Tamiasciurus Hudsonicus)

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2014-04-28 Patterns and Consequences of Parasitism in North American Red Squirrels (Tamiasciurus hudsonicus)

Patterson, Jesse

Patterson, J. (2014). Patterns and Consequences of Parasitism in North American Red Squirrels (Tamiasciurus hudsonicus) (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/27274 http://hdl.handle.net/11023/1436 doctoral thesis

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Patterns and Consequences of Parasitism in North American Red Squirrels (Tamiasciurus

hudsonicus)

Jesse Eric-Henry Patterson

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE

DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF BIOLOGICAL SCIENCES

CALGARY, ALBERTA

APRIL 2014

Abstract

Parasites are ubiquitous in nature and, by definition, impose harm on their hosts. The degree of harm is dependent upon many factors, such as co-evolutionary history, host immunity, and the magnitude of infection. Hosts must balance the costs of parasitism with investment in growth, development, and reproduction in order to maximize their fitness. As such, certain host traits may make certain individuals more susceptible to parasitism, which may affect observed patterns and costs of parasitism. For this thesis, I explored the patterns and consequences of parasitism using meta-analytical, experimental, and correlative approaches. Red squirrels

(Tamiasciurus hudsonicus) were used to explore hypotheses in a wild host population. I found that several host traits affected patterns of parasitism. Group size scaled positively with parasite infection intensity and prevalence across a wide-breadth of vertebrate species in a meta-analysis, but only for parasites that were contact transmitted. Adult male red squirrels had higher flea infection intensities than females, but only when reproductively active and only as adults.

Juveniles at birth had the highest flea intensities than at any other life-history stage. Nematodes had higher egg-shedding intensities in reproductively active hosts than in non-reproductive hosts, possibly owing to a trade-off between reproductive investment and immunity. The costs incurred by hosts appeared to be linked to the patterns of parasitism. For instance, through ectoparasite removal experiments, I found that parasitized juvenile red squirrels were lighter at emergence and less likely to survive from birth to emergence than their treated counterparts. Similarly, treated mothers spent significantly less time grooming than parasitized controls. The intensity of nematode egg shedding, but not flea intensity, was correlated with adult red squirrel body mass, suggesting a possible cost of endoparasitism to host condition. Finally, parasitism varied across

time, suggesting that the costs of parasitism may vary seasonally. My results indicate that parasites are distributed heterogeneously amongst their hosts, that parasites can alter the fitness and behaviour of their hosts, and that parasites, ultimately, have the potential to influence host life-histories and demography.

Preface

Contributions and Published Work

2013 J. E. H. Patterson and K. E. Ruckstuhl (Open Access). Reprinted with permission. JEHP developed the concepts and ideas, collected and analyzed the data, and wrote the manuscript;

KER provided feedback and edited the manuscript.

Chapter 3 contains a version of the unpublished manuscript by Patterson, J. E. H., P. Neuhaus, S.

J. Kutz, and K. E. Ruckstuhl. Patterns of ectoparasitism in North American red squirrels

(Tamiasciurus hudsonicus): sex-biases, temporal structure and effects on male body mass. JEHP designed the study, collected and analyzed the data, and wrote the manuscript. KER, PN, and

SJK provided feedback and edited the manuscript.

Chapter 4 was previously published as Patterson, J. E. H., P. Neuhaus, S. J. Kutz, and K. E.

Patterson, P. Neuhaus, S. J. Kutz, and K. E. Ruckstuhl (Open Access). Reprinted with permission. JEHP designed and performed the experiments, collected and analyzed the data, and wrote the manuscript; PN provided feedback and edited the manuscript; SK provided feedback;

KER provided feedback and edited the manuscript.

iii

Ethics Statement

The University of Calgary Life and Environmental Sciences Animal Care Committee (protocol

#AC11-0088) approved the use of all animals and procedures employed in this study. All research and animal use complied with provincial and federal regulations. Permits for this work were obtained from the Fish and Wildlife Division of the Ministry of Environment and

Sustainable Resource Development (Government of Alberta) and the Parks Division of the

Ministry of Tourism, Parks and Recreation (Government of Alberta).

Acknowledgements

I would like to extend my gratitude to all of the people who helped make this research program possible. Most notably, my supervisor, K. Ruckstuhl, and unofficial co-supervisor, P.

Neuhaus, who were constant sources of support, advice, encouragement, friendship, and inspiration throughout this entire process. My committee members, S. Kutz and R. Barclay, not only provided expert advice, lab space, supplies, and intelligent discussion, but also were instrumental in helping me formulate my ideas and keeping me focused. Although tasked with the under-appreciated role of having to examine me, I would like to extend a heartfelt thank you to L. Fedigan, R. Longair, M. Pavelka, and J. Waterman for serving on my candidacy and thesis defence committees. D. Whiteside helped with determining proper dosage for the anti-parasite medication and N. Parr assisted with fecal floatation training.

I have made a lot of good friends through this journey who were always there to lend insight when I needed to talk, share a joke when I needed to laugh, or enjoy a beverage at the

Grad Lounge when I was thirsty. Thanks to E. Baerwald, G. Beaudoin, B. Klug, L. Koren, L.

May, K. Mosdossy, J. Reimer, J. Smith, and G. Uhrig. My lab mates in the Ruckstuhl lab provided a stimulating environment and many interesting discussions: D. Andres, C. Corbett, B.

Edwards, B. Hoar, S. Liccioli, and P. McDougall. All of the researchers and staff at the R. B.

Miller Kananaskis Field Station (most notably, J. Buchanan-Mappin, S. Dobson, A. Fahlman, M.

Forrest, P. Neuhaus, A. Skibiel, and K. Yasuda) were always a great group of people to spend my field seasons with and were my family away from home. Of course, none of this would have been possible without the invaluable help from my very dedicated team of field and lab

assistants, to whom I am forever indebted: S. Majid, R. MacEachern, J. McMurray, and M.

Zabrodski. It was a pleasure working with and learning from every single one of you!

I gratefully acknowledge financial support from the Natural Sciences and Engineering

Research Council of Canada (Canada Graduate Scholarship), Bettina Bahlsen Memorial and

Queen Elizabeth II Scholarships, University of Calgary Thesis/Dissertation Grant, American

Society of Mammalogists Grants-in-Aid of Research, and Alberta Conservation Association

Grants in Biodiversity.

Finally, I thank my family for their unrelenting love and support.

To Oliver, for never ceasing to make me smile

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Table of Contents

Abstract ...... i Preface...... iii Contributions and Published Work ...... iii Ethics Statement ...... iv Acknowledgements ...... v Table of Contents ...... viii List of Tables ...... xi Epigraph ...... xiii

CHAPTER 1: INTRODUCTION ...... 1

CHAPTER 2: PARASITE INFECTION AND HOST GROUP SIZE: A META- ANALYTICAL REVIEW ...... 14 Introduction ...... 14 Methods ...... 18 Data collection ...... 18 Classification of data ...... 20 Sedentary hosts ...... 20 Mobile hosts ...... 21 Mode of parasite transmission ...... 21 Statistical analyses ...... 21 Results ...... 23 Relationships between host group size and parasite prevalence ...... 29 Relationships between host group size and parasite intensity ...... 29 Relationships between host group size and parasite species richness ...... 31 Discussion ...... 31 Parasite prevalence and intensity ...... 31 Parasite species richness ...... 35 Conclusions ...... 37

CHAPTER 3: PATTERNS OF ECTOPARASITISM IN NORTH AMERICAN RED SQUIRRELS (TAMIASCIURUS HUDSONICUS): SEX-BIASES, TEMPORAL STRUCTURE AND EFFECTS ON MALE BODY MASS...... 40 Introduction ...... 40 Materials and methods ...... 43 Results ...... 46 Adult sexual dimorphism ...... 46 Host body mass and ectoparasitism ...... 46 Temporal dynamics of ectoparasitism ...... 47 Ectoparasite removal ...... 47 Discussion ...... 49

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CHAPTER 4: PARASITE REMOVAL IMPROVES REPRODUCTIVE SUCCESS OF FEMALE NORTH AMERICAN RED SQUIRRELS (TAMIASCIURUS HUDSONICUS) ...... 56 Introduction ...... 56 Materials and methods ...... 58 Study area ...... 58 Study species ...... 58 Animal capture and experimental design ...... 59 Data analysis ...... 62 Results ...... 63 Flea prevalence and intensity ...... 63 Mother’s mass and survival ...... 63 Juvenile mass and survival ...... 64 Emergence date ...... 65 Discussion ...... 65

CHAPTER 5: EFFECTS OF ECTOPARASITISM ON THE ACTIVITY BUDGETS AND HOME RANGES OF FEMALE NORTH AMERICAN RED SQUIRRELS (TAMIASCIURUS HUDSONICUS) ...... 70 Introduction ...... 70 Methods ...... 75 Study area ...... 75 Animal capture ...... 76 Parasite removal ...... 77 Measuring behaviour in free-ranging red squirrels ...... 77 Home range size ...... 79 Statistical analysis ...... 79 Results ...... 80 Maternal activity budget ...... 80 Maternal home range ...... 82 Discussion ...... 82

CHAPTER 6: PATTERNS AND CORRELATES OF ENDOPARASITISM IN NORTH AMERICAN RED SQUIRRELS (TAMIASCIURUS HUDSONICUS) ...... 90 Introduction ...... 90 Methods ...... 97 Study area ...... 97 Study species ...... 98 Trapping and measuring red squirrels ...... 101 Parasite collection and recovery ...... 102 Home range estimation ...... 104 Statistical analyses ...... 105 Results ...... 107 Sampling and host-intrinsic factors ...... 107 Physaloptera sp...... 107

Citellinema bifurcatum ...... 115 Eimeria spp...... 121 Ectoparasite co-infection ...... 126 Discussion ...... 126

CHAPTER 7: SUMMARY AND CONCLUSIONS ...... 134 Sex-biases and temporal variation ...... 135 Transmission and control of parasite infection ...... 138 On the costs of parasitism ...... 140 Final words ...... 141

REFERENCES ...... 143

List of Tables

Table 2.1: Studies on the relationship between host group size and parasite prevalence included in the meta-analyses presented here, with number of groups examined (n), the correlation coefficient (r), host mobility (M = mobile, S = sedentary), transmission mode (I = indirect, D = direct, M = mobile)...... 24

Table 2.2: Studies on the relationship between host group size and parasite intensity included in the meta-analyses presented here, with number of groups examined (n), the correlation coefficient (r), host mobility (M = mobile, S = sedentary), transmission mode (I = indirect, D = direct, M = mobile)...... 26

Table 2.3: Studies on the relationship between host group size and parasite species richness included in the meta-analyses presented here, with number of groups examined (n), the correlation coefficient (r), host mobility (M = mobile), transmission mode (I = indirect, D = direct, M = mobile)...... 28

Table 2.4: Results from each separate meta-analysis regarding parasite infection in group- living species, with number of studies (n), overall mean correlation coefficient (Mean r), 95% confidence intervals, Z score, P-value of significance test and fail-safe numbers...... 30

Table 3.1: Median number of fleas and median number of fleas per unit mass of the hosts across the four life-history stages for male and female red squirrels in Sheep River Provincial Park, Alberta. Number of individual hosts in the sample (n) and 95% confidence intervals (CI) are reported...... 48

Table 5.1: Comparison of proportions of time spent by North American red squirrel (Tamiasciurus hudsonicus) mothers with experimentally reduced ectoparasite intensities and controls engaged in nine recorded behaviours in Sheep River Provincial Park, Alberta, Canada in 2011 and 2012. Means are reported ± standard deviation. U and P values were obtained from Mann-Whitney U-tests...... 81

Table 6.1: Parasite prevalence (%; 95% confidence levels in brackets) in North American red squirrels from Alberta, Canada for the full dataset, May-August 2010 and 2011...... 108

Table 6.2: Parasite prevalence (%; 95% confidence levels in brackets) in female North American red squirrels from Alberta, Canada for the reduced dataset, May-August 2010 and 2011...... 109

Table 6.3: Competitive generalized linear mixed models comparing parameters that affect Physaloptera sp. prevelance in North American red squirrels from Alberta, Canada, May-August 2010 and 2011 for the reduced and full datasets. Full dataset models controlled for random effects of ID and Year. Reduced dataset models controlled for random effects of ID...... 111

Table 6.4a: Mean intensity of Physaloptera sp. eggs shed per gram of feces for North American red squirrels in Alberta, Canada, May-August 2010 and 2011 for the full dataset. Means are reported for log10-transformed values ± 1 standard deviation (SD). N = number of samples...... 112

Table 6.4b: Median intensity of Physaloptera sp. eggs shed per gram of feces for North American red squirrels in Alberta, Canada, May-August 2010 and 2011 for the full dataset. Medians are reported with 95% confidence intervals. N = number of samples. ... 113

Table 6.5: Competitive linear mixed effect models comparing parameters that affect Physaloptera sp. intensity in North American red squirrels from Alberta, Canada, May- August 2010 and 2011 for the reduced and full datasets. All models controlled for random effects of ID and Year...... 114

Table 6.6: Competitive generalized linear mixed models comparing parameters that affect Citellinema bifurcatum prevelance in North American red squirrels from Alberta, Canada, May-August 2010 and 2011 for the reduced and full datasets. All models controlled for random effects of ID...... 116

Table 6.7a: Mean intensity of Citellinema bifurcatum eggs shed per gram of feces for North American red squirrels in Alberta, Canada, May-August 2010 and 2011. Means are reported for log10-transformed values ± 1 standard deviation (SD). N = number of samples...... 118

Table 6.7b: Median intensity of Citellinema bifurcatum eggs shed per gram of feces for North American red squirrels in Alberta, Canada, May-August 2010 and 2011. Medians are reported with 95% confidence intervals. N = number of samples...... 119

Table 6.8: Competitive linear mixed effect models comparing parameters that affect Citellinema bifurcatum intensity in North American red squirrels from Alberta, Canada, May-August 2010 and 2011 for the reduced and full datasets. All models controlled for random effects of ID...... 120

Table 6.9a: Mean intensity of Eimeria spp. oocysts shed per gram of feces for North American red squirrels in Alberta, Canada, May - August, 2010 and 2011. Means are reported for log10-transformed values ± 1 standard deviation (SD). N = number of samples...... 122

Table 6.9b: Median intensity of Eimeria spp. oocysts shed per gram of feces for North American red squirrels in Alberta, Canada, May - August, 2010 and 2011. Medians are reported with 95% confidence intervals. N = number of samples...... 123

Table 6.10: Competitive linear mixed effect models comparing parameters that affect Eimeria spp. intensity in North American red squirrels from Alberta, Canada, May- August 2010 and 2011 for the reduced and full datasets. Np = nematode presence/absence. All models controlled for random effects of ID...... 124

xii

Epigraph

“Now, here, you see, it takes all the running you can do, to keep in the same place.”

– Lewis Carroll

xiii

CHAPTER 1: INTRODUCTION

Organisms interact with each other in a variety of ways, such as through symbiosis, mutualism, commensalism, and parasitism (Ricklefs 2008). Parasitism is a remarkably common mode of interaction in nature. Some estimates suggest that as many as 50% of all known species are parasitic at some point in their life-cycle (Gunn & Pitt, 2012), although there is debate about what constitutes a species, especially among the prokaryotes. Parasites and their hosts have been described from all of the major groups of living organisms, including Bacteria, Fungi, Plantae,

Protozoa, invertebrates and vertebrates. Yet, despite the ubiquity of parasites in nature, parasitism remains a surprisingly difficult term/phenomena to define. According to Gunn and

Pitt (2012), “parasitism is a close relationship in which one organism, the parasite, is dependent on another organism, the host, feeding at its expense during the whole or part of its life.” The relationship between parasite and host is typically highly specific, with most parasites only capable of infecting a single host species or closely related host species due to the complex adaptations the parasite is required to evolve in order to identify, invade, and survive on or within the host. Any would-be parasite first needs to overcome the would-be host’s immune defences and then adapt to the host’s internal physiological environment before it can successfully parasitize that host. The host-parasite relationship always involves a degree of metabolic dependence on the part of the parasite upon its host, whether it is a flea acquiring a blood meal by puncturing the host’s epidermis or a cestode absorbing nutrients from the intestine of the host across a metabolically active tegument. By virtue of this metabolic dependence, parasites (by definition) cause harm to their hosts.

Undoubtedly, harm is also a difficult term to properly define, in large part because the degree of measureable harm is fully contingent upon the magnitude of the impact. An example of harm caused by a parasite may be analogous to the impact that a toy car running over a person’s foot may have compared to the impact that a large transport truck would have. That is to say, that a lone parasite within a large mammal, for example, may have no measureable impact on the host, but a large assemblage of the same parasite may lead to morbidity (a diseased state) and even mortality (death) in a host. Given the presumably long evolutionary history between hosts and their parasites, many ecologists and evolutionary biologists believed that the host-parasite interaction existed in a state of equilibrium. This was based on the notion that if one partner in the relationship could not adapt to the advances (i.e., immune defence, physiology) of the other, that the partnership would cease as the host would either be able to eliminate that parasite species or the parasite species would drive the host to extinction/extirpation. While there are examples of parasites causing mortality in their hosts and possibly even pushing hosts toward extirpation, most parasites generally do not gain from killing their host (and food supply). Therefore, biologists often regarded most parasites as not being too virulent so as to kill their host or lead to outwardly signs of clinical disease, with hosts not having to mount an intensive immune response in turn. This supposition of host-parasite equilibrium has been widely abandoned in favour of the “Red Queen hypothesis” which posits that hosts and parasites are engaged in a continual co-evolutionary arms race in which the parasite attempts to acquire more resources from the host in order to improve their fitness while the host evolves mechanisms for reducing its losses and eliminating the parasite (Ladle, 1992). Parasites exact harm on their hosts in many different, often elusive, ways, which impose costs to host reproductive success, survival, body condition, behaviour, susceptibility to other diseases, stress, and immune function as the host

attempts to fend off infection. Studied cases now abound in the published literature revealing the costs parasites impose on a wide breadth of host species (from plants and invertebrates to large mammals and fish), as well as the many trade-offs hosts are forced to make in order to compensate for the costs of parasitism (e.g., Allen & Little, 2011; Brown & Brown, 1986;

Derting & Compton, 2003; Hanssen et al., 2004; Hillegass et al., 2010; Møller, 1990; Møller,

1993; Neuhaus, 2003; Scantlebury et al., 2007; Schwanz, 2006). Generally, costs and effects of parasites are specific to the host and parasite species under investigation, and individual hosts vary with respect to their susceptibility, resulting in a general lack of consensus in the literature with regards to how and to what extent parasites affect their hosts. Although research is making good strides in this direction, clearly more study is well warranted.

Research into parasitism, its patterns, processes, consequences, and mitigation, has grown by leaps and bounds. In 2012 there were 34,779 studies gathered by the Web of Science containing the word “parasite”, up from the 23,999 published studies a decade earlier in 2002, and 15,911 published studies in 1992. Furthermore, there were 327,962 studies published between 2003 and 2013 listed on the Web of Science when using “parasite” as a search term.

This is up from 202,903 between 1992 and 2002, 140,182 between 1981 and 1991, and only

36,582 between 1970 and 1980. Clearly, researchers are paying far more attention to parasites today than they were in the past; perhaps, for good reason. Much of the increased attention being paid to parasites today (and in the recent decade or so) likely stems from the mounting array of emerging zoonotic diseases, particularly in the temperate parts of the world, which pose an ever increasing public health risk especially in the face of anthropogenic change. As such, there is an immediate and discernable need to recognize (emerging) infectious diseases while minimizing

the impacts that these parasites may have (and ultimately, the financial costs that they exact) on humans, livestock and wildlife worldwide. For instance, in 1979 the global cost of ticks and tick- borne diseases to humans and livestock alone was estimated at US$7 billion (McCosker, 1979), although more recent economic analyses have not been publicly published to my knowledge.

Ultimately, it wasn’t until 1978 when approaches to broadly understanding parasite transmission dynamics, parasite invasion and spread in a host population, and the stabilizing role of parasites on host population dynamics were well formalized (Anderson & May, 1978; May & Anderson,

1978); and, around the same time the intrinsic costs of parasitism in wild, free-ranging hosts began to gain more widespread attention and support in the academic literature through quantitative research (i.e., Brown & Brown, 1986; Hamilton & Zuk, 1982; Hoogland &

Sherman, 1976; Hudson, 1986; Kunz, 1976). Recently, scientific journals are seemingly inundated with research on the costs and consequences of parasitism. Mitigation and management of disease in humans and livestock has certainly driven a lot of the research in this field; however, for wildlife, much of the research is conducted if for no other reason than to gain a better understanding of the fundamental evolutionary and ecological relationships between parasite and host and, ultimately, the potential for parasites to constrain host fitness and generate observed phenotypic and genotypic variation (e.g., Chadwick & Little, 2005; Fitze et al., 2004;

Hamilton et al., 1990; Hillegass et al., 2008; Hochberg et al., 1992; Møller, 1993; Møller et al.,

2005; Neuhaus, 2003; Nunn & Heymann, 2005; Reckardt & Kerth, 2007; Richner, 1998;

Richner & Tripet, 1999; Scantlebury et al., 2010). But there is also a very important applied component to that research on wildlife-parasite interactions, including species-at-risk conservation and improving the management of diseases in wildlife (e.g., Haydon et al., 2006;

Joseph et al., 2013; Smyser et al., 2013; Torchin et al., 2003; Wobeser, 2002).

Parasites are not broadly and uniformly distributed amongst their hosts. In fact, parasites are often aggregated within their host populations, with most individuals harbouring low numbers of parasites and a smaller subset of the population hosting many (reviewed by Wilson et al., 2001). Therefore, the greatest costs of parasitism may be borne by only the most susceptible individuals and the transmission of parasites may be driven by a limited group of individuals in the host population. The resultant pattern of parasite infection and transmission is, in some cases, predictable. Age and sex are two of the most commonly cited host traits responsible for driving the observable patterns of parasitism, particularly in vertebrates. Two measures of parasitism

(richness and intensity; Chapter 2) tend to increase with age and may plateau in older animals, although juveniles of some species may be the most intensely parasitised, but also the most overlooked, age group. Further, males tend to be more heavily infected than females, possibly owing to differences in immune function and/or behaviour, although the mechanistic basis for sex biases in parasitism are still the topic of much investigation (e.g., Kiffner et al., 2013;

Scantlebury et al., 2010). In some species, males are also directly responsible for the majority of parasite transmission. Other factors that may contribute to observed heterogeneities in parasitism are host behaviours (i.e., home range/space use, gregariousness), genetics, host habitat associations, and seasonality. Understanding the forces driving the patterning and transmission of parasites in host populations may assist with managing the spread and outbreaks of disease through, for instance, targeted vaccine control programs.

My thesis, as presented here, aims to further uncover the role that parasites play in shaping the fitness and behaviour of their hosts, in addition to documenting and explaining

observable patterns (sex-biases, seasonality) of parasitism in wild animals. This research was achieved using a combination of meta-analytical literature review on a broad range of host and parasite taxa, as well as field and laboratory surveys of and experiments on the parasites and host-specific traits (i.e., behaviours, body mass, and reproductive success) of North American red squirrels (Tamiasciurus hudsonicus). Wild rodents, such as the red squirrel, present interesting biological systems through which studies on host-parasite interactions can be studied.

As with most rodents, the red squirrel is multiparous (giving birth to more than one offspring at a time), non-sexually dimorphic, easily observable and trappable, and host to a variety of parasite species (arthropods, nematodes, protozoa, and cestodes), thereby making them an appropriate species for the study of host-parasite interactions. While not of direct management concern, knowledge gained from studying the red squirrel system may be applicable to many other rodent species that are of management concern and/or host to vectors of zoonotic pathogens, such as plague. Red squirrels are commonly found in forested landscapes across Canada and are active year-round, relying on cached conifer cones (middens) during the winter months. Individuals establish and maintain separate territories containing at least one midden, which are then defended from conspecifics and competitors. Red squirrels are promiscuous mammals, and a female may mate with multiple males on the day of oestrous. Females generally only produce one litter per year following a 33-day gestation period, after which juveniles are fully weaned at approximately 70 days after birth. Red squirrel nests are fully accessible and traceable thereby allowing for the infection status (patterns) and impacts (consequences) of parasitism to be studied in juveniles from birth to emergence and compared to adults.

As indicated above, there are numerous hypotheses pertaining to the observed patterning and aggregation of parasites within host populations and species. Heterogeneous patterns in parasite infection are generated by variation between individuals in their risk of exposure and susceptibility to parasite infective stages (Anderson & May, 1978). Host, parasitological, ecological, and environmental factors, such as host age, host sex, host body mass/condition, time/seasonality, host behaviour (ranging and daily activity), co-infection with other parasite species, host reproductive status, parasite transmission mode, and the degree of host gregariousness/sociality are all thought to contribute to the observed heterogeneous patterns and, ultimately, the consequences of parasitism. I explore each of these ten potential explanatory variables quantitatively in this thesis, beginning with the role of group size, host mobility/ranging behaviour, and parasite transmission mode in generating heterogeneities in parasite infections. One of the most commonly cited costs of sociality, and group-living in general, is that parasites are more likely to be transmitted between members, such that infections amongst individuals within groups is predicted to be more severe than in solitary animals.

Clearly, for group-living to have been maintained through evolutionary time it must be a stable strategy with derivable benefits to the members of the group; however, according to Brown and

Brown (1986), in the absence of such benefits the costs of parasitism would quickly select for a solitary lifestyle (in a species of colonially nesting swallow; Hirundo pyrrhonota). For this chapter (Chapter 2), I used a meta-analytical approach to summarize quantitative results from seventy host-parasite assemblages gathered from the published literature. This chapter also serves to introduce important terms and concepts, such as: parasite prevalence, intensity and species richness; direct versus indirect transmission; and, the effects of host sex and ranging

behaviour on parasite infection risk – all of which make appearances throughout the subsequent chapters of this thesis.

Ultimately, disentangling the effects of parasites from the many other ecological and physiological factors that may influence reproductive success, survival, body condition, and behaviour of hosts requires careful experimentation to manipulate or control each of the potential mechanisms. Many correlative tests, while valuable at directing research, ultimately fail to disentangle the effects of parasites from other environmental and physiological variables simply due to the fact that not all variables can be or are controlled for. I used an experimental manipulation, occasionally in conjunction with correlative tests, to investigate the impacts of ectoparasites (i.e., parasites that spend all or part of their lifecycle on the exterior body surface of the host, such as fleas and ticks) on red squirrel hosts. Using this method, I experimentally reduced the ectoparasite intensity in a random sample of red squirrels and then explored whether this manipulation resulted in improved reproductive success, survival, and body mass, as well as altered activity budgets, home ranges, and co-infection status with endoparasites relative to unmanipulated controls. This approach should provide strong evidence as to whether or not ectoparasites are directly influencing the host (sensu Lehmann, 1992).

After exploring patterns of parasitism on a broader taxonomic scale in Chapter 2, Chapter

3 begins my foray into the patterns and consequences of parasitism in red squirrels. Chapter 3 presents the question: how are fleas (a ubiquitous ectoparasite of red squirrels, and rodents in general) patterned between the sexes and across various life-history stages (e.g., through time)?

