Theropithecus Gelada

Predictors, Costs, and Consequences of Larval Tapeworm Infection in Geladas

(Theropithecus gelada ).

India Schneider-Crease

Department of Evolutionary Anthropology Duke University

Date:______Approved:

______Charles L. Nunn, Co-Supervisor

______Leslie J. Digby, Co-Supervisor

______Jacinta C. Beehner

______Christine M. Drea

______Thomas T. Struhsaker

______R. Lee Reinhardt

Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Evolutionary Anthropology in the Graduate School of Duke University

2017

ABSTRACT

Predictors, Costs, and Consequences of Larval Tapeworm Infection in Geladas

(Theropithecus gelada ).

India Schneider-Crease

Department of Evolutionary Anthropology Duke University

Date:______Approved:

______Charles L. Nunn, Supervisor

______Leslie J. Digby, Supervisor

______Jacinta C. Beehner

______Christine M. Drea

______Thomas T. Struhsaker

______R. Lee Reinhardt

An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Evolutionary Anthropology in the Graduate School of Duke University

2017

Abstract

Parasitism is integral to primate evolution, contributing to major life history

tradeoffs with other processes critical to reproductive success and survival. Yet, few

studies have demonstrated the predictors and consequences of parasites that inflict

fitness costs on their wild hosts. In this dissertation, I investigate how infection with the

tapeworm Taenia serialis affects geladas ( Theropithecus gelada ) in the Simien Mountains

National Park, Ethiopia. After identifying T. serialis as the parasite causing protuberant cysts in geladas with the use of molecular tools, I described an overall cyst prevalence of

4.8% in the study population (Chapter 2). To identify infections that do not present as visible cysts, I adapted a non-invasive monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) to detect circulating Taenia spp. antigen in gelada urine

(Chapter 4). This assay detected Taenia antigen with high accuracy (98.4% specificity,

98.5% sensitivity, and an AUC (area under the curve) of 0.99). Implementing this assay in the study gelada population, I found that infection is substantially more widespread than would be predicted based on the occurrence of visible T. serialis cysts (16.5% of individuals of unknown status tested positive for antigen presence at least once).

Contrary to the female-bias observed in many Taenia -host systems, I found no significant sex bias in either cyst presence or antigen presence. Age, on the other hand, predicted cyst presence (older individuals were more likely to show cysts) but not antigen

presence (Chapter 4). This indicates that T. serialis may infect individuals early in life but result in visible disease only later in life. I found that cysts were strongly associated with decreased survival and reproductive success in adult geladas (Chapter 3). Counter to expectations, T. serialis cysts were not strongly associated with decreased fecal testosterone metabolite concentrations (Chapter 5). This suggests that the mechanisms underlying the wild T. serialis -gelada relationship differ from those observed in experimental systems. Together, the analyses contained in this dissertation offer novel insights into the predictors, costs, and consequences of a trophically-transmitted larval parasite in wild primates.

Dedication

To all of the feminists whose hard work and persistence brought me here.

To Ethiopia and its wildlife.

To Aberdeen Toast and Asheville.

To my mom and dad.

Contents

Abstract ...... iv

List of Tables ...... xi

List of Figures ...... xii

Acknowledgements ...... xiv

1. Introduction ...... 1

1.1 Parasitism in primates ...... 10

1.2 Host-parasite system ...... 14

1.3 Overview of dissertation ...... 22

2. Molecular identification of Taenia serialis coenurosis in gelada s ...... 27

2.1 Introduction ...... 27

2.2 Materials and Methods ...... 30

2.3 Results ...... 33

2.4 Discussion ...... 35

2.5 Conclusion ...... 37

3. High mortality and low reproductive success associated with parasitism in geladas (Theropithecus gelada ) in the Simien Mountains National Park, Ethiopia...... 39

3.1 Introduction ...... 39

3.2 Materials and Methods ...... 45

3.2.1 Subjects and study site ...... 45

3.2.2 Longitudinal data collection ...... 45

3.2.3 Adult mortality ...... 47

vii

3.2.4 Infant mortality ...... 49

3.2.5 Interbirth interval ...... 50

3.3 Results ...... 50

3.3.1 Cyst prevalence ...... 50

3.3.2 Adult mortality ...... 51

3.3.4 Infant mortality and interbirth interval ...... 54

3.4 Discussion ...... 56

3.4.1 Demographic variation in prevalence ...... 57

3.4.2 Impact of cysts on mortality ...... 58

3.4.3 Impact of cysts on reproductive success ...... 61

3.4.4 Future directions...... 63

3.6 Acknowledgements ...... 64

4. Identifying wildlife reservoirs of neglected taeniid tapeworms: non-invasive diagnosis of endemic Taenia serialis infection in wild primates ...... 66

4.1 Introduction ...... 66

4.1.1 Review: diversity and zoonotic potential of T. serialis ...... 67

4.1.2 An antigen ELISA to investigate larval T. serialis in wildlife...... 70

4.1.3 Antigen ELISA implementation in geladas ( Theropithecus gelada ) ...... 72

4.2 Materials and Methods ...... 74

4.2.1 Study site ...... 74

4.2.2 Urine sample collection ...... 75

4.2.3 Urine sample analysis ...... 76

viii

4.2.4 Receiver operating characteristic analysis ...... 78

4.2.5 Analysis of T. serialis infection predictors in urine samples ...... 79

4.3 Results ...... 80

4.3.1 Sample analysis...... 80

4.3.2 Statistical analysis of T. serialis predictors in urine samples ...... 85

4.4 Discussion ...... 90

4.5 Acknowledgements ...... 95

5. No evidence for deandrogenization of male geladas ( Theropithecus gelada ) naturally infected with Taenia serialis metacestodes...... 97

5.1 Introduction ...... 97

5.2 Methods ...... 101

5.2.1 Study subjects and sample collection ...... 101

5.2.2 Statistical analysis ...... 103

5.3 Results ...... 105

5.4 Discussion ...... 108

6. Conclusion ...... 111

6.1. Overview ...... 111

6.2 Taenia serialis: identification, diagnosis, and host diversity...... 111

6.3 Parasitism as a potential selective pressure ...... 113

6.4 Limitations and Future Directions ...... 115

6.5 Concluding Remarks ...... 118

References ...... 119

Biography ...... 156

List of Tables

Table 1: Prevalence of T. serialis cysts in geladas across age-sex classes a ...... 35

Table 2: Results of Cox models and scaled Shoenfeld residual tests...... 54

Table 3: Ag-ELISA results of gelada samples (true positive, true negative, unknown status) ...... 82

Table 4: AICc model selection for predictors of T. serialis cysts in geladas. The ‘top model set’ presented here includes all models within <2 Δ AICc points of the best model. Predictor coefficient intercepts, AICc values, Δ scores, and weights of each model are given...... 87

Table 5: AICc model selection for predictors of T. serialis antigen-positivity in gelada urine. The ‘top model set’ presented here includes all models within <2 Δ AICc points of the best model. Predictor coefficient intercepts, AICc values, Δ scores, and weights of each model are given. Models 2.1 and 2.2 include age as a categorical value (i.e., adult, subadult), and models 3.1 and 3.2 include age as a continuous variable...... 88

Table 6: Full model averaged coefficient estimates for the predictors of T. serialis cysts in geladas (Model 1), and the predictors of antigen-positivity (Models 2 & 3). Model 2 includes age as a categorical predictor, while Model 3 includes age as a continuous predictor. Adjusted standard errors (SE), z-values, and probability estimates (Pr(|>z|) for each estimate are given. Results are rounded to the nearest hundredth, and statistical significance is indicated by an asterisk (*)...... 89

Table 7: AICc model selection for predictors of T in geladas. The ‘top model set’ presented here includes all models within 2 Δ AICc points of the best model. Predictor coefficient intercepts, AICc values, Δ scores, and weights of each model are given...... 106

Table 8: Full model-averaged parameter estimates, adjusted standard errors (SE), z- scores, and probability estimates (Pr(|>z|) are presented for all variables that appeared in models within two AICc points of the top model. Statistical significance is indicated by an asterisk (*)...... 107

List of Figures

Figure 1: Female geladas with facial (A) and mammary (B) cysts indicative of infection with T. serialis (photographs by Jacinta C. Beehner)...... 29

Figure 2: Microscopic view (10x) of a scolex with a branching endogenous daughter cyst from a wild Ethiopian gelada. Photograph by Dr. James Flowers...... 31

Figure 3. Phylogenetic relationships between four species of Taenia (T. serialis, T. multiceps, T. crassiceps, T. pisiformis) and the sample obtained for this study (“sample from gelada”). The tree is based on maximum par- simony using published partial 12S and ITS-2 sequences obtained from GenBank (T. pisiformis: ITS-2 JX317674, 12S DQ104230; T. crassiceps: ITS- 2 DQ099564, 12S EU219547; T. multiceps: ITS-2 FJ886762, 12S JQ710642; T. serialis: ITS-2 DQ099575, 12S DQ104236). Numbers above branches represent the posterior support (max of 1) for each branch...... 34

Figure 4: Female gelada exhibiting a protuberant cyst characteristic of Taenia serialis on the right pectoral region...... 41

Figure 5: Hypothesized Taenia serialis life cycle in the gelada-canid system. Drawings by RHG...... 42

Figure 6: Scaled Schoenfeld residuals plotted against age for the cyst variable in Cox proportional hazards models for males (A) and females (B). Dashed lines represent confidence intervals. Deviations from a line with slope 0 indicate violations of the proportional hazard assumption. Schoenfeld residual tests indicate that the relationships depicted in both plots are significant deviations from the proportional hazard assumption...... 52

Figure 7: Log hazard ratios for cyst presence over age, estimated from the extended Cox model with time-varying coefficients for males (A) and females (B). The thick solid line represents the log hazard ratio, thin solid lines represent confidence intervals, light gray dotted lines represent 0 (i.e., no effect of cysts), and black dashed lines represent the constant log hazard ratio estimated from a standard Cox proportional hazard model. The time axis represents time since the first appearance of a cyst...... 53

Figure 8: Kaplan-Meier survival curves for infants born to mothers with and without cysts. The estimated curves in plot (A) are for all infants, while the curves in plot (B) were estimated for data excluding infants that died along with their mothers. Hatch marks indicate right-censoring, and dashed lines represent confidence intervals...... 56

xii

Figure 9. (A) Gelada with a larval T. serialis cyst protruding from the abdomen. (B) Internal view of coenuri in the cyst of an infected individual necropsied upon natural death...... 73

Figure 10: Receiver operator characteristic (ROC) curve of antigen ELISA detection of T. serialis infection in dried gelada urine. The optimal threshold cutoff index value (42.1) had an estimated specificity of 98.4% (95% CI: 95.1-1) and an estimated sensitivity of 98.5% (95% CI: 95.6-1)...... 81

Figure 11: Histogram showing counts of log sample index values (IVs) (the optical density of each sample indexed to the positive and negative controls on each plate) + a constant. Blue bars indicate samples from individuals without cysts, while grey bars indicate samples from individuals with cysts. The dotted line indicates the optimal threshold cutoff for positive samples indicating antigen presence calculated with the ROC analysis...... 84

xiii

Acknowledgements

I am indebted to an incredible network of support from friends, colleagues, mentors, and organizations that have facilitated the completion of this dissertation and shaped my intellectual growth during my graduate career at Duke University. My studies were funded with generous support from the National Science Foundation

Graduate Research Fellowship Program and the Duke University Graduate School. I offer profuse thanks to the NSF and Duke, as well as to the Primate Action Fund, the

Margot Marsh Biodiversity Foundation, Primate Conservation, Inc., Conservation

International, and the Nacey Maggioncalda Foundation for entirely funding the research presented in this dissertation. I thank my collaborators Dr. Pierre Dorny at the Institute of Tropical Medicine, Antwerp, and Drs. Patricia P. Wilkins and Sukwan Handali at the

Centers for Disease Control and Prevention for facilitating crucial parts of this research.

I thank Dr. Diane Doran-Sheehy for introducing me to primatology and catalyzing my desire to pursue fieldwork and the study of primate behavior and evolution. I very vividly recall the moment when Dr. Doran-Sheehy described mountain gorilla habituation and I thought: “I want to do that.” Similarly, I thank Dr. Patricia C.

Wright and the Centre ValBio team for my first foray into studying wild primates in their natural habitats, and Dr. Denise Gassner for hiring me as her field assistant while she studied ring-tailed lemur health and behavior in Madagascar. The multiple parasite

xiv

infections that I ended up with at the end of that field season sparked my passion for

parasitology and brought me to where I am now. It was also during that field season

that we climbed the Tsaranoro Massif and I was first acquainted with the sport that has brought me years of joy and sanity.

I thank my advisors at Duke University, Dr. Charles L. Nunn and Dr. Leslie J.

Digby, for taking a chance on someone with a Bachelor’s degree in Philosophy and

French Literature and a nascent passion for primatology and fieldwork. Their guidance

provided me with innumerable and invaluable lessons over the course of my studies.

Both advisors taught me to think critically and work independently, encouraged me to

take risks, and provided me with the freedom to pursue my intellectual passions. Drs.

Christine M. Drea, Thomas T. Struhsaker, and R. Lee Reinhardt provided critical and

productive feedback throughout the dissertation process. I am honored to have worked

with some of the best minds in primate behavior, health, endocrinology, and

conservation, and am sincerely grateful for their support over the past six years. I thank

my collaborators at the Centers for Disease Control and Prevention for mentoring me in

the laboratory with patience and humor. I am particularly grateful for the unwavering

support, guidance, and friendship of Lisa R. Jones, whose extensive knowledge and deft

navigation of the university system was indispensible throughout my graduate career.

Drs. Jacinta C. Beehner and Thore J. Bergman, Co-Directors of the University of

Michigan Gelada Research Project, welcomed me warmly into their project and tirelessly

mentored and guided me. I look forward to many years of collaboration with them,

tackling unanswered questions about gelada ecology and evolution and working to

conserve the habitat and wildlife of the Simien Mountains National Park. Dr. Noah

Snyder-Mackler is to thank for my initial engagement in this project, offering me the

opportunity to identify the cystic material excised from the cyst of a dead gelada and

mentoring me through the publication of my first paper (and beyond). I am fortunate to be able to continue working with Dr. Snyder-Mackler, along with Dr. Amy Lu, on the

effects of early life adversity in geladas in my postdoctoral career.

Everything I know about geladas, fieldwork, and Ethiopia, I learned from the

incredible UMGRP team. Nothing compares to life in field with the incredible Julie C.

Jarvey and Megan A. Gomery (or Per and Elsa, for that matter). Field life can be hard

and complicated, but, more importantly, it’s full of geladas, dancing, and pure joy. I am

so lucky to have spent time in the field with Dr. Marcela E. Benitez and Elizabeth T.

Johnson, both brilliant women and excellent field companions. No one knows the

geladas like Esheti Jejaw, Ambaye Fanta, and Setey Girmay, and their dedication to data

collection underpins these—and all—projects that come out of the Simien Mountains

National Park. The Ethiopian Wildlife Conservation Authority is owed an enormous

debt for permitting and facilitating this research, providing us with valuable experts

such as Belayneh Abebe. Tarik W/Aregay and his team introduced me to Addis Ababa

life and made me feel at home in a new city. I also thank Habtamu Demis at the

xvi

Ethiopian Television Network for enabling the dissemination of information about

geladas and working to raise awareness about endemic Ethiopian wildlife.

I thank my lab members Randi H. Griffin, Caroline R. Amoroso, Alexander

Vining, Debapriyo Chakraborty, and Joel Bray for their essential roles in my intellectual

and emotional growth. Randi, as my science life partner, taught me much of what I

know about the scientific process, critical thinking, analysis, and R. I count myself

incredibly lucky to be her friend and collaborator. It is only with the laughter,

misadventures, plan Bs (i.e., the Cuttery), political debates, and feelings nights (not to

mention the thousands of practice presentations) with Randi, Caroline, Alexander, and

Joel that I have been able to navigate the waters of academia. I thank all of the gifted

graduate students in Evolutionary Anthropology for their support and sense of

community.

The people with whom I share my life are owed an immense debt of gratitude.

The value of their friendships is immeasurable. I am extraordinarily fortunate to be part

of a team with the brilliant, hilarious, and accomplished Drs. Chris Krupenye and Emily

E. Boehm. From the cadaver lab to Calcutta, and everywhere in between, I can’t imagine

the past six years (or the future) without them. Jackson Spradley, Joseph T. Feldblum,

Sara J. Stevens, Aleah Bowie, Vlad Chituc, Paul Henne, and Rachna Reddy made

Durham home, and have shared adventures and misadventures with me from Motorco

to Knoxville, and from the Eno to Uganda. Since childhood, Dr. Carol Margolis and I

xvii

have been working towards making careers out of our shared love of animals: we’re finally there! Tessa Crosby has been my person and life travel partner since the moment we met in France, and I thank her for being her. I am lucky to have had Rachel Karasik in my life while finishing my dissertation, and I thank her immensely for being so fun, supportive, and unconditionally giving. Finally, I thank my climbing partners for our adventures to some of the most thrilling places on earth, and my NYC friends for always welcoming me home. All of these humans have made my life beautiful, even while finishing my dissertation.

And finally, I thank my family. My mom, Susan B. Schneider, is a gifted writer and editor and is my best friend. She has been an unflagging warrior for me, by my side since day one, through thick and thin. She taught me self-expression, communication, and generosity, and led by example. My dad, Robert P. Crease, is a brilliant philosopher and historian of science, and cultivated in me curiosity and critical thinking about the natural world (even when it meant dissecting frogs in the living room). Stephanie and

Alexander Crease have shown me unconditional love, support, and puns, and I am so lucky to have them in my family.

xviii

1. Introduction

Parasites are increasingly appreciated as drivers of wildlife ecology and evolution (Anderson & May 1978, Grenfell & Dobson 1995, Nunn & Altizer 2006). Wild animals are regularly exposed to a wide array of parasites, which are thought to affect reproductive success and survival ( Anderson & May 1978, Price 1980, Combes 2001,

Nunn & Altizer 2006, Poulin 2007, Tompkins et al . 2011 ). Parasites, defined as organisms that live in or on, and at some cost to, other living organisms (Thompson 1994, Cheng

1991, Toft 1991, Nunn & Altizer 2006), are suggested to be among the most numerous on earth (Toft 1986, May 1988, Thompson 1994, Windsor 1998, Dobson et al. 2008). For example, helminth parasites of vertebrates alone are estimated to number between

75,000-300,000 species (Dobson et al. 2008). All animals are thus faced with mitigating the costs of being parasitized while balancing the demands of other processes essential to their own fitness. This is a dynamic relationship that has resulted in the evolution of intricate immune responses and complex behaviors in hosts and reciprocal adaptations in parasites (i.e., coevolution; Keymer & Read 1991, Schmid-Hempel & Ebert 2003,

Poulin 2010). Thus, parasite-host relationships can be understood as essential components of evolution.

Life history theory holds that organisms balance energetic allocation to survival and reproductive success through tradeoffs between the behavioral and physiological

processes essential to each (Stearns 1992, Ricklefs & Wikelski 2002). Individuals

differentially allocate limited energy to maximize reproductive success, and must shift

allocation patterns based on extrinsic demands to processes essential to growth,

reproduction, and maintenance (Stearns 1992, Ricklefs & Wikelski 2002). Because

parasitism and associated immune defenses demand energetic resources, hosts are

expected to shift energetic allocation away from growth and reproduction and thus to

face diminished future reproductive success or survival (Booth et al. 1993, Forbes 1993,

Sheldon & Verhulst 1996, Svensson et al. 1998, Morand & Harvey 2000). Thus, parasitism is hypothesized to be one of the dynamic forces that organisms must balance in order to optimize their fitness.

Certain parasites, for example, can cause hosts to shift the timing of sexual maturation and reproduction in order to recoup fitness reductions incurred with shortened lifespan (Agnew et al. 2000, Hochberg et al. 2002). This was demonstrated in

the case of Tasmanian devils whose populations were ravaged by the extremely virulent

devil facial tumor disease. After the emergence of this contagious cancer, Tasmanian

devils exhibited significantly increased rates of sexual precocity that are hypothesized to

increase reproductive success given the likelihood of shorter lifespans (Jones et al. 2008).

While observing a shift in the timing of sexual maturation attributable to parasitism is

rare in the natural world, experimental work has demonstrated this effect in a number of

additional systems (Thornhill et al. 1986, Lafferty 1993, Polack & Starmer 1998, Krist

2001, Chadwick & Little 2005). More indirectly, parasites can siphon essential energetic

resources from the host or stimulate immunological responses that demand energetic

rerouting by hosts away from other essential processes (Booth et al. 1993, Delahay et al.

1995, Sheldon & Verhulst 1996, Demas et al. 1997, Svensson et al. 1998, Forbes et al. 2000,

Lochmiller & Deerenberg 2000, Nilsson 2003). Indeed, evidence points to tradeoffs between the immune system and reproduction (Greer 2008, Knowles et al. 2009), as well

as between the immune system and growth (McDade et al. 2008, Vijendravarma et al.

2009, van der Most et al. 2011 ). Alterations in behavior or activity budget tied to

parasitism, such as increases in self-grooming (Freeland 1981, Mooring 1995), may also

detract from the time available to the host for other behaviors essential to fitness (Poulin

1995, Schwanz 2006).

Over evolutionary time, parasite-mediated tradeoffs may lead to behavioral,

social, physiological, or immunological adaptations in host species ( Keymer & Read

1991, Hochberg et al. 2002). Sickness behaviors, for example, may be adaptations that permit animals to mitigate the fitness effects of parasitism stemming from the energetic and pathological consequences of infection (Johnson 2002, Ghai et al. 2015). Such behaviors, which include alterations in activity budget to increase resting and decrease food intake, may place constraints on other behaviors critical to fitness (Hart 1990,

Johnson 2002, Ghai et al. 2015). Other components of animal behavior and sociality may

also be affected by constraints related to the costs of parasitism ( Freeland 1976, Keymer

& Read 1991). Optimal group size and composition, for example, may be shaped by

constraints related to parasitism (Côté & Poulin 1995) in addition to the more

traditionally considered ecological forces of predation and competition for mates and

resources (Struhsaker 1969, Clutton-Brock & Harvey 1977, van Schaik 1983, Chapman &

Chapman 2000, Di Fiore 2002, Kappeler & van Schaik 2002, Reed et al. 2009). If group-

living increases transmission of certain parasites, adaptations to reduce transmission or

fitness effects of those parasites while retaining other beneficial elements of group-living

should emerge (Freeland 1976, 1979, 1980, Anderson & May 1992, Côté & Poulin 1995,

Reed et al. 2009, Patterson & Ruckstuhl 2013). Indeed, counter-strategies such as

fissioning, increasing group spread, and altering day range may decrease contact with

infected conspecifics or infectious agents (Snaith et al. 2008, Griffin & Nunn 2012,

Patterson & Ruckstuhl 2013, but see Reed et al. 2009).

In order for parasites to shape the evolution of host life history, behavior, and

physiology, they must inflict substantial costs on survival and reproductive success

(Stearns & Koella 2007). Robust bodies of clinical and scientific research have

demonstrated that parasites can cause pathological tissue damage (Holmes & Zohar

1990, Coop & Kyriazakis 1999, Engwerda et al. 2004), induce deleterious behaviors such

as anorexia (Arneberg et al. 1996, Kyriazakis et al. 1998, Coop & Kyriazakis 1999, Roberts

et al. 1999, Lochmiller & Deerenberg 2000, Mercer et al. 2000), and lead to reductions in

host body mass or condition (Gulland 1992, Booth et al. 1993, de Lope et al. 1998, Moller

et al. 1999, Forbes et al. 2000, Albon et al. 2002, Stien et al. 2002, Neuhaus 2003, Hawlena et

al. 2006, Ballestros et al. 2011). Immune defenses themselves can also inflict damage; for

example, chronic inflammation can lead to tissue damage as well as neurodegenerative

and cardiovascular disorders in later life (Ashley et al. 2012). These observations form the basis of the hypothesis that parasites decrease fitness in their natural hosts.

While parasites are hypothesized to negatively impact survival and lifetime reproductive success of hosts by increasing mortality or by decreasing fecundity or offspring survival, a review of the evidence suggests that the application of this hypothesis may be limited. Some studies have demonstrated positive effects of experimental removal or treatment on components of host reproductive success

(including longer time to conception, lower birth rates, smaller surviving offspring,

decreases in milk quality and lactation persistence, and fewer successful pregnancies,

e.g., Brown et al. 1995, Neuhaus 2003, Hillegass et al. 2010, Patterson et al. 2013, Lopes et

al. 2016), while others have reported no such relationship (e.g. Gauthier-Clerc et al. 2003,

Raveh et al . 2011, Hersh et al. 2015, Prendergast & Jenson 2011, Raveh et al. 2015).

Similarly, some studies have demonstrated significant negative effects of parasite

infection on fitness in natural parasite-host systems (e.g., Ratti 1983, Schall 1983,

Korpimaki et al. 1993, Dufva 1996, Marzal et al. 2005, Gooderham & Shulte-Hostedde

2011, Lachish et al. 2011), while others have failed to find such effects or have demonstrated positive associations between parasitism and reproductive success (e.g.

Lee & Clayton 1995, Dufva 1996, Brown et al. 2006, Lachish et al. 2011, Hersh et al. 2015,

Blackwell et al. 2015). Thus, it appears that certain parasites may negatively impact the fitness of certain hosts in certain conditions, but that this relationship is highly dependent on the study system and the condition of the host.

One explanation for the lack of observable fitness effects in some host-parasite

systems relies on arguments that parasites inevitably evolve to be less harmful to their

hosts over time (Price 1980, Toft & Karter 1990, Toft & Aeschilmann 1991, Gulland 1995,

Ewald 1995). For decades, the dominant paradigm of parasite ecology held parasites to be constrained in the extent of their deleterious effects on hosts because parasite survival

and reproduction depend on host survival (Baker 1974, Price 1980, Toft & Karter 1990,

Gulland 1995, Ewald 1995). In other words, a parasite that kills its host necessarily

diminishes its own fitness. In this view, genotypes of parasites with attenuated fitness

costs would have increased reproductive success compared to their more virulent

conspecifics, and selection would act to increase the frequency of those less-lethal

genotypes (Price 1980, Gulland 1995, Ewald 1995). Evolution towards such an

equilibrium (either where both host and parasite benefit, i.e., “mutualism”, or where

neither inflict substantial damage on the other, i.e., “commensalism”) likely occurs in

some systems (Toft & Aeschilmann 1991, Ewald 1995). However, this paradigm does not

take into account that parasites with high potential for transmission linked to

pathogenicity (e.g., viruses transmitted in the diarrhea they cause) can increase fitness by optimizing transmission efficacy instead of reducing their impact on host fitness

(Anderson & May 1982, Toft & Aeschilmann 1991, Ewald 1995, Ebert & Herre 1996, Day

2002, Galvani 2003, Robar et al. 2010). In other words, the success of parasites can be

understood as the number of propagules from a primary infection that result in

secondary infections (R 0), and parasites can optimize their R 0 for different levels of

virulence. Therefore, certain parasites can achieve high transmission with high host

mortality, and thus not incur selective pressure via decreased reproductive success to

mitigate the costs they inflict on hosts (Anderson & May 1982, Toft & Aeschilmann 1991,

Ewald 1995, Ebert & Herre 1996).

The paradigm of ‘parasite constraint’ also ignores the breadth of parasite species

that employ “predation-mediated transmission” (Robar et al. 2010) or “parasite-

increased trophic transmission” (Lafferty 1999) in their life cycles. Parasites that employ

this type of transmission are classified as having complex life cycles, in which parasites

utilize more than one host within a single life cycle; this is in contrast to parasites with

simple life cycles, in which parasites utilize a single host to complete a single life cycle

(Parker et al. 2003, Auld & Tinsley 2015). Parasites with complex life cycles typically rely on the ingestion of one host by another--and, accordingly, the death of the preyed-upon host (Lafferty 1999, Galvani 2003, Robar et al. 2010). Such parasites, particularly those that employ predation-mediated transmission, incur and inflict different costs than their counterparts with simple life cycles (Robar et al. 2010). The exploitation of a succession of host species during distinct life stages is common in a wide diversity of parasitic taxa

(Combes 2001, Moore 2002, Choisy et al. 2003, Mouritsen & Poulin 2003, Robar et al.

