Interspecific Competition Between Intraerythrocytic

INTERSPECIFIC COMPETITION BETWEEN INTRAERYTHROCYTIC

APICOMPLEXANS HEPATOZOON CLAMATAE AND

HEPATOZOON CATESBIANAE IN GREEN FROGS

Meghan Victoria Kerr

Thesis submitted in partial fulfilment of the

requirements for the Degree of

Bachelor of Science with

Honours in Biology

Acadia University

April, 2015

This thesis by Meghan Victoria Kerr

is accepted in its present form by the

Department of Biology

as satisfying the thesis requirements for the degree of

Bachelor of Science with Honours

Approved by the Thesis Supervisor

______Todd Smith Date

Approved by the Head of the Department

______Stephen Mockford Date

Approved by the Honours Committee

______Anthony Thomson Date

I, Meghan Kerr, grant permission to the University Librarian at Acadia University to

reproduce, loan or distribute copies of my thesis in microform, paper or electronic

formats on a non-profit basis. I, however, retain the copyright in my thesis.

______Signature of Author

______Date

iii

ACKNOWLEDGEMENTS

My journey through the honours program has been the most challenging, yet the most rewarding experience of my academic career. My positive experience would not have been what it was without the guidance, kindness, and enthusiasm from my supervisor Dr. Todd Smith. I will always remember your unfailing good humour in the face of seemingly infinite responsibilities, and your endless support and encouragement.

You exemplify the vital balance between a gentle authority figure and fellow research companion that anyone would be lucky to find in a supervisor. Thank you for all of your teachings.

I have also spent this past year in the company of some of the most hard-working students at Acadia, for without whom my days would have been considerably less enjoyable. Thank you Caoimhe McParland for your enormous help day after day, swamp after swamp, forever by my side in a matching pair of chest waders. Thank you to

Francine Heelan and Remington Winter for everything that you have done to help with our research, you are two of the most dedicated workers that I know. I must also thank

Amye Harrigan for her wisdom and advice in the early stages of the study, and for being such a positive role model for future honours students. A big thanks to Emma Gillis as well for entrusting me with healthy and happy green frogs, I could not have raised them better myself. Lastly, for never failing to put a smile on my face even during the most stressful of times, thank you to Mark Hanes for being such a wonderful office buddy.

This project would not have been possible without generous funding from the

Acadia Honours Summer Research Award and from the Atlantic Canada Society of

Microbial Ecology, for which I am grateful. I am similarly grateful for Tanya Morse and

Dawn Miner in Animal Care for their tremendous help and patience, you are both wonderful at your jobs and were wonderful to work with. We would not have been able to capture a large portion of our source frogs without the help of Phyllis Harvie, who so kindly gave us access to her pond. I would also like to thank Lisa Taul (for being Lisa

Taul), and Phil Taylor for his constructive statistics advice. For additional help with statistics, I cannot thank Laura Ferguson enough for taking the time out of her busy schedule to help a lowly honours student. To my second external reader, Dr. Juan-Carlos

Lopez, thank you for being such a helpful, kind, and positive presence throughout my journey. You have brightened up the biology department during your time at Acadia, and are not only a fantastic teacher, but a selflessly supportive mentor. Gracias por todo!

To all of my friends and family whom I love, thank you for your unfailing encouragement, support, patience, and comfort during this past year. Most of all, thank you for believing in me even when I didn’t always believe in myself.

Last, but certainly not least, I would like to thank the numerous froggies who so bravely sacrificed their freedom in the name of science.

TABLE OF CONTEN4TS

ACKNLOWLEDGEMENTS ...... iv

TABLE OF CONTENTS ...... vi

LIST OF TABLES ...... viii

LIST OF FIGURES ...... ix

ABSTRACT ...... x

INTRODUCTION ...... 1

Parasitism ...... 1

Phylum Apicomplexa ...... 2

Genus Hepatozoon ...... 3

Culex territans and green frogs (Rana clamitans) ...... 6

Models of multiparasite communities ...... 7

Exploitation, apparent, and interference competition ...... 11

Objectives ...... 13

MATERIALS AND METHODS ...... 20

Collection and maintenance of green frogs free of Hepatozoon infection ...... 20

Collection and maintenance of green frogs with pre-exising Hepatozoon infections 21

