). The exports of
semen started in Italy, Netherlands, Germany and France, with the later
implementation of an embryo program, in collaboration with the United

States, for the genetic improvement of the European dairy herds

(Philipsson, 1987; Nauta, 2001; Oltenacu & Broom, 2010).
The American Holstein-Friesian cows showed an extraordinary
productive potential when fed high quality diets. This led to the quick
development of highly specialized and technological intensive farms, using
good quality animal feed and a controlled/comfortable environment.
Within this scenario, the American Holstein-Friesian breed replaced
rapidly other local European breeds, such as the original Friesian cattle
(Philipsson, 1987; Brotherstone & Goddard, 2005). The global expansion
of this dairy farm model, led to an exponential increase of the global milk
production (Knaus, 2009; Oltenacu & Algers, 2005), mainly due to the
genetic improvements achieved in the Holstein-Friesian animals
(Brotherstone & Goddard, 2005; Oltenacu & Algers, 2005; Oltenacu &
Broom, 2010). This production strategy narrowed the breeding objectives
of the farmers to achieve high milk yields per animal, paying little or no
attention to other animal traits that could influence milk production; i.e.,
several functional traits of the animals could increase the efficiency of the
cows and reduce the cost of the inputs in the farm. These functional traits
englobe parameters related to the health, longevity and reproduction rates
of the animals (Roxström, 2001; Wall et al., 2003; Ahlman, 2010). The
genetic selection of the Holstein-Friesian cows, based solely on obtaining
high milk yields per animal, resulted in a low resistance of the herds to
certain diseases and fertility issues in the farms (Rauw et al., 1998;
Washburn et al., 2002; Haile-Mariam et al., 2004; Oltenacu & Algers,
2005; Jorjani et al., 2009; Bjelland et al., 2011). Thus, alternative animal
breeding strategies, focusing on the functional traits rather than on the milk
yield of the animals, are needed in both conventional and organic
production systems. These novel breeding plans are challenging, due to the
lack of agreement on the definition of functional traits and the low
heritability rates of these animal traits (Berglund, 2008).
Despite the huge expansion of the American Holstein-Friesian breed,
several countries applied alternative production strategies. New Zealand
farmers selected their own animals, based on the Friesian cattle imported
from the United States before 1925, leading to the creation of the New

Zealand Holstein-Friesian breed. The breeding strategy in New Zealand

regarded the selection of the animals based on the fat and protein contents
in the milk, and penalized the animals producing high milk yields
(Macdonald et al., 2008). In Austria and Switzerland, the dairy farms
raised mainly Fleckvieh and Brown Swiss cows (Baumung et al., 2001;
Haas & Bapst, 2004; ZAR, 2014); while in France, the Montbeliarde and
the Normande breeds, represented 17.5 and 8.3% of the total dairy cows
respectively (Institute d’Elevage & France Conseil Elevage, 2016).
The presence of the American Holstein-Friesian breed increased also
in organic dairy farms, although several countries promoted their local
breeds; i.e., in the Netherlands, 29% of the organic dairy farms raised
Holstein-Friesian animals and 51% of the organic dairy farmers owned
crossbred animals (Nauta et al., 2006c); in Sweden, the organic dairy farms
raised mainly Swedish Red cows (54.3%) and crossbred animals (7.7%)
(Ahlman, 2010); and more than 50% of the organic farms in Austria and
Switzerland raised the local breeds Brown Swiss and Fleckvieh (Haas and
Bapst, 2004).
AMERICAN-HOLSTEIN FRIESIAN COWS IN ORGANIC

FARMING - WHAT IS NOT WORKING?
The current animal breeding strategies, based on the genetic selection

of the cows by high milk yields per animal, are associated with a general
decrease in the health and reproductive efficiency of the herds (Rauw et al.,
1998; Pryce & Veerkamp, 2001; Veerkamp et al., 2001; Evans et al., 2002;
Brotherstone & Goddard, 2005; Oltenacu & Algers, 2005; Oltenacu &
Broom, 2010; Prendiville et al., 2011) that could make the current dairy
production system unsustainable by 2020 (Maas et al., 2009). The
reproductive performance of the American Holstein-Friesian cows
decreased when these animals were raised in other environments. This fact
aroused questions about the suitability of this breed in forage-based
systems, such as organic farms (Grétar & Hardarson, 2001; Kristensen &

Pedersen, 2001; Nauta et al., 2001, 2006b; Horn et al., 2012). The organic
farming systems (low input productions) require animals with high fertility
and reproductive rates, rather than animals with high milk yields (Dillon
et al., 2003b; Veerkamp et al., 2003). Numerous researchers concluded that
the cows from the conventional farms were not easily adaptable to the
organic production systems (Grétar & Hardarson, 2001; Harris & Kolver,
2001; Nauta et al., 2001; Dillon et al., 2003a; Pryce et al., 2004; Rozzi,
2012). These animals use a high proportion of their energy to achieve high
milk yields (Dillon et al., 2006); meeting their energetic requirements with
the input of good quality feed supplements. These higher dietary
requirements, not available in animals raised in pasture-based systems,
could produce metabolic disorders and poor fertility rates in the cows
(Knaus et al., 2001; Kristensen & Pedersen, 2001). In pasture-based
systems the Holstein-Friesian cows showed low survival rates, poor
fertility and body conditions; leading to a less profitable production
compared to other farms raising other local breeds (Harris & Kolver,
2001).
Genotype x Environment interactions (GxE) were described as the
changes in the response of the animals from selected genotypes to different
environmental conditions (Falconer & Mackay, 1996). GxE interactions
explained the differences in the reproductive performance between the
New Zealand and the North American cows in pasture-based farms (Harris
& Winkleman, 2000; Verkerk et al., 2000). Moreover, the GxE interactions
affected several functional traits (Nauta et al., 2006d) and the fertility rates
(Sundberg et al., 2010) of Holstein-Friesian cows raised in organic farms.
Thus, the bulls genetically selected for conventional systems might not be
suitable for organic farms due to these GxE interactions (Nauta et al.,
2006b); i.e., if a cow trait, like milk yield, is under the control of a different
set of genes in a different environment, it is possible that the genetic merit
of the animals could vary when the animals are raised in different
production systems (Pryce et al., 1999a). The organic farmers, using the
breeding strategies design for intensive farms, will not achieve the highest
potential from the breeding plans due to the GxE interactions (Nauta et al.,
2006a; Sundberg et al., 2010; Ahlman et al., 2011).

ANIMAL BREEDING STRATEGIES IN ORGANIC DAIRY

FARMING - WHAT CHANGE IS NEEDED?
Currently, the milk production yields are not the major income in the
modern dairy farms, due to the increased costs of maintenance of the
highly productive cows (Bluhm, 2009). The farmers managing low input
production systems, such as organic farms, prefer animals less susceptible
to health problems or more “robust”, than those selected in the intensive
dairy farms (Nauta, 2001). Countries with pasture-based dairy production,
such as New Zealand (Harris & Kolver, 2001) or Ireland (Dillon et al.,
2003a, 2003b), showed high profits raising local breeds; i.e., pasture-based
farms raising New Zealand Holstein-Friesian cows showed 12% more
profit than those using the American Holstein-Friesian breed (LIC, 1999).
Furthermore, the body condition score (Veerkamp et al., 2001) and the
longevity (Harris & Kolver, 2001) of the animals decreased with an
increase in the North American breed ancestry.
Cows could potentially live up to 20 years; however, in the modern
dairy farms, the animals do not exceed 6 years of age (Rushen & Passillé,
2013). The current genetic breeding strategies influenced negatively the
longevity of the cows (Xue et al., 2011), due to the negative association
between the age and the milk yields of the cows (Wall et al., 2003). The
longevity of the cattle in the dairy farms - measured as the time on-farm in
which the animals show acceptable production rates - is especially relevant
in organic production systems (Ahlman et al., 2011). In fact, this parameter
changed the breeding policies in the organic farms (Bluhm, 2009).
However, scientific studies evaluating the longevity and causes of culling
in the organic farms are scarce to date. “Fertility issues” were the main
cause for culling cows in organic and conventional farms in Canada (Rozzi
2012; Can West DHI, 2003) and Sweden (Ahlman, 2010; Ahlman et al.,
2011). A recent study concluded that the main reason for culling cows in
organic dairy farms in Galicia (Spain) was the age of the animals (73.2%),
followed by other health related issues such as infertility (14.3%), mastitis
(10.7%) and laminitis (1.8%) (Rodríguez-Bermúdez et al., 2016).

As previously mentioned, the organic farming systems are

continuously improving and diversifying their outputs. The organic farm is
currently being transformed into a “multifunctional system” to produce
milk, various dairy products (yogurt or cheese), leisure activities for the
tourists and the local community (i.e., training on animal handling or
courses to produce dairy products in the farms) and in-farm shopping
activities (i.e., milk and meat produced and sold within the farm) (van der
Ploeg & Renting, 2000; Ventura & Milone, 2000). These changing
business models could require the use of different animal breeding
strategies (Groen et al., 1995; Diepen et al., 2007); i.e., The Jersey cows
showed high fat and protein contents in the milk (Blake et al., 1980), useful
for farmers interested in cheese production (Auldist et al., 2004).
Multifunctional farming systems could also value animals capable to
produce high quality meat (Diepen et al., 2007; Nauta et al., 2009), such as
the Meuse Rhine Yssel, the Normande, the Fleckvieh, the Milking
Shorthorn and the Montbeliarde breeds. The good quality meat obtained
from the male calves of these breeds, but also from the heifers or cows
culled from the herds, could generate a valuable profit in multifunctional
farms (Diepen et al., 2007; Rengab-Genetics, 2013). Moreover, raising
local breeds could also be a marketing strategy, selling animal products
with sensorial characteristics distinguishable to consumers (Nauta et al.,
2009).
BREED SELECTION IN ORGANIC DAIRY FARMS
The selection of breeds in organic farming systems has generated a

great deal of attention in the scientific community. Diepen et al., (2007)
proposed a selection of the animals based on the resistance of the cows to
the most commonly experienced health problems in the current organic
farms. The ability of the cows to adapt to the local environment and to
maintain decent milk yields - while using poor quality diets (low in energy
and protein) and less antibiotics - should be valued in the animal breeding
strategies used in an organic farm (Rozzi et al., 2007).

There is no animal breeding program specialized in organic farms. The

selection of the animals in the current organic systems often results in a
lack of trust and in some cases, a dissatisfaction of the farmers with the
selected animals (Rodríguez-Bermúdez et al., 2016). Farmers managing
intensive and organic productions expressed similar goals and expected
outputs in relation to the animals’ production, conformation and
functionality (Martin-Collado et al., 2015; Slagboom et al., 2016).
However, the animal breed preference in organic farms is more varied than
the general agreement on the Holstein-Friesian breed in the intensive
systems. This could be due to the lack of information on the productivity
and performance of different breeds raised in organic production systems.
The scientific information available on several purebred and crossbred
animals (Holstein-Friesian crossed with other rustic breeds) and the
possibilities of each breed in organic farming systems is summarized
below.
Purebred Animals (the Local Breeds and the Rustic

Holstein-Friesian Breed)
As previously mentioned, the local breeds are generally more “robust”

and better adapted to the local environment than the Holstein-Friesian
cows (Sundrum, 2001; Diepen et al., 2007). Thus, different local breeds
could be good candidates to be raised in organic farms (Ahlman, 2010).
The organic farms could also be used to maintain a valuable genetic
diversity in the dairy sector (i.e., genes related to the resistance to climatic
stresses, parasites and diseases) for its use in later animal breeding
strategies (Nauta, 2001).
Overall, the rustic breeds seemed to be better adapted to the local
environment, with better longevities and reproductive performances than
the Holstein-Friesian animals raised in organic farms (Pryce et al., 1999b,
1999c; Roesch et al., 2005; Horn et al., 2012; Haas et al., 2013; Rodríguez-
Bermúdez et al., 2017). Also, the local breeds produce lower milk yields
and higher percentages of protein and fat in the milk than the Holstein-

Friesian cows. These breeds could be used to increase the profit in organic
farms from countries that pay the milk on the basis of the total solid
content (Davis et al., 2001). Moreover, the Jersey breed could be useful in
organic farms interested in cheese production, as the milk from these
animals coagulated faster and formed a firmer curd than the milk from the
Holstein-Friesian cows (Auldist et al., 2004). However, one of the main
limitations when choosing a local breed, different than the Holstein-
Friesian, is the lack of information available on the selected animals (i.e.,
lack of a herd book or a selection program). Thus, the farmer is in charge
of studying the progeny of their herds, assuming all the risks associated to
the implementation of an animal breeding plan. I.e., an improvement in the
“fitness” of the cows could decline the milk production and the udder
conformation of the animals (Rozzi, 2012).
The rustic Holstein-Friesian cows showed a reasonable reproductive
performance and milk production yields when raised in pasture-based
systems. The Holstein-Frisian animals showed to be the most profitable
breed, in both intensive and organic production systems, when the
appropriate strain was selected (Brotherstone & Goddard, 2005).
Baudracco et al., (2010) obtained the maximum profits raising the rustic
Holstein-Friesian breed in a pasture-based system with moderate amounts
of commercial feed. The New-Zealand Holstein-Friesian breed showed to
be the best fit to pasture-based production farms to date. The breeding
strategy in this strain focused on the selection of animals based on the milk
composition (fat and protein contents in milk), the economic efficiency and
the cost of maintenance of the cows (Harris, 1998). In New Zealand, 90%
of the milk is produced in pasture-based systems and transformed into
different dairy products, being the farmers paid on the basis of the
composition of the milk (Harris & Kolver, 2001; Clark et al., 2006). In
general, the New Zealand Holstein-Friesian animals showed higher levels
of protein and fat in the milk, better fertility rates and an overall economic
performance comparable to the North American Holstein-Friesian breed
(Harris & Kolver, 2001; Horan et al., 2005). Moreover, the New Zealand
Holstein-Friesian cows showed low maintenance requirements and dry

matter intakes in early lactation (Patton et al., 2008). In Europe, certain

strains of Holstein-Friesian cows in the Netherlands still maintain their
rusticity. The Dutch Friesian cows were selected following a family
breeding system (Baars & Nauta, 2001) developed to maintain the original
Dutch Friesian breed (Fries-Hollands) (Endendijk & Baars, 2001).
Crossbred Animals (Holstein-Friesian and Other Rustic Breeds)
Traditionally, the crossbreeding of the Holstein-Friesian cows with

other local breeds offered little or no advantages to intensive dairy farmers,
due to the lower milk production potential of all the local breeds in
comparison to the Holstein-Friesian purebred animals (Prendiville et al.,
2011a). However, the new breeding strategies, based on improving the
functional traits of the cows (such as fertility and health parameters),
showed the high genetic potential of the crossbred animals. There is a
renewed interest on implementing animal crossbreeding strategies in the
farms (Funk, 2006; Hansen, 2006; Walsh et al., 2007; Blöttner et al.,
2011). The successful implementation of animal crossbreeding plans could
eliminate animal inbreeding (mating of genetically related animals) (Rozzi
et al., 2007), while increasing the heterosis or “hybrid vigor” in the farms
(improvements in the performance of crossbred animals over their
purebred parents) (Brotherstone & Goddard, 2005; Rozzi et al., 2007;
Begley et al., 2009; Oltenacu & Broom, 2010; Bjelland et al., 2011;
Blöttner et al., 2011; Xue et al., 2011).
The crossbreeding, of Holstein-Friesian animals and the local breeds,
is a frequent strategy in organic farming systems (Rozzi et al., 2007). The
implementation of an animal crossbreeding plan could be challenging for
the farmers, especially after the production of the first generation of
crossbred animals. Thus, the production of a first generation of crossbred
animals, using semen from selected local bulls while maintaining the
purebred cows in the herd, could be a feasible crossbreeding plan for most
organic farmers. Jersey and Brown Swiss bulls are commonly used in the

crossbreeding strategies in both intensive and medium-high productive

organic farms, while most organic farmers prefer to use the Dutch Belted,
the Milking Shorthorn and the Fleckvieh breeds. The crosses of Holstein-
Friesian cows with other European breeds, normally resulted in
intermediate milk yield levels (Haas et al., 2013) and improvements in the
protein and fat contents in the milk (Rodríguez-Bermúdez et al., 2017).
However, several researchers found no differences in the somatic cell
counts in milk between the cross- and pure-breeds, as these differences
seemed to be primarily related to the management practices in the farms
(Orjales et al., 2016; Rodríguez-Bermúdez et al., 2017).
THE USE AND DEVELOPMENT OF GENETIC MERIT

INDEXES FOR ORGANIC FARMING
The main goal of any animal breeding strategy is to provide the type of
animal that could better satisfy the needs of the farmers, guiding them
along the breeding process (Bluhm, 2009). Thus, successful breeding
strategies take into consideration the farmers’ expectations in relation to
the outputs of the farms (i.e., milk or multifunctional farm). The genetic
merit indexes try to link the reproductive value and the favorable traits of
individual animals of a breed, helping farmers and stakeholders to compare
and to rank the best animals for their breeding plans (Xenética Fontao,
2010; CONAFE, 2016).
Multiple genetic merit indexes have been proposed by different
countries over the years, reflecting the cultural differences in farming goals
and the changes in animal breeding strategies (VanRaden, 2002; Powell
et al., 2003). Initially, the generic merit indexes were calculated trying to
increase the milk production of the cows. In the calculation of these
indexes, the production traits of the animals accounted for 59, 40 and 51%
of the total merit indexes proposed in Spain, Canada and the United States
respectively. The functional traits in the previously mentioned indexes

represented a lower percentage (6, 20 and 22% in Spain, Canada and the
United States) compared to the indexes proposed by the Netherlands
(45%), the United Kingdom (49%) and Denmark (57%).
The breeding strategies based on those genetic merit indexes resulted
in a reduction of the fertility rates of the cows (Powell et al., 2003) and in
health issues in dairy herds (i.e., inbreeding) (Kearney et al., 2004;
Brotherstone & Goddard, 2005). The animal breeding strategies in organic
farms should be based on animals evaluated in organic farming conditions,
allowing the selection of the proper bull in the crossbreeding plans (Nauta
et al., 2006c). The genetic merit indexes of the bulls used in organic farms
should be re-ranked, as the genetic merit of the animals will vary due to the
previously described GxE interactions (Falconer & Mackay, 1996; Nauta
et al., 2006c). Several associations of organic farmers emphasized the
importance of the incorporation of multiple functional traits in the
calculation of new genetic merit indexes, especially the longevity of the
cows (Harder et al., 2004). The genetic merit indexes developed in
multiple countries and the animal traits used in the calculation of the
indexes are summarized Table 1.
Several researchers developed merit indexes specific for organic dairy
farmers: (1) the “Ecological Total Merit Index” valued the production
persistence, the health and the vitality of the animals (Postler, 1998); (2)
the “Biological Total Merit Index” for the Simmental, the Brown Swiss
and the Gelbvieh breeds valued animal traits such as fertility (25%),
longevity (15%) and lactation persistency (10%) (Krogmeier, 2003); and
(3) the “Ecological Breeding Index” developed for the Brown Swiss, the
Fleckvieh and the Holstein-Friesian animals (Bapst, 2001; Rozzi et al.,
2007). Recently, Rozzi (2012) suggested that the genetic merit indexes for
organic dairy production should value traits such as fat, protein and milk
yield (28%), udder health and conformation (23%), feet and legs of the
animals (14%), animals’ capabilities (11%), lactation persistency (8%),
longevity (8%) and calving ease (8%).

Table 1. Genetic merit indexes developed in various countries. The contribution of several parameters of milk
composition and functional traits in the animals for the calculation of the different indexes
(expressed as % of the index) is also summarized
Merit Index Country Milk composition (%) Functional traits in the animals (%) References
Protein Fat Yield Conformation Productive Udder Fertility Other health
life health parameters
Profitable United 21.9 12.4 - 10.9 5.6 21.1 5.5 18.5 4.1 Bluhm, 2009
Lifetime Kingdom
Index
Economic Ireland 25 5 - 12 - 11 - 23 24
Breeding
Index
Total Merit Scandinavia 20 5 -5 13 4 14 13 26 Oltenacu &
Index Broom, 2010
Profit Australia 36.8 14 - 18.5 - 8.2 7.1 5.1 10.3 Bluhm, 2009
Ranking
Index
Breeding New 40 12 - 14 - 6 -7 8 - 13 Prendiville
Worth Zealand et al.,
2009, 2011b

CONCLUSION
The organic dairy farming sector is a heterogeneous production system

involving multiple management practices (variable feeding resources and
environmental conditions) and a wide variety of production expectations or
outputs (i.e., milk, cheese or multifunctional farms). Thus, the
implementation of an adequate animal breeding practice should be
evaluated on a “one by one basis,” as one breed alone cannot fit all the
possible scenarios and expected outcomes of the current organic dairy
farms. In general, the use of several local breeds could be beneficial when
implementing new breeding strategies in the organic farms. Multiple
purebred and crossbred animals should be evaluated in each particular farm
involved in a breeding program. Also, the Genotype x Environment
interactions described in the literature emphasize the need to develop novel
genetic merit indexes specific for animals raised in organic farms, to
increase the efficiency of the animal breeding strategies in the future.
ACKNOWLEDGMENTS
Ruth Rodríguez-Bermúdez is in receipt of a pre-doctoral contract

(Ref.14/01476) from the Ministry of Education, Culture and Sport (Spain).
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Chapter 3
THE ECOLOGY AND CONSERVATION OF THE

WEST AFRICAN LION
Michael Campbell*
Camosun College, Victoria, BC, Canada
ABSTRACT
The conservation of the lion (Panthera leo), a dominant apex

predator, indispensable ecological mainstay and popular wildlife symbol,
is one of the most important issues in current conservation biology
research and indeed is a relevant topic for other disciplines such as
applied ecology, biogeography and political ecology. The conservation
issues for the West African lion subspecies or variant (Panthera leo
senegalensis or Panthera leo leo) are particularly important, because the
ecology and status of this subspecies are less researched than those of the
Southern, Eastern, Asiatic and even extinct North African lions. The
West African lion is considered endangered and its changing populations,
history, human relations and current prospects are little known beyond
presence data. This chapter conducts a status assessment, based on a
comprehensive literature review, of the conservation issues of the West
*
Email: ecol5@hotmail.com.

74 Michael Campbell
African lion. A key issue is the lack of analysis of the human dimensions
of lion presence in the increasingly human dominated landcover of West
Africa, and lack of applications of relevant knowledge from studies of
lions and other large carnivores in other contexts and the
multidisciplinary theoretical analyses that could be applied to this issue. It
is concluded that without an intensive effort to rectify the factors for the
rarity or extirpation of the West African lion (extremely high human
population growth and urbanization, desertification, decline of food
sources, cattle herding, hunting and farming) and increased public
acknowledgement of this comparatively neglected lion subspecies, the
West African lion has a bleak future. A common, cross-border
conservation policy among the West African nations will be required, not
only for lions but for prey species and human landusers.
INTRODUCTION
The West African lion is seriously endangered in its current range, and
it lacks a strong captive population and serious conservation support
(Bauer et al., 2003; Bauer & Van Der Merwe, 2004). Henschel et al.,
(2014) argues that the “lion has undergone a catastrophic collapse in West
Africa.” Three key issues underpin any discussion of the status of the West
African lion: its classification (compared to the North, South and East
African lions, and the Asiatic lion); comparisons between the relevant
West African socio-cultural, political/economic and environmental
parameters and those of the more studied southern and Eastern African
contexts; and the possibility of the extinction of this subspecies. A major
problem concerns the relative neglect of this subspecies or variant
compared with the other lion subspecies in the research and popular
literature, compared with the lion populations in the Serengeti (Tanzania),
Amboseli and Tsavo (Kenya) and Kruger (South Africa) national parks and
game reserves (Chardonnet et al., 2010; Campbell, 2017). The West
African lion occurred and in some cases still occurs principally in French
speaking countries (Senegal, Gambia, Guinea, Mali, Niger, Cote d’Ivoire,
Chad and Burkina Faso) and to a lesser extent English speaking Nigeria
and Ghana (Burton et al., 2010). These countries are less scrutinized for
their wildlife than East Africa (Kenya, Uganda, Tanzania) and South

The Ecology and Conservation of the West African Lion 75
Africa (Bauer & Van Der Merwe, 2004). As argued by Mallon (2017),
West Africa “is much less well-known, at least in the English-speaking
world, than similar habitats in East Africa.” However, there are serious
reasons why the West African lion is at least as important a study as its
near relatives across the continent (Ogutu & Dublin, 2002; Bauer et al.,
2003; Burton et al. 2010).
Numerous studies have pointed to the threatened status of the West
African lion (Burton et al., 2010). Bauer & Van Der Merwe (2004) give
evidence that in terms of lion presence, African countries may be divided
into four categories: nations were lions are extinct (North Africa); nations
where at the time records were few (Sudan and Somalia); nations where
lions roam outside and inside protected areas (most Central, Eastern and
Southern African countries); and those countries where the few existing
lions were restricted to protected areas (some West African countries).
Henschel et al., (2015, 4) argue that “in West Africa, the lion
subpopulation is currently estimated at just above 400 animals (<250
mature individuals).” This subpopulation continues to decline. These
authors give the following country occurrences for lions: “native for Benin,
Burkina Faso, Niger, Nigeria and Senegal; possibly extinct for Ghana and
Guinea; and regionally extinct for Côte d'Ivoire, Gambia, Guinea-Bissau,
Mali, Mauritania, Sierra Leone and Togo. The United States Fish and
Wildlife Service (2017), citing (Bauer et al., 2015) gives a classified list of
the West African and other countries regarding lion status, distinguishing
between “lions extirpated” (Algeria. Egypt, Libya, Morocco, Tunisia);
“lions considered recently extirpated”; Côte d'Ivoire, Gabon, Gambia,
Guinea-Bissau, Mali, Mauritania, Niger, Nigeria, Senegal, Sierra Leone;
“lions considered possibly extirpated (Ghana, Guinea, Togo) and lions still
occurring (Benin, Burkina Faso, Cameroon, Central African Republic,
Chad, Congo, Democratic Republic of the Congo, Liberia, Niger, Nigeria,
Senegal) (Figure 1). The West African lion is compared with the other
subspecies in eastern and southern Africa, where it still occurs (Angola,
Botswana, Ethiopia, Kenya, Malawi, Mozambique, Namibia, Somalia,
South Africa, Sudan/South Sudan, Swaziland, Tanzania, Uganda, Zambia,
Zimbabwe); is possibly extinct (Burundi, Djibouti, Eritrea and Lesotho); or

76 Michael Campbell
recently extinct (Rwanda) (Bauer et al., 2015; United States Fish and
Wildlife Service, 2017).
Note: The lion habitats are in the Guinea and Sudan savanna.
Source: Lion conservation areas (Bauer et al., (2015). The area in Ghana is disputed
(Bauer & Van Der Merwe, 2004; Burton et al., 2010; Angelici et al., 2015;
Henschel et al., 2015).
Figure 1. The countries of West Africa, vegetation zones and lion conservation areas.
One recent survey result suggests that only 404 (269-583) lions remain
in West Africa (Henschel et al., 2014). It is listed as Critically Endangered
on the International Union for the Conservation of Nature (IUCN) Red
List, with a population in the hundreds and its occupied range less than 1.1
percent of its historic range. Moreover, this occupied range is composed of
isolated patches with no suitable corridors, with no recent recorded
occurrences in Ghana and Guinea (Bauer & Van Der Merwe, 2004;
Angelici et al., 2015; Henschel et al., 2015). One of the most important
groups of habitat areas is the WAP-Complex, which is mostly composed of
W, Arli, and Pendjari National Parks in Burkina Faso, Benin, and Niger.