2010, Parker et al. 2015). Complex life cycles generally involve parasitic exploitation of an established predator-prey transmission route; the sexually or hermaphroditically reproducing adult parasite infects the predator species (the host in which the parasite reproduces sexually or hermaphroditically is referred to as the “definitive” host), and the non-sexually or asexually reproducing larval parasite infects the prey species (the host in which the parasite does not reproduce or asexually reproduces are referred to as the “intermediate” host) (Lafferty 1999, Lafferty & Kuris 2002, Parker et al. 2015). The sexually reproducing or hermaphroditic adult parasite releases its propagules into the environment in the feces of the definitive host, where they are encountered by the intermediate host (Lafferty 1999). The larval stage that subsequently develops in the intermediate host must be ingested by the definitive host for the life cycle to achieve

completion (Lafferty 1999, Choisy et al. 2003). Because such life cycles are contingent upon the ingestion of one or more intermediate hosts, these parasites are under selection to increase the likelihood of transmission events.

Parasites employing predator-mediated transmission frequently optimize their fitness by manipulating the behavioral, morphological, or physiological phenotypes of their intermediate hosts (Combes 1991, Moore 2002, Poulin 1995, Lafferty et al. 2000,

Lefèvre et al. 2009, Poulin 2010, Parker et al. 2015). Two well-known examples of such manipulation are the lancet liver fluke ( Dicrocoelium dendriticum ), which causes infected ants to travel to the tips of grass blades to be consumed by the definitive sheep host

(Wickler 1976, Esch & Fernandez 1993, Manga-González et al. 2001, Moore 2002), and the protozoan Toxoplasma gondii , which stimulates attraction to cat urine in its rodent intermediate hosts (Berdoy et al. 2000). In these systems, increasing host mortality and predation risk in intermediate hosts is adaptive for the parasite (Ewald 1995, Poulin et al.

2005, Poulin 2007, Parker et al. 2015), and fitness consequences of parasites employing parasite-mediated transmission are significantly more likely to be observed than parasites with simple life cycles (Robar et al. 2010).

Thus, while many factors are likely to be involved, the difficulty of demonstrating direct fitness effects of parasitism in wild host-parasite systems may arise partly from the focus of many ecological studies on parasites with simple life cycles. A

renewed focus on parasites with complex life cycles in ecological studies is essential to developing a coherent understanding of how parasites directly affect the evolution of their hosts.

1.1 Parasitism in primates

Parasitism is thought to have played a significant role in the evolution of primate species (Nunn & Altizer 2006). Infectious disease has been argued to be perhaps the most important component of human evolution, with disease-associated mortality surpassing all other sources of mortality (Dobson & Carper 1996, Oldstone 1998,

Anderson & May 1992, Nunn & Altizer 2006). Accordingly, infectious disease has driven selection on immune- and defense-related genes in humans (Fumagalli et al. 2011,

Karlsson et al. 2014) as well as psychological and cultural components of human culture

(for example, the tendency towards collectivism or individualism, Fincher et al. 2008).

The long and intimate shared evolutionary histories of many parasite and host taxa make these relationships useful for shaping inferences about host evolutionary history, phylogeny and dispersal (Brooks & Glen 1982, Hugot 1999, Reed et al. 2009). For example, an early cladistic analysis of pinworm species infecting primates led to the suggestion that the Homo genus should be considered a sister taxon to the Pan, Pongo, and Gorilla genera (Brooks & Glen 1982, Hugot 1999), and molecular analysis of

Helicobacter pylori diversity has been used to reconstruct likely migrations throughout human history (Falush et al. 2003, Moodley et al. 2012).

Studies of parasitism in nonhuman primates have generally focused on elucidating host traits, adaptations, and tradeoffs related to parasitism (reviewed in

Nunn & Altizer 2006, Huffman & Chapman 2009) or demonstrating the effects of highly virulent viruses and bacteria on hosts. Virulent viruses and bacteria have intermittently emerged in nonhuman primate populations, often with devastating effects (Lloyd &

May 2001, Huijbregts et al. 2003, Walsh et al . 2003, Leendertz et al . 2004, Leroy et al. 2004,

Bermejo et al. 2006, Caillaud et al . 2006, Leendertz et al. 2006, Nunn & Altizer 2006, Walsh et al. 2009). These pathogens typically emerge as epidemics in primate populations, causing widespread mortality before dying out when they deplete the availability of susceptible hosts (Boots & Sasaki 1999, Galvani 2003). For example, the Zaire strain of

Ebola virus (ZEBOV) swept through western gorilla (Gorilla gorilla ) populations in

Congo, and was estimated to have killed between 3500-5000 gorillas over 2 years

(Bermejo et al. 2006). Studies of epidemics in primates are crucial for understanding and managing rapid disease spread, particularly in light of an expanding human-wildlife interface. Less commonly studied are endemic parasites, which are defined by their regular and extended occurrence in hosts, low levels of virulence, and slower

reproductive rates than epidemic parasites (Anderson & May 1979, Randall et al. 2013; but see Leggett et al. 2017 for a new perspective).

In contrast to the large body of research on the fitness effects of epidemics in

primates, relatively few studies have demonstrated explicit effects of endemic parasites

on host survival and reproductive success (Nunn & Altizer 2006, Budischak et al. 2012).

Milton (1996) and Keele et al. (2009) provide perhaps the most compelling evidence for

parasite-associated declines in survival and reproductive success in primates. Milton

(1996) demonstrated that monthly mortality rates in a population of howler monkeys

(Alouatta palliata ) were strongly correlated with bot fly ( Alouattamyia baeri ) prevalence

and relative density (prevalence*mean intensity). Keele et al. (2009) found that wild

chimpanzees ( Pan troglodytes ) infected with SIVcpz, a simian immunodeficiency virus

related to HIV, incurred a 10-16 fold increase in mortality hazard and significant

decreases in fecundity and offspring survival. These two studies provide essential

information on the fitness consequences of parasites in primates. However, given the

attention increasingly bestowed on parasitism in primates and the mounting evidence of

fitness effects of parasitism in other mammalian hosts, it is surprising that these two

studies constitute much of the available information on the effects of endemic parasites

on primate hosts.

Research on endemic parasites in primates generally focuses on parasite taxa with simple life cycles that are easily identified and quantified using non-invasive fecal samples (i.e., gastrointestinal helminths and protozoa) or visual counts (i.e., ectoparasites) (Hausfater & Watson 1976, Hausfater & Meade 1982, Muller-Graf 1997,

Gillespie et al. 2005, Snaith et al. 2008, Dupain et al. 2009, Hernandez et al. 2009, Huffman et al. 2009, Wright et al. 2009, MacIntosh et al. 2012). As discussed above, parasites with simple life cycles typically require a single host in which to reproduce; thus, selection on these parasites and their hosts may have worked to attenuate their costs. Primates infected with these parasites may thus only incur subtle costs, which are particularly difficult to detect in long-lived species (Nunn & Altizer 2006) (although it is worth noting that coinfections with multiple parasite species are expected to increase virulence,

May & Nowak 1995, Alizon et al. 2013). The focus on parasites with simple life cycles in studies of primates presents an obstacle to understanding the full breadth of how parasites can affect primate ecology and evolution. To overcome this obstacle, we should look to parasites with complex life cycles in which parasites are under selection to maximize costs on host fitness.

1.2 Host-parasite system

Geladas ( Theropithecus gelada ) native to the Ethiopian highlands (Dunbar and

Dunbar 1975; Dunbar 1980; Iwamoto 1993, Dunbar 1984) are regularly infected with the larval stage of Taenia serialis , a tapeworm parasite with a complex life cycle that employs

predation-mediated transmission. This parasite-host relationship provides a valuable

opportunity to investigate the predictors, costs, and consequences of a parasite in

primates that is under selection to increase the mortality of its hosts.

These primates are characterized by traits that are remarkable among primates

and have facilitated their inclusion as “intermediate hosts” in the T. serialis life cycle (i.e.,

hosts to the larval stage of T. serialis ). Geladas’ activity budget is dominated by feeding; between 45-65% of each day is spent feeding or foraging on grass, a diet which is

supplemented by roots, rhizomes, and seasonal flowers during the dry season (Iwamoto

and Dunbar 1983; Dunbar 1984). The significant amount of time spent feeding on a

notoriously difficult-to-digest resource has selected for a suite of morphological

adaptations in geladas. Cranial adaptations include a robust and anteriorly placed

temporalis muscle, which is critical to the regular mastication of grasses (Jablonski 1993).

Postcranial adaptations include high finger opposability and robusticity, which permit

efficient and selective foraging of grasses (Jablonski 1993; Krentz 1993; Dunbar 1984),

and elongated, flexible, and robust limbs, which permit geladas to squat and shuffle

while feeding, thereby reducing physical effort and maximizing grass intake (Krentz

1993; Jablonski 1993; Dunbar 1984). Together, these adaptations to terrestriality and

herbivory have provided the mechanisms through which geladas ingest infective T.

serialis eggs. Geladas are the only known primate species to serve as intermediate hosts for T. serialis , which is likely due to their extreme degree of terrestriality and herbivory; however, numerous other primate species exhibit varying degrees of terrestriality and herbivory and may thus also be at risk for T. serialis infection.

Taeniid tapeworms are geographically and phylogenetically widespread,

infecting a staggering number of mammalian species and causing enormous mortality,

morbidity, and economic losses in humans on nearly every continent (Sciutto et al. 2000,

Craig & Pawlowski 2002, Hoberg 2002). Like all Cyclophyllidean tapeworms, taeniids

require two host species to complete a single life cycle: a definitive host for the sexually

reproducing or hermaphroditic adult stage and an intermediate host for the non-

sexually reproducing or asexually reproducing larval stage (Abuladze 1964, Loos-Frank

2000, Hoberg 2002). Unlike other parasites such as pinworms (Brooks & Glen 1982,

Hugot 1999), taeniids do not co-speciate with their hosts (Hoberg et al. 2000, 2001,

Hoberg 2002). Rather, shifts between host species that are not closely related occur when

such species share ecological guilds and thus utilize common resources (Hoberg 2002).

For example, humans are the definitive host for at least three taeniid species ( Taenia

solium, T. saginata, and T. asiatica ) (Craig & Pawlowski 2002, Hoberg 2002), joining classic

carnivorous definitive hosts in the canid, felid, hyaenid, mustelid, and viverrid families

(Lieby & Dyer 1971, Loos-Frank 2000, Hoberg 2002). This transition is postulated to have

emerged when ancestral hominids shifted in diet from herbivory to omnivory,

positioning them to join the carnivorous guild feeding on prey species infected with

taeniid larval stages (Hoberg et al. 2000, 2001, Hoberg 2002).

While taeniids are among the most well-studied of all tapeworm parasites

(Abuladze 1964, Loos-Frank 2000, Hoberg 2002), disagreement and controversy have

surrounded the elucidation of their taxonomic statuses and phylogenetic relationships

(Hoberg et al. 2000, Hoberg 2002). Early authors assigned separate genera and species to

adult and larval stages (e.g., Taenia saginata as the adult stage and Cysticercus bovis as the larval stage of what is now referred to as T. saginata ; Hydatigera taeniaeformis as the adult

stage and Strobilocercus fasciolaris as the larval stage of what is now referred to as T.

taeniaeformis , reviewed in Hoberg et al. 2000, Hoberg 2002). Genera and species

designations were traditionally made on the basis of traits related to parasite

morphology, geographic occurrence, and host identity, all of which have been rejected

in recent years as insufficient for species or genus identification because of

morphological and behavioral plasticity exhibited by many taeniids. These approaches

have been increasingly replaced with genetic tools (Padgett et al. 2005, McManus 2006,

Zhang et al. 2007, Jeon et al. 2009, Jia et al. 2010, Avcioglu et al. 2011 ). In the words of

Combes (2001), “molecular techniques have reduced the abusive synonymizing of

species distinguished according only to hosts or other uncertain characters” (pg. 54).

Molecular identification of Taenia species has been conducted using both nuclear and mitochondrial DNA. Of particular importance to the identification of unknown taeniid isolates are the protein-coding mitochondrial gene 12S and the hypervariable region of the internal transcribed spacer (ITS1 and ITS2) (Eom et al. 2002, Padgett et al.

2005, McManus 2006). These regions are particularly useful for distinguishing between closely related taxa because of their fast rates of evolution and manageable sequence lengths (Eom et al. 2002, Padgett et al. 2005, McManus 2006). Indeed, these regions have been thoroughly sequenced and are available through the open-access sequence database GenBank. The availability of sequences from multiple closely related taeniid species facilitates the species identification of unknown isolates through the construction of nucleotide substitution models.

Within Cyclophyllidea, Taenia species are considered to be singular in their

infection of mammalian species in both larval and adult stages (Hoberg 2002). In

carnivorous definitive host species, the adult tapeworm is attached to the intestinal tract by means of the hooks and suckers of its scolex (anterior end) (Wardle 1975, Flisser

1991). The scolex produces proglottids, which are the hermaphroditic segments that

comprise the tapeworm body. Proximal proglottids mature as they progress distally,

and the most distal proglottids drop off as they become gravid. Gravid proglottids

migrate to the host anus and are expelled in feces during excretion (Wardle 1975, Flisser

1991). As proglottids disintegrate outside of the host body, they release eggs in numbers

that can range from 50,000 to 100,000 per proglottid, depending on the taeniid species

(Gregory 1976, Flisser 1991). These microscopic eggs are dispersed across the landscape

and within the soil profile by environmental elements such as rain, wind, and

mechanical vectors, where they can remain viable for up to 50 months (Gemmell 1977,

Lawson & Gemmell 1990, Torgerson et al. 1992, 1995, Craig & Macpherson 2000). Eggs are enclosed in adhesive proteinaceous shells (Conn & Swiderski 2008, Jabbar et al. 2009)

that permit them to stick on vegetation, which is crucial for their transmission to the next

host. Herbivorous artiodactyl, rodent, and lagomorph species incidentally ingest eggs

during foraging, upon which the second stage of the taeniid commences (Loos-Frank

2000, Hoberg 2002).

Amid the gastric juices of the intermediate host stomach, the protective layers of

the taeniid egg are worn away (Conn & Swiderski 2008). The erosion of these layers,

which include the keratin shell, embryophore, and embryonic envelopes, releases the

precursor to the taeniid larval form, the hooked hexacanth oncosphere (Heath 1971,

Conn & Swiderski 2008, Jabbar et al. 2009). The oncosphere protects the hooked

hexacanth embryo as it invades the villi of the intestinal wall, using its musculature to

engage in vigorous thrusting into the intestinal lining with the help of secretory

products from oncospheral penetration glands (Jabbar et al. 2009). After burrowing

through the intestinal wall, the hexacanth is picked up by the circulatory systems (Heath

1971, Marty & Neafie 2000, Jabbar et al. 2009). Depending on the species, hexacanths can

settle in a number of places around the body, including somatic tissue and the central

nervous system, to begin the larval, asexually budding stage of their development. In

coenurosis, the metacestode (i.e. larval) stage of T. serialis and T. multiceps , hexacanth

embryos develop into fluid-filled exogenously budding capsules containing multiple

protoscolices (the immature form of the scolex, the proximal end of the adult tapeworm

in the definitive host) (Meyers et al. 2001, Bowman 2009). Metacestodes asexually bud

through the branching and invagination of endogenous daughter cysts (Marty & Neafie

2000). This morphology is generally distinct from that of cysticerci (sing., cysticercus),

the metacestode stage of taeniid species such as T. saginata and T. solium . In contrast to

the multiple protoscolices contained inside each fluid-filled coenurus, each cysticercus

contains a single protoscolex (Marty & Neafie 2000).

The taeniid larval form can only achieve adulthood when larvae are ingested by

the appropriate definitive host ( Craig & Pawlowski 2002, Bowman 2009). Upon predation of an infected intermediate host by a definitive host, each of the scolices in the

intermediate host tissue can develop into adult tapeworms in the gastrointestinal tract of the definitive host. The immature scolices attach to the intestinal wall and begin producing proglottids that, as they mature, produce the infectious eggs that are shed into the environment to be infected by herbivorous intermediate hosts. Larval taeniids are virtually singular among parasites in their increased predilection for female hosts

(Morales-Montor & Larralde 2005). Broadly, estrogens directly stimulate larval taeniid proliferation and induce a permissive immune response, while androgens inhibit proliferation and induce a restrictive immune response (Esch 1967, Sciutto 1990, Huerta et al . 1992, Morales et al . 2002, Morales-Montor & Larralde 2005, Morales-Montor et al.

2006). In males, taeniid larvae induce deandrogenization by catalyzing aromatization of testosterone into estradiol, which creates a favorable environment for larval growth

(Terrazas et al. 1999a, b, Escobedo et al. 2004, Morales-Montor et al. 2004, Morales-Montor

& Larralde 2005, Pena et al. 2007). The inhibition of testosterone production accordingly reduces associated male sexual behaviors (Lin et al. 1990, Morales-Montor & Larralde

2005).

Taeniid infections have been shown to reduce host reproductive success through somatic damage and neuroendocrinological modulation leading to irregularities in or inhibition of sexual behavior, estrous cycling, and placental maintenance (Esch 1967, Lin et al. 1990, Sciutto et al. 1991, Terrazas et al. 1994, Larralde et al. 1995, Morales-Montor et

al. 2002, 2004, Gourbal & Gabrion 2004, Morales-Montor & Larralde 2005, Arteaga-Silva et al. 2009). In females, infection-induced endocrinological imbalances can cause irregularities in estrous cycling, reductions in receptivity and attractivity, and declines in successful pregnancies (Lin et al. 1990, Gourbal & Gabrion 2004, Arteaga-Silva et al.

2009), all of which can lead to unusually long interbirth intervals due to reductions in mating behavior or interference with implantation or placental maintenance.

Geladas are intermediate hosts to T. serialis , likely ingesting eggs as they forage on grasses. After hatching in and burrowing through the gelada gastrointestinal tract, hexacanth embryos begin developing in somatic, muscular, and visceral tissue. Here, they asexually bud, resulting in the massive cysts observed over the past century in wild-caught captive geladas in European and North American zoos (Scott 1926,

Schwartz 1926, Schwartz 1927, Urbain & Bullier 1935, Elek & Finkelstein 1939, Rodhain

& Wanson 1954, Bertolino 1957, Clark 1969) and wild geladas in the Ethiopian highlands

(Ohsawa 1979, Dunbar 1980, Nguyen et al. 2015). The presence of visible cysts in a wild population of habituated geladas under long-term monitoring provides an important opportunity to examine the dynamics of a parasite potentially under selection to increase the mortality of its hosts.

1.3 Overview of dissertation

In this dissertation, I investigate the predictors, costs, and consequences of

naturally acquired infection with the larval form of the complex tapeworm T. serialis in

geladas in the Simien Mountains National Park (SMNP), Ethiopia. In this system, I seek

to address how infection with a parasite employing a complex life cycle affects the

survival and reproductive success of its intermediate hosts. After identifying and

describing the distribution of the parasite responsible for the protuberant cysts observed

in wild geladas, I describe its physiological and fitness consequences on its gelada hosts.

Chapter 2: Molecular identification of Taenia serialis coenurosis in geladas.

In this chapter, I use molecular methods and phylogenetics to identify the

parasite behind the protuberant cysts observed in geladas using cystic material from a

dead gelada. I performed an informal morphological assessment on cystic material

extracted from a dead gelada that revealed thin-walled polycephalic structures

characteristic of coenurosis (the metacestode stage of T. serialis and T. multiceps ).

Comparison of the 12s and ITS-2 regions of the cyst genome to those available for

taeniid species revealed the most likely identity of this parasite to be T. serialis . I

conducted a cross-sectional demographic analysis of T. serialis , which revealed cysts to be significantly more common in adults than in juveniles or infants, but to be equally

prevalent in males and females. Collaborators on this project included Noah Snyder

Mackler (Duke University), who performed the genetic analysis, Julie C. Jarvey

(University of Michigan), who procured the cystic material, and Thore J. Bergman

(University of Michigan), who provided access to the site and project guidance.

Chapter 3: High mortality and low reproductive success associated with parasitism in geladas

(Theropithecus gelada) in the Simien Mountains National Park, Ethiopia

To examine the effects of protuberant T. serialis cysts on fitness, I integrated

longitudinal data on gelada reproductive success, survival, and cyst presentation. I

found that geladas exhibiting T. serialis cysts experienced significant increases in mortality, and that females with cysts exhibited decreases in reproductive success via decreased infant survival. This is consistent with the hypothesis that the reliance of T.

serialis on predation-mediated transmission places the parasite under selection to

increase the vulnerability of its intermediate host to predation through manipulation of

host behavioral or morphological phenotypes. The physical growth of T. serialis larvae

inside the muscular or somatic tissue can directly kill the host, allowing the infected

cadaver to be scavenged upon by the carnivorous definitive hosts, or can simply

enhance its vulnerability to predation by impeding limb or organ function.

Collaborators on this project included Randi Griffin (Duke University), who contributed

to analysis, Megan A. Gomery (University of Michigan), who contributed to data collection and project planning, Thore J. Bergman (University of Michigan), and Jacinta

C. Beehner (University of Michigan), both of whom provided access to longitudinal data and project guidance.

Chapter 4: Identifying wildlife reservoirs of neglected taeniid tapeworms: non-invasive diagnosis of endemic Taenia serialis infection in wild primates

I adapted an Ag-ELISA to non-invasively diagnose T. serialis infection in geladas, which permits the identification of infections that do not present as visible cysts and the investigation into patterns of infection without relying on visual cues. This assay was successfully used to diagnose active T. serialis infection in geladas with high accuracy, and revealed a larger percentage of infected individuals than those that present with visible cysts. Additionally, sequences of urine samples taken from the same individuals suggested that some individuals are able to fight off infections before they become established. Finally, while age was significantly associated with the presence of cysts, no host traits were significantly associated with urine samples positive for T. serialis antigen. Collaborators on this project included Patricia P. Wilkins, John C. Noh, Holly

M. Chastain, and Sukwan Handali (Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention), who provided materials and training in laboratory

methods and guidance in project design, Randi Griffin, Noah Snyder-Mackler, and

Charles L. Nunn (Duke University), who provided analytical and conceptual support,

Megan A. Gomery, Thore J. Bergman, and Jacinta C. Beehner (University of Michigan), who provided demographic data on the study population and contributed to project design and data collection, and Pierre Dorny (Institute of Tropical Medicine, Antwerp), who provided reagents, protocols, and conceptual support.

Chapter 5: No evidence for deandrogenization of male geladas (Theropithecus gelada) naturally infected with Taenia serialis metacestodes.

To test the hypothesis that larval T. serialis infection in geladas modulates the hormonal environment of the host to increase its reproductive success, I evaluated the effect of T. serialis cysts on the fecal testosterone metabolite concentrations in male geladas over ten years. Including other potential predictors of fecal testosterone metabolite concentration variation in geladas, such as age, dominance status, and climatic variables, I found that cysts had weak explanatory power. The lack of effect of cysts on fecal testosterone metabolite concentration is counter to my expectations, and prompts additional questions about the mechanisms of behavioral and physiological manipulation of T. serialis and other, better studied, larval taeniids. Collaborators on this project included Thore J. Bergman (University of Michigan), who provided access to the

study site and longitudinal data, Randi H. Griffin (Duke University), who contributed

analytical and conceptual support, and Jacinta C. Beehner (University of Michigan ), who provided access to the study site and longitudinal data, and contributed conceptual

support.

2. Molecular identification of Taenia serialis coenurosis in gelada s

The work presented in this chapter is published in a paper entitled “Molecular identification of Taenia serialis coenurosis in a wild Ethiopian gelada (Theropithecus gelada )” (Schneider-Crease et al. 2013).

2.1 Introduction

Taeniid parasites globally exploit mammalian predator-prey relationships, requiring a carnivorous definitive host for the adult form and an herbivorous intermediate host for the larval form (the “coenurus”) (Meyer, 1955). Although primates are not known to regularly act as intermediate hosts, the larval stage of one taeniid has been described in the terrestrial and herbivorous Ethiopian gelada ( Theropithecus gelada )

(Schwartz 1927, Urbain & Bullier 1935, Elek & Finkelstein 1939, Rodhain & Wanson

1954, Clark, 1969, Ohsawa, 1979, Dunbar, 1984).

Geladas become infected when they ingest tapeworm eggs shed in the feces of the definitive host. In intermediate hosts, the oncospheres in the eggs are released into the intestinal tract, burrow through the intestinal mucosa, and settle in the connective tissue or intermuscular fascia. Each oncosphere develops into a coenurus, a mass of multiple invaginated scoleces in a fluid-filled membrane that expands through the branching and invagination of endogenous daughter cysts. In geladas, this infection

(coenurosis) can result in large, protuberant masses (hereafter: “cysts”) (Fig. 1). When a carnivore ingests the infected tissue, each scolex from within the coenurus attaches to the intestinal mucosa to sprout the strobila, a series of hermaphroditic and self- fertilizing segments that constitute the tapeworm body (Meyer 1955).

Figure 1: Female geladas with facial (A) and mammary (B) cysts indicative of infection with T. serialis (photographs by Jacinta C. Beehner).

This parasite has been identified morphologically and experimentally in captive geladas with conflicting results: classifications have included Coenurus tumours (Scott

1926), Multiceps serialis (Schwartz 1926), Multiceps serialis var. theropitheci (Schwartz

1927), and Multiceps multiceps (Rodhain & Wanson 1954). Thus, the addition of genetic information to species identification is critical. A genetic identification of the parasite and an analysis of the patterns of disease in a wild population of geladas in the Simien

Mountains National Park (SMNP), Ethiopia, are performed here.

2.2 Materials and Methods

A multiloculated cyst from a dead 13-year-old male gelada in the SMNP was procured in November 2011. One of the authors ( J.C.J.), extracted tissue from a protuberant coenurus on the left ventral forelimb, and stored it in RNAlater (Applied

Biosystems/Ambion, Austin, TX, U.S.A.).

Figure 2: Microscopic view (10x) of a scolex with a branching endogenous daughter cyst from a wild Ethiopian gelada. Photograph by Dr. James Flowers.

DNA was extracted from the cestodes using the Qiagen DNeasy Blood and

Tissue Kit (Qiagen). 347bp of the mitochondrial rDNA (12S) region and 434bp of the second internal transcribed spacer of nuclear rDNA (ITS-2) were amplified and sequenced using the primers and PCR protocol described in Padgett et al . (2005).

Sequences were aligned using Sequencher v5.0 (Gene Codes Corporation, Ann Arbor,

MI) and blasted against the NCBI nucleotide data bank.

For the phylogenetic analysis, two nucleotide substitution models were selected using AIC and BIC as implemented in jmodeltest (Darriba et al. 2012). Both models were run using the Bayesian program BEAST v1.7.5 (Drummond et al . 2012). The MCMC chain length was set to 10 8 and the state was recorded every 103, resulting in 10 5 trees.

The effective sample size for all parameters was greater than 130. After discarding the first 10 2 trees as burn-in, the remaining trees were summarized into one consensus tree using TreeAnnotater v1.7.5, which was imported into FigTree v1.4

(http://tree.bio.ed.ac.uk/ ) for visualization (Fig. 3).

To measure the disease prevalence in the SMNP, researchers from the University of Michigan Gelada Research Project surveyed 291 geladas for visible cysts. The

University of Michigan Gelada Research Project has collected behavioral data on this population for seven years, and researchers are trained to recognize individuals with

100% accuracy based on morphological traits. Researchers recorded the presence/absence, size, and location of cysts for each known individual. Recorded cysts were confirmed by two independent observers.

The prevalence rates given are of obvious signs of disease (i.e., cysts), not infection, since infected individuals may not exhibit cysts indicative of T. serialis infection. To ascertain patterns of cysts across ages and sexes, Pearson’s chi-squared tests were run using JMP statistical software (JMP, Version 7. SAS Institute Inc., Cary,

NC, 1989-2007).

2.3 Results

The BLAST search revealed a strong match between the sample sequences (ITS-2:

GenBank ID KF414738; 12S: GenBank ID KF414739) and published sequences for T. serialis ITS-2 (99% nucleotide identity; GenBank ID DQ099575) and 12S (99% nucleotide identity; GenBank ID DQ104236) regions (Jia et al ., 2010). The next most closely related species was T. multiceps , with which the sample had 95% and 90% nucleotide identity at

12S and ITS-2, respectively (GenBank IDs: GQ228818, FJ886762) (Padgett et al ., 2005).

Phylogenetic reconstruction with published Taeniid ITS-2 and 12S sequences confirmed that this sample is more closely related to T. serialis than to any other taeniid species (Fig. 2). AIC and BIC supported two different models, K81+I and K80+G;

however, both models supported the same phylogeny as implemented in BEAST v1.7.5

(Drummond et al . 2012).