Collection of mosquito larvae and pupae ...... 23

Inoculation of mosquitoes and frogs ...... 23

Determining parasitaemia in frogs ...... 25

Statistical analysis ...... 26

RESULTS ...... 30

DISCUSSION ...... 41

Infection dynamics of Hepatozoon species ...... 41

Exploitation competition and erythrocytic merogony ...... 42

Relative parasitaemia of Hepatozoon species and apparent competition ...... 43

Mass of green frogs ...... 44

Variation in parasitaemia within infected frogs ...... 44

Exploitation competition by coinfection vs superinfection ...... 47

Experimental challenges ...... 48

Future studies ...... 49

REFERENCES ...... 51

vii

LIST OF TABLES

Table 1. The five experimental cohorts with ideal inoculation ratios of

H. clamatae to H. catesbianae ...... 27

Table 2. The three experimental cohorts with actual inoculation ratios of

H. clamatae to H. catesbianae ...... 28

Table 3. Statistical output for the linear mixed-effects model investigating

differences in mean total parasitaemia of Hepatozoon species within and

among cohorts 1, 4 and 5 over time ...... 32

Table 4. Statistical output for the linear mixed-effects model investigating

differences in mean merozoite intensity of Hepatozoon species within and

among cohorts 1, 4 and 5 over time ...... 33

Table 5. Statistical output for the linear mixed-effects model investigating

differences in mean parasitaemia of H. clamatae and H. catesbianae within

and among cohorts 4 and 5 over time ...... 34

viii

LIST OF FIGURES

Figure 1. Method of host cell invasion by apicomplexan parasites ...... 15

Figure 2. Cytopathological effects of intraerythrocytic gamonts of

Hepatozoon clamatae and Hepatozoon catesbianae ...... 16

Figure 3. Life cycle of Hepatozoon catesbianae in ranid frogs and in the

mosquito, Culex territans ...... 17

Figure 4. Population dynamics of a mixed infection of Plasmodium vivax

and Plasmodium falciparum infection over 361 days ...... 18

Figure 5. Exploitation competition between two species of parasites ...... 19

Figure 6. Cytopathological features of various stages in the life cycle of

Hepatozoon species ...... 29

Figure 7. Mean total parasitaemia of Hepatozoon species in three cohorts

of infected frogs over time ...... 35

Figure 8. Mean total merozoite intensity of Hepatozoon species in three cohorts

of infected frogs over time ...... 36

Figure 9. Mean parasitaemia of mixed 2:2 (H. clamatae to H. catesbianae)

infections in cohort 4 (n=2) over 121 days ...... 37

Figure 10. Mean parasitaemia of mixed 3:1 (H. clamatae to H. catesbianae)

infections in cohort 5 (n=5) over 121 days ...... 38

Figure 11. Proportion of gamonts of H. catesbianae to H. clamatae in

cohorts 4 and 5 over time ...... 39

Figure 12. Mean mass of green frogs in three cohorts over time ...... 40

ABSTRACT

Hepatozoon clamatae and Hepatozoon catesbianae are intraerythrocytic apicomplexans that infect the red blood cells of green frogs and bullfrogs of Nova Scotia.

Although H. clamatae and H. catesbianae have demonstrated mild host specificity in green frogs and bullfrogs, respectively, both species of Hepatozoon may simultaneously infect either species of frog. The purpose of this study is to investigate the dynamics of a dual infection of sexual stages (gamonts) of H. clamatae and H. catesbianae in the green frog, Rana clamitans, and to determine the existence of competition between these two species. Mosquitoes were fed on naturally infected green frogs, purposefully selected based on their desired parasitaemia (intensity) of Hepatozoon species. Numbers of mosquitoes to derive specific ratios of H. clamatae: H. catesbianae were fed to uninfected laboratory-reared green frogs, which were subsequently bled to assess the development of relative parasitaemia of the two species. Hepatozoon species were differentiated based on their unique cytopathological effects. Due to the unexpected persistence of erythrocytic merogony in all cohorts, species distinction was not attempted until 80 dPI in mixed infections, and merozoite intensity over time was recorded in all cohorts. Based on a linear mixed-effects model, it was determined that day of bleeding has a significant effect on parasitaemia and merozoite intensity in all cohorts. In addition, there is a trend toward a significant difference between the infection dynamics of cohort 1 and cohort 5. These results suggest that an acquired immune response may be responsible for the decline in parasitaemia over time, and with a larger sample size per cohort, the effect of competition between species may be detected by statistical methods.