Other areas are the Niokolo-Koba National Park in Senegal and the Kainji
Lake National Park and the Yankari Game Reserve in Nigeria (Henschel et
al., 2010, 2014). Although Riggio et al., (2016, E107) argue that the
extinction threat is exaggerated “of nine West and Central African lion
populations, the largest are stable (Bénoué, 200) or increasing (Pendjari,
∼100) …The declining populations are the smallest, with starting
populations of fewer than 70” in a critique of Bauer et al., (2015), the latter
researchers provide substantial evidence to defend their thesis.
Howard (2014) argues that “lions may soon disappear entirely from
West Africa unless conservation efforts improve, a new study predicts”
and cites studies that “found an estimated total of only 250 adult lions
occupying less than one percent of that historic range. The lions form four
isolated populations: one in Senegal; two in Nigeria; and a fourth on the
borders of Benin, Niger, and Burkina Faso. Only that last population has
more than 50 lions.” It is noted that the habitat in the Sahel and Sudan
savanna may still be suitable for lions, but the lion populations are still
declining or locally extinct.
Work by the IUCN and Henschel et al., (2014) on 17 protected areas
found that lions were present in three of the surveyed protected areas,
namely those in Senegal (Niokolo-Koba National Park, with less than 60
lions), Benin, Burkina Faso and Niger (the tri-national W-Arly-Pendjari
Complex, with 246 to 466 lions) and in Nigeria (the Kainji Lake National
Park, 23 to 63) (Henschel et al., 2014). Records were based on the
identification of tracks. These authors note that the claims of lion presence
in Mole National Park in Ghana and in Guinea’s Haut Niger and Kankan
Reserves, were not supported by investigations, and lions appeared to be
extinct in Togo, Guinea-Bissau, Mali and Côte d’Ivoire. The results of this
study suggest that there may be only between 269 and 583 lions in West
Africa, of these 121 to 375 being mature individuals, with “continuing
calamitous declines in prey populations are almost certainly causing
concomitant declines in lions” (Henschel et al., 2014).
This evidence supports the listing of the West African lion as Critically
Endangered, within the Criterions C2a(ii) and possibly C1. The former is
defined by the IUCN as when the “Population size estimated to number

78 Michael Campbell
fewer than 250 mature individuals,” there is a “continuing decline,

observed, projected, or inferred, in numbers of mature individuals” and
“(ii) at least 90% of mature individuals in one subpopulation.” C1 is
defined as when there is “an estimated continuing decline of at least 25%
within three years or one generation, whichever is longer, (up to a
maximum of 100 years in the future).” This chapter looks at the context of
the West African Region as the basis for the status and survival of the West
African lion, and the issues relevant to knowledge of this huge and
important carnivore.
THE CLASSIFICATION OF THE WEST AFRICAN LION
Is the West African lion a distinct subspecies, or a population derived

from North, East or Southern Africa? This has been long disputed, as it has
been variously classified over the past centuries. The initial assumption
was that it was a separate subspecies. The first classification was as Felis
leo senegalensis, defined by Johann Nepomuk Meyer in 1826, who
referred to a specimen from Senegal. Later, it was described as Felis leo
gambiana (Gray, 1843), by John Edward Gray, based on a Gambian
specimen located in the British Museum of Natural History. In 1900,
Matschie classified a Cameroonian specimen as Felis leo kamptzi
(Hemmer, 1974; Haas et al., 2005). Still later, they were considered
synonymous with Panthera leo senegalensis. Bertola et al., (2016) citing
Hemmer (1974) give the name Panthera leo senegalensis and Mazák
(2010) (using craniometric data), Barnett et al., (2006) and Bertola et al.,
(2011a, b, c) argue that the genetic research indicates the Western and
Central African lions might be more related to Asian lions than to lions
from southern or eastern Africa. Other researchers argue for the
distinctiveness of this subspecies by referring to its unique physical
features; noted points are its comparatively small size, less dense or
smaller manes, differently shaped skull and less social behavior (Schoe et
al., 2010; Bertola et al., 2011a, b). For example, in the Pendjari National

Park area, most males were recorded to have sparse or even no manes
(Schoe et al., 2010).
Antunes et al., (2008) examined samples of DNA (deoxyribonucleic
acid) of 11 lion populations, among which were hybrid mixtures from
southern, western and central Africa. The conclusion of this study was that
some West African and Moroccan specimens shared the mitochondrial
haplotypes H5 and H6 and jointly shared with some Asiatic specimens the
mtDNA grouping lineage III. This lineage was hypothesized to have
developed in East Africa and spread northwards and then westwards
forming H5 and H6 in Africa and H7 and H8 in Western Asia (Antunes et
al., 2008). More recent studies using further genetic analysis may suggest
significant differences between the West and Central African lions and
those of the rest of Africa and similarities with Asiatic lions (Bertola et al.,
2011a, b, c). Henschel et al., (2014) also note that “lions in West Africa
contain mtDNA haplotypes not found in other lion populations, elevating
the conservation significance of the few remaining West African
population.” These researchers point out that the creation of a distinct
subspecies could encourage more conservation efforts, through concern
over the loss of a unique variant. However, in 2017, the lion populations in
Northern, Western and Central Africa and Asia were all described as
Panthera leo leo (Kitchener et al., 2017).
Henschel et al., (2015) note that the West African Lion has been
classified as a separate subspecies since 2004, citing Chardonnet (2002),
Bauer and Nowell (2004) and Bauer and van der Merwe (2004) the reason
being the lack of breeding exchange between this and other subspecies.
These assessments were supported by molecular analyses, which may have
improved upon previous assessments which were based on national
borders from Senegal to Nigeria. An important position was that the lion
dispersal across West Africa was limited by the lower Niger River, this
isolating the eastern populations in Central Africa and central/eastern
Nigeria from those in West Africa (where West Africa is defined as west
of the lower Niger River). The lions in Yankari Game Reserve in central
Nigeria have been found to be closer genetically to those in Cameroon,
while western lions in the Kainji Lake National Park (NP) in Western

80 Michael Campbell
Nigeria are closer to those of Benin and Senegal (Bertola et al.,

unpublished, is cited). The final argument is that the results of these
genetic analyses may support the classifications based on national borders,
generally used by the IUCN (Henschel et al., 2015).
THE WEST AFRICAN REGION
West Africa is a distinct sub-region, composed of bands of vegetation

aligned in a west-east orientation and a rapidly expanding, urbanizing
human population (Figure1) (Campbell, 2013). In terms of vegetation, the
area of most concern in terms of lion habitat is the open savanna, divided
in terms of increasing tree cover, into the Sahel, Sudan and Guinea types
respectively (Table 1). As defined by Laris (2011, 1067) “the savanna
biome is defined by the codominance of grasses and trees, the precise ratio
of which is a function of numerous variables, both natural and
anthropogenic.” Lions generally avoid the thicker deciduous and rain
forests, partly due to the general absence of larger herbivores in the
contexts. Deforestation is not therefore a key issue (Campbell, 1998).
Table 1. Vegetation Zones of West Africa
Classification Rainfall (mm) Vegetation Fauna

Semi Desert < 500 Grass clumps Camels, mesofauna
Sahel < 1000 Grass, few trees Mesofauna
Sudan 1000-1500 Grass, shrubs, trees Megafauna
Guinea 1500-2000 Forest, shrubs, grass Megafauna
Forest >2000 Continuous canopy Mesofauna
See also Figure 1 for the map of these areas. Mesofauna = small/medium mammals.
Declining populations of large prey herbivores such as antelopes,

gazelles and African buffalo, the increased intensity of human landuse,
including hunting, farming, livestock grazing and urbanization and the
possible eradication of lions, due to the perception of their danger to

humans and livestock are more important issues for the survival of the
West African lion. As these issues are important for all lion populations all
over Africa, the relevant topics are those that differentiate West Africa
from other African regions in terms of lion status and environmental
variables (Campbell, 2017).
Table 2. Comparison of Conserved Areas in Southern,

Eastern and Central Africa
Country Area No. N. Park Conserved Lion

NP Area % Presence
Southern Africa
South Africa 1,221,037 41 40,000 3.3 Yes
Zambia 752,614 19 2,25784 30
Zimbabwe 390,757 10 27,177.1 7
Namibia 825,418 28 130,000 15.7
Mozambique 801,590 17 185,000 23
Botswana 581,726 12 68,336 11.7
Eastern Africa
Uganda 236,040 20 76,600 35.5 Yes
Kenya 580,367 36 71,966 12.4
Malawi 118,484 5 19,905 16.8
Tanzania 945,203 15 304,355 32.2
Ethiopia 1,104,300 15 203,191 18.4
Sudan 1,861,484 8 31,645 1.7
Central Africa
C.A Republic 622,984 10 31,066 5 Yes
Cameroon 475,442 13 23,920 5
Chad 1,284,000 2 228,552 17.8
Sources: Drewniak et al., (2012). Note the figure for Cameroon includes 9615 km2 in
the forest area outside normal lion habitat. African Wildlife Foundation (2011),
International Bank for Reconstruction and Development/The World Bank (2015),
Partenariat Régional pour la Conservation de la zone côtière et Marine en Afrique
de l'Ouest (2017). Note that some of these numbers vary, according to the
definition of National Park, Conservation Area and Nature Reserve.

82 Michael Campbell
Table 3. Comparison of Conserved Areas in Western Africa
Western Africa
Country Area No. N. Park Conserved % Lion
NP Area Presence
Ghana 238,534 7 36,019 15.1 No
Cameroon 475,442 12 51,823 10.9 Yes
Côte d'Ivoire 322,463 6 13,500 4 No
Mauritania 1 1600
Gambia 10,380 2 436 4.2 No
Liberia 111,369 1 2,784 2.5 Yes
Mali 1,240,192 1 104,176 8.4 No
Niger 1,267,000 1 222,992 17.6 Yes
Senegal 196,723 6 49,574 25.2
Sierra Leone 71,740 4 7,820 10.9 No
G.-Bissau 36,125 5 5,021 13.9
Sources: Drewniak et al., (2012). Note G.- refers to Guinea Bissau. The figure for
Cameroon includes 9615 km2 in the forest area outside normal lion habitat.
African Wildlife Foundation (2011), International Bank for Reconstruction and
Development/The World Bank (2015), Partenariat Régional pour la Conservation
de la zone côtière et Marine en Afrique de l'Ouest (2017). Note that some of these
numbers vary, according to the definition of National Park, Conservation Area
and Nature Reserve.
DeMarco (2015) notes that currently, the status of lions may be

overestimated, as some evidence points to lower population levels than
previously estimated. One key issue concerns a perceived difference
between the status of the southern populations and those of West, Central
and East Africa, with the former being better managed and hence more
durable against badly regulated or illegal sports hunting, and decimated
habitat and prey animals. The inclusion of the East African populations is
debated, as some research indicates these populations are recovering.
DeMarco (2015) quotes a study by Bauer on 47 lion populations across
Africa that argues that in West and Central Africa the human population is
higher and the conservation management is less intensive, hence there was
a 67% chance that the lion population would decline by 50% over the next

20 years. For the lions in eastern Africa, there was a 37% chance that the
population would decline by 50% in the next twenty years. Lions in
southern Africa were comparatively well off and even increasing in some
areas. Factors were the competent management based on fencing of the
southern African populations, contrasted with the free ranging system of
eastern Africa. These assessments were supported by the infrastructure, as
shown in Table 1 for southern, eastern and central Africa, and in Table 2
for western Africa.
URBANIZATION AND THE WEST AFRICAN LION
West Africa, with a human population of 362 million (out of 1.216

billion for Africa, hence West Africa has 30 percent of Africa’s
population) has the fastest human population growth rate of any of the
regions of Africa and the world (United Nations, 2017). The average
distance between agglomerations with more than 10,000 residents has
declined from 111 km to 33 km (Hitimana et al., 2011). However,
urbanization rates vary widely across countries and this region is also the
second slowest in Africa in terms of urbanization, as much of the growth is
also in rural areas (Aniah, 2006). As argued by Mallon (2017) “the major
factors” for the decline of wildlife are “hunting and habitat destruction due
to conversion of extensive areas of original habitat to agriculture and
overgrazing…The region is under tremendous pressure from the needs of a
rapidly growing human population: 337 million in 2013 and projected to
reach 800 million by 2050 and more than 1.5 billion in 2100.”
Urbanization and human population growth has already been blamed
for the decimation or extinction of several large carnivores. These include
the North African lion Panthera leo leo Linnaeus, 1758, which was due to
the long Roman and other habitation of North Africa (Nowell & Jackson,
1996; Black et al., 2013); the Bali tiger Panthera tigris balica Schwarz,
1912 (Jackson & Nowell 2008); the Javan tiger Panthera tigris spondaica
Temminck, 1844, extinct in the 1970s in densely populated Java (Mazák &
Groves, 2006; Jackson & Nowell, 2008); and the Caspian tiger Pantherid

84 Michael Campbell
tigris virgata Illiger, 1815 (Seidensticker et al., 1999; Jackson & Nowell
2008). In North America, the Eastern Cougar Puma concolor Linnaeus
1771 was declared extinct in 2011 (U.S. Fish and Wildlife Service, 2011).
The jaguar Panthera onca Linnaeus, 1758 is largely extinct from the
United States, excepting (possibly) Arizona; is also extinct in densely
populated El Salvador and declining in Mexico and most of Central
America (Hatten et al., 2005). Only very adaptive large carnivores survive
in urban areas (Campbell, 2014).
The most serious threats to African lions through urbanization are the
declining spaces between urban centers, massive development of
connecting transport networks in formerly rural contexts, the increased
exploitation of landcover and herbivore wildlife, and the overall greater
proximity of human presence. Although few studies have examined the
ranges of West African lions, their ranges may be hypothesized by
examining those of lions in other ranges of Africa. For example, Loveridge
et al. (2009) give evidence that lion ranges increased as the food sources
declined and also the size of lion social groups (prides) increased. This
result has dire implications for lions across Africa, as it indicates that the
declining numbers of prey species (due to urbanization, agriculture,
hunting and transport), would require increased ranges for lions, clearly an
impossibility as those human activities also take up more space.
Additionally, as more lions would require more space, any successful
recovery of the lion population through conservation efforts would have to
be matched by more supporting landcover, another impossibility due to
increased human land usage.
The common solution of setting up national parks is questioned, as
partly because of the landuse conflicts with urban expansion and partly
because of the cost (Campbell, 2013). As argued by Kinzig and Mcshane
(2015), landcover and habitat conservation general involves the exclusion
of pre-existing settlers from the required areas, and these people “have
limited understanding of its purpose, derive little or no benefit from its
creation, and hence do not support its existence.” The result of this is that
the local excluded peoples do not have trust in the agencies that establish
or maintain the conserved areas “in part because of the lack of attention

those authorities, supported by conservationists, have traditionally paid to

the link between ecology, the survival of wildlife, and the livelihoods of
displaced people (Kinzig & Mcshane, 2015’; see also McShane & Wells,
2004).
AGRICULTURE AND THE WEST AFRICAN LION
The problems of land conflict between large predators and agricultural

systems is like that between urbanization and large predators (danger to
people and sometimes companion animals), but in the former case there is
also the possibility of killing of commercial livestock (Campbell, 2012,
2017). This issue of lions killing livestock, especially cattle, is a key issue
in Africa (Campbell, 1998; Schwartz, 2017). The dominant agricultural
systems of the West African savanna are based on nomadic cattle herding
and small livestock rearing (termed pastoralism) and cereal cultivation, and
sometimes also shifting cultivation, in contrast to the shifting cultivation,
bush fallowing and intensive commercial cultivation in the southern
forests. Traditional cattle herding is a subsistence lifestyle, which may
produce meat, milk, blood, manure and traction. Cattle rearing in Africa
has been practiced for millennia, following the domestication or semi-
domestication of the breeds of Bos taurus indicus Linnaeus, 1758, the
humped Zebu which originated in southern Asia (Deshler, 1963). Evidence
of cattle herding includes fossil, archaeological, historical and social
sources (Doutressoulle, 1947; Epstein, 1971; Smith, 1980; Muzzolini,
1983; Epstein & Mason, 1984; Shaw & Hoste, 1987; Clutton-Brock, 1989;
Blench, 1993). In West Africa, most of the cattle herders are ethnic Fulani,
which may also tend herds for other tribes such as the Mande in Mali, the
Mossi in Burkina Faso and Ghana and the Hausa in Nigeria and Niger.
Fulani herdsmen who own cattle generally were and often still are
nomadic, moving north into the Sahel and northern Sudan savanna during
the rainy season, and southwards into the southern Sudan savanna during
the dry season (Campbell, 1998, 2013).

86 Michael Campbell
A major factor for the cattle herder’s avoidance of the more forested
regions is the disease trypanosomiasis, which kills livestock, wild
mammals and people, caused by the protozoa Trypanosoma brucei, which
is carried by the tsetse fly, genus Glossina, family Glossinidae (Murray et
al., 1982). Species that transmit the disease include Glossina morsitans, G.
swynnertoni, G. pallidipes, G. palpalis, G. actinides and G. fascines. This
protozoan occurs in the host animal’s blood and is transmitted among
animals through insect bite. This may result in the concentration of
livestock rearing in the savanna regions that comprise the main lion
habitat. Some cattle breeds, such as the Hamitic Longhorns (N'Dama) and
the Shorthorns are trypanotolerant or resistant to trypanosomiasis and
hence could theoretically cope with the southern forest ecosystems. The
N’Dama is indigenous to the Fouta-Djallon highlands of Guinea and may
have been domesticated about 8,000 years ago (Foy, 1911; Chandler, 1952;
Desowitz, 1959; Murray et al., 1982). Other breeds such as the Zebu are
less resistant and may be killed by trypanosomiasis (Dwinger et al., 1992;
Campbell, 1998). The more resistant Shorthorn cattle, occur in the Guinea
savanna belt.
Goats and sheep, mostly trypanotolerant are also reared in cattle
herding areas and are obvious candidates for lion kills. The two main
varieties are the West African Dwarf goats Capra aegagus hircus and
sheep Ovis aries (Adeoye, 1984). Goats and sheep are smaller, cheaper and
faster breeders than cattle, so in many areas there are the main protein and
supplementary income sources for farmers and nomadic herders. They are
particularly important for their flexible foraging ability, which cancels the
need for feeding management, the use of their manure as plant fertilizer
and sales for income for farm supplies (Campbell, 1998).
Regarding lion conservation, there is a conflict between income from
cattle that of tourism; “along with elephants, lions are Africa’s biggest
tourist draw, and what’s more, a natural heritage.” But for many pastoral
communities, wilderness is livestock’s larder. And like a rabid dog loose in
an American suburb, Panthera leo is considered a threat” (Schwartz,
2017). The killing of lions in retaliation for livestock has become a major
factor for the eradication of the lion from human dominated or even human

visited landcover. Several researchers have noted the increasing

importance of cattle for the income of rural stakeholders in West Africa,
especially as cattle are not only a source of immediate meat, but long-term
sources of status and capital (Campbell, 2013). The notion that lions kill
livestock is not unfounded; for example, the Daily Telegraph (2012) a
reliable British newspaper, noted “Lion ‘on a rampage’ kills two in
Nigeria” and further that the lion “killed two herders and 30 livestock in a
settlement in northeastern Nigeria's Yobe State,” this based on the
environment commissioner’s statement. This underscores the problem of
landsharing between people (in this case agriculturists) and large predators.
Chardonnet et al., (2010: 12) also note that “in Mali, for example, lion
predation on cattle is one of the main reasons for the drastic reduction in
the number of lions. In the national parks of northern Central African
Republic, the decrease in lion numbers is largely a result of systematic
shooting by pastoralists during the dry season.” Stephens (2015: 14754)
argues that “sparing …extensive tracts is unlikely to be possible in many
parts of the world, so we must usually choose between coexistence
(sharing lands with large mammals) and the confinement of species in
areas too small for them to play a meaningful ecological role.” In the case
of lions, every attack on people or livestock make cohabitation less likely,
especially with the lack of funding, increasing land-dependent human
population and the vast areas needed for lion foraging.
HUMAN-KILLING AND THE WEST AFRICAN LION
The tensions between lions and people on farmland lead naturally to

the issue of lions killing people. Chardonnet et al., (2010) note that the
problems between lions and people have probably been in existence for
thousands of years, but these may have increased in recent times due to
increased human populations in the Sahel and Sudan type savannas (see
also Cumming, 1982; Bourn and Blench, 1999). They quote the (Ugandan
Game Department Archives, 1962–1963): “The lion population continues
to be fairly strong and well distributed but, because of the necessity to

88 Michael Campbell
shoot cattle-killers and man-eaters, lion must be carefully looked after or

else they will become rare.” Although they mention eastern and southern
Africa as the key problem areas, especially southern Tanzania and northern
Mozambique, Western Africa shares many of the problems (population
growth, human intrusion into lion habitats, killing of wildlife, local non-
cooperation with conservation policy) of these regions (Campbell, 2013,
2017). Lions also have similar predatory habitats towards people
throughout their range (Treves & Karanth, 2003; Quigley & Herrero,
2005).
Löe and Röskaft (2004) ranked lions as third behind tigers Panthera
tigris Linnaeus, 1758 and leopards Panthera pardus Linnaeus, 1758 in
terms of killing people during the twentieth century. Lion attacks on people
are less common in West Africa than in the rest of Africa, possibly because
of the large population of lions in Tanzania., where 563 people were killed
and 308 injuring by lions between 1990 and 2006, Packer et al., (2005,
2006). In Uganda, lions attacked 275 people from 1923 and 1994
(Ugandan Game Department archives, 1923–1994). In West Africa, the
lower lion population and possibly the fewer reliable records may be
factors for the low number of reported kills, although some acknowledge
that isolated, non-reported attacks may occur (Chardonnet et al., 2010).
HUNTING AND THE WEST AFRICAN LION
A further conflict occurs when the lion’s prey animals are killed for the
bushmeat trade or lions themselves are killed by hunters. One contributory
factor is the reduction of other protein sources such as fish stocks and the
general economic decline in local areas, which encourage people to exploit
bush meat sources. As noted by Bauer et al., (2015: 14897) “Lion trends
are consistent with time series data on their main prey species: whereas
herbivore population sizes increased by 24% in southern Africa, herbivore
numbers declined by 52% in East Africa and 85% in West Central Africa
between 1970 and 2005, a position supported by Craigie et al., (2010).
Henschel et al., (2014) note that the killing of the lion’s prey animals is an

even bigger problem than the killing of lions for livestock predation.
Loomis (2014) suggests that in Senegal, the bush meat trade has decimated
prey especially Niokolo Koba's buffalo and roan antelope by 95 percent
over the past 20 years. Table 2 below shows the main prey species of lions
in West Africa. Almost all have severely reduced populations.
Table 4. Status of some lion prey species
English name Scientific name Status

Roan Antelope Hippotragus equinus Least Concern, declining in
(É. Geoffroy Saint-Hilaire, West Africa.
1803)
African Syncerus caffer (Sparrman, Least Concern, declining in
Buffalo 1779) West Africa.
Common Phacochoerus africanus Least Concern, declining in
Warthog (Gmelin, 1788) West Africa.
WA Giraffe Giraffa camelopardalis Threatened, isolated
peralta Thomas, 1898 populations.
WA Alcelaphus buselaphus Least concern, sharply
Hartebeest (Pallas, 1766 A. b. major declining, locally extinct.
(Blyth, 1869)
WA Topi Damaliscus lunatus Vulnerable to endangered.
ssp.korrigum (Ogilby, 1837)
Dama Gazelle Nanger dama (Pallas, 1766) Critically Endangered
Dorcas Gazelle Gazella Dorcas (Linnaeus, Vulnerable, declining
1758)
Red-fronted Eudorcas rufifrons (Gray, Least concern, declining
Gazelle 1846)
Oribi Ourebia ourebi Zimmermann, Least concern, declining.
1782
Western Giant Tragelaphus derbianus Much rarer than Eastern
Eland derbianus (Gray, 1847) Giant Eland, declining.
Kob Kobus kob (Erxleben, 1777) Least concern, vulnerable.
Sources: International Union for the Conservation of Nature IUCN (2008a, b, c, d;
2016a, b, c, d); de Jong et al., (2016); Fennessy et al., (2016).