Figure 3. Phylogenetic relationships between four species of Taenia (T. serialis, T. multiceps, T. crassiceps, T. pisiformis ) and the sample obtained for this study (“sample from gelada”). The tree is based on maximum par- simony using published partial 12S and ITS-2 sequences obtained from GenBank ( T. pisiformis : ITS-2 JX317674, 12S DQ104230; T. crassiceps : ITS- 2 DQ099564, 12S EU219547; T. multiceps : ITS-2 FJ886762, 12S JQ710642; T. serialis : ITS-2 DQ099575, 12S DQ104236). Numbers above branches represent the posterior support (max of 1) for each branch.

The prevalence of cysts in this population was 4.8% (Table 1). The prevalence

rate was 9.9% (13/131) among adults (over ~ 3 years old), and 0.9% among juveniles

(approx. 1.5-3 years old) (1/110). Cysts were not observed in infants (0-1.5 years old)

(n=50). Adults were significantly more likely to display cysts than juveniles and infants

(Pearson’s c 2= 13.661, P = 0.001, d.f. = 2). The prevalence rate for adult females was 10.8%

(10/93), and for males was 7.9% (3/38). There was no significant difference in the disease

prevalence between males and females (Pearson’s c 2= .247, p=0.620, d.f.=1).

Table 1: Prevalence of T. serialis cysts in geladas across age-sex classes a

Sex Age

Infant Juvenile Adult All

Female 0% (0/17) 1.8% (1/55) 10.8% (10/93) 6.7% (11/164)

Male 0% (0/33) 0% (0/55) 7.9% (3/38) 2.4% (3/126)

Both 0% (0/50) 0.9% (1/110) 9.9% (13/131) 4.8% (14/291)

a Number of individuals with cysts/total individuals in that age-sex class.

2.4 Discussion

The contribution of a genetic component to the identification of T. serialis in

geladas is imperative because morphological approaches have produced inconsistent

results. The first studies on this parasite classified it under the genus Multiceps ; now

Multiceps and Taenia are understood to be synonymous, and Taenia is the preferred nomenclature (Meyer, 1955; Benger et al ., 1981). While five studies used scolex arrangement and the size and number of rostellar hooks for identification (Schwartz

1927, Urbain & Bullier 1935, Elek & Finkelstein 1939, Rodhain & Wanson, 1954, Clark

1969), others used the morphology of the hook guards (Schwartz 1927) or hook blades

(Meyer 1955). However, other parasitologists assert that no morphological criterion

reliably diagnoses Taenia species (Beveridge et al . 1976).

The demographic analysis shows that T. serialis cysts are significantly more likely

to be found in adult than in subadult or infant geladas, corroborating two previous

studies of this population (Ohsawa 1979, Dunbar 1980). Adults may experience higher

cyst prevalence because of the immunosuppressive effects of chronic physiological and

social stresses related to adulthood (Muehlenbein & Bribiescas, 2005). Alternatively, the

time necessary for development of noticeable cysts may exceed the geladas’ juvenile

period, and adults will have had more time to become infected than non-adults.

Taenia serialis coenuri can displace viscera and exert mechanical pressure on nerves and arteries, causing spastic limb paralysis, muscle atrophy, increased vulnerability to predation, and death (Scott 1926, Elek & Finkelstein, 1939). Coenuri may also impact fitness by stimulating an energetically costly immune response, requiring an

individual to prioritize feeding and resting over reproductive behavior (Ohsawa 1979).

The presence and consequences of T. serialis in this population will continue to be

monitored.

Other diseases (e.g., cancer) may present similarly to T. serialis cysts, and, if so,

were erroneously included in this analysis. However, given the history of cysts whose

gross morphology in geladas is similar to those observed at this site (Fig. 3) and which have been identified morphologically as Taenia or Multiceps serialis , we are confident that the cysts reported are indicative of T. serialis infection. This interpretation is strengthened by the genetic confirmation of T. serialis from a characteristic cyst in this population.

2.5 Conclusion

A genetic analysis confirms the presence of T. serialis in geladas, and a demographic analysis shows that adults are more likely to exhibit cysts than non-adults.

However, the biology of this T. serialis life cycle is not yet entirely understood. We speculate that the black-backed jackal ( Canis mesomelas ), golden jackal ( Canis aureus ), spotted hyena ( Crocuta crocuta ), or domestic dog ( Canis familiaris ) may serve as definitive hosts in this iteration of the T. serialis life cycle, because all of these species may prey on geladas or scavenge their corpses and are known to carry the T. serialis adult stage

(Loos-Frank 2000, Hürni & Stiefal 2003, Bowman 2009). Future work should identify the definitive host and elucidate the risks for T. serialis infection in other primates.

Anthropogenic habitat change may increase geladas’ vulnerability to parasites, and changes in host-parasite dynamics may put humans at risk for emerging infectious

diseases. Understanding the population dynamics of the parasites in this region will be essential to predicting and controlling future outbreaks.

2.6 Acknowledgements

We thank the Ethiopian Wildlife Conservation Authority, the Simien Mountains

National Park, and the University of Michigan Gelada Research Project. We thank

Alison Von Striver, Caitlin Barale, Charles Nunn, Jacinta Beehner, James Flowers, Jenny

Tung, Leslie J. Digby, and Paul Durst for their help in the field, the laboratory, and during manuscript preparation, and three anonymous reviewers for their helpful comments. Research was approved by the University Committee on Use and Care of

Animals (UCUCA no. 09554) at the University of Michigan, the University of

Pennsylvania Institutional Animal Care and Use Committee (IACUC no. 802996), and adhered to the laws and guidelines of Ethiopia.

3. High mortality and low reproductive success associated with parasitism in geladas ( Theropithecus gelada ) in the Simien Mountains National Park, Ethiopia.

3.1 Introduction

Parasites may influence the reproductive success of their hosts (Anderson & May

1978, Tompkins et al. 2011) and the persistence of endangered species and populations

(McCallum & Dobson 1995, Smith et al. 2006). However, few studies have demonstrated fitness consequences of endemic helminth parasites on wild primate hosts. This dearth can largely be attributed to the scarcity of detailed, long-term field data on primate life history and parasitism that would permit the detection of fitness costs. Additionally, the long evolutionary relationships between many parasites and hosts may result in selection against extreme fitness costs, both because parasites that rely on host survival and reproduction are under selection to temper their costs (Ewald 1983) or because hosts are under selection to mitigate such costs (Boots et al. 2009, Roy & Kirchner 2000, Wenk

& Renz 2012).

For decades, geladas ( Theropithecus gelada ) inhabiting the Ethiopian highlands have been observed with protuberant cysts characteristic of infection with the tapeworm

Taenia serialis (Fig. 4), a parasite that exploits predator-prey relationships. Taenia serialis requires separate adult and larval stages that infect predator (“definitive”) and prey

(“intermediate”) hosts to complete a single life cycle (Nagaty & Ezzat 1946, Meyer 1955).

Canid definitive hosts harbor hermaphroditic adult tapeworms and deposit infective

eggs in feces, and herbivorous intermediate hosts harbor intrasomatic asexually budding

larvae (coenuri; sing., coenurus) that develop when herbivores ingest parasite eggs

(Nagaty & Ezzat 1946, Meyer 1955). Upon canid predation or scavenging of infected

herbivores, the larvae develop into adult tapeworms in the canid gastrointestinal tract,

and the cycle is perpetuated (Nagaty & Ezzat 1946, Meyer 1955) (Fig. 5). Wild-caught

captive geladas have been described with protuberant cysts characteristic of T. serialis beginning in the early 20th century (Scott 1926, Schwartz 1926, Schwartz 1927, Urbain &

Bullier 1935, Sandground 1937, Elek & Finkelstein 1939, Rodhain & Wanson 1954,

Bertolino 1957, Clark 1969), and recent application of molecular techniques confirmed

the diagnosis of T. serialis as the etiological agent behind the cysts (Schneider-Crease et

al. 2013, Nguyen et al. 2015). Herbivorous geladas are currently the only primate species

known to be parasitized by the larval stage of T. serialis .

Figure 4: Female gelada exhibiting a protuberant cyst characteristic of Taenia serialis on the right pectoral region.

Figure 5: Hypothesized Taenia serialis life cycle in the gelada-canid system. Drawings by RHG.

Taenia serialis can cause severe damage in its intermediate hosts, leading both to increased mortality and decreased reproductive success. Mortality associated with T. serialis cysts can result directly from physical damage to somatic tissue, with larvae causing muscle atrophy, spastic limb paralysis, and organ failure in captive geladas

(Scott 1926, Urbain & Bullier 1935, Elek & Finkelstein 1939). These pathologies can also indirectly increase mortality by enhancing the vulnerability to predation of infected individuals. Taeniid-induced reductions in reproductive success can result from mechanical damage to the reproductive tract or from neuroendocrinological modulation leading to irregularities in or suppression of sexual behavior, estrous cycling, and placental maintenance (Esch 1967, Lin et al. 1990, Sciutto et al. 1991, Terrazas et al. 1994,

Larralde et al. 1995, Morales-Montor et al. 2002, 2004, Gourbal & Gabrion 2004, Morales-

Montor & Larralde 2005, Arteaga-Silva et al. 2009). These perturbations can result in lengthened interbirth intervals in females and curtailed dominance tenure (and thus reproductive success) in males. Perhaps most directly, T. serialis can impede offspring growth and survival through parasite-induced maternal death; infants are exceedingly vulnerable, and rarely survive without their mothers in the wild (Nowak et al. 2000).

In the first study on the consequences of T. serialis cysts in wild geladas, Nguyen et al. (2015) found that T. serialis cysts were associated with increased mortality and decreased reproductive success. The authors used likelihood ratio tests to compare mortality rates between individuals with and without cysts across 6 years and found that a greater proportion of individuals with cysts died. The authors also used survival models to demonstrate that offspring of mothers with cysts had higher mortality than offspring of mothers without cysts. This result persisted even after the authors excluded

infants that died along with their mothers, suggesting an indirect effect of maternal cysts on offspring survival.

In the present study, we investigated the impact of T. serialis cysts on adult mortality and reproductive success of geladas inhabiting a distinct region of the

Ethiopian highlands, the Simien Mountains National Park (SMNP). First, we report the prevalence of cysts among demographic categories (age and sex) in our study group.

Second, we assess how T. serialis cysts impact adult survival, extending the approach used by Nguyen et al. (2015) by implementing survival analyses using estimated and known dates of birth for all adults. This allows us to more precisely quantify the relative risk associated with T. serialis cysts across the gelada lifespan. Finally, we examine the consequences of cysts on reproductive success by comparing offspring survival and interbirth interval length between females with and without cysts. Following Nguyen et al. (2015), we compare the impact of maternal T. serialis cysts on offspring whose mothers perished and on offspring whose mothers survived. This study both replicates and extends the study of Nguyen et al. (2015) in a population under longer study with more powerful statistical tools.

3.2 Materials and Methods

3.2.1 Subjects and study site

We studied geladas in the Sankaber area of the SMNP (13.1833’N, 38.0667’E). All

subjects derived from three bands (Snyder-Mackler et al. 2012b) totaling 351 females and

336 males of all ages (387 adults and 300 immatures) in ~40 reproductive units observed

over 10 years. The exact number of reproductive units in the study was variable due to

the fusion, fission, or disappearance of units (exact sample sizes are indicated with each

statistical test below).

3.2.2 Longitudinal data collection

Researchers with the University of Michigan Gelada Research Project collected

near-daily behavioral and demographic data across 10 years (Jan 2006 - Dec 2015).

Individuals born within the study period have known birthdates (n = 383), while those born prior to the study or outside of study groups were assigned estimated birth dates

according to maturational milestones and morphological traits (Barale et al. 2015,

Beehner et al. 2015, Roberts et al. 2017). Although maturational milestones vary between

and within the sexes (Barale et al. 2015, Roberts et al. 2017), we use 4 years of age as the

cutoff for inclusion in our analyses for both males and females as this minimizes

background rates of mortality related to infancy and juvenility. Study animals were

recognizable based on corporeal idiosyncrasies such as skin and hair patterns and

injuries, and are identified by researchers with near-perfect accuracy ensured by

intensive trainings and frequent interobserver reliability evaluations.

Because geladas are female-philopatric (Dunbar 1984, le Roux et al. 2011), females

that disappeared from their natal unit were assigned a date of death at the time of

disappearance. By contrast, male deaths were more difficult to assign with certainty because males move between units and bands throughout their lives (Dunbar 1984,

Pappano 2013). Thus, we adopted the conservative approach taken by Nguyen et al.

(2015). In brief, male disappearances were only classified as deaths if the disappearance

was accompanied by a major injury or an obvious decline in health, or if the individual

was an infant (and still dependent on his mother for nutrition).

Census data, including unit composition and health, were collected weekly.

Changes in leader males, infant presence/absence, body condition, and emergence of T.

serialis cysts were recorded on a near-daily basis. The high degree of proximity to

habituated study geladas available to researchers (~1.5 m), along with minimum

monthly requirements for data collection on individuals, ensured that UMGRP

researchers were able to observe cysts within ~1 month of emergence. Importantly, all

analyses performed here utilize the presence of visible cysts characteristic of T. serialis

infection, which may underestimate actual prevalence by ignoring infections that do not

present externally.

3.2.3 Adult mortality

We employed survival models to evaluate the impact of T. serialis cysts on adult

gelada mortality because these models can account for the timing of cyst appearance and

death and are therefore sensitive to how long individuals survive after cysts appear.

Additionally, survival analysis can incorporate data on censored individuals that only

contribute to estimates of model parameters during the portion of the study for which

they were observed. Our data are well suited to survival analysis since we have fairly

accurate ages at birth, first cyst appearance, and death for all study subjects, and many

of our subjects were right-censored (i.e., individuals that did not die during the study

period). Survival analyses were performed in R (R Core Team 2015) with the package

“survival” (Therneau 2015).

We used Cox proportional hazards models, which are flexible, robust, and do not

require specification of a probability distribution for survival times. Because male and

female geladas are subject to different demographic processes that likely result in a biased sample of adult males being observed in a given study group (i.e., we are more

likely to observe males that successfully immigrate into the study group and less likely

to observe male deaths), we fit separate Cox models for adult males (n = 170, 92.4% right-censored) and females (n = 216, 72.2% right-censored), including the presence of one or more cysts (cyst = 1) as a time-dependent covariate (Cox 1972). Because cysts rarely appeared in immature individuals, data were left-truncated at four years of age, excluding from the analysis any individuals that did not survive to at least four years of age. In addition, we right-truncated data at 24 years of age because beyond this age, small sample sizes and high age-related mortality resulted in poor model fit. Our Cox models took the form h(t|X) = h0(t) exp( β1X1(t)), where h(t|X) is the instantaneous hazard rate at time t; h0(t) is the baseline hazard rate; β1 is the log hazard ratio associated with cysts; and X1(t) is 0 if t < tc, where tc is the time of first cyst appearance, and 1 if t ≥ tc.

Proportional hazards models assume that model covariates have a constant, proportional (i.e., multiplicative) effect on the baseline risk of death. However, the proportional hazards assumption is violated when the effect of a covariate on the hazard rate changes over time. Using a scaled Schoenfeld residual test, we found that the proportional hazards assumption was violated in our model, and fitted extended Cox models of the form h(t|X) = h0(t) exp( f1(t)X1(t)). This differs from the Cox model in that the coefficient β1 is replaced by a linear function of time, f1(t), allowing the effect of the covariate to change through time.

3.2.4 Infant mortality

To assess the impact of maternal cysts on offspring survival, we compared infant survivorship to three years of age among mothers with and without cysts using Kaplan-

Meier survival curves and Cox proportional hazard models. We analyzed the full dataset (36 infants born to 27 cyst mothers, 339 born to 154 non-cyst mothers), as well as a subset of the data that excluded infants that died along with their mothers (leaving 25 infants born to 17 cyst mothers, and 325 born to 149 non-cyst mothers) to assess whether maternal mortality was the primary driver of infant death. Our Cox models took the form h(t|X) = h0(t) exp( β1X1), where h(t|X) is the instantaneous hazard rate at time t; h0(t) is the baseline hazard rate; X1 is 0 if the infant’s mother had a cyst at the time of birth and 1 if not; and β1 is the hazard ratio associated with maternal cysts. Scaled Schoenfeld residual tests revealed no violation of the proportional hazards assumption. To account for potential non-independence of infants born to the same mother, we fit Cox models that included a frailty term (i.e., a random effects term for mother ID) (Therneau,

Grambsch & Pankratz 2003). The frailty term has a multiplicative effect on the baseline hazard, and allows the offspring of different mothers to have different baseline hazard rates.

3.2.5 Interbirth interval

Due to a surprisingly low sample size of seven complete interbirth intervals

(IBIs) observed for females with cysts over 10 years, we were unable to statistically analyze the impact of cysts on IBI. The majority of cyst females died before completing a single interbirth interval (i.e., giving birth to two subsequent infants). Four of the seven recorded IBIs came from a single female that possessed a small cyst on her chest throughout her adult life.

3.3 Results

3.3.1 Cyst prevalence

Our dataset included 386 adult individuals: 170 males and 216 females. Over 10 years, 54 adults across three study bands exhibited cysts characteristic of T. serialis , producing a 14% period prevalence. Thirty-eight of 216 females exhibited cysts during the study period (17.6%), while 16 of 170 males exhibited cysts during the study period

(9.4%). Only 2 of 407 immature individuals (i.e., < 4 years of age) ever exhibited cysts

(0.05%).

3.3.2 Adult mortality

Survival analyses revealed reduced survival for males and females with cysts, although the effect was only significant for females (Table 2). The Cox proportional hazards model showed a positive effect of cysts on the hazard ratio for males and females, with estimated hazard ratios of 5.77 (95% CI: 3.3, 10.22) in females and 1.81

(95% CI: 0.4, 7.4) in males. Scaled Schoenfeld residuals tests revealed a significant deviation from the proportional hazard assumption for females but not males (Table 2), and plots of scaled Schoenfeld residuals against time indicated a clear negative relationship between time and the coefficient for cysts in males and females (Fig. 6). We therefore fit extended Cox models with time-varying coefficients for the cyst variable, specifying a linear relationship between time and the effect of cysts on the log hazard ratio. For females, this yielded a significant positive effect of cysts on the hazard ratio and a significant negative relationship between the effect of cysts and time (Table 2, Fig.

6B). While the same pattern was observed for males, small sample size likely inhibited the achievement of statistical significance (Table 2, Fig. 6A). Together, these results indicate that cysts reduce survival in adult geladas, with diminishing strength over time.

Table 2: Results of Cox models and scaled Shoenfeld residual tests.

Cox model Cyst Time -transform Schoenfeld residual test, coefficient coefficient cyst p-value Standard - Males 0.6 ( -0.81, 2) NA p = 0.18 Standard - 1.75 (1.8, 2.33) *** NA p < 0.001*** Females Extended - 3.85 -0.27 ( -0.63, NA Males (-0.18, 7.87) 0.07) Extended - 4.8 (3.24, 6.34) *** -0.19 ( -0.28, - NA Females 0.1) ***

3.3.4 Infant mortality and interbirth interval

Infants born to mothers with cysts experienced higher mortality than those born

to mothers without cysts (Fig. 8A). The frailty term for mother ID was not significant ( p =

0.76), so we report results for a Cox proportional hazards model without a frailty term.

The median was two births per mother, and the maximum was seven births per mother.

The model revealed that infants born to cyst mothers experience a ~2.5-fold increase in

hazard rate (hazard ratio = 2.49, CI = 1.47-4.23, p < 0.001). To assess whether maternal

mortality drove patterns of infant mortality among offspring of mothers with cysts, we

fit a Cox model to infant data that excluded offspring of mothers that died with their

infants (4% of non-cyst mothers, 30.6% of cyst mothers). We found that maternal cysts

no longer had a significant effect on infant survival (hazard ratio = 1.37, CI = 0.59-3.17, p

= 0.467; Fig. 8B). Offspring survival was only affected by maternal cysts when infants

that died along with their mothers were included.

Low sample size precluded quantitative analysis of interbirth interval data. Four

of the seven IBIs to females with cysts came from a single female (Mary) who possessed

a small cyst on her chest for most of her adult life. Of the five infants born to Mary while

she had a cyst, only one survived to sexual maturity, three died, and one was still

dependent at the end of the study. The other three complete IBIs (marked by the time between two subsequent births) were to three different females; one lost the infant at the start of the IBI and the infant at the end of the IBI, another died along with her second infant, and the third successfully weaned both offspring.

3.4 Discussion

Cysts characteristic of the tapeworm Taenia serialis were associated with increased mortality in geladas in the SMNP. While this effect was statistically significant only for females, the striking similarity between the magnitude of the estimated effect and the age-dependent pattern in both sexes suggest that similar processes are occurring in both males and females. Offspring of mothers with cysts had significantly higher

mortality, which was driven by high maternal mortality. In fact, maternal mortality was so high that females rarely survived the duration of an interbirth interval, precluding statistical analysis of interbirth intervals. These results contribute to the sparse literature demonstrating explicit fitness costs of an endemic helminth parasite infection in a wild primate, and expand the nascent body of research on T. serialis in wildlife.

3.4.1 Demographic variation in prevalence

Among adults, cyst prevalence over 10 years was 14%, with 18% prevalence in females and 9.4% prevalence in males. We refrained from statistically comparing prevalence rates between the sexes because males and females are subject to different demographic processes, which likely results in an underestimation of prevalence among males. Natal males disperse at sexual maturation to roving all-male groups (AMGs), from which they challenge dominant males (“leaders”)- generally in non-natal bands- for control of reproductive units (Dunbar 1984, Pappano 2013). Following takeovers, deposed leaders can become subordinate “followers” in their units, rejoin AMGs, or join other bands (Dunbar 1984, Pappano 2013). Thus, we only observe natal males until they disperse (when they are “lost” to data collection), and adult males for the period in which they are leaders or followers in study groups. Furthermore, as suggested by

Nguyen et al. (2015), T. serialis infection may preclude the ascension of infected males to

dominance, which would prevent males with cysts from ever entering into data collection. If infected males are less likely to enter into data collection than uninfected males, the true prevalence of T. serialis cysts among males would be further underestimated.

Over the same period, 14% of adults and ~0.5% of immatures exhibited T. serialis cysts. A similarly strong age bias was reported by Nguyen et al . (2015) for their geladas, and is consistent with evidence from other taeniid species that age is associated with higher infection risk, that infection persists throughout the host’s lifetime, and that hosts do not develop immunity with prolonged exposure (Gemmell et al. 1987, Torgerson et al.

1998).

3.4.2 Impact of cysts on mortality

The direct, extreme parasite-induced mortality demonstrated by our survival analyses is rarely observed in wild primates or other long-lived mammalian hosts (see

Milton 1996, Keele et al. 2009 for notable exceptions). While this effect was only significant for females, a similar trend was observed for males. This is likely due to our small sample size for males, but may also arise from the inclusion of both natal and non- natal males that may experience different background mortality rates. We hypothesize that the unusual degree of mortality demonstrated by our analysis results from a

parasitic life cycle that requires the demise of the intermediate host for its survival and reproduction. This phenomenon, “parasite increased trophic transmission”, may be an adaptation to high host predation, permitting parasite survival through infection of predators (Poulin 1994a, b , Lafferty 1999, Lafferty & Kuris 2002), or to low host densities

(Choisy et al. 2003). As in the intermediate stages of many tapeworm species, Taenia serialis larvae must be ingested by the carnivorous definitive host to reach sexual maturity (Parker et al. 2015). Thus, T. serialis and related tapeworms should be under selection to increase the harm inflicted on their intermediate hosts (Lafferty 1999, Parker et al. 2015).

Indeed, geladas in our study population were observed with cysts that appeared to impede breathing, movement, and mating, and often became infected after bursting.

Thus, T. serialis larvae appear to act similarly to those of other taeniid species, causing mechanical injury to muscular, visceral, neural, and connective tissue, physical impingement of nerves, and obstruction of arterial and venous flow (Sharma & Chauhan

2006, Godara et al. 2011, Oryan et al. 2014). Beyond directly precipitating death, these pathologies can thwart the predation-protection ostensibly offered by large social groups (van Schaik 1983, Janson & Goldsmith 1994) by hampering the ability of infected geladas to keep pace with their groups. Thus, the damage caused by T. serialis likely

facilitates the transmission of larvae to the definitive host, either through scavenging of

infected corpses or predation of lagging individuals.

Our survival analysis revealed an unexpected time-dependent relationship between age and mortality risk in adults, with the impact of cysts on mortality declining

with age. In other words, younger geladas with cysts faced higher mortality risk than

older geladas with cysts. This effect could be due to a time-dependent effect of cysts on

mortality, in which earlier stages of infection are more virulent than later stages.

However, this effect could also be an artifact of unmodeled individual-level

heterogeneity, in which individuals differ in their innate response to infection (Raberg,

Graham & Read 2009) or in the severity of their infections. If severity is related to cyst

location, for example, individuals with cysts in more benign locations (e.g.,

subcutaneous connective tissue) will survive longer than individuals with cysts in more

harmful locations (e.g., viscera). Similarly, if the adult males that maintain extended

tenures as leaders in study groups are less likely to have cysts in harmful locations, they

will survive longer than those with cysts in more benign locations. Unfortunately, our

dataset is too small to explore these potential explanations, and future work should

explore individual variation in T. serialis -associated mortality with fine-grained

descriptive data on cysts and immunological measures.

Another potential source of the unmodeled heterogeneity suggested by the results of our survival analysis is variation in individual susceptibility. Variation in susceptibility may be tied to the interaction between T. serialis and the endocrinological and immunological profiles of its gelada hosts. Larval taeniids are virtually singular among parasites in their predilection for female hosts and deandrogenization of male hosts (Lin et al. 1990, Morales-Montor & Larralde 2005). In fact, estrogens directly stimulate larval taeniid proliferation and induce a permissive immune response, while androgens inhibit proliferation and induce a restrictive immune response (Esch 1967,

Huerta et al. 1992, Morales et al. 2002, Morales-Montor & Larralde 2005). Future research should identify such potential drivers of individual susceptibility to T. serialis.

3.4.3 Impact of cysts on reproductive success

Consistent with evidence from experimental work showing taeniid-induced reductions in host reproductive success, offspring of female geladas with T. serialis cysts incurred a ~2.5-fold increase in mortality. Many parasite infections demand energetic resources and immune activation at the cost of reproductive effort (Forbes 1993, Ilmonen et al. 2000, Hurd 2001), or incite immunological or endocrinological changes that interfere with implantation and pregnancy (Lin et al. 1990, Krishnan et al. 1999, Gourbal

& Gabrion 2004, Arteaga-Silva et al. 2009). In this system, however, cyst-associated

declines in reproductive success take the shape of heightened offspring mortality for mothers with cysts, and this effect appears to be driven primarily by cyst-associated maternal death. When infants whose mothers perished were excluded from the analysis, no effect of maternal cysts was detected. This is in contrast to the findings of Nguyen et al. (2015), which demonstrated elevated mortality among offspring of gelada mothers with cysts even after excluding offspring that died with their mothers. One explanation for the divergence in results between the two sites is that cyst-associated mortality is higher in the SMNP than at Guassa. Thus, the effect and frequency of maternal mortality in the SMNP geladas may be too strong to detect alternate mechanisms of offspring mortality that might be at play. Future work should look to parasite-related decreases in milk quality and lactation persistence as candidate mechanisms driving infant mortality not related to maternal mortality in the Guassa population (Lopes et al. 2016).

Remarkably, mortality of cyst mothers in the SMNP was so high that we were unable to analyze the impact of cysts on interbirth interval (IBI). Over 10 years of data collected on this population, only 7 IBIs were observed for 31 mothers with cysts. This is in stark contrast to the Guassa population studied for 6.5 years by Nguyen et al. (2015), in which 27 IBIs were observed for 31 mothers with cysts. Although we were unable to directly compare cyst-associated mortality rates between the SMNP and Guassa geladas due to divergent statistical approaches, the increased rates of maternal death in the

SMNP suggest that cyst-associated mortality may be higher at this site. This pattern could arise through a number of mechanisms, including the evolution of infection tolerance (Hayward et al. 2014) in the Guassa geladas, hormonally modulated immunosuppression induced by increased anthropogenic stressors (Lafferty & Holt

2003) in the SMNP, comorbidity with other parasites (Pullan & Brooker 2008), or the presence of a more lethal strain of T. serialis in the SMNP. However, additional analyses must be conducted in order to directly compare mortality rates between the two populations.