I hypothesized that males would be more intensely parasitized than their female counterparts

across time owing to differences in physiology (testosterone is an immunosuppressant) and behaviour (larger home ranges, higher degree of risk-taking behaviour). Since body size dimorphisms are not present in red squirrels, any observed bias would not be attributable to size differences, although mass was considered in the analysis. I also hypothesized that juveniles would have higher flea intensities than adults while still in the nest environment. Juvenile rodents do not develop innate immunity until several weeks after birth and are unable to auto- groom, which should result in them being relatively defenceless to fleas during the time that they are in the nest. Additionally, the nest environment provides fleas with a relatively stable environment in terms of food supply (juveniles are always present) and temperature/humidity.

Given the presence of such favourable conditions, there is evidence that the population cycle of rodent-specific fleas actually coincides with the presence of juveniles (and a lactating mother) in the nest, although the mechanism for this phenomena is not well established (e.g., Haukisalmi &

Hanski, 2007). The topics of sex-biases, age-biases, and temporal variation are all explored in

Chapter 3. Furthermore, given the potential for male-biased flea infection intensity, I was interested in determining whether ectoparasites affected the body mass of male red squirrels. I used an experimental manipulation approach to answer this question in Chapter 3.

Experimental tests of the effects of ectoparasites on host reproductive success have been rather scant to date, however those that have been published show an interesting consensus: across vertebrate host taxa, ectoparasites have the potential to reduce the reproductive success of their hosts. Experimental manipulation of ectoparasites on two species of ground squirrels

(Urocitellus columbianus and Xerus inauris) resulted in mothers raising more offspring than controls (Hillegass et al., 2010; Neuhaus, 2003). As well, experimental reduction of ectoparasites

in the nests of several species of birds (Hirundo rustica, H. pyrrhonota, and Parus major) ultimately improved survivorship and condition of nestlings (Brown & Brown, 1986; Møller,

1990; Richner et al., 1993). While the results of the experimental manipulations have been largely unanimous in revealing a measureable cost of parasitism to host reproductive success, results from correlative tests have proven to be far more equivocal (e.g., Allander, 1998; de Lope

& Møller, 1993; de Lope et al., 1998; Harriman & Alisauskas, 2010; Johnson & Albrecht, 1993;

Møller, 1990; Møller, 1993; Moss & Camin, 1970; Oppliger et al., 1994; Pacejka et al., 1998), possibly due to an inability to control for a wide range of variables, such as environmental conditions and host-parasite coevolutionary ties. Using the experimental manipulation approach outlined above, I hypothesized that ectoparasite removal would allow red squirrel mothers to maintain or improve their body condition over time, increase their overwinter survival, produce heavier juveniles, and allow for a higher percentage of juvenile survival to emergence. All contingent, of course, upon ectoparasites reducing the amount of energy available to mothers for supporting lactation and to juveniles for supporting their own growth and development. This is the topic of exploration and discussion in Chapter 4.

Parasites are capable of manipulating their hosts’ behaviour in a variety of spectacular

(and less spectacular) ways. Correspondingly, the behaviour of the host can influence its likelihood of acquiring and maintaining a parasite infection (i.e., nest switching, selective foraging). Many of the more interesting behavioural modifications imposed by parasites on their hosts lead to increased opportunities for transmission, often between the intermediate and definitive hosts. For instance, the protozoan parasite Toxoplasma gondii is transmitted between rodents (intermediate host) and cats (definitive host), with the asexual stage encysting within the

intermediate hosts’ nervous tissue and often reaching very high levels of infection intensity.

Infection in the intermediate host results in rodents becoming more active, losing their normal fear of new objects, and losing their aversion to and even becoming attracted to the scent of cats

(Webster, 2007). Consequently, rodents infected with T. gondii are more likely to be captured by their definitive hosts. Examples of this sort of behavioural manipulation by parasites are plentiful in the literature (see Barber et al., 2000; Moore, 2002; and Poulin et al., 1994 for examples and reviews). Additional effects of parasites on host behaviour may include nest switching, allo- and auto-grooming, self-medication, and movement/migration (Moore, 2002). Of particular relevance to the red squirrel-ectoparasite system under investigation here is the role that ectoparasites play in altering the daily activity budget of their hosts, with particular interest in the grooming, maternal, and ranging behaviours of lactating females. Grooming provides beneficial effects to host fitness by directly eliminating ectoparasites from the body surface, in addition to removing debris and serving a social/maternal function. Animals that have been experimentally prevented from grooming have been found to experience elevated ectoparasite intensities

(Clayton, 1991), which may lead to declines in their reproductive success (Booth et al., 1993;

Marshall, 1981). As with all behaviours, the time spent by an individual engaged in one behaviour, such as grooming, trades-off directly with the amount of time available to invest in other behaviours, such as foraging, territoriality, and nursing offspring. Ultimately, any behaviour that reduces the amount of time a mother spends engaged in maternal behaviours has the potential to affect the phenotype of the offspring. Using the experimental approach outlined above, I predicted that parasitized mothers would spend more time foraging and grooming than non-parasitized mothers in order to compensate for the negative effects of ectoparasites. With ectoparasites removed from a group of mothers, I predicted that those mothers would spend more

time in the typically heavily parasitized nest environment nursing their offspring. Furthermore, I explored the potential for ectoparasites to affect home range size in the same lactating female red squirrels. I hypothesized that if parasite removal reduced the proportion of time spent foraging by red squirrels, treated animals would respond by maintaining a smaller home range (compared to controls) and more vigorously defending their reproductive investment. Alternatively, parasitized mothers may have less energy and time to invest in traveling/movement/territory defence, which would be reflected in smaller home ranges and more time spent resting. The topics of ectoparasitism and their effects on host behaviour/activity budgets are explored in greater detail in Chapter 5.

Endoparasites (i.e., parasites that live inside their hosts for at least a portion of their lifecycle) are thought to vary in their prevalence and intensity of infection in much the same way as the ectoparasites discussed in Chapter 3, that is by season, host sex, host age, and host reproductive status. In Chapter 6, I explore the patterns of endoparasite egg shedding in red squirrels. Endoparasite eggs are shed in the feces of their host and surveys of egg shedding prevalence and intensity represent a non-invasive means by which to assess infection status.

There are drawbacks to this approach and these are discussed in Chapter 6. Seasonality appears to be an important determining factor of endoparasite egg shedding in a wide variety of host taxa, probably because this is the point in the parasite lifecycle at which eggs are exposed directly to the external environment – so conditions must be right for larval development/survival. Environmental variables such as temperature, rainfall and humidity can affect the development and survival of free-living parasite larvae and the desiccation/viability of eggs. Effects of environmental seasonality are, however, difficult to separate from temporal

variability in host behaviour, diet, and physiology. For instance, nematode egg shedding in female domestic sheep peaks during the warm, humid spring and summer months, however this timing also coincides with host parturition (Waller et al., 2004) – so which factor, the external environment or the host’s biology, is driving the observed pattern? Seasonality is undoubtedly difficult to narrow down and tease apart because it is so ubiquitous in nature. In addition to seasonality, I also explore the potential roles of host sex, reproductive status, home range size, mass, and parasite co-infection on the prevalence and intensity of egg/oocyst output by two species of nematode and one protozoan in red squirrels.

The increased attention paid by ecologists and evolutionary biologists to parasites in the last decade or so has led to many exciting discoveries about host-parasite dynamics and the pressures exerted by parasites on their hosts. The available body of knowledge, while growing, remains small and still many questions regarding the mechanisms driving observable patterns of parasitism and the costs imposed by parasites on their hosts remain unanswered. When I began my PhD in 2008 only a small handful of publications on red squirrel parasites existed, and of those, only one attempted to expound the mechanisms governing the observed patterns of parasites in a wild population of host squirrels (Gorrell & Schulte-Hostedde, 2008). Since then, only one other study, mine excluded, has provided additional insight into the world of red squirrel-parasite interactions (Gooderham & Schulte-Hostedde, 2011). It is hoped, then, that my thesis continues to further advance the study of red squirrels, their parasites, and the fields of parasitology, behavioural ecology, and evolutionary biology by providing new data, fresh insights, and raising even more questions!

CHAPTER 2: PARASITE INFECTION AND HOST GROUP SIZE: A META-

ANALYTICAL REVIEW

Introduction

By definition, parasites impose costs to their hosts and are partially responsible for shaping the phenotype, genotype, and life-history traits of host organisms. In many host species, the more heavily parasitized individuals are less likely to mate (Hamilton & Zuk, 1982), more likely to raise inferior offspring if they do mate (Neuhaus, 2003), suffer from reduced life expectancy (Walter & Proctor, 1999) and display modified behaviours (Poulin, 1992). Parasites have been found to affect various parameters of host social organization, such as group size

(reviewed by Côté & Poulin, 1995 and Altizer et al., 2003), sexual segregation (Ferrari et al.,

2010), dominance hierarchies (Ezenwa, 2004) and contact networks (van Baalen, 2002). Parasite transmission rates typically scale positively with the density of the host population (density- dependent transmission) or with the frequency of interactions between the host and the parasite’s infective stage (frequency-dependent transmission). As such, host behavioural and ecological traits that increase host proximity to infective stages and local population size and density, such as social group size, should increase parasite infection intensity (number of parasites per individual host; Bush et al., 1997), parasite prevalence (the presence or absence of a parasite species on or in an individual host; Bush et al., 1997) and parasite species richness (the number of parasites on or in a host individual, population or species; Bush et al., 1997). The combination of these three terms will generally be referred to herein as ‘parasite infection’.

Host sociality (gregariousness) is a widespread phenomenon spanning many taxa including insects, fish, birds and mammals. Sociality has been the topic of much empirical and theoretical research concerning both ultimate and proximate causes for its existence. In essence, for gregariousness to have evolved and to have remained evolutionarily stable, the benefits of forming groups must consistently outweigh the costs. All social species experience both costs and benefits of living in groups. The benefits accrued to individuals living in groups are plentiful and variable and may include enhanced predator avoidance (i.e., dilution effects; Ostfeld &

Keesing, 2000), increased foraging efficiency, improved thermoregulation and more options for mate choice (Krause & Ruxton, 2002). Alexander (1974) noted that there are no automatic or universal benefits gained by individuals through group formation, but there are automatic and universal costs, including, but not limited to, increased intraspecific competition for limited resources, predator attraction and an increased probability of misdirecting parental care (Krause

& Ruxton, 2002). When the costs are exceedingly large, group formation may be discouraged altogether. Given that most parasite transmission is either density-dependent or frequency- dependent, epidemiological models predict host density and local population size as key contributing factors controlling the transmission dynamics of infectious diseases because these variables often set the threshold for successful parasite invasion and spread (Anderson & May,

1978). Increased parasite infection is widely interpreted as a cost of gregariousness (Côté &

Poulin, 1995). The magnitude of the cost depends on the degree to which infection negatively affects host fitness.

Alexander (1974) proposed that group formation and increased group size should lead to enhanced parasite infection among group members through increased transmission. Hoogland

and Sherman (1976) and Freeland (1977) were the first researchers to empirically test

Alexander’s idea. Hoogland and Sherman addressed this question with contagious parasites

(fleas) of colonial bank swallows (Riparia riparia) and Freeland examined this question with mobile, biting flies in primate (Cercocebus albigena, Cercopithecus ascanius, Piliocolobus badius) social groups. Hoogland and Sherman found a significant relationship between group size and both flea prevalence and intensity, lending support for Alexander's (1974) hypothesis.

Freeland’s data, on the other hand, did not support Alexander’s hypothesis. Freeland found evidence of a dilution effect: group size increased when attacks from biting flies increased, suggesting that grouping may be beneficial in terms of reducing per capita attack rates. Further testing of effects of group size on parasite intensity and prevalence has corroborated these original findings. For instance, Kunz (1976) observed that bat fly (Trichobius corynorhini) intensities and group sizes of Townsend's big-eared bats (Corynorhinus townsendii) were positively correlated. Similar to Freeland (1977), Rubenstein and Hohmann (1989) found that dipteran biting fly intensities were negatively correlated with group size in feral horses (Equus caballus). The feral horses also appeared to form more tightly clustered aggregations when flies were more abundant, active or bothersome, suggesting a behavioural adaptation for dealing with dipteran parasitism (Rubenstein & Hohmann, 1989). This select cross-section of studies highlights the importance of understanding basic host and parasite biology when developing hypotheses and interpreting results with respect to the interplay between parasite infection and host gregariousness.

The risk of parasitism by contagious contact-transmitted parasites (i.e., parasites whose infective stages are directly transmitted via contact between hosts and are immediately infective

– most ectoparasites; herein referred to as direct transmission) is predicted to positively correlate with group size as host proximity and the number and duration of conspecific contacts increases.

A contagious parasite in which the infective stages occur off of the definitive host (i.e., in an intermediate host) and which are not immediately infective (i.e., most endoparasites; herein referred to as indirect transmission) should follow the same patterns as directly transmitted parasites if host groups are sedentary and exposure to infective stages is consistent. Host species that are highly mobile and range more widely are not predicted to acquire more indirectly transmitted infective stages in larger groups because such groups are able to move away from contaminated areas into more parasite-free environments (Côté & Poulin, 1995), thereby reducing group-member-to-group-member transmission of infective stages. However, this need not always be the case. For instance, if large mobile groups of predators forage more efficiently on certain prey species than smaller groups, and prey species are the intermediate hosts, then variation in predator group size may control the variation in transmission rate of parasites.

However, in general, the parasite infection intensity in mobile groups is predicted to show weak or absent correlations with group size. Mobile parasites (i.e., biting flies, mosquitoes, aquatic crustaceans) represent an interesting case: once a parasite has detected a group of hosts, the dilution effect predicts that as group size increases the probability that each group member has of being attacked decreases, resulting in an expected inverse relationship between group size and parasite intensity. However, mobile parasites can track their hosts’ movement patterns, so host mobility is not expected to affect the relationship between mobile parasites and their hosts.

Earlier meta-analyses have generally supported the existence of an association between parasite infection and group size (Côté & Poulin, 1995; Rifkin et al. 2012). These meta-analyses

found, for instance, that contagious parasite infections increase with group size; however, Rifkin et al. (2012) did not support Côté & Poulin’s (1995) finding that mobile parasite intensity decreased with increasing group size. Rifkin et al. (2012) did not explore the effects of host mobility, but Côté & Poulin (1995) did not find any correlations between either parasite intensity or prevalence and host mobility. Côté & Poulin (1995) did not incorporate studies of parasite species richness, nor did they consider differences in direct and indirect parasite transmission modes in their analyses. Rifkin et al. (2012) did evaluate the effect of group size on parasite species richness but did not find any effect. Since Côté & Poulin's (1995) meta-analysis, several studies elucidating the relationship between group size and parasite infection risk have been published and meta-analytical techniques have improved. While Rifkin et al. (2012) offer a phylogenetic analysis of various measures of parasite infection and host group size, as well as taxon specific analyses, they do not evaluate several biologically meaningful explanatory variables, such as host mobility. In this article, we re-evaluate the literature and examine the relationship between host group size and parasite infection. Since our dataset spans a broad range of taxa, life-histories and evolutionary lineages, we made the following assumptions: first, we assumed that host individuals are similarly susceptible to parasitism across group sizes, which

Møller (1987) showed might not always be the case. Second, we assumed an even distribution of immunity against parasites across group sizes within species (Wilson et al., 2003).

Methods

Data collection

All data were collected from an extensive search of the literature published on or before

June 26 2012. A complete search of ISI Web of Science, Google Scholar, BIOSIS and PubMed was performed using all possible combinations of the terms: ‘parasite’, ‘parasitoid’, ‘pathogen’,

‘disease’ and ‘infection’ in conjunction with ‘group size’, ‘group’, ‘colony size’, ‘colony’,

‘colonial’, ‘gregarious’, ‘social’ and ‘communal’. References within collected published studies and all publications citing Côté & Poulin (1995) were searched for pertinent data. These sources of information should provide comprehensive coverage of the published data on this topic. To be included in the analysis, reported results had to meet several main criteria. First, studies were included in the data set if they reported a correlation coefficient (either Pearson or Spearman) between group size and one of three different measures of parasite infection risk: prevalence, intensity or species richness. These three measures of infection are most often predicted to correlate with gregariousness and group size in social animals. Pearson and Spearman correlation coefficients are commonly used as measures of effect size in meta-analyses (i.e., Côté & Poulin,

1995; Gontard-Danek & Møller, 1999) and are appealing because their squared value represents the variance in the response variable explained by the predictor. Second, published data were only included if the sample size (i.e., number of groups observed) was reported. Third, we chose not to mix single species and phylogenetic studies in our analyses. For example, in Table 2.1

(parasite prevalence) we chose to omit studies of cross-species relationships between mean group size and mean prevalence (e.g., Gregory et al., 1991, where n = 86 phylogenetically independent contrasts but is actually based on 100+ bird species). Mixing single species and phylogenetic studies is problematic in that there are obvious potential differences between within-species and cross-species patterns and the weighting of studies is thrown into question as, for example, 86 buffalo groups (i.e., one species) would be as heavily weighted as the 86 independent contrasts

from 100+ bird species. For Table 2.3 (species richness), we chose to omit the one single species study (Snaith et al., 2008) since all other studies were cross-species analyses. Additionally, where data on the same host-parasite interaction were reported in the same study population across several years (e.g., Brown et al., 2001), a composite mean effect was used in the analyses so as to deal with a possible lack of independence between the data. Finally, because different taxonomic groups of hosts contributed unequally to our dataset, we included higher taxa of hosts as a moderator in the models to control for any bias due to a taxonomically unbalanced dataset.

Classification of data

The data set was divided to test several hypotheses regarding host behaviour and parasite transmission dynamics. A separate meta-analysis was carried out to test each hypothesis where the data allowed.

Sedentary hosts

Sedentary hosts were considered as species or populations associated with a specific area for the duration of the original study, such as nests, burrows, or a highly restricted range (i.e., prairie dogs that remain on their group’s territory, colonially nesting birds that remain in the colony, nests or nestlings that remain stationary).

Mobile hosts

Mobile hosts on the other hand were considered to be those species or populations which range widely or whose location was not spatially predictable during the course of the original study (i.e., most ungulates with large home ranges, free-swimming fish).

Mode of parasite transmission

We looked at directly-transmitted parasites (contact-transmitted, immediately infective), indirectly-transmitted parasites (most parasites that require off-host development) and mobile parasites (parasites that can freely move and actively seek new hosts, often by flying or swimming: flies, some swimming crustaceans, many microparasites and their vectors).

Statistical analyses

Meta-analysis is widely regarded as a powerful method by which to quantitatively test hypotheses using previously published results (Cadotte et al., 2012; Nakagawa & Poulin, 2012).

Meta-analysis involves weighting effect size against the sample size. This is accomplished by transforming test statistics into a common metric called the effect size, which is a “standardized measure of the strength of a relationship between two variables of interest” (Møller & Jennions,

2001). The measures of effect size used here were obtained from Pearson and Spearman correlation coefficients drawn from the pertinent literature. Correlation coefficients were transformed by means of Fisher’s transformation to Z values (Sokal & Rohlf, 1995) and these Z

values were used as the effect size in subsequent analyses. Mixed effects models were used to analyze each of the data sets with host species entered as a moderator variable (Hunter &

Schmidt, 2000). Mixed effects models are random effects models with a moderator variable, and can be preferable in ecological studies where variation in observed effects is not due solely to sampling error and effect sizes are expected to vary between taxa and ecosystems (Jennions &

Møller, 2002). By allowing for random sources of variation in effect sizes between studies, as well as in sampling error, random effect models can account for random variance caused by stochastic and biological processes (Jennions & Møller, 2002). Confidence intervals (95%) are reported and the mean effect size can be interpreted as being significantly different from zero if the 95% confidence intervals do not overlap zero. This relationship is also reflected in the p- values (i.e., p < 0.05).

Publication bias can be problematic for analyses that rely on previously published findings. In order to overcome the potential hurdles imposed by publication bias, several statistical methods have been developed for use in meta-analyses to both detect and adjust for any bias in the data (Møller & Jennions, 2001), although there is only one true way of testing for publication bias and that is to compare published and unpublished studies. Here, we estimated the potential for publication bias in the data by use of rank correlation tests (Begg & Mazumdar,

1994). The rank correlation test uses Spearman rank correlations to investigate the relationship between the effect size and sample size. Plotting effect size against sample size should reveal a funnel-shaped plot (funnel plot): larger variance in effect sizes when sample size is small with decreasing variance as sample size increases. If the funnel plot is significantly skewed (i.e., asymmetric) in any way, the rank correlation test will show this (i.e., p < 0.05) and appropriate

measures can be taken to correct for the bias. The rank correlation test is regarded as being a fairly powerful test for investigating publication bias (Møller & Jennions, 2001). Publication bias was not detected for any of the tests in this study. We also determined fail-safe numbers for each of the meta-analyses using the Rosenthal method (Rosenthal, 1979). Fail-safe numbers represent the number of null results needed to eliminate an effect, or to change a significant result to a non- significant one (Møller & Jennions, 2001).

Meta-analyses were carried out in R version 2.15.0 (R Development Core Team, 2010) using the “metafor” package (Viechtbauer, 2010) and the “psychometric” package (Fletcher,

2008). The “metafor” package was used to generate the meta-analyses using maximum likelihood techniques, create funnel plots, conduct the rank correlation publication bias test, and to generate the fail-safe numbers. The “psychometric” package was used to convert reported correlation coefficients to Z values for use in the meta-analyses.

Results

Seventy correlations were found that met the selection criteria. Nineteen of these reported data on parasite prevalence and group size (Table 2.1), 34 correlations reported data on parasite intensity and group size (Table 2.2) and 17 reported data on parasite species richness and group size (Table 2.3). The average sample size across all studies was n = 22.8 groups (range: 4 – 86

Host Transmission Host/parasite system Study n r mobility mode Swallows/arbovirus- Brown et al., 2001 S D 17 0.52 swallow bug

Aphid/fungus Cappuccino, 1988 S D 30 0.47

Grant's gazelle/strongyle Ezenwa, 2004 M I 15 0.1

Impala/strongyle Ezenwa, 2004 M I 15 0.1

Grant's gazelle/coccidia Ezenwa, 2004 M I 15 0.141

Impala/coccidia Ezenwa, 2004 M I 15 0.141

Eland/strongyle Ezenwa, 2004 M I 15 0.224

Buffalo/strongyle Ezenwa, 2004 M I 15 0.374

Hartebeest/strongyle Ezenwa, 2004 M I 15 0.447

Thomson's Ezenwa, 2004 M I 15 0.624 gazelle/coccidia

Eland/coccidia Ezenwa, 2004 M I 15 0.624

Hartebeest/coccidia Ezenwa, 2004 M I 15 0.819

Buffalo/coccidia Ezenwa, 2004 M I 15 0.843

Hoogland & Sherman, Swallow/flea S D 22 0.53 1976

Host Transmission Host/parasite system Study n r mobility mode Swallow/swallow bug Møller, 1987 S D 32 0.87

Stickleback/copepod Poulin, 1999 M D 14 0.511

Swallow/swallow bug Shields & Crook, 1987 S D 4 0.96

Red colobus Snaith et al., 2008 M I 9 -0.934 monkey/endoparasite

Black howler Trejo-Macías et al., 2007 M I 8 0.645 monkey/endoparasite

Host Transmission Host/parasite system Reference n r mobility mode Marmot/mite Arnold & Lichtenstein, 1993 S D 35 0.03

Swallow/flea Brown & Brown, 1986 S D 6 0.48

Swallow/swallow bug Brown & Brown, 1986 S D 13 0.63

Swallow/arbovirus- swallow bug Brown et al., 2001 S D 30 0.6

Sunfish/fungus Côté & Gross, 1993 S M 22 -0.55

Eland/coccidia Ezenwa, 2004 M I 15 0.671

Eland/strongyle Ezenwa, 2004 M I 15 0.077

Grant's gazelle/coccidia Ezenwa, 2004 M I 15 0.48

Grant's gazelle/strongyle Ezenwa, 2004 M I 15 0.265

Impala/coccidia Ezenwa, 2004 M I 15 0.2

Thomson's gazelle/coccidia Ezenwa, 2004 M I 15 0.794

Thomson's gazelle/strongyle Ezenwa, 2004 M I 15 0.2

Buffalo/coccidia Ezenwa, 2004 M I 15 0.52

Buffalo/strongyle Ezenwa, 2004 M I 15 0.071

Hartebeest/coccidia Ezenwa, 2004 M I 15 0.458

Hartebeest/strongyle Ezenwa, 2004 M I 15 0.648

Host Transmission Host/parasite system Reference n r mobility mode Impala/strongyle Ezenwa, 2004 M I 15 0.089

Ground squirrel/ectoparasite Hillegass et al., 2008 S D 18 -0.16

Ground squirrel/endoparasite Hillegass et al., 2008 S I 18 0.14

Swallow/flea Hoogland & Sherman, 1976 S D 22 0.75

Prairie dog/flea Hoogland, 1979 S D 9 0.28

Prairie dog/flea Hoogland, 1979 S D 10 0.72

Badger/flea Johnson et al., 2004 S D 21 0.413

Bat/bat fly Kunz, 1976 M D 6 0.94

Swallow/swallow bug Loye & Carroll, 1991 S D 8 0.03

Stickleback/branchiuran Poulin & Fitzgerald, 1989 M M 4 -0.81

Stickleback/branchiuran Poulin & Fitzgerald, 1989 M M 4 -0.72

Stickleback/crustacean Poulin, 1999 M M 14 -0.465

Stickleback/copepod Poulin, 1999 M D 14 0.706

Rubenstein & Hohmann,

Horse/nematode 1989 M I 5 0.91

Rubenstein & Hohmann,

Horse/dipteran 1989 M M 6 -0.94

Horse/dipteran Rutberg, 1987 M M 42 -0.25

French grunt/monogenean Sasal, 2003 M I 4 0.878

Swallow/flea Shields & Crook, 1987 S D 4 0.86

Host Transmission Host/parasite system Reference n r mobility mode Hoofed mammal/arthropod Ezenwa et al., 2006 M D 64 -0.32

Hoofed mammal/helminth Ezenwa et al., 2006 M I 64 -0.33

Hoofed mammal/microparasite Ezenwa et al., 2006 M - 64 -0.08

Primate/protozoan Freeland, 1979 M I 11 0.91

Birds/cestode Gregory et al., 1991 M I 86 -0.1

Birds/nematode Gregory et al., 1991 M I 84 0.2

Birds/trematode Gregory et al., 1991 M I 83 -0.06

Teleost fish/metazoan Luque et al., 2004 M - 8 0.0491

Cyprinidae/ectoparasites Poulin, 1991a M D 33 -0.21

Cyprinidae/endoparasites Poulin, 1991a M I 33 0.062

Percidae/ectoparasites Poulin, 1991a M D 10 -0.174

Percidae/endoparasites Poulin, 1991a M I 10 0.77

Salmonoid/ectoparasites Poulin, 1991a M D 17 0.455

Salmonoid/endoparasites Poulin, 1991a M I 17 0.435

Cyprinidae/mobile parasites Poulin, 1991a M M 33 -0.106

Percidae/mobile parasites Poulin, 1991a M M 10 -0.073

Salmonoid/mobile parasites Poulin, 1991a M M 17 0.314

groups), for parasite prevalence studies average sample size was n = 15.8 groups (range: 4 – 32 groups), for parasite intensity studies average sample size was n = 14.6 groups (range: 4 – 42 groups) and average sample size for parasite species richness studies was n = 37.9 groups (range:

8 – 86 groups).