90 Michael Campbell
Lions may also be the victims of hunting; the motives for this may be
cattle killing, trophy extraction and use of their flesh for traditional Asian
medicine (Platt, 2011). Chardonnet et al., (2010, 42) note that in
comparison with other African regions “in west Africa there is currently
much less lion hunting, though pastoralists tend to shoot and poison lions
to protect their stock. Little information is available on the effects of
hunting in the region.” Platt (2011) gives an interesting report on the
possibility of introducing “conditioned taste aversion” which entails
conditioning lions to reject the taste of beef, an approach which has
“worked with some other endangered species, including Mexican wolves
and quolls in Australia.” This idea is based on the work of Denver Zoo
research associate Bill Given on captive lions in the Grassland Safari
Lodge in Botswana. Here lions, previously cattle killers were fed beef
treated with high doses of the deworming agent thiabendazole, which
resulted in temporary indigestion, and later rejection of beef and possibly
the scent of cattle.
THEORIES ON THE HUMAN/LARGE

CARNIVORE RELATIONS
Few studies have documented the relations between people and lions
in West Africa (Campbell, 2017). Nevertheless, there is a comprehensive
methodology on the relations between people and large, especially
potentially dangerous wildlife, encompassing gender and age-related
attitudes to wildlife presence in shared micro-spaces, possible differences
on impacts on adults, men, women and children, livestock and companion
animals and livelihood activities, species reintroduction policies and
methods for predator control. Few studies of this depth have been
conducted on the West African lion. Some other species have been better
covered, and a cursory examination of the main ideas and applications may
inform the possibilities for the West African lion, especially when direct
policy actions are being considered. The studies mentioned in this section

fall within many subjects, such as biogeography, conservation biology,

political ecology and rural geography (Campbell, 2013, 2017).
From the perspective of the current study, the science of biogeography
is particularly important as the overarching discipline, due to the spatial
considerations in the lion’s range. Biogeography is necessarily a well
linked, yet spatially diverse and multi-paradigmatic subject and therefore
has some of the necessary tools for the examination of the complex
relations in a region such as West Africa (Campbell, 1998). This diversity
and cohesion of biogeography as a theoretical and applied discipline is
apparent in attempts to define the subject. Huggett (2004: 3) argues that
“biogeographers address a misleadingly simple question: why do
organisms live where they do?”
The broad socio-environmental factors for the distribution of most
organisms require biogeographical researchers to connect with and even
‘borrow’ from other disciplines, especially those with cross-disciplinary,
interdisciplinary and multidisciplinary analytical tools and practical
applications. These include conservation biology, which is an even broader
subject than biogeography with strong links to socioeconomic, legal and
political dynamics (Campbell, 2017). Conservation biology’s “main
thread” has been defined as “the description, explanation, appreciation,
protection, and perpetuation of biological diversity”; working towards
“wilderness protection, sustained yield, wildlife protection and
management, the diversity-stability hypothesis, ecological restoration,
sustainability, and ecosystem health” (Meine, 2010: 7). In terms of
applications, conservation biology may also be perceived as “a mission
driven discipline comprising both pure and applied science”; and “a new
field, or at least a new rallying point for biologists wishing to pool their
knowledge and techniques to solve problems” (Soulé & Wilcox, 1980).
From the social sciences side of investigation, which is missing from
the studies of the West African lion, political ecology offers a reasonable
groundwork, in that it deals with social and political, group related power
dynamics, especially in applications to scenarios of conflicts in
perspectives and access nature-society relations. Political ecology may be
defined as “an approach to, but far from a coherent theory of, the complex

92 Michael Campbell
metabolism between nature and society” (Watts, 2009: 545), which “seeks
to understand the complex relations between nature and society through a
careful analysis of what one might call the forms of access and control
over resources and their implications for environmental health and
sustainable livelihoods” (Watts, 2009: 257). This discipline, like
biogeography and conservation biology, is broad and therefore an incisive
baseline for the study of human-carnivore relations (Campbell, 2013,
2017).
Political ecology as originally designed by geographers and
anthropologists used techniques explored by the disciplines of political
economy, cultural ecology and anthropology, which would be applicable to
socio-environmental problems at both macro and micro levels (Wolf 1972;
Blaikie, 1985, 1999; Blaikie & Brookfield, 1987; Greenberg & Park, 1994;
Robbins 2004). It further advanced interdisciplinary methods to avoid the
structuralist disciplinary boundaries intrinsic to older disciplines and
hampered holistic knowledges and discourses that served as the basis for
integrated projects (Robbins, 2004; Biersack & Greenberg, 2006; Elwood
2010). Political ecology addressed natural resource conservation;
environmental conflicts; scaled resource use politics; power group
marginalization; politics of consumption and production; identities and
claims; externalities of state incompetence; and property access and rights,
all of which would be relevant to the complexities of lion conservation in
West Africa, especially as this would include international ‘First World’
issues (McCarthy, 2002, 2005, 2006; Walker, 2005, 2006; Robbins, 2004;
Pomeroy, 2012; Campbell, 2017). Within the field of geographical
perspectives on conservation, advocates of political ecology designed re-
assessments of the methods of inquiry, “conceptualizing and articulating
the relationships between structural processes and local contexts, and
clearly establishing which methods and data may be best used to get at
which aspects of these relationships” (Elwood, 2010, 104).
Studies of large wildlife emanating from these methodologies include
those of Dickman, 2010, Campbell and Torres Alvarado (2011) and
Campbell (2012). These studies found that the conservation and presence
of large carnivores is particularly challenging, due to the size, strength and

consequently more media coverage, and financial support. Although some

studies show similar public attitudes to different species of large carnivores
(for example, Morzillo et al., (2007, 418) argues “people tend to respond
quite similarly to different large carnivore species regardless of ecological
and behavioral differences,” a point echoed by Kellert et al., 1996; Kleiven
et al., 2004), others show differences in perceptions dependent of on real or
imagined dangers, generational and gender related differences among the
appraisers and the positions of the public media (Campbell, 2012).
Campbell and Torres Alvarado (2011) and Campbell (2012) posed
hypotheses derived from the literature that older people and women have
greater fear for large carnivores, women and young people have more
support for carnivore conservation, and people are more afraid of those
carnivores with more dangers reputations (such as tigers, cougars and
brown bears, compared with black bears). The results of these studies in
Canada and Central America were that carnivore size was as important as
the popular perceptions on dangerous behavior and that the age and gender
of the respondents were significant for attitudes towards conservation and
tolerance of carnivores. These points were also relevant for species
reintroductions, where the large carnivores were previously extinct, but
people had formed opinions based on the general media, shared knowledge
with people living in shared carnivore habitats and older people with
memories of carnivore presence (Campbell, 2017). Attitudes to black bears
largely concern their role in property damage and nuisance behavior
(Morzillo et al., 2007; Freedman et al., 2003; Herrero & Higgins, 1999;
Decker et al., 1981). For cougars, the issues are, fear of attacks on people,
especially children and general uneasiness (Beier, 1991, 1993; Turner et
al., 1990; Herbert, 1989).
As these studies tested human reactions to brown and black bears,
cougars and jaguars, it may be implied that opinions of the lion may be
more negative, as lions have a far stronger record on human killing than
even the brown bear and the jaguar in the Americas, which may be more
dangerous than the black bear and cougar (Campbell & Lancaster, 2010).
Recent studies have classified carnivores according to their human killing
propensity (Löe & Røskaft, 2004; Quigley & Herrero, 2005). Linnell and

94 Michael Campbell
Alleau (2016) argue that of the human killing animals documented in the
literature, “only tigers, leopards, sloth bears, lions and brown bears kill
people on a regular basis (i.e., annually).” The listed species included
twelve that commonly kill people and five more that do so only rarely.
Including the literature sources, the twelve included five felines (tigers
Panthera tigris, lion Panthera leo, leopard Panthera pardus, cougar Puma
concolor, jaguar Panthera onca) (Neto et al., 2011; Mattson et al., 2011;
Bloomgaard, 2001; Dhanwatey et al., 2013; Chomba et al., 2012;
Yamazaki & Bwalya, 1999; Packer et al., 2005; Athreya et al., 2011;
Goyal, 2001), four ursines (polar bear Ursus maritimus, brown bear U.
arctos, American black bear U. americanus, sloth bear Melursus ursinus
(Bargali et al., 2005; Rajpurohit & Krausman, 2000; Fleck & Herrero,
1989; Clark et al., 2012; Chestin, 1993; Gunther & Hoekstra, 1998) and
three canids (grey wolf, Canis lupus Linnell et al., 2002; Fritts et al.,
2003), dingo Canis dingo and coyote Canis latrans). The less dangerous
species were the spotted hyaena (Crocuta crocuta), striped hyaena
(Hyaena hyaena), Asiatic black bear (Ursus thibetanus), sun bear
(Helarctos malayanus) and spectacled bear (Tremarctos ornatus)
(Linnell & Alleau, 2016).
The issues above document the more negative side of human-large
carnivore relations. There are also more positive issues. For example,
commenting on the work of the IUCN, Ridolfi, the Director at the
Sustainable Growth and Development program at the European
Commission Directorate for International Cooperation and Development,
argued (Vergnaud & Bradley, 2017):
The role and importance of large carnivores is recognized as being of

critical significance for the protection of fragile equilibriums of entire
ecosystems. Yet, increasing pressures on land and water resources are
leading to conflicts between man and animals and eventually the
irreversible degradation of whole landscapes. The involvement of local
communities as forefront actors in the conservation of threatened
carnivore species is of crucial importance and has proven to be a long-
underestimated key to success when it comes to sustainability and
efficiency.

Breitenmoser concurs and argues that “conserving lions, leopards and

cheetahs will help us conserve other species. Meanwhile, we will have to
address a broad range of threats and conflicts and involve many parts of
society in different ways depending on the species in question” (Vergnaud
& Bradley, 2017). However, despite these laudable objectives, and the
acknowledgment of local involvement, frequent criticisms are made that
local people are not partnered sufficiently, jeopardizing the results. This
would be particularly relevant in West Africa, considering the inferior
research findings, media promotion and funding compared with other parts
of Africa. The funding aspect is very important, albeit frequently neglected
in the current literature. For example, the commentary by Stephens (2015)
correctly points out the common points of large carnivore conservation
(comparing wolves in the United States and Scandinavia, with lions in
Africa), but such comparisons must be aware of the differences in funding
availability between Africa and the United States.
An analysis of the different types of participation may shed light on
this problem. For example, (Pretty, 1995) described seven forms of
participation, the first four (manipulative, passive, consultative and
material incentive based) are defined as “non-participation,” with “no
positive, lasting effect on people’s lives” (Pretty 1995, 1252). The
manipulative method controls local people’s input even they are present;
the passive method allows less control, but no shared participation; the
consultative method includes an agenda set and controlled by
professionals, with no power devolution to local actors; the incentive
method gives rewards but not power devolution of real consultative power
to local respondents. The last three, possibly better forms of participation
are: functional participation, which sharing objectives and some decision
making with local actors; interactive participation, which encourages more
joint actions between local and outside actors; and self-mobilization which
allows independent action for local actors, possible with requested help
from outside (Pretty & Shah, 1997).
In the West African context, the last three forms of participation
clearly be the most effect. An important caveat however, would be the
acknowledgement of the interest of the local people. Why should they

96 Michael Campbell
support these conservation actions, considering they need land, cattle and
their own safety? The difficulty would be to go beyond the mere self-
mobilization of the local people, which would be difficult without strong
commitment on their part, to sell the worth of the projects to the local
people’s context. As noted by Zinzig and McShane (2015) “conservation
remains one of the most visible and contentious areas of contact between
Africa and the West.” Basically, the argument would be that more lions,
and the encouragement of the ecological settings required, would mean
benefits and not problems for the local people. Considering the complexity
of the issues and the inter-connectedness of the biological, socio-economic
and political components at international, national and local levels, this
will be extremely difficult endeavor.
CONCLUSION
The evidence the literature shows that the West African lion is facing
serious problems for its survival. These include current regional problems,
such as human population increase and related developments in
urbanization, agriculture, landcover change, decimation of prey animals
and antagonization of people through livestock and human kills. But the
problems facing the lion in West Africa may also be seen to run deeper,
through the examination, not only of lion status in other regions of Africa,
but also of large carnivore/human relations concerning other species on
other continents. Evidently human and livestock killing, and public
attitudes to these and other nuisance behaviors are serious problems that
may militate against conservation efforts.
Concerning the problems faced with carnivore conservation in other
continents, concerning less dangerous mammals (such as the jaguar and all
the bear species), similarly dangerous species such as the leopard and the
slightly more dangerous tiger) and more adaptive species (such as the
cougar), and the fact that predator conservation may be more advanced and
socially supported in other continents, lion conservation may be an uphill
battle. West Africa faces more serious problems than the rest of Africa,

because of fewer conserved areas, less international support and funding

and fewer available research findings. The initial actions would be more
detailed research, deriving parameters from studies on other species and in
different regions and issues peculiar to the lion in West Africa. The next
set of actions would concern political support from both West Africa and
international sources, and social support from local people. Finally, local
ecological work would require the support, not only of lions but of the prey
animals upon which the lion depends.
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Chapter 4
GIRLS OF A FEATHER FLOCK TOGETHER:

SPATIAL PROXIMITY IN ADULT KIN IN
GREYLAG GEESE
(ANSER ANSER)
Isabella B. R. Scheiber1,* and Brigitte M. Weiß2,3

1
Department of Behavioural Biology, University of Vienna,
Vienna, Austria
2
Behavioural Ecology Research Group, University of Leipzig,
Leipzig, Germany
3
Max Planck Institute for Evolutionary Anthropology,
Leipzig, Germany
ABSTRACT
In birds, the formation of stable social bonds beyond a mated pair has
only been reported in a few monogamous species with a complex social
system, such as in corvids. Also, in various species of waterfowl, like
*
Corresponding Author Email: isabella_ scheiber@t-online.de.

112 Isabella B. R. Scheiber and Brigitte M. Weiß
swans, ducks and geese, such bonds are established not only between
pairs, but also within families, which may last far beyond independence
of offspring. Surprisingly, the story does not end here: Adult females
maintain bonds with their collateral female kin, i.e., sisters, throughout
life [1]. Sisters benefit particularly during the breeding season, if their
long-term bonding partners help in raising offspring, thereby reaping
benefits via kin selection. However, this does not explain the persistence
of these bonds outside the reproductive season. Whereas organization into
matrilines is a common pattern in group-living mammals, female-
centered clan structures are quite unusual for birds. Yet, the exceptional
characteristic features of waterfowl breeding systems, such as early pair
formation, female-biased philopatry, long-term monogamy, and precocial
development of offspring, may explain the formation of long-term adult
bonds among females. In this chapter we will first review the knowledge
of extended family bonds between females in mammals and birds and
will then dwell on why, based on their breeding system, waterfowl are
particularly predisposed to female-centered kin clans. Second, we will
present a hitherto unpublished experiment investigating whether lineal
kin, i.e., mothers and their adult daughters, form long-term bonds in a
well-studied model organism within waterfowl, the greylag goose (Anser
anser). We found that female and male greylag geese form long-term
social bonds with their parents, measured as close proximity. Long-term
social bonds are prevalent in waterfowl and stand, hence, in stark contrast
to other birds. We finish this chapter by proposing mechanisms enabling
the special social structure of waterfowl, which includes long-term social
bonds.
Keywords: greylag goose, Anser anser, female-centered clans, flock

dynamics, parent-offspring bonds, social support
“A son is a son till he takes him a wife, a daughter is a daughter all

of her life”1.
INTRODUCTION
Social Organization in Birds and Mammals
The complexity of social systems in vertebrates is defined by the

extent and nature of (repeated) social interactions with differently familiar
1
Irish Saying.

Girls of a Feather Flock Together 113
conspecifics [2] and is generally structured through cohesion within pairs,

families, or similar alliances [3, 4]. Many mammalian societies, and
particularly those that are considered highly complex, are frequently
organized around female kin, such as cetaceans [5, 6], ungulates [7, 8],
carnivores [9, 10], rodents [11], bats [12] and monkeys (reviewed in 13].
This also seems to be the ancestral state in human societies [14]. Those
social groups tend to be matrilines, where descendants can be traced
through the female line, resulting in high levels of female relatedness [15
and references therein). One underlying reason for this finding is that most
mammals have male-biased dispersal, where young males move away from
their places of origin [e.g., 16], whereas females display a tendency to
breed at or near their own birth site (female natal philopatry, [17, 18]) in
order to lower intra-sexual competition or the chance of inbreeding [19,
20]. Thus, in mammals, females form large clan-families which result in
the formation of social bonds between females that consist of several
generations living in the same place.
In the majority of bird species, however, social bonds are centered
around the reproductive pair and are often limited to the breeding season. It
is estimated that just 10% of the extant bird species form cooperative
breeding groups, in which independent young or unrelated individuals stay
with a parental pair to help raise the next generation of offspring, thereby
maintaining extended social relationships [reviewed in e.g., 21, 22].
Contrary to mammals, females are the dispersing sex in birds, while males
are philopatric [17, 23, 24]. Clarke et al., [24] describe the few exceptions
of male dispersal in a total of 22 passerine and non-passerine species,
representing 12 families. In most of those species some degree of male
dispersal is found only within a subset of the populations, e.g., males that
lost their mates, in certain locations but not others, or in some years and
not others [24]. True male dispersal patterns were described for only a very
small number of species, including white-browed sparrow weavers
(Plocepasser mahali), white-throated magpie-jays (Calocitta formosa), and
brown jays (Cyanocorax morio, [25-27]). From a functional point of view,
and similar to mammals, fitness consequences of philopatry in birds may
come about either directly, (i) if family groups have a higher chance of

survival, (ii) if an individual can increase its own reproduction within

family groups and/or (iii) indirectly by helping raising young of kin [28].
There are also cases in which kin clustering is the result of a preference for
a location rather than being based on descent, a phenomenon termed
matrilocality in case of mammalian female groups in social anthropology,
[29] or social vicinity in behavioral ecology [30].
Social Organization in Waterfowl
In contrast to the above-mentioned general pattern of avian female

dispersal, and hence quite unique among birds [31], is the dispersal pattern
of waterfowl (Anatidae). Here, without exception, all species show female-
biased philopatry [32]. Most species of waterfowl exhibit mate defense
mating systems [24], where mate selection takes place on the winter
grounds [24, 33]. Pairs arrive at the breeding grounds together, with males
following their mates to their hatching areas [32, 34]. The most commonly
accepted hypothesis that explains female philopatry in waterfowl is the
local knowledge hypothesis, which posits that females take advantage of
familiarity with a successful breeding area, including, for example,
knowledge of irregular food resources, predator routines or location of
conspecifics [35-37]. Female philopatry is thought to be the major
determinant to open the route to female-centered clans in waterfowl.
Kin-based female social structure in Anatidae has been described both
via behavioral observations of banded birds [e.g., 38-40] and using genetic
markers [e.g., 34, 41, 42]. We will here provide an overview of the nature
and extent of those kin clusters. In lesser snow geese (Anser caerulescens
caerulescens) Cooke et al., found a tendency for females from the same
age cohort to cluster together when nesting [43, 44]. However, due to the
lack of knowledge concerning the social background of wild individuals, it
remained uncertain whether clustering was due to social attachment or
reflected philopatry. Long-term studies in barnacle geese (Branta
leucopsis, [40, 45, 46]) suggested that individuals reared in the same place
at the same time moved and established breeding colonies together. Female

common eiders (Somateria mollissima) can rear their broods alone, but are
one of the best examples of several females cooperating to raise
‘amalgamated’ broods. Initially Öst et al., [47] found that coalitions were
as likely to consist of kin as of non-kin, yet a closer look revealed that this
was not always the case. Whereas coalitions of related females decreased
the larger the groups became, older females preferred smaller coalitions
with higher degrees of related individuals [48].
Kin selection, where females increase their fitness indirectly by
contributing to raising the young of relatives, is facilitated by female
philopatry in waterfowl [49-51, see also 52 for a recent review]. Although
not necessarily the case [51, 53-55], many species preferentially transfer
parental duties either before (via conspecific brood parasitism) or after
hatching (via adoptions, créches or communal rearing) to female relatives
[49, 56-58]. Thereby they potentially gain benefits from kin selection, e.g.,
the black swan (Cygnus atratus) [59], and common (Bucephala clangula,
[60]) and Barrows goldeneye (B. islandica, [61]) as representatives of
swans and ducks, as well as the greater white-fronted goose (Anser
albifrons frontalis) [62], Greenland white-fronted goose (A. a. flavirostris)
[63], and barnacle goose [54, 64] as representatives of geese. In Canada
geese (Branta canadensis), subadult (i.e., one-year-old) females helped
parents to raise subsequent young, whereas males never did [65]. Although
kin recognition is not absolutely required, it has been repeatedly suggested
that this may be facilitated by an ability of various waterfowl species to
differentiate between kin and non-kin [56, 66, 67].
Long-Term Kin Bonds in Waterfowl
All above mentioned benefits of sex-biased philopatry are in the

broadest sense directly related to successful reproduction in a given year. It
does not explain, however, why waterfowl do not break family bonds when
young birds become independent, but extend family bonds well beyond
this point, leading to long-term social bonds. Unlike many other species of
birds, waterfowl families frequently do not break up shortly after the young

fledge, but last until the onset of the next breeding season, and in some
species of geese, family bonds are maintained until the young sexually
mature at two or three years of age [39, 46, 63, 68-73]. Extending family
bonds beyond independence of the offspring is, per definition, found in all
territorial cooperative breeders [21, 74]. Cooperative breeding, however, is
rare in waterfowl [75], with the most common breeding systems being
uniparental female care in ducks and biparental care in geese and swans
[33]. Furthermore, contrary to many passerine species, where altricial
young need extended parental care after hatching, all waterfowl species are
precocial, with young being relatively mature, mobile and self-sufficient in
feeding shortly after hatching [2, 33]. So, why are waterfowl so vastly
different from the majority of birds with respect to philopatry and extended
family bonds? Using greylag geese (Anser anser) as a model, we aim to
shed light on these questions, not only from a functional, but also from a
mechanistic point of view.
Female-philopatric greylag geese are long-term monogamous, with
both parents caring for the precocial young. They exhibit a highly complex
social system [76-80] and flock for most of the year [81, 82]. Migrating
and wintering flocks include families, pairs without offspring, and
singletons [38]. In greylag geese, male and female young-of-the-year
usually stay with their parents until the onset of the following breeding
season, and may rejoin their parents for another year if the parents fail to
reproduce in the consecutive breeding season [81-83]. It is well established
that goose families benefit from social support, i.e., the stress-reducing
effect gained from the presence of social allies, as long as they are part of a
family [76, 83-88]. We suggested this to be the mechanistic explanation of
the extended family bonds in greylag geese, where we termed independent
offspring as ‘physiological helpers’. Notably, it is mainly the females that
benefit from the presence of family in terms of stress reduction [83, 86].
Furthermore, strong family bonds exist between female collateral kin,
i.e., sisters throughout life (Figure 1), whereas such relationships are not
maintained between male collateral kin, i.e., brothers [1]. These bonds are

expressed not only within [76], but also outside the breeding season [1];
therefore, cooperation in raising present offspring can only partly explain
these bonds.
Figure 1. Close proximity of greylag goose sisters during brood rearing.
Throughout the year, adult sisters, but not brothers, can be found
resting in closer proximity to each other irrespective of their age, pair bond
status and preferences for particular resting locations [1], suggesting
female clustering due to social attachment. This is supported by the fact
that greylag geese can truly distinguish between individual siblings from
an early age onwards [89]. In a perpetual study, we wanted to extend this
idea to lineal kin, i.e., parent-offspring dyads, by addressing the following
questions: (i) Do adult lineal kin rest closer to each other than other non-
kin members of the flock and, if so, does this reflect a common preference
for a particular site, or, alternatively, is such clustering of kin due to social

bonding? (ii) Is there a difference between sons and daughters in proximity

to their father and mother? (iii) Finally, is there an effect of age or social
status, i.e., paired versus single geese, and their distance to kin?
STUDY AREA, STUDY POPULATION

AND EXPERIMENTAL DESIGN
The Grünau Flock of Greylag Geese
In 1973, Konrad Lorenz and co-workers established a non-migratory,

free-roaming flock of greylag geese in the valley of the River Alm in
Grünau, Upper Austria ([90] for details), where the flock is still found
today. The geese roam the area around the Konrad Lorenz Forschungs-
stelle (KLF, now renamed Core Facility for Behaviour and Cognition of
the University of Vienna, 47°48’50.16’’N, 13°56’50.92’’E) and a lake
approximately 10 km to the south, where they roost at night, year round.
The flock is supplemented with pellets and grain twice daily on the
meadows around the research station, with low quantities from spring to
fall, and with sustaining amounts during winter. Individual life history data
and social backgrounds of all individuals have been monitored
continuously since 1973. Every few years a few geese are carefully hand-
raised to keep the flock accessible to humans. Both hand-raised and goose-
raised flock members are habituated to the close presence of humans [91]
and do not show avoidance behavior, elevated levels of immuno-reactive
corticosterone metabolites [92] or significant heart rate changes when
familiar humans approach [93]. Many studies pertaining to the social life
of geese, in an attempt to decipher even subtle costs and benefits to
sociality, have been performed with this flock, and we summarized those
findings in a book recently [77]. At the time of data collection for the lineal
kin study, the flock consisted of around 140 geese, all of which were
individually marked with colored leg bands.

Table 1. Life history parameters and parents of 32 focal individuals.