3.4.4 Future directions

The presence of T. serialis cysts in geladas under intensive study at two sites in the Ethiopian highlands with drastically different degrees of anthropogenic habitat change provides an exciting opportunity to evaluate the impact of habitat disturbance on an ecologically complex parasite. The relatively pristine Guassa Plateau is home to substantial rodent, lagomorph, and canid populations (Ashenafi et al. 2012) that may serve as additional T. serialis hosts. By contrast, land cultivation in the SMNP has eliminated rodent habitat and density (Ashenafi et al . 2012, Yihune & Bekele 2014).

Smaller host populations could lower the environmental risk of exposure of geladas to

T. serialis eggs. Further work should quantify anthropogenic habitat change at each site,

describe intermediate and definitive host identity and infection prevalence, and assess

environmental T. serialis risk.

Most emerging infectious diseases in humans are caused by pathogens that infect

multiple mammalian orders (Cleaveland et al. 2001, Pedersen et al. 2005). Although the

larval stage of T. serialis is frequently characterized as a pathogen of lagomorphs and

rodents (e.g., Bowman 2009), it has been documented in intermediate hosts as

taxonomically diverse as marsupials (e.g., Hough 2000), felines (e.g., Jull et al. 2012), bovids (e.g., Nevenic & Markovic 1951), and humans (e.g., Tappe et al. 2016). Given the

expanding human-wildlife interface in the Ethiopian highlands, understanding the

dynamics of T. serialis in wildlife is essential to managing its emergence in humans and

other non-traditional hosts.

3.6 Acknowledgements

This research was supported by the National Science Foundation ( IOS-1255974, BCS-

0715179), the Leakey Foundation, the National Geographic Society (Gr. #8989-11, Gr.

#8100-06), Primate Conservation, Inc., Conservation International , the Nacey

Maggioncalda Foundation , and the Margot Marsh Biodiversity Foundation . We thank

the Ethiopian Wildlife Conservation Authority and officials who facilitated our research,

and past and present field crew of the University of Michigan Gelada Research Project

for their dedication to ten years of data collection. We thank CL Nunn, LJ Digby, CM

Drea, TT Struhsaker, N Snyder-Mackler, AA Tositi, and all reviewers for their invaluable feedback. IASC/RHG acknowledge support from the National Science Foundation’s

Graduate Research Fellowship Program. All research was approved by the University

Committee on Use and Care of Animals at the University of Michigan (UCUCA #09554), the Duke University Institutional Animal Care and Use Committee (IACUC #A218-13-

08, and followed all laws and guidelines in Ethiopia.

4. Identifying wildlife reservoirs of neglected taeniid tapeworms: non-invasive diagnosis of endemic Taenia serialis infection in wild primates

4.1 Introduction

Tapeworm parasites of the genus Taenia are globally distributed in numerous mammalian hosts, frequently exploiting predator-prey relationships and posing considerable risk to humans. Although the life cycles and zoonotic potential of some taeniids are among the most well known of all tapeworms due to their importance in human health and evolution (Hoberg 2002), the descriptions of other taeniids have been neglected. Particularly enigmatic is Taenia serialis , conventionally thought to infect dogs in its adult stage and rodents and lagomorphs in its intermediate stage (Marty & Neafie

2000). Over the past century, extensive taxonomic and morphological confusion and disagreement have made it difficult to identify the geographic and phylogenetic distribution of this parasite (Loos-Frank 2000, Hoberg 2002). Thus, we begin by providing what is, to our knowledge, the first thorough review of T. serialis biology and zoonotic potential by synthesizing previous case reports. We then describe the antigen enzyme-linked immunosorbent assay (ELISA) that we validated for use with gelada urine samples. Finally, we demonstrate its application in a free-living population of

Ethiopian geladas ( Theropithecus gelada ), the only known primate host of the larval stage

of T. serialis , and provide recommendations for future implementations of this assay in wildlife systems.

4.1.1 Review: diversity and zoonotic potential of T. serialis

Singular among cyclophyllidean tapeworms, taeniid species parasitize mammals

in both their adult and larval stages (Hoberg 2002). Taeniid adult stages infect humans

and carnivorous species that include canids, felids, hyaenids, mustelids, and viverrids

(Loos-Frank 2000, Hoberg 2002) and cause few severe symptoms in healthy hosts

(Schmidt 1986, Eckert 1991, Marty & Neafie 2000). By contrast, taeniid larval stages

(metacestodes) generally infect herbivorous artiodactyl, rodent, and lagomorph species

(Loos-Frank 2000, Hoberg 2002) and regularly cause extensive muscular and visceral

damage (Schmidt 1986, Leiby & Dyer 1971, Marty & Neafie 2000, Mertz 2016).

Intermediate hosts become infected when they ingest eggs shed by adult tapeworms

harbored in the definitive host, and definitive hosts become infected when, via

predation or scavenging, they ingest larvae in infected intermediate hosts (Loos-Frank

2000, Hoberg 2002).

The scientific study of T. serialis is marked by a tendency to make species level

designations that may not be warranted and, consequently, to underestimate the range

of hosts that T. serialis infects. The T. serialis metacestode is a thin-walled, translucent

structure (coenurus) containing multiple protoscolices, the precursor to the mature

scolex that constitutes the attachment end of the adult tapeworm in the definitive host

(Leiby & Dyer 1971). This metacestode morphology is indistinct from that of T. multiceps , a zoonotic parasite found primarily in sheep (Marty & Neafie 2000). Before the relatively recent emergence of molecular tools (Padgett et al . 2005, McManus 2006, Zhang et al .

2007, Jeon et al . 2009, Jia et al . 2010, Avcioglu et al . 2011), cases of coenurosis were ascribed to either T. serialis or T. multiceps based on now-outdated metrics related to larval morphology (Marty & Neafie 2000) or infection site predilection (e.g., central nervous system or subcutaneous tissue) (Brumpt 1936, Templeton 1968, Marty & Neafie

2000). Furthermore, some researchers employed synonyms for T. serialis (e.g., T. brauni,

T. glomeratus ) based on geographic location or occurrence in a non-rodent or lagomorph

host (Fain 1956, Templeton 1968).

In addition to taxonomic confusion surrounding metacestode identification, the

occurrence of coenurosis ascribed to T. serialis in non-rodent or lagomorph hosts has been largely overlooked. Although parasitological texts invariably refer to T. serialis as a

parasite of rodents and lagomorphs in its larval stage, it has been reported in a wide

range of phylogenetically and geographically diverse hosts. Case studies have described

T. serialis coenurosis in three rodent species (Dollfus 1948, Bracken & Olsen 1950,

Newberne & Bernett 1951, Lacasse et al . 2005, Holmberg et al . 2007), domestic cats

(Georgi et al . 1969, Hayes & Creighton 1978, Kingston et al . 1984, Smith et al . 1988,

Slocombe et al . 1989, Huss et al . 1994, Jull et al . 2012), two marsupial species (Dunsmore

1968, Hough 2000), two lagomorph species (Hamilton 1950, Goudswaard & Thomas

1991, Bennett 2000, Fountain 2000, Wills 2001, O’Reilly et al . 2002), and two nonhuman

primate species (the greater spot-nosed guenon ( Cercopithecus nictitans ) (Sandground

1937), and the gelada (Scott 1926, Elek & Finkelstein 1939, Urbain & Buller 1935,

Schneider-Crease et al . 2013, Nguyen et al . 2015, Schneider-Crease et al . in review). To our knowledge, only two studies of naturally occurring T. serialis coenurosis have used molecular tools for species identification (Schneider-Crease et al . 2013, Nguyen et al .

2015). Given the lack of confirmed T. serialis diagnoses in the literature, including for the

cases in ‘standard’ rodent and lagomorph hosts, it stands to reason that T. serialis may be more widespread and flexible in its selection of intermediate hosts than previously described.

The historic difficulty of definitively diagnosing T. serialis coenurosis may have led to an underestimation of its zoonotic potential. Coenurosis has been recorded in humans across the globe (Lescano & Zunt 2013, Tappe et al . 2016), including Europe

(Bonnal et al . 1933, Brumpt 1936, Roger et al . 1942, Buckley 1947, Landells 1949, Ranque

& Nicoli 1955, Bertrand et al . 1956, D’Andrea et al . 1964, Michal et al . 1977, Pau et al . 1987,

Pau et al . 1990, Sabattani et al . 2004), Africa (Becker & Jacobson 1951a/b, Watson & Laurie

1955, Raper & Dockeray 1956, Wilson et al . 1972, Malomo et al . 1990, the Middle East

(Benifla et al . 2007, El-On et al . 2008), and the Americas (Correa et al . 1962, Ing et al . 1998).

Certain authors declined to assign a species (Templeton 1968, Wilson et al . 1972), while the others ascribed infection to T. serialis or T. multiceps based on morphological analysis.

Only one study used molecular tools, identifying T. serialis coenurosis in a man in

Nigeria (Tappe et al . 2016). In sum, the taxonomic uncertainty of coenurosis occurring in animals, including humans, has led to a fragmented record of the global occurrence and distribution of T. serialis and a potential underestimation of its zoonotic potential and importance to public health.

4.1.2 An antigen ELISA to investigate larval T. serialis in wildlife.

As humans come into increasing contact with wildlife, understanding the biology and zoonotic potential of T. serialis is crucial to preventing its transmission to humans and domestic animals. Little is known about the natural dynamics of Taenia spp. in wildlife hosts, largely because of the impracticality of obtaining and storing biological samples or performing medical imaging in remote settings and on wildlife. To obtain a more accurate assessment of the prevalence of larval T. serialis infection in wildlife host species, we adapted an existing monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Taenia antigen in dried urine samples

(Brandt et al . 1992, Van Kerckhoven et al . 1998, Dorny et al . 2000). The monoclonal

antibodies (B158C11 and B60H8) used in this assay are specific to the Taenia genus, which permits its use in the detection of larval infections of all taeniid species. Indeed, this assay has been used as an epidemiological tool, often complementary to other diagnostic methods, in studies of porcine, bovine, and human cysticercosis (Van

Kerckhoven et al . 1998, Dorny et al . 2000, Garcia & Del Brutto 2000, Nguekam et al . 2003,

Dorny et al . 2003, Dorny et al . 2004a, Dorny et al . 2004b, Allepuz et al . 2012, Eichenberger et al . 2011, Castillo et al . 2009, Mwape et al . 2011, Gabriël et al. 2012). Because this assay

detects circulating larval antigens, it identifies active infections rather than past

exposure identified by antibody assays (Dorny et al . 2004a).

Despite the success of this assay in studies of cysticercosis in livestock, the

difficulty of obtaining blood or serum samples from humans limited its use in human

populations (Castillo et al . 2009, Mwape et al . 2011). Thus, two teams adapted the

monoclonal antigen test to non-invasively diagnose these diseases in urine (Castillo et al .

2009, Mwape et al . 2011). However, these protocols are still impractical for

implementation in wildlife studies because they require that procured urine samples be

stored at -20°C until processing (Castillo et al . 2009, Mwape et al . 2011). Because many

wildlife studies are carried out in areas where electricity is absent or inconsistent, the

need for refrigeration limits the practicality of these tests in remote areas. We therefore

validated the use of dried urine with a modified protocol to investigate sylvatic cycles of

Taenia transmission.

4.1.3 Antigen ELISA implementation in geladas ( Theropithecus gelada )

Geladas - herbivorous primates endemic to the Ethiopian highlands – are known

to exhibit protuberant cysts characteristic of infection with the larval stage of T. serialis

(Fig. 9). Coenuri have been recorded in wild-caught captive geladas for nearly a century

and were ascribed to T. serialis based on morphological cues (Scott 1925, Schwartz 1926,

Schwartz 1927, Urbain & Bullier 1935, Elek & Finkelstein 1939, Rodhain & Wanson 1954,

Bertolino 1957, Clark 1969). Recently, this identification was confirmed with molecular diagnosis of cystic material obtained from protuberant cysts (Schneider-Crease et al .

2013, Nguyen et al . 2015). Prevalence of T. serialis -associated cysts in geladas ranges from

4-13% in an ecologically disturbed area (Dunbar 1980, Schneider-Crease et al . 2013,

Schneider-Crease et al . in review) to 30% in an ecologically intact area (Nguyen et al .

2015), and cysts in both areas are associated with significant increases in mortality and

decreases in reproductive success (Nguyen et al . 2015, Schneider-Crease et al . in review).

However, not all infections necessarily manifest as conspicuous cysts, a point illustrated by the presence of non-protruding cysts revealed during necropsies on infected captive

geladas. Thus, prevalence of T. serialis in geladas based on protuberant cysts alone is likely to be underestimated.

Figure 9. (A) Gelada with a larval T. serialis cyst protruding from the abdomen. (B) Internal view of coenuri in the cyst of an infected individual necropsied upon natural death.

We implemented the monoclonal antibody-based sandwich ELISA in a wild population of geladas in the Simien Mountains National Park (SMNP), Ethiopia, where individuals are known to be parasitized with T. serialis (Schneider-Crease et al . 2013).

Recent work in this population demonstrated sex- and age- biases in the distribution of

T. serialis cysts, with higher prevalence in adults and females (Schneider-Crease et al . in

review). This sex bias may reflect either patterns of data collection that bias towards

observing infected females and uninfected males, or the estrogen affinity exhibited by

the larvae of many taeniid species (Terrazas et al . 1998, Morales et al . 2002, Escobedo et al .

2004, Morales-Montor et al . 2004, Morales-Montor & Larralde 2005, Pena et al . 2007). The

increased prevalence of T. serialis cysts in adults compared to immatures may arise

either from increased susceptibility of adults due to the immunosuppressive effects of

hormones related to sexual maturity, or as a function of the time required for infection to

develop into observable cysts. The adaptation of the urine antigen ELISA to non-

invasively diagnose T. serialis in gelada urine allowed us to investigate the demographic

predictors of infection that cannot be detected solely by using observable cysts.

4.2 Materials and Methods

4.2.1 Study site

We conducted our study in the Sankaber area of the SMNP, Amhara Region, Ethiopia.

The SMNP was established in 1969 and has been classified as a UNESCO World

Heritage Site in Danger since 1996 due to substantial anthropogenic impact (Debonnet et

al . 2006). The park covers 13,600 hectares, is characterized by Afro-montane and Afro-

alpine habitats, and contains a number of mammals of potential importance to the T. serialis life cycle. These include the black-backed jackal (Canis mesomelas ), the golden jackal ( Canis aureus ), the spotted hyena ( Crocuta crocuta ), the Ethiopian wolf ( Canis

simiensis ), Starck’s hare ( Lepus starcki ), and the gelada monkey (Debonnet et al . 2006) The substantial human population in the SMNP has contributed to the loss of natural vegetation and the expansion of crops and grazing seen in many areas of the park

(Debonnet et al . 2006, pers. obs.). Dogs, jackals, hyenas, and Ethiopian wolves are among the carnivores living in the SMNP that potentially prey on or scavenge the corpses of geladas (Debonnet et al . 2006), and are thus of potential importance for the T. serialis life cycle.

4.2.2 Urine sample collection

From August 2014 to June 2015, we collected a total of 527 urine samples from

204 geladas (117 females, 87 males; 37 infants, 60 subadults, 107 adults) living in the

SMNP. We conducted sampling in two habituated gelada groups under long-term study by the University of Michigan Gelada Research Project (UMGRP). Individual geladas in the habituated groups are each assigned a three-letter code and are identifiable with near perfect accuracy based on suites of morphological characteristics.

Thus, all samples collected in this population were from known individuals, with most individuals sampled more than once over time (n=97 individuals; median: 2 samples/individual, range: 1-10). Sampling included 58 samples from 10 individuals exhibiting the cysts characteristic of T. serialis infection to serve as ‘true positives’, and 57

samples from 37 unweaned infants to serve as ‘true negatives’ (unweaned infants are unlikely to ingest eggs because they do not yet eat grass, see below for further explanation). All other samples (412 from 158 individuals) were collected for evaluation in the ag-ELISA as samples of ‘unknown status’ (median=2, range: 1-10). These included

94 females and 64 males; 60 subadults and 98 adults.

Urine samples were collected from the ground immediately after urination using

Whatman ® Qualitative Filter Papers (Grade 4, 11.0 cm). After urination, as much urine as possible was soaked up from the ground with a filter paper. The filter paper was folded and stored in a 2 oz. Whirl-Pak ® bag, which was labeled with the unique code associated with the individual, date, and time. Approximately 1 g of indicating silica desiccant was added to each bag to ensure samples remained dry and to prevent mold growth.

4.2.3 Urine sample analysis

Samples were processed and analyzed using the B158/B60 ELISA in the

Immunochemistry Laboratory of the Division of Parasitic Diseases and Malaria at the

Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia. To aid in identifying urine stains on the filter papers, we viewed each paper under a UV light

(long-wave, 365 nm; Spectroline Model ENF-240c) and used an office hole puncher to

remove four circles (~6 mm diameter) from the part of each filter paper that was soaked

on both sides. The hole puncher was sterilized and dried after each use to prevent cross-

contamination. The four circles taken from each sample were placed into a single labeled

2 mL sample tube. We ensured that each circle was taken from an area on the filter

paper that was soaked on both sides. Each sample was reconstituted with 1 mL blocking buffer (PBS-Tween 20 + 1% newborn calf serum (NBCS)) and vortexed.

Polystyrene ELISA plates (Nunc Maxisorp ® flat-bottom 96 well) were coated

with the capture antigen antibody (B158C11A1 monoclonal antibody) in a sensitization buffer (carbonate bicarbonate buffer, pH 9.5) and were incubated for 30 minutes at 37°C

on a shaker. After a single wash with PBS-Tween 20, wells were blocked with 150

µL/well of blocking buffer and incubated for 15 minutes at 37°C on a shaker. Without

washing, plates were loaded with 100 µL from each sample. Eighty unknown samples

per plate were tested, along with four known negative human urine samples, and two

positive control samples created by spiking known negative human samples with 0.125

µg antigen/1 mL urine T. crassiceps antigen (soluble protein extract). Plates were incubated for 45 minutes at 37°C on a shaker.

After washing four times with PBS-Tween 20 (hereafter: ‘wash’), 100 µL of detecting antibody dilution (B60H8A4 + blocking buffer) was added to each well, and plates were incubated for 15 minutes at 37°C on a shaker. After another wash, 100 µL of

Streptavidin-HRP dilution (Streptavidin-HRP conjugate + blocking buffer) was added to each well and plates were incubated for 15 minutes at 37°C while shaking. After another wash, wells were loaded with 100 µL of Tetramethylbenzidine (TMB) and plates were shaken at room temperature for two minutes. After the addition of 100 µL of stop solution (1N sulfuric acid; H2SO4) to each well, the optical densities (OD) of samples were read in the VersaMax™ ELISA Microplate Reader (Molecular Devices) at 450 nm.

If more than one control on a plate failed, the entire plate was repeated. We calculated the index value (IV) for each sample relative to the positive and negative controls on each plate using the following formula:

4.2.4 Receiver operating characteristic analysis

We assessed the sensitivity and specificity of the Ag-ELISA with a receiver operating characteristic (ROC) curve (Greiner 2000). The nature of working in a wild system precludes establishing a negative ‘gold standard’ because we are unable to confirm negative diagnoses with serological or imaging techniques. Thus, we used unweaned infants as ‘true negatives’ (n=57 samples) because they do not yet consume grass and are thus minimally exposed to T. serialis eggs and can be considered likely to

be negative. We used individuals presenting with T. serialis cysts as ‘true positives’ (n=58

samples). We selected the point on the ROC curve at the shortest distance from the

coordinate (0, 1) as the optimal threshold IV for classifying a sample as positive or

negative. ROC analysis was performed with the package “pROC” (Robin et al . 2011) in R

(R Core Team 2016).

4.2.5 Analysis of T. serialis infection predictors in urine samples

To investigate if sex and age predicted the occurrence of cysts among adults and

subadults (n=158 individuals), we used logistic regression implemented in the ‘glm’

function in the R package ‘stats’ (99). We coded age as a continuous variable based on

known or estimated birthdates for individuals. Model selection was performed with

Akaike information criterion (AICc), which selects the optimal model based on

maximum likelihood (Akaike 2011) with a finite sample size (Burnham & Anderson

2011).

To investigate if sex and age predicted the occurrence of antigen-positive

samples (i.e., those with an IV greater than the IV threshold from the ROC analysis)

among adults and subadults without cysts (n=412 samples, 158 individuals), we used a

generalized linear mixed effects model (GLMM) implemented with the ‘glmer’ function

in the ‘lme4’ package in R (Bates et al . 2014). We used binomial errors with a logit link

function, and included age and sex as fixed effects. Because individuals were sampled at

varying intensities and may have had different individual risks of infection, we included

individual identity as a random effect. We coded age in the following two ways: (1) as a

continuous variable based on known and estimated birthdates and (2) as an ordered

categorical variable with two levels based on developmental stage (i.e., subadult or

adult). Continuous age is expected to be a relevant predictor of infection if accumulated

exposure to T. serialis eggs in the environment drives risk, whereas categorical age based on developmental stages may be more relevant if hormonal factors are a major driver of risk. We compared the fit of the continuous age and categorical age models using AICc and calculated averaged coefficients for each variable using model averaging.

4.3 Results

4.3.1 Sample analysis

Our measurement of infection status using the described Ag-ELISA was highly

accurate. The ROC analysis revealed the optimal threshold IV to be 42.1, with 98.4%

specificity (95% CI: 95.1-1), 98.5% sensitivity (95% CI: 95.6-1) and an area under the

curve (AUC) of 0.99 (95% CI: 0.9937-1; Fig. 10). We identified only one likely false

negative (i.e., an infant with a positive sample) (98.2%, 56/57), and one false positive (i.e.,

an individual with a cyst that tested negative in one sample) (98.3%, 57/58, Table 3).

Table 3: Ag-ELISA results of gelada samples (true positive, true negative, unknown status)

True positives True negatives Unknown status

Ag-ELISA: Positive 57 (98.3%) 1 (1.8%) 50 (12.1%)

Ag-ELISA: Negative 1 (1.7%) 56 (98.2%) 362 (87.9%)

Total sample # 58 57 412 True positives= samples from individuals with cysts (n=10 individuals). True negatives= samples from unweaned infants (n=37 individuals). Unknown status=samples from individuals without cysts, excluding infants (n=158 individuals). Positive/negative assigned based on antigen presence above the optimal threshold IV cutoff (an indexed optimal density of 42.1) determined by the ROC analysis.

Twenty-six of our 158 non-cyst individuals (16.4%) tested positive at least once.

This included 14 females, 12 males, and 6 subadults, 20 adults. Overall, individuals with

cysts generally had higher logged index values (IVs) than individuals without cysts (all but one sample from a cyst individual fell above the optimal cutoff; Fig. 11).

Importantly, two individuals without cysts that tested antigen-positive developed

observable cysts within seven months of sampling. One of these individuals had one

negative and one positive sample in the 3 months prior to exhibiting an observable cyst,

after which all of his samples were positive. The other individual had one positive

sample seven months before exhibiting an observable cyst, after which all of her samples were positive.

To search for evidence of established T. serialis infection in non-cyst individuals, we focused on individuals that were sampled at least five times during the study period

(21 adults, 2 subadults). We found that some non-cyst individuals were consistently positive for T. serialis antigen, others were consistently negative, and still others switched between antigen-positivity and antigen-negativity throughout the study period. Twelve individuals showed no antigen-positive samples, two showed a clear majority of positive samples (one with 8/9 positive samples, one with 9/10 positive samples), and seven individuals had a single positive sample within a sequence of negative samples. The remaining two individuals showed an interesting mixture of positive and negative samples: one individual tested positive in three consecutive months and then negative seven months later. The other displayed a sequence of negative and positive samples within six months.

4.3.2 Statistical analysis of T. serialis predictors in urine samples

We investigated the predictors of visible cysts, focusing on age, sex, and the interaction between these two variables. AICc model selection revealed the models with the most support (ΔAICc < 2) to include age (in years), sex, and an interaction between age and sex as predictor variables, with the model including only age garnering the most support (Table 4). The importance of age and lack of effect of sex were reinforced with the results of full model averaging, which showed that increasing age was the strongest predictor of cysts across all models (Table 6).

We then investigated the predictors of antigen positivity in urine samples, again including age, sex, and the interaction between these two variables as predictors. One analysis included age coded categorically, whereas the other included age coded continuously, and both included individual ID as a random intercept to account for repeated sampling from individuals. In the first analysis (categorical age), AICc model selection showed that the model with the most support included only the random intercept (individual ID) and no fixed effects (i.e., no effect of age, sex, and the interaction on Taenia antigen-positivity in samples, Table 5). A model including age and the random intercept was less supported than the model containing only the random intercept (Table 5). Full model averaging revealed age to be a weaker predictor of antigen-positivity than the random intercept (Table 6). Results were similar for the

analysis that used age coded as a continuous variable. The model with the most support included only the random intercept and no fixed effects (Table 5), which was also reflected in the model averaging estimates (Table 6).

Model Intercept Years Male Male:Years AICc Δ weight

1.1. Cyst ~ Years -5.00 0.19 NA NA 70.9 0 0.528

1.2. Cyst ~ Male + Years -5.42 0.21 0.56 NA 72.4 1.51 0.248 87 1.3. Cyst ~ Male + Years + Male:Years -4.79 0.17 -2.23 0.23 72.6 1.75 0.22

Model Intercept Years Age AICc Δ weight

Categorical Age 2.1. Positive ~ (1 | Name) -7.76 NA NA 220.12 0 0.73 2.2 Positive ~ Age + (1 | Name) -7.88 NA 0.21 222.11 1.99 0.27

Continuous Age 3.1. Positive ~ (1 | Name) -7.76 NA NA 220.12 0 0.73 3.2. Positive ~ Years + (1 | Name) -7.54 -0.03 NA 222.1 1.98 0.27 88

Model Variable Estimate SE (adj) z-value Pr(|>z|) 1. Cyst ~ Male * Years Intercept -5.06 0.97 5.22 2e -07 *

Years 0.19 0.06 3.06 <0.01* Male 0.35 0.60 0.22 0.83 Male:Years 0.05 0.13 0.39 0.70

89 2. Positive ~ Male * Age + (1| ID) Intercept -7.79 1.15 6.77 <2e -16 * Age (Adult) 0.06 0.55 0.10 0.92 Male NA NA NA NA

3. Positive ~ Male * Years + (1| ID) Age (Adult) -7.70 1.22 6.34 <2e -16 * Male -0.01 0.07 0.11 0.91

4.4 Discussion

We provide the first evidence for widespread T. serialis infection in individuals that do not exhibit external cysts with a novel and highly accurate use of a monoclonal antibody-based sandwich ELISA protocol (Brandt et al . 1992, Dorny et al . 2000, Dorny et al . 2003, Allepuz et al . 2012, Castillo et al . 2009, Mwape et al . 2011). Our results indicate that T. serialis infection is more widespread than are cysts, with 18% of the sampled population testing positive for Taenia antigen where only 4.8% exhibited cysts. However, our results demonstrate the occurrence of short-term antigen presence in individuals sampled multiple times, suggesting that individuals may eliminate initial infections with

T. serialis and that a single positive sample may not necessarily indicate an established infection (as do cysts).

Because this assay identifies active infection by detecting glycoproteins produced by taeniid metacestodes, not oncospheres, positive antigen samples are highly likely to

reflect active larval growth (i.e., true infections) and not merely the presence of eggs

passing through the gastrointestinal tract (Dorny et al . 2004a/b, Santamaria et al . 2002).

We postulate that individuals without cysts that presented with high log(IV) samples should be considered positive for Taenia antigen and are likely to harbor active infections that are not visible as cysts to observers, whether because (1) the infection is young and has not yet had time to develop into a visible cyst or (2) the infection is

advanced but is located deep in the abdominal cavity or tissues and will never become

visible. It is highly unlikely that the samples positive for antigen presence are all false

positives: based on the false positive rate of 1.79% calculated using the “known

negative” infant set (in which 1 out of 57 samples from unweaned infants tested

positive), the expected number of false positives is 8.4, and the probability of observing

50 or more false positives in 412 samples is less than p=10 -25 . These two possibilities – that positive assay results indicate young infections or fully developed internal cysts – are not mutually exclusive.

In support of the interpretation of a positive antigen result as (1) reflecting the presence of young cysts that are not yet observable externally, two individuals that tested positive with no external cysts at the time developed cysts within a year of sampling. In support of the interpretation of a positive antigen result as (2) reflecting the presence of advanced infections in deep tissue that will never become visible to observers, early necropsies of wild-caught captive geladas revealed fully developed, non-protruding cysts in the abdominal cavities, deep musculature, and viscera (Scott

1926, Schwartz 1926, Urbain & Bullier 1935, Elek & Finkelstein 1939, Rodhain & Wanson

1954, Bertolino 1957, Clark 1969). Thus, positive assay results in the absence of observable cysts may reflect either young infections or advanced infections in undetectable locations.

