Leontocebus Weddelli)

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Summer 2020

The Seasonality and Parasite Richness and Prevalence of the Weddelli’s Saddleback Tamarin (Leontocebus Weddelli)

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Recommended Citation Banda, Krista, "The Seasonality and Parasite Richness and Prevalence of the Weddelli’s Saddleback Tamarin (Leontocebus Weddelli)" (2020). All Master's Theses. 1391. https://digitalcommons.cwu.edu/etd/1391

This Thesis is brought to you for free and open access by the Master's Theses at ScholarWorks@CWU. It has been accepted for inclusion in All Master's Theses by an authorized administrator of ScholarWorks@CWU. For more information, please contact [email protected]. THE SEASONALITY AND PARASITE RICHNESS AND PREVALENCE OF THE

WEDDELLI’S SADDLEBACK TAMARIN (LEONTOCEBUS WEDDELLI)

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A Thesis

Presented to

The Graduate Faculty

Central Washington University

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In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Primate Behavior

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Krista Emaly Banda

July 2020 CENTRAL WASHINGTON UNIVERSITY

Graduate Studies

We hereby approve the thesis of

Krista Emaly Banda

Candidate for the degree of Master of Science

APPROVED FOR THE GRADUATE FACULTY

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Dr. Gabrielle Stryker, Committee Chair

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Dr. Lori K. Sheeran

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Dr. Mrinalini Watsa

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Dr. Kristina Ernest

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Dr. Kevin Archer, Dean of Graduate Studies

ABSTRACT

THE SEASONALITY AND PARASITE RICHNESS AND PREVALENCE OF THE

WEDDELLI’S SADDLEBACK TAMARIN (LEONTOCEBUS WEDDELLI)

Krista Emaly Banda

July 2020

This study surveyed the intestinal helminths (parasitic worms) of Weddelli’s saddleback tamarins (Leontocebus weddelli), focusing on seasonality in parasite prevalence and richness.

The collaborative study with Field Projects International took place at the Estación Biológica Rio

Los Amigos (EBLA) in southeastern Peru. Fecal samples were collected by following semi- habituated groups of tamarins, yielding 16 samples in the dry season of 2015 and 11 samples in the wet season of 2015-2016. Findings were interpreted to understand trends for parasite prevalence and richness between the two seasons; however, novel helminths for the study species were observed. Trends were interpreted with consideration to the helminth’s longevity, study organisms’ home range use, and host behaviors.

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ACKNOWLEDGMENTS

This research was partially supported by the School of Graduate Studies and Research of

Central Washington University, Ellensburg, Washington. Funding for this research was provided by a Graduate Student Summer Research Grant awarded by the School of Graduate Studies and

Research, a conference travel grant awarded by the School of Graduate Studies and Research, and the Arlen and Debra Prentice Scholarship for Primate Research. I dedicate this thesis to future students and researchers for whom it may benefit.

Many thanks to my graduate committee for assisting me every step of the way; Dr.

Gabrielle Stryker, Dr. Lori K. Sheeran, Dr. Kristina Ernest, Dr. Mrinalini Watsa. I would like to thank Dr. Mrinalini Watsa and Dr. Gideon Erkenswick at Field Project International, without which this project would not be possible. A huge thank you to my field and laboratory assistants:

Sabrina Moore, Falon Sanderson, Alexandra Sheldon, and Imani Russell. I am grateful to have wonderful coworkers at Mt Stuart Animal Hospital for their moral support these past few years.

Thank you to my loving partner, Tasha Gentry, for your continued love and support.

I will forever be thankful for two important moments on this journe: the day Dr. Gabrielle

Stryker took me on as a student and the day we ran into one another again which reset this project in motion. No amount or combination of words can ever be enough to express my gratitude to Dr. Stryker for all she has done. The amount of advocacy and guidance she provided truly surpasses the role of a mentor, even a great one. Thank you, Dr. Stryker. I appreciate you.

TABLE OF CONTENTS

Chapter Page

I INTRODUCTION ...... 1

Parasitology ...... 3

II LITERATURE REVIEW ...... 10

Primate Parasite Ecology ...... 10 Seasonality and Primates ...... 12 Intestinal Parasites of Tamarins ...... 16

III METHODS ...... 22

Study Organisms and Study Site ...... 22 Fecal Sample Collections ...... 25 Ethics Note ...... 28

IV RESULTS ...... 29

V DISCUSSION ...... 38

Parasite Prevalence ...... 38 Parasite Richness ...... 39 Acanthocephala, Prostenorchis sp...... 39 Cestoda, Hymenolepsis sp ...... 40 Nematoda, Ancyclostoma/Necator sp ...... 42 Nematoda, Ascaris sp ...... 43 Nematoda, Larval Forms ...... 44 Nematoda, Oxyuridae ...... 44 Nematoda, Oxyuridae Unknown ...... 45 Nematoda, Spiruridae ...... 46 Nematoda, Strongyloidae ...... 46 Trematoda, Fasciola/Fasciolopsis sp...... 47 Range Use Between Seasons ...... 49 M2 ...... 49

VI CONCLUSION ...... 51

Recommendations for Future Research ...... 52

TABLE OF CONTENTS (continued)

Chapter Page

REFERENCES ...... 54

LIST OF TABLES

Table Page

1 Parasite Studies of Callitrichids of the Past 20 Years ...... 18

2 Total Fecal Samples Collected ...... 29

3 Description of Parasite Categories ...... 30

4 Parasite Richness of Individuals Observed in Both Seasons ...... 33

5 Parasite Prevalence ...... 34

6 Parasite Burden for All Fecal Samples ...... 36

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LIST OF FIGURES

Figure Page

1 Life Cycles of the Protozoan Parasites Giardia, Cryptosporidium,

Plasmodium, and Trypanosoma brucei...... 5

2 Life Cycle of Strongyloides fuelleborni...... 7

3 Comparison of Larval Rhabditiform and Filariform Stages of

Representative Hookworm and Strongyloides ...... 8

4 Filariform Stage Comparison Between Hookworm and Strongyloides ...... 9

5 Location of Field Site and Nearby Field Sites ...... 22

6 Trail Map of the Estación Biológica Rio Los Amigos ...... 24

7 Range Use in the Dry vs the Wet Season for One Group of

Leontocebus weddelli ...... 37

8 Life Cycle of Fasciola sp...... 48

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CHAPTER I

INTRODUCTION

Understanding the environmental factors that influence parasite prevalence and diversity is imperative for long-term primate and ecological conservation. Global climate change and anthropogenic influences simultaneously bring about unprecedented change in tropical ecosystems while also influencing the ecological conditions for disease proliferation (Wiederholt

& Post, 2009). Parasitological studies in primates require thorough, long-term monitoring along with standardized collection and analyzing protocols to allow valid comparisons among studies

(Stuart & Strier, 1995). Many studies that have focused on the intestinal parasites of wild populations of African apes (Kalema-Zikusoka et al., 2005; Petrášová et al., 2010), baboons

(Ravasi, 2009; Howells et al., 2011; Drewe, 2012), colobus (Gillespie et al., 2005; Chapman et al., 2006), and howlers (Stoner, 1996; Stuart et al., 1998; Rondón et al., 2017). However, outside of these groups, the intestinal parasites of other taxa are scarce beyond brief surveys.

In this study, we examined the seasonality of gastrointestinal parasites of saddleback tamarins (Leontocebus weddelli) residing in the Estación Biológica Rio Los Amigos (EBLA) in the Madre de Dios Department of southeastern Peru. The study aimed to evaluate seasonality by

1) surveying the parasite prevalence, defined as proportion of infected hosts (Margolis et al.,

1982; Gillespie, 2004), and 2) identifying parasite richness, defined as the number of different species per host (Margolis et al., 1982; Bush et al. 1997).

Tamarins are small, neotropical primates distributed throughout the Amazon Basin and utilize mixed forest types (Peres, 1992; Rylands, 1996). Weddelli’s saddleback tamarin

(Leontocebus weddelli) received two consecutive reclassifications in recent years from comprehensive genetic analyses; originally from Saguinus fuscicollis weddelli to Saguinus

1 weddelli (Matauschek et al., 2011) to Leontocebus weddelli (Rylands et al., 2016). Saddleback tamarins are in the family Callitrichidae (tamarins and marmosets), known for cooperative breeding and habitual twinning. Callitrichids maintain cooperative breeding systems through the combination of polyandry (Terborgh, 1983; Goldizen et al., 1996) and reproductive suppression of other females (Savage et al., 1988). Saddleback tamarins have one dominant, breeding female with one or more adult males (breeding and nonbreeding) and offspring. Both males and females disperse, and the nonbreeding individuals are believed to be offspring of the dominant female

(Garber et al., 1993). Multiple studies have explored this unique breeding system in saddleback tamarins, emperor tamarins (Saguinus imperator), cotton-top tamarins (S. oedipus), and moustached tamarins (S. mystax) populations in both captive and wild settings (Terborgh, 1983;

Garber, 1984; Terborgh & Goldizen, 1985; Goldizen et al., 1996).

Due to their small sizes, tamarins have a fast metabolism requiring a nutrient rich diet

(Rylands, 1996). Tamarins are omnivorous and feed primarily on fruit and invertebrates (Garber et al., 1993; Rylands, 1996). Their home range depends heavily on the resources available. In nutrient poor areas, their home ranges can encompass marshlands to terra firme. Due to their eating patterns, tamarins display territorial behaviors with conspecifics (Terborgh, 1983;

Goldizen, 1989; Soini, 1987) but are observed to be sympatric with other species of Saguinus and Callicebus (Terborgh, 1983; Sussman & Kinzey, 1984; Porter, 2004; Nadjafzadeh &

Heymann, 2008; Watsa, 2013).