M = male, F = female, S = single, LP = loosely paired, P = paired,
P+ = paired with offspring. Missing cells in the Mother, Father or
Hatch Year columns indicate that the respective individual was
missing or dead at the time of data collection
Individual Hatch Sex Social Mother Hatch Father Hatch

Year Status Year Year
Beleriand 2003 F S Bwejuu 2000 Tipi 1999
Mohican 2004 F S Little Indian 1993 Mario 1986
Jaspis 2000 F LP Jessie 1996 Herbert 1998
Large 2004 F LP Levi 1998 Plum 1998
Baggins 2003 F P Balu 1993 Sinus 1992
Barbados 2004 F P Bwejuu 2000 Tipi 1999
Jewel 2003 F P Jade 2000 Cheyenne 1999
Joey 2004 F P Jessie 1996 Herbert 1998
Levi 1998 F P Lestate 1995
Algonquin 2004 F P Little Indian 1993 Mario 1986
Theoden 2003 F P Timber 1999 Murphy 1999
Timber 1999 F P Tlingit 1996
Lorbeer 2003 F P Iwan 1993
Bwejuu 2000 F P+ Balu 1993 Sinus 1992
Bombadil 2003 M S Balu 1993 Sinus 1992
Balin 2003 M S Bwejuu 2000 Tipi 1999
Jacaranda 2004 M S Jacky 1999 Tristian 1999
Jamaica 2004 M S Jade 2000 Cheyenne 1999
Jordan 2004 M S Jessie 1996 Herbert 1998
Little 2004 M S Levi 1998 Plum 1998
Uncas 2004 M S Little Indian 1993 Mario 1986
Neptun 1994 M S Rocky Grün 1990
Bob 1998 M P Balu 1993 Sinus 1992
Boston 2004 M P Bwejuu 2000 Tipi 1999
Joshua 2003 M P Jessie 1996 Herbert 1998
Lesley 1998 M P Lestate 1995
Lindon 2003 M P Levi 1998 Plum 1998
Nenya 2003 M P Nasse 1995 Verdi 1996
Tarek 1999 M P Tlingit 1996
Bailey 2000 M P Nestor 1990
Dylan 1998 M P Punki 1992
Tipi 1999 M P+ Tlingit 1996

Focal Individuals
For this study, we collected data from 32 individuals, of which either

both parents (n = 23) or one parent (n = 5 mothers only, n = 4 fathers only)
were still alive during the time of data collection (December 2006 to
February 2007, Table 1). We only considered sexually mature animals
older than two years. To avoid pseudoreplication on the level of the family,
we did not consider offspring of the same sex and social status more than
once per parent. In total, we analyzed data from nine paired females, nine
paired males, eight single males, two single females, one paired female
with offspring, one paired male with offspring and two loosely paired
females. This represented all offspring in the flock at the time meeting the
above criteria.
Control individuals were matched in age and social status as closely as
possible to parental birds, but were unrelated to the focal individuals and,
consequently, to their parents. If a parental bird had more than five
possible control animals in the flock, we chose five of them at random. The
age of the focal geese ranged from 2 to 12 years ( X ± SE: 4.22 ± 0.47),
the age of mothers ranged from 6 to 13 years ( X ± SE: 9.27 ± 0.79), the
age of fathers ranged from 7 to 20 years ( X ± SE: 11.3 ± 1.23); age of the
control females deviated from mothers by 0.51 ± 0.16 years ( X ± SE), age
of control males deviated from fathers by 0.8 ± 0.23 years ( X ± SE).
Data Collection
After the morning and afternoon feeding, the geese rested on the
meadows in front of the research station, that is, an area of more than 2,000
m2. Starting approximately 30 minutes after feeding, we identified the
position of each focal goose, its parents, as well as the five respective
control females and males. If leg bands were not visible we identified
sitting geese from head and body shape, which is possible for humans who
are well-acquainted with the geese, and later confirmed their identities after

the individuals got up. Once we discovered relevant individuals, i.e., focal,
parental and control birds, we marked their positions with removable
nametags mounted on a wooden stick. The geese were so human-
acquainted (see above) that we could place nametags without disturbing
the positioning of the geese. Once all positions were marked, we measured
distances between each relevant dyad (focal birds to parent(s) and control
individuals) with a Leica®, DistoTM A2 Laser Distance Meter to the
closest decimeter. In total, each focal bird was recorded 15 times. To
obtain independent samples, we avoided consecutive observations of the
same individuals during the same resting period. We only collected
distance data if focal birds, parents, and at least four of the five control
individuals were present.
Figure 2. Map of the study site around the Konrad Lorenz Research Station, Core
Facility for Behaviour and Cognition of the University of Vienna, in Grünau im
Almtal, Austria with division into 15 subunits. (Source: “Konrad Lorenz
Forschungsstelle”. 47°48'50.41"N and 13°56'50.93"E. Google Earth. Image © 2009
Geoimage Austria. 30.01.2009).

Furthermore, to investigate the effects of place preference during rest,

we collected data on the resting location of all 78 participating geese; that
is focal individuals, parents, and control individuals. The roaming area of
the geese surrounding the KLF was divided into 15 subunits determined by
the composition of the terrain around the station (Figure 2). Overall, we
recorded each individual's resting location 24.5 ± 1.47 times ( X ± SE;
range 10 – 65).
Data Analyses
We analyzed dyadic distances by computing a Linear Mixed Model

(LMM) in R [version 3.4.1, 94] using the lme4 package [version 1.1-14,
95]. We used square-root transformed distances as the Gaussian response
variable and fitted the effect of dyad type (mother, father, control female or
control male) as three two-way interactions of dyad type with either the
focal's sex, social status (single, paired, or parental) or age. This allowed us
to assess whether any effect of dyad type on resting distances differed
between sons and daughters, individuals of different social status, or age.
We controlled for the identity of the focal individual, the identity of the
dyad partner, the dyad (i.e., unique combination of focal bird and dyad
partner) and the observation period (1-15 per focal bird) by fitting these
terms as random effects. To achieve more accurate p-values, we further
fitted all random slopes showing sufficient variation within each random
effects level [96]. The model fulfilled assumptions of normal residuals and
homoscedastic variance and showed no indications of collinearity between
predictors or presence of influential cases. P-values were determined with
Likelihood Ratio Tests (LRT) comparing the full model with the null
model lacking all test predictors, i.e., the interactions, or a reduced model
lacking the specific term of interest. Non-significant interactions were
removed to facilitate interpretation of the main terms, while non-significant
main terms were kept in the model to avoid multiple testing issues.
To detect individual preferences for a particular resting location, we
calculated individual 2-Goodness-of-Fit Tests for each of the 78

individuals separately. All tests are given two-tailed and significance level
was set to p = 0.05.
RESULTS
Lineal Kin in Greylag Geese – Proximity to Parents
The type of dyad significantly affected resting distances of our focal

individuals (Full-null model: LRT, 2 = 46.382, df = 19, p = 0.0004),
whereby our focal individuals generally rested closer to parents than to
unrelated controls (LRT parents vs. controls: 2 = 14.67, df = 1, p =
0.0001, X ± SE: focal – mother 19.1 ± 1.4 m, range 9.7 – 36.6; focal –
female controls 25.3 ± 1.47 m, range 13.0 – 36.6; focal – father 18.9 ± 1.45
m, range 10.1 – 36.8; focal – male controls 21.6 ± 1.37 m, range 14.2 –
36.8). Mean individual resting distances were shorter to parents than to
unrelated controls for 25 out of the 32 focal individuals (Table 2). Resting
distances to mothers and fathers were generally similar (LRT mothers vs.
fathers: 2 = 0.035, df = 1, p = 0.851, see Table 2, Figure 3), which is not
surprising given that for 23 out of 32 focals both parents were still alive,
and paired geese, in this case the parents of our focals, are always found in
close proximity to each other. Notably, the effect of the dyad type on
resting distances did not significantly differ between the sexes (interaction
dyad type * focal sex: LRT, 2 = 1.72, df = 3, p = 0.633), which implies
that the pattern of resting closer to parents than to unrelated controls was
similar for both, females and males.
Also, social status did not affect the relationship between the type of
dyad and resting distances (interaction: dyad type * focal individual`s
social status: LRT, 2 = 7.835, df = 6, p = 0.251): Single individuals
showed the same resting patterns as paired, loosely paired, and parental
individuals. As loosely paired and parental birds were represented in the
data by only few individuals, these results primarily reflect single and
paired individuals. A closer look at the resting distances of the individuals

from the underrepresented social classes showed that the two loosely
paired females (Table 2, Jaspis and Large), both pursuing potential mates
at the time of data collection, were not closer to their mother than to
control females. Yet, one appeared to maintain a closer proximity to her
father relative to control males. Finally, the only paired female with
offspring (Table 2, Bwejuu) showed closer proximity to both her mother
and father than to control individuals, whereas her mate, the only paired
male with offspring (Table 2, Tipi) did not show this proximity to his
mother, and his father was dead at the time of data collection.
Unlike sex or social status, age tended to modulate the resting patterns
with respect to the different dyads (interaction dyad type * focal age: LRT,
2 = 7.385, df = 3, p = 0.061). Resting distances generally became larger
in older individuals, but did so more strongly for father-offspring dyads
than for mother-offspring dyads (Figure 3). As a result, the closer
proximity to the father relative to control males disappeared in the oldest
focals, all of which were males.
Figure 3. Dyadic resting distances of focal individuals to their mothers, control

females, fathers and control males. Symbols indicate mean raw values per dyad type
and age (parents black and controls grey, females circles and males triangles), the lines
represent model estimates for each dyad type (parents black and controls grey, females
solid and males hatched), controlled for individual ID, dyad partner ID, dyad and
observation.

Table 2. Mean distances to parents and control individuals for 32 focal

individuals. M = male; F = female, S = single, LP = loosely paired,
P = paired, P+ = paired with offspring. X = mean distances (meters)
to Mother, Control Females, Father, or Control Males. Missing cells
indicate that this individual was missing or dead at the time
of data collection
Individual Sex Social

X Mother X Control X Father X Control
Status
Females Males
Beleriand F S 17.3 26.2 17.6 21.6
Mohican F S 33.5 32.6 33.9 33.1
Jaspis F LP 24.8 25.3 26.0 19.8
Large F LP 15.6 16.9 15.4 24.0
Baggins F P 20.4 31.5 20.6 32.0
Barbados F P 13.8 18.7 14.0 17.4
Jewel F P 15.7 19.2 15.9 17.9
Joey F P 21.4 27.8 21.5 21.2
Levi F P 18.5 31.3
Algonquin F P 23.4 36.6 28.4 36.8
Theoden F P 11.2 18.8 11.4 14.2
Timber F P 21.5 29.6
Lorbeer F P 28.6 22.0
Bwejuu F P+ 21.4 33.6 21.4 33.8
Bombadil M S 24.1 35.9 24.2 33.2
Balin M S 19.1 19.3 18.9 22.6
Jacaranda M S 12.7 14.9 12.9 20.0
Jamaica M S 9.7 22.8 10.1 20.0
Jordan M S 17.7 30.5 17.9 22.0
Little M S 18.0 13.0 18.3 25.0
Uncas M S 30.0 32.4 30.4 31.7
Neptun M S 33,5 24,0
Bob M P 20.2 25.2 20.4 20.4
Boston M P 14.9 16.9 15.0 18.2
Joshua M P 23.2 26.7 23.3 21.0
Lesley M P 36.8 29.5
Lindon M P 16.7 18.6 16.4 18.7
Nenya M P 36.6 26.9 36.8 32.5
Tarek M P 26.1 18.9
Bailey M P 26.9 28.1
Dylan M P 36.8 30.0
Tipi M P+ 27.9 27.9

Locations
The majority of geese showed no significant place preference. Only 29

of 78 (37%) geese preferred particular resting sites, rather than being
distributed over the 15 locations at random (Table 3). Among those geese
with a place preference, particularly, locations 5 (area with a little stream
used for drinking) and 9 (feeding area, Figure 2) were often used for
resting.
Table 3. Individual results of 2-Goodness-of-Fit Tests for all 78

individuals, which participated in the study. M = male; F = female,
S = single, LP = loosely paired, P = paired, P+ = paired with offspring.
Test statistics (), degrees of freedom (df) and P values are given.
Significant results, which indicate a place preference,
are marked in bold
Individual Sex Status 2 df p Individual Sex Status  df P

Algonquin F P 3.20 6 0.78 Laura M P 19.91 9 0.28
<
Aphrodit M P 9.73 6 0.14 Leanne F P 18.90 6 0.00
Baggins F P 3.67 7 0.82 Leda M P 15.00 5 0.01
Bailey M P 10.83 10 0.37 Lesley M P 4.00 8 0.86
Balin M S 4.13 6 0.66 Lestate F P 13.47 6 0.04
<
Balu F P 79.73 7 0.00 Lester M P 26.62 10 0.03
<
Barbados F P 7.87 6 0.25 Levi F P 53.71 11 0.00
Beleriand F S 10.20 5 0.07 Lindon M P 2.27 6 8.89
<
Bob M P 50.09 9 0.00 Little M S 9.60 8 0.29
Bombadil M S 0.47 7 1.00 Lorbeer F P 2.20 5 0.82
<
Boston M P 4.00 4 0.41 Luise F P 16.29 4 0.00
<
Bwejuu F P+ 77.50 8 0.00 Mario M P 7.00 5 0.22
<
Cheyenne M P 9.50 5 0.09 Marlin F P 47.54 8 0.00
Corrie M P 2.60 7 0.92 Mohican F S 4.73 7 0.69
Daisy F P 17.07 9 0.05 Murphy M P 2.60 7 0.92
<
Daniel M P 31.09 8 0.00 Nalle M P 12.13 6 0.06

Individual Sex Status 2 df p Individual Sex Status  df P

< <
Dora M P 26.13 10 0.00 Nasse F P 62.58 8 0.00
Dylan M P 16.57 6 0.01 Nenya M P 9.73 6 0.14
<
Freja F P 62.29 7 0.00 Neptun M S 1.30 9 1.00
<
Fremder2 M P 10.20 5 0.07 Nestor M P 34.95 4 0.00
<
Gantenbein F P 4.77 6 0.57 Noodles F P 61.08 9 0.00
Herbert M P 11.06 8 0.20 Pierre M P 15.24 11 0.17
Ida F P 17.70 6 0.01 Plum M P 6.00 6 0.42
<
Iwan M P 11.56 6 0.07 Punki M P 30.42 9 0.00
Jacaranda M S 6.87 7 0.44 Rockygrün M P 12.18 7 0.10
Jacky F P 3.00 9 0.96 Sepp M P 18.24 7 0.01
Jade F P 12.66 7 0.08 Silva M P 5.27 3 0.15
<
Jamaica M S 9.73 6 0.14 Sinus M P 63.11 7 0.00
Jaspis F LP 14.11 9 0.12 Skgrün M P 2.00 4 0.74
Jessie F P 7.63 6 0.27 Tarek M P 4.67 4 0.32
Jewel F P 0.40 6 1.00 Theoden F P 3.67 7 0.82
Joey F P 4.73 7 0.69 Timber F P 15.55 9 0.77
<
Jordan M S 8.60 5 0.13 Tipi M P+ 56.39 6 0.00
<
Joshua M P 13.47 6 0.04 Tlingit F P 28.47 8 0.00
Judith F P 6.69 8 0.57 Tristan M P 3.25 10 0.98
Keiko M P 8.60 5 0.13 Tsitika M P 9.56 7 0.22
<
LittleIndian F P 36.13 9 0.00 Uncas M S 7.71 7 0.36
< <
Lafayette M P 27.22 9 0.00 Verdi M P 14.60 3 0.00
Large F LP 2.60 7 0.92 Wendelin M P 16.68 5 0.01
DISCUSSION
Lineal Kin in Greylag Geese
The findings of this experiment give further support for the

maintenance of long-term kin bonds in greylag geese. Our results show
that mature sons and daughters, regardless of their pairing status, rest

closer to their parents than to randomly chosen control flock members.

Whereas the closer proximity of offspring to mothers than to controls did
not appear to change over time as progeny got older, distances to fathers
tended to increase and become similar to those of controls in focal birds
older than 8 years.
Most of these older focal individuals were males, which might point
towards a longer-lasting maintenance of close proximity to parents in
daughters than in sons, but sample size in this age class was too low
(particularly for females) to draw firm conclusions. Yet, these results
support the idea that the underlying social structure of greylag geese is
centered around females. We suggest that this effect may be even more
pronounced in migrating wild goose populations. Here, philopatric females
will take their mates along to their own hatching areas for breeding which,
in turn, increases the chances that sons will not be able to associate with
parents once they are paired. This idea is backed by studies on Greenland
white-fronted geese, where sons tend to leave the family unit at an earlier
age than daughters [97]. In our semi-tame flock, however, where
immigrations and emigrations are rare, sons also have a chance of
extending bonds with their parents well into adulthood. Nevertheless, in
this flock, chances to remain with parents also are higher for mature female
than male offspring, a phenomenon we described as ‘tertiary family’ units.
Previous research on these individuals has shown that of the seven
instances in the flock, where mature offspring maintained tight bonds with
parents long into adulthood, five involved daughters [98, section 6.1.2].
Due to our limited sample size of offspring with only one parent alive,
it was not possible to distinguish between distances of sons and daughters
to either parent. Validly, it might be argued that parental distances are not
independent from one another, i.e., parents cannot be considered
independent ‘spatial entities’. The nine individuals with only one parent
alive during data collection, however, further support the idea of female-
centered clans. In these instances, only daughters were closer to their
mother, whereas this was not the case for sons to mothers, or daughters and
sons to fathers.

When referring to female kin relationships in waterfowl, authors

generally cannot distinguish between collateral and lineal first-order
relatives without having both genetic and observational data on kinship,
i.e., siblings or parent-offspring dyads, as the genetic make-up alone does
not allow for such a fine-scale evaluation [34, 41, 42, 52, 56, 61, 99-103,
see also 67 for a recent review). Our study allowed us to assess this
difference between collateral and lineal kin, because we used a similar
experimental design on the same flock as Frigerio et al., [1]. The direct
comparison of the two studies indicates that distances between lineal kin,
in particular mothers and daughters (this study), are larger than between
collateral kin, i.e., sisters [1, 98]. Similar evidence for closer proximity
between individuals with their sister rather than with their mother comes
from barnacle geese, which were shown to nest close to their parents and
sisters when breeding on the same island. However, when parents and
sisters nested on different islands, females stayed closer to their same-age
sisters than to their parents [40]. One possible reason for this pattern may
be that coordination with a relatively small number of sisters from the
same age cohort is easier than that among mothers and her offspring from
several years. And, as clan size grows, the costs through resource
competition may outweigh the benefits of being surrounded by close
relatives.
Two possible scenarios might account for the proximity between
(female) relatives. On the one hand, there might be a preference for a
particular common resting location. Or, it shows persisting social
attachment between individuals. However, few attempts have been made to
tease apart effects of preferences for certain sites or proximity to kin,
particularly outside the breeding season [1, 40, 104]. In the present study
we found that some geese indeed preferred certain resting positions, but
similar to the study by Frigerio et al., [1] only about 1/3 of the geese
showed a place preference. Therefore, only a minor part of our results can
be explained by place preferences. Our data support the notion of adult
lineal kin maintaining high levels of social tolerance towards each other
that leads to the observed close proximity and are a prerequisite for
continued social attachment.

Evolutionary Aspects of the Maintenance of Long-Term

Female Bonds
Rohwer & Anderson already pointed out the exceptional position of

migratory waterfowl with respect to early pairing on the wintering grounds
and female-biased philopatry [33]. Here we will now summarize the
evolutionary aspects of how this might result in female-kin centered clans,
as shown in many waterfowl including this study, and suggest a
mechanistic basis for the maintenance of long-term adult female bonds as
exemplified again by studies on greylag geese.
Contrary to many passerine species, where females obtain nutrients to
produce eggs on a day–to–day basis [105], most female waterfowl adopt a
‘capital breeding strategy’, i.e., produce eggs from stored body reserves,
which they obtained for the most part on the wintering grounds prior to, or
partially during, spring migration [106-110, but see 111, 112]. During
breeding, the primary reproductive effort in waterfowl is high. Without
exception, they produce large, energy-dense eggs with generous amounts
of yolk to prepare their precocial young optimally before hatching [113].
Additionally, waterfowl clutches are large, and although some might
question that clutch size per se is dependent upon body reserves acquired
during the winter [114], there is good evidence that females do rely on
stored reserves during egg production. This notion is supported by geese
nesting during a time where food resources are still scarce early in the
season or at higher latitudes, where the breeding season is short and
clutches have to be initiated as soon as possible [115]. Here, stored
reserves at least partially determine clutch size [107, 110, 116, 117].
Furthermore, large energy stores are also important once clutches are
completed. Female waterfowl, which incubate exclusively, only very
briefly leave their nests for feeding bouts over the whole incubation period.
Females in better body condition are known to take fewer and shorter
feeding recesses [110, 118, 119, for a recent review 117], thereby greatly
reducing egg predation [117, 120-122].

Those previous points indicate that successful reproduction in

waterfowl is linked quite closely to what happens before the breeding
season (see [123] for a recent review), and conditions prior to actual egg
production will have profound effects on the annual (and life-time)
reproductive success of female waterfowl [107]. Therefore, it does not
come as a surprise that waterfowl are also highly philopatric with respect
to their wintering areas, and many of the hypotheses brought forward to
explain philopatry to breeding areas also apply to the wintering grounds
[35]. Hence, during winter, when our data collection took place, increased
proximity between related females might contribute to better conditions for
females in the spring. For this we will provide a possible mechanistic
explanation, which may be one of the underlying driving forces behind the
female-centered social organization in geese throughout the year.
A strict dominance hierarchy in geese, where families outrank pairs
and pairs dominate singletons has been documented numerous times
[46, 124, 125]. A dominance hierarchy is also maintained during winter
[126-130], and greylag geese are no exception [83, 86, 131]. Behaviorally,
parental and paired females acquire benefits from increased vigilance by
males and offspring, allowing them to accumulate fat stores during winter
through longer feeding bouts [46, 87, 132-137]. Physiologically,
individuals in better condition show lower levels of glucocorticoids (GCs,
138], resulting in increased fitness ('the cort-fitness hypothesis', 139, 140].
Generally, GCs are a group of steroid hormones that are released by the
adrenal cortex as the final products of one of the major neuroendocrine
systems, the hypothalamic-pituitary-adrenal (HPA) stress axis. Besides its
major role in returning the body to homeostasis in response to stress, the
HPA axis controls many body processes, including energy storage and
expenditure, fat and carbohydrate metabolism, and channeling energy into
behavior in the case of need [141-143]. We have shown repeatedly that
females benefit from a reduction in fecal corticosterone metabolites
through social support, the stress-reducing mechanism gained through the
presence of social allies, which we deem a prime mechanism for efficient
individual stress management [83, 86]. During winter, this is noticeable not
only in mothers with juvenile young [86], but, particularly, also in mothers

and their subadult daughters [83]. Females, who produce and incubate eggs
solely from body reserves, are energetically more dependent than males
[144] and any reduction of stress is, therefore, advantageous, particularly
for females. Favoring resting in close proximity to more tolerant next-of-
kin, is thus likely to reduce exposure to severe aggressive acts [98]. As a
result, these females may benefit from lower baseline corticosterone levels
throughout winter, and – following the cort-fitness hypothesis [140] –
should be in better condition overall, and, particularly, once the next
breeding cycle starts. This may explain why in geese successful
reproduction increases, rather than decreases, the prospects for successful
breeding attempts [90, 145].
CONCLUSION
Our study supports the idea of female-centered clan structures in

greylag geese and expands the maintenance of long-term bonds from
collateral to lineal kin. Our data also suggest that under certain
circumstances (such as lack of migration as in the observed flock), these
bonds may not only include daughters, but also sons. We provide further
evidence that the observed proximity among adult kin is not just an effect
of a preference for certain resting locations but, in fact, reflects social
bonds that are maintained into adulthood. Social support provides a
possible explanation for the observed spatial patterns and should be
considered in future studies on kin-biased social organization. It is unlikely
that the observed clan structures in greylag geese are biased enough to lead
to an ecologically significant matrilineal flock structure, yet, a recent study
on the age-dependent kin relations in female eiders promoted the authors to
suggest that “the combination of female philopatry, local relatedness,
active kin discrimination, long life, reproductive success, and parental
care for offspring of relatives may suggest a social system in female eiders
that in some respects resemble that of long-lived matriarchical mammals”
[67, 90]. As all these factors are also true for greylag geese, a similar claim
might be permissible.

ACKNOWLEDGMENTS
We are indebted to K. Kotrschal for his continued unfaltering support.

C. Wascher and I. Dunduc (nèe Nedelcu) helped to collect data. D.
Frigerio, J. Hemetsberger, K. Hirschenhauser, I. Dunduc, C. Schloegl, and
C. Wascher promoted discussions on the topic. We gratefully acknowledge
financial support by the FWF-Project 18601-B17, the “Verein der Förderer
der Konrad Lorenz Forschungsstelle”, the “Herzog von Cumberland
Stiftung” and a grant (190132335) contributed by the University of
Groningen (The Netherlands) to J. Komdeur, who supported IBRS during
writing of this chapter. We are grateful to A. Poehlman and J. LaFrancois
for correcting the English. The conducted study complies with all current
Austrian laws and regulations concerning the work with wildlife.
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Reviewed by: Manon Schweinfurth, School of Psychology &

Neuroscience, St Mary's Quad, South Street, St Andrews, KY16 9JP,
Scotland

Chapter 5
ESTIMATING ASSOCIATIONS BETWEEN

AMBLYOMMA TICKS AND WILD BIRDS IN
A NEOTROPIC FOREST FROM SURVEY DATA
Ralph Maturano1,*, Ronaldo R. Bastos2, Erik Daemon3

and João L. H. Faccini1
1
Programa de Pós-Graduação em Ciências Veterinárias,
Universidade Federal Rural do Rio de Janeiro,
Seropédica, Rio de Janeiro, Brazil
2
Departamento de Estatística,
Universidade Federal de Juiz de Fora,
Juiz de Fora, Minas Gerais, Brazil
3
Programa de Pós-Graduação em Ciências Biológicas –
Comportamento e Biologia Animal,
Universidade Federal de Juiz de Fora,
Juiz de Fora, Minas Gerais, Brazil
*
Corresponding Author Email: ralphmaturano@gmail.com.

150 Ralph Maturano, Ronaldo R. Bastos, Erik Daemon et al.
ABSTRACT
The Atlantic Forest biome is one of the most biodiverse, and

threatened in the world. Most studies on bird parasitism by ticks in Brazil
have been carried out in forest fragments of this biome, and primarily
focused on pathogens the ticks carry than the relationships between ticks
and birds. Accordingly, in this study we conducted a quantitative
analysis, by means of multiple correspondence analysis (MCA), on the
published data about parasitism by Amblyomma ticks on birds of the
Atlantic Forest. The hypothesis tested was that ecological and
morphological variables of the host (habitat occupation, eating habits, site
of occurrence, mass and length) are associated with parasitism by ticks.
Based on 403 tick/bird associations we found that the most prevalent
species associated with Atlantic Forest birds are Amblyomma longirostre,
Amblyomma nodosum, Amblyomma calcaratum, Amblyomma aureolatum
and Amblyomma parkeri. All these species showed association with birds
inhabiting the forest understory and midstory. In addition, both immature
stages of A. aureolatum and nymphs of A. calcaratum were associated
with birds with terrestrial habits. Regarding diets, the most common
association was with insectivorous birds, followed by frugivorous and
nectarivorous birds. All tick species were associated with forest border
birds, while A. aureolatum was also associated with birds which forage in
open areas. As for bird length and mass, those with up to 40 cm and 40 g
are the most associated with tick larvae and nymphs. In terms of hosts,
the Thraupidae, Thamnophilidae and Furnariidae families were the most
parasitized.
Keywords: wild birds, tick ecology, specificity, habitat occupation,

Ixodidae
1. INTRODUCTION
The Atlantic Forest biome covers an area of over 1,300,000 km2,

classified into five types of forest formations, as well as the transition areas
between them, which are called ecological tension zones since they consist
of a mixture of floral species from different forest formations (SOSMA
and SOS, 2014). This biome is fragmented due to deforestation, with only

Estimating Associations between Amblyomma Ticks … 151
7-8% of forests remaining today. Despite this reduction, the Atlantic Forest
is still home to over 60% of the terrestrial species on the planet, being
considered a hot spot (Galindo-Leal and Câmara, 2003). Approximately
891 species of birds are found in the Atlantic Forest, with just over 140
species performing seasonal migrations, and 213 species are native (Lima,
2014). These birds, as in other biomes, are important hosts for ticks,
especially at their immature stages (Luz and Faccini, 2013).
Many pathogens are involved in bird-tick association, especially
viruses and bacteria (Arnal, et al., 2014; Capligina, et al., 2014; Estrada-
Peña and de la Fuente, 2014; Ogrzewalska, et al., 2012). Some of these
pathogens have zoonotic potential, resulting from their spread by migratory
and/or peridomestic bird species due to contact with humans, which mainly
occurs as a result of habitat destruction (Hasle, 2013; Ogrzewalska, et al.,
2011). Thus, the knowledge regarding the relationships between birds and
ticks occurring either in natural or in peridomiciliar environments is a
major issue to be taken into consideration when monitoring such
pathogens.
When we analyse papers on tick prevalence in birds, we can see that
some species are known to be more prone to parasitism than others
(Maturano, et al., 2015; Ogrzewalska, et al., 2009; Ogrzewalska, et al.,
2010; Ogrzewalska, et al., 2011). Although this fact points to the existence
of possible parasite-host specificity, the hypothesis that the environment
shared by hosts and ticks greatly influences this relationship is currently
accepted (Klompen, et al., 1996; Nava and Guglielmone, 2013). The
results obtained by authors who have studied tick-bird relationships in the
Atlantic Forest (Labruna, et al., 2007; Lugarini, et al., 2015; Ogrzewalska,
et al., 2009) strengthen this hypothesis.
We test this prediction by using environmental, behavioral and
morphological variables of the hosts which would be influencing
parasitism by ticks. We used multiple correspondence analysis (MCA) for
data reported in literature and data collected by the authors over the years
2014-2015 on the relationship between birds and Amblyomma ticks in the
Atlantic Forest in Brazil.