Interestingly, we observed switches in infection status within individuals

without cysts (i.e., positive to negative and vice versa). Among 23 well-sampled

individuals without cysts (i.e., five or more samples), only two had a clear majority of

antigen positive samples, whereas twelve had no positive samples, seven had just one

positive sample, and the remaining two flipped from positive to negative during the

study period. The observed switches in infection status may reflect either (1) the

inability of some larvae to persist, or (2) the ability of hosts to control or eliminate their

infections through calcification (although caveats in data certainty must also be

considered, such as incorrect individual identification during sample collection). A

similar phenomenon was described in humans with T. solium cysticercosis, with 3.5% of

867 participants exhibiting a single positive sample in between two negative samples

(Mwape et al . 2013). The authors postulated that this short-term antigen presence could

owe to incomplete parasite formation or to effective host defenses that enable clearance

of the parasite.

In geladas, short-term antigen presence may indicate low T. serialis egg viability

or highly effective host immune responses that result in stunted infections or incomplete

parasite establishment. Indeed, experimental infection of swine with T. solium eggs demonstrated low rates of infection establishment even with high infectious doses

(Santamaria et al . 2002). Attempts by the host immune system to control infection may

not always be successful; for example, one individual tested positive once and negative

once in the three months before developing an external cyst, after which he consistently

tested positive. This may indicate a process in which the host attempted to mount an

immune response and was fleetingly able to control the infection before succumbing.

Early stages of infection may also release antigens less reliably, which would make early

infection difficult to detect. Future work that combines frequent longitudinal urine

sampling from known individuals while monitoring for external signs of disease is

needed to better understand the frequency and health consequences of transient T.

serialis infections.

The higher occurrence of cysts among older individuals is consistent with

previous studies on T. serialis cyst prevalence in geladas (Nguyen et al . 2015, Schneider-

Crease et al . in review), whereas the lack of support for a strong relationship between age and antigen-positive samples was unexpected. Together, these results suggest that susceptibility to infection does not vary strongly with age, but that cysts may take years to develop to a stage at which they protrude and are visible to observers. Contrary to our predictions based on the increased female susceptibility observed in other larval taeniid systems (Terrazas et al . 1998, Morales et al . 2002, Escobedo et al. 2004, Morales-

Montor et al . 2004, Morales-Montor & Larralde 2005, Pena et al . 2007) or the female-bias in data collection, we found no evidence for a sex bias in either T. serialis cysts or antigen

positivity in samples. The lack of support for increased susceptibility with age or sex

suggest that susceptibility to T. serialis in geladas may not be hormonally modulated, but

further work is needed to elaborate the mechanisms of susceptibility in this system.

The use of dried urine for larval Taenia infection diagnosis provides the

substantial benefits of not requiring refrigeration or invasive procedures; thus, this

method is well suited to the identification of Taenia infections in wildlife inhabiting

remote areas. However, this approach also has one notable drawback. Mainly, this assay

is genus-specific, not species-specific, and will pick up antigens from any Taenia species.

Thus, other methods must be used for species-level identification. If possible, tissue from

an infected individual can be used (from a dead individual, as in Schneider-Crease et al .

(2013), or from leaked cystic material, as in Nguyen et al . (2013)). Non-lethal traps may be used in studies of smaller species, and fecal analysis of carnivore hosts sympatric

with the target intermediate host species may used to determine which taeniid species

are present in a given area. Future researchers that use dried urine samples for Taenia

antigen detection should consider outlining the saturated area of each filter paper to

facilitate urine retrieval from samples on filter paper.

In conclusion, the global distribution and flexibility in intermediate host selection

of many taeniid species makes them critically important to monitor for global human

and animal health. However, the sylvatic life cycles of taeniids remain largely unknown

because diagnosing larval taeniid infection typically requires obtaining sera or

performing somatic imaging. Such invasive procedures are often prohibited or

otherwise unfeasible in protected species and remote settings. Thus, the adaptation of a

serum protocol for the detection of Taenia infections for use with dried urine samples is a useful and pioneering step towards a complete understanding of the dynamics of Taenia infection in wildlife. Although this assay cannot be used as a standalone diagnostic technique, particularly given its genus-wide specificity, it holds great value for studies of infection dynamics in host populations for which regular invasive monitoring is impractical and in areas where sample storage prohibits the collection of wet urine samples.

4.5 Acknowledgements

Long-term gelada research was supported by the National Science Foundation

(IOS-1255974, BCS-0715179), the Leakey Foundation, and the National Geographic

Society (Gr. #8989-11, Gr. #8100-06). Support for project-specific fieldwork and

laboratory analysis was provided by Primate Conservation, Inc., Conservation

International , the Nacey Maggioncalda Foundation , the Margot Marsh Biodiversity

Foundation . We thank the Ethiopian Wildlife Conservation Authority, the Amhara

National Regional Parks State Development and Protection Authority, and all park

officials who have permitted and aided our research. We thank all past and present field crew of the University of Michigan Gelada Research Project for their dedication to over a decade of data collection. We thank Dr. Siddhartha Mahanty (NIH) for the T. crassiceps antigen extract . We thank LJ Digby, CM Drea, TT Struhsaker, RL Reinhardt, and AA

Tositi for helpful feedback throughout this project. IASC and RHG acknowledge support from the National Science Foundation’s Graduate Research Fellowship

Program. All research was approved by the University Committee on Use and Care of

Animals at the University of Michigan (UCUCA protocol #09554), the Duke University

Institutional Animal Care and Use Committee (IACUC protocol #A218-13-08, and followed all laws and guidelines in Ethiopia.

5. No evidence for deandrogenization of male geladas (Theropithecus gelada ) naturally infected with Taenia serialis metacestodes. 5.1 Introduction

Males are generally considered to be the “sicker” of the two sexes, exhibiting

increased susceptibility to parasite infection and disease across many taxa (Poulin 1996,

Schalk & Forbes 1997, Muehlenbein & Bribiescas 2005, Zuk 2009, Cordoba-Aguilar &

Munguia-Steyer 2013, Guerra-Silveira & Abad-Franch 2013). Ultimate explanations for

this pattern point to selection on males for increased investment in mating effort relative

to immunity, whereas proximate explanations revolve around endocrinological or

immunological mechanisms that lead to increased male susceptibility (Zuk & McKean

1996, Zuk 2009, Cordoba-Aguilar & Munguia-Steyer 2013). A prominent proximate

mechanism linked to higher male susceptibility to parasitism is androgen-induced

immunosuppression, and higher androgen hormone concentrations have been

associated with diminished immune function and increased parasitism across a wide breadth of taxa (Poulin 1996, Zuk 2009). However, a number of parasite-host

relationships deviate from this pattern, with females exhibiting increased parasitism

with certain parasitic taxa –for example, those in the Taenia genus.

The unusual phenomenon of preferential taeniid parasite establishment in female

hosts (murine T. crassiceps and swine T. solium ) has been attributed to increased larval

taeniid proliferation in estrogen-rich environments and restricted proliferation in

androgen-rich environments. Taeniid larvae, which are the asexually reproducing or

non-sexually reproducing taeniid “metacestode” stage, are armed with sex steroid

receptors that bind to estrogens and androgens (Escobedo et al. 2004, Escobedo et al.

2010b). In T. crassiceps , ovarian hormones (17 b-estradiol, E2, and progesterone, P4,

stimulate larval proliferation upon binding to sex steroid receptors on the metacestode

(Escobedo et al. 2004, Escobedo et al. 2010b), whereas androgens (testosterone, T, and

dihydrotestosterone, DHT) inhibit it upon binding (Vargas-Villavicencio et al. 2005,

Ibarra-Coronado et al. 2011). Indeed, larvae treated in vitro with E 2 and P 4 exhibit enhanced growth as compared to those treated with T and DHT (Escobedo et al. 2004,

Escobedo et al. 2010a, Ambrosio et al. 2015). This differential growth is due to the activity of AP-1 complex genes (c-Fos and c-jun ), which underlie processes of cell proliferation and thus are important players in larval asexual reproduction (Morales-Montor et al.

1998, Escobedo et al. 2004). Estrogens increase c-Fos and c-jun activity, leading to increased larval cell proliferation and thus increased larval growth, whereas androgens decrease larval cell activity, leading to cell apoptosis and thus inhibited larval growth

(Escobedo et al. 2004). Thus, sex steroid hormones mediate the female-biased establishment of taeniid larvae in hosts.

Perhaps because of their increased growth in estrogen-rich environments, some

larval taeniids are able to upregulate estrogen secretion and downregulate androgen

secretion in their hosts. This process, known as parasite-driven deandrogenization

(Terrazas et al. 1998, Morales-Montor & Larralde 2005), works by increasing the

synthesis of aromatase (enzyme aromatase cytochrome P-450), an enzyme that catalyzes

the conversion of T to E 2 (aromatization) (Simpson et al. 1994, Morales-Montor et al.

1999, Morales-Montor et al. 2001). Taeniid infection stimulates the production of substances critical to the induction and activation of P-450 aromatase in hosts: follicle stimulating hormone (FSH) and the cytokine IL-6 (Nakai et al. 1988, Spangelo et al. 1989,

Morales-Montor & Larralde 2005). For example, male mice and swine infected with T. crassiceps and T. solium, respectively, exhibited increased FSH and IL-6 production, higher aromatase activity, higher estradiol concentrations, lower testosterone concentrations, and increased larval growth (while treatment with aromatase inhibitors blocked this process) (Larralde et al. 1995, Morales et al. 1996, Morales-Montor et al.

1999, 2001, Gourbal et al . 2002, Morales-Montor et al. 2002, Vargas-Villavicencio et al.

2006, Pena et al. 2007). Thus, parasite-driven deandrogenization may be an adaptive manipulation by the parasite that permits taeniid larvae to optimize proliferation in male hosts.

The larval stage of T. serialis , a taeniid closely related to T. crassiceps , infects herbivores ranging from rodents and lagomorphs to primates. Geladas ( Theropithecus

gelada ), which are primates endemic to the Ethiopian highlands and the only known

natural primate hosts for a larval taeniid species, exhibit protuberant cysts indicative of

advanced T. serialis infection (Schneider-Crease et al. 2013, Nguyen et al. 2013). These

cysts, which appear in ~4.8-30% of geladas, are associated with decreased survival for

infected individuals as well as for offspring of infected mothers (Nguyen et al. 2015,

Schneider-Crease et al. in review). Interestingly, no sex differences were found in the

prevalence of T. serialis cysts (Nguyen et al. 2015) or T. serialis antigen presence in urine

(Schneider-Crease et al. submitted) in two gelada populations. However, sex differences

in mortality were found (see Chapter 2), with cyst-associated mortality observed only in

females. Because T. serialis appears to establish equally well in male and female geladas,

we hypothesize that T. serialis may deandrogenize its male gelada hosts to facilitate its

success. Alternately, the presence of a sex difference in mortality but not in prevalence

suggests that T. serialis may establish equally well in male and female hosts, but may fail

to deandrogenize male hosts and may thus be less successful in those hosts. Here, we

test these hypotheses using a decade of data on T. serialis cyst emergence and fecal

testosterone metabolite concentrations in a gelada population inhabiting the Simien

Mountains National Park (SMNP), Ethiopia.

100

5.2 Methods

5.2.1 Study subjects and sample collection

We studied a population of habituated geladas (~300 individuals) in the SMNP

(13° 11'N, 38° 04'E), which covers 22,000 hectares of high-altitude Afromontane and

Afroalpine habitat in the Amhara region of Ethiopia (Asrat et al. 2012). Geladas are

herbivorous and terrestrial cercopithecine primates endemic to the Ethiopian highlands

(Dunbar and Dunbar 1975; Dunbar 1980; Iwamoto 1993, Dunbar 1984). The gelada social

system is multi-tiered; multiple one-male, multi-female reproductive units (OMUs)

aggregate to form “foraging bands” (Dunbar and Dunbar 1975, Snyder-Mackler et al.

2012b). Females within OMUs are related, and males disperse upon sexual maturity to join roving all-male groups as “bachelors”. Adult, non-natal bachelor males compete for

the ‘one-male’ position (“leader” of an OMU, Dunbar and Dunbar 1975, Snyder-Mackler

et al. 2012a, b). When leader males are “overthrown” by other adult males, they often

remain in the OMU as “follower” males and occasionally sire offspring (Snyder-Mackler

et al. 2012a). Thus, OMUs can contain up to three males; however, there is one clear

“leader” male that sires most of the offspring during his tenure as leader (Snyder-

Mackler et al. 2010, 2012).

Since 2006, researchers with the University of Michigan Gelada Research Project

(UMGRP) have collected data on the study population. These data include male status

101

changes, the emergence of visible T. serialis cysts, and fecal testosterone (T) and

glucocorticoid (GC) metabolite concentrations. Geladas are individually identifiable based on idiosyncratic suites of morphological traits, and ages are assigned based on

known dates of birth or estimated based on validated maturational milestones and age-

specific traits (Barale et al. 2015, Beehner et al. 2015, Roberts et al. 2017). Taenia serialis

cysts were recorded upon their first visible emergence, fecal samples from known males

in study groups were collected on a rotating schedule from 2006-2015, and assays to

measure hormone concentrations were performed according to Beehner et al. (2009) and

Pappano & Beehner (2015). Our sample set included 2400 fecal samples from 87 males

(including 218 samples from 14 males with cysts). Because males often switched between “leader” and “not-leader” status, this dataset included samples from 58 leaders

(n=1523 samples) and 54 non-leaders (n=877 samples). Seven leaders with cysts were

included (n=52 samples), as were 14 non-leaders with cysts (n=166 samples).

In brief, hormone extraction was conducted between 6-8 hours of collection at the

field site, and samples were transported to the University of Michigan Core Assay

Facility for hormone metabolite analysis. Samples were analyzed for T using two

different modified commercially available T RIA kits (Diagnostics Systems Laboratories;

Pantex) validated for use in geladas by Beehner et al. (2009) and Pappano & Beehner

2015. A subset of samples was run with both T antibodies (n=84 samples) to obtain a

102

correction factor that was then applied to all T values of the smaller dataset (note that

despite this correction factor, we still control for methods-based differences in our

statistical analyses). Samples were assayed for GCs with a modified commercially

available corticosterone RIA kit (MP Biomedicals) validated for use in geladas by

Beehner & McCann (2008). All hormone metabolite concentrations are expressed in ng/g.

5.2.2 Statistical analysis

We modeled log-transformed fecal T metabolites (fTM) as a function of the

presence of T. serialis cysts and other potential predictors of T: age, dominance status, log-transformed fecal GC metabolites, rainfall, temperature (fixed effects) and individual

ID (random effect). This analysis was performed using generalized linear mixed models

(GLMMs) with model selection and model averaging in the statistical platform R (R

Core Team 2016), using the ‘lme4’ and ‘MuMIn’ packages (Barton 2013, Bates et al. 2014).

We performed model selection using Akaike’s information criterion (Akaike 1998, 2011) with a correction for small sample size (AICc) (Hurvich & Tsai 1989, Burnham et al.

2011), which ranks models based on maximum likelihood (Akaike 1998, 2011). We averaged all models within two AICc points of the most likely model (Burnham &

Anderson 2002, Grueber et al. 2011).

We used a standardized measurement of log fTM as the outcome variable in our

103

models. Because two different T antibodies were used across the study period, standardizing these data allowed us to control for variation stemming purely from methods-based differences. We included two weather variables shown to be important predictors of fTM in a previous study (Pappano & Beehner 2015): rainfall and temperature. Rainfall was coded as the median amount of rainfall for the 30 days preceding sample collection, which functions as a proxy for food availability (with increased rainfall associated with increased food availability) and thus energy balance for geladas (Pappano & Beehner 2015). Temperature was coded as the midpoint (in C°) for the day of sample collection to reflect the average temperature, which is known to affect GCs in geladas (Beehner & McCann 2008). We also included the dominance status of the individual on the day of collection (coded as ‘leader’ or ‘not leader’ because a preliminary analysis revealed that only leaders differed significantly in their fTMs), the cyst status of the individual on the day of collection (i.e., whether the individual exhibited a visible T. serialis cyst on the day of collection), and fecal glucocorticoid metabolite (fGCM) concentrations. Log fGCMs were also standardized to control for methods-based variation. Because multiple samples were taken from each individual

(mean of 27 per individual, range of 1-86), we included individual ID as a random effect.

104

5.3 Results

Counter to expectations, our analysis revealed that the presence of a T. serialis cyst was not a strong predictor of decreased fTMs. Individual-level traits, including age, dominance status, fGCMs, and an ecological variable (rainfall) were the strongest predictors of fTMs (top model set presented in Table 7). Higher dominance status, fGCMs, and rainfall were all associated with increased fTM concentrations (full model averages presented in Table 8). The ‘cyst’ variable appeared in the least-supported of the top models, and full model averaging revealed weak associations between cyst presence and fTMs among subordinate males (slight increase) and dominant males (slight decrease) (Table 8).

105

Intercept Age Leader Rain fGCM Cyst Cyst: Leader AICc Δ weight

Model 1 -0.07 -0.04 0.17 0.04 0.37 NA NA 5644.66 0 0.47 2 -0.09 NA 0.23 0.04 0.37 NA NA 5645.49 0.83 0.31 3 -0.12 -0.04 0.22 0.04 0.37 0.24 -0.32 5646.1 1.44 0.23

1: Z_t ~ Age + Leader + Rain+ Z_gc + (1 | Name) 106 2: Z_t ~ Leader + Rain + Z_gc + (1 | Name) 3: Z_t ~ Age + Cyst + Leader + Rain + Z_gc + Cyst:Leader + (1 | Name)

Table 8: Full model-averaged parameter estimates, adjusted standard errors (SE), z-scores, and probability estimates (Pr(|>z|) are presented for all variables that appeared in models within two AICc points of the top model. Statistical significance is indicated by an asterisk (*).

Variable Estimate SE (adj) z-value Pr(|>z|) Intercept -0.09 0.05 1.58 0.11 Age -0.03 0.02 1.30 0.19

Leader 0.20 0.06 3.4 <0.01 * 107 Rainfall 0.04 <0.01 12. 89 < 2e -16 * fGCM 0.37 0.02 22.96 < 2e -16 * Cyst 0.05 0.11 0.49 0.62 Cyst:Leader -0.07 0.15 0.48 0.63

5.4 Discussion

Our results did not provide adequate support for the hypothesis that T. serialis

induces deandrogenization in gelada males, at least in the case of advanced infection

indicated by protuberant cysts. In the least-supported of the top models, cysts were

weakly associated with variation in fTMs, with the effect differing depending on the

dominance status of the individual. Cysts were associated with decreased fTMs for

dominant males and with increased fTMs for non-dominant males (followers and bachelors). Possible explanations for the divergent effect of cysts on dominant and

subordinate males should be further explored; however, we wish to emphasize that

there was only thin support for any effect of cysts regardless of status. This weak

support for the deandrogenizing effect of T. serialis on gelada males is surprising given the robust in situ and in vitro evidence for deandrogenization during infection with two taeniid species closely related to T. serialis (T. crassiceps in mice and T. solium in swine).

We found that increased fGCMs were strongly associated with increased fTMs.

This positive association, which runs counter to the general understanding that glucocorticoids inhibit testosterone production (Dixson 1998), may be mediated by seasonal variables. Rainfall and temperature were previously shown to be important predictors of testosterone and glucocorticoids in geladas, with cold temperatures associated with increased fGCMs and increased rainfall associated with increased fTM

(Beehner & McCann 2008, Pappano & Beehner 2015). Thus, the positive association

108

between fTMs and fGCMs presented here may reflect the effects of seasonal variables on both hormones. Alternatively, the dry season (low rainfall, higher temperatures) may be

a period of energy conservation for geladas, leading to restrictions in hormone

production in time periods characterized by low rainfall and high temperatures.

Another possible explanation for the positive association between fTMs and fGCMs is

that the metabolic demands of male reproductive behavior require mobilization of

energetic resources via increases in glucocorticoids (Sapolsky 1993). Indeed, in systems

with metabolically costly male reproductive behavior, males can exhibit high

concentrations of both testosterone and glucocorticoids (McDonald et al. 1986, Emerson

& Hess 2001, Bercovitch & Ziegler 2002, Lynch et al. 2002, Gesquire et al. 2011). Gelada male dominance is contingent upon physical displays and challenges that are likely mediated by testosterone (Pappano & Beehner 2015), which may explain the positive association between fTMs and fGCMs.

Although T. serialis does not appear to manipulate the hormonal profiles of its hosts, two major caveats are worth considering. First, the sample size of males with cysts was small (n=14), which weakens the power of our analyses to detect differences related to cysts. Second, the presence of external cysts may be an insufficient measure of T. serialis infection. Recent research has identified individuals that are positive for T. serialis antigen (i.e., active infection) yet do not exhibit observable protuberant cysts, suggesting that cysts may grow internally without ever becoming visible to observers (Schneider-

109

Crease et al. submitted). For example, 2.4% of 168 males exhibited visible T. serialis cysts, whereas 9.5% of the same males exhibited antigen-positive urine samples. Thus, it is possible that nearly four times as many males that presented with cysts harbored T. serialis infections, which would obscure significant differences between males.

Future analyses should compare fTMs of individuals that test positive for Taenia antigen to those of individuals that test negative for infection and should track variation in males over the course of infection (including pre-infection). The development of an assay to detect estrogen concentrations in males would further deepen the investigation into deandrogenization in geladas. Similarly, future analyses should evaluate the impact of T. serialis on females; if T. serialis does indeed deandrogenize its hosts, infected females should exhibit higher estrogen concentrations than uninfected. Finally, in vitro studies should describe and compare the cytokine profiles and aromatase activity of infected and uninfected individuals. These analyses will enhance the understanding of the T. serialis -gelada relationship and provide insight into how this relationship compares to those of closely related taeniid species.

110

6. Conclusion 6.1. Overview

In this dissertation, I investigated the predictors, costs, and consequences of a tapeworm parasite on the survival, reproductive success, and physiology of geladas. At the onset of this investigation, I used molecular tools to ascertain the identity of the parasite behind the protuberant masses observed for decades in both captive and wild geladas as the larval stage of the tapeworm Taenia serialis . Using long-term data on a wild gelada population in the Simien Mountains National Park (SMNP), Ethiopia, I illustrated that T. serialis cysts decrease the reproductive success and survival of female geladas. I then adapted a non-invasive assay to detect T. serialis infection in non- invasively collected urine samples. Using this diagnostic tool, I showed that T. serialis infections do not always present as protuberant cysts visible to observers, and that certain individuals may be able to combat initial infection. Finally, I demonstrated that

T. serialis deviates from the taeniid pattern of deandrogenizing its male hosts and has little effect on the testosterone concentrations of its male hosts. Here, I review the overarching themes of the dissertation.

6.2 Taenia serialis: identification, diagnosis, and host diversity

The work presented in this dissertation resolved nearly a century of uncertainty surrounding the identity of the parasite behind the protuberant masses observed in

111

geladas with the use of molecular diagnostic tools, which further deepened the current

understanding of the T. serialis life cycle. The history of taeniid identification is marked by disagreements and confusion, with species distinctions drawn on the now-rejected basis of morphological traits, global occurrence, infection site predilection, or host

choice. The implementation of genetic tools for species identification in this study

permitted the definitive diagnosis of this parasite as T. serialis , and opened the door for a

revision of commonly accepted tenets surrounding the host breadth and zoonotic

potential of this parasite by confirming that intermediate host species identity of T.

serialis is not constrained to rodents and lagomorphs.

The synthesis of research on T. serialis demonstrated that the general understanding of T. serialis host diversity and estimation of its zoonotic potential are

insufficient. This insufficiency is largely due to the fragmented record of T. serialis in

wildlife (contained mostly in case reports that, until now, have never been synthesized).

The morphological similarity between the larval stages of T. serialis and T. multiceps ,

paired with the historic lack of genetic tools available for species identification, have also

contributed to the poor understanding of T. serialis host breadth, global occurrence, and

zoonotic potential. The regular occurrence of T. serialis coenurosis in geladas, as

indicated by the detection of Taenia antigen in gelada urine, suggests that the life cycle of

T. serialis may be more flexible and more widespread than previously thought. These

findings call for a revision to our knowledge of the T. serialis life cycle, with implications

112

for the understanding of taeniid evolution and ecology. The review and revision of T.

serialis ecology provided here furthermore suggests that the zoonotic potential of this

parasite may be underestimated, with T. serialis posing a greater threat to humans than

previously appreciated because of its ability to infect primates.

6.3 Parasitism as a potential selective pressure

The inability of many descriptive studies, both in primates and, more generally

in mammals, to demonstrate fitness effects of naturally occurring parasites on their hosts

in the wild is due both to logistical and biological obstacles. Logistically, evaluating the

effects of parasites on host fitness non-experimentally is challenging because extensive

lifetime data are needed for such endeavors. Ecologically, parasite-host coevolution may

attenuate the detrimental effects of parasites over time, making effects subtler and more

difficult to detect in the wild. However, certain parasites with complex life cycles that

require the ingestion of one host to complete a single life cycle should be under selection

to increase their virulence in the host that must die for life cycle completion (the

“intermediate host”).

In support of this perspective that virulence is important in considerations of

host fitness effects, larval T. serialis significantly decreases the survival of its gelada intermediate hosts. I argue that virulence, manifested as increased morbidity, is adaptive for T. serialis because it facilitates the transmission of the parasite to its final

113

(“definitive”) host during predation or scavenging. Larval T. serialis cysts significantly reduce the reproductive success of affected mothers, but this effect is entirely driven by maternal mortality in the SMNP. Thus, I argue that the effect of T. serialis infection on reproductive success in the SMNP gelada population is a byproduct of the primary effects on survival, and is not adaptive for the parasite itself. Thus, increased virulence can have consequences for hosts that are both adaptive and not adaptive for the parasite.

However, a prior study of T. serialis in geladas inhabiting a more pristine environment found a direct effect of maternal T. serialis cysts on offspring mortality, with offspring of females with cysts significantly more likely to die even when offspring that died along with their mothers were excluded. This divergence suggests that different mechanisms may be at play in the two populations.

The complexities of parasite-host relationships go beyond the fairly straightforward effects on host fitness. Parasite-induced behavioral changes in hosts are considered to be adaptive for a parasite when they increase its reproductive success

(Poulin 2010). For parasites that thrive in estrogen-rich environments and atrophy among androgen-rich environments, such as the larval stages of some taeniids, adaptations involve parasitic manipulation of the host hormonal phenotype to favor estrogens and disfavor androgens (i.e., deandrogenization). Both T. solium and T.

crassiceps have been shown to do precisely that; larval infections are associated with striking decreases in male testosterone concentrations and increases in estradiol in hosts

114

(Larralde et al . 1995, Morales et al . 1996, Morales-Montor et al . 1999, 2001, Gourbal et al.

2002, Morales-Montor et al . 2002, Vargas-Villavicencio et al . 2007). However, the analyses

I present failed to provide evidence for deandrogenization in geladas infected with the

larval stage of T. serialis . This potentially interesting deviation from the expected pattern

may indicate that there are differences in estrogen affinities between taeniid species that

may result from the specific evolutionary trajectory of host-parasite pairs. Further

research is needed to elaborate the host and parasite mechanisms that shape the T.

serialis -gelada relationship.

6.4 Limitations and Future Directions

Investigations of disease dynamics in long-lived primate hosts necessarily

depend on the ability to observe and collect data on relevant markers of disease and

fitness components over time. The decade of data that permitted my analysis of the

effects of T. serialis cysts on gelada fitness components, while extensive, reflects only a

portion of the full lives of geladas. Additionally, analyses were shaped by obstacles that

arose from the intersection of data collection protocol and form of the gelada social

system. The University of Michigan Gelada Research Project (UMGRP) collects data on

two focal gelada ‘foraging bands’, which are conglomerates of one-male, multi-female

reproductive units (Dunbar 1984, Snyder-Mackler et al. 2012). Geladas are female philopatric, meaning that males disperse upon sexual maturity (Dunbar 1984). After

115

dispersal, adult males appear for discrete windows of time in reproductive units as

“leader” (dominant) or “follower” (subordinate) males. Evaluating the effects of T.

serialis infection on male reproductive success with these data is thus largely constrained

to inferences based on tenure length in study bands, and is unable to address

reproductive success of males before or after their time in the study bands. Furthermore,

adult males are only visible to data collection when they have successfully overtaken

dominance from a previous male; otherwise, they are not followed. Thus, if T. serialis

infection prevents males from becoming dominant (as T. crassiceps infection does in male mice, Gourbal et al. 2002), data collection is biased against observing infected males.