Early parasitological literature has consistently highlighted the importance of host population density and correlated density with the abundance of intestinal parasites (Arneberg et al., 1998; Freeland, 1979). Primates generally have close-knit social groups which may facilitate parasite transmission (Arneberg et al., 1998; Chapman et al., 2012; Freeland 1979). More studies

2 are appreciating parasitology through ecological influences. Seasonal variation is one of the most prominent external sources influencing ecological systems. Seasonal variation can influence parasite-host interactions through the regular, cyclic fluctuations in temperature, humidity, and rainfall (Weiderholt & Post, 2009). Considering the naturally occurring external pressure of seasonal variation as a mediating factor, a more comprehensive, ecological context can better help us understand the relationship between behavioral changes (i.e., feeding preference and grooming frequency) and observed parasite prevalence and richness.

Habitat structure may also influence hosts’ susceptibility to certain parasites. Variables such as humidity, vegetation type, and invertebrate vectors can influence prevalence (proportion of infected populations for a given parasite) and incidences (number of new infections) of helminths (Appleton & Henzi, 1993; Stoner, 1996; Weiderholt & Post, 2009). While varying ecological zones may encourage different intestinal parasites, it is possible that only a small proportion of the host population may harbor most of the helminth population, especially in fragmented or degraded habitats (Gillespie & Chapman, 2008). Therefore, longitudinal research on all individuals within a population is necessary to ascertain any correlation with parasite prevalence and abundance.

Parasitology

A parasite is an organism that lives in/on another organism, known as the host, at the host’s expense (Centers of Disease Control, 2016). There are three general categories of parasites: protozoans, helminths/endoparasites, and ectoparasites (Centers of Disease Control,

2016). However, many include eukaryotes and viruses as parasites. For the purposes of this paper, we will exclude those arguments and focus primarily on intestinal helminths

(endoparasites) while also providing an overview on literature including protozoans and

3 ectoparasites. The overall general description of a parasite in this paper is a symbiotic organism living at the detriment, usually physical and/or nutritional, of its host.

Protozoans are unicellular eukaryotes that are usually commensal or enteric. Protozoans pass through several broad stages in their life cycle transmission; trophozoites and cysts are normally the pathogenic phase that actively feed and multiply. The simple Giardia life cycle

(Figure 1a) includes a pathogenic trophozoite stage in the digestive tract where the Giardia cysts are shed into the environment and survive until ingested by another host (Bowman, 1999). The

Cryptosporidium life cycle (Figure 1b) adds complexity as reproduction can be both sexual and asexual before shedding into the environment as an oocyst. Hemoparasites such as Plasmodium

(Figure 1c) and Trypanosoma brucei (Figure 1d) include both sexual and asexual stages along with an intermediate host.

Not all parasites require the assistance of intermediate hosts in their development, but for

Plasmodium and Trypanosoma spp. it is a necessity. Some parasites, including many helminths, ectoparasites (e.g., Dermatobia hominis), and protozoans, have complicated life cycles that can be developed only with the help of a specific intermediate host. The life cycle of some parasites relies on infecting a single host species, many can be ingested by unintended host species, otherwise known as a dead-end (accidental or incidental) host species, thereby halting parasite transmission to other individuals. For example, Trypanosoma brucei requires development in the

Tsetse fly before transmission to a human host to reach its final developmental stage (Bowman,

1999).

Figure 1. Life Cycles of the Protozoan Parasites Giardia, Cryptosporidium, Plasmodium, and

Trypanosoma brucei (Goodman & McFadden, 2008).

Understanding the life cycles of parasites provides key information in understanding the ecological influences on parasite infections and encounters. Cryptosporidium and Giardia are associated with contaminated water. These two protozoan parasites are transmitted through the fecal-oral route, implying that infection with this parasite is through fecal-contaminated food or water. Giardia cysts and Cryptosporidium oocysts can remain infective in the environment for up to three months. Knowing these life cycle influences can help researchers understand how host species can encounter these parasites in their environment.

Ectoparasites include arthropods that can take quick blood meals from a host (e.g. mosquitos) but more generally describe organisms that latch or burrow onto a host for extended periods of time (e.g. ticks, lice, and botflies) lasting anywhere from days to a lifetime (Centers for Disease Control, 2016). These arthropods can also act as a vector for other parasites, as seen in the relationship between the protozoan Trypanosoma brucei requiring transmission through the ectoparasite Tsetse fly to find its way to a human host (Figure 1d). Most early primatology literature on parasites focused on primate aggregation patterns and ectoparasites. Several studies analyzed the associations between primate population density and ectoparasite infections, essentially questioning if higher parasite infections were observed in denser host populations

(Seyfarth, 1977; Freeland, 1979). Others added grooming strategies as a mediating factor, positing grooming as a social function to mitigate parasitic infection (Seyfarth, 1977; Freeland,

1981; Dunbar, 1991; Akinyi et al, 2013).

Helminths are worms that live inside the host. The Centers for Disease Control (2016) divides helminths in three broad categories: flatworms (i.e., platyhelminths that include trematodes and tapeworms), roundworms (i.e., nematodes), and thorny-headed worms (i.e., acanthocephalans). The life cycles of these parasites may be complex and vary among those

6 three categories. The most abundant helminths, nematodes, generally follow the same cycle of egg, rhabditiform, infective filariform, and finally adult worm (Figure 2).

Figure 2. Life Cycle of Strongyloides fuelleborni (Centers for Disease Control, 2019 July 30)

Microscopically, helminths can be broadly identified through tracking their life cycle stages. Unfortunately, many helminths are morphologically indistinguishable, necessitating molecular analysis for definitive identification. Any morphological analysis solely on helminth eggs should be proceeded with caution. Fecal samples that include various stages of development including eggs, rhabditiform, and filariform provide more information on the parasites’ identities. For example, a comparison of larval rhabditiform and filariform stages of hookworms and Strongyloides show no discernable differences (Figure 3). Only in the filariform stage are minute differences noticeable in the shape of the mouth, length of the mouth-to-esophagus, and tail shape. Even then, the differences are difficult to discriminate at this current stage, adding to

7 the necessity for caution in morphological interpretation. Figure 4 illustrates how morphologically similar the filariform stages are to one another. Nevertheless, the differences between the buccal cavity, the length of the esophagus, and the shape of the tail provide key distinctions between various parasites.

Figure 3. Comparison of Larval Rhabditiform and Filariform Stages of Representative

Hookworm and Strongyloides (Centers for Disease Control, 2019 July 30).

Figure 4. Filariform Stage Comparison Between Hookworm and Strongyloides (Parasites In

Humans, 2015)

This thesis provides a baseline survey of helminths in wild Peruvian tamarins collected during both the wet and dry seasons. Without molecular analysis, morphological interpretations must taken with caution. It is still invaluable to providing a baseline understanding of what parasites are found in the study population.

CHAPTER II

LITERATURE REVIEW

Primate Parasite Ecology

Infectious disease is a growing concern in conservation work, with trends suggesting that anthropogenic activities influence disease transmission (Stuart & Strier, 1995; Poulin, 2001;

Nunn et al., 2003; Smith et al., 2012; Rondón et al., 2017). Potential cross-species disease transmission presents a concern to both humans and nonhuman primates, as human settlements increasingly expand throughout the world. For primates threatened by habitat loss and fragmentation, anthropogenic activity and host density affects parasite prevalence and richness

(Mbora & McPeek, 2009). An understanding of epidemiology and parasite ecology is necessary to mitigate infectious disease in nonhuman primates and decrease pathogen pressure. Social interactions and habitat use can be used to evaluate patterns of parasitism, although relatively few experimental studies on wild populations exist (Janson, 2000). Due to small sample sizes, findings and interpretations may only be relevant for that study population. Any generalizable observations obtained from these studies must be cautiously analyzed.

Analyzing past primate ecology data is difficult due to varying methodologies between studies (e.g., fecal flotation and sedimentation methods). Gillespie (2006) called for standardization of methods in field parasite research. Gillespie noted the different fecal collection methods of previous research and argued the necessity to collect fresh fecal samples to avoid accidentally collecting free living parasites. Most importantly, Gillespie called for fecal analysis standardization to allow for consistent analysis throughout different studies. Current primate parasite studies report findings as frequencies while others are percentages within the population, some studies analyze parasite abundances incorrectly, or use solutions that may

10 degrade the parasites. Since primate studies tend to have relatively small sample sizes due to the nature of studying wild primates and reliably collecting identifiable fecal samples, many primate studies are immediately ineligible for direct comparison with other studies and only hold internal validity within that singular population. By creating a consistent standard, field researchers can add to the previous studies with stronger reliability.

Despite these hardships, it is still important to study the parasites of primates. Humans and nonhuman primates share a close phylogenetic relationship resulting in a high potential for pathogen exchange (Lafferty & Holt, 2003; Gillespie et al., 2005; Kalema-Zikusoka et al., 2005;

Mbora & McPeek 2009; Drewe et al., 2012). As humans continue to encroach into the habitat of nonhuman primates and irreversibly influence climate change, more studies have focused on emerging infectious diseases with either human or nonhuman primate origins (Stoner, 1996;

Lafferty & Kuris, 2005; Gillespie, 2006; Chapman et al., 2012; Figueiredo et al., 2017; Hanes et al., 2018). Providing baseline data on naturally occurring parasites in nonhuman primates, ecological variables influencing host-parasite relationships, and behavioral adaptations influencing parasite susceptibility, are imperative to alerting conservationists to threats to primate and ecological conservation.