INTRODUCTION

Parasitism

The evolution of parasitology as a science traces back to antiquity, when parasites and their afflictions were first discovered and described in humans. The symptoms of malaria, emerging as violent and periodic fevers, were first described in early Chinese writings from 2700 BCE and have surfaced within every ensuing civilization in areas endemic for the disease (Cox, 2002; Goater et al., 2014). Hippocrates was the first to provide a detailed description of malaria by associating fever and chills with the disease

(Sallares and Gomzi, 2001; Cox, 2002). The invention of the microscope by Antony Von

Leeuwenhoek in the 17th century unveiled the microbial world, and the discipline of microbiology was further developed by the disproval of spontaneous generation, synthesis of germ theory by Louis Pasteur, and by the derivation of Koch’s Postulates in the late 19th century (Cox, 2002; Goater et al., 2014). It was not until the 20th century, however, that parasitology was established as a distinct science (Marquardt et al., 2000;

Goater et al., 2014).

Parasitism is a type of symbiotic relationship in which the smaller organism, or parasite, lives inside or on the surface of, and consequently has adverse effects on, the larger organism, or host (Marquardt et al., 2000). The coevolution of parasite and host gives rise to morphological and physiological adaptations in the parasite so it may better utilize host metabolic resources, consequently reducing host fitness (Price, 1977;

Combes, 2005). Due to complex and intricate host-parasite relationships, many parasites are specific to their host. As parasite and host travel through evolutionary time, they battle in a constant arms race; as the host develops greater resistance and immunity to the

parasite, the parasite evolves greater virulence or avirulence (Gunn and Pitt, 2012). More advanced parasites from an evolutionary perspective are those that cause the least amount of harm to the host, as they have developed strategies to remain metabolically dependent whilst preventing excessive harm during prolonged exploitation (Levin and Pimentel,

1981; Lenski and May, 1994; Ebert and Herre, 1996).

As many as 30 to 50% of animal species are parasitic at some period in their life, thus parasitism is a widely used strategy of acquiring nutrients, and host-parasite interactions are ubiquitous across all 35 animal phyla (Marquardt et al., 2000; Goater et al., 2014). To a parasitic agent, hosts are collections of ecological niches available for exploitation (Marquardt et al., 2000; Roberts and Janovy, 2005). Parasites with indirect life cycles, or those that pass through more than one host species throughout their lifespan, require definitive and intermediate hosts. A definitive host houses the parasite during stages of sexual development, whereas the intermediate host harbours the parasite during changes in morphology and asexual growth (Goater et al., 2014). A host may also be termed a vector if it is a smaller invertebrate that transmits the parasite between larger vertebrate hosts (Marquardt et al., 2000).

Phylum Apicomplexa

The phylum Apicomplexa is a protozoan phylum of great size and significance to humans and other animals. The phylum consists of species of obligate intracellular parasites of vertebrates and invertebrates that are characterized by motile, invasive stages, or zoites, featuring a morphologically unique apical complex, composed of specialized organelles, at the anterior end (Morrissette and Sibley, 2002; Gunn and Pitt, 2012). This apical complex is indispensable to stages of apicomplexans that invade cells, and consists

of two secretory organelles, micronemes and rhoptries, to facilitate motility, host cell adhesion, penetration, and formation of an intracellular vacuole in which the developing parasite resides (Fig. 1) (Dubremetz et al., 1998; Morrissette and Sibley, 2002; Shen and

Sibley, 2012). Phylum Apicomplexa is a diverse assemblage of exclusively parasitic species that have elaborate interactions with one to three species of hosts due to hundreds of millions of years of co-evolution, and some species parasitize only one or a small group of related hosts due to their high level of specificity (Marquardt et al., 2000;

Morrison, 2009). Apicomplexans develop via alternation of generations, reproducing sexually by fusion of haploid gametes (syngamy) and asexually by multiple fission

(sporogony and merogony) (Marquardt et al., 2000). This phylum contains many species of blood-dwelling parasites, most notably human malaria parasites of the genus

Plasmodium, but also feature less familiar groups, including six genera collectively referred to as haemogregarines. These intraerythrocytic or intraleukocytic parasites undergo development in vertebrate blood and various tissues of a wide range of invertebrates (Siddall, 1995; Smith, 1996; Gunn and Pitt, 2012).