2. MATERIAL AND METHODS
2.1. Data Compilation
Searches for terms such as “ticks,” “bird,” “Atlantic Forest” and

“Brazil” were performed, both in Portuguese and in English, covering
every year to 04-09-2015, in Scopus, Web of Science, SciELO, PubMed
and Google Scholar indexing databases. All results reporting the
occurrence of Amblyomma ticks in birds of the Atlantic Forest in Brazil
were compiled, except those regarding domestic fowl, captive birds, and
also those accounts where the identification of ticks and host species were
doubtful. In order to homogenize the sample, the data from each article
regarding the bird-tick species relationships at a certain stage were
considered only once. This is due to the fact that not all of the studies
provide detailed information on the prevalence of tick species. In total, we
used 381 datasets published in 16 papers and 22 datasets collected by the
authors (recently published in Zeringóta et al., 2017), summing up 403
bird-tick relationships.
2.2. Database Preparation
The geographical coordinates of each published location of bird-tick

relationships were plotted on a map containing a vector layer of the forest
formations, available on the Ministry of the Environment’s website
(www.mma.gov.br, accessed on 05/05/2015). Those locations whose
geographic coordinates were unclear, and thus likely to cover more than
one forest formation, were considered as missing data. Therefore, three
different forest formations were identified (Dense Ombrophilous Forest,
Mixed Ombrophilous Forest and Semideciduous Seasonal Forest) and two
areas of ecological tension (Savannah/Seasonal Forest and Pioneer
Formations) in 198 occurrences (Figure 1).
A worksheet was filled in with the database on tick-bird relationships.
Bird nomenclature and classification follow the Brazilian Ornithological

Records Committee - CBRO (2014) and Sigrist (2014) whereas tick

classification follows Guglielmone and Nava (2014) and Nava, et al.,
(2014).
Figure 1. Localities of the occurrence of Amblyomma ticks found in wild birds on the
Atlantic Forest in Brazil.
The following variables were treated as independent binary (1 for

“yes,” 0 for “no”): bird diet (“carnivorous,” “insectivorous,” “frugivorous,”
“granivorous” and “nectarivorous”), places birds frequent (“forest
interior,” “border” and “open area”) and foraging stratum (“terrestrial,”
“understory,” “midstory”and “canopy”). Variables regarding parasitism by
ticks for each stage were also treated as binary (1 for “yes,” 0 for “no”).
Variables regarding bird morphology were categorized into class intervals:
length (“up to 20 cm,” “between 20 and 40 cm” and “greater than 40 cm”)
and mass (“up to 20 g,” “between 20 and 40 g,” “between 40 and 80 g” and
“greater than 80 g”). The selection of variables for each bird species was
based on Ridgely and Tudor (2009); Sick (1997); Sigrist (2014); Stotz, et
al., (1996); The Cornell Lab of Ornithology (2015). A single nominal

categorical variable was created with regard to forest formation (“Dense

Ombrophilous Forest,” “Mixed Ombrophilous Forest,” “Semideciduous
Seasonal Forest,” “Savannah/Seasonal Forest” and “Pioneer Formations”).
2.3. Statistical Analysis
Multiple Correspondence Analysis (MCA), implemented in the

FactoMineR package (Lê, et al., 2008), of the R statistical program (R
Development Core Team, 2014), was used to assess possible associations
of morphological, behavioral and locality variables and parasitism by
species of Amblyomma ticks at different stages. For this, the names of the
bird species have been omitted from the worksheet. Variables regarding
bird family and tick stage/species were treated as supplementary, with only
the tick identifications at species level being considered, while the other
variables were treated as active in order to assess their structuring action
when compared to the supplementary variables, which do not affect the
MCA solution. Ninety-five percent confidence ellipses were obtained by
simulation for variable coordinates and their respective categories
represented in the main plane. Confidence ellipses correspond to a defined
area on the main plane where a particular variable may be located, thus the
smaller the ellipse the greater the accuracy of the variable location. A
derivative test of the F-test was performed, in order to see which variables
have influence on each axis, as well as the chi-squared test to assess the
association between categories of different variables. A hierarchical
clustering analysis (HCPC) was carried out from the MCA result,
according to Husson, et al., (2010), taking the first five dimensions
(axes 1 to 5) into consideration.
3. RESULTS
Considering reports which Amblyomma ticks were classified to

species, the tick-bird relationships were observed in 29 bird families from

10 orders, with the order Passeriformes being the most common, with 19
parasitized families (Figure 2). Thraupidae was the most common family,
with 76 occurrences distributed among 19 species. Thamnophilidae was
the second most common family, with 51 occurrences among 14 species,
followed by Furnariidae, with 34 occurrences among 11 species. All of the
non-Passeriformes orders showed a number of species and occurrences
lower than five. The ratio of number of occurrences in the family/number
of species in the family was less than five for all families except for the
Conopophagidae and Platyrinchidae families (Figure 2).
Figure 2. Bird families, number of species per family and number of tick
occurrence/species parasitized ratio by Amblyomma ticks in the Atlantic Forest in
Brazil.
Figure 3. Occurrences of species and stages of Amblyomma ticks found in wild birds in
the Atlantic Forest in Brazil.

Figure 4. Distribution of the stages of the four main species of Amblyomm a ticks on
bird families in the Atlantic Forest in Brazil. The thickness of the lines connecting the
tick species and bird families are proportional to the number of occurrences.
Regarding the species of Amblyomma recorded on wild birds in Brazil,

Amblyomma longirostre (Koch, 1844) is the most common, followed by
Amblyomma nodosum Neumann, 1899, Amblyomma aureolatum (Pallas,
1772) and Amblyomma calcaratum Neumann, 1899 in both larval and
nymphal stages. Amblyomma parkeri Fonseca and Aragão, 1952 was the

most common species in the larval stage, while Amblyomma coelebs

Neumann, 1899, Amblyomma ovale Koch, 1844 and Amblyomma sculptum
Berlese, 1888 were the most common species in the nymphal stage. The
occurrence of parasitism in the adult stage was recorded only for A.
longirostre (Figure 3). Figure 4 shows the interactions of the stages of the
four main species of tick and the bird families. A. longirostre was the
species with the broadest spectrum of parasitism in bird families, with the
Thraupidae, Pipridae, Rhynchocyclidae and Dendrocolaptidae families
showing the highest occurrence of larvae while the Thraupidae,
Thamnophilidae, Pipridae and Turdidae were the most parasitized by
nymphs. A. nodosum was recorded on 15 bird families, and the
Thamnophilidae and Conopophagidae families were the most
representative for larvae and the Thamnophilidae and Thraupidae were the
most parasitized by nymphs. A. aureolatum was recorded on eight families,
evenly distributed between larvae and nymphs. A. calcaratum was
recorded on 14 families, with the highest records of nymphs on the
Thraupidae, Thamnophilidae and Conopophagidae.
Results obtained by MCA as seen in axes 1 and 2 (main plane) explain
21.45% of data variability (Figure 5). Variables regarding bird morphology
contributed most to the formation configuration of both axes, followed by
variables related to bird “sites,” “foraging stratum” and “diet” for axis 1
and bird “diet” and “sites” for axis 2. In terms of the categories’
contribution to axis configuration, ''open areas,” “body mass up to 20 g,”
“body mass between 40 and 80 g,” “canopy” and “length greater than 40
cm” contributed the most to axis 1 and “length greater than 40 cm,” “body
mass greater than 80 g” and “carnivore” contributed the most to axis 2,
which allows us to infer, for example, that the categories “carnivore” and
“body mass greater than 80g” are related to the family Strigidae, since the
points representing these variables and categories have similar coordinates
when the same axis is taken into consideration, and so forth (Figure 5).
As for the categories of active variables treated as binary, they were all
separated by both axes; however, the categories “understory,” “midstory,”
“insectivorous” and “border” retained coordinates close to zero in both
axes. As for the variable “forest formation,” the categories “Savannah/

Seasonal Forest” and “Semideciduous Forest” were separated from the

categories “Dense Ombrophilous Forest,” “Pioneer Formations,” “Mixed
Ombrophilous Forest” for axis 1 and “Savannah/Seasonal Forest” and
“Semideciduous Seasonal Forest” were separated from “Dense
Ombrophilous Forest,” “Pioneer Formations” and “Mixed Ombrophilous
Forest” for axis 2, which allows us to infer that the parasitized birds
located in the first three forest formations differ in morphological and/or
ecological terms from those located in the last two forest formations.
Figure 5. Distribution of active variables and clusters in the main plane of the MCA.

Regarding additional variables related to tick stages/species, the

categories “presence” and “absence” were separated by both axes in all
species; however, there was ellipse overlap for A. sculptum larvae and A.
longirostre, A. nodosum, A. calcaratum, A. aureolatum, A. ovale and A.
coelebs nymphs. Great ellipses were generated for A. sculptum larvae, A.
coelebs, A. sculptum and A. ovale nymphs due to the low number of
individuals parasitized by the respective stages of these species. Because a
maximum of two individuals parasitized by A. ovale and A. coelebs larvae,
A. parkeri nymph and A. longirostre adults were recorded, no confidence
ellipses were generated for these categories (not showing in Figure 5).
With regard to the additional variable “Family,” a cluster was formed near
the zero value of axis 2, with ellipse overlapping (not showing in Figure 5),
which indicates a similarity among bird families in ecological and
morphological terms, mainly regarding the order Passeriformes. The
following categories were plotted away from this cluster for both axes
“Columbidae,” “Strigidae,” “Turdidae,” “Momotidae,” “Corvidae,”
“Cariamidae,” “Ramphastidae,” “Cracidae” and “Cuculidae.” Among these
categories, with the exception of “Strigidae” and “Turdidae,” none of the
others had confidence ellipses generated due to the low number of
individuals. Most Passeriformes retained a negative coordinate for axis1,
indicating similarity among the families of this order that are correlated
with this axis.
Cluster analysis generated four different groups (Figure 5), which
allowed us to differentiate some families according to the variables taken
into consideration in the analysis. The variables “family” (p = 1.07-95 – df =
90), “weight” (p = 5.77-90 – df = 12), “length” (p = 5.59-63 – df = 9) and
“open area” (p = 4.81-33 – df = 3) were those which most characterized the
formation of the four groups. In terms of family, those placed in a single
cluster were: Bucconidae, Conopophagidae, Onychorhynchidae, Parulidae,
Platyrhinchidae, Rhynchocyclidae, Troglodytidae, Tityridae, Scleruridae
and Xenopidae (Cluster 1); Trochilidae and Vireonidae (Cluster 2);
Cariamidae, Momotidae, Cuculidae, Cracidae, Corvidae, Ramphastidae
(Cluster 3). Cluster 4 contained families included in the other clusters. The
individuals closest to the center of gravity, that is, those in the center

position of each cluster and which were most distant from the other
clusters belong, respectively, to the Furnariidae and Conopophagidae
families, for Cluster 1, Thraupidae and Dendrocolaptidae for Cluster 2,
Ramphastidae and Cariamidae for Cluster 3 and Turdidae, in both cases,
for Cluster 4. That is to say that these families that are close to the center
of gravity are the most representative of their respective groups. For
Cluster 1, mainly small birds were included (weight less than 20g and
length less than 20 cm), while Cluster 2 encompassed birds of small and
intermediate size (weight up to 40 g and length up to 40 cm). Cluster 3 was
primarily comprised of terrestrial birds frequenting open areas.
Table 1 summarizes the relationships found by MCA among tick
stages/species and the active variables. This table is the result of the
analysis of the contributions and correlations of the categories, as well as
the contributions of the variables in relation to axes 1 and 2, categorical
description of the variables and categories for each axis and links among
categories of different variables. Only stages of the species with at least 10
occurrences are shown. The A. longirostre, A. nodosum, A. calcaratum and
A. parkeri species are those which were most similar at the larval stage
with regard to the variables related to the birds’ diet, foraging stratum and
morphology. As for A. aureolatum larvae, this stage differred from the
aforementioned species, since it is more common in birds weighing
between 40 and 80 g, with terrestrial habits and which frequent open areas.
Birds that frequent open areas are also prone to parasitism by A.
aureolatum and A. longirostre nymphs, as well as birds weighing between
40 and 80 g, as terrestrial habits are also associated with the latter.
Regarding the species A. nodosum and A. calcaratum in nymph stage, there
were differences concerning the association to the frugivorous habits of the
host, which is absent in A. nodosum and present in A. calcaratum. As for
the association with bird morphologic variables, with the exception of A.
calcaratum nymphs, these two species showed similarity with regard to
bird weight and length. Concerning species associations in terms of forest
formations, A. longirostre showed relationship with the Dense
Ombrophilous Forest in the larval stage, while A. nodosum and A.
calcaratum showed relationship with the Semideciduous Seasonal Forest

in both immature stages. In addition, A. nodosum, was also associated with

the Savannah/Seasonal Forest ecological tension zone in the nymph stage.
Birds with insectivorous habits, frequenting borders, with lengths up to 20
cm and body mass up to 20 g were associated with stages of all species
listed in Table 1.
4. DISCUSSION
This is the first detailed analysis of tick parasitism in birds of the

Atlantic Forest in Brazil. The choice for analyzing only this environment is
due to two reasons: (i) the Brazilian Atlantic Forest is the biome with the
highest number of reports of tick parasitism in wild birds; (ii) variations
between biomes, particularly vegetation type, may interfere with the way
tick species occur. Thus, we believe that the various ecosystems of the
Atlantic Forest and their inherent characteristics are excellent setting to
study how ticks and wild birds relate in the Neotropical region.
Although MCA suggests relationships between bird habit and species
occurrence and tick stage, bias in the analysis can not be ruled out. For
instance, one shoud take into account two important points: (i) the
relationships found in Table 1 are not exclusive, that is, other relationships
are likely to be found in nature, but in low frequency; (ii) the absence of
relationships in Table 1 does not necessarily mean a given relation is non-
existent, it can be an artifact caused by the sample or sample size. Thus,
due to the very few reports on the occurence of bird parasitism by tick
species not listed in Table 1, it was not possible to draw conclusions by
means of MCA for all of the species and stages reported.
Amblyomma longirostre was the tick species with the greatest number
of occurrences in both immature stages, although the dominance of this
species is not a rule for all areas studied (Ogrzewalska et al., 2009;
Maturano et al., 2015).
In addition, the two only records of adult ticks of the genus
Amblyomma found in birds in the Atlantic Forest belong to this species.
This species also shows the broadest spectrum of parasitism among bird

families; however, the strong associatons of larvae and nymphs with the
Thraupidae are notorious, which could be the result of specific ecological
characteristics of this bird family.
Table1. Associations between the ecological variables of wild birds in

the Atlantic Rainforest in Brazil and the main tick species and stages
Larva Nymph
A. aureolatum
A. aureolatum
A. calcaratum
A. calcaratum
A. longirostre
A. longirostre
A. nodosum
A. nodosum
A. parkeri
Carnivorous
Insetivorous + + + + + + + + +
Frugivorous + + + + + + +
Granivorous
Diet
Nectarivorous +
Forest interior + + + + + +
Border + + + + + + + + +
Site
Open area + + +
Up to 20 g + + + + + + + +
Between 20 and 40 g + + + + + + + + +
Weight
Between 40 and 80 g + + +
Greater than 80 g
Up to 20 cm + + + + + + + + +
Length
Between 20 and 40 cm + + + + + + + +
Greater than 40 cm
Terrestrial + + +
Understory + + + + + + + + +
Stratum
Midstory + + + + + + + + +
Canopy + +
Dense Ombrophilous Forest +
Semideciduous Seasonal Forest + + + +
Formation
Mixed Ombrophilous Forest

Forest
Pioneer Formations
Savannah/Seasonal Forest +
Cross markings (+) represent those matches found after completion of MCA.

Indeed, host switching has been considered common in shaping

parasitic associations among ticks (Klompen, et al., 1996). On the other
hand, thraupids are most sampled birds (Maturano, et al., 2015), which
might also account for the most published tick-birds associations in the
Atlantic Forest. Larvae and nymphs of this species parasitize insectivorous
and frugivorous birds that frequent the understory and midstory vertical
strata, as well as arboreal rodents of the Coendou and Sphiggurus genera,
suspected of being the main hosts for this tick in its adult stage (Labruna,
et al., 2007; Oliveira and Bonvicino, 2006; Onofrio, et al., 2006).
Regarding the size of the hosts, A. longirostre showed preference for small
birds (up to 40 cm and 40 g), while nymphs were also associated with
greater body mass birds (80 g).
Amblyomma nodosum and A. calcaratum are closely related species,
sharing the same host in adult life (Anteaters, Xenarthra:
Myrmecophagidae) (Guglielmone, et al., 2014; Onofrio, et al., 2006).
These hosts frequent the ground and trees for feeding and resting (Medri,
et al., 2006). Regarding the families of parasitized birds, similarities were
observed between the two species. However, some ecological differences
have been identified by MCA for the two species, such as the association
with frugivorous and insectivorous birds for A. calcaratum and only for
insectivorous birds for A. nodosum. There are reports on the occurrence of
both A. nodosum and A. calcaratum in birds feeding on the ground or near
the ground and also in the understory (Labruna, et al., 2007; Maturano, et
al., 2015; Ogrzewalska, et al., 2009). However, with MCA, an association
with the birds which forage on the ground was only observed for A.
calcaratum nymphs. However, in tick collection by dragging on soil
(Sabatini, et al., 2010; Szabó, et al., 2009), none of these species have been
found, except by Szabó et al., (2007), who collected one single individual
of A. nodosum. Thus, further collections are needed to clarify this issue.
Unlike other species mentioned, A. aureolatum showed a relationship
not only with birds of arboreal habits, but also with those of terrestrial
habits and frequent in open areas, which are also shared by wild and
domestic canids, common hosts in the adult stage (Costa, 2011;
Guglielmone, et al., 2003; Guglielmone, et al., 2014; Guimarães, et al.,

2001). In fact, free-living adults of this species have been found on the
ground (Sabatini, et al., 2010; Szabó, et al., 2009). The presence of A.
aureolatum on the ground or in open areas may be the reason why this
species has a narrower spectrum among bird families when compared to A.
longirostre, A. nodosum and A. calcaratum, since most of the bird species
frequent the understory and midstory. Unlike the aforementioned species,
A. parkeri was mainly reported in larval stage, with only one report for
nymph stage. Rodent species of the Erethizontidae, which have
predominantly arboreal habits as birds parasitized by larvae of this tick
species, are apparently common hosts for adult ticks of this species
(Guglielmone, et al., 2014; Labruna, et al., 2009; Oliveira and Bonvicino,
2006; Onofrio, et al., 2006).
The last four species of ticks A. sculptum (formely A. cajennense), A.
coelebes, A. ovale and A. naponense have not been included in MCA
analysis because reports of their associations with wild birds in the Atlantic
forest is still too scarce.
The cluster analysis showed that some bird species belonging to the
same family have different susceptibility to parasitism by ticks. This
finding supports the hypothesis tested in this study, according to which
birds with similar habits tend to be parasitized by the same tick species. In
general, parasitism by certain tick species is observed in phylogenetically
distant hosts that frequent similar sites (Klompen, et al., 1996; Nava and
Guglielmone, 2013). In addition, it is noteworthy that size and degree of
fragmentation could affect bird abundance, and consequently increase
parasitism by ticks (Ogrzewalska, et al., 2011).
It is also plausible to infer that, in this scenario, generalist birds, in
terms of habitat occupation, are more susceptible to infestations/
coinfestations. Finally, taking into consideration the extent, biodiversity
and heterogeneity of the Atlantic Forest, this review clearly shows how
scarce our knowledge is regarding the diversity of ticks on birds. There are
still several gaps in terms of geographic distribution in this biome, which
was once continuous, but is now fragmented. Some forest formations have
not yet been explored and others must be better studied. In addition, forest
fragments have special features, which make them unique ecosystems, in

the sense that generalizations at the macro-ecological level do not apply to

all fragments of the same biome. More detailed information will only be
possible after long-term systematic surveys in order to investigate not only
the association among birds and ticks, but also the role of other members
of the local wildlife as hosts for these ectoparasites, as well as the
composition of the local vegetation, a hitherto neglected variable.
ACKNOWLEDGMENTS
This research was funded by CNPq and CAPES.
CONFLICT OF INTEREST
The authors declared that there is no conflict of interest.
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Chapter 6
CRYPTOSPORIDIOSIS IN
NON-HUMAN PRIMATES
Sérgio Diniz Garcia1,*, PhD,

Bruno Criado de Araújo Mendes2,
Débora Barbosa Bruno1, Jaqueline da Silva Borégio2,
Marcela Dastre2, Vinícius Camarena Borges2
and Katia Denise Saraiva Bresciani3,4, PhD
1
Departamento de Clínica, Cirurgia e ReproduçãoAnimal,
Universidade Estadual Paulista (Unesp),
Faculdade de Medicina Veterinária, Araçatuba, São Paulo, Brasil
2
3
Departamento de Apoio, Produção e Saúde Animal,
*
Corresponding Author Email: sergio@fmva.unesp.br.

172 S. Diniz Garcia, B. C. de Araújo Mendes, D. Barbosa Bruno et al.
4
Departamento de Medicina Veterinária Preventiva e Reprodução
Animal, Universidade Estadual Paulista (Unesp), Faculdade de Ciências
Agrárias e Veterinárias, Jaboticabal, São Paulo, Brasil
ABSTRACT
The criptosporidiosis is known since 1907, but there is no much data

compilation about it. 152 animal species have been described with this
parasite. With all the researches found, it is necessary a review about the
disease, for a great understanding about the Cryptosporidium. This
research has the purpose to show all the information found in a systematic
review of articles about cryptosporidiosis in non-human primates. For a
better understanding, this article describe the historic, taxonomy,
epidemiology cycle, clinical signs, diagnostic, treatment and prevention.
Keywords: monkeys, cryptosporidiosis, protozoan, Cryptosporidium,

zoonosis and primates
INTRODUCTION
All living things are subject to some kind of parasitic infection.

Cryptosporidium is one of the most important and one of the few studied
or neglected, especially in relation to wild animals.
This review aims to condense the maximum possible information from
several authors about the occurrence of Cryptosporidium in non-human
primates. With the purpose of facilitating the diagnosis, treatment and even
new studies on this topic.
In the search for information, we have found several uninformed or
contradictory reports, or even from unreliable sources. This work aimed at
copying and aggregating the most reliable sources and important
information in a single work. In order to provide an objective and easy
consultation for anyone who needs information about cryptosporidium in
non-human primates. Several researchers from various areas of our faculty
have come together to research and obtain this data.

Cryptosporidiosis in Non-Human Primates 173
HISTORIC
The Cryptosporidium parasite was first reported in 1907 by the

American doctor and parasitologist Ernest Edward Tyzzer (1875-1965).
This protozoon was found in gastric glands of laboratory mice (Mus
musculus). The parasite received the name Cryptosporidium in 1910,
suggesting the denomination of the specie described as Cryptosporidium
muris. In 1912, Tyzzer proposed a new species, Cryptosporidium parvum.
As in 1955, the Cryptosporidium spp. started to gain the interest of
cientists with the description of a new species – Cryptosporidium
meleagridis – which was associated with lethal infections in turkeys
(Meleagris ocellata) (Slavin 1955, 262-63). Cryptosporidium spp. started
to show importance in veterinary medicine when the species C. parvum
was associated with lethal diarrhea in cattle (Panciera, R. J., R. W.
Thomassen and F.M. Garne 1971, 479-84).
Several protozoa are observed in neotropical primates, including the
Cryptosporidium spp. In a research performed in the Barcelona Zoo with
mammals, with 29 species of primates, oocysts of this parasite were
detected in Ateles belzebuth (Geoffroy 1806) (Cebidae) and Macaro
mayottensis (Linnaeus 1766) (Lemuridae) (Gomez 1992, 619).
In fecal samples of primates seized by the authorities and kept in
CETAS (Wild Animals Center), located in Seropedica, Rio de Janeiro,
Brazil, Crypstosporidium was detected in howler monkeys (Alouatta fusca)
and marmosets (Callithrix spp.). (Filho et al., 2006, 354-55-58).
This coccidia was observed in several species of monkeys (Alouatta
caraya, Ateles paniscus, Saimiri boliviensis and C. apella) at the Zoo of
Buenos Aires, Argentina (Venturini et al., 2006, 90-91-92-93).
Even with the large number of hosts and the wide geographical
distribution of this parasite, the first case of parasitism by Cryptosporidium
spp. in a primate of the species Callithrix penicillata was identified in a
zoo and a conservationist establishment located in the municipalities of
Cachoeira do Sul and Santa Maria, Rio Grande do Sul, Brazil. (Silva 2008,
2659).