Public health and conservation efforts have focused on the One Health approach, which calls for a collaborative, “trans-disciplinary approach—working at the local, regional, national and global levels—with the goal of achieving optimal health outcomes recognizing the interconnections between people, animals, plants, and their shared environment” (Centers for

Disease Control, 2020). This approach necessitates the expertise and collaboration of researchers, veterinarians, public health officials, academics, ecologists, cultural anthropologists, and rangers (Bardosh et al., 2014; Woldehanna & Zimicki, 2015; Kelly et al., 2016).

Veterinarians and epidemiologists have been on the forefront of the approach, from firsthand observation of the human-animal health interface (Kelly et al., 2016) to the inclusion of ecosystem health (Cook et al., 2004; Gibbs, 2013). This perspective focuses on anthropogenic and ecological variables in regards to organism health.

Seasonality and Primates

Early parasitological studies on nonhuman primates examined host density mediated by parasite prevalence and diversity (Morand & Poulin, 1998; Packer et al., 1999). Recent ecological studies have shown that habitat characteristics may be a better predictor of parasite infection than is primate density (Stuart et al., 1993; Gilbert, 1997; Gillespie, 2004; Gillespie &

Chapman, 2005). Environmental variables can cause indirect or direct changes to host-parasite relationships (Weiderholt & Post, 2009). Changes in elevation and rainfall can indirectly influence those relationships by influencing host and vector migration and aggregation (Appleton

& Henzi, 1993; Bennett et al., 2016). Soil attributes (e.g., humidity, chemical make-up, and temperatures) can influence the parasite eggs’ survival. Certain parasite eggs, for example

Ascaris spp., are extremely hardy able to survive low temperatures, varying soil humidity levels, and chemical changes, but not all parasites are as sturdy. More direct changes like temperature and humidity can influence host susceptibility to disease. Rainfall fluctuations impact resource availability and indirectly influence host migration patterns. These ecological variables are pertinent considerations in studying host-parasite relationships of wild populations.

Forest structure influences the home range use and health of nonhuman primates.

Fragmented forests harbored primates exhibiting a lowered parasite load than those in continuous forests (Gillespie & Chapman, 2008; Bublitz et al., 2014). Differences in gastrointestinal parasite abundance between chacma baboon (Papio cynocephalus) was observed populations

12 living at different elevations (Appleton & Henzi, 1993). Several groups of mantled howler monkeys (Aloutta palliata) living in more humid environments of Costa Rica was found to have increased parasite diversity as opposed to their arid forest counterparts (Stoner, 1996). While

Rondón et al. (2017) did not find a statistically-significant relationship between parasite prevalence and seasonality (p = 0.066), they did find an effect on parasite richness (i.e., number of different parasite species per host) due to seasonality (p = 0.0001) for red howler monkeys

(Alouatta seniculus), brown spider monkeys (Ateles hybridus), and varied white-fronted capuchins (Cebus versicolor).

Seasonal variation impacts the availability of food sources. This in turn influences optimal foraging strategies and indirectly affects parasite richness and encounters. Mantled howler monkeys in the humid habitat of La Selva, Costa Rica have greater intestinal parasite prevalence than their counterparts in the drier habitat of La Pacifica, Costa Rica (Stoner, 1996).

The humid environment may provide the necessary habitat for the nematode and trematode eggs to survive extended periods in the external environment. The Stoner study suggested that the relationship between the hosts and parasitic infections are influenced by other factors beyond host density as popularized by Freeland’s (1979) Biological Island Hypothesis. Host-parasite relationships are more complex and requires critical analysis of the role of home range and movement patterns in influencing parasite prevalence and intensity.

Parasitological and epidemiological studies are moving away from the notion that host density is the strongest indicator for parasite transmission. In fact, extreme anthropogenic activities have led to the realization that ecological factors play a key role in parasite transmission. For example, there is a notable decline of population size in endangered red colobus (Procolobus rufomitratus) living in forest fragments compared to their counterparts in

13 unfragmented forests in Kibale National Park, Uganda (Gillespie & Chapman, 2008). Gillespie and Chapman found increased parasite prevalence in fragmented groups. As anthropogenic influences are driving rapid habitat loss, it is imperative to consider how habitat use and home ranges can influence parasite incidence (Chapman et al., 2005; Gillespie & Chapman, 2008;

Bublitz et al., 2014; Pafčo et al., 2018).

Parasitological studies in primates have provided information on both evolutionary and ecological relationships. Primate-parasite relationships are affected by climatic variation, anthropogenic influence (i.e., habitat fragmentation, logging, hunting), and diet among others. As a result, there is an increasing call to combine parasitology and ecology to address conservation biology and management concerns. Due to close social groups and high densities, primates are particularly susceptible to parasitic infections (Freeland, 1979; Arneberg et al., 1998; Chapman et al., 2012). As a result, they are highly susceptible to habitat disturbance and climatic variation, which may also influence incidences of parasitism. Bublitz et al. (2014) found an association with anthropogenic disturbance and increased incidence of enterobacteria in wild lemur populations. Edge areas along human settlement and agriculture tend to have increased infection rates. Anthropogenic activities that contaminate water sources could extend human influences further than the immediate areas around human settlements.

Seasonal variation impacts ectoparasite and endoparasite loads differently. Ectoparasites

(e.g., lice and ticks) live outside the host and are susceptible to ecological conditions. They can decrease host fitness and act as vectors for viruses, bacteria, protozoa, helminths, and cestodes

(Barrett et al, 2013). Wright et al. (2009) noted that lice infection intensity of Milne-Edward’s sifaka (Propithecus edwardsi) was higher during the warm/wet seasons compared to the dry season. The researchers also found that the subset populations living in disturbed areas had even

14 greater ectoparasite infection intensity during the warm/wet season. A study by Hokan et al.

(2018) observed only strongyle egg sheds during the known rainy season for the Western woolly lemur (Avahi occidentalis) with nearly 37.5% prevalence (n = 29), while a sympatric species of

Milne-Edward’s sportive lemur (Lepilemur edwarsi) observed Lemuricola spp. and Strongylida eggs in both seasons (n = 28).

Endoparasites are also sensitive to ecological changes. Differences in prevalence of intestinal endoparasites between seasons are indicative of a shorter lifespan. Intestinal parasites with longer lifespans could survive within a host for years, with no differences in egg outputs between seasons. Schwitzer et al. (2010) found that Callistoura sp. eggs of blue-eyed black lemurs (Eulemur flavifrons) were more abundant in the wet seasons than the dry seasons.

Populations living in secondary forests tended to have higher incidences of parasitism between seasons, similar to Wright et al. (2009). This suggests that living in disturbed environments could amplify naturally occurring cycles.

The gray mouse lemur (Microcebus murinus) showed higher gastrointestinal parasite abundance and diversity during the hot season as compared to the cold season (Raharivololona &

Ganzhorn, 2010). This difference was attributed to both the harsh environment of the cold season and the gray lemur’s ability to enter torpor and hibernate, which would limit new parasite encounters and possibly influence parasite metabolism and reproduction.

Increased nematode infection was found among chimpanzees of the Mahale Mountains in

Tanzania during the rainy season (Huffman et al., 1997). Over the course of five years, a population of approximately 90 individuals were studied and fecal samples were collected from

~48 individuals. While parasite egg abundance of Oesophagostomum stephanostomum was significantly higher in the rainy season (Fisher’s exact test, p = 0.0001) than the dry season (p =

0.01), prevalence of two other intestinal helminths (Trichuris trichuria and Strongyloides fuelleborni) showed no significant seasonal trends.

Stoner (1996a) found that howler monkeys (Aloutta palliata) living in moist environments (primary forest with swamps) had higher intestinal parasite intensity than their counterparts that did not have swamps in their home ranges. Stoner suggested that the differences could be due to microclimatic factors, ranging patterns, and home range size (1996a,

1996b). This was reaffirmed at the same field site by Maldonado- Lopez et al (2013) which studied the same howler monkey troops along with sympatric spider monkeys (Ateles geoffroyi).

The sympatric troops shared multiple similar parasite infections and prevalence, but unlike the earlier Stoner articles (1996a, 1996b), statistically significant differences of seasonality were not observed.

Studies in primate parasitology are still few and far in between. Researchers must consider ecological and behavioral influences that would affect or indicate seasonality. The

Maldonado-Lopez (2013) study updating the ongoing research on Stoner’s (1996a, 1996b) Costa

Rican howler monkey troops is a prime example of how micro-climatic instances (moist environment versus dry environment) can influence parasite prevalence and encounters, even if there were no statistically significant seasonal influence. It is pertinent to be cautious when analyzing for seasonality, as many seasonal attributes can also be attributed to micro-climatic changes.

Intestinal Parasites of Tamarins

Comprehensive, long term studies in wild primates are relatively rare due to the difficulty of field research in the Neotropics. It is incredibly difficult to study tamarins outside of the laboratory or captive setting (del Portillo & Damian, 1986) due to their small size and habitat

16 preferences. Tamarin home ranges are of mixed forest types, including bamboo forests, woodland forests, floodplain, and even palm swamps. Studies on intestinal helminths of neotropical primates have been collected in a laboratory setting but have recently shifted to wild populations to manage primate population health. The endemic Peruvian red uakari monkey

(Cacajao calvus ucayalii) have found eggs for Trypanoxyuris sp., Strongyloididae, Spirurid, and

Taeniidae (Conga et al., 2013). This population is threatened by increasing deforestation and habitat fragmentation. Peruvian red uakari’s small population size may also influence the parasite diversity and load found in this study.