Relationships between host group size and parasite prevalence

Among comparisons of parasite prevalence, the effect size estimate was high and significantly different from zero (Table 2.4a). Thus, a strong positive correlation was found between overall parasite prevalence and group size. Similar positive correlation trends were also found between group size and both directly transmitted parasites (Table 2.4a) and indirectly transmitted parasites (Table 2.4a). No studies were found that reported data on group size and parasite prevalence for mobile parasites (Table 2.1). Due to high covariation between studies on mode of transmission and host mobility, we did not test for effects of host mobility on parasite prevalence.

Relationships between host group size and parasite intensity

Larger groups had more intense parasite infections than expected by chance (Table 2.4b).

The intensities of directly transmitted parasites (Table 2.4b) and indirectly transmitted parasites

(Table 2.4b) also showed significant positive trends with increasing group size (Table 2.4b). The relationship between mobile parasite intensity and group size showed a significant negative trend

(Table 2.4b). Host mobility played a role in determining parasite infection intensity, with larger

Table 2.4: Results from each separate meta-analysis regarding parasite infection in group-living

species, with number of studies (n), overall mean correlation coefficient (Mean r), 95%

confidence intervals, Z score, P-value of significance test and fail-safe numbers.

n Mean r 95% CI Z P Fail-safe A. Parasite Prevalence 19 0.500 0.049; 0.114 4.950 <0.0001 104

Directly Transmitted Parasites 6 0.770 0.116; 0.382 3.669 0.0002 28

Indirectly Transmitted

Parasites 13 0.369 0.033; 0.130 3.269 0.0011 15

B. Parasite Intensity 34 0.331 0.013; 0.048 3.376 0.0007 105

Mobile Parasites 6 -0.540 -0.107; -0.009 -2.322 0.0202 13

Directly Transmitted Parasites 13 0.550 0.017; 0.066 3.369 0.0008 54

Indirectly Transmitted

Parasites 15 0.424 0.017; 0.078 3.031 0.0024 49

Mobile Host 21 0.225 -0.001; 0.054 1.886 0.059 23

Sedentary Host 13 0.325 0.010; 0.059 2.742 <0.0099 14

C. Parasite Species Richness 17 0.070 -0.023; 0.078 1.066 0.287 0

Directly Transmitted Parasites 4 -0.099 -0.175; 0.193 0.097 0.923 0

Indirectly Transmitted

Parasites 8 0.212 -0.026; 0.090 1.085 0.278 0

groups of sedentary hosts more likely to have higher parasite intensities (Table 2.4b). Mobile hosts did not show a significant effect of group size and parasite intensity (Table 2.4b).

Relationships between host group size and parasite species richness

Finally, for overall parasite species richness the effect size was low, making it likely that the observed mean could have been obtained by chance (Table 2.4c). No trend was obtained for the relationship between directly or indirectly transmitted parasite species richness and group size (Table 2.4c). All available studies were from mobile hosts so distinctions between mobile and sedentary hosts could not be made (Table 2.1).

Discussion

Parasite prevalence and intensity

We have noted general trends spanning a variety of taxa that support a positive relationship between group sizes in social animals and the infection intensity and prevalence of many parasites. These findings are consistent with previous meta-analyses (Côté & Poulin, 1995;

Rifkin et al., 2012). This result is not surprising when we consider that all contagious (i.e., non- mobile) parasites require direct contact between susceptible hosts and infective stages and that most parasites exhibit density-dependent or frequency-dependent transmission. Accordingly, the results presented here are supported by predictions of epidemiological models and social evolution theory (Alexander, 1974; Anderson & May, 1978). Epidemiological models predict

that parasite transmission will be enhanced in group-living species due to increased proximity and contact with infective stages (Anderson & May, 1978) resulting in elevated parasite infections amongst group members. As such, increased parasite infection intensity and prevalence appear to be costs of group-living across a broad range of host and parasite species.

Where the costs of parasitism are great, parasites may thus put constraints on optimal group sizes and the evolutionary stability of social groups.

In contrast, we have also shown that forming groups may be beneficial to individuals faced with infection by mobile parasites (e.g., dipterans, mosquitoes), possibly through the encounter-dilution effect. When confronted with mobile parasites, group formation is predicted to reduce the attack rate per individual group member (Freeland, 1977). To date, no studies have been conducted to test the relationship between group size and the prevalence of mobile parasites. Studies of mobile parasite intensity are more common and there is good support for a dilution effect (grouping benefits) when mobile parasites are present. For instance, Poulin &

Fitzgerald (1989) experimented with a mobile crustacean ectoparasite that parasitizes stickleback fish and found that attack rates increased less than linearly so that each individual in the group was less at risk when in a larger group. In mammals, Freeland (1977), Duncan & Vigne (1979),

Espmark & Langvatn (1979), Rutberg (1987) and Rubenstein & Hohmann (1989) have all found positive effects of grouping through reduced attack rates of mobile parasites. The results presented here agree with Côté & Poulin (1995) in terms of the overall trend observed between group size and mobile parasite intensity, but differ from those of Rifkin et al. (2012), possibly owing to differences in analytical techniques.

Interestingly, the mobility of the host appears to be important. Mobile hosts are able to move away from infected areas or hosts and this ability to escape may mitigate the effect of group size on parasite infection intensity. Mobile hosts may also have less rigid group structures than sedentary hosts, which may lead to highly variable group sizes over time and may confound studies looking for group size effects. Additionally, larger, less stable groups have been shown to experience increased social stress related to crowding and/or less rigid dominance hierarchies

(Sapolsky, 2005). The majority (15 of 21) of the group-living species included in the mobile host

– parasite intensity meta-analysis presented here are species that exhibit some form of hierarchical social structure. Dominant individuals generally have higher levels of androgen hormones (e.g., testosterone), which suppress host immune function (Nunn et al., 2009;

Sapolsky, 2005). Additionally, dominant individuals that frequently engage in physical combat to maintain rank may be more likely to acquire directly (contact) transmitted parasites.

Submissive individuals typically have higher levels of cortisol, a stress hormone that has also been linked to a reduced immune function (Gulland, 1992). Subordinates may also have fewer possibilities to select grazing locations and may incur a higher risk of parasitism by grazing in less preferred, contaminated locations (Hutchings et al., 2002). As a result, group-living animals that form dominance hierarchies may experience variable or skewed parasite intensities depending on the social system and physiological responses of the species or population, which could not be addressed in our study.

In sedentary groups, the social contact networks and transmission patterns are spatially and temporally stable, which may foster continual transmission of parasites between neighbours.

Most studies of sedentary hosts consider nesting or burrowing species. Nests and burrows are

spatially fixed features that hosts are ‘attached’ to, at least for the duration of study. For instance, prairie dogs typically hold the same burrow location throughout their lifetime and interact with the same set of neighbours most frequently (Hoogland, 1995). Transmission of parasites is likely then facilitated by repeated interactions between neighbours and by continuous host exposure to infective stages, such that parasites are readily shared between adjacent and nearby hosts. In some cases, neighbours are kin and related hosts may have common genetic dispositions to infection (i.e., weak immune systems). As such, we should expect that patterns of parasite infection intensity would hold for sedentary hosts, and indeed we found that they do.

If hosts are faced with a virulent parasite and higher contact rates induce greater parasite transmission, evolutionary forces should drive the host to respond with lower rates of contact to a point where the costs of group-living are minimized. Bonds et al. (2005) point out the conspicuous absence of published studies that have found a negative correlation between group size and contagious parasite prevalence. This is substantiated here as we could only find one study that reported a negative relationship between group size and prevalence that met the selection criteria. Bonds et al. (2005) question that if animal behaviours change in response to infection risk, why is it that we have not seen evidence of more substantial evolutionary influences on social structure? It is clear that the evolutionary relationship between hosts and parasites is not unidirectional, and that parasites should be expected to evolve mechanisms to overcome host adaptations and vice versa (i.e., Decaestecker et al., 2007; Duffy & Sivars-

Becker, 2007; Hamilton et al., 1990; Morran et al., 2011; Webster et al., 2004). Importantly, the evolutionary history and the fitness costs of infection should be explicitly stated in all studies of host-parasite interactions. Host specificity of the parasite also needs to be taken into

consideration. Studies have shown that parasite species able to exploit many taxonomically unrelated hosts often achieve higher intensities than specialist parasites, possibly because host species have developed particular defenses for coping with specialist parasites, but not generalists (Krasnov et al., 2004). Ezenwa (2004) showed that gregarious species of hosts were more likely to be infected than were solitary species with generalist species of strongyles (a gastrointestinal parasite). For strongyles, contact between heterospecific and conspecific hosts could both affect the rates of exposure to this group of parasites (Ezenwa, 2004). Finally, intersexual differences may drive the parasite transmission dynamics, but often sex ratios are not explicitly considered in studies of group size. Sex ratios can be extremely biased in many groups, such as harems and sexually segregated groups. In studies of group-living species it is pertinent to acknowledge that males and females are often not infected by parasites in the same manner and the transmission dynamics and infection risk can be affected by the sex ratio. In most vertebrate species, males are more likely to be infected with parasites (higher prevalence) and carry a higher intensity of infection than are females (Ferrari et al., 2004; Hillegass et al., 2008;

Klein, 2004; Moore & Wilson, 2002; Poulin, 1996; Schalk & Forbes, 1997; Zuk & McKean,

1996). Therefore, groups with male-biased sex ratios may be at an elevated risk of acquiring parasites and maintaining infections. One hypothesis that is especially pertinent to group-living species, but which has received little attention, is that males and females may sexually segregate as a means to avoid parasite transmission (Ferrari et al., 2010).

Parasite species richness

Hosts with higher mobility or those that live in open societies with intergroup member exchange are widely hypothesized to have higher parasite species richness because contact with a diversity of individuals and environments should promote the acquisition of novel parasites. A large group is also more likely to contain a group member (or members) with rare parasite fauna.

Freeland (1979) found more protozoan species in larger mangabey (Cercocebus albigena) and blue monkey (Cercopithecus mitis) groups inhabiting the same area. However, results from subsequent studies testing associations between group size or group-living and parasite species richness have varied considerably, with some studies showing positive relationships, some showing negative relationships, and others showing no relationship at all (Gregory et al., 1991;

Nunn et al., 2003; Poulin, 1991a; Poulin, 1991b; Ranta, 1992; Tripet et al., 2002; Vitone et al.,

2004; Watve & Sukumar, 1995). Our results support the conclusion that no broad general trends between parasite species richness and group size appear to exist in the available literature, counter to our original prediction. These findings are in concordance with Rifkin et al. (2012).

Given the complexities of host social behaviour, it is likely that the degree and directionality of the effect of host group size on parasite species richness depends on the specific social system of the host group/species under consideration and on other elements of host behaviour that affect contact rates, such as dominance hierarchies (Wilson et al., 2003).

Some authors have argued that sociality should lower the risk of parasite transmission if increased clustering of individuals into relatively permanent groups effectively quarantines parasites into discrete host patches (Hess, 1996; Watve & Jog, 1997; Wilson et al., 2003).

Freeland (1979) proposed that individuals that remain within a group are less likely to become infected with parasites to which they have not previously been exposed than are individuals that

frequently engage in extra-group social relationships. In this sense, closed social groups or more sedentary groups may act as barriers to parasite transmission between groups or extra-group members (Loehle, 1995), leading to reduced species richness.

Conclusions

1. Sociality is thought to be one of the last major evolutionary transitions (Jackson &

Hart, 2009) and group-living generates many advantages, such as diluted predation risk, extended parental care and enhanced potential for information sharing (Krause & Ruxton, 2002).

However, group-living also imposes costs, particularly a predicted increase in the risk of parasite infection. Local population density and social group size can increase rates of host-host contact and host interaction with parasite infective stages. While the role of parasites in the evolution of group formation and sociality are far from clear, empirical evidence appears to support the hypothesis that parasites can exert selective pressures on group size and group formation, and may have driven, at least in part, the evolution of social behaviours. We have shown here that parasite intensity and parasite prevalence generally trend positively with increasing group size, suggesting an increased cost associated with forming larger groups.

2. The results presented here are correlational and do not allow explicit derivations of causative mechanisms. Future research should focus on the causes of increased parasite intensity and prevalence in groups and the selective forces imposed by parasites on group formation and stability. In order to fully assess these problems, the costs of parasitism must be explicitly determined a priori. If parasites do not impose appreciable costs to host fitness (e.g., reduced

offspring growth and survival, reduced mating success, reduced lifespan), then parasitism should not be expected to function as an evolutionary pressure influencing group size. It is apparent that many parasites do impose costs on the fitness of their hosts and, in this regard, parasites may constrain group size. In extreme situations intense parasitism could lead to a reduction of social behaviour or the elimination of group-living altogether; however, the latter situation is highly unlikely. For group-living to be evolutionarily stable and persistent, the costs of living in groups must not outweigh the benefits. When the risk of parasite transmission increases, the costs to group-living also increase. Clearly, in these cases, benefits of group formation must be great.

According to Brown & Brown (1986), ‘‘...without compensating benefits of coloniality, the cost of ectoparasitism would quickly select for solitary nesting in Cliff Swallows.’’

3. Here we see that parasitism can be one of the most important costs associated with sociality and group-living. However, in the case of mobile parasites, group-living appears to be beneficial for reducing parasite intensities. Therefore, when grouping leads to increased parasite risk, group size will be down regulated to achieve an optimal group size that balances the costs of parasitism with the other benefits of group-living. However, in cases where grouping reduces parasite attack rates, lower parasitism will be a benefit of group-living and group sizes may be larger than expected.

4. Ultimately, group-living has probably evolved several times and for several different reasons (Alexander, 1974), so parasite risk must be considered along with other factors that favour or discourage gregariousness, such as predation and competition, in a complete cost- benefit model. If we are to fully understand why animals live in groups we must employ a

holistic approach that incorporates multiple explanatory factors, in particular host-parasite phylogenies, host sex, host group structure, parasite specificity and parasite transmission mode, in comparative analyses that will allow us to examine the links between host sociality and parasite infection in a broader context. Even after 15 years of intense study, it is still necessary to echo Côté & Poulin's (1995) call for future studies that focus on experimental manipulations of parasite infection risk in gregarious species to elucidate the causative factors modulating optimal group sizes in free-ranging animals. Additionally, studies on the relationship between parasite prevalence and group size are needed specifically for sedentary species and indirectly transmitted parasites.

CHAPTER 3: PATTERNS OF ECTOPARASITISM IN NORTH AMERICAN RED

SQUIRRELS (TAMIASCIURUS HUDSONICUS): SEX-BIASES, TEMPORAL

STRUCTURE AND EFFECTS ON MALE BODY MASS

Introduction

Parasites can affect the body condition, reproductive success, survival and physiology of their hosts (Booth et al., 1993; Brown & Brown, 2004; Hillegass et al., 2010; Khokhlova et al.,

2004; Neuhaus, 2003; Scantlebury et al., 2007; Vaughan et al., 1989), often imposing significant energetic costs (Booth et al., 1993; Careau et al., 2010; Kam et al., 2010; Khokhlova et al.,

2002; Scantlebury et al., 2007), which can result in parasite-induced evolutionary shifts in life- history traits (Chadwick & Little, 2005; Fredensborg & Poulin, 2006; Ohlberger et al., 2011;

Richner, 1998; Richner & Tripet, 1999). Parasitism is often biased or structured by age, size, gregariousness, or sex of the host (e.g., Patterson & Ruckstuhl, 2013; Rifkin et al., 2012; Schalk

& Forbes, 1997). Specifically, in many vertebrate species, males are commonly found to be the more intensely parasitized sex (Bacelar et al., 2011; Cowan et al., 2007; Gorrell & Schulte-

Hostedde, 2008; Harrison et al., 2010; Moore & Wilson, 2002; Poulin, 1996; Schalk & Forbes,

1997; Waterman et al. 2014) and, thus, males may be accountable for much of the parasite transmission in many species and populations (Ferrari et al., 2004; Perkins et al., 2003; Perkins et al., 2008; Skørping & Jensen, 2004). There have been many hypotheses offered to explain the phenomenon of male-biased parasitism. First, observed patterns of male-biased parasitism may be due to sexual dimorphisms in species where the male is the larger or more ornamented sex.

This trend is common in species with polygynous mating systems where sexual selection favours

larger or more ornate males (Clutton-Brock et al., 1977). These individuals may offer more resources (i.e., nutrients, space) to parasites (“well-fed host” hypothesis; Hawlena et al., 2005), may simply be larger targets for parasites (Moore & Wilson, 2002) and therefore easier to find, or, because of energetic constraints, larger/more ornate individuals may trade-off growth at the expense of immune function (Rolff, 2002). Second, males are generally associated with a more risky lifestyle, particularly in polygynous species (Kraus et al., 2008), and these behaviours may increase the opportunities for males to acquire and transmit parasites (Moore & Wilson, 2002).

Related to this, in mammals, males are the sex most likely to engage in natal dispersal and males often have larger home ranges than females (Cockburn et al., 1985; Dobson, 1982; Lane et al.,

2009; Pusey, 1987; Wolff et al., 1988). As such, it has been hypothesized that male hosts offer their parasites better opportunities for dispersal and inbreeding avoidance, thereby improving the parasite’s fitness. Third, levels of testosterone produced by males usually exceed those produced by females and testosterone has known immunosuppressive qualities (Zuk & McKean, 1996), which may reduce the ability of males to fend off parasite infections (immunocompetence handicap hypothesis), although support for this hypothesis is varied (Bilbo & Nelson, 2001;

Roberts et al. 2007). Ultimately, trade-offs likely exist between combating an infection and the allocation of energy to growth, reproduction, and metabolism (Folstad & Karter, 1992; Owens,

2002).

Here we explore the patterns and consequences of ectoparasitism in a population of asocial rodents, North American red squirrels (Tamiasciurus hudsonicus), across four life-history stages (birth, emergence, adult mating, adult post-mating). Male-biased parasitism has been observed previously in this species from a geographically distant population (Gorrell & Schulte-

Hostedde, 2008). Male red squirrels do not possess secondary sexual characters (i.e., antlers, bright colours) that may affect parasitism and their parasite infection status can be tracked both from birth through to emergence and as adults. As such, red squirrels are a good species for exploring the topic of sex-biased parasitism, particularly since any observed male-bias in adult parasite infection intensity should result from differences in behaviour or physiology. Red squirrels are widely considered to be non-sexually dimorphic, however males are generally 5-

10% heavier than females (Boutin & Larsen, 1993; see Results). We predicted that males would display higher ectoparasite intensities than females throughout the study period if body size dimorphism drives ectoparasite infection intensity; however, since any sexual size dimorphism is slight in this species we did not expect to find any difference in ectoparasite intensity based on size alone. If ectoparasites preferentially select larger (heavier) hosts, then we expected heavier adult squirrels to host greater intensities of ectoparasites. Alternatively, larger animals may be larger because they have greater immunocompetence and, therefore, would be expected to have fewer parasites (within the same sex). Additionally, if males are preferred hosts due to their propensity to disperse and distribute contact-transmitted ectoparasites, then we predicted that males would be more heavily parasitized at the time of natal dispersal (emergence) or at the time of mating, when rates of host-host contact (i.e., in the nest, between mating individuals) and off- territory ranging movements are greatest (Lane et al., 2009; Larsen & Boutin, 1994). Finally, we experimentally removed ectoparasites from male red squirrels to investigate the effects of ectoparasite infection on host body mass. If male hosts are the more heavily parasitized sex and there is an energetic cost associated with parasitism (Booth et al., 1993; Scantlebury et al.,

2007), parasites should decrease the amount of energy available to support body maintenance

(Speakman, 2008), with the prediction that ectoparasites would negatively affect the body mass of male red squirrels across time.

Materials and methods

This study was conducted in Sheep River Provincial Park, Alberta, Canada (110° W, 50°

N; 1500 m) between April and October 2010 and 2011. Red squirrels were captured in coniferous-dominated forest by use of live-traps (XLF15, H. B. Sherman Traps, Inc.,

Tallahassee, Florida) baited with peanut butter. One grid (10×4 trap pattern) was established at each site with forty traps spaced 50 m apart at three distinct sites (~6.75 ha per site) within a 5 km radius. Upon capture, each squirrel was ear tagged (Monel #1, National Band and Tag Co.,

Newport, Kentucky), weighed using a spring scale (±1 g; Pesola AG, Baar, Switzerland), and the zygomatic breadth (skull width) was measured using a dial caliper to the nearest 0.1 mm

(Scienceware #134160001, Bel-Art Products, Wayne, New Jersey). For consistency, all body masses used in the analyses were collected in the early morning (between 7h00 and 10h00) and masses collected on consecutive days were averaged. In both years, pregnant females were radiocollared (N = 12; SOM-2190, Wildlife Materials, Inc., Murphysboro, Illinois) and tracked to their nests at night following parturition to gain access to their offspring. Offspring were marked with unique ear notches shortly after birth and, where possible, the nest was revisited

~25 days later to mark the offspring with numbered ear tags (Monel #1, National Band and Tag

Co., Newport, Kentucky).

At each capture all individuals were systematically searched for 2 minutes to count fleas

(Orchopeas caedens, Monopsyllus vison [Mahrt & Chai, 1972]) using a flea comb and by visual inspection following the methods described by Patterson et al. (2013). Detailed flea counts from both adult males and females were collected in 2011, while counts from juveniles were taken in

2010 and 2011. In 2010, adult males were only inspected for the presence or absence of fleas using the aforementioned search technique at first capture to ensure that the removal experiment described below was not biased in any way. Fleas were counted, but never removed from untreated control animals. All juveniles were inspected for fleas at the time of first nest entry

(birth) and at emergence from the nest (~40 days after parturition) when juvenile squirrels begin to explore, disperse, and settle away from the natal territory (Rusch & Reeder, 1978). While likely present, we did not quantify prevalence or intensity of lice, mites or ticks.

Data on flea intensities were divided into four groups based on life-history stage: mating adult, post-mating adult, juvenile birth and juvenile emergence. When mating, male red squirrels have scrotal testes, which become abdominal when mating ceases (Layne, 1954). Only reproductively mature males were included in this study. In 2011, mating was delayed compared to the two previous years, possibly due to a deep snow pack, late winter weather, poor food availability, and/or an impending spruce cone mast (J. Patterson, pers. obs.; Fletcher et al.,

2013). For determining sexual dimorphism, we chose mass and zygomatic breadth of adults (>1 year) in Spring 2011 (May) and Fall 2011 (September-October). We only used individuals that we had trapped in previous years to ensure that yearlings were not included in the analysis.

Although males were scrotal in May 2011, none of the females displayed signs of pregnancy via

palpation or lactation in May 2011. In the September-October 2011 dimorphism analysis, we only included females who had ceased lactation.

For the parasite removal experiment, we captured reproductively active (i.e., scrotal) adult males at the beginning of May 2010. Males were initially placed into treatment and control groups based on the flip of a coin and alternating thereafter. Males in the treatment group were each given 0.15 ml/kg of Bayer K9 Advantix (8.8% imidacloprid, 44.0% permethrin; Bayer

HealthCare LLC, Animal Care Division, Shawnee Mission, Kansas) applied directly to the skin between the shoulder blades. Treatment was repeated every 30 days. At the dosage used, this combination of drugs has been shown to be highly effective at killing arthropod parasites with a low toxicity in mammals, including squirrels (Larsen et al., 2005; Metzger & Rust, 2002).

Control and treated animals were both handled and trapped in the same manner and with equal effort; however, controls were not given the medication. We explored differences in average mass as well as mass gains and losses across time between treated and control males at 30-day intervals: day 0 (when treatment started: first two weeks of May 2010), day 30 (30 days after initial treatment: first two weeks of June 2010) and day 90 (90 days after initial treatment: first two weeks of August 2010).

Where multiple flea counts or squirrel masses were obtained in the same period for the same individuals we took the average value for use in the analysis. All measures of median intensity of ectoparasites, as well as their associated 95% confidence intervals (CI), were determined using Quantitative Parasitology version 3.0 (Rózsa et al., 2000). All data were tested for normality and data that were normally distributed were analyzed using a two-sample t-test or

a generalized linear model (GLM). Non-normal data were difficult to properly transform, so in these cases Mann-Whitney tests and a full model GLM fitted with a quasi-Poisson distribution were used to compare the data. All means are reported  1 standard deviation (SD) unless otherwise stated. All statistical analyses were conducted using R version 2.12.0 (R Development

Core Team, 2010).

Results

Adult sexual dimorphism

Adult male red squirrels ( x =239.117.7 g, n=33) averaged 19.7 g heavier than adult females ( =219.4 10.85 g, n=32) in May 2011 (t58.7=-5.12, p<0.001), resulting in a mass ratio  of 1.09. The dimorphism in body mass persisted across time, as adult males ( =237.520.9 g, n=16) were, on average, 22.6 g heavier than adult females ( =214.916.3 g, n=17) in the fall season of 2011 (t28.4=3.45, p=0.002), producing a mass ratio of 1.11. Average adult male zygomatic breadth ( =27.41.0 mm, n=33) did not differ from that of adult females (

=27.10.7 mm, n=32) in May 2011 (t41.7=-1.21, p=0.232).

Host body mass and ectoparasitism

Adult male red squirrel body mass during both the breeding period (t=0.071, p=0.944) and the post-mating period (t=-1.173, p=0.259) did not predict the intensity of fleas during those

time periods in 2011 when entered in a GLM. Similarly, adult female red squirrel body mass during both the breeding period (t=-0.840, p=0.407) and the post-mating period (t=0.950, p=0.355) in 2011 did not predict the intensity of fleas in a GLM.

Temporal dynamics of ectoparasitism

Ectoparasitism in red squirrels was male-biased, however this bias was temporally structured. No significant sex bias existed at either birth (Table 3.1; U=371, p=0.119) or emergence (Table 3.1; U=56.5, p=0.934). However, during the mating/pre-parturition period, adult males had significantly more fleas than adult females (Table 3.1; U=892, p=0.001).

Additionally, flea intensities were significantly female-biased during the lactation period (Table

3.1; U=198, p=0.021). Juveniles at birth were the most intensely parasitised host life-history stage for both sexes (Table 3.1). Males in the mating period had significantly higher flea intensities than males in the post-mating period (U=79, p<0.0001). There was no difference between flea intensities in adult females during and after their reproductive period (U=257.5, p=0.768). In a GLM, life history stage was an important predictor (t=-3.442, p=0.0007), but sex was not significant (t=1.158, p=0.248) and there were no interaction effects (t=-1.151, p=0.251).