TAXONOMY
The protozoa Cryptosporidium is an eukaryote, being first classified on

the phylum Apicomplexa as an obligatory intracellular parasite, belonging
to the Coccidea class and the Cryptosporidiiae Family (Rose, Huffman e
Gennaccaro 2002, 113). Later considered a local intracellular parasite,
despite that extracitoplasmatic (Barta e Thompson 2006, 464; Rosales et
al., 2005, 74), nowadays it is considered its epicellular location
(Valigurová et al., 2008, 921). However, extracellular stages have been
observed in the biological cycle of this agent, showing up that the
Cryptosporidium is not mandatorily intracellular (Barta e Thompson 2006,
466).
There is a morphological identification difficulty – through
microscopy – of the species from genus Cryptosporidium due mainly to the
oocysts size – measuring about 4 to 6 µm, which are seen inside feces such
as its distinction from other stool componentes as leaven, vegetables, and
algae (Fayer, Morgan e Upton 2000, 1305), oocysts conservation, isolation
techniques, in addtion to the limited analysis from length and width
characteristics (Fall 2003, 401). Its spherical appearance and its inner
structures of hard observation make even harder to distinguish the species
(Fayer, Morgan e Upton 2000, 1305). Due to identification difficulties, it
was established that beyond the occyst’s morphometry must be analyzed
the natural specificity of hosts and the genetical and molecular distinction
(Xiao et al., 2004a, 84).
Based on genetical, molecular and biological analysis, it was
demonstrated that the Cryptosporidium belongs to a distinct class of
Coccidea, the gregarinas (Hijjawi 2002; Rosales et al., 2005). The
taxonomic proximity and other similarities made base to stablish a new
order to the Cryptosporidium, the Gregarinomorphea Class (Ryan et al.,
2016, 310-11). The similarities observed at the Cryptosporidium,
responsable for the taxonomic class amendment is the Cryptosporidium
skill of completing its life circle on the lack of a host, extracellular stages
of the cycle and capacity of environment adaptation, changing its cellular
structure depending on the environment (Ryan et al., 2016, 307).

EPIDEMIOLOGY
The Cryptosporidium parasite affects mainly the gastrointestinal tract

(Rose, Huffman and Gennaccaro 2002, 113; Valigurová et al., 2008, 913),
being responsable of human enteritis diarrhoeal (Hijjawi 2002, 1719; Xiao
et al., 2004b, 483) and animals (Rose, Huffman and Gennaccaro 2002,
113; Thompson, Koh and Clode 2016, 3), being considered a cosmopolitan
disease (Fayer, Morgan and Upton 2000, 1315). This genus has a powerful
ability of infection, not only in humans but also in a variety of animals
(Fayer 2010, 90).
Due to the Cryptosporidium capability to infect several animals and
the presence of infective oocysts in the environment (Tzipori and Widmer
2000, 2), there is a vast environmental spread, becoming human beings
likely to infection through many ways of transmission, such as the wind
(Fayer, Morgan and Upton 2000, 1308), direct contact with infected people
or animals, ingestion of contaminated food or water, the main way of
dissemination of Cryptosporidium (Xiao 2010, 80). Even the water used
for recreation, such as swimming pools may carry the oocysts (Rose,
Huffman and Gennaccaro 2002, 116-17), where applicable emphasize its
great chlorine resistance, chemical compound largely used for bacteria
control of potable water and recreational activities (Dillingham, Lima and
Guerrant 2002, 1060).
Oocysts also have been observed in raw vegetables in the market,
probably transported by contamined watering containing stool or by lack of
hygiene by touching food (Fayer, Morgan and Upton 2000, 1310). The
lack of sanitation – sewage treatment and animal waste – it is the important
factor of water contamination, favoring the environmental dissimination
even more (Rose, Huffman and Gennaccaro 2002, 115).
The most affected groups – due to the opportunist Cryptosporidium
characteristic – are the children (Xiao et al., 2000, 287). The
immunosuppression is observed in cases of AIDS patients, transplanted,
chemoterapy treated and immunosuppresed diseases (Fayer, Morgan and
Upton 2000, 1308).

In addition to the public health worsen is the Crypstosporidum species

capacity of cross infection (Xiao et al., 2000, 288; Xiao, 2004a, 74),
becoming an important question of health and animal conservation, as in
wild life animals as in captive animals.
In natural conditions, the infection is rarely seen, however in captive
animals, which might suffer from nutritional stress, contaminated water
ingestion or living in agglomerated conditions – leading to immunity issues
– becoming more vulnerable to parasitism (Malan et al., 1997, 142-149).
There are several reports of Cryptosporidium infections in primates kept in
captivity (Dubey, Markovits and Killary 2002; Gracenea 2002; Silva 2003;
Levecke 2007; Cordón 2008).
Human interventions can also cause stress, hydric and nutritional
restriction in wild animals, especially when natural areas are fragmented.
(Malan et al., 1997, 148).
The information about this parasite involvement with monkeys are
uncertain, showing the importance of epidemiologic studies about possible
zoonotic diseases involved, considering the human and monkey interaction
in the ambiental exploration and fauna conservation. New studies must be
conduced from the new taxonomic classification, searching for more
understanding about the Cryptosporidiosis, involving its risk for
individuals and environment contamination (Thompson, Koh and Clode
2016, 6; Ryan et al., 2016, 309).
CYCLE
Cryptosporidium oocysts are released in the feces in their sporulated

(infective) form, presenting four sporozoites. The infection occurs by the
oocyst ingestion, the sporozoites are later released in the intestine,
affecting epithelial cells. The sporozoites are internalized and have an
asexual reproduction and later a sexual reproduction with the formation of
macro and micro gametes, forming the zygote and later the oocyst (Tzipori
and Widmer 2000, 10-11).

CLINICAL SIGNS
The criptosporidiosis manifests as acute and self-limiting

gastroenteritis in immunocompetent individuals, and may be fatal in
immunocompromised animals, with higher risk in young, elderly or
immunosuppressed individuals (Moreno 2013, 396-97). Asymptomatic
infection may be common in wild species (Santos 2011, 37-39), and this
animals may have clinical sequelae, increasing the probability of non-
obvious health problems (Moreno 2013, 396-97). Clinical signs are related
to the host´s immune status, pathogenicity of the infecting species and
agent concentration in the organism. The non-human primates are more
sensible to the protozoa infection, expressing clinical signs in adults and
immunocompetent individuals (Santos 2011, 37-39). Cryptosporidium
infections in chimpanzees are similarly to humans (Moreno 2013, 396-97).
The principal signs are intermittent diarrhea, predominantly liquid and
profuse, dehydration, fever, abdominal pain and distention, vomiting,
malabsorption, apathy, (Codices 2013, 20) lethargy, progressive weight
loss and anorexia (Santos 2011, 37-38). Several cases can lead to enteritis,
cystitis, hepatitis, pancreatitis and respiratory diseases such as sinusitis and
pneumonia (Santos 2011, 38). Pathogeny is due to the lysis of the host´s
gastrointestinal epithelial cells, leading to villous atrophy, apoptosis of
epithelial cells, destruction of intestinal epithelial cells, increasing
intestinal permeability and causing malabsorption of nutrients.
Anatomopathological findings include hyperemia and emaciation of the
intestinal mucosa and edema of the cecal wall; atrophy of intestinal villi
and decrease of microvilli; intestinal crypt cell hyperplasia; mononuclear
inflammatory infiltrate in lamina propria and enterocytes degeneration
(Santos 2011, 39).
DIAGNOSIS
Several diagnostic methods can be applied to determine the occurrence

of Cryptosporidium spp., varying in speed, specificity and cost (Codices

2013). Clinical signs, coproparasytological, immunological, endoscopic

and molecular techniques represent an important diagnostic method for
Cryptosporidium infection (Codices 2013, 43). Microscopic techniques are
indicated for an early diagnosis, complemented by molecular techniques,
to distinguish the infection agent species and genotypes (Santos 2011,
112). Some Brazilian primates, Callithrix jacchus, Ateles belzebuth e
Saimiri sciureus were reported as Cryptosporidium spp. hosts (Santos
2011, 34-45). The genotypes described in non-human primates encompass:
Cryptosporidium hominis, Cryptosporidium muris, Cryptosporidium
parvum, Cryptosporidium cunicullus and Cryptosporidium ubiquitum. C.
hominis, is mainly reported in non-human primates (Karim 2014, 07). C.
hominis lb and li subtypes have been found in Macaca mulatta, Macaca
fascicularis, Lorisidae family’s, macacos foliates de François (Karim
2014, 07) IdA (idA19) and ibA (ibA9G3) subtypes in Macaca mulatta,
Macaca fuscata, Pan troglodytes and gibbons (Youung 2016, 846-853).
C. parvum A type and B type and C. muris. Were found in Pongo
(Mynarova 2016). C. cuniculus e C. ubiquitum, were reported in
chimpanzees and gorillas (Butel, C., Steve, AM, Pascal, D., Donika, K.,
Eitel, MN, Michele, M., Eric, D., Martine, P., Sabrina, L., 2015, 09, 10).
And Cripto sp. Rat genotype III in Pithecia sp and crypto sp. Deer mouse
genotyoe IV in Alouatta caraya (Santos 2011, 149).
Diagnose Methods
Microscopic Coproparasytological
Examination of biological product by non-invasive methods (feces),
indicated due to its sensitivity, speed and cost. Oocyst concentration
techniques can be use such as flotation with saturates sucrose solution, zinc
sulfate or sodium chloride (NaCl) (Bosa 2014, 79); gradients separation by
percoll gradients separation by cesium, if necessary, fat removal of the
samples can be realized (Codices 2013, 45); or sedimentation techniques:
Hoffman, pons and janer and centrifugal-sedimentation with formaldehyde
-ether are also used. Phase contrast microscopy is performed to visualize

Cryptosporidium oocysts. Kinyoun and Ziehl Nielsen coloring techniques

are most commonly used, others techniques include Giemsa, Safranina,
methylene blue and malachite green. Samples are places on slides, covered
by coverslips and observed under magnification of 100x, 200x or 400x
(Santos 2011, 46-47-48).
Samples can also be fixed with methanol and stained with phenolic
basic fuscine (Codices 2013, 44), or can be used the sheater flotation
technique and lugol spot method (Karim 2014, 02).
It´s important to pay attention to the proper collection and storage of
fecal samples, avoiding contamination. The samples can be preserved six
to nine months for later PCR with the use of 2.5% potassium dichromate
and refrigeration at 4ºC, however, this condition impairs the
immunological diagnosis, because it changes the oocyst wall of this
parasite (Santos 2011, 126).
Hystologic
This diagnose method is realized by the histologic observation of the
intracellular forms in intestinal tissue biopsy material, in the staining of
Giemsa and Hematoxlyn Eosin with electronic microscopy. In this case,
the parasite is observed in the inside of a vacuole. However, this method is
not very widely used because of its low sensitivity (Codices 2013, 43).
Immunologic
This method include the latex agglutination reaction, direct
immunofluorescence with polyclonal or monoclonal antibodies labeled
with fluorescein isothiocyanate, widely used in wild animals (Santos 2011,
112), ELISA (enzyme-linked immunosorbent assay), immunochromato-
graphy and immunomagnetic separation. Specific anti-Cryptosporidium
immunoglobulins M and G can be detected in the ELISA (Bosa 2014, 19).
The limitation include: cross-reactions with other microorganisms, false
positive results and high costs (Codices 2013, 45-46), on the other hand,
this method advantages include high sensitivity and specificity (Bosa 2014,
19).

Flow Cytometry
The technique evaluate multiple parameters of individual cells in
heterogenic populations, combine fluid, lasers, optical and detectors
systems and a data and computer systems and storage. It´s used for
immunophenotyping (immunofluorescence associated with monoclonal
antibodies labeling), cell counting, organelle analysis, functional studies
(membrane potential, cell replication, calcium flux), among others
(Codices 2013, 47-49). It´s used by several authors for the oocysts
detection and counting in faecal and environmental samples. It has greater
sensitivity compared to conventional immunofluorescence.
Molecular Techniques
Molecular analysis by the DNA fragments extraction and
amplification, PCR and its variations: NESTED PCR, RFLP-PCR, RT-
PCR, reverse transcriptase PCR, multiplex PCR, it works for the molecular
characterization and identifying the epidemiologic agents in species and
genotypes. The genes used in the amplification comprehend SSU rDNA or
18S rDNA, glycoprotein 60(gp60), actina, HSP70 and Cryptosporidium
oocyst wall protein (COWP). Primers are more suitable for use with SHP1,
SHP2, SHP3 e SSU-R3 (Santos 2011, 112).
Differential diagnosis must be realized in any animal that shows acute
and persistent diarrhea (Codices 2013, 43).
TREATMENT
Cryptosporidiosis, a disease that affects primates and other animals,

does not have a specific therapeutic protocol for the treatment and
eradication of Cryptosporidium spp. being onl performed when
symptomatic treatment is necessary (Coutinho, 2008.21; Spósito Filha,
2009. 19).
The use of medications such as paramycin, azithromycin (antibiotics
that reduce oocyst excretion) and nitazoxanide (antiprotozoal) have been
tested in animals and can be indicated in some cases, but they not provide

total elimination of the protozoan. Monitoring the patient's clinical status,

parasitological tests and an adequate nutritional support are necessary
(Bosa 2014, 18; Galvão 2012, 24).
Due to the common diarrhea in immunocompetent individuals,
treatment based on rehydration and replacement of salts is satisfactory
(Bosa 2014, 18). However, in cases where the individual is immuno-
compromised, Coutinho (2008, 21) and Bosa (2014, 18) antiretroviral drug
therapies can be used, which act to stimulate lymphocyte production and
result in clinical improvement of the animal.
CONTROL
The cryptosporidiosis occurrence with high morbidity is related to the

high resistance that the oocysts possess, surviving after the use of several
disinfectants and water treatment methods such as chlorination and
filtration, through this, a good sanitary management with hygienic
measures is necessary in captive primates. As soon as the confirmation of
the infection and possible oocysts elimination, the animal must be isolated,
preventing the contamination of younger and immunosuppressed animals
(Filha 2009, 17-18).
Biosecurity maneuvers are necessary for prophylaxis of the disease,
due to the difficult elimination of the protozoan after parasitizing the
animal. Some fundamental measures for the infection control are the
protection of water sources, potability of water after research of
Cryptosporidium spp. and control of industrial and agricultural effluents
(Silva 2008, 2660 and Filha 2009, 17-18).
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URL: http://www.bioone.org/doi/full/10.1638/2015-0301.1.

Chapter 7
THE EFFECT OF BLUEBERRIES

AND RASPBERRIES ON THE REPRODUCTIVE
BEHAVIOR AND FECUNDITY OF DROSOPHILA
SUZUKII FEMALES
Elizabeth Krohn, Jonathan Fingerut

and Scott McRobert
Department of Biology, Saint Joseph’s University,
Philadelphia, PA, US
ABSTRACT
Drosophila suzukii, a fruit fly species native to Southeast Asia, has

become an invasive species and threat to agricultural production in many
parts of Europe and North America. Differing from other Drosophilias,
D. suzukii’s impact as a formidable agricultural pest, stems from the

Department of Biology, Saint Joseph’s University, 5600 City Ave. Philadelphia, PA. USA
19131, smcrober@sju.edu.

190 Elizabeth Krohn, Jonathan Fingerut and Scott McRobert
ability of the females to use ripe fruit that is still on the vine as sites for
oviposition. Developing a greater understanding of the lifecycle and
behavior of D. suzukii may be key in controlling this pest. In this study,
exposure to two fruits, blueberries and raspberries, was analyzed for
possible effects on sexual behavior and fecundity of D. suzukii females.
Neither blueberries nor raspberries had significant effects on copulation
frequency, latency to copulation, nor copulation duration. However,
females exposed to whole blueberries or whole raspberries both prior to,
and after, mating yielded significantly more offspring than females from
other housing conditions. These results add to our understanding of the
life history of a major agricultural pest.
Keywords: Drosophila suzukii, diet, mating behavior, fecundity
INTRODUCTION
Origin and Dispersal
The invasive agricultural pest Drosophila suzukii (Diptera

Drosophilidae), or Spotted Wing Drosophila (SWD), is believed to have
originated in the Kashmir region (Korea, Thailand and India) but was first
recorded and described by Matsumura in 1931, not long after the species
was introduced to Japan. The fly was later found in other parts of Asia,
including China, Taiwan, Eastern Russian, Pakistan and Myanmar
(Cini et al., 2012). In 1980, D. suzukii was collected for the first time on
U.S. soil in Oahu, Hawaii and several other Hawaiian Islands but was not
reported to be causing damage to local crops. It was not until September of
2008 that the fly was known to have reached the mainland when the
Entomology Department of the Plant Pest Diagnostics Center of the
California Department of Food and Agriculture (CDFA) in Sacramento
received a sample of a fly from Santa Cruz County, CA that was collected
in a raspberry field. The specimen was identified as Drosophila, but since
no members of this genus were considered to be a pest at the time
(Drosophila typically utilize rotting fruit, not live fruit, as a food source), it
was not identified more specifically. What the CDFA did not know,

The Effect of Blueberries and Raspberries … 191
however, was that the raspberry crops, and the nearby strawberry crops,
were infested with Drosophila larvae that were damaging the fruit. The
farm advisor who sent the initial report, dissatisfied with the CDFA’s
identification, sought other help, but received misinformation from a non-
taxonomist who identified the species as D. biarmipes, a species with
similar physical characteristics to D. suzukii, but which is not an
agricultural pest (Hauser, 2011).
In the spring of 2009 the CDFA was continuing to receive records of
fly larvae, this time found in cherry crops that were suspected to be the
western cherry fly, Rhagoletis indifferens. As reports of larvae in crops
became more frequent from counties neighboring Santa Cruz, the CDFA
realized that it was imperative to properly identify the species, as they may
be the primary cause of the massive crop damage being seen. Comparing
its genetic sequence to data in GenBank (http://www.ncbi.nlm.nih.gov/
genbank/), the species was identified as Drosophila, but only when the
morphological characteristics were studied in more detail was this fly
finally identified as Drosophila suzukii (Hauser, 2011).
Since then, D. suzukii has spread rapidly throughout the United States
(Cini et al., 2012). In 2009, D. suzukii was recorded in 20 counties across
the entire state California, as well as Oregon, Washington, British
Columbia and in Florida. By 2010, D. suzukii had reached Utah, Louisiana,
North Carolina, South Carolina, Wisconsin, Michigan, Alberta, Manitoba,
Ontario and Quebec. Of these regions, Florida was the only place other
than California that reported widespread D. suzukii populations. In Florida,
unlike California, D. suzukii were not reported to cause wide spread
damage, which was probably due to Florida’s agricultural practices that
targeted other adult pest species and, in turn fortuitously inhibited the
growth of D. suzukii populations (Hauser, 2011).
Reports of D. suzukii were not limited to North America, as the fly
began to be reported in Europe starting in October of 2008 (Hauser, 2011)
and in South America, particularly southern Brazil, in 2012 (Deprá et al.,
2014). In Europe, D. suzukii first appeared in Spain and later in Italy and
France. However, Northern Italy was the only region to experience

significant crop damage (Hauser, 2011). The northern humid regions of

Europe have been predicted to be more suitable ecosystems for D. suzukii
compared to drier environments like the Mediterranean, since desiccation
is known to be a limiting factor for D. suzukii (Walsh et al., 2011). By
2010-2011 the range of D. suzukii had increased to include Switzerland,
Slovenia, Croatia, Austria, Germany, and Belgium, and today researchers
believe that all of continental Europe is at risk of invasion (Cini et al.,
2012).
IDENTIFICATION
Drosophila suzukii closely resembles the common laboratory fly

Drosophila melanogaster in both size and appearance. D. suzukii belongs
to the D. suzukii species subgroup (subgenus Sophophora, D. melanogaster
species group), comprised primarily of Asian species and one species from
Africa (Hauser, 2011). Drosophila biarmipes, also in the melanogaster
subgroup, is considered to be the sister species to D. suzukii (Cini et al.,
2012).
The nickname Spotted Wing Drosophila (SWD) derives from the
single black spot found on each wing of D. suzukii males. However,
confusion may arise in identification, as both D. biarmipes or D.
subpulchrella have similar wing spots (Cini et al., 2012; Hauser, 2011). D.
suzukii males are also known for having two sets of black tarsal combs -
one on the first, and one on the second, tarsal segments (Hauser, 2011).
D. suzukii females are distinct from most other drosophilids in having
a large, long, ovipositor which they use to penetrate the soft skin of certain
fruits. Other Drosophila females possess ovipositors, but D. suzukii
females can be distinguished from these by comparing the size of the
ovipositor to their two spherical, mushroom-cap shaped spermathecae
(receptacles in which sperm is stored after mating). In D. suzukii, the
ovipositor is 6-7 times as long as the spermathecae whereas in most other
drosophilids, who by utilizing rotting fruit do not have to pierce the skin,
the ovipositor is only 2-4 times as long as the spermathecae.

AGRICULTURAL IMPACT
D. suzukii females’ ability to exploit ripe fruit makes it an agricultural

pest as compared to other species of Drosophila that utilize decaying fruit
(Bellamy et al., 2013). Out of approximately 1500 Drosophila species, D.
suzukii is one of only two drosophila species known to oviposit in fruit that
is still on the vine (the other being the Japanese species D. pulchrella)
(Walsh et al., 2011). The insertion of the ovipositor causes physical
damage to the fruit and allows other forms of infecting agents to enter,
including filamentous fungi, yeasts and bacteria that may increase the
fruit’s deterioration and lead to further crop losses. The main form of
damage, however, is due to the hatched larvae, which feed on the inside of
the fruit and thus render it soft and completely unsuitable for harvest
(Cini et al., 2012).
What makes D. suzukii so pervasive and concerning is its ability to
utilize a large variety of different hosts, including strawberries, blueberries,
raspberries, blackberries, cranberries, boysenberry, table and wine grapes,
nectarines, apricots, cherries, plums, peaches, pears, kiwis, persimmons,
and figs, as well as injured apples and oranges. They are also extremely
opportunistic, utilizing alternate resources including spoiled fallen fruit,
oak tree sap, and even flowers, in the absence of ripe fruit. This ability to
use a wide variety of resources to lay eggs is of great concern in regions
like U.S. Pacific coastal states, where there are a wide variety of
ornamental and native fruiting plants that ripen at different times
throughout the year, enabling D. suzukii population numbers to stay high
all year long (Walsh et al., 2011).
In the United States, the majority of small fruit production occurs in
the Pacific Coast states. California, Oregon, and Washington account for
76% of the commercial production of raspberries, blackberries,
strawberries, blueberries and cherries, or approximately $2.6 billion in
value. Though there is very little known about the cumulative potential
damage that D. suzukii could cause, one estimate claimed that if there were

20% yield losses (which is considered possible) for the five main crops in
these Pacific Coast states, then damages from D. suzukii could amount to
$511 million annually (Walsh et al., 2011).
CURRENT MANAGEMENT MEASURES
Many different approaches are currently being investigated. Several

insecticides have been shown to be effective against D. suzukii. However,
this species’ preference for ripening fruit causes problems regarding
application timing (amount of days needed between pesticide applications
and harvesting) and also the possibility of negative impacts on beneficial
plant pollinators. Traps (typically yeast, sugar and soapy water) are
effective at catching D. suzukii, but it is unknown whether this practice
results in a significant reduction in crop damage. The parasitoid wasps
Braconidae and Cynipidae are thought to be the primary predators on other
Drosophila species and research is underway to examine the possibility of
using these animals as biological control agents against D. suzukii.
Research has also been conducted on Orius insidiosus, which has been
reported to feed on D. suzukii larva in raspberries and blueberries (Walsh
et al., 2011).
This Study
The objective of this study was to analyze how exposure to fruit,

blueberries or raspberries, before and after mating affected reproductive
behaviors and offspring production. It was expected that the presence of
fruit would have a significant impact on offspring production, however, it
was unclear whether the presence of fruit prior to mating would make a
difference in offspring production. These studies were conducted with the
purpose of adding to the existing knowledge of D. suzukii biology in order
to aid efforts to control this important pest species.

MATERIALS AND METHODS
Stocks
D. suzukii stocks were maintained in glass vials containing Carolina

Instant Drosophila Media (Formula 4-24), mixed with a 0.05% propionic
acid solution, plus yeast and a single whole blueberry. A small piece of
moistened paper was also added to each vial. The vials were maintained
between 25-27°C under a 16:8 light-dark cycle. Water was pipetted into
each vial 2-3 times/week to ensure that the paper towel remained moist.
When pupal casings were noted inside the vials, usually within 2 weeks
after flies were transferred to the vial, the adult flies were transferred to
new stock vials.
Courtship Behavior and Lifecycle
Drosophila utilize a ‘female choice’ sexual system, in which females

determine whether or not mating will occur. To increase female
receptivity, Drosophila males perform complex courtship displays (see
Greenspan and Ferveur, 2000; McRobert, 2004). These displays typically
include: orientation of the male’s body so that he is facing the female,
following the female if she moves away, tapping the abdomen of the
female with the fore-tarsi (possibly to pick up chemical cues), extension
and vibration of the wings (presumably to produce a courtship song),
extension of both wings while facing the female (possibly showing the
wing spots to the female), and attempted copulation (McRobert and
Jackson, 1989). Mating will ensue only if the female is sufficiently
stimulated by the male’s display.
Similar to other Drosophila species, the D. suzukii life cycle is short,
moving from egg to egg-laying female in just over seven days at 21.1°C, to
12-15 days at 18.3°C (Kanzawa 1939, Walsh et al., 2011). Newly-eclosed
flies reach maturity in one to two days (Lin et al., 2014, Cini et al., 2012),
and the adult life span can be between 3 and 9 weeks. In Japan, D. suzukii

can have between 10 to 13 generations/year and in California they survive,

on average, for 10 generations/year (Kanzawa 1939, Walsh et al., 2011). A
single female may lay between 200 and 600 eggs in her lifetime
(Cini et al., 2012).
Mating Assays
Virgin flies were collected within six hours of eclosion and sexed
under CO2 anesthesia. Newly-eclosed males, which lack wing spots, were
identified by their dark pigmented genitalia and small body size (compared
to females). Females were identified by their larger body size and by the
presence of an ovipositor. After collection, virgin males were housed,
individually, in vials containing Drosophila media, 0.05% propionic acid
solution and yeast. Virgin females were separated into two groups. One
group was housed, individually, in vials containing Instant Drosophila
media (4-24), 0.05% propionic acid solution and yeast. The other group
was housed, individually, in vials containing Drosophila media, 0.05%
propionic acid solution, yeast and one whole piece of fruit: either a whole
blueberry or a whole raspberry.
All mating assays were conducted in the morning (0700 – 1200), and
flies were 3-5 d old when tested. For each assay, single male-female pairs
were aspirated into glass vials (containing Instant Drosophila Media, but
no fruit) and observed for 60 minutes. During this time, copulation
frequency, copulation latency (time until copulation commenced) and
copulation duration (length of the time the flies were in copula) were
recorded.
Offspring Production
Virgin flies were collected within six hours of eclosion and sexed
under CO2 anesthesia. Virgin males were housed, individually, in vials
containing Drosophila media, 0.05% propionic acid solution and yeast.