A survey of the primate population in Peru’s Tambopata National Reserve have shown that primates with minimal direct contact with humans and in an area with no history of human activity shared similar parasite burden across species (Phillip et al., 2004). Five of the eight species of primates sampled had Strongyloides eggs, commonly found in nonhuman primates, which may indicate shared habitat and ranging behaviors. The saddleback tamarin (Saguinus weddelli) group that was followed in this study had four individuals. The researchers found eggs of unidentified strongyles, Trichuris sp., and the protozoan cysts of Iodamoeba sp. in their fecal samples.

The ecological impact of parasitism in urban tamarins are explored by Gordo et al. (2013) and Soto-Calderon et al. (2016), respectively on the pied tamarin (Saguinus bicolor) and white footed tamarin (Saguinus leucopus). Low parasite diversity was found in urban dwelling tamarins compared to their rural counterparts (Soto-Calderon et al., 2016). The body condition of the urban dwelling primates were rated higher than the rural-dwelling tamarins, suggesting that the lower nutritional stress may influence the urban dwelling tamarins’ ability to fight off new infection. Alternatively, the urban environment may provide fewer opportunities for infection.

Previous parasitological surveys on wild tamarins have documented their intestinal parasites. All tamarin parasite studies within the last 20 years (Table 1) used different modifications of fecal sedimentation methods except for Michaud et al. (2003), who opted for a direct smear.

Table 1. Parasite Studies of Callitrichids of the Past 20 Years

Primate Species Parasites Found Studies

Callithrix sp. (hybrid) Primasubulura jacchi (de Oliveira Tavela et al., 2013)

Prosthenorchis sp. (de Oliveira Tavela et al., 2013)

Ancyclostomatidae (de Oliveira Tavela et al., 2013)

Dilepididae (de Oliveira Tavela et al., 2013)

Callithrix jacchus Primasubulura jacchi (dos Santos Sales et al., 2010)

Callithrix geoffroyi Pterygodermatities (Sato et al., 2003)

nycticebi

Leontocebu weddelli/ Trichuris trichiura (Phillips et al., 2004)

Saguinus fuscicollis

Iodamoeba buetschii (Phillips et al., 2004)

Entamoeba spp. (Phillips et al., 2004)

Unidentified (Phillips et al., 2004)

strongyle

Hymenolepsis (Wenz et al., 2009)

(cebidarum)

Trypanosoma (Solorzano-Garcia & Perez-Ponce

minasense de Leon, 2018)

Table 1. (Continued)

Primate Species Parasites Found Studies

Leontocebus weddelli/ Dipetalonema sp. (Solorzano-Garcia & Perez-Ponce Saguinus fuscicollis de Leon, 2018) Mansonella mariae (Solorzano-Garcia & Perez-Ponce de Leon, 2018) Cestoda (Wenz et al., 2009)

Prosthenorchis sp. (Erkenswick et al., 2018)

Spirurid-like (Wenz et al., 2009; Erkenswick et al., 2018) Nematode larva (Wenz et al., 2009)

Strongylid (Wenz et al., 2009)

Saguinus labiatus Athesmia (Michaud et al., 2003)

heterolecithoides

Prosthenorchis (Michaud et al., 2003) elegans Filariopsis barretoi (Michaud et al., 2003)

Primasubulura jacchi (Michaud et al., 2003)

Saguinus mystax Primasubulura jacchi (Michaud et al., 2003)

Prosthenorchis (Michaud et al., 2003) elegans Filariopsis barretoi (Michaud et al., 2003)

Hymenolepsis (Michaud et al., 2003; Mucke, diminuta 2011; Wenz et al., 2009)

Table 1. (Continued)

Primate Species Parasites Found Studies

Saguinus mystax Spirometra spargana (Michaud et al., 2003)

Cryptosporidium sp. (Mucke, 2011)

“Large spirurid” (Mucke, 2011)

“Small spirurid” (Mucke, 2011)

Strongylid (Mucke, 2011)

Nematode larva (Mucke, 2011)

Leontopithecus rosalia Ancyclostomatidae (Monteiro et al., 2007)

Ascarididae (Monteiro et al., 2007)

Tripanoxyuris (Monteiro et al., 2007)

minutus

Onicola sp. (Monteiro et al., 2007)

Trichostrongylidae (Monteiro et al., 2007)

Spiruridae (Monteiro et al., 2007)

Fecal sedimentation is a process that passes the feces through a filtration process to separate fiber, debris, and seeds from the feces. This method makes it easier to spot and study just the parasites found in the samples. Fecal smears contain more artifact, making it harder to spot and accurately count the parasites in the sample. Fecal flotation can be done before fecal sedimentation. Flotation is performed by mixing the feces in a sugar solution so cysts, eggs, and/or trophozoites with a lighter density float to the top and adhere to a coverslip for analysis.

These lighter parasites would normally be missed in the fecal sedimentation method. The

20 staining technique for fecal smear, flotation, and sedimentation could also influence the ability of the researcher to spot certain parasites.

Protozoans are single-celled organisms. Many are free-living in nature while others live as commensals or parasites (Bowman, 1999; Centers for Disease Control, 2016). Protozoans are infrequently described in nonhuman primate literature, although enteric protozoans like amoebae, Cryptosporidium, and Giardia are prevalent globally. This is likely because of the collection and fecal analysis methods. Most field stations are not able to immediately analyze fecal samples and protozoans tend to quickly degrade in harsh fecal flotation and sedimentation solutions. Nevertheless, small traces of protozoans, such as Cryptosporidium sp., have been found in wild populations of the mustached tamarin (Saguinus mystax) in the Peruvian Amazon

(West et al., 2013). While the observed Cryptosporidium prevalence is low, this is the first documented observation in Amazonian primates. Cryptosporidium is a highly pathogenic waterborne protozoan parasite capable of infecting both humans and nonhuman primates.

Cryptosporidium is common in contaminated water, suggesting tamarins will drink from bodies of water.

Studies of the parasites of wild tamarins are scarce, and the majority are surveys (Phillips et al., 2004; Monteiro et al., 2007; Wenz et al., 2009; West et al., 2013) of the primate’s parasite prevalence and intensity. These studies did not explore other external variables that could influence parasite burden, but more studies are being now exploring those ecological variables.

Those original parasitological surveys are necessary to establish a baseline and apply ongoing behavioral and ecological data.

CHAPTER III

METHODS

Study Organisms and Study Site

The collaborative study with Field Project International was conducted at the Estación

Biológica Rio Los Amigos (EBLA), also known as the Centro de Investigación y Capacitación

Río Los Amigos (CICRA), in the Madre de Dios Department of southeastern Peru (Figure 5).

The station is on the confluence of the Madre de Dios and Los Amigos rivers. It boasts over

4000 recorded species and 11 primate species (ACCA, 2020). The field site was previously a logging site until it was acquired by the Asociación para la Conservación de la Cuenca

Amazónica (ACCA).

Figure 5. Location of Field Site and Nearby Field Sites (Plata & Lücking, 2012).

There are five broad forest types (Figure 6): mature woodland forest, secondary woodland forest, bamboo growth, palm swamps, floodplain, and successional forest (Pitman et al., 2001; Watsa, 2013). Mature woodland forests at EBLA averages 35m while secondary woodland growth have emergent trees averaging 25m. The forest composition for mature woodland is different from secondary growth woodland forests. Mature woodland forests are primarily comprised of Moraceae, Fabaceae, and Cecropiaceae, while secondary growth tend to consist of Cecropiaceae, Euphorbiaceae, and Melanstromataceae. Bamboo forests consist mostly of Guadua spp. typically found close to the field station and along the Río Los Amigos, evidence of the field site’s previous logging history. Palm swamps are primarily found by the oxbow lakes and are areas permanently flooded through terra firme forest. Floodplain emergent trees average

35m and are flooded part of the year. Successional forests are areas of secondary growth typically found along the riverbank and comprised of Cecropiaceae.

The project focused on one group of semi-habituated saddleback tamarins (Leontocebus weddelli) during two distinct seasons: dry (April to September) and wet (October to March) characterized by daily rainfall. All individuals are identifiable through Field Project

International’s (FPI) mark-recapture program that was implemented in 2009 (Watsa et al., 2015).

Tamarins are morphologically and nearly indistinguishable from one another from a quick physical observation. Adults measure 7-10 inches and offspring obtain adult size within a year.

The animals were all captured and released by group, typically taking up the entirety of the day.

Groups are captured annually and identified with their assigned HomeAgain microchip that allows researchers to identify individuals regardless of dispersal patterns and loss of temporary identification throughout years of study. The mark and recapture program have shown no negative consequences on the animals themselves even after long-term follows to evaluate post-

23 sedation well-being (Watsa, 2013), anecdotally evidenced by the tamarins immediately and repeatedly returning to the capture site.

Figure 6. Trail Map of the Estación Biológica Rio Los Amigos (ACCA, 2020).

Individuals are given two forms of individually unique, but temporary identification known by all researchers of Field Projects International. The tails of all individuals are bleached with distinctive banding to allow for intra-group identification, and beaded collars for intergroup identification (Watsa, 2013). The bleach banding lasts two to three months until the hair regrows. All individuals except for the dominant female are given beaded collars visible in the front and back of the neck. The visible collars allow the researchers to determine group membership, sex, and individual identification. The beaded collars are fitted to accommodate for weight fluctuations throughout the year and fall off once snagged. The only exception from the

24 collars are the dominant female of the group, who is instead fitted with a beaded radio collar.