Genus Hepatozoon

One of these six genera of haemogregarines is Hepatozoon, which contains species that infect the visceral organs and blood of tetrapod vertebrates and the gut, haemocoel and Malpighian tubules of hematophagous arthropods (Boulianne et al.,

2007). Vertebrates such as mammals, reptiles, birds, and amphibians serve as intermediate hosts for these heteroxenous parasites, whereas mosquitoes, fleas, mites and ticks serve as definitive hosts (Kim et al., 1998; Boulianne et al., 2007). These intraerythrocytic, or intraleukocytic apicomplexans are characterized by the presence of

large multisporocystic oocysts in the haemocoel of arthropods, as opposed to the smaller asporocystic oocysts in the gut wall of leeches, which is characteristic of species of the closely related genus Haemogregarina (Desser et al., 1995).

Two well-described species of Hepatozoon that infect frogs in eastern Canada are

Hepatozoon clamatae, which preferentially inhabits the erythrocytes of green frogs (Rana clamitans) and Hepatozoon catesbianae, which preferentially inhabits the erythrocytes of bullfrogs (Rana catesbeiana) (Desser et al., 1995; Kim et al., 1998), although both species may be found in either species of frog (Boulianne et al., 2007). In a sample of green frogs from Nova Scotia, the presence of Hepatozoon was at a staggering 88.4%, with a 75.4% prevalence of H. clamatae and a 29.0% prevalence of H. catesbianae

(Boulianne et al., 2007). In a sample of bullfrogs, the presence of Hepatozoon was a mere

23.5%, with a 5.9% prevalence of H. clamatae and a 17.6% prevalence of H. catesbianae

(Boulianne et al., 2007). Mixed infections were recorded in green frogs, but not observed in bullfrogs (Boulianne et al., 2007). The nature of the weak host specificity for each of these Hepatozoon species for the two species of frogs is unknown, although host specificity is not determined at the gamont stage that enters erythrocytes (Dickson et al.,

2013). Furthermore, these species may induce changes in the phenotype of anuran hosts to increase transmission success by mosquitoes (Ferguson et al., 2013).

Hepatozoon clamatae and H. catesbianae have identical life cycles, and both use the mosquito Culex territans as a definitive host and vector. These two closely related species were initially differentiated based on their distinct cytopathological effects, first observed by Barta and colleagues (1984). The presence of H. clamatae fragments the erythrocytes nucleus, whereas the presence of H. catesbianae displaces the erythrocyte

nucleus laterally (Fig. 2) (Desser et al., 1995; Kim et al., 1998). Additionally, although both species infect green frogs and bullfrogs, H. clamatae also infects northern leopard frogs, Rana pipiens, whereas H. catesbianae also infects wood frogs, Rana sylvatica

(Hammer, 2012). The discovery of single-nucleotide polymorphisms at six sites of the internal transcribed spacer (ITS-1) region of the nuclear genome of each parasite distinguishes species at the molecular level (Boulianne et al., 2007).

The life cycle of H. clamatae and H. catesbianae in ranid frogs and C. territans requires one round of asexual development in hepatocytes, optional rounds of asexual development in erythrocytes, and sexual development in the haemocoel of the mosquito

(Desser et al., 1995; Kim et al., 1998; Smith et al., 2000). More specifically, when a frog ingests an infected mosquito, sporozoites emerge from the insect and migrate to the hepatocytes of the liver, which becomes the site of a type of multiple fission called merogony. The resulting merozoites leave hepatocytes, enter the circulation, invade erythrocytes, and differentiate into female or male gamonts, which are subsequently taken up by a mosquito during a blood meal. In some cases, Hepatozoon species may form erythrocytic meronts and undergo further rounds on asexual division in the blood before differentiating into gamonts (Smith et al., 2000). Once in the mosquito midgut, gamonts emerge from erythrocytes, move to the Malpighian tubules, and undergo gametogenesis to form female or male gametes. Fusion of gametes during fertilization is immediately followed by meiosis to restore the haploid state in the form of an oocyst. In another type of multiple fission called sporogony, the oocyst differentiates into hundreds of sporocysts, each containing four sporozoites. Sporozoites within mature oocysts are

then infective to frogs when these amphibians eat infected mosquitoes (Fig. 3) (Kim et al., 1998).