Ectoparasite removal

No effect of parasite removal on average body mass was observed in adult males between

May (Day 0) and August (Day 90) 2010. Also, no effect was observed in body mass gains or

Table 3.1: Median number of fleas and median number of fleas per unit mass of the hosts across the four life-history stages for male and female red squirrels in Sheep River Provincial Park,

Alberta. Number of individual hosts in the sample (n) and 95% confidence intervals (CI) are reported.

No. of Range No. of Life-history stage n ectoparasites (min- 95% CI ectoparasites (median) max) (median)/g*100 Juvenile male birth 28 5.0 0-18 3.0-7.0 11.09

Juvenile female birth 21 4.0 0-12 2.0-9.0 9.82

Juvenile male emergence 11 3.0 0-8 1.0-5.0 2.65

Juvenile female emergence 10 2.0 0-6 1.0-4.0 1.86

Adult male mating 36 3.5 0-17 2.0-4.0 1.48

Adult female pre-parturition 34 1.6 0-6 1.0-2.0 0.72

Adult male post-mating 17 1.0 0-2 1.0-2.0 0.43

Adult female lactation 19 2.5 1-6 2.0-3.0 1.07

losses of control (n=12) and treated (n=12) individual males across the treatment periods.

Between Day 0 and Day 30 (June) control males lost an average of 4.29 g (SD=13.12), while treated males lost an average of 5.93 g (SD=7.56; t11.4=0.309, p=0.763). Between Day 30 and

Day 90 control males lost an average of 14.07 g (SD=10.66), while treated males lost an average of 7.93 g (SD=11.72; t11.9=-1.026, p=0.325). Over the course of the entire study (Day 0 to Day

90) control males lost an average of 14.75 g (SD=9.75), while treated males lost an average of

13.06 g (SD=8.36; t11.7=-0.349, p=0.733).

Discussion

We have shown that parasitism of red squirrels by fleas is temporally sex-biased, with adult males facing increased infection intensities during the mating period and adult females incurring higher flea intensities than males during the lactation period. Fleas may be exploiting the behaviour and life-history strategies of their hosts for transmission and reproduction. For instance, fleas are transmitted through direct host-host contact, which in red squirrels typically only takes place between adults during the mating season (Steele, 1998) and in the natal nest.

Females are in oestrous for only one day and during that time they may mate with an average of seven males (McFarlane et al., 2011). Males engage in a scramble competition for females and during coitus the male holds the female around the posterior abdomen while resting his head and anterior abdomen on her back (Steele, 1998). Copulations generally last for less than 60 s (Lane et al., 2009; McFarlane et al., 2011). Direct contact during mating increases the potential for transmission of contact-transmitted parasites and may be the only opportunity for ectoparasite transmission outside of the natal nest. Females, on the other hand, are exposed to higher

intensities of fleas in the nest environment when rearing their young (Table 3.1) and, consequently, carry significantly more fleas during the lactation period than their adult male counterparts. Lactation and pregnancy are energetically demanding events for all female mammals (Speakman, 2008), during which maternal immunity is reduced (Jones et al., 2012;

Lloyd et al., 1983) and increased susceptibility to parasite infection is common (e.g., Festa-

Bianchet, 1989; Shubber et al., 1981), although flea intensities did not appear to differ based on female reproductive status in red squirrels. There is no indication that adult females pay a personal cost to their body mass through ectoparasitism, although higher ectoparasite intensities in the nest appear to negatively affect the growth and survival of offspring (Patterson et al.,

2013; Chapter 4).

On their own, transmission dynamics may not fully explain the observed male-bias in flea abundance. During the reproductive period males of many species generally experience elevated levels of circulating testosterone (Boonstra et al., 2001; Brockman et al., 1998; Cavigelli &

Pereira, 2000), with testosterone concentrations positively correlated with reproductive success in some species (Alatalo et al., 1996; Hutchison & Hutchison, 1983). Testosterone has been linked to lower immune function and higher parasite infection intensities in many species (Cox

& John-Alder, 2007; Decristophoris et al., 2007; Folstad & Karter, 1992; Folstad et al., 1989;

Mougeot et al., 2004; Mougeot et al., 2006; Saino et al., 1995). The link between testosterone and parasite infection is often cited as a key driver of male-biased parasitism as testosterone mediates sex differences in immune function and risky behaviour (Grear et al., 2009; Hughes &

Randolph, 2001; Klein et al., 2004; Zuk & McKean, 1996). However, testosterone may also lead to elevated levels of circulating glucocorticoids and, as such, its effects may be correlated with

stress-induced immunosuppression (Evans et al., 2000). Male red squirrels search extensively for receptive females during the mating period, while females generally stay on their territories during this time (Lane et al., 2009). Males may also spend less time grooming during the breeding period as they allocate energy and time to other functions, such as mate searching.

Therefore, differences in ranging behaviour and activity budgets during the mating period could also explain the observed differences in flea intensities. Ultimately, male red squirrels during the reproductive period may experience elevated testosterone and stress hormone levels, lowered immune function, higher rates of intraspecific contact, increased ranging behaviour and reduced auto-grooming, all of which may contribute to increasing their reproductive success (Gooderham

& Schulte-Hostedde, 2011; Lane et al., 2009) and flea infection intensities concomitantly.

Gooderham and Schulte-Hostedde (2011) found that male red squirrels with higher ectoparasite intensities had higher reproductive success, suggesting a trade-off between investment in reproduction and exposure to parasites. All of these factors may combine to produce the observed biases in male ectoparasitism during the reproductive period as fleas exploit host behaviour and physiology. Our findings seemingly contrast with those of Gorrell and Schulte-

Hostedde (2008), who found that male red squirrels only had more fleas than females during the month of August; however, males in their population had become secondarily scrotal and reproductively active in August with subsequently higher testosterone levels than in July. As such, while our temporal patterns of ectoparasitism differ for adult males, the hypothesis that fleas favour their male hosts during the mating period is still supported across these two geographically distinct populations.

While we did not investigate the sex of the fleas, Gorrell and Schulte-Hostedde (2008) found that male red squirrels were more likely to be parasitised by male fleas. If dispersal and inbreeding avoidance are the objectives, then male fleas should prefer male red squirrels, but only during the breeding period or during natal dispersal. Higher flea intensities on males during the reproductive period suggest that male red squirrels may be responsible for transmitting infective adult fleas between susceptible hosts. Male-driven parasite transmission has been observed in other rodent species (Ferrari et al., 2004; Perkins et al., 2003). But why might males be so important for the transmission of parasites? Perhaps, it may be due to sex-related differences in behaviour and home range size which are common amongst mammals

(Greenwood, 1980). The potential roles of immunocompetence and androgen hormones also cannot be understated. Ultimately, the role of host sex in parasite transmission dynamics and the underlying mechanisms require further study.

Our finding that male-biased parasitism does not exist in juveniles at either birth or emergence from the nest suggests that fleas are not exploiting juvenile males for their ability to disperse in order to reduce possible effects of inbreeding. In red squirrels, dispersal is rather limited with most juveniles choosing to settle on or near their mother’s territory (Larsen &

Boutin, 1994), although long-range natal dispersal has been observed (Haughland & Larsen,

2004; Sun, 1997). Dispersal in red squirrels does not appear to be significantly sex-biased

(Boutin & Larsen, 1993); however, pre-settlement ranging behaviour by dispersing males and females has not been adequately studied. Interestingly, in our population, males at birth experience, on average, the highest flea intensities than at any other life-history stage. This effect is even more pronounced when we take body mass into account (Table 3.1). While in the nest,

fleas distribute themselves more or less equally amongst male and female neonates, yet the high infestation intensities experienced by neonates suggest that, in general, fleas are preferentially exploiting the relatively defenceless hosts for nutritional resources. This was corroborated by the

GLM which showed life-history stage to be a more important predictor of flea intensity in host red squirrels than host sex. It is unclear why flea intensities decline from birth to emergence in juveniles, although we speculate that this decline may be the result of immune system development, which can take several weeks in rodents (Grindstaff et al., 2003; Hasselquist &

Nilsson, 2009), juveniles learning to auto-groom, and/or the life cycles of the ectoparasites. At northern latitudes, fleas parasitizing arboreal squirrels display annual cyclic population fluctuations, with each flea species possessing its own non-overlapping population peak (Day &

Benton, 1980; Haukisalmi & Hanski, 2007). For flea species common during the host’s reproductive period, a single annual population peak typically coincides with host lactation and the presence of host neonates in the nest (Day & Benton, 1980; Haukisalmi & Hanski, 2007).

Overall, by selecting adult males during the breeding season and by preferring neonates to adults, fleas may be actively selecting their hosts to increase their likelihood of transmission or obtaining a meal or a mate. As such, a picture of flea development and transmission in red squirrel hosts begins to emerge coinciding with the life cycles of both host and parasite within which males appear to play an important role in the transmission of fleas, while juveniles of both sexes likely play an important role in the development and reproduction of fleas. Given the immunosuppressant effects of reproduction in both sexes, the time to acquire immunity in neonate rodents, and the annual lifecycles of fleas in arboreal squirrels at northern latitudes, fleas may be sequentially exploiting the immunosuppressed: first males, then females, and then their young.

Sexual dimorphism (observed here as heavier males) likely does not explain the male- bias in parasite infection intensity, as male-biased parasitism was only evident during the mating period, while the dimorphism persisted across time. The sexually-dimorphic mass ratios observed in this red squirrel population are consistent with those found in other, more northerly, geographic regions (Boutin & Larsen, 1993). Similarly, there was no apparent effect of host mass on flea intensity, suggesting that fleas do not choose larger hosts. Alternatively, this finding could be taken to provide evidence that fleas do not affect the mass of their adult hosts. Taken as a whole, fleas of red squirrels do not appear to select for host size, regardless of host sexual dimorphism (Gorrell & Schulte-Hostedde, 2008), and, as such, these results do not support the

“larger host” class of hypotheses (Moore & Wilson, 2002). This finding is generally supported by several other studies indicating that sex biases in ectoparasitism are not driven by body size sexual dimorphisms in small mammals (Krasnov et al., 2005; Morand et al., 2004; Scantlebury et al., 2010; Waterman et al., 2014).

The results of the parasite removal experiment did not support our hypothesis that ectoparasites affect the ability of adult males to maintain their body mass. Raveh et al. (2011) found no effect of ectoparasite removal on male body mass in Columbian ground squirrels

(Urocitellus columbianus). A lack of any observable effect of ectoparasite removal on male body mass, however, does not mean that ectoparasites have no effect on males in this red squirrel population. In fact, quite the opposite may be true. Removal of energetically consumptive parasites may have freed energy and time for males to invest in other fitness-improving activities, such as territory defence, mate searching, food acquisition, sperm production, and

vigilance. There may also have been effects on larder hoard depletion, which we did not assess.

Furthermore, body mass measures may hide some of the underlying parasite-driven variation in the mass of certain organs (e.g., spleen) and fat storage (Scantlebury et al., 2010), although body mass does appear to be a good predictor of body fat storage in red squirrels (Becker, 1992;

Humphries & Boutin, 1999). Additionally, if parasites could be permanently removed, non- parasitized males may experience improved survival by not having to mount a costly immune response to ectoparasites (Hanssen et al., 2004) and, therefore, their possibility of improved lifetime reproductive success may be enhanced. As mate search tactics are under positive sexual selection pressure in male red squirrels (Lane et al., 2009), parasite removal prior to and during the mating period may allow males to invest more energy into mating effort, thereby leading to improved mating success. However, male red squirrels appear to trade-off reproductive investment and parasite infection (Gooderham & Schulte-Hostedde, 2011) and male Columbian ground squirrels did not experience increased reproductive success when ectoparasites were experimentally removed (Raveh et al., 2011). Variability in host and parasite genetics, parasite virulence, parasite co-infection, host demography, and environmental conditions may influence the effects of parasites on host reproductive success, body mass, and transmission dynamics, and these potential relationships require further study. Red squirrels represent an interesting system for further testing hypotheses about the effects of parasites on male reproductive success and sexual selection on male traits.

CHAPTER 4: PARASITE REMOVAL IMPROVES REPRODUCTIVE SUCCESS OF

FEMALE NORTH AMERICAN RED SQUIRRELS (TAMIASCIURUS HUDSONICUS)

Introduction

Two of the central tenets of life-history theory are that natural selection maximizes fitness and that fitness-related traits are constrained by trade-offs given limited resources

(Stearns, 1992). By imposing considerable energetic costs on their hosts (Booth et al., 1993;

Careau et al., 2010; Scantlebury et al., 2007), parasites have been implicated in the development, expression and evolution of many life-history traits (Chadwick & Little, 2005; Ohlberger et al.,

2011). Of particular interest to evolutionary ecologists is the role that parasites play in shaping the fitness of their hosts. In mammals, an increase in maternal investment can increase offspring condition and/or mass, which in turn may improve offspring fitness (Trivers, 1974). Parasites impose considerable energetic and behavioural costs on their hosts (Giorgi et al., 2001;

Khokhlova et al., 2002; Ritter & Epstein, 1974; Scantlebury et al., 2007). Hosts therefore have to deal with trade-offs between current reproduction, future reproduction, maintenance, survival and the costs associated with parasites. To address these complex trade-offs, parents may adjust the number and quality of their offspring to optimize their own reproductive success.

There have been relatively few experimental tests of the relationship between host reproductive success and parasitism, especially in mammals (Hillegass et al., 2010; Neuhaus,

2003). Neuhaus (2003) and Hillegass et al. (2010) both used anti-parasitic insecticides to experimentally remove ectoparasites from female ground squirrels (Urocitellus columbianus and

Xerus inauris, respectively). These experimental removals resulted in significant increases in the reproductive success of the studied females (Hillegass et al., 2010; Neuhaus, 2003). Correlative tests have been more ambiguous in mammals. For instance, ectoparasite intensity was positively correlated with the reproductive success of adult male North American red squirrels

(Tamiasciurus hudsonicus; Gooderham & Schulte-Hostedde, 2011), while ectoparasite intensity did not correlate with the reproductive success of muskrats (Ondatra zibethicus; Prendergast &

Jensen, 2011). However, greater ectoparasite intensity has been correlated with reduced overwinter survival, and thus recruitment, of juvenile alpine marmots (Marmota marmota;

Arnold & Lichtenstein, 1993). Comparatively more work has been done with ectoparasite interactions in birds; nonetheless, the experimental and correlative results with respect to host reproductive success appear similarly equivocal (Allander, 1998; de Lope & Møller, 1993; de

Lope et al., 1998; Harriman & Alisauskas, 2010; Johnson & Albrecht, 1993; Møller, 1990;

Møller, 1993; Moss & Camin, 1970; Oppliger et al., 1994; Pacejka et al., 1998). The effects of ectoparasites on host reproductive success and physiology are variable and dependent on environmental conditions, host susceptibility, host life-history, and host-parasite coevolutionary ties.

To evaluate the impact of ectoparasites on the reproductive success and survival of North

American red squirrels, we experimentally removed ectoparasites from reproductive females shortly after mating. We compared juvenile mass, juvenile survival from birth to emergence, and juvenile emergence dates between control and treatment litters, as well as the body mass and overwinter survival of parasitized and non-parasitized mothers. Since ectoparasites can reduce the amount of energy available to support lactation, body maintenance, offspring growth, and

survival (Speakman, 2008), we predicted that ectoparasite removal would result in heavier mothers, increased overwinter survival of mothers, heavier juveniles at emergence, and a higher percentage of juvenile survival between two important life-history stages (birth and emergence).

If parasite removal resulted in faster-growing juveniles, we predicted that these juveniles, while not necessarily heavier than control juveniles, would experience advanced emergence dates compared to controls. Earlier emergence dates can convey important advantages to juvenile red squirrels, such as increased overwinter survival (Kerr et al., 2007). Measuring the effects of ectoparasites on female reproductive success, offspring mass and offspring survival will contribute to our understanding of the coevolution of host and parasite life-history traits.

Materials and methods

Study area

We conducted this study in Sheep River Provincial Park, Alberta, Canada (110° W, 50°

N; 1500 m) in the foothills of the Rocky Mountains between 2010 and 2011. Study sites were composed of mature second-growth subalpine forest (~80-100 years old) dominated by white

(Picea glauca) and black (P. mariana) spruce, and interspersed with aspen (Populus tremuloides), balsam fir (Abies balsamea) and lodgepole pine (Pinus contorta var. latifolia).

Study species

Red squirrels are small (<250 g) rodents common in coniferous habitat throughout much of North America. They defend food-based territories containing at least one central food cache

(midden) year-round (Smith, 1968). Red squirrels are promiscuous and they exhibit a scramble- competition mating system wherein a female mates with an average of 7 males in a single day

(McFarlane et al., 2011). Females are in oestrus for one day and males congregate on her territory to compete for access (Steele, 1998). Reproductive females generally produce a single litter each year following a mean gestation period of ~33 days (Steele, 1998). Juveniles emerge from the nest approximately 40 days after birth and are fully weaned and independent at about 70 days following birth (Steele, 1998). Mean litter sizes range from 3.2 to 5.4 depending on geographic location and food availability (McAdam et al., 2007; Steele, 1998). This species displays a small degree of sexual dimorphism, with adult males being slightly (5-10%) heavier than females (Boutin & Larsen, 1993).

Red squirrels in the Sheep River population host at least three species of fleas

(Orchopeas caedens, Monopsyllus vison, Taropsylla coloradensis), two species of lice

(Hoplopleura sciuricola, Neohaematopinus sciurinus), and one species of tick (Ixodes angustus;

Mahrt & Chai, 1972).

Animal capture and experimental design

We captured squirrels from late April to late September in 2010 and 2011 at three ~6.75 ha sites by use of live-traps (H. B. Sherman Traps, Inc., Tallahassee, Florida) baited with peanut butter. One grid (10 x 4 trap pattern) was established at each site with forty traps spaced 50 m

apart. Fleas were counted, but never removed, from all captured animals used in the study by systematic searching and combing with a fine metal flea comb. We searched the entire body of each squirrel for two-minutes: dorsally from the tip of the tail to the ears and ventrally from the tip of the tail to the forelimb in 1 cm swatches by combing and blowing on the fur to expose the skin while counting any observed fleas. We paid particular attention to the ears, groin/genitals and underarms, as fleas appeared to favour these areas. Any fleas found in the handling bag following the release of the animal were also included in the prevalence and intensity estimates.

Prevalence (number of infected individuals in a sample of hosts; Bush et al., 1997) and intensity

(number of fleas per infected individual; Bush et al., 1997) were determined for all mothers and their offspring. We did not quantify prevalence or intensity of lice, mites or ticks.

We captured adult females, weighed them to the nearest gram using a spring scale

(Pesola AG, Baar, Switzerland), attached unique ear tags (Monel #1, National Band and Tag Co.,

Newport, Kentucky) with unique combinations of coloured washers (National Band and Tag Co.,

Newport, Kentucky) and radio-collared them (SOM-2190, Wildlife Materials, Inc.,

Murphysboro, Illinois). In 2010 and 2011, we randomly assigned mothers to either the treatment or control group, initially based on the flip of a coin and alternating thereafter. We treated all females in the treatment group once every thirty days with 0.15 ml/kg of K9 Advantix (8.8% imidacloprid, 44.0% permethrin; Bayer HealthCare LLC, Animal Care Division, Shawnee

Mission, Kansas) applied to the skin between the shoulder blades. This drug combination is highly effective at killing ectoparasites (including fleas, lice, mites and ticks) and has low toxicity in mammals, including squirrels, at the topical doses used (Larsen et al., 2005; Metzger

& Rust, 2002). We initiated the treatment of squirrels 2-3 weeks prior to parturition to ensure

that the medication had sufficient time to remove ectoparasites from the mother and from the nest. We handled and trapped the control animals in exactly the same manner and with equal effort as the treatment animals, but did not give them the medication. We conducted all trapping in the morning (7h00 – 10h00) to ensure consistency in body mass measurements. If individuals were captured on consecutive days, the average mass was used in the analysis.

Shortly after parturition, we located nests using radio-telemetry. We weighed (± 0.5 g;

Pesola AG, Baar, Switzerland), sexed and aged (Boutin & Larsen, 1993) all offspring. We then gave offspring unique ear notches and reentered the nest when the offspring were ~25-30 days old to ear-tag them. Several days before juvenile emergence was expected, we tracked mothers to the nest and observed the nest area for any emergent juveniles twice daily until emergence occurred. When juveniles emerged from the nest (mean: 41.5 days after birth, range: 38-44 days after birth) we captured and weighed the mother and her offspring. To control for the mass of individual mothers, we compared mass differences (mass loss or gain) across subsequent life- history events (birth, emergence and weaning). We did not include cases of complete post- parturition litter loss in the analysis.

We determined overwinter survival of mothers by setting traps on and in the vicinity

(within 50 m) of her territory and by trapping the same grids used the previous summer between

May 1 and June 15, 2011. A trapping pattern of three days open, three days closed was followed

(totalling approximately 840 trap-nights per individual). Territory ownership rarely changes across years with the exception being when mothers bequeath their territories to one of their offspring and move to an unoccupied nearby territory or when territory owners die (Berteaux &

Boutin, 2000; Larsen & Boutin, 1995; Price & Boutin, 1993). As such, trapping and observations of squirrels on and around known territories should give an accurate representation of overwinter survival of adult squirrels.

Data analysis

All data were checked for normality and transformed if necessary. Where data could not be normalized, nonparametric tests were used. Proportional data were normalized using an arcsine transformation. All measures of prevalence and median intensity of fleas, as well as their associated 95% confidence intervals (CI), were determined using Quantitative Parasitology version 3.0 (Rózsa et al., 2000). We used Sterne’s exact method to determine the 95% CIs for parasite prevalence (Rózsa et al., 2000). All other statistical analyses were done in R version

2.12 (R Development Core Team, 2010). We tested for year effects with respect to offspring survival, offspring mass at emergence, and emergence dates (number of days from birth) using generalized linear models (GLM) with year, parasite treatment and mother’s mass at emergence as random variables. When no year effects were found, the data were pooled as treatment and control groups in comparative analyses. All comparisons between treatment and control groups were tested using two sample t tests, except for comparisons of mother’s mass differences, which were analyzed using nonparametric Mann-Whitney U tests. In each year, we followed 12 mothers from parturition through to juvenile emergence and, in each year, 6 mothers were treated and 6 were left as controls. For some of the analyses, we either pooled mothers and litters (n=24) to control for certain effects of interest (i.e., year) or we pooled, where possible, the control

(n=12) and treatment (n=12) groups for comparison tests. All means are reported ± 1 standard

deviation, unless otherwise noted. For non-significant results, we provide 95% CIs for the effect size as a way of describing the range of effect sizes supported by the data (Colegrave & Ruxton,

2003). For parametric data these were based on Cohen’s d and for nonparametric data these are based on Cliff’s ∆, and were computed using the “orddom” package in R (Rogmann, 2012).

Results

Flea prevalence and intensity

Mothers were only entered into the experiment, as either a control or a treatment animal, if they were found to host at least one flea at the outset. No fleas were observed on treated mothers two weeks following the initial treatment and throughout the study period. Prevalence of fleas on the litters of treated mothers was 0.0% (95% CI: 0.0-24.0%; n=12 litters) and 100.0% for control mothers (95% CI: 76.0-100.0%; n=12 litters) at the time of birth. Prevalence of fleas amongst individual offspring from control mothers at birth was 79.6% (95% CI: 66.0-89.0%, n=49), when data from 2010 and 2011 were pooled. Median intensity of fleas per individual juvenile from control mothers at birth was 5.0 (95% CI: 4.0-7.0; n=39). The median intensity of fleas in the entire litter at birth from control mothers was 19.5 (95% CI: 10.0-27.0, n=12 litters), when data from 2010 and 2011 were pooled. Median intensity of fleas per juvenile from control mothers at emergence was 3.0 (95% CI: 1.5-4.5; n=19), when 2010 and 2011 data were pooled.

Mother’s mass and survival

Control mothers did not lose or gain significantly more mass than treated mothers between measurement periods. Between parturition and emergence, control mothers lost a mean mass of 1.75±10.69 g and treated mothers lost 1.5±24.12 g (Mann-Whitney U=6.0, p=0.629,

∆=0.25, 95% CI for effect size: -0.66-0.86) in 2010, while in 2011, control mothers gained

2.0±17.3 g and treated mothers gained 10.4±5.4 g (U=3.5, p=0.800, ∆=0.22, 95% CI for effect size: -0.75-0.89). Between emergence and weaning, control mothers lost 24.0±16.5 g and treated mothers lost 16.8±25.0 g (Mann-Whitney U=10.0, p=0.686, ∆=-0.25, 95% CI for effect size: -

0.86-0.65) in 2010, while in 2011, control mothers lost 36.7±17.4 g and treated mothers lost

28.4±10.1 g (U=5.0, p=0.176, ∆=0.58, 95% CI for effect size: -0.30-0.93). Between parturition and weaning, control mothers lost 25.8±7.9 g and treated mothers lost 18.3±6.2 g (Mann-

Whitney U=4.0, p=0.314, ∆=0.50, 95% CI for effect size: -0.44-0.92) in 2010, while in 2011, control mothers lost 25.8±5.6 g and treated mothers lost 13.4±10.6 g (U=2.0, p=0.229, ∆=0.67,

95% CI for effect size: -0.39-0.97). Overwinter survival of mothers did not differ amongst treatment and control groups between 2010 and 2011, as two control mothers and two treated mothers from 2010 were not found in 2011.

Juvenile mass and survival

There was no effect of year or mother’s mass at emergence on the mass of juveniles at emergence (t=-0.608, p=0.546, n=24) or on offspring survival from birth to emergence (t=1.959, p=0.074, n=24) when treatment, mother’s mass and year were jointly entered into a GLM. As such, data were pooled across years. Treated mothers raised offspring that were, on average, 11.6 g (11.1%) heavier at emergence ( x =116.2±14.6 g, n=12 mothers) than control juveniles (

64 

=104.6±14.2 g, n=12 mothers; t=2.547, p=0.014 when controlling for mother’s mass, mother’s

ID, and year in a GLM). On average, 75.2%±17.5% of juveniles from treated mothers (n=12) survived from birth to emergence, whereas only 51.3%± 22.3% of juveniles from control mothers (n=12) survived the same interval (t14.98=-2.350, p=0.033).

Emergence date

Year (t=0.776, p=0.450), mother’s mass (t=-0.148, p=0.884) and parasite treatment (t=-

0.251, p=0.805) were not significant predictors of juvenile emergence date in a GLM (n=24 litters). When treated (n=12) and control (n=12) mothers were compared, treatment was not found to affect the date of litter emergence (t14.65=-0.215, p=0.833, d=-0.01, 95% CI for effect size: -0.52-0.87). Average number of days from birth to emergence for treatment and control litters was 41.4±1.9 days and 41.6±1.1 days, respectively.