Radio collars are given to dominant females of groups because tamarin groups are centered around one breeding female and thereby are unlikely to disperse. The radio collar weighs less than 3% of the average adult tamarin (Watsa, 2013). Procedures in radio collaring mammals suggest that the recommended limit of the radio collar should not exceed 5% of the animal’s body weight (Cuthill,1991). Whenever a known tamarin defecated, my field researchers and I determined their identification using both tail and beaded markers whenever possible. The radio collared individual allowed us to determine which group we are observing as they all have their own set frequencies.

Fecal Sample Collections

Through Field Projects International, I had a team of field assistants for both field seasons. We obtained fecal samples collected during the dry (June – September 2015) and wet

(December 2015 – January 2016) seasons. A fecal sample is defined as one fecal sample deposit by the one individual. All samples were collected opportunistically using both full (sleep-site to sleep-site) and half-day follows. A “follow” is defined as a single session in which the group is observed. Several focal groups were initially attempted before later focusing on one targeted follow group due to time and researcher limitations. Sleep-site to sleep-site follows allowed researchers to track the study group’s daily movements with minimal gap in knowledge, whereas half day follows allowed researchers to follow multiple groups from sleep-site to leisurely afternoon sessions then switching to another habituated group to determine their sleep-site for the next day’s follow. All follows were tracked with GPS units every 10 seconds, allowing researchers to notate time of observation, unique behaviors observed, and complete follow time.

Semi-habituated saddleback tamarin groups are familiar with the presence of humans, but do not venture close to the researcher. These primate groups are well studied by Field Projects

International and observed by other researchers at the Estación Biológica Rio Los Amigos, making noninvasive fecal collection possible, but requires intimate knowledge in identifying all individuals. These methods allowed researchers to monitor and noninvasively collect fecal samples during times subjects are most known to defecate (i.e., upon waking up and before choosing a sleep site). Researchers waited and observed the study organisms until they defecated. Upon which they there immediately identified by tail and/or beaded markers and collected from the forest floor. Fecal samples are placed and marked in individual plastic bags and then placed in a chilled thermos until returning to basecamp. Each sample was given an individual ID, noting time of collection, consistency, and study individual. All fecal samples used in the study are from known observed subjects verified by myself and my field assistants during the follow. Samples were fixed in 10% neutral buffered formalin upon the return to basecamp at the end of the day’s follow until export to the laboratory of Dr. Patricia Parker at the

University of Missouri-St. Louis for analysis.

I analyzed the fecal samples using fecal sedimentation to isolate cysts, eggs, and potential larva. A modified fecal flotation method with sodium nitrate (Specific Gravity 1.2) was used for all samples once and read immediately (Zajac and Conboy, 2012). I mixed 1g of the fecal sample with 10mL of 10% buffered formalin and poured the mixture into a 15mL centrifuge tube. I then added ethyl acetate until the tube is full. The tube is then capped and shaken approximately 50 times, then placed in the centrifuge for 5 minutes at 500 x g. There are three distinct layers at the end of this stage: (1) a top layer of ethyl acetate, debris, and fat; (2) a middle layer primarily of the formalin supernatant; and (3) the bottom layer with the sediment. I decant the supernatant to

26 get the bottom sediment. The rest of the bottom sediment is homogenized and then one drop is placed on a microscope slide, coverslip, and examined.

I read each fecal sedimentation samples twice and averaged for intra-rater reliability.

Photographs and measurements were taken of the first appearance of each helminth egg, protozoan cyst, larva, artifact, etc. for each sample for review and external reliability. For identification of larval nematodes, photographs captured the mouth, esophagus, tail, and finally the entirety of larva. This is necessary since nematodes have similar morphology as larvae, with subtle differences. All slides were read at 100x magnification and positive samples were followed up with 400x photography for identification.

All observations were cross referenced with previous parasitological publications along with discussion with Field Projects International. Identifications were classed to the most specific level possible. Some parasites observed were morphologically unique enough that they were able to be classified to the genus level, while several others could only safely be described to the family level. Caution was taken in the interpretation for all observations, but particularly with those classified to family.

Parasite prevalence (proportion of infected hosts) of all categories is shown in Table 5.

Parasite prevalence is the percentage of parasitism of a particular parasite found in a population.

This is scored by the presence/absence of the parasite in at least one sample obtained in each individual / total number of subjects. In my study, I have written it as both proportion and percentages because my sample sizes are different in both seasons. Parasite richness is the number of different parasite categories observed. Average parasite egg abundance is the number of observations for that particular parasite category (total observed in all samples / number of total samples).

Ethics Notes

This study does not involve direct contact with any of the primate subjects. Central

Washington University’s Institutional Animal Care and Use Committee approved the following and collection of dropped feces for parasite examination (A041510 & A051603). This is a collaborative study with Field Projects International, a Memorandum of Understanding (12094) was drawn and signed by Krista Emaly Banda (Master’s candidate at Central Washington

University), Dr. Kevin Archer (Dean of Graduate Studies and Research at Central Washington

University), and Dr. Mrinalini Watsa (President of Field Project International). A collection permit was obtained through the collaboration with Field Projects International to collect and export approved biological samples (193-2015-SERFOR/DGGSPFFS).

CHAPTER IV

RESULTS

A total of 27 fecal samples were collected during both field seasons from a combined 11 individuals (Table 2). One semi-habituated group of saddleback tamarins was followed during both dry and wet seasons yielding 16 samples in the dry from three subjects and 11 samples in the wet season from four subjects; several samples from other sympatric groups were obtained opportunistically during each follow yielding six samples in the dry season from five individuals and one sample from one individual in the wet season. Samples were opportunistically collected in total from four female subjects and five male subjects.

Table 2. Total Fecal Samples Collected

Subject Dry Season Samples Wet Season Samples F1 2 3 F2 0 2 F3 2 1 F4 1 0 M1 4 2 M2 3 3 M3 1 0 M4 1 0 M5 2 0 Note: Total number of individual fecal samples collected from all individuals during both the dry and wet seasons. Subjects identified by female (F) and male (M). For example, Subject F1 is female. A total of two fecal samples were collected during the dry season and three samples were collected during the wet season.

The parasites observed were placed into 10 separate categories and described based on morphological descriptions (Table 3). We were able to differentiate five parasites to a likely species or genus (Hymenoleopsis sp., Fasciola/Fasciolopsis sp., Ascaris sp., and

Ancyclostoma/Necator sp.), three to the level of family (Oxyuridae, Spiruridae, and

Strongylidae), and two morphologically distinct unknown categories (larval forms and unknown

Oxyuridae). Unknown nematodes were documented, measured, and photographed. These unknown nematodes are differentiated by morphological categories based on visual characteristics and measurements. During microscopic identification, representative photos were taken during each slide reading. Several parasite eggs are morphologically distinct allowing for easy categorization, but various stages of parasite develop was also noted that makes identification difficult and requires caution. As shown in Figure 3 and Figure 4, morphological differences in rhabditiform and filariform are minute. All individual parasites in these stages are lumped together to prevent a false identification.

Table 3. Description of Parasite Categories

Ancyclostoma/Necator spp. Characteristics: Colorless, ovoid, thin membrane. Clear vitelline layer with 2-8 distinct blastospheres.

Related: Ancyclostoma spp. and Necator spp. eggs cannot be differentiated without molecular analysis.

Literature: Regularly described in nonhuman primates (de Oliveira Tavela et al., 2013) 60-76µm by 35-40µm Ascaris spp. Characteristics: Round or oval egg with a thick, rough, brown shell. Fertilized eggs are more oval whereas unfertilized eggs are irregularly shaped.

Related: Ascaris sp. found in old and new world primates.

Literature: Observed in colobus monkeys (Gillespie et al., Fertilized egg: 45 – 70µm x 35 – 2005), proboscis monkeys (Klaus et al., 2017), and various 45µm Peruvian primates (Michaud et al., 2003). Unfertilized egg: 85 – 95µm x 35 – 45µm Fasciola/Fasciolopsis spp. Characteristics: Eggs are yellow and light brown, with an ellipsoidal, thin shell with a small, distinct operculum. Umembryonated eggs possess germinal cells within the yolk.

Related: Fasciola hepatica and Fasciolopsis buski are not easily morphologically distinguished and would require molecular analysis to be differentiated.

Fasciola hepatica: 120 – 150µm Literature: Fasciola hepatica described in the black-tufted x 63-90µm marmoset (Callithrix penicillata) Fasciola hepatica was Fasciolopsis buski: 130-159µm described in an indigenous community living near this study’s x 78-98µm field site (Cabada et al., 2016).

Table 3. (Continued)

Hymenolepsis spp. Characteristics: Oval, usually colorless eggs possessing two distinct membranes. Hooked oncosphere, less than six visible hooks.

Related: Besides the size difference in H. diminuta vs H. nana, H. diminuta eggs lack polar filaments (CDC, 2012).

Literature: Hymenolepsis cebidarum (Wenz et al., 2009). Hymenolepsis sp. without polar filaments observed in white- headed capuchin monkeys (Martin-Solano et al., 2017). Hymenolepis nana: 40-60µm x 30-50µm H. diminuta: 70-86µm x 60- 80µm Larval forms Characteristics: Lump category once the eggs have hatched including larval stages (L2-5) and immature adults. Lump category may include multiple different species as larval forms are morphologically similar until reaching adult form.

Related: All forms are lumped together to minimize incorrect identification. It is difficult to differentiate between larval stages of hookworms and strongyloides.

Oxyuridae Characteristics: Elongate-oval eggs with a clear vitelline membrane and a pill-shape germinal cell.

Related: Trypanoxyuris sp., Trichospirura sp.

Literature: Three Trpanoxyuris spp. observed in red howler monkeys (Solórzano-García et al, 2011).