Such a bias would diminish our ability to investigate sex-driven effects or consequences of T. serialis infection. Additionally, the moment of death is rarely observed, and deaths are nearly invariably inferred from disappearances. While such inferences are done carefully and are based in years of observer experience, these caveats must be taken into account in interpretations. Advances in data collection techniques, such as the addition of GPS collars and additional observers for bachelor groups, will facilitate the collection of fine-grained data on males throughout their lives.

Gelada susceptibility to T. serialis appears to be shaped by factors other than sex and age. One potential such driver, suitable for future research, is the early life experience. Adversity during early life may impact the immunological composition and thus strength of the immune response of individuals (Repetti et al. 2002, Miller & Chen

116

2010, Shonkoff et al. 2012, Tung et al. 2016). Geladas exhibit high infanticide rates as a

result of a complex social structure in which males compete to lead stable groups of

related females (the ‘reproductive unit’) (Dunbar 1984, Snyder-Mackler et al. 2012a).

Because leader males father most of the offspring in their units (Snyder-Mackler et al.

2012b), a successful challenger male benefits from killing dependent offspring fathered by the former leader by accelerating the pace at which unit females return to receptivity

(Beehner & Bergman 2008). Even more frequent than infanticide itself are infanticide

attempts, in which new leaders attack offspring after takeovers. Thus, gelada infants are

regularly exposed to early life adversity during a crucial developmental window. Paired

with the ongoing longitudinal data collection on geladas in the SMNP, this system

provides the opportunity to investigate the long-term effects of early life adversity on

metrics of susceptibility.

Importantly, geladas are infected with two types of pathogens that each recruits

a disparate immune response. Infections with gastrointestinal nematode parasites are

prevalent, with strongyle-type eggs (family Strongylida ) found in 99% of sampled

individuals in a preliminary survey (unpub. data, n=100). This type of parasite is

typically met with antibody activity (adaptive immunity) (Finkelman et al. 1997, Gause

et al. 2003), which is a highly specific response that becomes more effective over time. By

contrast, T. serialis infection triggers generalized immune cell activity (innate immunity)

to combat initial infections (Terrazas et al. 1998, Toenjas et al. 1999). Thus, geladas are

117

infected with two sets of pathogens that each trigger a different set of immune

mechanisms with disparate costs. This provides a natural experiment in which to

evaluate how individuals can cope with early life adversity by prioritizing different

components of the immune response, and how early life adversity shapes susceptibility

to infection and disease.

6.5 Concluding Remarks

In this thesis, I investigate the predictors, costs, and consequences of a larval

tapeworm with a complex life cycle on its gelada hosts. I offer a new perspective of the

host breadth and zoonotic potential of T. serialis , postulating that the generally accepted understanding of the T. serialis metacestode as a parasite of rodents and lagomorphs

with little zoonotic potential is incorrect. The new perspective of the T. serialis

metacestode as a parasite with great host flexibility and zoonotic potential has

implications for public health policies and global health research. I extend the use of a

non-invasive antigen ELISA for Taenia diagnosis with its first implementation in wild

primates, opening the door for its use in wildlife systems. My analysis of the impact of T.

serialis cysts on male geladas did not detect a deandrogenizing effect of cysts, which

deviates from the pattern exhibited by T. crassiceps and T. solium . This calls for further

research that incorporates additional avenues of evidence to establish whether T. serialis

truly diverges from its sister taxa in stimulating deandrogenization in its hosts. I present

118

evidence that parasitism by T. serialis is a major driver of gelada mortality, providing

one of the first explicit demonstrations of mortality driven by an endemic helminth

parasite in a wild primate population. Together, these results emphasize the importance

of considering parasite transmission mode and life cycle in investigations of the effects

of parasitism on wild populations.

References

Abuladze, K.I. (1964). Taeniata of animals and man and diseases caused by them. In K. I. Skrjabin (Ed.), Essentials of Cestodology (Vol. 4). Akademiia Nauk SSSR, Gel’mintologicheskaia Laboratoriia, Izdatel’stvo Nauka, Moskva, (English translation, Israel Program for Scientific Translations, Jerusalem, 1970).

Agnew, P., Koella, J. C., & Michalakis, Y. (2000). Host life history responses to parasitism. Microbes and Infection , 2(8), 891-896.

Akaike, H. (1998). Information theory and an extension of the maximum likelihood principle. In Selected Papers of Hirotugu Akaike (pp. 199-213). Springer: New York.

Akaike, H. (2011). Akaike’s information criterion. In M. Lovric (Ed.), International Encyclopedia of Statistical Science (pp. 25). Springer: Berlin.

Albon, S. D., Stien, A., Irvine, R. J., Langvatn, R., Ropstad, E., & Halvorsen, O. (2002). The role of parasites in the dynamics of a reindeer population. Proceedings of the Royal Society of London B: Biological Sciences, 269(1500), 1625-1632.

Alizon, S., de Roode, J. C., & Michalakis, Y. (2013). Multiple infections and the evolution of virulence. Ecology Letters , 16 (4), 556-567.

Allepuz, A., Gabriël, S., Dorny, P., Napp, S., Jansen, F., Vilar, M. J., ... & Casal, J. (2012). Comparison of bovine cysticercosis prevalence detected by antigen ELISA and visual inspection in the North East of Spain. Research in Veterinary Science, 92 (3), 393-395.

119

Ambrosio, J. R., Valverde-Islas, L., Nava-Castro, K. E., Palacios-Arreola, M. I., Ostoa- Saloma, P., Reynoso-Ducoing, O., ... & Morales-Montor, J. (2015). Androgens exert a cysticidal effect upon Taenia crassiceps by disrupting flame cell morphology and function. PloS One, 10 (6), e0127928.

Anderson, R. M., & May, R. M. (1978). Regulation and stability of host-parasite population interactions: I. Regulatory processes. The Journal of Animal Ecology , 219-247.

Anderson, R. M., & May, R. M. (1979). Population biology of infectious diseases: part I. Nature, 280 (5721), 361–367.

Anderson, R. M., & May, R. M. (1982). Coevolution of hosts and parasites. Parasitology, 85 (02), 411-426.

Anderson, R. M., May, R. M., & Anderson, B. (1992). Infectious Diseases of Humans: Dynamics and Control (Vol. 28). Oxford University Press: Oxford.

Arneberg, P., Folstad, I., & Karter, A. J. (1996). Gastrointestinal nematodes depress food intake in naturally infected reindeer. Parasitology , 112 (02), 213-219.

Arteaga-Silva, M., Vargas-Villavicencio, J. A., Vigueras-Villaseñor, R. M., Rodríguez- Dorantes, M., & Morales-Montor, J. (2009). Taenia crassiceps infection disrupts estrous cycle and reproductive behavior in BALB/c female mice. Acta Tropica, 109 (2), 141-145.

Ashenafi, Z. T., Leader-Williams, N., & Coulson, T. (2012). Consequences of human land use for an Afro-alpine ecological community in Ethiopia. Conservation and Society, 10 (3), 209-216.

Ashley, N. T., Weil, Z. M., & Nelson, R. J. (2012). Inflammation: mechanisms, costs, and natural variation. Annual Review of Ecology, Evolution, and Systematics, 43, 385-406.

Asrat, A., Demissie, M., & Mogessie, A. (2012). Geoheritage conservation in Ethiopia: the case of the Simien Mountains. Quaestiones Geographicae, 31 (1), 7-23.

Auld, S. K., & Tinsley, M. C. (2015). The evolutionary ecology of complex lifecycle parasites: linking phenomena with mechanisms. Heredity , 114 (2), 125-132.

120

Avcioglu, H., Yildirim, A., Duzlu, O., Inci, A., Terim, K. K., & Balkaya, I. (2011). Prevalence and molecular characterization of bovine coenurosis from Eastern Anatolian region of Turkey. Veterinary Parasitology, 176 (1), 59-64.

Ballesteros, M., Bårdsen, B. J., Langeland, K., Fauchald, P., Stien, A., & Tveraa, T. (2012). The effect of warble flies on reindeer fitness: a parasite removal experiment. Journal of Zoology, 287 (1), 34-40.

Barale, C. L., Rubenstein, D. I., & Beehner, J. C. (2015). Juvenile social relationships reflect adult patterns of behavior in wild geladas. American Journal of Primatology, 77 (10), 1086-1096.

Bartoń, K. (2009). MuMIn: Multi-model inference. R package, version 0.12.2. http://r- forge.r-project.org/projects/mumin/ .

Bates, D., Mächler, M., Bolker, B., & Walker, S. (2014). Fitting linear mixed-effects models using lme4. arXiv preprint arXiv :1406.5823.

Becker, B. J. P., & Jacobson, S. (1951). Infestation of the human brain with Coenurus cerebralis : report of a fourth case. The Lancet, 258 (6696), 1202-1204.

Beehner, J. C., Gesquiere, L., Seyfarth, R. M., Cheney, D. L., Alberts, S. C., & Altmann, J. (2009). Testosterone related to age and life-history stages in male baboons and geladas. Hormones and Behavior, 56 (4), 472-480.

Beehner, J. C., Gesquiere, L., Seyfarth, R. M., Cheney, D. L., Alberts, S. C., & Altmann, J. (2015). Corrigendum to “Testosterone related to age and life-history stages in male baboons and geladas" [Hormones and Behavior 56(4), 472-480, (2009)]. Hormones and Behavior , 80 , 149.

Beehner, J. C., & McCann, C. (2008). Seasonal and altitudinal effects on glucocorticoid metabolites in a wild primate ( Theropithecus gelada ). Physiology & Behavior, 95 (3), 508-514.

Benger, A., Rennie, R. P., Roberts, J. T., Thornley, J. H., & Scholten, T. (1981). A human coenurus infection in Canada. The American Journal of Tropical Medicine and Hygiene, 30 (3), 638-644.

Benifla, M., Barrelly, R., Shelef, I., El-On, J., Cohen, A., & Cagnano, E. (2007). Huge

121

hemispheric intraparenchymal cyst caused by Taenia multiceps in a child: case report. Journal of Neurosurgery: Pediatrics , 107 (6), 511-514.

Bennett, H. (2000). Coenurus cyst in a pet rabbit. The Veterinary Record, 147 (15), 428.

Bercovitch, F. B., & Ziegler, T. E. (2002). Current topics in primate socioendocrinology. Annual Review of Anthropology, 31 (1), 45-67.

Berdoy, M., Webster, J. P., & Macdonald, D. W. (2000). Fatal attraction in rats infected with Toxoplasma gondii . Proceedings of the Royal Society of London B: Biological Sciences , 267 (1452), 1591-1594.

Bermejo, M., Rodríguez-Teijeiro, J. D., Illera, G., Barroso, A., Vilà, C., & Walsh, P. D. (2006). Ebola outbreak killed 5000 gorillas. Science, 314 (5805), 1564.

Bertolino, P. (1957). Studio clinico ed anatomo-patologico su due casi di cenurosi in Theropithecus gelada . Profilassi, 30 , 3-10.

Bertrand, I., Callot, J., Terrasse, J., Janny, P., & Perol, E. (1956). A new case of cerebral coenurosis. La Presse Médicale , 64 (15), 333.

Beveridge, I., & Rickard, M. D. (1975). The development of Taenia pisiformis in various definitive host species. International Journal for Parasitology, 5 (6), 633-639.

Blackwell, A. D., Tamayo, M. A., Beheim, B., Trumble, B. C., Stieglitz, J., Hooper, P. L., ... & Gurven, M. (2015). Helminth infection, fecundity, and age of first pregnancy in women. Science, 350 (6263), 970-972.

Bonnal, G., Joyeux, C., & Bosch, P. (1933). Coenurosis in man due to Multiceps serialis (Gervais). Bulletin de la Société de Pathologie Exotique, 26 (8), 1060-1071.

Booth, D. T., Clayton, D. H., & Block, B. A. (1993). Experimental demonstration of the energetic cost of parasitism in free-ranging hosts. Proceedings of the Royal Society of London B: Biological Sciences, 253 (1337), 125-129.

Boots, M., & Sasaki, A. (1999). ‘Small worlds’ and the evolution of virulence: infection occurs locally and at a distance. Proceedings of the Royal Society of London B: Biological Sciences , 266 (1432), 1933-1938.

122

Boots, M., Best, A., Miller, M. R., & White, A. (2009). The role of ecological feedbacks in the evolution of host defence: what does theory tell us? Philosophical Transactions of the Royal Society of London B: Biological Sciences, 364 (1513), 27-36.

Bowman, D. D. (2009). Georgis' Parasitology for Veterinarians . Elsevier Health Sciences.

Bracken, F. K., & Olsen, O. W. (1950). Coenurosis in the chinchilla. Journal of the American Veterinary Medical Association, 116 (879), 440-443.

Brandt, J. R. A., Geerts, S., De Deken, R., Kumar, V., Ceulemans, F., Brijs, L., & Falla, N. (1992). A monoclonal antibody-based ELISA for the detection of circulating excretory-secretory antigens in Taenia saginata cysticercosis. International Journal for Parasitology, 22 (4), 471-477.

Brooks, D. R., & Glen, D. R. (1982). Pinworms and primates: a case study in coevolution. Proceedings-Helminthological Society of Washington, 49 (1), 76-S5.

Brown, C. R., Brown, M. B., & Rannala, B. (1995). Ectoparasites reduce long-term survival of their avian host. Proceedings of the Royal Society of London B: Biological Sciences , 262 (1365), 313-319.

Brumpt, E. (1936). Précis de parasitologíe , 5th Ed. Masson: Paris.

Budischak, S. A., Jolles, A. E., & Ezenwa, V. O. (2012). Direct and indirect costs of coinfection in the wild: linking gastrointestinal parasite communities, host hematology, and immune function. International Journal for Parasitology: Parasites and Wildlife, 1 , 2-12.

Buckley, J. J. C. (1947). Coenurus from human spinal cord. Transactions of the Royal Society of Tropical Medicine and Hygiene, 41 (1), 7.

Burnham, K. P., & Anderson, D. R. (2003). Model selection and multimodel inference: a practical information-theoretic approach . Springer Science & Business Media.

Burnham, K. P., Anderson, D. R., & Huyvaert, K. P. (2011). AIC model selection and multimodel inference in behavioral ecology: some background, observations, and comparisons. Behavioral Ecology and Sociobiology, 65 (1), 23-35.

123

Caillaud, D., Levréro, F., Cristescu, R., Gatti, S., Dewas, M., Douadi, M., ... & Ménard, N. (2006). Gorilla susceptibility to Ebola virus: the cost of sociality. Current Biology, 16 (13), R489-R491.

Castillo, Y., Rodriguez, S., García, H. H., Brandt, J., Van Hul, A., Silva, M., ... & Gilman, R. H. (2009). Urine antigen detection for the diagnosis of human neurocysticercosis. The American Journal of Tropical Medicine and Hygiene, 80 (3), 379-383.

Chadwick, W., & Little, T. J. (2005). A parasite-mediated life-history shift in Daphnia magna. Proceedings of the Royal Society of London B: Biological Sciences, 272 (1562), 505-509.

Chapman, C. A., & Chapman, L. J. (2000). Determinants of group size in primates: the importance of travel costs. In S. Boinski & P. A. Garber (Eds.), On the move: how and why animals travel in groups (pp. 24-42) . University of Chicago Press: Chicago.

Cheng, T. C. (1991). Is parasitism symbiosis? A definition of terms and the evolution of concepts. In C. A. Toft, A. Aeschilmann, & L. Bolis (Eds.), Parasite-Host Associations, (pp. 15-36). Oxford University Press: Oxford.

Choisy, M., Brown, S. P., Lafferty, K. D., & Thomas, F. (2003). Evolution of trophic transmission in parasites: why add intermediate hosts? The American Naturalist, 162 (2), 172-181.

Clark, J. D. (1969). Coenurosis in a gelada baboon ( Theropithecus gelada ). Journal of the American Veterinary Medical Association, 155 (7), 1258-1263.

Cleaveland, S., Laurenson, M. K., & Taylor, L. H. (2001). Diseases of humans and their domestic mammals: pathogen characteristics, host range and the risk of emergence. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 356 (1411), 991-999.

Clutton-Brock, T. H., & Harvey, P. H. (1977). Primate ecology and social organization. Journal of Zoology, 183 (1), 1-39.

Combes, C. (1991). Ethological aspects of parasite transmission. The American Naturalist, 138 (4), 866-880.

124

Combes, C. (2001). Parasitism: The Ecology and Evolution of Intimate Interactions . University of Chicago Press: Chicago.

Conn, D. B., & Swiderski, Z. (2008). A standardised terminology of the embryonic envelopes and associated developmental stages of tapeworms (Platyhelminthes: Cestoda). Folia Parasitologica, 55 (1), 42.

Coop, R. L., & Kyriazakis, I. (1999). Nutrition–parasite interaction. Veterinary Parasitology, 84 (3), 187-204.

Córdoba-Aguilar, A., & Munguía-Steyer, R. (2013). The sicker sex: understanding male biases in parasitic infection, resource allocation and fitness. PloS One , 8(10), e76246.

Correa, F., Ferriolli Jr., F., Forjaz, S., & Martelli, N. (1962). Cerebral coenurosis: report of a human case. Revista do Instituto de Medicina Tropical de Sao Paulo , 4(1), 38-45.

Côté, I. M., & Poulin, R. (1995). Parasitism and group size in social animals: a meta- analysis. Behavioral Ecology, 6 (2), 159-165.

Cox, D. R. (1972). Regression models and life tables. Journal of the Royal Statistical Society , 34 (2), 187-220.

Craig, P. S., & Macpherson, C. N. L. (2000). Dogs and cestode zoonoses. In C. N. Macpherson, F. X. Mesli, & A. I. Wandeler (Eds.), Dogs, Zoonoses and Public Health (pp. 149-211). CAB International: Oxon, UK.

Craig, P., & Pawłowski, Z. (2002). Cestode zoonoses: echinococcosis and cysticercosis: An emergent and global problem (Vol. 341). IOS Press: Amsterdam.

Cudicini, C., Lejeune, H., Gomez, E., Bosmans, E., Ballet, F., Saez, J., & Jégou, B. (1997). Human Leydig cells and Sertoli cells are producers of interleukins-1 and-6. The Journal of Clinical Endocrinology & Metabolism, 82 (5), 1426-1433.

D’Andrea, F., & Morello, G. (1964). La cenurosi cerebrale. Acta Neurologica , 19 , 245-257.

Darriba, D., Taboada, G. L., Doallo, R., & Posada, D. (2012). jModelTest 2: more models, new heuristics and parallel computing. Nature Methods, 9 (8), 772.

125

Day, T. (2002). Virulence evolution via host exploitation and toxin production in spore-producing pathogens. Ecology Letters, 5 (4), 471-476.

De Lope, F., Møller, A. P., & De la Cruz, C. (1998). Parasitism, immune response and reproductive success in the house martin Delichonurbica . Oecologia, 114 (2), 188- 193.

Debonnet, G., Melamari, L., & Bomhard, B. (2006). Reactive monitoring mission to Simien Mountains National Park Ethiopia. WHC/IUCN Mission Report, UNESCO, Paris and IUCN, Gland .

Delahay, R. J., Speakman, J. R., & Moss, R. (1995). The energetic consequences of parasitism: effects of a developing infection of Trichostrongylus tenuis (Nematoda) on red grouse (Lagopus lagopus scoticus ) energy balance, body weight and condition. Parasitology, 110 (04), 473-482.

Demas, G. E., Chefer, V., Talan, M. I., & Nelson, R. J. (1997). Metabolic costs of mounting an antigen-stimulated immune response in adult and aged C57BL/6J mice. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology, 273 (5), R1631-R1637.

Di Fiore, A. (2002). Predator sensitive foraging in ateline primates. In L. E. Miller (Ed.), Eat or Be Eaten: Predator Sensitive Foraging Among Primates (pp. 242-267). Cambridge University Press: Cambridge.

Dixson, A. (1998). Primate Sexuality . John Wiley & Sons, Ltd, New Jersey.

Dobson, A. P., & Carper, E. R. (1996). Infectious diseases and human population history throughout history the establishment of disease has been a side effect of the growth of civilization. Bioscience, 46 (2), 115-126.

Dobson, A., Lafferty, K. D., Kuris, A. M., Hechinger, R. F., & Jetz, W. (2008). Homage to Linnaeus: how many parasites? How many hosts? Proceedings of the National Academy of Sciences, 105 (Supplement 1), 11482-11489.

Dollfus, R. (1947). Coenurose de la cavité abdominale chez un écureuil ( Sciurus vulgaris ) á Richelieu (Indre-et Loire). Annales de Parasitologie Humaine Comparee , 22 , 143- 147.

Dorny, P., Vercammen, F., Brandt, J., Vansteenkiste, W., Berkvens, D., & Geerts, S.

126

(2000). Sero-epidemiological study of Taenia saginata cysticercosis in Belgian cattle. Veterinary Parasitology, 88 (1), 43-49.

Dorny, P., Brandt, J., Zoli, A., & Geerts, S. (2003). Immunodiagnostic tools for human and porcine cysticercosis. Acta Tropica, 87 (1), 79-86.

Dorny, P., Brandt, J., & Geerts, S. (2004a). Immunodiagnostic approaches for detecting Taenia solium (letter).

Dorny, P., Phiri, I. K., Vercruysse, J., Gabriël, S., Willingham, A. L., Brandt, J., ... & Berkvens, D. (2004b). A Bayesian approach for estimating values for prevalence and diagnostic test characteristics of porcine cysticercosis. International Journal for Parasitology, 34 (5), 569-576.

Drummond, A. J., Suchard, M. A., Xie, D., & Rambaut, A. (2012). Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular Biology and Evolution, 29 (8), 1969-1973.

Dufva, R. (1996). Blood parasites, health, reproductive success, and egg volume in female Great Tits Parus major. Journal of Avian Biology, 83-87.

Dunbar, R. I. M. (1980). Demographic and life history variables of a population of gelada baboons ( Theropithecus gelada ). The Journal of Animal Ecology , 49 (2), 485-506.

Dunbar, R. I. M. (1984). Reproductive decisions: An economic analysis of gelada baboon social strategies . Princeton University Press: New Jersey.

Dunbar, R., & Dunbar, P. (1975). Social dynamics of gelada baboons. Contributions to Primatology . Karger: Basel.

Dunsmore, J. D., & Howkins, A. B. (1968). Coenurus serialis in a grey kangaroo. Australian Journal of Science, 30 (11).

Dupain, J., Nell, C., Petrzelková, K. J., Garcia, P., Modry, D., & Gordo, F. P. (2009). Gastrointestinal parasites of bonobos in the Lomako Forest, Democratic Republic of Congo. In M. A. Huffman & C.A. Chapman (Eds.), Primate Parasite Ecology , (pp. 297-310). Cambridge University Press: Cambridge.

Ebert, D., & Herre, E. A. (1996). The evolution of parasitic diseases. Parasitology Today, 12 (3), 96-101.

127

Eckert, J. (1991). Interactions between cestodes and their vertebrate hosts. In C. A. Toft, A. Aeschlimann, & L. Bolis (Eds.), Parasite-host associations: Coexistence or conflict? (pp. 201-227). Oxford University Press: Oxford.

Eichenberger, R. M., Stephan, R., & Deplazes, P. (2011). Increased sensitivity for the diagnosis of Taenia saginata cysticercus infection by additional heart examination compared to the EU-approved routine meat inspection. Food Control, 22 (6), 989- 992.

Elek, S. R., & Finkelstein, L. E. (1939). Multiceps serialis infestation in a baboon. Report of a case exhibiting connective tissue cystic masses. Zoologica, 24 , 323-328.

El-On, J., Shelef, I., Cagnano, E., & Benifla, M. (2008). Taenia multiceps : a rare human cestode infection in Israel. Veterinaria Italiana , 44 (4), 621-631.

Emerson, S. B., & Hess, D. L. (2001). Glucocorticoids, androgens, testis mass, and the energetics of vocalization in breeding male frogs. Hormones and Behavior, 39 (1), 59-69.

Engwerda, C. R., Ato, M., & Kaye, P. M. (2004). Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis. Trends in Parasitology, 20 (11), 524-530.

Eom, K. S., Jeon, H. K., Kong, Y., Hwang, U. W., Yang, Y., Li, X., ... & Rim, H. J. (2002). Identification of Taenia asiatica in China: molecular, morphological, and epidemiological analysis of a Luzhai isolate. Journal of Parasitology, 88 (4), 758-764.

Esch, G. W. (1967). Some effects of cortisone and sex on the biology of coenuriasis in laboratory mice and jackrabbits. Parasitology, 57 (1), 175-179.

Esch, G. W., & Fernández, J. C. (1993). A Functional Biology of Parasitism . University of California Press: Sheffield.

Escobedo, G., Larralde, C., Chavarria, A., Cerbón, M. A., & Morales-Montor, J. (2004). Molecular mechanisms involved in the differential effects of sex steroids on the reproduction and infectivity of Taenia crassiceps . Journal of Parasitology, 90 (6), 1235-1244.

Escobedo, G., Camacho-Arroyo, I., Hernández-Hernández, O. T., Ostoa-Saloma, P., García-Varela, M., & Morales-Montor, J. (2009a). Progesterone induces scolex

128

evagination of the human parasite Taenia solium : evolutionary implications to the host-parasite relationship. BioMed Research International , 2010.

Escobedo, G., Lopez-Griego, L., & Morales-Montor, J. (2009b). Neuroimmunoendocrine modulation in the host by helminth parasites: a novel form of host-parasite coevolution? Neuroimmunomodulation, 16 (2), 78-87.

Escobedo, G., De León-Nava, M. A., & Morales-Montor, J. (2010). Sex differences in parasitic infections: beyond the dogma of female-biased resistance. In S. L. Klein, & C. W. Roberts (Eds.), Sex hormones and immunity to infection (pp. 187-204). Springer: Berlin.

Ewald, P. W. (1983). Host-parasite relations, vectors, and the evolution of disease severity. Annual Review of Ecology and Systematics, 14 (1), 465-485.

Ewald, P. W. (1995). The evolution of virulence: a unifying link between parasitology and ecology. The Journal of Parasitology , 81 (5), 659-669.

Fain, A. (1956). Coenurus of Taenia brauni in man and animals in the Belgian Congo and Ruanda-Urundi. I. Coenurus in wild animals, with cerebral localization. Annales de la Societe Belge de Medecine Tropicale, 36 (5), 673-7.

Falush, D., Wirth, T., Linz, B., Pritchard, J. K., Stephens, M., Kidd, M., ... & Yamaoka, Y. (2003). Traces of human migrations in Helicobacter pylori populations. Science, 299 (5612), 1582-1585.

Fincher, C. L., Thornhill, R., Murray, D. R., & Schaller, M. (2008). Pathogen prevalence predicts human cross-cultural variability in individualism/collectivism. Proceedings of the Royal Society of London B: Biological Sciences, 275 (1640), 1279- 1285.

Fitzpatrick, S. L., & Richards, J. S. (1994). Identification of a cyclic adenosine 3', 5'- monophosphate-response element in the rat aromatase promoter that is required for transcriptional activation in rat granulosa cells and R2C Leydig cells. Molecular Endocrinology, 8 (10), 1309-1319.

Flisser, A. (1991). Taeniasis-cysticercosis: an introduction. Southeast Asian Journal of Tropical Medicine and Public Health , 22 , 233-235.

129

Folstad, I., & Karter, A. J. (1992). Parasites, bright males, and the immunocompetence handicap. The American Naturalist, 139 (3), 603-622.

Forbes, A. B., Huckle, C. A., Gibb, M. J., Rook, A. J., & Nuthall, R. (2000). Evaluation of the effects of nematode parasitism on grazing behaviour, herbage intake and growth in young grazing cattle. Veterinary Parasitology, 90 (1), 111-118.

Forbes, M. R. (1993). Parasitism and host reproductive effort. Oikos , 444-450.

Fountain, K. (2000). Coenurus serialis in a pet rabbit. The Veterinary Record, 147 (12), 340.

Freeland, W. J. (1976). Pathogens and the evolution of primate sociality. Biotropica, 8 (1), 12-24.

Freeland, W. J. (1979). Primate social groups as biological islands. Ecology, 60 (4), 719-728.