Discussion

Our study shows that the removal of ectoparasites can directly affect host reproductive success. Our results suggest that ectoparasites have a substantial and persistent negative effect on the reproductive success of red squirrel mothers, in that we detected costs to reproductive output due to parasitism across years and despite a small sample size. Not only did our broad ectoparasite removal affect the mass of juveniles at emergence, but it also enhanced survival probabilities of juveniles between successive life-history stages (birth and emergence). Similarly, access to food resources has been found to influence the survival of juvenile red squirrels from

birth to emergence, although food supplementation only affected juvenile survival and did not improve the body mass of juvenile red squirrels at emergence (Kerr et al., 2007). However, much like access to food, parasites factor into the energetic equation of their hosts. Parasitised offspring, by losing energy directly to parasites (Khokhlova et al., 2002), likely have less energy to invest into their own growth and condition regardless of the mother’s investment. While we only quantified fleas in this study, we do not attribute our findings solely to fleas since lice, mites and ticks also parasitize red squirrels, and all of these ectoparasites would have been removed from treated mothers through our anti-parasite treatment.

Our results are consistent with previous findings, which show that parents are able to produce more, higher-quality offspring when ectoparasites are removed (Brown & Brown, 1986;

Hillegass et al., 2010; Neuhaus, 2003). For instance, removal of ectoparasites from female

Columbian ground squirrels (Urocitellus columbianus) resulted in an increase in total litter mass at weaning and number of emergent juveniles (Neuhaus, 2003). Similarly, Cape ground squirrel

(Xerus inauris) mothers with experimentally reduced parasite intensities raised more offspring than controls (Hillegass et al., 2010). Comparable findings have also been reported for birds. In barn swallows (Hirundo rustica), cliff swallows (Hirundo pyrrhonota) and great tits (Parus major), experimental treatment of nests against ectoparasites resulted in improved survivorship of nestlings between hatching and fledging, as well as heavier chicks at fledging (Brown &

Brown, 1986; Møller, 1990; Richner et al., 1993). Juvenile barn swallows had longer nestling periods than did controls when ectoparasites were experimentally removed from the nest

(Møller, 1990). Parasitized nestlings may have fledged earlier as a form of parasite escape, despite facing reduced survival probabilities as a result (Møller, 1990). We did not find any

effect of parasite treatment on the number of days to emergence for red squirrel juveniles, contrary to our prediction that non-parasitized juveniles might emerge earlier if their growth- rates were accelerated. For red squirrels, early emergence conveys advantages such as greater access to available territories, a longer period of time in which to acquire and store food resources, and a higher likelihood of overwinter survival (Kerr et al., 2007). Despite the advantages of earlier emergence, the actual number of days between birth and emergence does not appear to be very flexible. Instead, mothers in good condition may mate earlier in the season, thereby ensuring their young benefit from early emergence (Kerr et al., 2007). Our experimental design did not manipulate the date of mating as all females were randomly entered into the experiment after mating had occurred.

In highly variable environments, it may be beneficial for females of short-lived iteroparous species, such as red squirrels (female red squirrels surviving to 1 year of age have an average lifespan of 3.5 years; McAdam et al., 2007), to invest heavily in current reproduction if reproductive conditions (i.e., food availability, population density, parasitism) are favourable, even if it does trade off with future reproduction or survival (Hirshfield & Tinkle, 1975).

However, we did not find any effect of parasitism on the overwinter survival of mothers, which is consistent with other red squirrel populations (Humphries & Boutin, 2000). In red squirrels, reproductive success appears to vary in response to prevailing environmental conditions, such as food availability (Descamps et al., 2007) and, as shown here, parasitism. Our findings suggest that female red squirrels maintain an optimal body mass regardless of their parasite infection or reproductive output, possibly to optimize their future survival and reproductive potential. If they lack the energy required for bodily maintenance and reproduction, females appear to invest less

into their current offspring. This would explain the impact of the experimental parasite removal on juvenile size and survival, and the lack of impact on mother’s mass and survival. Unlike other squirrel species, red squirrels store much of their energy in the form of conifer cones in a midden rather than as body fat. Thus, it is not surprising that we could not detect an effect of ectoparasites on female body mass, although the same trend was also observed in Columbian ground squirrels, who do store their energy as fat (Neuhaus, 2003). There may also have been effects on hoard depletion, which we did not measure. While our non-significant findings are biologically relevant and supported by findings in other red squirrel populations, readers should interpret these results with caution. Due to small sample sizes, our power to detect an effect where one may have been present was low, especially for mother’s mass differences, as shown by the effect size confidence intervals (Colegrave & Ruxton, 2003). The hypotheses tested here for which significant differences between treatment and control groups were not found may in fact be Type II error. However, the effect size and corresponding confidence interval for days to emergence are consistent with the null hypothesis of no effect of parasitism and, therefore, our conclusion is supported by our data. Future research on the effects of ectoparasitism on adult survival, midden depletion, and juvenile emergence dates, as well as on the effects of endoparasites on various life-history components of red squirrels, is warranted.

Many populations and species may be faced with increased parasite infection risk due to range expansions of pathogens and hosts, changes in parasite communities, and anthropogenic environmental change (Daszak et al., 2000; Kutz et al., 2005). Ecologists rarely consider the effects of parasites when predicting population demographics and informing management practices; however, parasites have important implications for individual host reproductive

success, host life-histories, and host population viability (Fitze et al., 2004b; Gooderham &

Schulte-Hostedde, 2011; Hillegass et al., 2010; Hudson et al., 1998; Neuhaus, 2003). Our study is a rare investigation of the consequences of parasitism on observed host life-history traits in a natural population of free-ranging mammals and is, to our knowledge, the first study to directly assess the effects of ectoparasites on the survival of mammalian offspring from birth through to emergence.

CHAPTER 5: EFFECTS OF ECTOPARASITISM ON THE ACTIVITY BUDGETS AND

HOME RANGES OF FEMALE NORTH AMERICAN RED SQUIRRELS

(TAMIASCIURUS HUDSONICUS)

Introduction

All animals must allocate their time and energy to the various activities required for maintenance, growth, survival and reproduction to improve their fitness. Through selective pressures on survival and reproductive success, the behaviour of individuals can drive the evolution and expression of various life-history traits (Dingemanse & Réale, 2005). Many behavioural traits, including maternal behaviours, are heritable and are expected to influence fitness, and are thereby a potential target of natural selection (van Oers et al., 2005). Mothers are forced to make trade-offs between various behaviours, such as time spent feeding their young, time spent foraging, time spent autogrooming and allogrooming, and time spent defending territories or forging social bonds, all of which can contribute to their lifetime reproductive success. Behaviours necessarily impose costs to the individual performing them in terms of time and energy lost, but any costs should be compensated for by the direct benefits of engaging in that behaviour (e.g., eating more food or removing more ectoparasites).

Parasites are a major cost to fitness in many species (e.g., Albon et al., 2002; Booth et al.,

1993; Hillegass et al., 2010; Møller, 1990; Møller, 1993; Neuhaus, 2003). It is therefore imperative for individuals to reduce these costs by preventing infections or by attempting to reduce infection intensities once infected. Animals engage in a wide variety of behaviours to

reduce infection intensities, such as self-medication (Clayton & Wolfe, 1993), nest switching

(Hausfater & Meade, 1982; Roper et al., 2002), selective foraging (Brambilla et al., 2013;

Ezenwa, 2004b), conspecific avoidance (Freeland, 1979), and grooming/preening. Grooming

(referred to as preening in birds) serves a variety of functions, such as removal of debris and bacteria from the body surface (Kohari et al., 2009; Simmons, 1964), social bonding (Arnold &

Whiten, 2003; Dunbar, 1991; Kutsukake & Clutton-Brock, 2010), and coping with stress

(Henson et al., 2012), but it also serves to control harmful ectoparasites (Clayton, 1991; Hart,

1992; Marshall, 1981; Tanaka & Takefushi, 1993). There is considerable evidence that grooming provides beneficial effects to host fitness via ectoparasite removal. For instance, when prevented from self-preening, rock doves (Columba livia) suffer from increased ectoparasite intensities

(Clayton, 1991), which reduce their fitness (Booth et al., 1993; Marshall, 1981). Similarly, debeaked domestic chickens (Gallus gallus) are unable to preen efficiently and their ectoparasitic lice (Menacanthus stramineus) intensities increased dramatically as a result of the experimental manipulation compared to control animals with intact beaks (Brown, 1972). In mammals, house mice (Mus musculus) suffer from increased lice infestation levels when they are housed singly and their grooming ability is impaired, but when impaired individuals are housed with unimpaired individuals, infection intensities are reduced through allogrooming (Bell & Clifford,

1964). The extent to which grooming behaviours are displayed appears to be related to the pressures exerted by ectoparasites. For instance, nestling barn swallows (Hirundo rustica) responded to experimentally elevated haematophagous mite (Ornithonyssus bursa) intensities by increasing the amount of time they spent preening themselves (Møller, 1991). Similarly, time spent preening/grooming is positively correlated with ectoparasite intensity in several species of

birds (e.g., Brown, 1974; Cotgreave & Clayton, 1994) and mammals (e.g., Hillegass et al., 2010;

Madden & Clutton-Brock, 2009; Mooring, 1995).

In addition to grooming, parasites also influence the amount of time spent by hosts engaging in other behaviours, such as foraging (Pelletier & Festa-Bianchet, 2004), vigilance

(Mooring & Hart, 1995), territoriality (Fox & Hudson, 2001), singing/calling (Madelaire et al.,

2013; Møller, 1991a), and nursing/food provisioning offspring (Festa-Bianchet, 1988; Tripet &

Richner, 1997), many of which trade-off directly with maternal care, reproductive success, and survival. For instance, impala (Aepyceros melampus) with higher ectoparasite intensities dedicate a greater proportion of their daily activity budget to grooming, which trades-off directly with the amount of time spent being vigilant for predators with potential implications for predation and survival (Mooring & Hart, 1995). These effects are not limited to ectoparasitism. For example, bighorn sheep (Ovis canadensis) mothers that are heavily parasitized by a lungworm

(Protostrongylus spp.) suckle their lambs for less time during mid- and late-lactation and engage in nuzzling less frequently than parasitized mothers (Festa-Bianchet, 1988). A reduction in nursing behaviour may be driven in part by reduction in milk yield and milk quality caused by parasitism (Hinde, 2007; Hoste & Chartier, 1993) and/or parasitised mothers spending more time engaged in other activities, such as resting. Undoubtedly, all characteristics and conditions that potentially affect the mother’s reproductive investment, including her activity budget, are important sources of variation in the offspring phenotype.

To date, few studies have examined the effects of parasites on maternal activity budgets during lactation in free-ranging mammals. One method by which to investigate the impact of

parasites on hosts in natural populations is by the experimental manipulation of parasite infection. A random sample with an experimentally reduced parasite load, that then shows improved fitness or altered behaviour relative to non-treated controls, provides evidence that parasites are directly influencing the host (sensu Lehmann, 1992). Using this experimental approach, I investigated the effects of ectoparasites on maternal behaviours in free-ranging female North American red squirrels (Tamiasciurus hudsonicus). I removed ectoparasites from half of the female red squirrels in the studied population and compared their activity budgets and home ranges to those of non-treated controls. Red squirrels are host to a variety of haematophagous ectoparasites (fleas, ticks, lice; Mahrt & Chai, 1972) that impose measureable fitness costs to females (Patterson et al., 2013, Chapter 4). Previous work on my study population found high natural infestation rates by fleas in the nests of lactating females (100% prevalence; Patterson et al., 2013; Chapters 3 and 4). Adult fleas bite and consume blood from red squirrel mothers and their offspring throughout the nesting period (Rothschild & Clay, 1952).

Red squirrel neonates rely exclusively on their mothers for grooming, feeding and defense, as well as to counteract the costs imposed by ectoparasites in terms of lost energy through food provisioning (lactation). The “parental food compensation hypothesis” predicts higher feeding rates in infested broods, and little to no detectable effect of parasites on offspring condition and survival (Tripet & Richner, 1997). However, effects on offspring mass and survival have been detected in this red squirrel-ectoparasite system (Patterson et al., 2013; Chapter 4); therefore, I did not predict that parasitized red squirrel mothers would spend significantly more time provisioning their offspring (nursing and/or foraging) than treated squirrels to compensate for the effects of parasites. Instead, red squirrel mothers appear to maintain their own condition, while putting additional resources towards reproduction when they are available to ensure a certain

level of offspring survival or condition (Fletcher et al., 2013; Humphries & Boutin, 1996;

Patterson et al., 2013). In this manner, red squirrel mothers may trade-off current reproduction for future reproduction and survival (McAdam & Boutin, 2003; McAdam et al., 2007).

Assuming that ectoparasites impose an energetic cost to red squirrels and that grooming removes ectoparasites, I predicted that control mothers would spend more time foraging and grooming compared to treated squirrels to maintain their body condition. Similarly, if parasite removal improves the energetic environment of the host and removes the consistent irritation of biting arthropods, particularly from the nest, I predicted that treated mothers would spend more time in the nest nursing their young to maximize their current reproductive effort (energetic resources are greater when not being lost to parasites, so the net energetic gain from parasite removal may be invested into offspring) and less time grooming themselves. I also predicted that control animals would spend more time resting outside of the nest (and away from the dual pressures of offspring and parasites). I also predicted that treated squirrels would invest more time (and energy) into defending their territory from conspecifics and intruders (higher rates of vocalization and vigilance), due to their apparently greater reproductive investment (Patterson et al., 2013; Chapter 4).

Red squirrels are highly territorial animals and defend food-based territories from conspecifics year round (Steele, 1998); however, red squirrels frequently forage off territory to pilfer an average of 25% of their daily food intake from neighbours’ food caches (Gerhardt,

2005). Parasitism alters territorial behaviours in some species, such as red grouse (Lagopus lagopus scoticus; Fox & Hudson, 2001; Mougeot et al., 2005), by reducing aggressiveness and increasing time spent resting. Home range sizes may also affect the risk of infection by parasites

in some systems (Bordes et al., 2009; Nunn & Dokey, 2006) as more widely ranging animals are perceived to have a greater risk of encountering novel parasites. I did not associate territorial behaviour (e.g., rattling, scent marking) to specific locations to define territorial boundaries, but rather I noted locations of squirrels throughout the study (i.e., ranging). Squirrels located away from the immediate vicinity of the nest and midden (food cache), were most often found to be foraging (J. Patterson, personal observation). As such, I hypothesized that if parasite removal reduced the proportion of time spent foraging by red squirrels then treated animals should have smaller home ranges than controls. Similarly, treated mothers should have smaller home ranges if they are more vigilant in defending their offspring and more territorial. Alternatively, if parasites reduce the amount of energy and time hosts have to devote to movement/traveling and increase the amount of time spent resting, then control squirrels may have smaller home ranges

(sensu Yeaton & Cody, 1974), as has been found in Australian sleepy lizards (Tiliqua rugosa;

Main & Bull, 2000). Tick intensities of Australian sleepy lizards were experimentally manipulated to produce a high-tick group and low-tick group: the high-tick group spent more time basking and less time walking than the low-tick group did, with the overall effect that high- tick individuals had smaller home ranges than low-tick individuals on average owing to reduced physical activity.

Methods

Study area

This study was conducted in southwestern Alberta, Canada along the eastern slope of the

Rocky Mountains in Sheep River Provincial Park (50o38’N, 114o39’W; elevation 1500 m). This region is composed primarily of mature coniferous-dominated mixed-wood forest interspersed with grassland meadows and wetlands. The dominant overstory trees are white (Picea glauca) and black spruce (P. mariana), lodgepole pine (Pinus contorta var. latifolia) and aspen (Populus tremuloides). Balsam fir (Abies balsamea) and balsam poplar (Populus balsamifera) also occur in lower proportions throughout the study area.

Animal capture

I trapped adult female red squirrels during gestation and before the onset of parturition during April and May 2011 and 2012 at three ~6.75 ha sites, using live-traps (H. B. Sherman

Traps, Inc., Tallahassee, Florida) baited with peanut butter. One grid (10 x 4 trap pattern) was established at each site with forty traps spaced 50 m apart. Upon initial capture, I weighed adult females to the nearest gram using a spring scale (Pesola AG, Baar, Switzerland), applied alphanumeric eartags with unique combinations of coloured washers (National Band and Tag

Co., Newport, Kentucky) and radio-collared them (SOM-2190, Wildlife Materials Inc.,

Murphysboro, Illinois). I captured females on a regular basis as part of various other ongoing studies, but these recaptures allowed me to check proper fit of the radiocollar and monitor reproductive status. Also, at each capture, I searched the entire body of each squirrel for ectoparasites for two-minutes: dorsally from the tip of the tail to the ears and ventrally from the tip of the tail to the forelimb in 1 cm swatches by combing and blowing on the fur to expose the

skin. I only radiocollared female squirrels for this study if fleas were present at the time of initial capture.

Parasite removal

For the experimental treatment, I randomly assigned mothers to either the treatment or control group, initially based on the flip of a coin and alternating thereafter. I treated all females in the treatment group once every thirty days with 0.15 ml/kg of K9 Advantix (8.8% imidacloprid, 44.0% permethrin; Bayer HealthCare LLC, Animal Care Division, Shawnee

Mission, Kansas) applied to the skin between the shoulder blades. As determined previously, this anti-parasitic drug is highly effective at killing ectoparasites at the topical doses used for small mammals (Larsen et al., 2005; Metzger & Rust, 2002) and, most notably, red squirrels (Patterson et al., 2013; Chapter 4). I started behavioural observations of female red squirrels a minimum of

3 weeks following the initial application of K9 Advantix to ensure that the medication had sufficient time to remove ectoparasites from the host and the nest. I handled and trapped the control animals in exactly the same manner and with equal effort as the treatment animals, but did not give them the medication.

Measuring behaviour in free-ranging red squirrels

I conducted 7-minute focal observations, during which instantaneous samples of the behaviour of breeding females were recorded at 30-second intervals (Altmann, 1974; Humphries

& Boutin, 2000). I conducted a total of 202 (n = 106 treated, n = 96 control) 7-minute

observation sessions on 26 female red squirrels (14 treated, 12 control) in 2011 and 2012 for a total of 1,414 minutes of observation time. This corresponded to an average of 7.8 observation sessions per female squirrel (range: 4-12 sessions). The interval between behavioural observation sessions on the same individual was at least 24 hours to minimize potential temporal autocorrelation. Behavioural observations on each female were conducted opportunistically during the reproductive period with the goal of maximizing the spread of observations for each female across different reproductive conditions. Behavioural focal sessions occurred from 07h00 to 20h00 and were evenly distributed throughout the day.

I categorized and analyzed female behaviour in a similar way as previous behavioural studies of red squirrels (Anderson & Boutin, 2002; Dantzer et al., 2011; Humphries & Boutin,

2000; Stuart-Smith & Boutin, 1995), including whether the squirrel was in or out of its nest, grooming, feeding, foraging, traveling, resting, vigilant, vocalizing (‘barking’ and ‘rattling’:

Smith 1968), or out of sight. For lactating squirrels, I interpreted time spent in the nest with offspring as an indirect measure of maternal behaviour as mothers are ensuring proper thermoregulation of offspring, grooming, nursing and interacting with their dependent offspring

(sensu Dantzer et al., 2011). Foraging and feeding observations were identified as separate behaviours in the field but were grouped together to calculate the proportion of time spent self- provisioning and I refer to the combination of these two behaviours simply as foraging. Rattling is the territorial vocalization of red squirrels, whereas barking is an alarm call (Smith 1968); however, for analytical purposes both calls were combined to the form the term ‘vocalizing’. Red squirrels become accustomed to human observers rapidly following a brief period of acclimation

(Smith 1968) and are therefore an appropriate species for the study of behaviour in free-ranging

populations. The approach used here does not quantify the full daily activity budget of each individual red squirrel but instead gives an adequate idea of how individuals in each group

(treatment vs. control) allocate their time to various activities.

Home range size

Radio-collared females were located for a variety of purposes (e.g., behavioural observations, nest searches). When females were located I recorded their spatial location on a

Garmin GPS 60 global positioning system (±5 m). Home ranges were determined using the adaptive local convex hull method (Getz et al., 2007) available in the “adehabitatHR” package

(Calenge, 2006) in R v3.0.2 (R Development Core Team, 2013). Home ranges represent the 95% isopleth and were computed using a minimum of ten locations per female (range: 10 – 22, mean

= 15.3).

Statistical analysis

To account for variation in the amount of time that each animal was observed, I summed the number of instantaneous behavioural observations for each individual over the course of the study year and divided that number by the total number of observations for that individual. I summarized behaviour during a focal session by calculating the proportion of each behaviour category recorded over the sampling points (7-min session, 30-sec intervals). I then averaged the proportions recorded during each focal session for each female to give the average proportion of time spent participating in each behaviour. This approach followed that of previous red squirrel

behavioural research (Anderson & Boutin, 2002; Humphries & Boutin, 2000; Stuart-Smith &

Boutin, 1995), thereby allowing for comparisons to be made between my findings and those of others.

Mann-Whitney U-tests were used to explore differences between the proportion of time spent by treated and control groups engaged in each of the reduced set of five explanatory behavioural variables. A Student’s t-test was used to assess if home range sizes were statistically different between treated and control squirrels.

All data were checked for normality prior to conducting statistical tests. All means are reported ± 1 standard deviation (SD) unless otherwise noted. All statistical tests were conducted in R version 3.0.2 (R Development Core Team, 2013).

Results

Maternal activity budget

Nesting was the most frequent behaviour reproductive female red squirrels engaged in overall; however, treated females spent significantly more time in the nest than did control females (Table 5.1). Control females also spent significantly more time autogrooming outside of the nest than their treated counterparts (Table 5.1). There were no statistical differences in the proportion of time spent foraging, resting, vocalizing, traveling, vigilant, out of sight, or chasing

Table 5.1: Comparison of proportions of time spent by North American red squirrel

(Tamiasciurus hudsonicus) mothers with experimentally reduced ectoparasite intensities and controls engaged in nine recorded behaviours in Sheep River Provincial Park, Alberta, Canada in

2011 and 2012. Means are reported ± standard deviation. U and P values were obtained from

Mann-Whitney U-tests.

Mean ± SD Mean ± SD Variable (Treated) (Control) U P (n = 14) (n = 12) Nesting 0.388 ± 0.27 0.156 ± 0.19 43 0.011

Grooming 0.016 ± 0.02 0.069 ± 0.08 141.5 0.036

Vigilant 0.163 ± 0.21 0.310 ± 0.28 63 0.117

Resting 0.109 ± 0.16 0.159 ± 0.17 115 0.422

Chasing 0.008 ± 0.03 0.001 ± 0.00 110 0.456

Vocalizing 0.023 ± 0.04 0.043 ± 0.07 112 0.489

Out of sight 0.018 ± 0.02 0.013 ± 0.01 110.5 0.553

Foraging 0.192 ± 0.19 0.170 ± 0.22 90 0.747

Traveling 0.082 ± 0.06 0.079 ± 0.06 92.5 0.831

between treated and control females (Table 5.1). Although, control squirrels spent 15% more of their time on average being vigilant than treated squirrels, the difference was not significant

(Table 5.1).

Maternal home range

Home range size was not affected by the parasite removal experiment (t18.3 = -0.190, p =

0.852). Mean home range size of treated animals was 0.64 ± 0.40 ha and that of controls was

0.67 ± 0.43 ha. Proportion of time spent foraging was not correlated with home range size (rs = -

0.43).

Discussion

Ectoparasites affected the maternal activity budget of free-ranging red squirrels. Control mothers spent significantly more time grooming and significantly less time in the nest when compared to mothers with experimentally reduced ectoparasite intensities. Grooming is an effective means by which animals can reduce their ectoparasite intensities (Akinyi et al., 2013;

Clayton, 1991; Tanaka & Takefushi, 1993). Assuming that grooming removes ectoparasites in red squirrels, I had predicted that control animals would groom more frequently than treated animals. I found that control red squirrel mothers spent approximately 7% of their activity budget engaged in grooming, compared to 1.5% for treated animals. The amount of time spent grooming by parasitized red squirrels in my study fell within the reported ranges for other species. Scantlebury et al. (2007) found that Cape ground squirrels (Xerus inauris) spent 7% of

their time grooming prior to ectoparasite removal and only 1% of their time grooming following the treatment. Some non-human primates have been found devoting upwards of 20% of their time towards grooming (Dunbar, 1991; Shutt et al., 2007); wild impala spend 5% of their time grooming, on average (but up to 40% for some individuals; Mooring, 1995); domestic cats (Felis domestica) spend an average of 8% of their non-sleeping/resting time in self-oral grooming

(Eckstein & Hart, 2000); parasitized bats spend between 10 and 15% of their time grooming, while non-parasitized bats spend as little as 5% of their time grooming (Giorgi et al., 2001); and, in a comprehensive review of grooming behaviour in 62 species of birds, Cotgreave and Clayton

(1994) found that those species spent an average of 9% of their daily activity budget engaged in grooming/maintenance behaviour (range: 0.3 – 25.4%). Grooming behaviour has not been well described in red squirrels previously, with only one study showing that juveniles spend approximately 1-2% of their time autogrooming (Anderson & Boutin, 2002). My findings support the hypothesis that grooming is governed in part by stimulation (e.g., irritation) from ectoparasites, while the retention of grooming behaviour by de-parasitized animals suggests that grooming also plays a role in maintaining dermal condition (e.g., removing dirt and debris). My findings agree with several previous studies showing that ectoparasites drive the rate of host grooming. For instance, impala in a tick-free region self-groomed significantly less than impala exposed to ticks (Hart et al., 1992). Furthermore, baboons (Papio cynocephalus) self-groomed for longer periods of time and more frequently as tick densities increased (Saunders, 1988). Also, moose (Alces alces), elk (Cervus elaphus), mule deer (Odocoileus hemionus), and white-tailed deer (O. virginianus) experimentally infested with ticks groomed more frequently than uninfected individuals (Samuel, 1991; Welch et al., 1991). Several experimental studies of rodents and viverrids have demonstrated that when ectoparasites are experimentally removed,

grooming rates decrease within social groups (Hawlena et al., 2008; Hillegass et al., 2010;

Madden & Clutton-Brock, 2009). Hillegass et al. (2010) observed a complete cessation of allogrooming behaviour between group members when ectoparasites were experimentally removed from Cape ground squirrels. These previous experiments provide several pieces of evidence supporting the idea that the presence of ectoparasites stimulates grooming behaviour and that grooming functions to reduce ectoparasite intensities.

Grooming activity is not without inherent costs, both direct and indirect. Grooming is a physical activity and as such requires energy. One of the only studies to date that has investigated the energetic requirement of grooming found that male king penguins (Aptenodytes patagonicus) devoted 9% of their daily energy budget (22% of their activity budget) to grooming

(Viblanc et al., 2011). Additionally, grooming may also lead to evaporative loss of water and electrolytes (Giorgi et al., 2001; Mooring, 1995). Grooming is also a time-consuming activity that fundamentally affects an individual’s activity budget and that trades-off with various other vital daily activities such as feeding, resting, and nursing. I found that parasitized female red squirrels compensated for the extra time invested in grooming by diminishing the time they spent in the nest but, surprisingly, not the amount of time they spent foraging. In mouse-eared bats

(Myotis myotis), time spent grooming, which was directly related to ectoparasite intensity, resulted in reducing the amount of time heavily parasitized individuals spent resting and sleeping

(Giorgi et al., 2001). Although I did not evaluate the effects of ectoparasites on sleeping behaviour, ectoparasites have been shown to reduce the quality and quantity of sleep in their hosts (Christe et al., 1996; Giorgi et al., 2001), which may impair host reproductive success, body mass, and survival (Elgar et al., 1988; Meddis, 1975). Furthermore, Mooring and Hart

(1995) found that ectoparasite-driven grooming behaviour reduced the amount of time impalas spent being vigilant. For grooming behaviour to be an adaptive trait, the benefits obtained by grooming (e.g., parasite removal, social bonding) must outweigh the costs. In particular, the energetic costs imposed by ectoparasites must be greater than the energetic costs of grooming, if grooming behaviour for the explicit purpose of ectoparasite removal is to remain an adaptive evolutionary trait, which appears to be the case in some species.