35µm x 63µm

Table 3. (Continued)

Prostenorchis sp. Characteristics: Ovoid, usually brown, typically possessing a thick outer shell.

Related: Eggs are passed from the female parasite as colorless and can easily be mistaken for other eggs before tanned.

Literature: Observed in tamarin populations in human settlements (Wenz et al, 2010). Readily observed in nonhuman primates, especially with known invertebrate consumption

39-45µm x 68-73µm (Falla et al., 2015; Catenacci et al., 2016). Consumption of Deropeltis sp. sequenced along with other insects in Weddelli’s tamarin (Mallot et al., 2014).

Spiruridae Characteristics: Eggs are small and kidney shaped, usually with a dark germinal cell filling the interior.

Related: Spirura delicata, Spirura guianensis

Literature: Description of morphologically similar Spirura sp. in the Brazilian gracile opposum (Torres et al., 2015). Spirura delicata described in Saguinus mystax (Vincente et al, 1992). Spirura tamarini described in Cosgrove et al, 1963. 33-37µm x 43-48µm Strongylidae Characteristics: Lump category at various stages of strongyle development. Ovoid, usually with blastospheres. Characterized by eight or more distinct blastospheres. Included in this category is the stage of indistinct blastulas forming into the larval L1 stage.

Literature: Various unidentified strongyles observed in white- headed capuchin monkeys (Martin-Solano, et al., 2017). Suspect Strongyloides stercoralis in moustached tamarins and saddleback tamarins (Wenz et al., 2009).

Table 3. (Continued)

Unknown Oxyuridae Characteristics: Egg is long, rectangular with a thin membrane. Egg possesses a vermiform embryo.

Literature: Morphologically similar to Habronema spp. and Draschia spp. which is explored in more detail in discussion.

17µm x 70µm Note: Description of all parasite categories described in this study.

Table 4. Parasite Richness of Individuals Observed in Both Seasons

ID Dry Season Wet Season F1 2 5 F2 4 4 M1 5 3 M2 4 8 Note: The total parasite categories observed in individuals wherein fecal samples are collected in

both seasons. A total of 10 separate categories were distinguished. Total Categories Observed /

Total Categories Described.

The samples sizes were too small to analyze, however, some trends were noted. Two

parasite egg types (unknown Spiruridae and suspected Ascaris sp.) were observed only during

the wet season while all other parasites categories were observed in both seasons (Table 5).

Table 5. Parasite Prevalence

Parasites Prevalence %

Phylum Genus or Family Dry (ntotal subjects=8) Wet (ntotal subjects =5)

Acanthocephala

Prostenorchis sp. 8/8 (100%) 5/5 (100%)

Nematoda

Ancyclostoma/Necator sp. 2/8 (25%) 2/5 (40%)

Strongyloides sp. 8/8 (100%) 5/5 (100%)

Larval Forms 7/8 (75%) 4/5 (80%)

Oxyuridae 3/8 (37.5%) 3/5 (60%)

Unknown Habronematidae 1/8 (12.5%) 1/5 (20%)

Ascaris sp. 0/8 (0%) 1/5 (20%)

Spiruridae 0/8 (0%) 1/5 (20%)

Platyhelminthes

Fasciola/Fasciolopsis sp. 1/8 (12.5%) 1/5 (20%)

Hymenolepsis sp. 4/8 (50%) 3/5 (40%)

Note: Parasite prevalence is measured by number of parasites observed in all subjects / total subjects.

Although attempts were made, not all individuals were sampled in both dry and wet season. Ascaris sp. and Spiruridae were observed only during the wet season both at a prevalence of 20%. The sole suspect Ascaris sp. observed is described in Table 3. The parasite is morphologically similar to Ascaris sp. but has not been confirmed by molecular analysis.

Fasciola/Fasciolopsis sp. is a similar predicament. The egg observed fits within the parameters and looks morphologically similar, but without molecular analysis, the identification is tentative.

All parasites described in this study are tentatively identified solely through morphology.

Since sample sizes are small, the data of all parasite observations is displayed in Table 6 to allow for transparency. Table 6 is a compendium showing the parasite burden (total number of parasites observed in each fecal sample) for all study subjects. For individual F1, (n=2,3) represents two fecal samples collected during the dry season and three in the wet season. In each category, the number of eggs observed for that parasite is documented for each sample. For example, all parasite observations in the first fecal sample collected in the dry season is documented. It shows no observations for Ancyclostoma/Necator sp., Ascaris sp.,

Fasciola/Fasciolopsis sp., Hymenolepsis sp., larval forms, Oxyuridae, and Unknown Oxyuridae.

Only Prostenorchis sp. (15 observations) and Strongylidae (two observations) were observed in the first sample for F1. Instances in which no fecal samples were collected during the season are marked null (-).

Table 6. Parasite Burden for All Fecal Samples

Ancyclostoma (Suspect) Fasciola / Hymenolepsis Larval Oxyuridae Prostenorchis Spiruridae Strongylidae Unknown /Necator sp. Ascaris sp. Fasciolopsis sp. Forms sp. Oxyuridae sp. Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet Dry Wet F1 0 0 0 0 0 2 0 0 0 0 0 12 15 99 0 0 2 2 0 0 (n = 2, 3) 0 0 0 0 0 0 0 0 0 7 0 0 77 0 0 0 7 0 0 0 0 0 0 0 0 1 0 0 56 0 F2 - 0 - 0 - 1 - 0 - 1 - 1 - 19 - 0 - 4 - 0 (n = 0, 2) 0 0 1 0 0 1 20 0 30 0 F3 0 0 0 0 0 0 0 10 6 0 0 1 2 73 0 0 3 12 0 0 (n = 2, 1) 0 0 0 0 0 0 0 0 0 1 F4 0 - 0 - 0 - 0 - 28 - 1 - 5 - 0 - 3 - 0 - (n = 1, 0) M1 0 1 0 0 0 0 0 0 1 0 0 0 16 0 0 0 2 6 0 0 (n = 4, 2) 0 0 0 0 0 0 2 0 0 13 0 0 36 0 0 0 3 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 M2 0 0 0 0 0 0 1 3 0 0 0 0 53 59 0 3 4 7 0 0 (n = 3, 3) 0 2 0 0 3 0 0 0 0 1 3 0 53 18 0 0 3 3 0 0 0 0 0 1 0 0 0 0 0 56 1 0 0 0 0 0 0 13 0 3 M3 0 - 0 - 0 - 0 - 2 - 0 - 10 - 0 - 4 - 0 - (n = 1, 0) M4 2 - 0 - 0 - 6 - 12 - 0 - 24 - 0 - 3 - 0 - (n = 1, 0) M5 0 - 0 - 0 - 0 - 13 - 1 - 10 - 0 - 9 - 0 - (n = 2, 0) 0 0 0 2 12 0 10 0 5 0 Note: Total amount of parasites observed for all individuals for each fecal sample collected. Wherein (n = samples collected in the dry season, samples collected in the wet season) and follows accordingly across rows for each corresponding parasite. For example, individual F2 (n = 0,2) states that no fecal samples were collected during the dry season, and two fecal samples were collected during the wet season. Along F2’s row, all of the Dry Season is marked null (-) and the following Wet Season columns marked each time that corresponding parasite was observed in each of the two samples.

A map detailing the range use of the focal group was created using QGIS version

3.10.7 (Figure 7). Only the GPS tracks that were marked with the group being “In Sight”

(immediately able to be identified and viewed) were included. The map shows the difference in home range use in the dry and wet season.

Figure 7. Range Use in the Dry vs the Wet Season for One Group of Leontocebus

Weddelli.

CHAPTER V

DISCUSSION

Because intestinal parasites are long lived, often carried over through years sometimes for a host’s entire lifetime, it is imperative to approach parasitological studies through an ecological, long-term perspective. This study aimed to evaluate seasonality by

1) surveying parasite prevalence and 2) identifying parasite richness. Many of the parasites observed in this survey were previously reported in tamarins (Table 1) and other

New World primates except for the suspect Fasciola/Fasciolopsis sp. finding. The data gathered from this study provided a survey of parasite categories found in the affected study population (Table 6) showed indiscriminate parasite infections between the two seasons surveyed. This is not unexpected given parasite lifespans, the overlap of foraging ranges of primates in the study area, close proximity to humans, and a varied diet consisting of plants and insects.

Each parasite category is described and further analyzed, discussing their life cycle and prepatent period (the duration from moment of infection to egg production) if known. It is important to evaluate since their full immunological health is not known, although ongoing studies by Field Projects International are establishing methods to do so

(Watsa et al., 2015; Erkenswick et al., 2017; Erkenswick et al., 2019; Sacco et al., 2020).

Parasite Prevalence

Parasite burden for all individuals appear similar for both seasons, with minimal differences in parasite prevalence (Table 5). Parasite prevalence did not differ greatly

between the two seasons. It was observed that parasitism observed during the dry season was similarly mirrored during the wet season.

The thorny-headed worm Prostenorchis sp. was observed in 38% of the samples

(ndry=8) obtained during the dry season and observed in 100% of the samples (nwet=5) in the wet season. It is commonly described in nonhuman primates (Table 1).

Spiruridae was observed in only one fecal sample during the wet season (Table 6).

The identification of the suspect Spurira sp. is based on the descriptions of Cosgrove et al. (1963), Blanchard & Eberhard (1985), Torres et al. (2015), and Erkenswick et al.

(2019).

Parasite Richness

Parasite richness, the of different species per host (Margolis et al., 1982; Bush et al. 1997), was difficult to interpret with a small sample size. Fecal samples were collected in both seasons from for four individuals (Table 4). No discernable differences in the total number of different parasites between the two seasons for each affected host nor between male and female hosts were found.