Virgin females were housed, individually, in vials containing Drosophila

media, 0.05% propionic acid solution and yeast, in one of 12 conditions
prior to, and following, mating (see Table 1).
Table 1. Housing conditions for females prior to, and following,

mating. + indicates the addition a whole piece of fruit (blueberry or
raspberry), – indicates no fruit was added to the basic food media
Fruit Condition Conditions before and after

mating
Blueberry Whole +/+
+/-
-/+
Raspberry +/+
+/-
-/+
Mated females were left in vials for 7 days, after which offspring were
counted. To count offspring, a 20% sucrose solution was added to each vial
and stirred into the food, which was then poured into a flat plastic dish.
Eggs and larvae, which floated in the sucrose solution, were then counted
(see Frank et al., 1995).
RESULTS
Mating Assays
Whole Berry Mating Assays

No statistical difference was found in mating success between those
raised with (58/81: 71.6%) or without (52/80: 65.0%) a blueberry (chi-
square test df = 1, 𝑋 2 = 0.53, p = 0.47) or with (55/84: 65.5%) vs. without
(48/88: 54.5%) a raspberry (chi-square test df = 1, 𝑋 2 = 1.7, p = 0.19).

Further, copulation latency and duration both showed a similar patter with
no statistical difference between the presence of blueberries or raspberries
(p > 0.5 for all comparisons). Flies showed a latency of 1331.5 ± 166.4s
(average ± standard deviation) with and 1479.3 ± 233.3s without
blueberries, while those with a raspberry had a latency of 385.4 ± 86.8s vs
657.3 ± 109.8s without. Duration results were 1323.9 ± 80.2s with a
blueberry vs. 1257.4 ± 51.8s without and 1322.1 ± 44.7s with a raspberry
vs 1226.8 ± 40.2s without.
Offspring Assays
Effect of Whole Berries on Offspring Production

The number of offspring was found to be significantly different
between blueberry treatments (one-way ANOVA, df = 3, F = 5.8, p < 0.01)
with B+/+ females producing the most offspring (42.9 ± 4.3), followed by
B+/- females (39.5 ± 4.9), B-/+ females (25.1 ± 3.2) and then the control
group (23.5 ± 3.5). Bonferroni Post hoc analysis showed that B+/+ and B
+/- were similar, but both were significantly different from the control
group as was B+/+ from B-/+.
Raspberry treatments resulted in a similar order of production with the
most offspring from the R+/+ females (30.7 ± 3.6), followed by R+/-
females (24.8 ± 4.3), R-/+ females (19.8 ± 3.1) and then the control group
(14.3 ± 3.1) and a similar statistical result (one-way ANOVA, df = 3, F =
5.8, p < 0.01). Bonferroni post-hoc analysis showed less internal
differences, with only the R+/+ females having significantly more
offspring than the control group (p < 0.01).
Finally, comparison of offspring production between the housing
conditions found B+/+ females had significantly more offspring than R+/+
females (two sample t-test: p = 0.03) and B+/- females had significantly
more offspring than R+/- females (two sample t-test: p = 0.03). No
significance difference was found, however, between the offspring
produced by B-/+ females and R-/+ females (two sample t-test: p > 0.05).

DISCUSSION
In this study, we have examined the effect of fruit (blueberries and

raspberries) on specific aspects of the reproductive life history of the
agricultural pest Drosophila suzukii. Our findings show that the availability
of whole blueberries or raspberries led to increased offspring production.
This is consistent with our expectations, as D. suzukii stocks are difficult to
maintain in captivity on Drosophila media alone. Addition of whole fruit
to the media leads to healthy stocks, presumably (from what has been
shown here) because the fruit increases offspring production. With regard
to sexual behavior, prior to and during copulation, the availability of fruit
had no effect on copulation frequency, copulation latency, or copulation
duration. This suggests that while female exposure to fruit affects the
production of healthy eggs, it has no effect on the CNS control of female
mating behavior.
With regard to copulation frequency, Drosophila utilize a female-
choice mating paradigm in which the males court indiscriminately, and
females assess courtship and determine whether mating will occur. Female
nutrition has been shown to affect their receptivity to courtship. For
example, the presence of yeast in the diet of D. melanogaster females has
been shown to increase copulation frequency, without increasing the sexual
attractiveness of the female (McRobert, 1986; Chapman and Partridge,
1996). Conversely, lack of nutritious food for D. melanogaster females has
been shown to inhibit copulation (Fricke and Bretman, 2009), possibly by
inhibiting oogenesis (Wheeler, 1996). In this study, however, we found
that while female nutrition affected offspring production, it did not affect
the likelihood that a female would mate.
Copulation duration, or time a pair of flies remain in copula, appears to
be under the control of the male. In studies on D. melanogaster it has been
shown that copulation duration is affected by mutations in genes that affect
male anatomical structures and certain aspects of courtship behavior in
males (Yamamoto and Nakano, 1999; Kuniyoshi et al., 2002), as well as
the activity of circadian clock genes in males (Beaver and Giebultowicz,

2004). Therefore, it is not surprising that we found no effect of female diet

on copulation duration.
With regard to offspring production, females provided whole fruit,
both before and after mating, produced the most offspring. The next
highest numbers of offspring were seen in the housing condition that only
provided fruit before mating, followed by the housing condition that only
provided fruit after mating, and then the control, which had the least
amount of offspring. Our data indicate that while there can be marginal
improvements in fecundity from fruit availability after fertilization, the
greatest effect comes from availability before copulation. This is likely due
to a dietary need for some aspect of reproductive physiology such as
oogenesis.
Our results support earlier findings that nutrition affects offspring
production in insects. In D. melanogaster it has been shown that slight
variations in nutritional components have significant effects on offspring
production. For instance, Lee et al., (2008) found that ingesting food with a
protein-to-carbohydrate ratio of 1:2 led to maximum egg-laying rates, and
a P: C ration of 1:4 maximized life-time egg production. What’s more, it
was shown that flies actively regulate their diet in a way that maximizes
their fitness. What’s interesting about the results of our study is that D.
suzukii females, known to utilize ripe fruit as an oviposition site, can also
benefit from the availability of fruit prior to mating. This indicates that ripe
fruit may provide an important nutrient source for D. suzukii adults as well
as their larvae.
CONCLUSION
Overall, exposure to soft skinned fruit, known to be impacted by D.

suzukii, had no significant effect on whether a D. suzukii female would
copulate, how quickly she copulated, nor long copulation continued, and
for how long. However, exposure to soft skinned fruit, both prior to, and
following, mating, led to significant increases in offspring production by
D. suzukii females.

From these studies, a number of questions arise, which will guide

future studies. In particular, what it is about blueberries or raspberries that
gives rise to the increased number of offspring. Our lab has started to
develop techniques to isolate or manipulate the chemical composition of
the food provided to the flies through the use of artificial “berries”. These
constructs have similar mechanical and structural characteristics to real
berries but can be made from any fraction of the juice from a berry, or
other solutions of our choosing. Further, we are looking at differences
between D. suzukii and its sister species D. biarmipes to determine if they
have differences in their reproductive behaviors and impacts that might
reflect or be illustrative of their relationship with fruit and its significance
in their lifecycle.
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Chapter 8
PHEROMONE COMMUNICATION SYSTEMS

IN THE NASONIA SPECIES COMPLEX
Jan Buellesbach, PhD

Department of Environmental Science Policy & Management,
University of California
Berkeley, CA, US
ABSTRACT
The parasitoid jewel wasp genus Nasonia, encompassing four

species, has proven to be an excellent model system to investigate
genetic, behavioral and ecological aspects of chemical communication. In
recent years, it has become one of the best-investigated hymenopteran
taxa concerning pheromone-based sexual signaling, mate choice and
assortative mating behavior. A hierarchy of three main pheromone
communication systems can be clearly differentiated in Nasonia:

Corresponding Author Email: jan.b@berkeley.edu.

206 Jan Buellesbach
 A long-range pheromone produced in the male abdomen to attract

virgin females from longer distances.
 Female cuticular hydrocarbons that act as short-range contact
pheromones eliciting male arrestment, courtship and copulation
behavior.
 A male-produced mouthpart pheromone released during
courtship, hypothesized to induce female receptivity, but so far
only demonstrated to terminate the female response to the long-
range sex attractant.
These three different systems are discussed in light of their

respective chemical basis, hierarchical interplay, species-specificity and
potential contribution to prezygotic reproductive isolation.
INTRODUCTION
Research on chemical communication in the Hymenoptera, one of the

largest and most prominent insect orders (approximately 115,000 reported
species so far, with estimates of up to 10 times more species in existence,
Grimaldi and Engel 2005; Sharkey 2007) has been mostly devoted to the
eusocial taxa. Particularly, the phenomenon of intraspecific nestmate and
colony recognition mediated by characteristic chemical profiles has been
most extensively studied for many decades (e.g., Gamboa et al., 1986; Van
der Meer and Werren 1998; Dani et al., 2005; Jandt and Gordon 2016).
However, chemical communication in non-social Hymenopteran taxa has
received less research attention so far, although they contain vast potential
for insights into the distinctive features fundamentally separating them
from the eusocial life style (Ayasse et al., 2001).
The jewel wasp genus Nasonia (Hymenoptera: Pteromalidae),
encompassing the four species N. vitripennis, N. giraulti, N. longicornis
and N. oneida, represents an exception as an excellent non-social
Hymenopteran model system with a well-established research history. All
Nasonia species parasitize the pupae of various Dipteran host species,
eventually consuming and killing their hosts (Whiting 1967; Darling and
Werren 1990), which classifies them as “parasitoids”, distinguished from
“parasites” that, in turn, are not naturally lethal to their hosts (Reuter

Pheromone Communication Systems … 207
1913). The host range of Nasonia includes economically relevant

agricultural pests, such as staple-, flesh- and blowflies, which has led to
their successful establishment and application as biological pest control
agents (Rutz and Scoles 1989; Fried et al., 1990; Kaufman et al., 2001).
The pursuit to improve their efficiency in controlling agricultural pests
has fostered considerable interest in closer investigations of their
reproductive behavior, instrumental for optimizing their implementation as
large-scale agricultural pest control (Benelli et al., 2014). Thus, their mate
assessment, mate choice and general mating behavior have been
exceptionally well-studied as opposed to other parasitoids in particular and
other non-social Hymenoptera in general (Barrass 1960; Matthews 1975;
van den Assem 1986). Concordantly, the elucidation of the chemical
compounds they use as sexual signals has been particularly successful,
resulting in several intriguing findings greatly contributing to our general
understanding of insect pheromone communication.
LONG-RANGE MALE SEX PHEROMONE
Notably, Nasonia vitripennis was the first parasitoid Hymenopteran

species where a male sex pheromone could be identified that attracts virgin
females (Ruther et al., 2007, Figure 1 a). Since Nasonia males develop
faster and eclose earlier than females (i.e., protandry, Morbey and
Ydenberg 2001), release of this long-range sex pheromone typically takes
place early on, after males have emerged from the host pupa wall and
compete to gain access for the later eclosing females (Whiting 1967). The
release of the pheromone has been hypothesized to influence the outcome
of these competitions, signaling mate quality through correlation with
sperm titers (Ruther et al., 2009) and body size (Blaul and Ruther 2012),
furthermore mediating territory marking behavior and site fidelity in the
males (Ruther et al., 2011).
Biosynthesized in the male rectal vesicle and released via the anal
orifice by dabbing movements of the abdominal tip (Abdel-latief et al.,
2008; Steiner & Ruther 2009), it consists of a diastereomeric mixture of

208 Jan Buellesbach
Erythro-(4R,5S)- and Threo-(4R,5R)-5-hydroxy-4-decanolides (HDL,

Ruther et al., 2007) and the synergizing trace component 4-
methylquinazoline (4-MeQ, Ruther et al., 2008).
Figure 1. Schematic of Nasonia vitripennis male (left) and female (right) representing
their respective contributions to the hierachical pheromone communication system.
a) The long-range sex pheromone consisting of the two main components

Erythro-(4R,5S)- and Threo-(4R,5R)-5-hydroxy-4-decanolide (HDL) and the
synergistic minor component 4-methylquinazoline (4-MeQ). Note that this
composition is unique to Nasonia vitripennis, as the other 3 species all lack
Threo-(4R,5R)-HDL.
b) Short-range (contact) pheromone comprised of the female cuticular

hydrocarbon (CHC) profiles which itself consist of over 75 different
compounds. Exemplary representations of the three main compound classed,
alkanes, alkenes and methyl-branched alkanes, are indicated.
c) Courtship pheromone released by the males after mounting the female. The
three fatty acid ethyl esters ethyl oleate, ethyl linoleate and ethyl α-linolenate
have been identified from male (and female) cephalic extractions and shown
to terminate the response of virgin females to the male long-range sex
pheromone. However, the receptivity inducing agent has not been isolated or
characterized as of yet, indicated by the question mark.
A correlation could be established between linoleic acid as a precursor

compound in the larval diet and higher pheromone titers in adults (Blaul

and Ruther 2011), although Nasonia has also been shown to be capable of
biosynthesizing the precursor compound themselves from oleic acid,
potentially through a putative ∆12-desaturase (Blaul et al., 2014).
Furthermore, the Epoxide Hydrolase Nasvi-EH1 has been shown to be
functionally involved in the biosynthesis of the diastereoisomers
(Abdel-latief et al., 2008).
The composition of the male sex pheromone blend varies between the
species (Niehuis et al., 2013): All species produce the main component
Erythro-HDL and the trace component 4-MeQ, whereas the diastereomere
Threo-HDL is exclusively produced by N. vitripennis. Virgin N. vitripennis
females show clear preference behavior towards blends containing the
species-exclusive Threo-HDL compound (Niehuis et al., 2013). This bears
some interesting implications on the potential evolutionary emergence of
this species-specific pheromone compound functioning in mate
discrimination and prezygotic reproductive isolation (Ruther et al., 2014).
The relatively simple genetic basis of the stereochemical difference
between the two main pheromone compounds corroborates these
implications, with three closely linked genes coding for short-chain
dehydrogenases/reductases constituting the main mediators (Niehuis et al.,
2013). Knockdown of either gene in N. vitripennis leads to an apparent
inability to produce the exclusive Threo-HDL pheromone compound,
effectively resulting in the pheromone phenotype characteristic for the
other three species, hypothesized to be the ancestral state. This signifies
both the importance of the synergy of all three genes for the biosynthesis
of the N. vitripennis-exclusive Threo-HDL compound as well as showing a
hypothetical and plausible scenario for the potential evolution of the
genetic and biosynthetic capability to produce novel pheromone
compounds (Niehuis et al., 2013; Ruther et al., 2014).
SHORT RANGE FEMALE CONTACT PHEROMONE
The next step in the hierarchy of pheromone-based sexual signaling

occurs when the potential mating partners come into closer proximity,

210 Jan Buellesbach
being mediated by female cuticular hydrocarbons (CHC) functioning as

close-range cues inducing arrestment and courtship behavior in the males
(Steiner et al., 2006, Figure 1 c). As the dominant fraction of the outer lipid
layer on the insect epicuticle primarily providing desiccation resistance
(Howard and Blomquist 1982; Lockey 1985), CHC are involved in a wide
variety of chemical signaling systems across all major insect taxa, most
prominently mediating sexual (e.g., Cobb and Jallon 1990; Simmons et al.,
2003; Grillet et al., 2006) and species recognition behavior (e.g., Singer
1998; Howard et al., 2003; South et al., 2008). In Nasonia, over 75
different CHC compounds could be detected, mainly composed of alkanes,
alkenes and methyl-branched alkanes, with the latter constituting the
dominant compound class in all Nasonia species (Buellesbach et al., 2013,
see Figure 1 c). A pilot study accessing the genetic basis of Nasonia CHC
variation found a rather complex pattern, with over 100 quantitative trait
loci (QTL) responsible for the variation in the different CHC compounds
between N. vitripennis and N. giraulti alone (Niehuis et al., 2011).
However, clusters of QTL governing the variation in structurally similar
compounds give first hints at shared biosynthetic pathways through
potentially closely linked co-localizing genes.
Female CHC profiles have been shown to display pronounced
differences between the four Nasonia species (Buellesbach et al., 2013).
These differences generally function in mediating preference behavior of
conover heterospecific females, but in populations of one particular
species, N. giraulti, they appear to mediate the opposite: Preference of
heterospecific female CHC profiles, while conspecific CHC profiles
apparently lost the sexual signaling function (Buellesbach et al., 2013).
This surprising finding is corroborated by an apparent shift in N. giraulti
female CHC profiles, distinguishing them the most from the other female
profiles. This hints at other sexual signaling modalities potentially taking
precedence over CHC in that particular species, further demonstrating the
importance of the interplay of several communication systems ensuring
successful mate recognition, acceptance and reproduction.

CEPHALIC MALE COURTSHIP PHEROMONE
For matings to ultimately occur in Nasonia, a third pheromone-

mediated mate assessment step appears to be crucial, relegating the role of
the choosy sex back to the females. After a male has mounted a potential
female mate, he initiates a stereotypic display of courtship behaviors,
consisting of a successive series of head nods with synchronous extrusions
of his mouthparts close to the female antennae (Barrass 1960). This series
of courtship behaviors ultimately leads to the female signaling receptivity
by lowering her antenna, raising her abdomen and extruding her genitalia.
It has been suggested that an air-borne chemical aphrodisiac is released
from the male mouthparts on the female antennae inducing receptivity (van
den Assem et al., 1980; Ruther et al., 2010). Males with glued mouthparts
lose the ability to render females receptive, but contact with air vented
from regularly courting and copulating mating pairs apparently induces
receptivity in females even when directly courted by males with their
mouthparts glued (van den Assem et al., 1980).
But so far, a chemical aphrodisiac directly inducing female receptivity
could neither be isolated nor characterized despite decades of intensive
research (Ruther, Schmitt & Werren, pers. comm.). However, it has
recently been demonstrated that three distinct fatty acid ethyl esters
identified from cephalic male extracts terminate the response of virgin
females to the male long-range sex attractant (Ruther and Hammerl 2014,
Fig 1 b). Interestingly, these compounds are not male-specific, as they have
also been identified in cephalic female extracts. It has been hypothesized
that they are exclusively released by the males however, since only males
display characteristic mouthpart extrusions during courtship. It is
interesting to note that the identified male cephalic pheromone which is not
active before actual courtship apparently directly influences the
hierarchically earlier acting female perception of the long-range
pheromone which functions primarily in initial mate finding and attraction
(Ruther et al., 2010; Ruther and Hammerl 2014).

212 Jan Buellesbach
CONCLUSION
This chapter discussed the chemically, functionally and behaviorally

very distinct features of different pheromone communication systems that
clearly form a sequential hierarchy in the Nasonia species complex. With
increasing proximity, different pheromones with divergent chemical
properties, dispersal modalities and perception functionalities are used for
attracting, assessing and ultimately courting potential mates, eventually
paving the way to ensure successful reproduction. The interplay of these
diverse pheromone communication systems presents multiple selective
regimes for both assuring mate quality (intraspecifically) as well as
assortative mating (interspecifically). The latter of which appears to be of
particular importance, as the Nasonia species are reproductively isolated in
nature by different species-specific strains of Wolbachia bacteria, causing
cytoplasmic incompatibility and hypothesized to inflict strong fitness costs
on interspecific matings (Breeuwer and Werren 1990; Bordenstein and
Werren 1998; Bordenstein et al., 2001). Hence, a rigorous, multi-level
selection of suitable mates for successful reproduction appears to be an
important facet of Nasonia mating behavior, potentially having contributed
to the evolution of its complex hierarchy of pheromone communication
systems.
ACKNOWLEDGMENTS
I would like to thank Elizabeth I. Cash, Thomas Schmitt and Brian A.

Whyte for reviewing this chapter and offering valuable suggestions, and
Christian Hanner for the schematic Nasonia drawings in the accompanying
figure. The chemical structures represented in Fig. 1 were created with
ChemDraw Direct, Version 1.5.1.190
(https://chemdrawdirect-cdn.perkinelmer.com/1.5.1/demo/index.html).

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Chapter 9
GIARDIASIS IN BIRDS
Elis Domingos Ferrari1,*,

Bruno César Miranda Oliveira2,3,
Mariele Fernanda da Cruz Panegossi2,
Gisele Moraes dos Santos Reginaldo²,
Walter Bertequini Nagata2 and
Katia Denise Saraiva Bresciani2, PhD
1
Departamento de Clínica, Cirurgia e Reprodução Animal –
Faculdade de Medicina Veterinária, Araçatuba,
São Paulo, Brasil
2
Departamento de Apoio, Produção e Saúde Animal -
Faculdade de Medicina Veterinária, Araçatuba,
São Paulo, Brasil
*
Corresponding Author Email: elisd.ferrari@yahoo.com.br.

220 E. D. Ferrari, B. C. M. Oliveira, M. F. da Cruz Panegossi et al.
³Tufts Cummings School of Veterinary Medicine,

North Grafton, Massachusetts, US
ABSTRACT
The protozoan Giardia spp. is a flagellate intestinal parasite and six

species are recognized, being G. agilis that affects amphibians, G. psittaci
and G. ardeae that infect birds, G. microti and G. muris that can be
present in rodents and G. duodenalis that can cause disease in humans
and some domestic animals, according to their assemblages, ranging from
A to H. The individual can be infected by consuming water and foods
with the presence of cysts. The frequently clinical signs seen in the
Giardia infection are delayed growth, dehydration, weight loss, and
recurrent diarrhea. The occurrence of Giardia spp. in birds from a
particular site demonstrates the extent of the disease, its specificity to its
hosts, its risk and transmission potential.
Keywords: Giardia, Birds, Pet Birds
INTRODUCTION
The protozoan Giardia spp. is a flagellate intestinal parasite belonging

to the class Zoomastigophorea and to the order Diplomonadida, whose
genus is omnipresent among vertebrate animals, but which may differ its
species depending on the class of the affected individuals (Thompson and
Monis 2004, 69-120; Abe et al. 2012, 1710-11). Based on what has already
been studied, it is known that the genotypes known usually be well adapted
to their hosts in nature, therefore, the same species of Giardia does not
normally affect individuals of different classes or orders. The zoonotic
potential of the parasite is related to the specificity and number of animals
that its species can reach (Xiao and Fayer 2008, 1243-44).
Six species are recognized in different hosts: Giardia agilis affects
amphibians, Giardia psittaci and Giardia ardeae infect birds, Giardia

Giardiasis in Birds 221
microti and Giardia murisis can be present in rodents and Giardia

duodenalis can cause disease in humans and some domestic animals,
according to their assemblages, ranging from A to H (Adam 2001, 447-70;
Ballweber et al. 2010, 180-87; Abe et al. 2012, 1710-11; Reboredo-
Fernández et al. 2015, 1-2).
LIFE CYCLE
In parasite life cycle, the host is infected by ingestion of contaminated

food or with cysts of the parasite. Giardia has two morphologies: cyst and
trophozoite (Farthing 1994, 15-7).
The trophozoite has a pyriform or ellipsoid shape, its body presents a
dorso-ventral flattening that forms the suctorial disc, being this structure
responsible for the adhesion in the intestinal epithelium of the host, causing
the clinical manifestations as diarrhea and malabsorption (Health Canada
2011, 5). The cyst has an oval or ellipsoid shape, and in its interior there
are two to four nuclei, a variable number of fibrils and the dark bodies
half-moon shaped located on the pole opposite the nuclei. This immobile
parasitic structure is the latent form of the parasite, and can survive for
several months in the environment (Health Canada 2011, 5). Mature cysts
with potential to form trophozoites are the forms normally found in the
feces of the infected host.
After ingestion of the cyst, the acidic conditions of the stomach and the
pancreatic enzymes stimulate the process of excystation and liberation of
the trophozoites, the active form of the parasite, that will multiply
asexually and, by binary division, they arrive at the light of the intestine,
where they adhere to the mucosa by suction mechanism, establishing the
infection. Under appropriate conditions these trophozoites are again
transformed into cysts. The formation of the cysts occurs when the parasite
transits the colon to be eliminated by stool (Payne and Artzer 2009, 6).

EPIDEMIOLOGY
In underdeveloped countries, where the disease becomes more

clinically relevant, since the basic sanitation and hygiene conditions are
precarious, allowing and facilitating the fecal-oral transmission of the cysts
(Thompson and Monis 2004, 69-120). Although some species of Giardia
spp. Are host-specific, G. duodenalis, assemblages A and B, can affect
both humans and animals and is, therefore, considered a zoonosis (Feng
and Xiao 2011, 110-32). Although this species is not recognized as causing
disease in birds, assemblages A, B, D and F have already been found in
wild birds, enabling that these cysts to contaminate streams and vegetation,
with the ability to infect humans, animals and other wild animals, mainly
because some birds have migratory habits (Upcroft et al. 1997, 408-10;
Reboredo-Fernández et al. 2015, 6-8).
Due to this, although the occurrence of Giardia spp. in poultry has
been little studied, this data from a particular site demonstrates not only the
extent of the disease and its specificity to its hosts, but also its risk and
transmission potential among other wild animals, to animals of production
and to humans.
CLINICAL SIGNS
Studies addressing Giardia spp. causing disease in birds are scarce.

The frequently clinical signs seen in the Giardia infection are growth
retardation, dehydration, weight loss and recurrent diarrhea, already found
in several parakeets, a cockatoo (Cacatua galerita) and ibis (Threskiornis
spinicolli), being caused by the following species of Parasite, respectively:
G. psittaci (Filippich et al. 1998, 247-48), G. duodenalis (Upcroft et al.
1997, 408-10) and G. ardeae (Mcroberts et al. 1996, 714-16).
The disease may also present as subclinical asymptomatic, however,
these birds may eliminate the infecting cysts in their feces, as well as in the
clinical form of the disease, participating in the spread of the disease.