Time spent in the nest during the day was interpreted as an indirect measure of maternal care (sensu Dantzer et al., 2011). I found that ectoparasites significantly reduced the amount of time red squirrel mothers spent in the nest caring for their young during the day. There are several explanations for this finding. First, parasitized mothers may simply be avoiding the nest because this is where the highest intensities of ectoparasites are found during the lactation stage

(Chapter 3). If ectoparasites reduce the condition of and impose energetic costs on mothers, then mothers should actively engage in parasite avoidance behaviour. Interestingly, control mothers did not spend more time in the nest grooming their offspring. Furthermore, mothers with high parasite infection intensities may be unable to produce additional milk because their energy resources are depleted by the parasites (Hinde, 2007; Hoste & Chartier, 1993) and as a result spend more time out of the nest during the day avoiding the pressures imposed by their young. In lactating dairy sheep, endoparasites reduced the quantity of milk produced by mothers by up to

44% and the protein content of the milk by 12% (on average), which also corresponded with reduced offspring mass (Cringoli et al., 2008; Cruz-Rojo et al., 2012). Similar results have also been reported for dairy cattle with a 7-15% increase in milk production after parasites were removed via anthelmintic treatment (Gross et al., 1999; Perri et al., 2011; Sanchez et al., 2004).

There has been little research conducted on the effects of parasites on maternal care, especially the effects on nursing bouts, milk quality/quantity, and possible impacts on offspring development in wild animals. Additionally, all published work on these topics deals only with the effects of endoparasites. In one such study, bighorn sheep mothers heavily parasitized by a species of lungworm showed significant declines in nursing rate, which correlated with low juvenile survival (Festa-Bianchet, 1988). Host metabolic rates are directly affected by ectoparasite infection (Booth et al., 1993; Careau et al., 2010; Kam et al., 2010; Møller et al.,

1994), which could lead to a decrease in the amount of resources available to support lactation.

The interplay between ectoparasitism, host energetics, and lactation performance was not directly explored here and warrants future attention by researchers.

Although I could not directly measure rates of nursing, parasitized red squirrel mothers appeared to spend less time during the day engaged in direct maternal care (e.g., grooming/licking, nursing, thermoregulation), as evidenced by their absence from the nest.

Instead of engaging in direct maternal care, parasitized mothers spent more time outside of the nest engaged in other activities, most notably grooming (as discussed previously), vigilance and, to a lesser extent, foraging. Control mothers spent, on average, 2% more time foraging than treated animals; however, this difference was not significant and supports my prediction that red squirrel mothers do not engage in “parental food compensation” to dampen the effects of ectoparasites on their offspring. Red squirrels store much of their energy in the form of spruce cones in a central midden for later use rather than as body fat, and, as such, parasitized mothers may not increase foraging effort to reduce the risk of depleting their stored energy reserves, which could affect their survival and future reproductive output. Mothers of both groups spent

similar amounts of time foraging to maintain their body condition and support lactation. The increased survival and mass of juvenile red squirrels (Chapter 4, Patterson et al., 2013) does not appear to result from a greater investment by females via increased foraging, but instead appears to be directly related to the direct energetic costs of parasitism.

Ectoparasite removal did not affect the ranging behaviour of lactating female red squirrels in this study. I predicted that if ectoparasite removal affected the activity budget of host red squirrels by reducing foraging, reducing resting and/or increasing defensive behaviours (e.g., vocalizing, vigilance) of host animals, that ranging behaviour would in turn be reduced. Since foraging, resting, vigilance and vocalizing behaviours were not affected by the experimental removal of ectoparasites, a lack of effect on home range size is not surprising. An increase in time spent foraging by parasitized mothers may have been interpreted as a form of the parental food compensation hypothesis, which, as discussed previously, does not seem to apply to red squirrels. Alternatively, I also predicted that if ectoparasites reduced the time spent traveling, control squirrels would have smaller home ranges; however, there was no effect of parasite removal on time spent traveling. Since female red squirrels appear to maintain their own body condition and invest surplus energy into their offspring, and since higher rates of movement/traveling and territory defense would increase energy expenditure and increase the risk of predation (Stuart-Smith & Boutin, 1995), individuals, regardless of their parasite infection status, may simply stay put and rely on the acquired and available resources in their home range regardless of its size. Similarly, Scantlebury et al. (2007) did not find any difference in the amount of time Cape ground squirrels spent moving when comparing groups of individuals before and after the removal of both ectoparasites and endoparasites. Although not tested here,

ranging behaviour and territoriality may change over time, particularly as: red squirrel behaviours shift from feeding on cached cones in the spring and summer to caching cones in autumn; densities of conspecific competitors change; and, portions of territories are bequeathed to offspring (Boutin et al., 2000; Price & Boutin, 1993; Vlasman & Fryxell, 2002). In previous studies, endoparasite removal increased aggressiveness and competitiveness in male red grouse, which led to larger territory sizes and an increased likelihood of establishing a territory (Fox &

Hudson, 2001; Mougeot et al., 2005). It is possible then that juvenile red squirrels from the treatment group possessed a competitive advantage over their parasitized juvenile conspecifics with regards to territory establishment in the autumn, possibly owning to a larger body mass

(Chapter 3), although this remains to be tested. Galapagos hawk (Buteo galapagoensis) territory ownership, for example, was inversely correlated with ectoparasite infection intensity (Whiteman

& Parker, 2004). In sleepy lizards, experimental manipulation of ectoparasites affected home range sizes, such that individuals with higher ectoparasite intensities had significantly smaller home range areas than individuals with lower intensities, owing to reduced activity, reduced movement, and higher levels of basking/resting (Main & Bull, 2000). To the best of my knowledge, I have provided the first study to test the effects of ectoparasitism on home range size in a mammalian system.

I have shown that behaviours of female red squirrels are plastic and can change depending on the degree of ectoparasite infestation. Red squirrel mothers responded to parasite removal by reducing the amount of time spent grooming and spending more time in the nest.

Ectoparasites appear to stimulate grooming behaviour in their female red squirrel hosts, although it is unclear how effective grooming is at actually removing ectoparasites. Grooming would be

adaptive in this system if it provides an important means by which individuals can control infections and minimize the detrimental effects of ectoparasites. More research is needed on the effects of grooming on ectoparasite removal in red squirrels.

CHAPTER 6: PATTERNS AND CORRELATES OF ENDOPARASITISM IN NORTH

AMERICAN RED SQUIRRELS (TAMIASCIURUS HUDSONICUS)

Introduction

Identifying the determinants and patterns of species abundance, fitness, and community interactions are three central problems in ecology (Damuth, 1987; Ricklefs, 2008). Likewise, a key issue in disease ecology concerns the factors that influence patterns of abundance and prevalence of parasite species in host animals. Numerous empirical studies have shown that, almost without exception, parasite abundances are patterned across host populations, often with the majority of the parasite population concentrated into a minority of the host population (see

Shaw et al., 1998 for a review). Heterogeneous patterns such as these are driven by variation between individuals in their exposure risk to infective stages of parasites and by their physiological susceptibility to maintaining an infection once it has been acquired (Wilson et al.,

2001). The manner by which parasites are distributed amongst their hosts within populations can have important implications for the dynamics of host populations through negative effects on host survival and fecundity (Albon et al., 2002; Anderson & May, 1978; Hudson et al., 1998;

Neuhaus, 2003; Chapter 3), as well as host-parasite co-evolution and disease outbreaks (Hudson et al., 1998; Møller et al., 2005; Richner, 1998).

In host populations, biased parasite distributions may be governed by a variety of host features, such as age, sex, body size, social networks, co-infection status, immunocompetence, diet, and behaviour (Arneberg et al., 1998a; Arneberg et al., 1998b; De Nys et al., 2013; Fenton,

2013; Hawlena et al., 2007; Moore & Wilson, 2002; Nunn & Dokey, 2006; Pedersen &

Antonovics, 2013; Poulin, 1996b). Individuals with a greater predisposition to parasite infection may be responsible for maintaining and transmitting parasites in host populations (Perkins et al.,

2003). A number of comparative studies have investigated sex-biased parasitism and reached the conclusion that, within vertebrate hosts, males tend to have significantly higher parasite prevalence and intensity than females (e.g., Ferrari et al., 2004; Poulin, 1996b; Schalk & Forbes,

1997; Waterman et al., 2014; Wilson et al., 2001), although these patterns are not ubiquitous

(Kiffner et al., 2013; Wirsing et al., 2007). Furthermore, male hosts regulate parasite transmission dynamics (Ferrari et al., 2004; Grear et al., 2012; Luong et al., 2009; Perkins et al.,

2008; Skørping & Jensen, 2004) and parasite persistence (Hughes & Randolph, 2001) in some systems. Male-biased parasitism is typically attributed to the immunosuppressive effects of androgens and glucocorticoids, such as testosterone and cortisol (Evans et al., 2000; Folstad &

Karter, 1992; Hughes & Randolph, 2001; Mougeot et al., 2006; Sapolsky, 2005), which weaken the immune system (Duffy et al., 2000) and render hosts more susceptible to parasite infection

(Mougeot et al., 2004). In North American red squirrels (Tamiasciurus hudsonicus), males exhibit higher levels of ectoparasitism only during the reproductive period when they are scrotal and, presumably, when testosterone levels and rates of parasite transmission through host-host contact are highest (Gorrell & Schulte-Hostedde, 2008; Chapter 2). Alternatively, male-biased parasitism has also been attributed to sexual size dimorphisms, such as body size, which are prevalent within mammalian species (Moore & Wilson, 2002; Sheldon & Verhulst, 1996). Body size, often measured as body mass (Poulin & George-Nascimento, 2007), is one of the most commonly reported correlates of parasite infection across a broad range of host taxa (Ezenwa et al., 2006; Gregory et al., 1996; Kiffner et al., 2011; Nunn et al., 2003; Sasal & Morand, 1998;

Vitone et al., 2004). Positive correlations between parasitism and body mass typically derive from differences in host behaviour, physiology and/or surface area (e.g., Pelletier & Festa-

Bianchet, 2004); however, negative correlations between parasitism and host body mass may indicate a cost of parasitism (e.g., Lourenco & Palmeirim, 2007).

Endoparasite population dynamics are thought to be influenced by seasonal environmental fluctuations, such as temperature, rainfall and humidity, which can affect the survival of free-living parasite larvae and the desiccation of excreted eggs (Moss et al., 1993).

Numerous studies have found or suggested a link between the output of endoparasite eggs in host feces and either month, season (wet vs. dry), location (arid vs. humid) or rainfall amount

(Hausfater & Meade, 1982; McGrew et al., 1989; Parr et al., 2013; Stuart & Strier, 1995; Stuart et al., 1990; Turner et al., 2012). Seasonal differences in parasitism may also be driven by variation in host behaviour, diet and physiology over time. For instance, western gorillas (Gorilla gorilla) had higher endoparasite egg-shedding intensities in the dry season compared to the wet season, possibly due to greater stress-levels imposed by reduced food availability, impaired nutritional status, reduced immune function, and/or altered foraging behaviour leading to increased exposure to parasite infective stages (Masi et al., 2012). Furthermore, infection intensity of a pulmonary protozoan (Pneumocystis carinii) peaked in late autumn in host field voles (Microtus agrestis) possibly due to seasonal changes in host immunity and/or density

(Laakkonen et al., 1999). In domestic sheep, intestinal nematode (particularly Haemonchus contortus) egg-shedding intensities peaked during spring and summer when warm temperatures and high humidity allowed for parasite development to take place outside of the host following a periparturient drop in host immunity (Waller et al., 2004). Seasonality of parasite egg-shedding

may also be coincident with the availability of intermediate hosts (e.g., arthropods). While seasonality in parasite infections has been well documented, the environmental and biological drivers of these observed patterns still require examination (Altizer et al., 2006).

Other factors that may play critical roles in host susceptibility and exposure to parasites include home range size and co-infection status. The geographical range hypothesis (Lindenfors et al., 2007; Morand, 2000) has been extended to individual home ranges and predicts that animals with larger home ranges will increase their probability of exposure to novel parasites, while animals with more compact home ranges will be exposed to a smaller subset of those parasites (Bordes et al., 2009; Nunn & Dokey, 2006). Alternatively, individuals with smaller, more compact home ranges may be exposed to higher levels of infective parasite stages due to consistent foraging around potentially parasite-contaminated resources (e.g., food caches), while animals with larger home ranges may be exposed to a lower abundance of infective stages due to a sort of dilution effect (spatial dispersion; Bordes et al., 2009; Nunn & Dokey, 2006).

Furthermore, maintaining (and defending) a larger home range may require more energy, higher levels of aggression and testosterone (Cavigelli & Pereira, 2000; Seivwright et al., 2005), and may indicate that the individual’s home range is of poorer quality than those occupying smaller home ranges (e.g., Banci & Harestad, 1990; Hulbert et al., 1996), thereby suggesting that animals with larger home ranges may invest less energy into parasite defense and immune response. Bordes et al. (2009) found that an increase in home range size led to a corresponding decrease in parasite species richness in carnivores and did not affect parasite diversity in ungulates, lending support to the spatial dispersion hypothesis. Similarly, I found that ectoparasitism did not affect home range size in female red squirrel hosts (Chapter 3).

Most animals are infected with multiple parasite species (Petney & Andrews, 1998).

Within hosts, these parasites have the potential to interact via competition and mutualism, with interactive or cumulative effects on host responses. Interactive effects may be due to direct influences of one parasite on another through manipulation of gut physiology (e.g., pH change), direct competition for resources, physical crowding, and altered host immune responses (Lello et al., 2004). For instance, mice infected concurrently with Echinococcus multilocularis and

Mesocestoides corti developed fewer antibodies to E. multilocularis than in a single-species infection (Hinz et al., 1990). Host immune responses depend on the type of parasite infection, which can be complicated by co-infection. Intracellular parasites (e.g., protozoan parasites) stimulate the immune system to produce T helper 1 (Th1) cells, while T helper 2 (Th2) cells mediate defenses against extracellular (e.g., nematodes) parasites (Abbas et al., 1996;

Yazdanbakhsh et al., 2002). The two pathways are cross-regulated, meaning that cytokines produced by Th1 cells suppress Th2 immune function and vice versa (Morel & Oriss, 1998). For example, mice infected with a filarial nematode parasite (Litomosoides sigmodontis) experienced more severe malaria infections when co-infected compared to those without nematode infections

(Graham et al., 2005). Furthermore, disease severity is affected by the community of parasites infecting individuals, with many major parasitic diseases of wildlife, domestic animals, and humans (e.g., HIV, tuberculosis, ascariasis, coccsidiosis, and lymphatic filariasis) affected by the presence and infection intensity of other parasite species (Fenton et al., 2010 and references therein). To date, most studies of parasite-parasite interactions have been based on measures of parasite occurrence correlated to the occurrence or intensity of another parasite in the community with the direction and strength of the relationship taken to reflect the importance of the

underlying biological relationship (Behnke et al., 2005; Lello et al., 2004; Tello et al., 2008).

Few experimental approaches have been used to test potential interspecifc parasite interactions in free-ranging mammals (except see Pedersen & Antonovics, 2013). Ultimately, through competition and indirect effects on immune function, parasite co-infection and the host immune response may have important effects on the occurrence and control of disease.

The purpose of this study was to assess several potential correlates (month, sex, reproductive status, co-infection, mass, and home range size) of endoparasitism in a free-ranging population of North American red squirrels (Tamiasciurus hudsonicus). All of these factors have the potential to constrain or promote egg shedding by nematodes and protozoan oocyst formation, and have been identified as risk factors in other host species. Seasonal fluctuations in egg shedding are broadly reported in nematodes, and I hypothesized that if parasites time their reproductive output to coincide with biological or environmental events (rainfall, host physiology, hypobiosis) then egg/oocyst shedding would display varying degrees of intensity and prevalence in the host red squirrel population over time. Further, given that many endoparasites appear to favour male squirrels (e.g., Waterman et al., 2014; Scantlebury et al., 2010), whether it be due to physiological or behavioural mechanisms, I predicted that male red squirrels would have higher parasite infection intensities and prevalence than females. In Chapter 3 I found that flea parasitism varied by sex, but also by the reproductive status of the host individual. Factors such as periparturient declines in immune function in females and greater production of immunosuppressant testosterone in reproductive males may trigger endoparasites to increase their reproductive output, which I predicted would result in higher egg/oocyst output in reproductively active males and females. The effects of reproductive immune suppression may

be very different between the sexes, and the effect of reproductive status on endoparasitism may be sex-specific. Because ectoparasites and endoparasites may interact with and stimulate their host’s immune responses in diverse ways, infection with one parasite type may affect the host response to the other. As such, I predicted that if co-infection with an ectoparasite impaired or altered the ability of the host to fend off infection by a nematode, that nematode egg shedding would increase in hosts that were jointly infected with ectoparasites. Similarly, if parasitism by either a nematode or an ectoparasite affected the ability of the host to defend itself against protozoan parasites, then oocyst prevalence and intensity would be higher in individuals harbouring nematodes and/or ectoparasites.

The influence of body mass may operate in two mutually exclusive directions. First, I predicted that endoparasitism would, if harmful enough to the host, lead to reductions in host body mass, such that higher intensities and prevalence of endoparasites would scale negatively with the mass of their hosts. Additionally, hosts in better condition may have greater energetic or genetic means for coping with parasites. Second, parasites may prefer “well-fed” hosts to malnourished hosts since those hosts may provide more energetic resources for the parasite

(Hawlena et al., 2005) or because larger individuals may trade-off growth with immune function

(Rolff, 2002), which I predicted would result in a positive relationship between host mass and parasite infection. Finally, I predicted that home range size would affect the probability of red squirrels acquiring parasite infective stages, which would be reflected by a positive relationship between home range size and parasite prevalence (Bordes et al., 2009; Nunn & Dokey, 2006).

Additionally, parasites can reduce territorial behaviours and the amount of energy that hosts have to invest in movement and travel (Fox & Hudson, 2001; Main & Bull, 2000; Mougeot et al.,

2005). Subsequently, highly parasitized hosts may spend more time resting, in which case I predicted that more intensely parasitized red squirrels would have smaller home ranges.

Alternatively, high infection intensities may deplete hosts of their energy stores more rapidly than hosts with low-level infections, which in turn may drive hosts to forage more often and, perhaps, forage at greater distances from the core of their home range to acquire necessary dietary resources.

Methods

Study area

I conducted this study on a population of North American red squirrels (Tamiasciurus hudsonicus) residing in Sheep River Provincial Park (SRPP), Alberta, Canada (110° W, 50° N;

1500 m) between May and August 2010 and 2011. This region was characterized by subalpine mature second-growth conifer-dominated forest along the eastern slope of the Rocky Mountains.

Closed-canopy forest in the study area was dominated by spruce (Picea glauca, P. mariana) and trembling aspen (Populus tremuloides), with smaller proportions of lodgepole pine (Pinus contorta var. latifolia), balsam fir (Abies balsamea) and balsam poplar (Populus balsamifera).

Highest rainfalls in SRPP in 2010 and 2011 occurred in June (total = 239.2 mm combined years), with lower rainfalls (150.3 mm) but higher snowfalls in May, and July (116.8 mm) and August

(100.6 mm) were drier than both May and June (Environment Canada, 2013).

Study species

North American red squirrels are small (generally <250 g) rodents common in coniferous-dominated closed-canopy forests throughout North America. Red squirrels are conifer seed specialists throughout most of the year, particularly during the winter in which they rely on seeds stored in closed cones in a central midden for survival (Larsen & Boutin, 1994; Steele,

1998). During the summer both sexes rely on stored conifer seeds (if available), conifer buds, pollen cones, insects, berries and fungi (Steele, 1998). Females enter estrous for one day each year, followed by a ~33 day gestation period and a ~70 day lactation period (McAdam et al.,

2007; Patterson et al., 2013; Steele, 1998). This species does not display prominent sexual dimorphism, although males are on average 5-10% heavier than females (Boutin & Larsen,

1993; Chapter 2).

Red squirrels in SRPP are host to a variety of endoparasites: Mahrt and Chai (1972) documented six species of cestodes, including Paranoplocephala primordialis (syn. Andrya primordialis); four species of nematodes, including Citellinema bifurcatum, Physaloptera sp., and Ascaris sp.; and three species of protozoa, including Eimeria tamiasciuri and E. toddi.

The genus Paranoplocephala (Cestoda: Anoplocephalidae) presently comprises at least

35 species, most of which are parasites of rodents in the Holarctic region (Haukisalmi et al.,

2006). Paranoplocephala cestodes appear to be predictably rare in rodents (Haukisalmi &

Henttonen, 2007), including red squirrels in Minnesota (~5% prevalence; Douthitt, 1915) and

Saskatchewan (8% prevalence; McGee, 1980), but not SRPP (26% prevalence; Mahrt & Chai,

1972). Paranoplocephala primordialis appears to be host-specific to red squirrels, which is the only sciurid from which it has been previously reported (Mahrt & Chai, 1972; McGee, 1980;

Rausch & Schiller, 1949; Rausch & Tiner, 1948). I did not consider Paranoplocephala in the analyses due to the potential for their reproductive strategies to bias the results. As with all cestodes, Paranoplocephala store fertilized eggs in a proglottid (segment containing a complete sexually mature reproductive system with both male and female reproductive organs) until it is full, at which point the entire proglottid is shed in the feces. Therefore, measures of parasite egg- shedding intensity would be far less reliable for Paranoplocephala cestodes.

Citellinema bifurcatum (Nematoda: Trichostrongylidae) is one of seven described species of Citellinema and one of the most commonly detected nematodes in North American squirrels

(Lichtenfels, 1971; Mahrt & Chai, 1972; McGee, 1980; Rausch & Tiner, 1948; Waterman et al.,

2014). All five species of the genus Citellinema are parasitic in rodents of the family Sciuridae

(Lichtenfels, 1971). Mahrt and Chai (1972) detected adult C. bifurcatum in sampled red squirrels in SRPP. Citellinema is a directly transmitted parasite, shedding eggs singly in the feces of their hosts, with adults commonly found attached to the small intestine (Dikmans, 1938). There is very little information on the life cycle and epidemiological consequences of Citellinema infection in rodents; however, as with other Trichostrongylid nematodes (e.g., Trichostrongylus spp.), infections are likely asymptomatic except when present in large numbers, which may result in diarrhea, emaciation and anemia, particularly in stressed or malnourished animals

(Bowman, 2008).

Physaloptera (Nematoda: Physalopteridae) is a widespread genus of nematode, parasitizing a broad range of definitive host species worldwide, including primates, rodents, cats, dogs, birds, amphibians, and reptiles. Physaloptera require an invertebrate intermediate host

(Olsen, 1986). Mature Physaloptera attach to the gastric mucosa of the primary host where they feed on the host’s blood. Eggs are generally laid singly and are larvated. Mahrt and Chai (1972) detected adult Physaloptera sp. in sampled red squirrels in SRPP, but did not identify their specimens to the species level. The Physaloptera genus has not been well described in squirrels.

To the best of my knowledge, Physaloptera massino is the only described species of the genus

Physaloptera from North American sciurids (Coyner et al., 1996; Ubelaker et al., 2010). I did not describe the species of Physaloptera in SRPP red squirrels from collected egg specimens

(formalin preservation prevented genetic analysis) and, following Mahrt and Chai (1972), I categorized all detected Physaloptera eggs as Physaloptera sp. In rodent hosts, the feeding activities of Physaloptera sp. cause the formation of chronic ulcers, which exhibit fibrosis, extensive leucocyte infiltration, sclerosis, and hyperplasia (Schell, 1952).

I assessed two species of protozoan coccidia in red squirrels for this study: Eimeria tamiasciuri and E. toddi (Apicomplexa: Eimeriidae). Oocysts of these parasites are typically found throughout the digestive tract of red squirrels from the anterior duodenum through the rectum, with considerable numbers present in the cecum (Bullock, 1959). Eimeria tamiasciuri is one of the most prevalent parasites of red squirrels, often occurring in 91-99% of individuals within studied populations throughout North America (Dorney, 1966; Mahrt & Chai, 1972;

Seville et al., 2005; Soon & Dorney, 1969), although a lower prevalence was reported in New

Mexico (26.6%; Patrick & Wilson, 1995). Eimeria tamiasciuri has been reported in seven

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species of sciurid and one cricetid rodent in North America (Hill & Duszynski, 1986 and references therein) and E. toddi has only been reported from red squirrels (Dorney, 1962; Hill &

Duszynski, 1986; Mahrt & Chai, 1972). For the purpose of this study, I did not differentiate between E. tamiasciurus and E. toddi. Instead, all Eimeria were pooled as Eimeria spp. in the analyses. Eimeriosis, often designated as coccidiosis, is the disease caused by Eimeria parasites resulting in severe mucosal damage, weight loss and, occasionally, death. The disease is widespread and found in many species such as domestic livestock, birds, and mammals (Innes &

Vermeulen, 2006).

Although detected, I did not analyze Ascaris sp. because it is doubtful that ascarids are actually able to mature and reproduce within red squirrel hosts (Rausch & Tiner, 1948). Instead, the detected eggs were likely ingested by red squirrels in SRPP and passed through in the feces without becoming infective. There have only been a few cases in which immature ascarids have been found in red squirrels (e.g., Rausch & Tiner, 1948) and, to the best of my knowledge, mature, reproductively active adult ascarids have never been found in red squirrels or other species of tree squirrel.

Trapping and measuring red squirrels

I trapped red squirrels on three grids within the study area with Sherman traps (H. B.

Sherman Traps, Inc., Tallahassee, Florida) spaced 50 m apart in a 10 x 4 pattern and baited with peanut butter. Captured squirrels were given unique metal ear tags with combinations of coloured washers (National Band and Tag Co., Newport, Kentucky) to aid in the identification of

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individuals. Animals were trapped and observed throughout the summer months (May-August) in 2010 and 2011. I determined the reproductive status of males (abdominal vs. scrotal testes) and females (pregnant [via palpation], lactating, or neither) during live-trapping, and radio- collared pregnant females (7g; SOM-2190, Wildlife Materials, Inc., Murphysboro, Illinois). I weighed animals to the nearest gram using a spring scale (Pesola AG, Baar, Switzerland) at each capture. In cases where an individual was captured on consecutive days or multiple times in the same day, I took an average of the masses for use in analyses. To test for effects of co-infection with ectoparasites, I experimentally removed ectoparasites and collected fecal samples from fourteen radio-collared female adult red squirrels (15 radio-collared females were used as controls; N = 29 fecal samples from treated animals and N = 41 fecal samples from controls) in

2010 and 2011 following the methods described in Chapters 3, 4 and 5. Only fecal samples from females who had received the ectoparasite treatment at least 30 days prior to fecal collection were used in the analysis.