Acanthocephala, Prostenorchis sp.

The thorny-headed worm Prostenorchis sp. is commonly described in captive primate necropsies (Porter, 1972; Middleton, 1996). The European Association of Zoo and Wildlife Veterinarians (EAZWV) have described the consumption of cockroaches as the mode of transmission (Lécu & Fiuza, 2019). Prostenorchis sp. has been described in primates known to consume insects, most particularly roaches. In tamarin studies,

Prostenorchis sp. eggs are a common sight and found in great abundance. This study

found all individuals were infected with Prostenorchis sp and release large numbers of eggs (Table 6).

A study by Mallot et al. (2014) analyzed fecal samples of Weddelli’s tamarins in northern Bolivia to determine insect consumption through DNA barcoding. It confirmed the omnivorous diet of Weddelli tamarins and identified 12 genera of insects. One genus of roach (Deropeltis sp.) was identified, providing evidence in consumption for Mallot et al.’s study population. It provides insight on the route of transmission for Prostenorchis elegans. A detailed life cycle that includes prepatency period (the duration from moment of infection to egg production) for Prostenorchis elegans has not been established.

Anecdotally, the study populations at ELBA (Estación Biológica Rio Los Amigos) were observed to consume Blattodea and Orthoptera. Prostenorchis sp. eggs were found in abundance in the study samples. Parasite prevalence (Table 5) was ndry = 8/8 (100%) and nwet = 5/5 (100%) and the average parasite egg abundance per fecal sample (total observed in all samples / number of total samples) observed was ndry = 19.44% and nwet =

26.18%. Total fecal egg count is interpreted with caution due to variability in egg shed.

This could be due to the parasite itself, shedding at different stages of its life cycle, environmental factors that can influence egg production, and the host’s own health.

Cestoda, Hymenolepsis sp.

Notable in the photos taken of suspect Hymenolepsis sp. is the lack of observable polar filaments. The samples observed average 38.7µm x 32.1µm (average from four photos taken), with less than six hooks in the oncosphere and a noticeable outer shell.

This contrasts from the well documented descriptions (Centers for Disease Control, 2017)

of Hymenolepsis diminuta (70-86µm x 60-80µm, six hooks on oncosphere, lacks a polar filament, and on an unstained slide can have a transparent yellow color) and H. nana (40-

60µm x 30-50µm, four to eight polar filaments, six hooks on oncosphere, and transparent). H. nana eggs can survive 10 days with the adult living for four to six weeks in the host; however, autoinfection allows H. nana infection to persist for years. H. diminuta has a prepatent period of 14 days. The suspect Hymenolepsis sp. observed in this study is markedly smaller and lacks pivotal morphological characteristics of the known H. diminuta and H. nana. Outside of this study population, suspect Hymenolepsis cebidarum and another unclassified cestode have also been observed in a survey by Wenz et al. (2009) in both Saguinus fuscicollis and Saguinus mystax in a field site close to this study. Molecular evaluation of the suspect Hymenolepsis sp. is needed for distinct classification. H. cebidarum was first named by Dunn (1963) and is described to have six distinct hooks on the oncophere.

While the parasite prevalence (Table 5) was ndry = 4/8 (50%) and nwet = 3/5 (40%), there was a noticeable difference in average parasite egg abundance per fecal sample (ndry

= 0.69%, nwet = 1.18%) which suggests potentiation of autoinfection. It is possible that initial infection could occur during the dry season and autoinfection could explain the increase during the wet season. The previously parasitized hosts continue to create more

Hymenolepsis sp. eggs internally. Unlike Hymenolepsis diminuta, H. nana’s life cycle does not necessitate an intermediate host to complete its life cycle.

Nematoda, Ancyclostoma/Necator sp.

Hookworm eggs of Ancyclostoma sp. and Necator sp. cannot be differentiated without molecular analysis as they are morphologically identical. Ancyclostoma/Necator sp. eggs are regularly described in nonhuman primates (de Oliveira Tavela et al., 2013;

Hasegawa et al., 2014). Without the molecular analysis for differentiation, the suspect nematoda has been referred to as Ancyclostoma/Necator sp. to avoid misidentification.

Their eggs are colorless ovoid shaped with a thin membrane, overall averaging 60-76µm x 35-40µm. They possess a clear vitelline layer with 2-8 distinct germinal cells.

It is highly possible that through molecular analysis, the presence of

Anyclostoma/Necator sp. may be greater. All strongyle-like eggs are lumped in this study as “Strongyles” as they are all morphological similar and various stages of embryonation could be observed. Only clearly distinguishable eggs with eight or less germinal cells were separated into the Ancyclostoma/Necator sp. category. As the embryonated

Ancyclostoma/Necator spp. develop, the once distinct germinal cells merge together, making them morphologically indistinguishable from other strongyle-like eggs.

Ancycostoma duodenale has been identified in great apes through molecular analysis (Hasegawa et al., 2014). A. duodenale and Necator americanus eggs hatch into free-living rhabditiform larva (Centers for Disease Control, 2019). The rhabditiform larva molts into infectious filariform which can survive in the environment for three to four weeks. The study subjects have been observed to forage on the forest floor, which is behavior that would allow exposure to the infectious filariform. While this appears

Necator americanus’s only route of transmission, Ancyclostoma duodenale infectious can

also occur by oral and transmammary routes. This means that the study subjects can ingest foods contaminated with the filariform larva or through the mammary glands of an infected mother to her breast-feeding infant (Miller, 1981). A. duodenale has a prepatent period of two to three weeks (Bowman, 1999). Upon maturation, adult worms secrete eggs daily. Necator americanus, also noted in primate literature (de Oliveira Tavela et al.,

2013), has a prepatent period of four to eight weeks (Bowman, 1999). In humans hosts, adult worms can live for five years, shedding eggs intermittently. This study duration and fecal collection period ran longer than the prepatent period of both Ancyclostoma sp. and

Necator sp., allowing potential newly infected individuals to be included.

In this study population, the suspect Ancyclostoma/Necator sp. is found in both dry and wet seasons (Table 6), but only in three male subjects (nmale total = 5). Parasite prevalence (Table 5) was ndry = 2/8 (25%) and nwet = 2/5 (40%) and the average parasite egg abundance per fecal sample observed was ndry = 0.19% and nwet = 0.27%.

Nematoda, Ascaris sp.

The presence of a single egg resembling Ascaris sp. is possibly an artifact, but

Ascaris sp. has been observed (although not described) in tamarin species in previous literature (Phillips et al., 2004; Wenz et al., 2009). The observed Ascaris-like egg measured 80µm x 42µm, which is well within the average size (78-105µm x 38-55µm) of an unfertilized Ascaris lumbricoides egg but lack the distinct misshapen elongated shape.

The unfertilized state of A. lumbricoides is elongated, thick brown shell and a rough outer membrane. A. lumbricoides has a prepatent period of eight weeks, adult worms can live in the host for 12-18 months, and sheds eggs daily (Centers for Disease Control, 2019). It

is highly possible this sample is an artifact and without repeated observations and molecular analysis, any identification is speculation.

Nematoda, Larval Forms

All hatched helminth larva are lumped in this broad category. The broad category may include multiple different species and genera as larval forms are morphologically similar until reaching adult form. As discussed earlier and illustrated in Figure 3 and

Figure 4, even identifying adult helminths are difficult due to morphological similarities.

Parasite prevalence (Table 5) is ndry = 7/8 (75%) and nwet = 4/5 (80%) and the average larval form count per fecal sample observed was ndry = 4.63% and nwet = 7.09%. Although there is a difference in average larval form count (ndry = 4.63%, nwet = 7.09%), one sample obtained during the wet season had a high parasite burden (Table 6). It is still possible that more larval forms are observed during the wet season due to 1) incubation period and 2) food abundance allowing the host species to maintain heavy parasite burden.

Nematoda, Oxyuridae

The broad classification of Oxyuridae in this paper is used to refer to distinct pill- shaped eggs, a clear vitelline membrane, and another pill-shaped germinal cell in various stages of development. Similar Trypanoxyuris sp. have been observed in red howler monkeys (Solórzano-García et al., 2011; Solórzano-García et al., 2018; Frias et al., 2019) and Trichospirura leptostoma has been observed in captive marmosets, Callitrix jacchus,

(Illgen-Wilcke et al., 1992; Hawkins et al., 1997). Trichospirura leptostoma has a prepatency period of eight to nine weeks, with adults living about two years (Illgen-

Wilcke et al., 1992). The eggs of both Trypanoxyuris sp. and Trichospirura sp. are morphologically similar and fit within the broad classification of this study. This is to not be confused with this study’s classification of “Nematoda, Spiruridae” wherein the eggs are morphologically different from “Nematoda, Oxyuridae,” although I believe both classifications are Spurira spp. Erkenswick et al. (2019) classified this morphology as

Gongylonematidae. Helenbrook et al. (2015) described a morphologically similar egg as

Trypanoxyuris sp. in mantled howler monkeys (Alouatta palliata aequatorialis) in the

Bilsa Biolgical Station in Ecuador.

Nematoda, Oxyuridae Unknown

The egg is long, rectangular (~17µm x 70µm) with a thin membrane and possesses a vermiform embryo. No known observation of this parasite has been observed in new world primates. The eggs are morphologically similar to the Habronematoidae superfamily including Habronema sp. and Draschia sp. (Bowman, 1999) which are normally observed in horses and similar ungulates. The life cycle of Habronematoidae require a muscid fly intermediate host to develop larva from L1 to L3 (Parasitipedia,

2018). Fly maggots consume the larva hatched in affected feces, wherein the larva develops inside the maggot from L1 to eventually L3 larval stage. Once at that infectious stage, the larva migrates to the mouths of the flies where they are passed and able to develop into the adult form. The prepatent period is typically two months.