Culex territans and green frogs (Rana clamitans)

Culex territans (Diptera: Culicidae) is widely distributed in unpolluted waters across the Northern Hemisphere, populating many permanent aquatic habitats including marshes, swamps, ponds, streams and wetland pools (Knight and Stone, 1977; Joy and

Clay, 2002; Bartlett-Healy et al., 2008). The abundance of Culex territans throughout

North America, Europe, and Asia makes it the most widely distributed of the 26 species of the subgenus Neoculex (Walter Reed Biosystematics Unit, 2001; Bartlett, 2009), populating the greatest variety of habitats (Linam and Nielson, 1970). In order to foster the development of their offspring, C. territans maintain a parallel distribution to green frogs (Crans, 1970), which are found in similar aquatic habitats across eastern North

America (Conant and Collins, 1991). By sharing habitats and ovipositing near this amphibian species, female C. territans ensure their offspring are close to future blood meals (Bartlett-Healy et al., 2008).

Green frogs are territorial and maintain a small home range, rarely venturing far from water to avoid predation and desiccation (Martof, 1953; Stockwell and Hunter,

1989). The growth of green frogs is highly dependent on food availability, thus green frogs opportunistically feed frequently during night and day (Hamilton, 1948; Martof,

1956). Green frogs are 5.7 to 9.0 cm in length, have a spotted or mottled green-brown dorsum with two distinctively raised dorsolateral folds, a creamy white venter, and dark transverse leg stripes (Conant and Collins, 1991). The tympanum, or eardrum, of males is larger than the eye, and the throat of males is yellow. The call of male green frogs is

explosive and reminiscent of a plucked banjo string, occurring as a single short blast, or repeated three to four times as progressively softer blasts (Conant and Collins, 1991).

Models of multiparasitic communities

Concomitant infections are characterized by the presence of two or more genetically different infectious agents coexisting in the same host, and may be a mix of different species or genetically diverse members of the same species (Cox, 2001). For example, there are four species of human malaria caused by parasites of the genus

Plasmodium and in areas of high transmission, over 80% of malaria cases contain more than one species (Babiker et al., 1999; Konaté et al., 1999; Bell et al., 2006). In addition to increasing our basic understanding of parasite and host ecology, the applied importance of untangling the web of interactions among host and coexisting parasites lies in its application to epidemiological studies, mainly the prediction and control of virulence evolution (Alizon and Baalen, 2008). Virulence describes the aggressive nature of a pathogen and its destructive behaviour toward the host, and is thus a feature of the parasite measured as a demographic parameter of the host (Alizon and Baalen, 2008).

Mixed infections may unfold as a coinfection or a superinfection: a coinfection describes the simultaneous inoculation of two genetically different pathogens, and a superinfection describes the successive inoculation of one pathogen after another (Nowak and May,

1994; May and Nowak, 1995). The order of parasite inoculation is crucial to the outcome of a mixed infection and may completely reverse competitive hierarchies (Cox, 2001; de

Roode et al., 2005).

The classical model of within-host competition is a negative relationship between genetic similarity and virulence of parasites. This kin selection model claims that lower

relatedness of parasite species promotes within-host competition and a battle for limited host resources, which selects for the more exploitative species. This more virulent strain outcompetes the less virulent strain, consequently promoting the evolution of increased virulence (Ebert and Herre, 1996; Frank, 1996; Chao et al., 2000; Brown et al., 2002;

West and Buckling, 2003; de Roode et al., 2005; Bell et al., 2006; Alizon and Baalen,

2008). Although the classical model of inter- and intra- species competition between parasites within a host has been shown with empirical and theoretical evidence, such as trials on Plasmodium chabaudi in rats (Bell et al., 2006), a plethora of alternative methods have been described, and the classical model is increasingly being challenged as equivocal.

Although the selection for increased virulence in mixed infections is a general phenomenon, it has also been suggested that less virulent clones are better able to evade the host immune system, which may increase transmission success compared to the more virulent clone, which draws more attention from the immune system (Chao et al., 2000;

Brown et al. 2002; de Roode et al., 2005; Bell et al., 2006). This avirulent model

(Alizon et al., 2009) is an indication that avirulent parasites may play a larger role in nature than typically assumed (West and Buckling, 2002; de Roode et al., 2005). Studies on the two most widespread of the four species of human malaria, Plasmodium vivax and

Plasmodium falciparum, revealed this type of interspecies inhibition (Mason and

Mackenzie, 1999). Plasmodium falciparum is the more lethal of the two species, but when inoculated after P. vivax, the peak parasitaemia of P. falciparum was greatly reduced (Mason and McKenzie, 1999). Although P. vivax could not maintain high densities after a coinfection with P. falciparum, it was able to thrive more successfully

after sequentially later superinfections by P. falciparum, reaching densities up to four times higher and lowering peak parasitaemia of P. falciparum by 28% (Fig. 4) (Mason and McKenzie, 1999).