Parasite collection and recovery

I used fecal egg/oocyst counts as a non-lethal, non-invasive method to estimate gastrointestinal parasite prevalence and intensity (Coltman et al., 2001; Ezenwa, 2004a; Gillespie

& Chapman, 2006). Although the relationship between fecal egg counts and actual parasite prevalence and intensity can vary, fecal egg counts have been shown to be a reliable estimate of relative gastrointestinal parasite infection across hosts (Cabaret et al., 1998; Seivwright et al.,

2004; Stear et al., 1995). I attempted to collect at least two fecal samples from each individual in the focal population between May and August in each year, where possible. Deaths, movement,

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trapability of animals, and inadequate amounts of feces (required minimum of 0.5 g for analysis;

Gorrell & Schulte-Hostedde, 2008) occasionally impaired my ability to collect repeated samples and led to a reduced overall sample size. Small mammals, such as red squirrels, generally produce low quantities of feces at one time, which can complicate studies involving fecal collection (e.g., Dorney, 1964). I collected samples from trapped individuals immediately after defecation and stored them in 15-ml plastic centrifuge tubes with screw caps. Within 4 h, I weighed (± 0.01g) and homogenized the sample and filled the centrifuge tube with 10% neutral- buffered formalin in a 10:1 ratio of formalin to feces. I stored samples at room temperature in the dark until I could transport them back to the laboratory for analysis.

I analyzed each fecal sample (N = 191) to recover parasite eggs and oocysts via centrifugal flotation in Sheather’s solution (specific gravity = 1.27). Faecal samples were first mixed with tap water and centrifuged for five minutes to remove small debris. After the supernatant was poured off, the sample was mixed with Sheather’s solution, additional

Sheather’s solution was added to create a positive meniscus on the test tube, and a cover slide was placed on top of the tube which was then centrifuged again for five minutes. The cover slide was removed and analyzed using a Leica DM-E (Leica Microsystems, Wetzlar, Germany) compound microscope. All slides were scanned systematically (Smith et al., 2007), all parasites were counted, and, when needed, measured using an ocular micrometer. Eimeria spp. oocysts were scanned at x400 magnification, while all other parasite eggs were scanned at x100 magnification. Parasites were identified to the lowest possible taxonomic level based on morphological characteristics. Due to the extremely high infection intensities of Eimeria spp., I adopted a sub-sampling procedure to avoid the necessity of counting such large numbers, often

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numbering into the tens of thousands. Following a modified version of the methodology developed by Dorney (1964), ten non-overlapping “rows” (one row comprised one field of view scanned horizontally, left to right, over the entire slide at x400 magnification) in the centre of the slide were scanned for Eimeria spp. and the infection intensity of the entire sample was determined by extrapolation. In the single case where Eimeria spp. was not detected, the entire slide was scanned.

For each parasite, I report its prevalence (number of infected samples/number of samples) as a percentage, and intensity (number of parasites/infected individual) in either eggs/gram of feces or oocysts/gram of feces (Bush et al., 1997). As I was unable to obtain multiple samples for all individuals over consecutive days and months, I acknowledge that some infections may have been missed and my reported prevalence and intensity values should be considered conservative estimates.

Home range estimation

I collected home range data using a Garmin GPS 60 handheld GPS unit (accuracy ≤ ±5 m). Females were tracked to their locations opportunistically throughout the day (between 7h00 and 20h00) as part of a larger project on red squirrel behaviour (Chapter 4) between May and

August 2010 and 2011. I calculated home range sizes using the adaptive local convex hulls method (Getz et al., 2007) available in the “adehabitatHR” package (Calenge, 2006) in R 3.0.2

(R Development Core Team, 2013). Home ranges represent the 95% isopleth and were computed using a minimum of six locations per female (range: 6 – 22, mean = 13.5).

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Statistical analyses

To determine if host mass, sex, reproductive status, home range size, and month

(independent variables) were correlated with the probability of infection by a specific parasite

(prevalence), I used a generalized linear mixed model with a binomial distribution (logit-link function) and parasite presence (0: absence of the parasite, 1: presence of the parasite) as the dependent variable. I log10-transformed all measures of parasite intensity to achieve normality and reduce heteroscedasticity. To determine if host mass, sex, reproductive status, home range size, and month (independent variables) were correlated with the intensity of parasite egg- shedding (intensity), I used a linear mixed effect model (Gaussian distribution) fit by maximum likelihood with the log10-transformed number of eggs/oocysts per gram of feces as the dependent variable. For the Eimeria spp. intensity analysis, I also included co-infection with a nematode

(either Physaloptera sp. or C. bifurcatum or both) as a binary explanatory variable. Because of the high collinearity between home range and sex (home range data were only available for females), I analyzed the effects of home range using the same methods and independent variables (aside from sex) described above, but with only a subset of the data for which home range information was available (N = 78 samples, N = 29 squirrels). Squirrel ID was controlled for in all analyses as a random effect. The effects of year were assessed separately prior to developing the models by use of either Mood’s median test (intensity) or Fisher’s exact test

(prevalence), and where yearly differences in intensity and prevalence were found (p ≤ 0.05), I included year as a random effect in the analysis. I developed Spearman rank correlation matrices for the full set of independent variables to assess multi-collinearity prior to entering terms into the model; however, none of the dependent variables were highly correlated (i.e., rs ≥ 0.6). I

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compared (non-transformed) infection intensities of Eimeria spp., Physaloptera sp., and C. bifurcatum between adult females without ectoparasites (experimentally removed) and controls using Wilcoxon rank-sum tests since the intensity data were not normally distributed. To account for individual and temporal variation in egg/oocyst shedding intensities, I took an average of the intensities from each fecal sample for each individual across the entire year for use in the ectoparasite co-infection analysis.

For each dependent variable I constructed and contrasted models that included all possible combinations of the predictor variables. I used the information theoretic approach

(Burnham & Anderson, 2002) wherein all models were ranked based on their second-order

Akaike information criterion (AICc) values and Akaike weights (wi). Models with low AICc and high wi values are considered to be the models with the greatest explanatory power (Burnham &

Anderson, 2002). Individual variables were ranked based on their relative importance value

(RIV), which provides a measure to determine the individual strength of each variable in predicting the outcome of the dependent variable in question (Burnham & Anderson, 2002).

All medians are reported with 95% confidence intervals (CI). I used Quantitative

Parasitology version 3.0 (Rózsa et al., 2000) to compute median intensity and parasite prevalence for all samples as well as their associated 95% CIs using Sterne’s exact method

(Reiczigel, 2003). I also used Quantitative Parasitology to compare sample median intensities using Mood’s median test and prevalence’s using Fisher’s exact test. I conducted all other statistical analyses (i.e., models and correlations) using R version 3.0.2 (R Development Core

Team, 2013). All means are reported ± 1 standard deviation (SD).

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Results

Sampling and host-intrinsic factors

I collected 191 fecal samples from 93 individuals in 2010 and 2011 (mean: 2.06 ± 1.08 samples/individual, range: 1-5; Table 6.1). Of those individuals, 62 were females (N = 132 samples) and 31 were males (N = 59 samples; Table 6.1). The average mass of all individuals in this study was 227.8 ± 20.0 g, with females averaging 227.9 ± 20.3 g (range: 184 – 291 g) and males averaging 227.5 ± 19.5 g (range: 186 – 274 g). Host mass and fecal sample mass were not correlated (Pearson’s r = 0.34, N = 191). Home range sizes were estimated for 29 females for which fecal samples (N = 78 samples) had also been collected (Table 6.2). Mean home range size was 0.60 ± 0.36 ha (range: 0.08 – 1.35 ha).

Physaloptera sp.

One hundred and eight of the 191 samples (56.5%) tested positive for Physaloptera sp.

(Table 6.1). Prevalence did not differ between males and females (Fisher’s exact test p = 0.207), but did differ by reproductive status (p < 0.0001), year (p < 0.0001), and month (p < 0.0001) for the full dataset (Table 6.1). For the reduced dataset (Table 6.2), prevalence differed with reproductive status (Fisher’s exact test p = 0.0002), year (p = 0.04), and month (p < 0.0001).

Year was included as a random effect in the models.

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August August 2011. and 2010

Parasite prevalence (%; 95% confidence levels in brackets) in North American red squirrels from squirrels from North in in red (%; American brackets) levels confidence 95% prevalence Parasite

Table 6.1 Table the for Canada May full Alberta, dataset,

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August 2010 and 2011. 2010 and August

Parasite prevalence (%; 95% confidence levels in brackets) in female North American red squirrels from red American from North female in in squirrels (%; brackets) levels confidence 95% prevalence Parasite

able able

T the May dataset, for Canada reduced Alberta,

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Both the reduced and full dataset AICc-selected models for the prevalence of

Physaloptera sp. included month as the best predictor (Table 6.3). For the reduced data set, month had a RIV of 0.83 and, for the full dataset month had a RIV of 1.0. None of the other variables had a RIV > 0.5 for the full dataset, while reproductive status had a RIV of 0.65 for the reduced dataset. The top model for the reduced dataset included month and reproductive status as its only two terms and the next nearest model was > 1 AICc value higher. For the full dataset, the top three models were all within 0.5 AICc values of each other; however, the variables of sex and reproductive status were not considered important explanatory variables (RIVs of 0.44 and

0.38, respectively). For the full dataset, month was present in all of the top eight models.

Prevalence was highest in June and lowest in August for both datasets (Tables 6.1 and 6.2).

Median intensity of Physaloptera sp. for the full dataset was significantly different between males and females (Mood’s median test p = 0.009), reproductive status (p < 0.0001) and year (p = 0.003), but not month (p = 0.377; Tables 6.4a and 6.4b). For the reduced dataset,

Physloptera sp. median intensity did not differ by reproductive status (Mood’s median test p =

0.101) and month (p = 0.923), but did differ by year (p = 0.049). Year was included as a random effect in all of the models.

Physaloptera sp. egg-shedding intensity was best explained by the model containing month and reproductive status as independent variables for both the full and reduced datasets

(Table 6.5). Reproductive status had RIVs of 0.998 and 0.845 for the full and reduced datasets, respectively. Month had RIVs of 0.981 and 0.899 for the full and reduced datasets, respectively.

Host mass, sex, and home range all had RIVs < 0.6 for both datasets and were generally only

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Table 6.3: Competitive generalized linear mixed models comparing parameters that affect

Physaloptera sp. prevelance in North American red squirrels from Alberta, Canada, May-August

2010 and 2011 for the reduced and full datasets. Full dataset models controlled for random effects of ID and Year. Reduced dataset models controlled for random effects of ID.

Model K AICc ΔAICc Wi Reduced Dataset (N=78) Month + Reproductive 2 81.08 0.00 0.244 Mass + Month 2 82.24 1.16 0.137 Month 1 82.55 1.47 0.117 Mass + Month + Reproductive 3 82.62 1.54 0.113 Home Range + Month + Reproductive 3 83.18 2.10 0.085 Reproductive 1 83.38 2.30 0.077 Home Range + Mass + Month 3 84.33 3.25 0.048 Mass + Reproductive 2 84.46 3.38 0.045 Home Range + Month 2 84.61 3.53 0.042 Home Range + Mass + Reproductive + Month (Global model) 4 84.63 3.55 0.041

Home Range + Reproductive 2 85.44 4.36 0.028 Home Range + Mass + Reproductive 3 86.29 5.21 0.018 Mass 1 89.50 8.42 0.004 Home Range + Mass 2 91.37 10.29 0.001 Home Range 1 98.24 17.16 <0.001

Model K AICc ΔAICc Wi Full Dataset (N=191) Month 1 223.42 0.00 0.222 Sex + Month 2 223.56 0.14 0.207 Month + Reproductive 2 223.86 0.44 0.178 Sex + Month + Reproductive 3 225.03 1.61 0.100 Mass + Month 2 225.06 1.64 0.098 Sex + Mass + Month 3 225.23 1.81 0.090 Mass + Month + Reproductive 3 225.83 2.41 0.067 Sex + Mass + Reproductive + Month (Global model) 4 226.92 3.50 0.039 Reproductive 1 247.72 24.30 <0.001 Mass + Reproductive 2 248.76 25.34 <0.001 Sex + Reproductive 2 249.76 26.34 <0.001 Sex + Mass + Reproductive 3 250.83 27.41 <0.001 Mass 1 260.02 36.60 <0.001 Sex + Mass 2 260.26 36.84 <0.001 Sex 1 265.52 42.10 <0.001

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transformed values ± 1 1 ± values transformed

ces for North American American in North red squirrels cesfor

sp. eggs shed per shed fe of eggs sp. gram

Physaloptera

August 2010 and 2011 for the full dataset. Means are reported for log10 for reported are Means dataset. for full 2011 the and 2010 August

ntensity of ntensity

Meani

able 6.4a: able

Alberta, Canada, May Canada, Alberta, T samples. of number (SD). = N deviation standard

112

ntervals. ntervals.

ggs shed per gram of feces for North American in North red per shed feces of squirrels for gram ggs

sp. e sp.

Physaloptera

August 2010 and 2011 for the full dataset. Medians are reported with 95% confidence i 95% with reported for 2011 Medians confidence 2010 and August are dataset. full the

Median intensity of intensity Median

6.4b:

Table Table May Canada, Alberta, samples. of number = N

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Table 6.5: Competitive linear mixed effect models comparing parameters that affect

Physaloptera sp. intensity in North American red squirrels from Alberta, Canada, May-August

2010 and 2011 for the reduced and full datasets. All models controlled for random effects of ID and Year.

Model K AICc ΔAICc Wi Reduced Dataset (N=56) Mass + Month + Reproductive 3 131.80 0.00 0.335 Month + Reproductive 2 132.39 0.59 0.249 Home Range + Mass + Reproductive + Month (Global model) 4 134.03 2.23 0.110

Home Range + Month + Reproductive 3 134.60 2.80 0.083 Month 1 135.01 3.21 0.067 Mass + Reproductive 2 136.21 4.41 0.037 Mass + Month 2 136.98 5.18 0.025 Home Range + Month 2 137.15 5.35 0.023 Home Range + Mass + Reproductive 3 137.75 5.95 0.017 Mass 1 137.85 6.05 0.016 Reproductive 1 138.55 6.75 0.011 Home Range + Mass + Month 3 139.15 7.35 0.008 Home Range 1 139.22 7.42 0.008 Home Range + Mass 2 139.63 7.83 0.007 Home Range + Reproductive 2 140.53 8.73 0.004

Model K AICc ΔAICc Wi Full Dataset (N=108) Month + Reproductive 2 237.20 0.00 0.517 Mass + Month + Reproductive 3 239.14 1.94 0.196 Sex + Month + Reproductive 3 239.15 1.95 0.195 Sex + Mass + Reproductive + Month (Global model) 4 241.14 3.94 0.072 Reproductive 1 245.56 8.36 0.008 Sex * Reproductive 2 245.61 8.41 0.008 Mass + Reproductive 2 246.25 9.05 0.006 Sex + Reproductive 2 247.43 10.23 0.003 Sex + Mass + Reproductive 3 248.22 11.02 0.002 Month 1 251.16 13.96 <0.001 Mass + Month 2 251.40 14.20 <0.001 Sex + Month 2 252.28 15.08 <0.001 Sex + Mass + Month 3 252.92 15.72 <0.001 Sex 1 254.73 17.53 <0.001 Mass 1 256.22 19.02 <0.001

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found in the lowest AIC ranked models. The model containing month and reproductive status was the best model for the full dataset and the next best model was almost 2 AICc values higher.

For the reduced dataset, the model containing mass, month and reproductive status was the best model; however the next nearest model (containing month and reproductive status) was only

0.59 AICc values higher, was more parsimonious (contained only 2 versus 3 variables), and mass had an RIV of 0.554 (Table 6.5). Therefore, the second model (month and reproductive status) was taken to represent the best model for the reduced dataset. Reproductive status was positively related to Physaloptera sp. intensity, such that egg-shedding intensity was higher in reproductive individuals (Tables 6.4a and b). Physaloptera sp. intensity was also highest in August and June

(Tables 6.4a and 6.4b).

Citellinema bifurcatum

Citellinema bifurcatum was recovered from 142 of 191 samples (74.3%), making it the most common non-protozoan parasite in the studied population of red squirrels (Table 6.1).

Prevalence of C. bifurcatum in the full dataset did not differ between males and females (Fisher’s exact test p = 1.0), reproductive condition (p = 0.868), or year (p = 0.679), but did differ between months (p = 0.003; Table 6.1). For the reduced dataset, prevalence did not differ by reproductive condition (Fisher’s exact test p = 0.541) and year (p = 0.747), but did differ by month (p =

0.0073). Year was not controlled for in any of the models.

For the full dataset, the most parsimonious model included only month (Table 6.6), while none of the measured variables sufficiently explained C. bifurcatum prevalence in the reduced

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Table 6.6: Competitive generalized linear mixed models comparing parameters that affect

Citellinema bifurcatum prevelance in North American red squirrels from Alberta, Canada, May-

August 2010 and 2011 for the reduced and full datasets. All models controlled for random effects of ID.

Model K AICc ΔAICc Wi Reduced Dataset (N=78) Home Range 1 78.40 0.00 0.124 Reproductive 1 78.50 0.10 0.118 Month 1 78.61 0.21 0.111 Mass 1 78.68 0.28 0.108 Month + Reproductive 2 78.98 0.58 0.093 Mass + Reproductive 2 79.41 1.01 0.075 Home Range + Reproductive 2 79.75 1.35 0.063 Home Range + Month + Reproductive 3 80.15 1.75 0.052 Home Range + Month 2 80.23 1.83 0.050 Mass + Month + Reproductive 3 80.25 1.85 0.049 Home Range + Mass 2 80.38 1.98 0.046 Mass + Month 2 80.63 2.23 0.041 Home Range + Mass + Reproductive 3 81.03 2.63 0.033 Home Range + Mass + Reproductive + Month (Global model) 4 81.86 3.46 0.022

Home Range + Mass + Month 3 82.37 3.97 0.017

Model K AICc ΔAICc Wi Full Dataset (N=191) Month 1 212.42 0.00 0.284 Month + Reproductive 2 213.46 1.04 0.169 Mass + Month 2 214.36 1.94 0.108 Sex + Month 2 214.46 2.04 0.102 Mass + Month + Reproductive 3 215.23 2.81 0.070 Sex + Month + Reproductive 3 215.33 2.91 0.066 Mass 1 216.22 3.80 0.043 Sex + Mass + Month 3 216.43 4.01 0.038 Reproductive 1 217.32 4.90 0.025 Sex + Mass + Reproductive + Month (Global model) 4 217.12 4.70 0.027 Sex 1 217.42 5.00 0.023 Sex + Mass 2 218.26 5.84 0.015 Mass + Reproductive 2 218.26 5.84 0.015 Sex + Reproductive 2 219.36 6.94 0.009 Sex + Mass + Reproductive 3 220.33 7.91 0.005

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dataset. For the reduced dataset, all four explanatory variables had RIVs < 0.5 and all of the single term models were within 0.3 AICc values of each other (Table 6.6). The RIV of month for the full dataset was 0.865 and all other models were > 1 AICc score higher than the model containing month as its only explanatory variable. In the full dataset, prevalence of C. bifurcatum was highest in July and lowest in May (Table 6.1). Sex, mass, home range and reproductive status had relatively little importance for either data set (Table 6.6) with each variable having a

RIV < 0.5.

Citellinema bifurcatum median intensity for the full dataset did not differ between males and females (Mood’s median test p = 0.856), reproductive status (p = 1.0), or year (p = 0.393); however, median intensity did differ by month (p = 0.029; Tables 6.7a and 6.7b). For the reduced data set, C. bifurcatum median intensity did not differ by reproductive status (Mood’s median test p = 0.287) or year (p = 0.07), but did differ by month (p = 0.001). Year was not controlled for in any of the models.

Citellinema bifurcatum intensity was best explained by the model containing month (RIV

= 0.986), reproductive status (RIV = 0.837), and host mass (RIV = 0.816) based on the full dataset (Table 6.8). This was also the top ranked model for the full dataset and the next best model was both less parsimonious and 1.87 AICc values higher. Month (RIV = 0.829) best explained C. bifurcatum intensity for the reduced dataset and was present in 8 of the top 9 models (Table 6.8).

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Table 6.7a: Mean intensity of Citellinema bifurcatum eggs shed per gram of feces for North

American red squirrels in

Alberta, Canada, May-August

2010 and 2011. Means are reported for log10- transformed values ± 1 standard deviation (SD). N =

number of samples.

transformed values ± 1 standard deviation 1 ± deviation values transformed standard

eggs shed per gram of feces for of squirrels American in for North red per shed feces eggs gram

Citellinema bifurcatu Citellinema

August 2010 and 2011. Means are reported for log10 for Means 2011. reported are 2010 and August

ntensity of ntensity

Meani

6.7a:

Table Table May Canada, Alberta, number samples. = N of (SD).

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Table 6.7b: Median intensity of

Citellinema bifurcatum eggs shed per gram of feces for

North American red squirrels in

Alberta, Canada, May-August

2010 and 2011. Medians are reported with 95% confidence intervals. N = number of

samples.

eggs shed per gram of feces for North American red squirrels in Alberta, shed red American for per North gramfeces of eggs

furcatum

of Citellinema of bi

August 2010 and 2011. Medians are reported with 95% confidence intervals. N = number intervals. = N samples. of are confidence 95% with Medians 2011. 2010 reported and August

Table 6.7b: Median intensity intensity Median 6.7b: Table May Canada,

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Model K AICc ΔAICc Wi Reduced Dataset (N=64) Month 1 156.87 0.00 0.255 Mass + Month 2 157.56 0.69 0.181 Home Range + Month 2 158.83 1.96 0.096 Month + Reproductive 2 158.95 2.08 0.090 Mass + Month + Reproductive 3 159.06 2.19 0.085 Mass 1 159.30 2.43 0.076 Home Range + Mass + Month 3 159.75 2.88 0.061 Home Range + Month + Reproductive 3 160.95 4.08 0.033 Home Range + Mass + Reproductive + Month (Global model) 4 161.32 4.45 0.028

Mass + Reproductive 2 161.37 4.50 0.027 Home Range + Mass 2 161.39 4.52 0.027 Reproductive 1 162.22 5.35 0.018 Home Range + Mass + Reproductive 3 163.54 6.67 0.009 Home Range 1 163.65 6.78 0.009 Home Range + Reproductive 2 164.11 7.24 0.007

Model K AICc ΔAICc Wi Full Dataset (N=142) Mass + Month + Reproductive 3 332.10 0.00 0.422 Sex + Mass + Reproductive + Month (Global model) 4 333.97 1.87 0.201 Month + Reproductive 2 335.87 3.77 0.078 Sex + Mass + Month 3 336.83 4.73 0.048 Mass + Month 2 337.16 5.06 0.041 Sex + Month + Reproductive 3 337.19 5.09 0.040 Sex + Month 2 337.40 5.30 0.036 Month 1 337.77 5.67 0.030 Mass 1 341.84 9.74 0.004 Sex + Mass 2 341.88 9.78 0.004 Mass + Reproductive 2 342.06 9.96 0.004 Sex + Mass + Reproductive 3 343.24 11.14 0.002 Sex 1 348.34 16.24 <0.001 Sex + Reproductive 2 349.98 17.88 <0.001 Reproductive 1 350.23 18.13 <0.001 Reproductive * Sex 2 350.44 18.34 <0.001

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For the reduced dataset, the model containing mass and month was 0.69 AICc values higher than the model containing only month, but the RIV of mass was < 0.5 (Table 6.8). Host mass was negatively related to infection intensity in the full dataset (t = -2.97). Egg-shedding intensity was highest in July and August (Tables 6.7a and 6.7b).

Eimeria spp.

Eimeria spp. ooscsyts were recovered from 99.5% of the samples. The ubiquity of

Eimeria spp. in the population did not permit for comparisons and tests on its prevalence.

Median intensity of Eimeria spp. did not differ between males and females (Mood’s median test p = 1.0), reproductive status (p = 1.0), year (p = 0.464), nematode co-infection (p = 0.551), or month (p = 0.773) for the full dataset (Tables 6.9a and 6.9b). Similarly, for the reduced dataset median intensity of Eimeria spp. did not differ between reproductive status (Mood’s median test p = 0.809), nematode co-infection (p = 1.0) or year (p = 0.801), but did differ significantly by month (p = 0.003). Year was not controlled for in any of the analyses.

The intensity of Eimeria oocysts for the full dataset was best predicted by co-infection with nematode parasites (Physaloptera sp. and C. bifurcatum; Table 6.10); however, the RIV of nematode co-infection was low (RIV = 0.546). Eimeria spp. intensity was not well predicted by any of the measured explanatory variables for the reduced dataset. All single term models were ranked highest, but all were within 0.15 AICc values of each other and RIVs for all five variables were < 0.39. Oocyst output was consistent across months as well as years, and didn’t vary with respect to sex or reproductive status (Tables 6.9a and 6.9b).

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transformedstandard 1 ± (SD). values deviation

of feces red feces squirrels American of in forNorth Alberta,

spp. oocysts shed per per gram shed oocysts spp.

Eimeria

ntensity of ntensity

August, 2010 and 2011. Means are reported for are log10 Means 2011. reported 2010 and August,

Table 6.9a: Mean i 6.9a: Table May Canada,

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gram of feces for North American red squirrels in Alberta, red of American in Alberta, North fecessquirrels for gram

spp. oocysts spp. per shed

Eimeria

August, 2010 and 2011. Medians are reported with 95% confidence intervals. N = number intervals. = N samples. of confidence 95% with reported are Medians 2011. 2010 and August,

Median intensity of intensity Median

9b:

able 6. able

T May Canada,

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Table 6.10: Competitive linear mixed effect models comparing parameters that affect Eimeria spp. intensity in North American red squirrels from Alberta, Canada, May-August 2010 and

2011 for the reduced and full datasets. Np = nematode presence/absence. All models controlled for random effects of ID.