This has not been documented in primate studies. It is unclear if this nematode classification is accurate, but it does resemble Habronematoidae until molecular analysis.

Nematoda, Spiruridae

Similar to the preceding “Nematoda, Oxyuridae” classification, Spiruridae in this study refers to the small kidney shaped eggs. It is morphologically similar to descriptions of Spirura delicata (Vincente et al., 1992) and Spirura guianensis (Cosgrove et al., 1963) but without molecular analysis, it is unclear if it is a true separate category or just a transitional stage for the broader “Nematoda, Oxyuridae.” This particular egg was only in one sample (Table 6) but has been observed in the same study populations by other Field

Project International researchers (Erkenswick, 2017).

Nematoda, Strongylidae

Strongyles are commonly described in all nonhuman primates (Bowman, 1999;

Viney & Lok, 2007). Several strongyles have been observed in new world primates;

Strongyloides stercoralis in Saguinus mystax mystax and Saguinus fuscicollis nigrifrons

(Wenz et al., 2009) and various unidentified Strongyloides spp. in Cebus albifrons

(Martin-Solano et al., 2017), Cacajao calvus ucayalii (Conga et al., 2013), Alouatta spp.

(Stoner, 1996; Stuart et al., 1998), and more. Because Strongyloides stercoralis and S. fuelleborni are pathogenic in humans and nonhuman primates (S. stercoralis is primarily a human parasite), the life cycle is well described (Bowman, 1999). S. stercoralis and S. fuelleborni are highly pathogenic with a prepatent period of 5-11 days. Maximum egg output is usually 20 days post infection and egg shed can continue for 11 weeks. S. stercoralis autoinfection can occur in humans, but is believed to be impossible for S. fuelleborni. Parasite prevalence (Table 5) was ndry = 8/8 (100%) and nwet = 5/5 (100%) and the average parasite egg abundance per fecal sample observed was ndry = 4.75% and

nwet = 12.18%. There is a noted increase in egg shed found during the wet season as opposed to the dry season. Since all individuals were parasitized in both seasons, the increased egg shed during the wet season could be attributed to the nutritional abundance.

Trematoda, Fasciola/Fasciolopsis sp.

The observation of a suspect Fasciola/Fasciolopsis sp. present a rare finding.

Only one study has described Fasciola hepatica in Callitrichids. Mendes et al. (2008) observed F. hepatica in black-tufted marmosets (Callithrix penicillata) and determined that the marmoset and nearby cattle (Bos taurus) are definitive hosts for the cestode.

Fasciola hepatica has also been described in an indigenous human population also living in the Madre de Dios region (Cabada et al, 2016). The general location of the indigenous population can be viewed in Figure 5. Fasciola hepatica, also known as “the common liver fluke,” is commonly associated with sheep or cattle and require a snail intermediate host. Cabada et al. discussed potential snail species that would act as a competent intermediate host in the area (2016). The potential Fasciola/Fasciolopsis sp. observed in the feces indicates that the tamarins utilize wet forest types. The study groups followed in this study have home ranges that include wet forest types that would allow them to be in contact with water sources (Table 7, Figure 7).

Current studies on the Weddelli tamarin’s insect consumption does not currently include snails (Mallot et al., 2014), but the consumption of the intermediate snail species is not necessary for infection. As portrayed in Figure 8, a snail intermediate host is necessary for the development for the free-living cercarciae. Once the cercarciae is encysted (metacercarciae), tamarins can ingest it through drinking the contaminated

water or vegetation. In future studies, an evaluation of the study groups home ranges and forest type utilizations would provide keen insight to the potential mode of

Fasciola/Fasciolopsis sp. transmission.

Figure 8. Life cycle of Fasciola spp (Centers for Disease Control, 2018).

Parasite prevalence (Table 5) was ndry = 1/8 (12.5%) and new t= 1/5 (20%) and the average parasite egg abundance per fecal sample observed was ndr y= 0.18% and nwet =

0.36%. Fasciola/Fasciolopsis sp. eggs were found in both seasons in two different individuals. Fasciola hepatica has a prepatent period of 8-12 weeks (Ballweber, 2014a);

Fasciola gigantica’s prepatent period is 10-16 weeks (Ballweber, 2014b; CDC, 2019).

Adult flukes can live in the host for many years, and the infectious metacercariae can survive for months before ingestion. Adult Fasciola hepatica is known to live in the bile duct while Fasciolopsis buski is known to live in the intestines.

The observation of a suspect Fasciola/Fasciolopsis sp. has pertinent especially for such a small bodied host. Leontocebus weddelli average 7-10 inches long while the adult

Fasciola hepatica fluke averages about an inch long. The small bile duct is the preferred location of F. hepatica. Even one adult fluke in the bile duct is enough to cause pathogeny.

Range Use Between Seasons

Home range use differed between the dry and wet season (Figure 7) for the focal group. The focal group was found to spend more time traveling during the dry season than they did during the wet season. This could be due to the increase of food availability during the wet season. These omnivorous primates do not have to travel far to obtain high quality foods. They have been observed to consume fruits, insects, and the occasional small invertebrate in both seasons. The notable difference in range use can help to understand how parasite encounters could be influenced by seasonality.

Only one individual was observed to be positive for all parasite categories (Table

6). M2 is suspect to be the offspring of the dominant breeding female. Tamarin groups are based around the dominant breeding female, her breeding males, and their offspring.

Both male and female offspring disperse once they reach adulthood, although it is

suggested that some adult offspring may stay with their maternal group longer.

Throughout both field seasons, behavioral scans were obtained for all individuals every

10 minutes. It is possible that M2 was observed before he dispersed out to a different group. Before adult offspring disperses, they slowly start venturing away from the group.

The increase range during this period of their lives could increase their parasite encounters.

CHAPTER VI

CONCLUSION

The study aimed to evaluate parasite prevalence and identify parasite richness between the dry and wet seasons in 2015-2016. There was not a statistically significant difference between the dry and wet seasons, but there are noticeable differences between seasons even with the small sample size. In future studies, this could be better explored with larger sample size. This study provides a quick survey, highlighting some of the helminths observed in semi-habituated free-ranging Weddelli’s saddleback tamarin.

Seasonal variation is well known to influence foraging behavior and range use.

Through the regular fluctuations in temperature, humidity, and rainfall, climatic changes can impact an animal’s susceptibility to parasites (Wiederholt & Post, 2009). As anthropogenic influence continues to impact climate change and alter the Amazon’s structure, it is important to consider its effects on the co-evolution of parasites and their hosts. Some intestinal helminths are long-lived in their definitive host species, anywhere from weeks to the host’s lifetime. Helminths that require specific definitive host species have co-evolved their host under set environmental systems. This co-evolution between parasite and host species fill a niche that allows both parties to function in their ecosystem (Thomas et al., 2004; Lafferty & Kuris, 2005; Carlson et al., 2006).

Conservation focusing on the continuation of endangered animals and their ecosystems have largely ignored the role of parasites in maintaining that ecosystem. The potential extinction of co-evolved parasites would allow novel parasites to fill that niche, leading to species “die-offs” (Carlson et al., 2006). This opens niches for novel, more pathogenic

(as compared to co-evolved parasites) parasites.

As humans continue to alter the ecosystem (e.g., unregulated logging, mining, wildlife trafficking, etc) new animals are introduced to disturbed ecosystems, and similarly, new parasites are also introduced (Lafferty, 2003; Lafferty & Holt, 2003;

Lafferty & Kuris, 2005; Bolt et al., 2019). Anthropogenic influenced stressors impairs host health, making them more susceptible to infectious diseases and co-infections

(Lafferty & Holt, 2003; Smith et al, 2005; Helenbrook et al., 2017). While most of the parasites observed in this survey may not be of public health concern to human populations, they are important for the well-being of the ecosystem.

Now more than ever, we are seeing the effects of the Covid-19 pandemic that was most likely brought about by multiple anthropogenic influences on the local and global environments. We must continue to work towards a collaborative and cohesive science to better understand and evaluate disease ecology. Just as the health of the study species is rarely independent of the ecology’s health, so are the parasites'.

Recommendations for Future Research

In future studies, I recommend increased sample size, suggest a longitudinal study, and an addition of molecular techniques. Gillespie (2006) suggest a minimum requirement of 60 independent fecal samples for 0.05 significance level. His suggested formula where a is the significance level and p is the population size:

n = ln (a)/ ln (1 − p)

Ideally there would be at least five individuals per study group with at minimum five fecal samples per individual per season. It is important to collect fecal samples six

weeks into the start of each season for 12 weeks over the course of several years. This would factor in the life cycles of various intestinal parasites and provide ample time for when most helminths shed novel parasite eggs.

Habitat attributes would provide details of potential seasonal parasite infections.

Behavioral data focusing on feeding, grooming, and ranging behavior would provide much needed additional data on potential parasite encounters. Behavioral scans and GIS data focused primarily on that could be added during fecal follows. In long-term studies, these could provide determining ecological scope influencing primate health and behavior.

A final additional molecular component would be necessary for identifying parasite species. This would provide key detail on the parasite’s life and pathogeny. It is nearly impossible to identify intestinal parasites earlier than the final adult form through visual examination at the species level. As noted throughout this study, some helminth eggs are morphologically identical. Molecular studies would reliably shed a light on differentiating these parasites.

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