When calculating the fitness of a megapopulation of parasites, the influence of parasite genotype on host survival must be considered (Levin and Pimentel, 1981). If interdemic selection, a type of natural selection that acts upon populations within a species, were to occur in a parasite community, there would not be a fight to overexploit the host. Rather, there would be a selection for traits beneficial to the collective group as opposed to the individual, trending towards lower levels of virulence to avoid overexploitation (Chao et al., 2000; Brown et al., 2002). This model of natural selection was observed in the myxoma virus introduced to the rabbit population of Australia in

1950. The initial virulent strains lethal in >99% of infected rabbits were soon ousted by less virulent strains. Due to the lower lethality rate (70-90%) and longer survival time

(17-28 days) caused by these attenuated viruses, they were more successful in being transmitted to potential vectors and subsequent vertebrate hosts (Fenner et al., 1956;

Fenner and Marshall, 1957). Further studies by Best and Kerr (2000) demonstrated how the less virulent strains of myxoma virus facilitated the development of innate immunity in rabbit populations, promoting the coevolution of host and parasite.

Another model of multiparasite dynamics is the trade-off model described by

Anderson and May (1979), which suggests there is a trade-off between virulence and transmission. This “tragedy of the commons” approach recognizes the benefit of balancing prudent exploitation with maximal reproductive rates to promote the fitness of the megapopulation (Harden, 1968; Frank, 1996). Although rapid proliferation promotes

fecundity of parasite species, it may be detrimental to the survival of the host, thus more prudent parasites promoting increased longevity are selected, and evolutionarily favourable intermediate levels of virulence are achieved (Bremermann and Pickering,

1983; Lenski and May, 1994; Frank, 1996). For instance, higher overall malaria prevalence in humans is characterized by the presence of mixed Plasmodium infections, and individuals suffering from these concomitant infections show fewer clinical symptoms than if they were infected by the constituent parasites alone (Mason and

McKenzie, 1999). Studies by Luxemburger and colleagues (1996) found severe malaria to be 4.2 times more common in individuals hosting only P. falciparum as opposed to individuals hosting a mixed Plasmodium infection, which could lead to earlier deaths of the intermediate hosts. This type of cooperation may increase both the growth rate and transmission success of all species in the mixed infection and may be seen in biofilms, immune suppression, and the production of iron-binding molecules, or siderophores

(West and Buckling, 2002). The formation of biofilms and cooperative suppression of the host immune system aid with colonization, and the production of siderophores allows the parasite community to more effectively scavenge iron, which is a limiting factor in their growth and development (West and Buckling, 2002).

Although the trade-off hypothesis offers a compromise to the kin selection model and the avirulent model, it is being challenged due to its simplicity and lack of evidence

(Lipsitch and Moxon, 1997). However, Alizon and colleagues (2009) assert that newly proposed species-specific models are insufficient in general virulence management applications and that the trade-off hypothesis provides the most promising model for

virulence management strategies. This provides an opportunity to improve upon the theory, as opposed to discarding it.

Exploitation, apparent, and interference competition

The dynamics of concomitant malarial infections may be difficult to elucidate due to the passage of parasites through two very different infracommunities, namely their definitive and intermediate hosts (Petney and Andrews, 1997). The above models of multiparasite dynamics (kin selection, avirulent, and trade-off) are best explained based on three types of competition: exploitation, apparent, and interference (Mideo, 2009).

Exploitation competition describes the conflict among parasites occupying overlapping ecological niches and the resulting fight for host resources (Mideo, 2009). The ecological factor most considered in malaria infections is the supply of erythrocytes, as these cells are the substrate for the blood stages of malaria parasites and the desired host resource

(McQueen and McKenzie, 2006; Mideo, 2009). Erythrocytes serve as shielded microenvironments for merogony, thus during a coinfection, host erythrocytes must be divided among the parasite community, which limits the growth rate of one or both of the cohabitants (Fig. 5) (Desser et al., 1995; Kim et al., 1998; de Roode et al., 2005). This will hinder both parasites during a coinfection, and greatly hinder the second inoculant in a superinfection, due to the depletion of uninfected erythrocytes available to be infected