Model K AICc ΔAICc Wi Reduced Dataset (N=78) Home Range 1 175.16 0.00 0.095 Month 1 175.22 0.06 0.092 Reproductive 1 175.23 0.07 0.092 Np 1 175.26 0.10 0.091 Mass 1 175.31 0.15 0.088 Mass + Reproductive 2 176.79 1.63 0.042 Mass + Month 2 176.79 1.63 0.042 Home Range + Mass 2 176.93 1.77 0.039 Home Range + Np 2 177.07 1.91 0.037 Home Range + Reproductive 2 177.12 1.96 0.036 Home Range + Month 2 177.13 1.97 0.036 Mass + Np 2 177.20 2.04 0.034 Month + Np 2 177.22 2.06 0.034 Np + Reproductive 2 177.24 2.08 0.034 Month + Reproductive 2 177.28 2.12 0.033 Home Range + Mass + Reproductive 3 178.51 3.35 0.018 Home Range + Mass + Month 3 178.54 3.38 0.018 Mass + Month + Reproductive 3 178.73 3.57 0.016 Mass + Month + Np 3 178.88 3.72 0.015 Home Range + Mass + Np 3 178.88 3.72 0.015 Mass + Reproductive + Np 3 178.90 3.74 0.015 Home Range + Month + Np 3 179.16 4.00 0.013 Home Range + Reproductive + Np 3 179.17 4.01 0.013 Home Range + Month + Reproductive 3 179.25 4.09 0.012 Month + Reproductive + Np 3 179.35 4.19 0.012 Home Range + Mass + Reproductive + Month 4 180.55 5.39 0.006 Home Range + Mass + Reproductive + Np 4 180.68 5.52 0.006 Home Range + Mass + Month + Np 4 180.68 5.52 0.006 Mass + Reproductive + Month + Np 4 180.92 5.76 0.005 Home Range + Reproductive + Month + Np 4 181.37 6.21 0.004 Home Range + Mass + Reproductive + Month + Np (Global model) 5 182.80 7.64 0.002 Home Range 1 175.16 0.00 0.095 Month 1 175.22 0.06 0.092

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Model K AICc ΔAICc Wi Full Dataset (N=191) Np 1 419.68 0.00 0.125 Sex 1 420.82 1.14 0.071 Month 1 420.98 1.30 0.065 Sex + Np 2 421.01 1.33 0.064 Month + Np 2 421.16 1.48 0.060 Reproductive 1 421.24 1.56 0.057 Np + Reproductive 2 421.33 1.65 0.055 Mass 1 421.44 1.76 0.052 Mass + Np 2 421.65 1.97 0.047 Sex + Month 2 422.36 2.68 0.033 Sex + Month + Np 3 422.52 2.84 0.030 Mass + Month 2 422.78 3.10 0.027 Sex + Reproductive 2 422.79 3.11 0.026 Sex + Mass 2 422.80 3.12 0.026 Mass + Month + Np 3 422.92 3.24 0.025 Sex + Reproductive + Np 3 422.93 3.25 0.025 Sex + Mass + Np 3 423.00 3.32 0.024 Month + Reproductive 2 423.00 3.32 0.024 Mass + Reproductive 2 423.05 3.37 0.023 Mass + Reproductive + Np 3 423.05 3.37 0.023 Month + Reproductive + Np 3 423.15 3.47 0.022 Sex + Mass + Month 3 424.18 4.50 0.013 Sex + Mass + Month + Np 4 424.30 4.62 0.012 Sex + Month + Reproductive 3 424.41 4.73 0.012 Sex + Reproductive + Month + Np 4 424.61 4.93 0.011 Sex + Mass + Reproductive 3 424.70 5.02 0.010 Mass + Month + Reproductive 3 424.75 5.07 0.010 Sex + Mass + Reproductive + Np 4 424.77 5.09 0.010 Mass + Reproductive + Month + Np 4 424.79 5.11 0.010 Sex + Mass + Reproductive + Month 4 426.27 6.59 0.005 Sex + Mass + Reproductive + Month + Np (Global model) 5 426.38 6.70 0.004

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Ectoparasite co-infection

There were no statistically detectable differences in mean egg/oocyst shedding intensities for Physaloptera sp. (W = 73.5, p = 0.409), C. birfurcatum (W = 119, p = 0.167), or Eimeria spp.

(W = 96.0, p = 0.699) between treated (N = 14 squirrels) and control (N = 15 squirrels).

Discussion

The nematode parasites of red squirrels in SRPP displayed strong seasonality in both their prevalence and egg-shedding intensity. Physaloptera sp. eggs were more likely to be detected (higher prevalence) in June and July, while intensities were highest in both June and

August. The second intensity peak in August may have been driven by small sample size, which can be problematic in parasitic studies because the wide variance in parasite infection intensities may not be adequately captured (Wilson et al., 2001). However, multiple annual peaks in nematode egg-shedding intensity are common (e.g., Moss et al., 1993). Citellinema bifurcatum was more likely to be present in June, July and August (median prevalence > 70% in all three months) than in May, with intensities peaking in July. Similar findings were reported by Gorrell and Schulte-Hostedde (2008), who found that egg-shedding intensity of a strongylid nematode parasite (Strongyloides robustus) in a geographically distinct population of North American red squirrels varied significantly by month, with intensities peaking in June and July. The observed seasonal differences may be the result of innate differences in the predisposition of individuals to infection, consistent differences in exposure to infective larvae, the reproductive biology of nematodes, or other differences in the environment and/or host behaviour/physiology.

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Seasonal variation in nematode egg-shedding is quite common, especially in temperate climates where the external environmental conditions are only conducive to larval development outside the host for a brief period of time in the summer. In response to seasonally unfavourable conditions (e.g., cold winters, arid seasons), many parasites have evolved adaptations such as seasonal reproduction and hypobiosis (an arrested stage of larval development; Gibbs, 1982).

While the red squirrel-nematode system requires more research to tease apart the drivers of seasonal variation in nematode egg-shedding, these topics have been explored in other species.

In red grouse, for instance, Trichostrongylus tenuis egg-shedding varies by month with frequent annual increases in egg-shedding by individual birds between March and April, a less frequent tendency to increase between April and July, a tendency to decline during October and no change between November and April (Moss et al., 1993). These seasonal increases in egg- shedding appear to be driven by the simultaneous maturation of both overwintering hypobiotic larvae and young worms in the host that begin producing eggs in early spring and summer (Moss et al., 1990; Moss et al., 1993; Shaw & Moss, 1989), and may not be directly related to environmental conditions. Decreases in egg-shedding in October are likely due to reproductive senescence imposed by the aging and death of mature worms (Moss et al., 1990; Shaw & Moss,

1989), although the potential for density-dependent regulation of nematode fecundity and effects of host immunity should not be ruled out (Luong et al., 2011). Year-to-year variability in nematode egg shedding by red grouse appears to be driven by rainfall the previous summer, probably because parasite recruitment was greatest during wet summers. In rodent definitive hosts, Physaloptera spp. mature to 3rd stage larvae at 30-35 days after hatching in an intermediate host and, once ingested by a definitive host, require an additional 73-90 days to reach sexual maturity and begin depositing embryonated eggs (Schell, 1952). Once mature, adult

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nematodes can survive for 3 to 10 months in their hosts; however, nematode life expectancy can vary substantially among hosts depending on the strength of the host’s immune response.

Regardless, this developmental phenology supports the hypothesis that adult and juvenile

Physaloptera worms either overwinter in red squirrels or as infective larva in the environment, maturing and commencing reproduction in late spring/early summer.

What triggers the onset of larval maturation, reproduction, and increased fecundity in adult nematodes is not well understood, but may be related to physiological changes in the host

(Babayan et al., 2010). Filarial nematodes develop faster and reproduce earlier in response to elevated immune responses in hosts, which the authors suggest is because mortality is perceived to be high under those conditions so nematodes compensate for a reduced life expectancy by investing in earlier and greater reproductive output (Babayan et al., 2010). This finding is counter to many other findings that suggest that nematodes increase their fecundity when host immune function is suppressed by, for example, raising offspring (i.e., lactation and the periparturient rise; Festa-Bianchet, 1989; Jones et al., 2012; Ross et al., 1993; Shanks et al.,

1997; Waldron & Revelo, 2009), nutritional stress (low or poor quality forage availability;

Athanasiadou, 2012; Hughes & Kelly, 2006; Keymer & Dobson, 1987; Smith et al., 2005; Tadiri et al., 2013), and elevated levels of testosterone (Braude et al., 1999; Decristophoris et al., 2007;

Grear et al., 2009; Mougeot et al., 2006). Late spring and early summer coincide with the period of reproduction in red squirrels and also the period of low and/or poor quality food availability

(over-winter food caches are becoming depleted and additional food sources [i.e., pollen cones, conifer buds, fungi, insects] are not readily available). Males also have higher levels of circulating immunosuppressant testosterone during this time (Gorrell & Schulte-Hostedde,

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2008). I also found reproductive status to be an important predictor of nematode prevalence and intensity, suggesting that physiological mechanisms/cues associated with reproduction

(testosterone, stress, immunosuppresion) may drive the fecundity and development of nematodes. Although the increased fecundity of nematodes in red squirrels peaked during or just subsequent to months with high rainfall in my study, it seems unlikely that rainfall directly drives nematode fecundity despite the presence of more ideal (i.e., humid) conditions for development, maturation and transmission of infective stages. Perhaps, also, adult Physaloptera respond to dietary cues, such as the presence of intermediate hosts (insects) in the diet of red squirrels, which would indicate ideal transmission conditions in the external environment. Teasing apart the influences of each potential causative factor on the seasonality of parasitism poses a challenge to disease ecologists. A key problem is that seasonality is so ubiquitous in nature that identifying the relevant ecological and physiological drivers, as well as the parameters they influence, becomes extremely difficult (Altizer et al., 2006).

Contrary to my predictions, there was no effect of sex or female home range size on endoparasitism in red squirrels, and only a very weak effect of nematode co-infection on Eimeria spp. shedding intensity. Male-biased parasitism is widely reported across host taxa and host sex has been found to affect patterns of parasite aggregation within host populations (e.g.,

Scantlebury et al., 2010), as well as parasite transmission dynamics (e.g., Ferrari et al., 2004).

Gorrell and Schulte-Hostedde (2008) found evidence of higher endoparasite egg-shedding intensities in males, but only in August, and this effect was negated when corrected for multiple statistical tests. This result also differs from that for ectoparasites in SRPP red squirrels that did exhibit sex-biased distributions, but only during certain life history stages (Chapter 3). My

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results contrast with those of Waterman et al. (2014), who found that nematode intensities were male-biased, but that flea intensities were not. Factors such as nematode life history, development, and maturation, as well as seasonal nutritional stress and reproductive immunosuppression, which appear to influence nematode fecundity, may apply correspondingly to both males and females in this population. Differences in host biology, such as behaviour, mating systems, and parasite co-evolutionary ties may account for differences among species.

Another possibility is that immunological differences between the sexes modulates parasite fecundity, adult nematode body size, and the rates of parasite development and survival, with males ultimately providing a better environment for parasite growth and reproduction than female hosts (Finkelman et al., 1997; Poulin, 1996a). Therefore, male red squirrels may actually have higher parasite intensities than females, but through density-dependent regulation of fecundity, the actual number of eggs shed in the feces may not differ between the sexes.

Although not tested here, a lack of observable difference in parasite egg/oocyst shedding between the sexes does not preclude male red squirrels from being responsible for the transmission of endoparasites in this system, as is the case for other rodent species (Ferrari et al.,

2004; Perkins et al., 2003).

Interestingly, C. bifurcatum intensity had a negative relationship with host body mass, suggesting that this parasite impairs the condition of its host when the intensity of infection is high. An alternative explanation for this relationship is that hosts in poor condition are less able to mount an appropriate immune response to C. bifurcatum. As is the nature of correlative tests, one cannot explicitly link results to causative factors; however, in this case both explanations are biologically valid and warrant further investigation. At high infection intensities, C. bifurcatum

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likely consumes more host resources and triggers costly immune responses, which can lead to both anemia and weight loss. Although little work has been conducted on C. bifurcatum and the

Citellinema genus as a whole, related Trichostrongylid nematode species (e.g., Ostertagia spp.,

Trichostrongylus spp. and Oesophagostomum spp.) have been found to reduce the body mass of their hosts (Irvine et al., 2006; Newey et al., 2004; Sweeny et al., 2011), as well as host food intake (Arneberg et al., 1996) and fecundity (Albon et al., 2002; Hudson, 1986).

I predicted that home range size would correlate with prevalence of endoparasites as larger home ranges may increase exposure to novel parasites, while smaller home ranges may lead to repeated exposure to infective stages at contaminated sites. Home range size may also affect the energy available to mount an immune response if maintaining larger home ranges incurs an energetic cost or if larger home ranges are of poorer quality than smaller home ranges.

While red squirrels are highly territorial animals (Larsen & Boutin, 1995; Price & Boutin, 1993), they do move off-territory regularly to forage and mate, which could increase exposure to parasites (i.e., cache pilfering; Gerhardt, 2005). While territory size does not appear to relate to territory quality in red squirrels (Larsen & Boutin, 1995; Vlasman & Fryxell, 2002), the degree of off-territory ranging behaviour may. My results, however, suggest that immune function and parasite acquisition are not affected by the ranging behaviour of individuals in this system. Given that the suite of endoparasites detected in red squirrel feces was low and prevalence of these parasites in hosts was high (>60%), it is not surprising that parasite prevalence was not affected by ranging behaviour.

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Other factors not explored here could also influence patterns of parasitism in free-ranging animals, such as: population density (Arneberg, 2002; Nunn et al., 2003; Stanko et al., 2002) and gregariousness (Altizer et al., 2003; Ezenwa, 2004a; Patterson & Ruckstuhl, 2013; Rifkin et al.,

2012), metabolism and immunity (Krasnov et al., 2004b; Morand & Harvey, 2000), age/longevity (De Nys et al., 2013; Krasnov et al., 2006; Lopez et al., 2007), and directly quantified measures of stress and testosterone (Decristophoris et al., 2007). Aside from direct measures of host physiology, age appears to be one of the most important predictors of parasitism in free-ranging animals. Unfortunately, I could not accurately estimate age in my red squirrel population given the short time-scale of my project. Red squirrels have a maximum life expectancy of 8 years in the wild (McAdam et al., 2007), with an average life expectancy of 3.5 years if they are able to survive the first year of life (McAdam et al., 2007). Parasite infection intensities and species richness typically increase during the early stages of life across a broad range of vertebrates, often peaking in the middle stages and then either declining or reaching a plateau as immunity is acquired and species richness is maximized (Nunn et al., 2003; Quinnell et al., 1995; Shaw et al., 1998). In European serins (a species of finch; Serinus serinus), the number of coccidia oocysts discharged in their faeces decreased as the birds aged (Lopez et al.,

2007). This trend suggests that the presence of coccidian parasites leads to the acquisition of immunity in S. serinus over time, as it does in other species (e.g., Lillehoj & Lillehoj, 2000; Rose

& Hesketh, 1982). Future research should focus on the effects of age, immune response, and immunosuppressive hormones (e.g., testosterone, cortisol) as determinants of endoparasitism in red squirrels. Additional work on C. bifurcatum and Physaloptera sp. life cycles in red squirrels is needed to properly assess the seasonal population and reproductive/transmission dynamics of these parasites and their effects on host body mass.

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Range distributions of parasites are shifting at unprecedented rates as a result of climate change, biodiversity loss and land use change (Chapman et al., 2005; Daszak et al., 2001; Kutz et al., 2005; Patz et al., 2000), leading to the emergence of infectious diseases that may pose major threats to human, animal and ecosystem health (Daszak et al., 2000). Ultimately, studying the patterns of parasite infection and host susceptibility will assist wildlife managers, parasitologists, and evolutionary ecologists in better understanding the risk factors that make parasite infection in hosts more likely. Thus, by identifying risk prone segments of the host population, managers can make better informed decisions and develop more appropriate strategies to mitigate the spread and persistence of infectious diseases in free-ranging animals

(Woolhouse et al., 1997).

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CHAPTER 7: SUMMARY AND CONCLUSIONS

Parasites are ubiquitous in nature and play an important role in both ecosystem function and stability (Hudson & Greenman, 1998; Hudson et al., 2006; Hudson et al., 1998). By definition, parasites are harmful to their hosts. As such, parasites have the potential to affect the reproductive success, survival, activity budget, body condition, and social organization of their hosts. However, not all individuals and populations are affected equally and patterns of infection are typically heterogeneous. The complex processes determining these heterogeneities are not fully understood, although they are often attributable to various host parameters, such as age and sex, as well as genetic differences in immunity, spatial and temporal differences in the distribution and survival of infective stages, and seasonal variation in the risk of exposure

(Wilson et al., 2001). Further, because hosts only have a limited resource pool from which to draw, and parasite resistance can be costly, trade-offs between immune function and fitness traits

(e.g., growth, mate searching) are expected (Allen & Little, 2011; Sheldon & Verhulst, 1996). In the preceding chapters, I have explored several ecological mechanisms that may explain the observed patterns of parasitism in wild animals, as well as some of the constraints and costs imposed by parasites on the fitness and behaviour of their hosts. I have shown that parasites display temporally- and sex-biased infection intensities with measurable consequences to the reproductive success and activity budgets of their red squirrel hosts. I have also shown that ecological and demographic phenomena, such as group size, have the potential to affect the susceptibility of hosts with notable implications for the observed patterns of parasitism in free- ranging animals.

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Sex-biases and temporal variation

In Chapter 3, I found that flea infection was sexually and temporally structured in the red squirrel population I studied. Juveniles in the nest environment were more heavily parasitised than at any other life-history stage, and adult males had significantly higher flea intensities than adult females, but only when males were reproductively active. There is some support that sex differences in parasitism, particularly in mammals, are driven by sex-specific life-histories, whereby females usually prioritize traits that enhance survival, such as immunity, and males prioritize traits that enhance shorter-term reproductive benefits, such as increased aggression

(Wilson & Cotter, 2013), although more research is needed. In species exhibiting promiscuous/polygynous mating systems, males also produce higher levels of testosterone during the mating season and exhibit greater levels of risk-taking and aggressive behaviour, which can reduce immune function through immunosuppression and increase the likelihood of acquiring/transmitting parasite infective stages. Male red squirrels may also be responsible for the horizontal transmission of fleas in this population since fleas are contact transmitted and adults only come in contact with each other during mating – the period when males are more intensely parasitized.

In Chapter 6, I did not find that endoparasitism was sex-biased. However, I did find that reproductive status is an important predictor of Physaloptera sp. prevalence and egg shedding intensity. Investment in current reproduction by both sexes likely leads to corresponding declines in immune function, although the timing and mechanisms are likely to be different between the sexes. Females likely experience reduced immune function and corresponding increases in

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endoparasitism following parturition, which may be driven by investment in energetically expensive lactation (i.e., periparturient rise). Males, on the other hand, invest in current reproduction during mating and increased parasitism during that time may be driven by elevated testosterone/stress and behavioural mechanisms. Interestingly, no difference between males and females in Physaloptera sp. egg shedding with respect to reproductive status was found, further suggesting that infection with Physaloptera sp. is not sex-dependent at different life stages.

Importantly, month also correlated with Physaloptera sp. and Citellinema bifurcatum egg shedding, suggesting a role for seasonality, possibly linked to the life cycle of the parasite and/or the physiology of the host.

In response to unfavourable seasonal environmental conditions, many parasites have evolved adaptations such as seasonal reproduction, hypobiosis (an arrested stage of larval development), and liver or deep tissue stages (e.g., Gibbs, 1982). Seasonality is a widespread phenomenon amongst parasites. Annual outbreaks of disease are common in many species, including humans (e.g., influenza virus), and understanding the drivers of these outbreaks is of utmost importance for the proper management of their impacts and transmission. For instance, mycoplasmal conjunctivitis (Mycoplasma gallisepticum) is an emerging epizootic infectious disease in wild house finches (Carpodacus mexicanus) that undergoes extensive outbreaks in the autumn and winter months, leading to rapid weight loss and death of host individuals

(Hochachka & Dhondt, 2000). Mycoplasmal conjunctivitis outbreaks may be driven by a multitude of factors including fall and winter flocking behaviour, annual variation in hormones and host defenses, or nutritional constraints (Altizer et al., 2004; Hosseini et al., 2004). Similar behavioural, physiological and nutritional factors may explain the observed temporal variability

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in endoparasite egg production and ectoparasite intensities in red squirrels. However, differences between ectoparasites and endoparasites in their infection patterns should be expected given that their transmission, reproduction, life cycles, and feeding systems are quite dissimilar. The population cycles of fleas parasitizing red squirrels, and other species, appear to be timed to coincide with the presence of neonates in the nest. Fleas spend almost all of their lifecycle inside the nest and overwinter in the nest as larvae feeding on the feces of adult fleas. In the presence of a host, flea larvae pupate and adults emerge to feed on their hosts and reproduce. The constant source of a bloodmeal and stable environmental conditions provided by neonates in the nest provide adult fleas with critical opportunities to feed and reproduce. As such, fleas demonstrate seasonality in their exploitation of hosts to maximize their fitness, which may also include transmission of infective stages (i.e., adult fleas) via reproductively active adult male red squirrels – a type of sexually transmitted infection. In addition to displaying a seasonal population cycle, fleas also contributed, along with all other ectoparasites, to a reduction in the mass and survival of neonate red squirrels.

A critical next step may involve experimentally perturbing processes that covary with season, such as hormones (via implants/injections), food availability, and reproduction (litter size manipulations, contraceptives), across replicated populations. This may help researchers better understand the temporal variability in parasite infection intensity, transmission dynamics, and the trade-offs faced by hosts. Using geographic variation within specific host-parasite systems may also provide insight into observed temporal dynamics of parasitism. Additionally, our understanding of host-parasite interactions would benefit from developing a complete description of the life cycles of both Physaloptera sp. and C. bifurcatum nematodes. Developing a more

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complete understanding of the role that temporal variation and seasonality play in affecting multiple processes, such as host behaviour, reproduction, immune function, and parasite transmission and survival in the environment, will become increasingly important, particularly with the prospect that global climate change may rapidly alter current patterns of seasonality.

Transmission and control of parasite infection

Transmission of most parasite infective stages is either density-dependent or frequency- dependent. That is to say that transmission should vary with the density of host individuals in a population and the frequency of host contact with infective stages. Several host factors can influence the successful invasion and spread of disease in a population, such as crowding, contact networks, mating opportunities, and gregariousness. Further, hosts must be susceptible to infection, which may be driven in part by genetics, previous exposure to parasites, co-infection with other parasite species, nutritional status/body condition, and behavioural factors. In Chapter

2, I show that group size leads to increased risk of acquiring and being infected by contagious parasites, such as nematodes and fleas. Group living then represents an important host behaviour that can influence the invasion and spread of parasites and diseases in wild host populations.

Interestingly, mobile parasites that are able to seek out their hosts do not exhibit the same relationship with host group size. Instead, larger groups act to dilute the intensities of infection by these parasites, likely to the benefit of the group. Within groups, some individuals may be more susceptible to infection than others due to dominance hierarchies (and associated differences in hormone levels, contact, mating, and grooming), differential investment in immune function, age, and previous exposure to parasites, amongst others. Sex-ratios in groups

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and populations of hosts may also be an important consideration when assessing parasite transmission dynamics. In many species, males are more heavily parasitized than females and are responsible for much of the transmission dynamics of certain parasite species (e.g., Ferrari et al.,

2004). As such, in male-biased groups and populations, rates of transmission may be higher than in female-biased groups/populations. When modeling the epidemiology of parasites and disease, the mode of transmission as well as the complexities of host behaviour and population structure should always be considered.

Transmission is generally determined by the rate of contact between an infected host/infective stage and a susceptible host (number of contacts/unit of time) and the actual risk or probability of the infective stage being transmitted. This relationship realizes that transmission depends on patterns of contact, degree of sociality, types of contact between hosts, as well as the biology of the parasite itself. For instance, if an infected individual frequently engages in contact with a susceptible individual and the parasite infective stage is present and contact-transmitted, then the probability of transmission occurring is high. Once transmitted, factors specific to the host will determine its degree of susceptibility, such as previous parasite exposure and investment in immune function. In systems where infection with a specific parasite or pathogen is of management concern, reducing the probability of transmission and/or reducing the susceptibility of uninfected individuals, possibly through targeted vaccination or culling programs, may aid in slowing or preventing the spread of parasites in a population.

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On the costs of parasitism

Key to the definition of parasitism is that parasites cause harm to their hosts. Harm can be difficult to measure in host species, although harm is often expressed as metabolic costs, immune activation, reductions in host condition and reproductive success, and mortality. By directly withdrawing nutritional resources from their hosts, causing irritation and pathologies, and triggering behavioural alterations in their hosts, parasites have been implicated in the loss of fitness (e.g., Albon et al., 2002; Møller, 1993; Neuhaus, 2003) and the evolution of certain traits, such as sexual reproduction (Hamilton et al., 1990), in their hosts. Numerous studies have implicated parasites in the reduction of host reproductive success, which can place important selective pressures on hosts to evolve mechanisms for parasite resistance or traits that convey parasite resistance to their mates. For instance, males of many species have evolved ornamentations, such as bright colours and horns, which act as honest signals of parasite resistance, immune function and body condition/nutritional status, which are selected for by their mates (e.g., Ezenwa & Jolles, 2008; Hamilton & Zuk, 1982; Møller & Alatalo, 1999; Møller &

Saino, 1994; Mougeot et al., 2004; Thompson et al., 1997; Zahavi, 1977). In red squirrels, male search tactics are under positive sexual selection (Lane et al., 2009). If ectoparasites increase the amount of time spent grooming and resting, as they do in female red squirrels and other species

(Chapter 5; Main & Bull, 2000), and reduce the energy available to males to invest in reproduction, then parasite resistance mechanisms should be selected for if males with strong parasite defenses have higher reproductive success and those resistance traits are heritable. In red squirrels, the most obvious harm was caused by ectoparasites and affected the mass and survival of juveniles between birth and emergence from the nest, when immune function and grooming –

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two important defence mechanisms – have not yet fully developed. In this sense, ectoparasites

(fleas, in particular) appear to be exploiting relatively defenseless juvenile hosts for resources required for their own growth and reproduction. Reduced body mass in juveniles can translate into reduced competitive success, territory acquisition, and overwinter survival compared to heavier individuals (Festa-Bianchet et al., 2000; Kerr et al., 2007). Female red squirrels parasitized by ectoparasites spend more time grooming themselves, presumably to remove ectoparasites, but do not appear to pay a personal cost to their body mass. While ectoparasites may not reduce body mass of adult red squirrels, perhaps due to increased grooming effort to reduce intensities, nematode egg shedding intensity does negatively correlate with adult body mass. Egg production by nematodes is an energetically demanding process for the parasite and may require additional host resources, which could be expressed as reduced body mass in hosts with high parasite egg counts. Alternatively, nematodes may opportunistically produce more eggs in hosts that are already in poor condition and who, as a result, may not be able to mount an appropriate immune response. Future research on the effects of parasitism on red squirrel body mass and fitness would benefit from experimental manipulations of nematode infections, studying the effects of infection on immune function, how individuals may trade-off immune function with fitness, continued study of the energetic and metabolic costs of parasitism, and the potential costs of parasites to male mating success.

Final words

There exists a great need to develop a complete understanding of the processes that produce heterogeneous patterns in parasite distributions within their host populations, and the

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consequences of these patterns and processes on the fitness of individual hosts. Through this thesis, I have attempted to provide additional answers to these problems through experimentation and correlative tests in a relatively well-understood species of rodent. Continued examination of these important topics in the future and application of my findings to other systems will undoubtedly provide necessary insights into host-parasite coevolution, life-history theory, and adaptive management of parasite and disease epizootics. We still have a lot to learn about host- parasite interactions, and it is my hope that research into parasites will continue to grow and mature in the coming years and decades without researchers having to make too many trade-offs.

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