Freeland, W. J. (1980). Mangabey ( Cerocebus albigena ) movement patterns in relation to food availability and fecal contamination. Ecology, 61 (6), 1297-1303.

Freeland, W. J. (1981). Functional aspects of primate grooming. The Ohio Journal of Science, 81 , 173–177.

Fumagalli, M., Sironi, M., Pozzoli, U., Ferrer-Admettla, A., Pattini, L., & Nielsen, R. (2011). Signatures of environmental genetic adaptation pinpoint pathogens as the main selective pressure through human evolution. PLoS Genetics , 7(11), e1002355.

Gabriël, S., Blocher, J., Dorny, P., Abatih, E. N., Schmutzhard, E., Ombay, M., ... & Winkler, A. S. (2012). Added value of antigen ELISA in the diagnosis of neurocysticercosis in resource poor settings. PLoS Neglected Tropical Diseases, 6(10), e1851.

Galvani, A. P. (2003). Epidemiology meets evolutionary ecology. Trends in Ecology & Evolution , 18 (3), 132-139.

Garcia, H. H., & Del Brutto, O. H. (2000). Taenia solium cysticercosis. Infectious Disease Clinics of North America, 14 (1), 97-119.

Gauthier-Clerc, M., Mangin, S., Le Bohec, C., Gendner, J. P., & Le Maho, Y. (2003). Comparison of behaviour, body mass, haematocrit level, site fidelity and

130

survival between infested and non-infested king penguin Aptenodytes patagonicus by ticks Ixodes uriae. Polar Biology , 26 (6), 379-382.

Gemmell, M. A., Lawson, J. R., & Roberts, M. G. (1987). Population dynamics in echinococcosis and cysticercosis: evaluation of the biological parameters of Taenia hydatigena and T. ovis and comparison with those of Echinococcus granulosus . Parasitology, 94 (01), 161-180.

Georgi, J. R., De Lahunta, A., & Percy, D. H. (1969). Cerebral coenurosis in a cat. Report of a case. The Cornell Veterinarian, 59 (1), 127-134.

Gesquiere, L. R., Onyango, P. O., Alberts, S. C., & Altmann, J. (2011). Endocrinology of year-round reproduction in a highly seasonal habitat: environmental variability in testosterone and glucocorticoids in baboon males. American Journal of Physical Anthropology, 144 (2), 169-176.

Ghai, R. R., Fugère, V., Chapman, C. A., Goldberg, T. L., & Davies, T. J. (2015). Sickness behaviour associated with non-lethal infections in wild primates. Proceedings of the Royal Society B: Biological Sciences , 282 (1814), 20151436.

Gillespie, T. R., Chapman, C. A., & Greiner, E. C. (2005). Effects of logging on gastrointestinal parasite infections and infection risk in African primates. Journal of Applied Ecology, 42 (4), 699-707.

Gooderham, K., & Schulte-Hostedde, A. (2011). Macroparasitism influences reproductive success in red squirrels ( Tamiasciurus hudsonicus ). Behavioral Ecology, 22 (6), 1195-1200.

Goudswaard, M. F., & Thomas, J. A. (1991). Coenurus serialis infection of a white rabbit. Veterinary Record, 129 (13), 295-295.

Gourbal, B. E. F., & Gabrion, C. (2004). A study of mate choice in mice with experimental Taenia crassiceps cysticercosis: can males choose? Canadian Journal of Zoology, 82 (4), 635-643.

Gourbal, B. E. F., Lacroix, A., & Gabrion, C. (2002). Behavioural dominance and Taenia crassiceps parasitism in BALB/c male mice. Parasitology Research, 88(10), 912-917.

131

Gourbal, B. E. F., Righi, M., Petit, G., & Gabrion, C. (2001). Parasite-altered host behavior in the face of a predator: manipulation or not? Parasitology Research, 87 (3), 186- 192.

Greer, A. W. (2008). Trade-offs and benefits: implications of promoting a strong immunity to gastrointestinal parasites in sheep. Parasite Immunology, 30 (2), 123- 132.

Gregory, G. G. (1976). Fecundity and proglottid release of Taenia ovis and T. hydatigena . Australian Veterinary Journal, 52 (6), 277-279.

Greiner, M., Pfeiffer, D., & Smith, R. D. (2000). Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Preventive Veterinary Medicine, 45 (1), 23-41.

Grenfell, B. T., & Dobson, A. P. (1995). Ecology of Infectious Diseases in Natural Populations (Vol. 7). Cambridge University Press: Cambridge.

Griffin, R. H., & Nunn, C. L. (2012). Community structure and the spread of infectious disease in primate social networks. Evolutionary Ecology, 26 (4), 779-800.

Grueber, C. E., Nakagawa, S., Laws, R. J., & Jamieson, I. G. (2011). Multimodel inference in ecology and evolution: challenges and solutions. Journal of Evolutionary Biology, 24 (4), 699-711.

Guerra-Silveira, F., & Abad-Franch, F. (2013). Sex bias in infectious disease epidemiology: patterns and processes. PloS One , 8(4), e62390.

Gulland, F. M. D. (1992). The role of nematode parasites in Soay sheep ( Ovis aries L. ) mortality during a population crash. Parasitology, 105 (03), 493-503.

Gulland, F. M. D. (1995). The impact of infectious diseases on wild animal populations: a review. Ecology of infectious diseases in natural populations (pp. 20-51). Cambridge University Press: Cambridge.

Hamilton, A. G. (1950). The occurrence and morphology of Coenurus serialis in rabbits. Parasitology, 40 (1-2), 46-9.

Hart, B. L. (1988). Biological basis of the behavior of sick animals. Neuroscience & Biobehavioral Reviews, 12 (2), 123-137.

132

Hausfater, G., & Watson, D. F. (1976). Social and reproductive correlates of parasite ova emissions by baboons. Nature , 262 (5570), 688-689.

Hausfater, G., & Meade, B. J. (1982). Alternation of sleeping groves by yellow baboons (Papio cynocephalus ) as a strategy for parasite avoidance. Primates, 23 (2), 287-297.

Hawlena, H., Khokhlova, I. S., Abramsky, Z., & Krasnov, B. R. (2006). Age, intensity of infestation by flea parasites and body mass loss in a rodent host. Parasitology, 133 (02), 187-193.

Hayes, M. A., & Creighton, S. R. (1978). A coenurus in the brain of a cat. The Canadian Veterinary Journal, 19 (12), 341.

Hayward, A. D., Nussey, D. H., Wilson, A. J., Berenos, C., Pilkington, J. G., Watt, K. A., ... & Graham, A. L. (2014). Natural selection on individual variation in tolerance of gastrointestinal nematode infection. PLoS Biology, 12 (7), e1001917.

Heath, D. D. (1971). The migration of oncospheres of Taenia pisiformis , T. serialis and Echinococcus granulosus within the intermediate host. International Journal for Parasitology, 1 (2), 145-150.

Hernandez, A. D., Macintosh, A. J., & Huffman, M. A. (2009). Primate parasite ecology: patterns and predictions from an ongoing study of Japanese macaques. In M. A. Huffman & C.A. Chapman (Eds.), Primate Parasite Ecology , (pp. 387-402). Cambridge University Press: Cambridge.

Hersh, M. H., LaDeau, S. L., Previtali, M. A., & Ostfeld, R. S. (2014). When is a parasite not a parasite? Effects of larval tick burdens on white-footed mouse survival. Ecology , 95 (5), 1360-1369.

Hillegass, M. A., Waterman, J. M., & Roth, J. D. (2010). Parasite removal increases reproductive success in a social African ground squirrel. Behavioral Ecology, 21 (4), 696-700.

Hoberg, E. P. (2002). Taenia tapeworms: their biology, evolution and socioeconomic significance. Microbes and Infection , 4(8), 859-866.

133

Hoberg, E. P., Jones, A., Rausch, R. L., Eom, K. S., & Gardner, S. L. (2000). A phylogenetic hypothesis for species of the genus Taenia (Eucestoda: Taeniidae). Journal of Parasitology , 86 (1), 89-98.

Hoberg, E. P., Alkire, N. L., Queiroz, A. D., & Jones, A. (2001). Out of Africa: origins of the Taenia tapeworms in humans. Proceedings of the Royal Society of London B: Biological Sciences , 268 (1469), 781-787.

Hochberg, M. E., Michalakis, Y., & De Meeus, T. (1992). Parasitism as a constraint on the rate of life-history evolution. Journal of Evolutionary Biology , 5(3), 491-504.

Holmberg, B. J., Hollingsworth, S. R., Osofsky, A., & Tell, L. A. (2007). Taenia coenurus in the orbit of a chinchilla. Veterinary Ophthalmology , 10 (1), 53-59.

Holmes, J. C., & Zohar, S. (1990). Pathology and host behaviour. Parasitism and host behaviour , 34-63.

Hough, I. (2000). Subcutaneous larval Taenia serialis in a ring-tailed possum (Pseudocheirus peregrinus ). Australian Veterinary Journal , 78 (7), 468-468.

Huerta, L., Terrazas, L. I., Sciutto, E., & Larralde, C. (1992). Immunological mediation of gonadal effects on experimental murine cysticercosis caused by Taenia crassiceps metacestodes. The Journal of Parasitology , 471-476.

Huffman, M. A., & Chapman, C. A. (2009). Primate Parasite Ecology. Cambridge University Press: Cambridge.

Huffman, M. A., Pebsworth, P., Bakuneeta, C., Gotoh, S., & Bardi, M. (2009). Chimpanzee-parasite ecology at Budongo Forest (Uganda) and the Mahale Mountains (Tanzania): Influence of climatic differences on self-medicative behavior. In M. A. Huffman & C.A. Chapman (Eds.), Primate Parasite Ecology , (pp. 331-350). Cambridge University Press: Cambridge.

Hugot, J. P. (1999). Primates and their pinworm parasites: the Cameron hypothesis revisited. Systematic Biology , 48 (3), 523-546.

Huijbregts, B., De Wachter, P., Obiang, L. S. N., & Akou, M. E. (2003). Ebola and the decline of gorilla Gorilla gorilla and chimpanzee Pan troglodytes populations in Minkebe Forest, north-eastern Gabon. Oryx , 37 (04), 437-443.

134

Hürni, H., & Stiefel, S. L. (2003). Report on a mission to the Simen Mountains National Park World Heritage Site, Ethiopia. NCCR North-South and the East & Southern Africa Partnership Programme of the Centre for Development and Environment, Basel, Switzerland .

Hurvich, C. M., & Tsai, C. L. (1989). Regression and time series model selection in small samples. Biometrika , 297-307.

Huss, B. T., Miller, M. A., Corwin, R. M., Hoberg, E. P., & O'Brien, D. P. (1994). Fatal cerebral coenurosis in a cat. Journal of the American Veterinary Medical Association , 205 (1), 69-71.

Ibarra-Coronado, E. G., Escobedo, G., Nava-Castro, K., Ramses, C. R. J., Hernández- Bello, R., García-Varela, M., ... & Pavón, L. (2011). A helminth cestode parasite express an estrogen-binding protein resembling a classic nuclear estrogen receptor. Steroids , 76 (10), 1149-1159.

Ilmonen, P., Taarna, T., & Hasselquist, D. (2000). Experimentally activated immune defence in female pied flycatchers results in reduced breeding success. Proceedings of the Royal Society of London B: Biological Sciences , 267 (1444), 665-670.

Ing, M. B., Schantz, P. M., & Turner, J. A. (1998). Human coenurosis in North America: case reports and review. Clinical Infectious Diseases , 27 (3), 519-523.

Iwamoto, T. (1993). The ecology of Theropithecus gelada . In N. G. Jablonski (Ed.), Theropithecus: The Rise and Fall of a Primate Genus, (pp. 441-452). Cambridge University Press: Cambridge.

Iwamoto, T., & Dunbar, R. I. M. (1983). Thermoregulation, habitat quality and the behavioural ecology of gelada baboons. The Journal of Animal Ecology , 357-366.

Jabbar, A., Swiderski, Z., Mlocicki, D., Beveridge, I., & Lightowlers, M. W. (2010). The ultrastructure of taeniid cestode oncospheres and localization of host-protective antigens. Parasitology , 137 (03), 521-535.

Jablonski, N. G. (1993). Evolution of the masticatory apparatus of Theropithecus . In N. G. Jablonski (Ed.), Theropithecus: The Rise and Fall of a Primate Genus, (pp. 299-330). Cambridge University Press: Cambridge.

135

Janson, C. H., & Goldsmith, M. L. (1995). Predicting group size in primates: foraging costs and predation risks. Behavioral Ecology , 6(3), 326-336.

Jeon, H. K., Chai, J. Y., Kong, Y., Waikagul, J., Insisiengmay, B., Rim, H. J., & Eom, K. S. (2009). Differential diagnosis of Taenia asiatica using multiplex PCR. Experimental Parasitology , 121 (2), 151-156.

Jia, W. Z., Yan, H. B., Guo, A. J., Zhu, X. Q., Wang, Y. C., Shi, W. G., ... & Littlewood, D. T. J. (2010). Complete mitochondrial genomes of Taenia multiceps , T. hydatigena and T. pisiformis : additional molecular markers for a tapeworm genus of human and animal health significance. BMC Genomics , 11 (1), 447.

Johnson, R. W. (2002). The concept of sickness behavior: a brief chronological account of four key discoveries. Veterinary Immunology and Immunopathology , 87 (3), 443-450.

Jones, M. E., Cockburn, A., Hamede, R., Hawkins, C., Hesterman, H., Lachish, S., ... & Pemberton, D. (2008). Life-history change in disease-ravaged Tasmanian devil populations. Proceedings of the National Academy of Sciences , 105 (29), 10023-10027.

Jull, P., Browne, E., Boufana, B. S., Schöniger, S., & Davies, E. (2012). Cerebral coenurosis in a cat caused by Taenia serialis : neurological, magnetic resonance imaging and pathological features. Journal of Feline Medicine and Surgery , 14 (9), 646-649.

Kappeler, P. M., & van Schaik, C. P. (2002). Evolution of primate social systems. International Journal of Primatology , 23 (4), 707-740.

Karlsson, E. K., Kwiatkowski, D. P., & Sabeti, P. C. (2014). Natural selection and infectious disease in human populations. Nature Reviews Genetics , 15 (6), 379-393.

Keele, B. F., Jones, J. H., Terio, K. A., Estes, J. D., Rudicell, R. S., Wilson, M. L., ... & Wroblewski, E. (2009). Increased mortality and AIDS-like immunopathology in wild chimpanzees infected with SIVcpz. Nature , 460 (7254), 515-519.

Keymer, A. E., & Read, A. F. (1991). Behavioural ecology: the impact of parasitism. In C. A. Toft, A. Aeschlimann, & L. Bolis (Eds.), Parasite-host associations, coexistence or conflict? (pp. 37-61). Oxford University Press: Oxford.

136

Kingston, N., Williams, E. S., Bergstrom, R. C., Wilson, W. C., & Miller, R. (1984). Cerebral coenuriasis in domestic cats in Wyoming and Alaska. Proceedings Helminthological Society of Washington , 51 , 309-314.

Knowles, S. C., Nakagawa, S., & Sheldon, B. C. (2009). Elevated reproductive effort increases blood parasitaemia and decreases immune function in birds: a meta-regression approach. Functional Ecology , 23 (2), 405-415.

Korpimaki, E., Hakkarainen, H., & Bennett, G. F. (1993). Blood parasites and reproductive success of Tengmalm's Owls: Detrimental effects on females but not on males?. Functional Ecology , 420-426.

Krentz, H. B. (1993). Postcranial anatomy of extant and extinct species of Theropithecus . In N. G. Jablonski (Ed.), Theropithecus: The Rise and Fall of a Primate Genus , (pp. 383-422). Cambridge University Press: Cambridge.

Krishnan, L., Guilbert, L. J., Wegmann, T. G., Belosevic, M., & Mosmann, T. R. (1996). T helper 1 response against Leishmania major in pregnant C57BL/6 mice increases implantation failure and fetal resorptions. Correlation with increased IFN- gamma and TNF and reduced IL-10 production by placental cells. The Journal of Immunology , 156 (2), 653-662.

Krist AC. (2001). Variation in fecundity among populations of snails is predicted by prevalence of castrating parasites. Evolutionary Ecology Research, 3 (2), 191-197.

Kyriazakis, I., Tolkamp, B. J., & Hutchings, M. R. (1998). Towards a functional explanation for the occurrence of anorexia during parasitic infections. Animal Behaviour , 56 (2), 265-274.

Lacasse, C., Travis, E., Gamble, K. C., & Craig, T. (2005). Cestode cysts in two African giant pouched rats ( Cricetomys gambianus ). Journal of Zoo and Wildlife Medicine , 36 (1), 95-99.

Lachish, S., Knowles, S. C., Alves, R., Wood, M. J., & Sheldon, B. C. (2011). Fitness effects of endemic malaria infections in a wild bird population: the importance of ecological structure. Journal of Animal Ecology , 80 (6), 1196-1206.

Lafferty, K. D. (1993). The marine snail, Cerithidea californica , matures at smaller sizes where parasitism is high. Oikos , 3-11.

137

Lafferty, K. D. (1999). The evolution of trophic transmission. Parasitology Today , 15 (3), 111-115.

Lafferty, K. D., & Holt, R. D. (2003). How should environmental stress affect the population dynamics of disease? Ecology Letters , 6(7), 654-664.

Lafferty, K. D., & Kuris, A. M. (2002). Trophic strategies, animal diversity and body size. Trends in Ecology & Evolution , 17 (11), 507-513.

Lafferty, K. D., Thomas, F., & Poulin, R. (2000). Evolution of host phenotype manipulation by parasites and its consequences. In R. Poulin, S. Morand, & A. Skorping (Eds.), Evolutionary biology of host–parasite relationships: Theory meets reality (pp. 117-127). Elsevier Science: Amsterdam.

Landells, J. W. (1949). Intra-medullary cyst of the spinal cord due to the cestode Multiceps multiceps in the coenurus stage: report of a case. Journal of Clinical Pathology , 2(1), 61.

Larralde, C., Morales, J., Terrazas, I., Govezensky, T., & Romano, M. C. (1995). Sex hormone changes induced by the parasite lead to feminization of the male host in murine Taenia crassiceps cysticercosis. The Journal of Steroid Biochemistry and Molecular Biology , 52 (6), 575-580.

Lawson, J. R., & Gemmell, M. A. (1990). Transmission of taeniid tapeworm eggs via blowflies to intermediate hosts. Parasitology , 100 (01), 143-146. le Roux, A., Beehner, J. C., & Bergman, T. J. (2011). Female philopatry and dominance patterns in wild geladas. American Journal of Primatology , 73 (5), 422-430.

Lee, P. L., & Clayton, D. H. (1995). Population biology of swift ( Apus apus ) ectoparasites in relation to host reproductive success. Ecological Entomology , 20 (1), 43-50.

Leendertz, F. H., Ellerbrok, H., Boesch, C., Couacy-Hymann, E., Mätz-Rensing, K., Hakenbeck, R., ... & Vigilant, L. (2004). Anthrax kills wild chimpanzees in a tropical rainforest. Nature , 430 (6998), 451-452.

Leendertz, F. H., Pauli, G., Maetz-Rensing, K., Boardman, W., Nunn, C., Ellerbrok, H., ... & Christophe, B. (2006). Pathogens as drivers of population declines: the importance of systematic monitoring in great apes and other threatened mammals. Biological Conservation , 131 (2), 325-337.

138

Lefèvre, T., Lebarbenchon, C., Gauthier-Clerc, M., Missé, D., Poulin, R., & Thomas, F. (2009). The ecological significance of manipulative parasites. Trends in Ecology & Evolution , 24 (1), 41-48.

Leiby, P. D., & Dyer, W. G. (1971). Cyclophyllidean tapeworms of wild carnivora. In J. W. Davis, & R. C. Anderson (Eds.), Parasitic diseases of wild animals (pp. 174-234). Iowa State University Press: Iowa City.

Leroy, E. M., Rouquet, P., Formenty, P., Souquiere, S., Kilbourne, A., Froment, J. M., ... & Zaki, S. R. (2004). Multiple Ebola virus transmission events and rapid decline of central African wildlife. Science , 303 (5656), 387-390.

Lescano, A. G., & Zunt, J. (2013). Other cestodes: sparganosis, coenurosis and Taenia crassiceps cysticercosis. Handbook of Clinical Neurology , 114 , 335.

Lin, Y. C., Rikihisa, Y., Kono, H., & Gu, Y. (1990). Effects of larval tapeworm ( Taenia taeniaeformis ) infection on reproductive functions in male and female host rats. Experimental Parasitology , 70 (3), 344-352.

Leggett, H. C., Cornwallis, C. K., Buckling, A., & West, S. A. (2017). Growth rate, transmission mode, and virulence in human pathogens. Philosophical Transactions of the Royal Society B, 372 (1719), 20160094.

Lloyd, A. L., & May, R. M. (2001). How viruses spread among computers and people. Science , 292 (5520), 1316-1317.

Lochmiller, R. L., & Deerenberg, C. (2000). Trade-offs in evolutionary immunology: just what is the cost of immunity? Oikos , 88 (1), 87-98.

Loos-Frank, B. (2000). An up-date of Verster's (1969) ‘Taxonomic revision of the genus Taenia Linnaeus’ (Cestoda) in table format. Systematic Parasitology , 45 (3), 155-184.

Lopes, F. C., de Paiva, K. A. R., Coelho, W. A. C., Nunes, F. V. A., da Silva, J. B., da Escóssia, C. D. G. M., ... & Batista, J. S. (2016). Lactation curve and milk quality of goats experimentally infected with Trypanosoma vivax . Experimental Parasitology , 167 , 17-24.

Lynch, J. W., Ziegler, T. E., & Strier, K. B. (2002). Individual and seasonal variation in fecal testosterone and cortisol levels of wild male tufted capuchin monkeys,

139

Cebus apella nigritus . Hormones and Behavior , 41 (3), 275-287.

MacIntosh, A. J., Jacobs, A., Garcia, C., Shimizu, K., Mouri, K., Huffman, M. A., & Hernandez, A. D. (2012). Monkeys in the middle: parasite transmission through the social network of a wild primate. PLoS One , 7(12), e51144.

Malomo, A., Ogunniyi, J., Ogunniyi, A., Akang, E. E. U., & Shokunbi, M. T. (1990). Coenurosis of the central nervous system in a Nigerian. Tropical and Geographical Medicine , 42 (3), 280-282.

Manga-González, M. Y., González-Lanza, C., Cabanas, E., & Campo, R. (2001). Contributions to and review of dicrocoeliosis, with special reference to the intermediate hosts of Dicrocoelium dendriticum . Parasitology , 123 (7), 91.

Marty, A. M., Neafie, R.C. (2000). In W. M. Meyers, R. C. Neafie, A. M. Marty, & D. J. Wears (Eds.), Pathology of Infectious Diseases. Volume 1: Helminthiases (pp. 185-196). Armed Forces Institute of Pathology: Washington, D.C.

Marzal, A., De Lope, F., Navarro, C., & Møller, A. P. (2005). Malarial parasites decrease reproductive success: an experimental study in a passerine bird. Oecologia , 142 (4), 541-545.

May, R. M. (1988). How many species are there on earth? Science , 241 (4872), 1441.

May, R. M., & Nowak, M. A. (1995). Coinfection and the evolution of parasite virulence. Proceedings of the Royal Society of London B: Biological Sciences , 261 (1361), 209-215.

McCallum, H., & Dobson, A. (1995). Detecting disease and parasite threats to endangered species and ecosystems. Trends in Ecology & Evolution , 10 (5), 190-194.

McDade, T. W., Reyes-García, V., Tanner, S., Huanca, T., & Leonard, W. R. (2008). Maintenance versus growth: investigating the costs of immune activation among children in lowland Bolivia. American Journal of Physical Anthropology , 136 (4), 478- 484.

McDonald, I. R., Lee, A. K., Than, K. A., & Martin, R. W. (1986). Failure of glucocorticoid feedback in males of a population of small marsupials ( Antechinus swainsonii ) during the period of mating. Journal of Endocrinology , 108 (1), 63-68.

140

McManus, D. P. (2006). Molecular discrimination of taeniid cestodes. Parasitology International , 55 , S31-S37.

Mercer, J. G., Mitchell, P. I., Moar, K. M., Bissett, A., Geissler, S., Bruce, K., & Chappell, L. H. (2000). Anorexia in rats infected with the nematode, Nippostrongylus brasiliensis : experimental manipulations. Parasitology , 120 (06), 641-647.

Mertz, G. J. (2016). Zoonoses: infectious diseases transmissible from animals to humans. Clinical Infectious Diseases , 63 (1), 148-149.

Meyer, M. C. (1955). Coenuriasis in varying hare in Maine, with remarks on the validity of Multiceps serialis . Transactions of the American Microscopical Society , 74 (2), 163- 169.

Michal, A., Regli, F., Campiche, R., Cavallo, R. J., de Crousaz, G., Oberson, R., & Rabinowicz, T. (1977). Cerebral coenurosis. Report of a case with arteritis. Journal of Neurology , 216 (4), 265.

Miller, G. E., & Chen, E. (2010). Harsh family climate in early life presages the emergence of a proinflammatory phenotype in adolescence. Psychological Science , 21 (6), 848-856.

Milton, K. (1996). Effects of bot fly ( Alouattamyia baeri ) parasitism on a free-ranging howler monkey ( Alouatta palliata ) population in Panama. Journal of Zoology , 239 (1), 39-63.

Møller, A. P., Christe, P., & Lux, E. (1999). Parasitism, host immune function, and sexual selection. The Quarterly Review of Biology , 74 (1), 3-20.

Møller, A. P., Sorci, G., & Erritzøe, J. (1998). Sexual dimorphism in immune defense. The American Naturalist , 152 (4), 605-619.

Moodley, Y., Linz, B., Bond, R. P., Nieuwoudt, M., Soodyall, H., Schlebusch, C. M., ... & Van der Merwe, S. W. (2012). Age of the association between Helicobacter pylori and man. PLoS Pathogens , 8(5), e1002693.

Moore, J. (2002). Parasites and the behavior of animals . Oxford University Press: Oxford.

Mooring, M. S. (1995). The effect of tick challenge on grooming rate by impala. Animal Behaviour , 50 (2), 377-392.

141

Morales, J., Larralde, C., Arteaga, M., Govezensky, T., Romano, M. C., & Morali, G. (1996). Inhibition of sexual behavior in male mice infected with Taenia crassiceps cysticerci. The Journal of Parasitology , 82 (5), 689-693.

Morales, J., Velasco, T., Tovar, V., Fragoso, G., Fleury, A., Beltran, C., ... & Larralde, C. (2002). Castration and pregnancy of rural pigs significantly increase the prevalence of naturally acquired Taenia solium cysticercosis. Veterinary Parasitology , 108 (1), 41-48.

Morales-Montor, J., Rodríguez-Dorantes, M., Mendoza-Rodríguez, C. A., Camacho- Arroyo, I., & Cerbón, M. A. (1998). Differential expression of the estrogen- regulated proto-oncogenes c-fos, c-jun, and bcl-2 and of the tumor-suppressor p53 gene in the male mouse chronically infected with Taenia crassiceps cysticerci. Parasitology Research , 84 (8), 616-622.

Morales-Montor, J., Gamboa-Dominguez, A., Rodriguez-Dorantes, M., & Cerbon, M. A. (1999a). Tissue damage in the male murine reproductive system during experimental Taenia crassiceps cysticercosis. The Journal of Parasitology , 85 (5), 887- 890.

Morales-Montor, J., Rodríguez-Dorantes, M., & Cerbon, M. A. (1999b). Modified expression of steroid 5α-reductase as well as aromatase, but not cholesterol side- chain cleavage enzyme, in the reproductive system of male mice during ( Taenia crassiceps) cysticercosis. Parasitology Research , 85 (5), 393-398.

Morales-Montor, J., Baig, S., Mitchell, R., Deway, K., Hallal-Calleros, C., & Damian, R. T. (2001). Immunoendocrine interactions during chronic cysticercosis determine male mouse feminization: role of IL-6. The Journal of Immunology , 167 (8), 4527- 4533.

Morales-Montor, J., Baig, S., Kabbani, A., & Damian, R. T. (2002). Do interleukin-6 and macrophage-migration inhibitory factor play a role during sex-associated susceptibility in murine cysticercosis? Parasitology Research , 88 (10), 901-904.

Morales-Montor, J., Hallal-Calleros, C., Romano, M. C., & Damian, R. T. (2002). Inhibition of P-450 aromatase prevents feminisation and induces protection during cysticercosis. International Journal for Parasitology , 32 (11), 1379-1387.