(de Roode et al., 2005). Studies by McQueen and McKenzie (2006) confirm that erythrocyte supply is not a limiting factor for pure P. falciparum infections, yet mixed- species infections of P. falciparum and P. vivax promote competitive suppression of one species by the other. Another outcome from dual Plasmodium infections is the exchange of transmissible, non-replicating gamont stages for non-transmissible, replicating

schizont stages (McKenzie and Bossert, 1998; McQueen and McKenzie, 2006). Asexual merozoite stages are destructive to host erythrocytes, thus multiple infections select for reduced gamont conversion rates in order to maximize acquisition of free, undamaged erythrocytes to invade (Mideo and Day, 2008).

A fundamental determinant of the outcome of any form of mixed infection is the host immune system, thus apparent competition refers to the indirect interactions of parasites via the host immune response (Mideo, 2009). The innate immune response is responsible for parasite clearance during the acute phase of infection, whereas the acquired immune response controls parasite numbers during the chronic phase of infection, targeting the parasite that initially triggered immunity (Bell et al., 2006). The acute phase may trigger strain-transcending immunity to cross-react with all infectious agents until strain-specific immunity targets a single genotype (de Roode et al., 2006).

However, immune responses may affect various genotypes asymmetrically in cases where the immune response induced by each antigenically different parasite affects the other with varying severity (de Roode et al., 2005; Bell et al., 2006). A possible consequence may be the immunosuppression of one infectious agent and ensuing immune avoidance of the other, demonstrated in model populations of Plasmodium chabaudi. In a study by Raberg and colleagues (2006), immunocompetent mice asymmetrically suppressed avirulent clones of P. chabaudi, whereas immunosuppression of the avirulent clones was alleviated in immunodeficient mice. Another outcome of apparent competition may be the prevention of superinfection by genetically similar yet novel pathogens, a phenomenon known as premunition (Cox, 2001; Vardo et al., 2006).

This disadvantage was shown by the inoculation of Plasmodium mexicanum in pre-

exposed lizard hosts and resulting poor clonal establishment compared to previous inoculations in naïve hosts (Vardo et al., 2006; Vardo-Zalik and Schall, 2009).

Interference competition refers to the direct inhibition of growth, reproduction, or transmission of competitors (Mideo, 2009). This includes all forms of allelopathy, which is the secretion of biochemical inhibitors by an organism to impair the growth of a competitor, and has been extensively studied in prokaryotic organisms, such as the production of multiple bacteriocins in Escherichia coli (Mideo, 2009; Gordon and

O’Brien, 2006). These antimicrobial peptides, produced by 99% of all bacteria, have a relatively narrow killing spectrum and only target bacteria closely related to the producing strain (Riley and Wertz, 2002).

Objectives

The purpose of this study is to investigate the infection dynamics of infections of both H. clamatae and H. catesbianae in one of their ranid hosts, the green frog.

Specifically, the first objective of the study will be to monitor parasitaemia levels of

Hepatozoon infections in experimentally infected green frogs over time. The second objective of the study will investigate the possibility that competition occurs between two

Hepatozoon species in experimentally infected green frogs. This objective will be explored by determining the potential amplification of infection intensity by asexual division (merogony) in the erythrocytes of frogs infected with one or two species, and by comparing the relative parasitaemia of H. clamatae and H. catesbianae in mixed infections over time. The final objective will involve monitoring the mean mass of green frogs in each cohort over time as a marker for deleterious effects of infection on the health of frogs. A previous study by Trites (2013) determined that the parasitaemia of H.

clamatae (12.4%) increased significantly compared to H. catesbianae (1.6%) in a green frog inoculated simultaneously with both species of parasite. Additionally, the parasitaemia of H. catesbianae (0.3%) in a green frog inoculated only with this parasite was lower than the parasitaemia of H. catesbianae (1.6%) in a mixed infection (Trites,

2013). These results indicate that interspecies competition may be occurring, but due to the small sample size of two test frogs, further studies must be carried out with more frogs in a greater number of experimental cohorts. For this study, we propose five cohorts featuring different simultaneous inoculums of the two species of parasites: 1) pure H. clamatae, 2) pure H. catesbianae, 3) low parasitaemia of H. clamatae and high parasitaemia of H. catesbianae, 4) moderate infections of both species, and 5) high parasitaemia of H. clamatae and low parasitaemia of H. catesbianae.