DIAGNOSIS
Due to the intermittent elimination in the faeces of their hosts, when

Giardia is among the clinical suspicions, the collection of feces should be
done three times and analyzed in a pool to increase the accuracy of the
diagnosis.
The morphology of Giardia spp. Does not vary remarkably among its
species, being that between the assemblages of G. duodenalis it is
impossible the differentiation, necessitating to realize the molecular
characterization (Cacciò et al. 2008, 1523-24).
The most simple and low cost technique used is microscopy after
purification and concentration of cysts by centrifugation in zinc sulfate
solution. Immunological methods for fecal antigen search are also
available, but none yet standardized for performing diagnostic in bird
samples. The most sensitive technique available is the detection and
molecular characterization by polymerase chain reaction (PCR) for the
detection of parasite DNA in feces, but it is a high-cost test that requires
skilled people and sophisticated equipment for its achievement. Thus,
microscopy is usually the option of choice for diagnosis in the clinical
routine.
TREATMENT
Treatment should be instituted from an accurate diagnosis of

giardiasis, with the aim of eliminating clinical signs and stopping the
elimination of cysts. Asymptomatic animals also require treatment, as the
infection may lead to decreased immunity and susceptibility to other
diseases (Bush et al. 2011, 1261–1272).
There are few drug studies available for the treatment of Giardiasis in
birds, and those available are very old. Thus, we have few data on drug
resistance on this genus of parasites in birds. In order to avoid the

indiscriminate use of antiparasitics, the ideal is that prevention and

prophylaxis be done adequately, as well as treatment.
Metronidazole administered orally is still the recommended treatment
for the treatment of giardiasis. Due to the stress caused by birds on
handling, risks and difficulty in administering medications to birds, the
most used way to administer the drug is to dilute it in drinking water at the
dosage of 800 milligrams per liter of water for five days (Filippich et al.
1998, 246-249). Another drug is dimetridazole, but it is little used and
unavailable in many countries.
PROPHIILAXY
Prevention is the appropriate way to avoid contamination of the

environment and infection of other animals and also of humans. This
practice is based on the good hygiene of the cages, do not let these feces
remain in the environment, avoid the contact of fomites with the excrement
of the birds and the elevation of the water and food to avoid contamination
(Filippich et al. 1998, 246-249), since birds can perch in the containers and
defecating on their own supplies.
A way to minimize environmental contamination with Giardia spp. is
the process of raising awareness among bird owners and showing how
important it is to conduct tests on these animals (Farlen et al. 2007, 265-
268), since it is common for bird owners have many animals, several of
them including in the same cage or nursery.
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006.

INDEX
Amblyomma sculptum, 157, 168

A
amphibians, xiii, 7, 220
anatidae, 114, 138
adaptability, ix, 44, 63
animal breeding, vii, ix, 44, 47, 48, 50, 51,
adaptation, 14, 59, 174
52, 53, 55, 56, 58, 62, 65
adults, 9, 90, 159, 164, 177, 200, 208
animal welfare, 45
Africa, x, 74, 75, 78, 79, 81, 82, 83, 84, 85,
Anser anser, v, xi, 111, 112, 116, 133, 140,
86, 88, 90, 95, 96, 98, 99, 101, 102, 103,
141, 142, 145
105, 108, 109, 110, 192
antelope, 89, 104, 105
African buffalo, 80, 89
anthropologists, 92
aggressiveness, 100
anthropology, 92, 114
agricultural pest, xii, 189, 190, 193, 199,
Argentina, 43, 173
207
Asia, 79, 85, 190
agriculture, 61, 69, 83, 84, 96, 108
Austria, 13, 18, 45, 48, 71, 111, 118, 121,
Alaska, 10, 143
142, 169, 192
alkenes, 208, 210, 214
avian, viii, 2, 3, 4, 9, 10, 12, 14, 15, 17, 18,
Amblyomma, vi, viii, xi, 149, 150, 151, 152,
19, 20, 22, 23, 24, 25, 27, 28, 33, 36, 37,
153, 154, 155, 156, 161, 163, 166, 167,
38, 41, 114, 135, 136, 167
168, 170
Amblyomma aureolatum, xii, 150, 156, 166
Amblyomma coelebs, 157 B
Amblyomma longirostre, xii, 150, 156, 161
Amblyomma nodosum, xii, 150, 156, 163 Baars, 54, 59, 61, 65
Amblyomma ovale, 157, 166 bacteria, 20, 21, 151, 175, 193, 212
Amblyomma parkeri, xii, 150, 156, 167 bacterial infection, 24

228 Index
Barbados, 119, 125, 126 carnivores, x, 74, 83, 92, 93, 94, 106, 110,
barnacle geese (Branta leucopsis, 114, 129, 113
136, 137, 143, 144, 145 cattle, vii, ix, x, 22, 44, 45, 46, 47, 50, 59,
barrows goldeneye (B. islandica, 115 61, 62, 63, 65, 66, 67, 68, 69, 70, 71, 74,
Benin, 75, 76, 77, 80, 109 85, 86, 88, 90, 96, 100, 101, 102, 108,
biogeography, x, 73, 91, 92, 100, 106 109, 173
biological control, 194, 213 cell culture, 27, 31, 34
biparental care, 116 Central African Republic, 75, 87
birds, vi, vii, viii, x, xi, xiii, 2, 3, 7, 8, 9, 10, Chad, 74, 75, 81
11, 12, 13, 14, 15, 16, 17, 18, 19, 23, 24, cheese, 45, 51, 53, 58
25, 26, 27, 29, 30, 32, 34, 35, 36, 38, 39, chemical communication, viii, xiii, 205,
111, 112, 113, 114, 115, 120, 121, 123, 206, 214
128, 133, 135, 136, 137, 140, 150, 151, chemical properties, 212
152, 153, 155, 156, 158, 160, 161, chemical structures, 212
162,163, 164, 165, 166, 167, 168, 169, children, 90, 93, 175, 185
170, 214, 219, 220, 222, 223, 224, 226 classification, 3, 74, 78, 98, 152, 176, 184,
black swan (Cygnus atratus), 115, 138 216
blueberries, vi, viii, xii, 189, 190, 193, 194, clinical signs, viii, ix, xii, xiii, 2, 10, 19, 20,
198, 199, 201 22, 24, 25, 26, 27, 30, 172, 177, 178,
body size, 196, 207 220, 222, 223
body weight, 144 close proximity, xi, 112, 117, 123, 128, 129,
Botswana, 75, 81, 90 132
Brazil, xi, 13, 32, 107, 149, 150, 151, 152, cluster, 114, 159, 164
153, 155, 156, 161, 162, 167, 168, 169, clustering, 114, 117, 136, 154, 166
170, 173, 186, 191 coalitions, 115, 137
breeding, vii, ix, xi, 10, 16, 26, 44, 47, 48, collateral kin, 116, 129
49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60, common (Bucephala clangula), x, xi, xii,
61, 62, 63, 65, 66, 71, 79, 112, 113, 114, 10, 16, 20, 21, 29, 38, 39, 40, 41, 74, 84,
116, 117, 128, 129, 130, 132, 135, 137, 88, 95, 112, 115, 116, 117, 129, 137,
138, 139, 140, 143, 144, 145, 215 139, 142, 144, 150, 155, 156, 160, 163,
breeding goal, 65 177, 181, 186, 192, 224
brood parasitism, 115, 136, 137, 138, 139, common eiders (Somateria mollissima, 115,
142, 143 137, 144
Burkina Faso, 74, 75, 76, 77, 85 communal rearing, 115
communication, viii, xiii, 205, 206, 208,
210, 212, 214, 216
C
communication systems, viii, xiii, 205, 210,
212
Cameroon, 75, 79, 81, 82, 182
complex social system, x, 111, 116, 140
Canada geese (Branta canadensis), 115,
Congo, 75, 182
139, 143, 144, 145
capital breeding, 130

Index 229
conservation, vii, x, 73, 74, 76, 77, 79, 82, direct observation, 135
84, 86, 88, 91, 92, 93, 94, 95, 96, 98, 99, discrimination, 132, 137, 139, 209
103, 104, 105, 108, 109, 174, 176 diseases, viii, ix, 2, 3, 14, 32, 35, 38, 44, 47,
consumption, 69, 92 52, 97, 175, 176, 177, 187, 223
contaminated food, 8, 18, 29, 175, 221 disinfection, 24
contaminated water, 176 drinking water, 23, 224, 225
contamination, 175, 176, 179, 181, 224 Drosophila, vi, viii, xii, 189, 190, 191, 192,
copulation, xii, xiii, 190, 195, 196, 198, 199, 193, 194, 195, 196, 199, 201, 202, 203,
200, 201, 202, 206 214
cort-fitness hypothesis, 131 Drosophila suzukii, viii, xii, 189, 190, 191,
cost, 47, 53, 84, 144, 145, 146, 177, 178, 192, 193, 194, 195, 199, 200, 201, 202,
223 203
Côte d’Ivoire, 77 drugs, ix, 23, 24, 33, 36, 37, 44
cougar, 84, 93, 96, 98, 100, 104, 107, 110
courtship, xiii, 29, 195, 199, 202, 206, 208,
E
210, 211, 213, 214, 215, 216
créches, 115
ecological restoration, 91
crop, viii, 2, 8, 19, 20, 21, 22, 40, 191, 192,
ecology, x, 73, 85, 91, 92, 99, 103, 107,
193, 194
108, 110, 114, 144, 150, 166, 214
cryptosporidiosis, vi, xii, 171, 172, 176,
economic efficiency, 53, 62
180, 181, 182, 185, 187
economic performance, 53
Cryptosporidium, viii, xii, 26, 172, 173,
ecosystem, 91
174, 175, 176, 177, 178, 179, 180, 181,
egg, 130, 131, 139, 143, 144, 195, 200
182, 183, 184, 185, 186, 187, 225, 226
Egypt, 28, 32, 75
cuticular hydrocarbons, xiii, 206, 210, 214,
El Salvador, 84, 100
216
endangered, ix, x, 2, 14, 25, 30, 31, 73, 74,
cytoskeleton, 4, 5, 7, 40
89, 90, 104, 110
endangered species, ix, 2, 14, 30, 90
D environment, 47, 49, 51, 52, 58, 65, 69, 87,
142, 151, 161, 174, 175, 176, 221, 224
data collection, 118, 119, 120, 124, 125, environmental conditions, 49, 58
128, 131 environmental contamination, 224
dehydration, viii, xiv, 177, 220, 222 environmental factors, 91
Democratic Republic of Congo, 75, 182 environmental sustainability, 102
diagnostic, viii, xii, 20, 23, 29, 172, 177, environmental variables, 81
223 environmentalism, 102
diarrhea, viii, xiv, 173, 177, 180, 181, 185, enzyme-linked immunosorbent assay, 179
220, 221, 222 epidemic, ix, 2, 10, 11, 32, 36, 37, 41
diet, 18, 19, 26, 36, 143, 153, 157, 160, 162, epidemiology, vii, ix, xii, 2, 11, 12, 30, 166,
190, 199, 200, 208 168, 172, 175, 182, 183, 185, 187, 222,
diplomonadida, 220 225, 226

230 Index
epithelial cells, 20, 36, 176, 177

G
Ethiopia, 75, 81
etiology, 17, 23, 33
gazelle, 89
eukaryote, 174
genotype, 25, 26, 27, 28, 29, 36, 37, 58, 59,
evolution, 135, 137, 138, 139, 209, 212,
178, 185, 213
214, 215
genus, viii, xiii, 5, 31, 86, 161, 174, 175,
extended social relationships, 113
190, 205, 206, 220, 223
extinction, vii, 16, 74, 77, 83, 99, 108
Germany, 35, 46, 111, 192
Giardia, viii, xiii, 184, 185, 220, 221, 222,
F 223, 224, 225, 226
Giardia psittaci, 220, 224
families, x, xii, 30, 112, 113, 115, 116, 131, giardiasis, vi, 219, 223, 224, 225
139, 140, 141, 150, 154, 155, 156, 157, Giraffe, 89, 102
159, 162, 163, 164 glucocorticoids, 131, 146
farmers, ix, 44, 45, 47, 48, 49, 50, 51, 52, goose, vii, xi, 112, 115, 116, 117, 118, 120,
53, 54, 55, 56, 64, 65, 69, 86 128, 136, 138, 139, 140, 141, 144, 146
farms, vii, ix, 28, 44, 45, 47, 48, 49, 50, 51, granulomas, viii, 2, 20, 21
52, 53, 54, 55, 56, 58, 59, 62, 68, 70 grazing, 59, 70, 80
fecundity, vi, viii, xii, 189, 190, 200 greater white-fronted goose (Anser albifrons
female natal philopatry, 113 frontalis), 115
female relatedness, 113 Greenland white-fronted goose
female-biased philopatry, xi, 112, 114, 130, (A. a. flavirostris), 115
136 greylag goose, vii, xi, 112, 117, 140, 141
female-centered clans, 112, 114, 128 Guinea, 74, 75, 76, 77, 80, 82, 86, 108
fertility, 47, 49, 53, 54, 56, 59, 62, 64, 67, Guinea-Bissau, 75, 77
70
fertility rate, 49, 53, 56
H
fidelity, 139, 207, 216
flock dynamics, 112
habitat, xi, 76, 77, 80, 81, 82, 83, 84, 86, 98,
food, vii, x, 8, 14, 16, 17, 21, 23, 24, 26, 30,
103, 143, 150, 151, 164, 169
74, 84, 114, 130, 146, 175, 190, 197,
habitat occupation, xi, 150, 164
199, 200, 201, 224
health, ix, 41, 44, 46, 47, 48, 50, 51, 54, 56,
food intake, 146
57, 59, 64, 66, 67, 70, 91, 92, 176, 177
forest ecosystem, 86
health problems, 50, 51, 177
forest formations, 150, 152, 158, 160, 164
heterogeneity, 33, 64, 164
forest fragments, xi, 150, 164
history, viii, x, xiii, 73, 98, 99, 103, 106,
France, 18, 33, 45, 46, 48, 63, 166, 191
118, 119, 135, 137, 141, 190, 199, 206
fruits, viii, xii, 190, 192, 201
hormones, 70, 131, 146
human, vii, x, xii, 5, 73, 80, 82, 83, 84, 86,
87, 92, 93, 94, 96, 97, 101, 102, 106,

Index 231
109, 113, 121, 134, 172, 175, 176, 177,

K
178, 184
hunting, vii, x, 74, 80, 82, 83, 84, 90
Kenya, 74, 75, 81
hymenoptera, 206, 207, 213, 214, 215, 216
kin cluster, 114, 136
hypothalamic-pituitary-adrenal (HPA), 131
kin recognition, 115, 135, 139
kin selection, xi, 112, 115, 133, 137
I kin-based female social structure, 114, 137
kinship, 129, 133, 134, 137, 142
inbreeding, 54, 56, 62, 113, 135
incubation period, 130
L
India, 97, 102, 103, 108, 190
individuals, 26, 30, 40, 75, 77, 78, 113, 114,
lactation, 54, 56, 63, 67
118, 119, 120, 121, 122, 123, 124, 125,
landscapes, 94, 97, 103, 109
126, 128, 129, 131, 159, 176, 177, 181,
larvae, xii, 150, 157, 159, 160, 162, 164,
220
191, 193, 197, 200, 202
infection, viii, xiii, 8, 14, 15, 16, 17, 18, 19,
latency, xii, 190, 196, 198, 199
20, 22, 24, 25, 26, 28, 36, 41, 168, 175,
lesions, viii, 2, 3, 9, 14, 16, 17, 18, 20, 21,
176, 177, 178, 181, 220, 221, 222, 223,
24, 25, 26, 27, 28, 29, 30, 36, 37
224, 225
Liberia, 75, 82
infertility, 50
life cycle, 8, 10, 184, 195, 221
infestations, 164, 168
likelihood ratio tests (LRT), 122, 123, 124
ingestion, 8, 18, 175, 176, 221
lineal kin, vii, xi, 112, 117, 118, 123, 127,
insect, 86, 201, 206, 207, 210, 213, 214,
129, 132
215, 216
lion, v, vii, x, 73, 74, 75, 76, 77, 78, 79, 80,
intensive, vii, ix, x, 44, 45, 47, 49, 50, 52,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
53, 54, 55, 62, 74, 82, 85, 211
92, 93, 96, 97, 98, 99, 100, 101, 103,
International Bank for Reconstruction and
104, 108, 109, 110, 134
Development, 81, 82, 104
liver, 8, 21, 22, 31, 40
invasive species, xii, 189
livestock, 44, 64, 66, 69, 80, 85, 86, 89, 90,
ixodidae, 150, 166, 167, 168, 169
96, 99, 109, 184
local breed, 44, 48, 49, 50, 51, 52, 54, 58
J long-term monogamous, 116
jaguar, 84, 93, 96, 103

M
Jamaica, 119, 125, 127
Japan, 190, 195
malabsorption, 177, 221
jewel wasp, viii, xiii, 205, 206, 214
Malawi, 75, 81
Jordan, 119, 125, 127, 135
male-biased dispersal, 113, 135
Mali, 74, 75, 77, 82, 85, 87, 106

232 Index
mammals, vii, xi, 80, 86, 87, 96, 112, 113, Norway, 18, 144
132, 133, 135, 137, 173 nutrients, 28, 130, 177
mating, xiii, 54, 114, 135, 190, 192, 194, nutrition, 63, 67, 199, 200, 201
195, 196, 197, 199, 200, 203, 205, 207,
209, 211, 212, 213, 215, 216
O
mating behavior, xiii, 190, 199, 205, 207,
212
organic, v, vii, ix, 38, 43, 44, 45, 46, 47, 48,
matrilocality, 114
49, 50, 51, 52, 53, 54, 55, 56, 58, 59, 60,
Mauritania, 75, 82
61, 62, 63, 64, 65, 66, 67, 68, 69, 71
meat, 51, 68, 85, 87, 88
organism, vii, xi, 12, 112, 177
media, 22, 93, 95, 196, 197, 199
Oropharyngeal trichomonosis, vii, viii, 2, 4,
medicine, 36, 90, 173, 187
22
Mediterranean, 14, 165, 192
microorganisms, 30, 179
microscopy, 7, 31, 174, 178, 179, 223 P
migration, 10, 11, 130, 132, 143, 145, 146
monkeys, 113, 172, 173, 176, 182, 183 pairing status, 127
morphology, 3, 5, 7, 40, 153, 157, 160, 223 pancreatitis, 177
mortality, 10, 11, 15, 25, 35, 39, 40, 41 parasite, viii, xii, xiii, 2, 7, 8, 14, 19, 22, 23,
Mozambique, 75, 81, 88, 104 25, 26, 27, 28, 29, 36, 40, 102, 137, 138,
mucosa, 8, 21, 177, 221 142, 151, 172, 173, 174, 175, 176, 179,
multifunctional farm, x, 44, 51, 55, 58 184, 220, 221, 223
multiple correspondence analysis, xi, 150, parasitic diseases, 35
151, 154 parasitic infection, 16, 172
parasitoid, viii, xiii, 194, 205, 207, 213, 214,
215, 216
N parental care, 116, 132, 139
parent-offspring bonds, 112
Namibia, 75, 81
parents, xi, 54, 112, 115, 116, 119, 120,
Nasonia, vi, viii, xiii, 205, 206, 207, 208,
122, 123, 124, 125, 128, 129, 138
209, 210, 211, 212, 213, 214, 215, 216,
pasture, ix, 44, 49, 50, 53, 60, 62, 67, 70
217
pasture-based system, ix, 44, 49, 53, 62
national parks, 74, 84, 87
pet birds, 220
Netherlands, 46, 48, 54, 56, 65, 133
pheromone, vi, viii, xiii, 205, 206, 207, 208,
New Zealand, 47, 49, 50, 53, 57, 59, 62, 64,
209, 211, 212, 213, 214, 215, 216, 217
66, 70
philopatry, 113, 114, 115, 131, 132, 135,
Niger, 74, 75, 76, 77, 79, 82, 85
136, 143
Nigeria, 74, 75, 77, 79, 85, 87, 97, 101
physiological helpers’, 116
non-social, 206, 207
place preference, 122, 126, 129
North Africa, x, 73, 75, 83, 101
political ecology, x, 73, 91, 92, 99, 100,
North America, ix, xii, 2, 46, 49, 50, 53, 62,
103, 107, 109, 110
66, 84, 100, 105, 138, 189, 191, 214, 226

Index 233
population, vii, x, 3, 10, 11, 15, 18, 25, 33, sexual behavior, viii, xii, 190, 199, 202
38, 40, 60, 63, 71, 74, 76, 77, 78, 79, 80, sexual reproduction, 176
82, 83, 84, 87, 88, 96, 97, 101, 107, 109, siblings, 117, 129, 133, 141
134, 136, 137, 139, 144, 193 Sierra Leone, 75, 82
population growth, vii, x, 74, 83, 88 Simplicomonas, 13
precocial, 112, 116, 130, 133, 138, 143 sisters, xi, 112, 116, 117, 129
predation, ix, 2, 87, 89, 109, 130 snow geese (Anser caerulescens
predator, x, 14, 73, 90, 96, 114 caerulescens), 114, 137, 143, 145, 147
prevention, viii, ix, xii, 23, 44, 103, 108, social allies, 116, 131, 141
172, 224 social attachment, 114, 117, 129
prezygotic reproductive isolation, viii, xiii, social behavior, 78
206, 209 social bonds, x, 111, 113, 115, 132
primates, vi, xii, 171, 172, 173, 176, 177, social group, 84, 113
178, 180, 181, 183, 184, 186 social status, 118, 120, 122, 123, 124, 146
production, ix, xii, 17, 43, 44, 45, 46, 47, social structure, xi, 112, 114, 128, 137, 140
48, 49, 50, 51, 52, 53, 54, 55, 56, 58, 59, social support, 97, 112, 116, 131, 132, 140,
60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 141
71, 92, 109, 130, 131, 136, 143, 181, South Africa, 74, 75, 81, 110, 184
189, 193, 194, 196, 198, 199, 200, 213, South America, 166, 167, 169, 191, 201
214, 222 Southeast Asia, viii, xii, 189
protected areas, 75, 77, 101, 102, 108 Spain, ix, 1, 2, 3, 11, 13, 14, 15, 16, 19, 37,
protozoan, xiii, 3, 9, 14, 15, 16, 29, 40, 86, 38, 41, 43, 45, 50, 55, 58, 68, 70, 182,
172, 181, 220 191, 226
species, viii, x, xii, xiii, 2, 4, 6, 9, 10, 11, 12,
13, 14, 15, 17, 18, 19, 22, 23, 24, 27, 28,
R
29, 31, 34, 35, 36, 37, 63, 74, 84, 87, 88,
89, 90, 93, 94, 95, 96, 111, 113, 114,
rainforest, 169
115, 130, 138, 150, 151, 152, 153, 154,
raspberries, vi, viii, xii, 189, 190, 193, 194,
155, 156, 159, 160, 161, 162, 163, 164,
198, 199, 201
167, 168, 170, 172, 173, 174, 176, 177,
reproduction, 47, 59, 61, 70, 114, 115, 130,
178, 180, 183, 189, 190, 191, 192, 193,
132, 140, 144, 176, 202, 210, 212
194, 195, 201, 205, 206, 207, 208, 209,
reproductive behavior, vi, 189, 194, 201,
210, 212, 213, 214, 216, 220, 222, 223,
207
226
resources, ix, 44, 58, 61, 92, 114, 130, 143,
specificity, viii, xiv, 150, 151, 168, 174,
193
177, 179, 220, 222
stress, 11, 25, 26, 116, 131, 141, 146, 176,
S 224
stress axis, 131
Senegal, 15, 74, 75, 77, 78, 79, 82, 89, 106 stress management, 131
sex, xiii, 113, 115, 120, 122, 123, 124, 135, stress response, 141
206, 207, 208, 209, 211, 213, 215, 216 stress-reducing mechanism, 131

234 Index
Sudan, 75, 76, 77, 80, 81, 85, 87 USA, 3, 9, 10, 13, 15, 17, 19, 31, 32, 34, 35,
survival, 25, 30, 49, 61, 62, 78, 81, 85, 96, 36, 38, 39, 1, 46, 47, 55, 75, 84, 95, 98,
114, 136, 139 108, 110, 37, 138, 140, 189, 191, 193,
survival rate, 25, 49 202, 213
susceptibility, 10, 25, 41, 107, 164, 223
sustainability, 44, 91, 94, 102
V
Sweden, 18, 45, 48, 50
Switzerland, 48, 192
vegetables, 174, 175
vegetation, 76, 80, 161, 165, 222
T
W
T. canistomae-like, 13
T. tenax-like, 13
waterfowl, vii, x, 111, 114, 115, 129, 130,
Tanzania, 74, 75, 81, 88, 108, 184
136, 138, 144, 145
taxonomy, viii, xii, 106, 172
weight loss, viii, xiv, 177, 220, 222
tick ecology, 150
welfare, ix, 44, 64, 65, 66
ticks, vi, viii, xi, 149, 150, 151, 152, 153,
West Africa, v, vii, x, 73, 74, 75, 76, 77, 78,
154, 155, 156, 161, 163, 164, 165, 166,
79, 80, 81, 83, 84, 85, 86, 87, 88, 89, 90,
167, 168, 169, 170
91, 92, 95, 96, 97, 98, 100, 101, 102,
tiger, 83, 96, 102, 109
103, 104, 106, 109
Togo, 75, 77
wild animals, 172, 176, 179, 187, 222
tourism, 69, 70, 86
wild birds, v, vi, 1, 3, 11, 12, 15, 16, 18, 32,
traits, ix, 44, 47, 49, 54, 55, 56, 57, 59, 62,
35, 36, 38, 149, 150, 153, 155, 156, 161,
64, 65, 67, 69, 70
162, 164, 168, 170, 222, 226
treatment, vii, ix, xii, 2, 3, 23, 44, 172, 175,
wilderness, 86, 91
180, 181, 223, 224
wildlife, viii, x, 2, 3, 11, 14, 16, 17, 19, 37,
treatment methods, 181
73, 74, 83, 84, 85, 88, 90, 91, 92, 101,
Trichomonas gallinae, viii, 2, 4, 30, 31, 32,
102, 106, 108, 110, 133, 165, 187
33, 34, 35, 36, 37, 38, 39, 40, 41
Trichomonas gypaetinii, 11, 36
Trichomonas stableri, 9, 33 Z
Tritrichomonas blagburni n. sp.-like, 11
trypanosomiasis, 86, 100 Zambia, 75, 81, 101, 110
Zimbabwe, 75, 81, 97
zoonosis, 172, 183, 222
U
Uganda, 74, 75, 81, 88

United Kingdom, 45, 56, 57
United Nations, 83, 110
urbanization, vii, x, 74, 80, 83, 84, 85, 96,
97, 104

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