142

Morales-Montor, J., Chavarria, A., De Leon, M. A., Del Castillo, L. I., Escobedo, E. G., Sanchez, E. N., ... & Larralde, C. (2004). Host gender in parasitic infections of mammals: an evaluation of the female host supremacy paradigm. Journal of Parasitology , 90 (3), 531-546.

Morales-Montor, J., & Larralde, C. (2005). The role of sex steroids in the complex physiology of the host-parasite relationship: the case of the larval cestode of Taenia crassiceps . Parasitology , 131 (3), 287-294.

Morales-Montor, J., Escobedo, G., Vargas-Villavicencio, J. A., & Larralde, C. (2008). The neuroimmunoendocrine network in the complex host-parasite relationship during murine cysticercosis. Current Topics in Medicinal Chemistry , 8(5), 400-407.

Morand, S., & Harvey, P. H. (2000). Mammalian metabolism, longevity and parasite species richness. Proceedings of the Royal Society of London B: Biological Sciences , 267 (1456), 1999-2003.

Mouritsen, K. N., & Poulin, R. (2003). Parasite-induced trophic facilitation exploited by a non-host predator: a manipulator's nightmare. International Journal for Parasitology , 33 (10), 1043-1050.

Muehlenbein, M. P., & Bribiescas, R. G. (2005). Testosterone-mediated immune functions and male life histories. American Journal of Human Biology , 17 (5), 527-558.

Müller-Graf, C. D. M., Collins, D. A., Packer, C., & Woolhouse, M. E. J. (1997). Schistosoma mansoni infection in a natural population of olive baboons ( Papio cynocephalus anubis ) in Gombe Stream National Park, Tanzania. Parasitology , 115 (06), 621-627.

Mwape, K. E., Praet, N., Benitez-Ortiz, W., Muma, J. B., Zulu, G., Celi-Erazo, M., ... & Gabriël, S. (2011). Field evaluation of urine antigen detection for diagnosis of Taenia solium cysticercosis. Transactions of the Royal Society of Tropical Medicine and Hygiene , 105 (10), 574-578.

Mwape KE, Phiri IK, Praet N, Speybroeck N, Muma JB, Dorny P, Gabriël S. (2013). The incidence of human cysticercosis in a rural community of Eastern Zambia. PLoS Neglected Tropical Disease s, 7(3): e2142.

Nagaty, H. F., & Ezzat, M. A. E. (1946). On the identity of Multieeps multiceps (Leske, 1780), M. gatgeri Hall, 1916, and M. serialis (Gervalis, 1845), with a review of these

143

and similar forms in man and animals. Proceedings of the Helminthological Society of Washington , 13 (2), 33-44.

Neuhaus, P. (2003). Parasite removal and its impact on litter size and body condition in Columbian ground squirrels ( Spermophilus columbianus ). Proceedings of the Royal Society of London B: Biological Sciences , 270 (Suppl 2), S213-S215.

Nevenic V & Markovic D. (1951). La chèvre comme un hôte intermédiaire pour Taenia serialis (Gervais 1847). Acta Veterinaria, 1(1), 128-131.

Newberne, P. M., & Burnett, S. (1951). Cestodiasis in the chinchilla. Veterinary Medicine , 46 (4), 156-157.

Nguekam, J. P., Zoli, A. P., Zogo, P. O., Kamga, A. C. T., Speybroeck, N., Dorny, P., ... & Geerts, S. (2003). A seroepidemiological study of human cysticercosis in West Cameroon. Tropical Medicine & International Health , 8(2), 144-149.

Nguyen, N., Fashing, P. J., Boyd, D. A., Barry, T. S., Burke, R. J., Goodale, C. B., ... & Miller, C. M. (2015). Fitness impacts of tapeworm parasitism on wild gelada monkeys at Guassa, Ethiopia. American Journal of Primatology , 77 (5), 579-594.

Nilsson, J. Å. (2003). Ectoparasitism in marsh tits: costs and functional explanations. Behavioral Ecology , 14 (2), 175-181.

Nowak, R., Porter, R. H., Lévy, F., Orgeur, P., & Schaal, B. (2000). Role of mother-young interactions in the survival of offspring in domestic mammals. Reviews of Reproduction , 5(3), 153-163.

Nunn, C., & Altizer, S. M. (2006.) Infectious diseases in primates: Behavior, ecology and evolution . Oxford University Press: Oxford.

Oldstone, M. B. (1998). Molecular mimicry and immune-mediated diseases. The FASEB Journal , 12 (13), 1255-1265.

O'Reilly, A., McCowan, C., Hardman, C., & Stanley, R. (2002). Taenia serialis causing exophthalmos in a pet rabbit. Veterinary Ophthalmology , 5(3), 227-230.

Ohsawa, H. (1979). The local gelada population and environment of the Gish area. In M. Kawai (Ed.), Ecological and sociological studies of gelada baboons (pp. 4-45). Karger Publishers: Tokyo.

144

Padgett, K. A., Nadler, S. A., Munson, L., Sacks, B., & Boyce, W. M. (2005). Systematics of Mesocestoides (Cestoda: Mesocestoididae): evaluation of molecular and morphological variation among isolates. Journal of Parasitology , 91 (6), 1435-1443.

Pappano, D. J. (2013). The Reproductive Trajectories of Bachelor Geladas (Doctoral dissertation). The University of Michigan, Ann Arbor.

Pappano, D. J., & Beehner, J. C. (2015). Harem-holding males do not rise to the challenge: androgens respond to social but not to seasonal challenges in wild geladas. Royal Society Open Science , 1(1), 140081.

Parker, G. A., Chubb, J. C., Ball, M. A., & Roberts, G. N. (2003). Evolution of complex life cycles in helminth parasites. Nature , 425 (6957), 480-484.

Parker, G. A., Ball, M. A., & Chubb, J. C. (2015). Evolution of complex life cycles in trophically transmitted helminths. II. How do life-history stages adapt to their hosts? Journal of Evolutionary Biology , 28 (2), 292-304.

Patterson, J. E., Neuhaus, P., Kutz, S. J., & Ruckstuhl, K. E. (2013). Parasite removal improves reproductive success of female North American red squirrels (Tamiasciurus hudsonicus ). PloS One , 8(2), e55779.

Patterson, J. E., & Ruckstuhl, K. E. (2013). Parasite infection and host group size: a meta- analytical review. Parasitology , 140 (7), 803.

Pau, A., Turtas, S., Brambilla, M., Leoni, A., Rosa, M., & Viale, G. L. (1987). Computed tomography and magnetic resonance imaging of cerebral coenurosis. Surgical Neurology , 27 (6), 548-552.

Pau, A., Perria, C., Turtas, S., Brambilla, M., & Viale, G. (1990). Long-term follow-up of the surgical treatment of intracranial coenurosis. British Journal of Neurosurgery , 4(1), 39-43.

Peña, N., Morales, J., Morales-Montor, J., Vargas-Villavicencio, A., Fleury, A., Zarco, L., ... & Sciutto, E. (2007). Impact of naturally acquired Taenia solium cysticercosis on the hormonal levels of free ranging boars. Veterinary Parasitology , 149 (1), 134-137.

145

Polak, M., & Starmer, W. T. (1998). Parasite–induced risk of mortality elevates reproductive effort in male Drosophila . Proceedings of the Royal Society of London B: Biological Sciences , 265 (1411), 2197-2201.

Poulin, R. (1994a). The evolution of parasite manipulation of host behaviour: a theoretical analysis. Parasitology , 109 (S1), S109-S118.

Poulin, R. (1994b). Meta-analysis of parasite-induced behavioural changes. Animal Behaviour , 48 (1), 137-146.

Poulin, R. (1995). “Adaptive” changes in the behaviour of parasitized animals: a critical review. International Journal for Parasitology , 25 (12), 1371-1383.

Poulin, R. (1996). How many parasite species are there: are we close to answers? International Journal for Parasitology , 26 (10), 1127-1129.

Poulin, R. (2007). Evolutionary Ecology of Parasites . Princeton University Press: Princeton.

Poulin, R. (2010). Parasite manipulation of host behavior: an update and frequently asked questions. Advances in the Study of Behavior , 41 , 151-186.

Poulin, R., Fredensborg, B. L., Hansen, E. K., & Leung, T. L. (2005). The true cost of host manipulation by parasites. Behavioural Processes , 68 (3), 241-4.

Prendergast, J. A., & Jensen, W. E. (2011). Consequences of parasitic mite infestation on muskrat ( Ondatra zibethicus ). Western North American Naturalist , 71 (4), 516-522.

Price, P. W. (1980). Evolutionary biology of parasites (Vol. 15). Princeton University Press.

Pullan, R., & Brooker, S. (2008). The health impact of polyparasitism in humans: are we under-estimating the burden of parasitic diseases? Parasitology , 135 (07), 783-794.

R Core Team. (2015). R: A Language and Environment for Statistical Computing . R Foundation for Statistical Computing, Vienna, Austria.

R Core Team . (2016). R: A Language and Environment for Statistical Computing . R Foundation for Statistical Computing, Vienna, Austria.

Råberg, L., Graham, A. L., & Read, A. F. (2009). Decomposing health: tolerance and resistance to parasites in animals. Philosophical Transactions of the Royal Society of

146

London B: Biological Sciences , 364 (1513), 37-49.

Randall, J., Cable, J., Guschina, I. A., Harwood, J. L., & Lello, J. (2013). Endemic infection reduces transmission potential of an epidemic parasite during coinfection. Proceedings of the Royal Society of London B: Biological Sciences , 280 (1769), 20131500.

Ranque, J., & Nicoli, R. N. (1955). Parasitological observations on cerebral coenuriasis, with reference to a new case. Annales de Parasitologie Humaine et Comparee , 30 (1-2), 22-42.

Raper, A. B., & Dockeray, G. C. (1956). Coenurus cysts in man: five cases from East Africa. Annals of Tropical Medicine & Parasitology , 50 (2), 121-128.

Rätti, O., Dufva, R., & Alatalo, R. V. (1993). Blood parasites and male fitness in the pied flycatcher. Oecologia , 96 (3), 410-414.

Rau, M. E. (1983). The open-field behaviour of mice infected with Trichinella spiralis . Parasitology , 86 (2), 311-318.

Raveh, S., Heg, D., Dobson, F. S., Coltman, D. W., Gorrell, J. C., Balmer, A., ... & Neuhaus, P. (2011). No experimental effects of parasite load on male mating behaviour and reproductive success. Animal Behaviour , 82 (4), 673-682.

Raveh, S., Neuhaus, P., & Dobson, F. S. (2015). Ectoparasites and fitness of female Columbian ground squirrels. Philosophical Transactions of the Royal Society B, 370 (1669), 20140113.

Reed, D. L., Toups, M. A., Light, J. E., Allen, J. M., & Flannigan, S. (2009). Lice and other parasites as markers of primate evolutionary history. In M. A. Huffman & C.A. Chapman (Eds.), Primate Parasite Ecology , (pp. 231-250). Cambridge University Press: Cambridge.

Repetti, R. L., Taylor, S. E., & Seeman, T. E. (2002). Risky families: family social environments and the mental and physical health of offspring. Psychological Bulletin , 128 (2), 330.

Ricklefs, R. E., & Wikelski, M. (2002). The physiology/life-history nexus. Trends in Ecology & Evolution , 17 (10), 462-468.

147

Robar, N., Burness, G., & Murray, D. L. (2010). Tropics, trophics and taxonomy: the determinants of parasite-associated host mortality. Oikos , 119 (8), 1273-1280.

Roberts, H. C., Hardie, L. J., Chappell, L. H., & Mercer, J. G. (1999). Parasite-induced anorexia: leptin, insulin and corticosterone responses to infection with the nematode, Nippostrongylus brasiliensis . Parasitology , 118 (1), 117-123.

Roberts, E. K., Lu, A., Bergman, T. J., & Beehner, J. C. (2017). Female Reproductive Parameters in Wild Geladas ( Theropithecus gelada ). International Journal of Primatology , 31 (1) , 1-20.

Robin, X., Turck, N., Hainard, A., Tiberti, N., Lisacek, F., Sanchez, J. C., & Müller, M. (2011). pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics , 12 (1), 77.

Rodhain, J., & Wanson, M. (1954). Un nouveau cas de coenurose chez le babouin, Theropithecus gelada RUPPELL. Rivista di Parassitologia , 15 (4), 613-620.

Roger, H., Sautet, J., & Paillas, J. E. (1942). Un cas de cénurose de la fosse cérébrale postérieure. Revue Neurologique , 74 , 319-321.

Roy, B. A., & Kirchner, J. W. (2000). Evolutionary dynamics of pathogen resistance and tolerance. Evolution , 54 (1), 51-63.

Sabattani, S., Marliani, A. F., Roncaroli, F., Zucchelli, M., Zini, A., Calbucci, F., & Chiodo, F. (2004). Cerebral coenurosis. Case illustration. Journal of Neurosurgery , 100 (5), 964.

Sandground, J. H. (1937). On a coenurus from the brain of a monkey. The Journal of Parasitology , 23 (5), 482-490.

Santamaría, E., Plancarte, A., & Aluja, A. S. (2002). The experimental infection of pigs with different numbers of Taenia solium eggs: immune response and efficiency of establishment. Journal of Parasitology , 88 (1), 69-73.

Sapolsky, R. M. (1993). The physiology of dominance in stable versus unstable social hierarchies. In W. A. Mason & S. P. Mendoza (Eds.), Primate Social Conflict, (pp. 171-204). State University of New York Press: Albany.

148

Schalk, G., & Forbes, M. R. (1997). Male biases in parasitism of mammals: effects of study type, host age, and parasite taxon. Oikos , 67-74.

Schall, J. J. (1983). Lizard malaria: cost to vertebrate host's reproductive success. Parasitology , 87 (01), 1-6.

Schmid-Hempel, P., & Ebert, D. (2003). On the evolutionary ecology of specific immune defence. Trends in Ecology & Evolution , 18 (1), 27-32.

Schmidt, G. D. (1986). CRC Handbook of Tapeworm Identification . CRC Press, Inc.

Schneider-Crease, I. A., Griffin, R. H., Gomery, M. A., Bergman, T. J., Beehner, J. C. High mortality and low reproductive success associated with parasitism in geladas (Theropithecus gelada ) in the Simien Mountains National Park, Ethiopia. In review.

Schneider-Crease, I. A., Griffin, R. H., Gomery, M. A., Nunn, C. L., Bergman, T. J., Beehner, J. C . Identifying wildlife reservoirs of neglected taeniid tapeworms: non-invasive diagnosis of endemic Taenia serialis infection in wild primates. In preparation.

Schneider-Crease, I. A., Snyder-Mackler, N., Jarvey, J. C., & Bergman, T. J. (2013). Molecular identification of Taenia serialis coenurosis in a wild Ethiopian gelada (Theropithecus gelada ). Veterinary Parasitology , 198 (1), 240-243.

Shonkoff, J. P., Garner, A. S., Siegel, B. S., Dobbins, M. I., Earls, M. F., McGuinn, L., ... & Committee on Early Childhood, Adoption, and Dependent Care. (2012). The lifelong effects of early childhood adversity and toxic stress. Pediatrics , 129 (1), e232-e246.

Schwanz, L. E. (2006). Schistosome infection in deer mice ( Peromyscus maniculatus ): impacts on host physiology, behavior and energetics. Journal of Experimental Biology , 209 (24), 5029-5037.

Schwartz, B. (1926). A subcutaneous tumor in a baboon due to Multiceps larvae. Journal of Parasitology , 12 , 159.

Schwartz, B. (1927). A subcutaneous tumor in a primate caused by tapeworm larvae experimentally reared to maturity in dogs. Journal of Agricultural Research, 35 (5), 471–480.

149

Sciutto, E., Fragoso, G., Diaz, M. L., Valdez, F., Montoya, R. M., Govezensky, T., ... & Larralde, C. (1991). Murine Taenia crassiceps cysticercosis: H-2 complex and sex influence on susceptibility. Parasitology Research , 77 (3), 243-246.

Sciutto, E., Fragoso, G., Fleury, A., Laclette, J. P., Sotelo, J., Aluja, A., ... & Larralde, C. (2000). Taenia solium disease in humans and pigs: an ancient parasitosis disease rooted in developing countries and emerging as a major health problem of global dimensions. Microbes and Infection , 2(15), 1875-1890.

Scott, H. H. (1926). Report on deaths occurring in the Society's Gardens during the year 1925. Proceedings of the Zoological Society of London, 96(1), 231-244.

Sheldon, B. C., & Verhulst, S. (1996). Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology. Trends in Ecology & Evolution , 11 (8), 317- 321.

Simpson, E. R., Mahendroo, M. S., Means, G. D., Kilgore, M. W., Hinshelwood, M. M., Graham-Lorence, S., ... & Mendelson, C. R. (1994). Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis. Endocrine Reviews , 15 (3), 342- 355.

Slocombe, R. F., Arundel, J. H., Labuc, R., & Doyle, M. K. (1989). Cerebral coenuriasis in a domestic cat. Australian Veterinary Journal , 66 (3), 92-93.

Smith, K. F., Sax, D. F., & Lafferty, K. D. (2006). Evidence for the role of infectious disease in species extinction and endangerment. Conservation Biology , 20 (5), 1349- 1357.

Smith, M. C., Bailey, C. S., Baker, N., & Kock, N. (1988). Cerebral coenurosis in a cat. Journal of the American Veterinary Medical Association , 192 (1), 82-84.

Snaith, T. V., Chapman, C. A., Rothman, J. M., & Wasserman, M. D. (2008). Bigger groups have fewer parasites and similar cortisol levels: a multi-group analysis in red colobus monkeys. American Journal of Primatology , 70 (11), 1072-1080.

Snyder-Mackler, N., Alberts, S. C., & Bergman, T. J. (2012a). Concessions of an alpha male? Cooperative defence and shared reproduction in multi-male primate groups. Proceedings of the Royal Society of London B: Biological Sciences , 279 (1743), 3788-3795.

150

Snyder-Mackler, N., Beehner, J. C., & Bergman, T. J. (2012b). Defining higher levels in the multilevel societies of geladas ( Theropithecus gelada ). International Journal of Primatology , 33 (5), 1054-1068.

Stearns, S. C. (1992). The Evolution of Life Histories (Vol. 249). Oxford University Press: Oxford.

Stearns, S. C., & Koella, J. C. (2007). Evolution in health and disease . Oxford University Press.

Steinkampf, M. P., Mendelson, C. R., & Simpson, E. R. (1987). Regulation by follicle- stimulating hormone of the synthesis of aromatase cytochrome P-450 in human granulosa cells. Molecular Endocrinology , 1(7), 465-471.

Stien, A., Irvine, R. J., Ropstad, E., Halvorsen, O., Langvatn, R., & Albon, S. D. (2002). The impact of gastrointestinal nematodes on wild reindeer: experimental and cross-sectional studies. Journal of Animal Ecology , 71 (6), 937-945.

Svensson, E., Råberg, L., Koch, C., & Hasselquist, D. (1998). Energetic stress, immunosuppression and the costs of an antibody response. Functional Ecology , 12 (6), 912-919.

Struhsaker, T. T. (1969). Correlates of ecology and social organization among African cercopithecines. Folia Primatologica , 11 (1-2), 80-118.

Tappe, D., Berkholz, J., Mahlke, U., Lobeck, H., Nagel, T., Haeupler, A., ... & Poppert, S. (2016). Molecular identification of zoonotic tissue-invasive tapeworm larvae other than Taenia solium in suspected human cysticercosis cases. Journal of Clinical Microbiology , 54 (1), 172-174.

Templeton, A. C. (1968). Human coenurus infection: a report of 14 cases from Uganda. Transactions of the Royal Society of Tropical Medicine and Hygiene , 62 (2), 251-255.

Terrazas, L. I., Bojalil, R., Govezensky, T., & Larralde, C. (1994). A role for 17-β-estradiol in immunoendocrine regulation of murine cysticercosis ( Taenia crassiceps ). The Journal of Parasitology , 563-568.

151

Terrazas, L. I., Bojalil, R., Govezensky, T., & Larralde, C. (1998). Shift from an early protective Th1-type immune response to a late permissive Th2-type response in murine cysticercosis ( Taenia crassiceps ). The Journal of Parasitology , 84(1), 74-81.

Terrazas, L. I., Bojalil, R., Rodriguez-Sosa, M., Govezensky, T., & Larralde, C. (1999a). Taenia crassiceps cysticercosis: a role for prostaglandin E2 in susceptibility. Parasitology Research , 85 (12), 1025-1031.

Terrazas, L. I., Cruz, M., Rodriguez-Sosa, M., Bojalil, R., Garcia-Tamayo, F., & Larralde, C. (1999b). Th1-type cytokines improve resistance to murine cysticercosis caused by Taenia crassiceps . Parasitology Research , 85 (2), 135-141.

Therneau T. (2015). A Package for Survival Analysis in S. version 2.38. http://CRAN.R- project.org/package=survival

Therneau, T. M., Grambsch, P. M., & Pankratz, V. S. (2003). Penalized survival models and frailty. Journal of Computational and Graphical Statistics , 12 (1), 156-175.

Thompson, J. N. (1994). The coevolutionary process . University of Chicago Press.

Thornhill, J. A., Jones, J. T., & Kusel, J. R. (1986). Increased oviposition and growth in immature Biomphalaria glabrata after exposure to Schistosoma mansoni . Parasitology , 93 (03), 443-450.

Toft, C. A. (1986). Communities of species with parasitic life-styles. In J. Diamond, & T. J. Case (Eds.), Community ecology (pp. 445-463). Harper and Row: New York.

Toft, C. A. (1991). An evolutionary perspective: the population and community consequences of parasitism. In C. A. Toft, A. Aeschilmann, & L. Bolis (Eds.), Parasite-Host Associations, (pp. 319-343). Oxford University Press: Oxford.

Toft, C. A., & Karter, A. J. (1990). Parasite-host coevolution. Trends in Ecology & Evolution , 5(10), 326-329.

Tompkins, D. M., Dunn, A. M., Smith, M. J., & Telfer, S. (2011). Wildlife diseases: from individuals to ecosystems. Journal of Animal Ecology , 80 (1), 19-38.

Torgerson, P. R., Gulland, F. M. D., & Gemmell, M. A. (1992). Observations on the epidemiology of Taenia hydatigena in Soay sheep on St Kilda. Veterinary Record , 131 (10), 218-219.

152

Torgerson, P. R., Pilkington, J., Gulland, F. M. D., & Gemmell, M. A. (1995). Further evidence for the long distance dispersal of taeniid eggs. International Journal for Parasitology , 25 (2), 265-267.

Torgerson, P. R., Williams, D. H., & Abo-Shehada, M. N. (1998). Modelling the prevalence of Echinococcus and Taenia species in small ruminants of different ages in northern Jordan. Veterinary Parasitology , 79 (1), 35-51.

Tung, J., Archie, E. A., Altmann, J., & Alberts, S. C. (2016). Cumulative early life adversity predicts longevity in wild baboons. Nature Communications , 7.

Urbain, A., & Bullier, P. (1935). Un cas de cenurose conjonctive chez un gelada. Bulletin de l’Académie Vétérinaire de France , 8(6), 322-324.

van der Most, P. J., de Jong, B., Parmentier, H. K., & Verhulst, S. (2011). Trade-off between growth and immune function: a meta-analysis of selection experiments. Functional Ecology , 25 (1), 74-80.

Van Kerckhoven, I., Vansteenkiste, W., Claes, M., Geerts, S., & Brandt, J. (1998). Improved detection of circulating antigen in cattle infected with Taenia saginata metacestodes. Veterinary Parasitology , 76 (4), 269-274.

Van Schaik, C. P. (1983). Why are diurnal primates living in groups? Behaviour, 87 (1), 120-144.

Vargas-Villavicencio, J. A., Larralde, C., De León-Nava, M. A., & Morales-Montor, J. (2005). Regulation of the immune response to cestode infection by progesterone is due to its metabolism to estradiol. Microbes and Infection , 7(3), 485-493.

Vargas-Villavicencio, J. A., Larralde, C., De León-Nava, M. A., Escobedo, G., & Morales- Montor, J. (2007). Tamoxifen treatment induces protection in murine cysticercosis. Journal of Parasitology , 93 (6), 1512-1517.

Vijendravarma, R. K., Narasimha, S., & Kawecki, T. J. (2009). Effects of parental larval diet on egg size and offspring traits in Drosophila. Biology Letters , rsbl20090754.

Walsh, P. D., Abernethy, K. A., Bermejo, M., Beyers, R., De Wachter, P., Akou, M. E., ... & Lahm, S. A. (2003). Catastrophic ape decline in western equatorial Africa. Nature , 422 (6932), 611-614.

153

Walsh, P. D., Bermejo, M., & Rodriguez-Teijeiro, J. D. (2009). Disease avoidance and the evolution of primate social connectivity: Ebola, bats, gorillas, and chimpanzees. In M. A. Huffman, & C. A. Chapman (Eds.), Primate parasite ecology: The dynamics and study of host-parasite relationships (pp. 183-198). Cambridge University Press.

Wardle, R. A., McLeod, J. A., & Radinovsky, S. (1975). Advances in the zoology of tapeworms, 1950-1970 . University of Minnesota Press.

Watson, K., & Laurie, W. (1955). Cerebral coenuriasis in man. The Lancet , 266 (6904), 1321- 1322.

Wenk, P., & Renz, A. (2013). Parasitism and evolution: opposing versus balancing strategies. Historical Biology , 25 (2), 251-259.

Wickler, W. (1976). Evolution-oriented Ethology, Kin Selection, and Altruistic Parasites. Ethology , 42 (2), 206-214.

Wills, J. (2001). Coenurosis in a pet rabbit. The Veterinary Record , 148 (6), 188.

Wilson, V. C. L. C., Wayte, D. M., & Addae, R. O. (1972). Human coenurosis—The first reported case from Ghana. Transactions of the Royal Society of Tropical Medicine and Hygiene , 66 (4), 611-623.

Windsor, D. A. (1998). Controversies in parasitology, most of the species on Earth are parasites. International Journal for Parasitology , 28 (12), 1939-1941.

Wright, P. C., Arrigo-Nelson, S. J., Hogg, K. L., Bannon, B., Morelli, T. L., Wyatt, J., ... & Ratelolahy, F. (2009). Habitat disturbance and seasonal fluctuations of lemur parasites in the rain forest of Ranomafana National Park, Madagascar. In M. A. Huffman & C.A. Chapman (Eds.), Primate Parasite Ecology , (pp. 311-330). Cambridge University Press: Cambridge.

Yihune, M., & Bekele, A. (2012). Diversity, distribution and abundance of rodent community in the Afro-alpine habitats of the Simien Mountains National Park, Ethiopia. International Journal of Zoological Research , 8(4), 137-149.

Zhang, L., Hu, M., Jones, A., Allsopp, B. A., Beveridge, I., Schindler, A. R., & Gasser, R. B. (2007). Characterization of Taenia madoquae and Taenia regis from carnivores in Kenya using genetic markers in nuclear and mitochondrial DNA, and their

154

relationships with other selected taeniids. Molecular and Cellular Probes , 21 (5), 379- 385.

Zuk, M. (2009). The sicker sex. PLoS Pathogens , 5(1), e1000267.

Zuk, M., & McKean, K. A. (1996). Sex differences in parasite infections: patterns and processes. International Journal for Parasitology , 26 (10), 1009-1024.

Zuk, M., & Stoehr, A. M. (2010). Sex differences in susceptibility to infection: an evolutionary perspective. In S. L. Klein, & C. W. Robert (Eds.), Sex hormones and immunity to infection (pp. 1-17). Springer: Berlin.

155

Biography

India Schneider-Crease was born in New York, New York on August 31 st , 1987.

She obtained a Bachelor of Arts ( summa cum laude) in 2009 from Stony Brook University with a double major in Philosophy and French Language and Literature. She has published the first chapter of this dissertation ( Molecular identification of Taenia serialis coenurosis in a wild Ethiopian gelada (Theropithecus gelada ) in Veterinary Parasitology . In

2012, Schneider-Crease was awarded the Graduate Research Fellowship from the

National Science Foundation (NSF). She received additional research funding throughout her graduate career from the Department of Evolutionary Anthropology at

Duke University (2012, 2013, 2015), the Primate Action Fund (2013), the Margot Marsh

Biodiversity Foundation (2014), the Nacey Maggioncalda Foundation (2014), and

Primate Conservation International (2016). She has been awarded the National Science

Foundation Postdoctoral Research Fellowship to continue research on health, immunity, and early life adversity in geladas.

156