Figure 1. Method of host cell invasion by apicomplexan parasites. 1. When a zoite

(represented by an oval) comes into contact with a target host cell (represented by a rectangle), the presence of the cell is communicated via signal transduction (star, arrow) to the apex of the zoite. 2. The detection of a target cell triggers re-orientation of the zoite so the apical complex is facing the host cell surface, at which point micronemes are exocytosed to induce apical binding, and the moving junction is formed. 3. Host cell invasion begins as rhoptries are exocytosed and the zoite penetrates the host cell surface, causing the moving junction to slide back as the zoite enters the parasitophorous vacuole.

The exocytosed micronemal material extends and coats the zoite surface untouched by the moving junction. 4. The parasitophorous vacuole scavenges host cell plasmalemma material as it enlarges to accommodate the invading zoite, until the moving junction seals the vacuole behind the successfully penetrated zoite. Dense granules are exocytosed in the vacuoloar space. (Dubremetz et al.,1998).

Figure 2. Cytopathological effects of intraerythrocytic gamonts of Hepatozoon clamatae and Hepatozoon catesbianae. A. Erythrocyte of green frog containing gamont of H. clamatae (arrow) with fragmented host nucleus (Kim et al., 1998). B. Erythrocyte of green frog containing gamont of H. catesbianae with intact host nucleus (Desser et al.,

1995).

Figure 3. Life cycle of Hepatozoon catesbianae in ranid frogs and in the mosquito,

Culex territans. a. Gamonts emerging from erythrocytes ingested by the mosquito are released in the midgut. b. Gamonts migrate to the Malpighian tubules, enter tubule cells and associate in pairs. c. Male gamonts undergo gametogenesis; one of two resulting male gametes fertilizes female gamete to form a diploid zygote. d. Zygote develops into an oocyst, which segments to form sporoblasts. e. Mature oocyst is multisporocystic.

f. Each sporocyst contains four sporozoites. g. When frog ingests an infected mosquito, sporozoites are released into the intestine. h. Sporozoites migrate to hepatocytes and undergo multiple fission to produce meronts. i. Merozoites emerge from hepatic meronts and enter bloodstream of the frog to invade erythrocytes, where they will mature into gamonts capable of infecting subsequent mosquitoes (Smith, 1996).

Figure 4. Population dynamics of a mixed infection of Plasmodium vivax and

Plasmodium falciparum over 361 days. A. Simultaneous infection of P. falciparum and

P. vivax. B. Superinfection in which P. falciparum was inoculated 10 days after P. vivax was established, resulting in a reduced peak parasitaemia of P. falciparum (7 400 parasites/µL) compared to the peak parasitaemia in the coinfection (10 100 parasites/µL)

(Mason and McKenzie, 1999).

Figure 5. Exploitation competition between two species of parasites. The two species are distinguished based on colour (red vs. green) and the host cell resource is represented as larger, grey circles. The “–” signs indicate a negative influence on host cell resources.

In the case of two Hepatozoon species, indicated by the presence of small red or green circles within the larger grey circles, the acquired resources take the form of host cell erythrocytes. A species may choose to undergo further asexual division, in this case erythrocytic merogony, to produce merozoites and infect a greater number of erythrocytes, or undergo gamogony to form transmissible gamonts, indicated by curved lines, which may be taken up by a mosquito vector. In the case of this schema, the green species produces greater quantities of invasive asexual stages, which will acquire more host cell erythrocytes, compared to the red species, which has a higher conversion rate to transmissible stages. In this way, exploitation competition may facilitate the selection for lower conversion rates in Hepatozoon infections (Mideo, 2009).

MATERIALS AND METHODS

Collection and maintenance of green frogs free of Hepatozoon infection

All handling and maintenance of animals followed the guidelines set forth by the

Canadian Council on Animal Care (CACC), and all research on animals was first approved by the Acadia Animal Care Committee. Tadpoles of green frogs, Rana clamitans, were collected using dip nets from various ponds in Kings County, Nova

Scotia from May to July 2013. Tadpoles were transported in screen-covered propylene tubs filled with pond water to Acadia University’s Animal Care Facility. Tadpoles were housed in long, flat, and transparent propylene tubs (58.42 cm x 42.25 cm x 15.24 cm) filled with geothermal water, maintained at 25 C on a light:dark cycle of 16 hr:8 hr, and fed boiled organic romaine lettuce, boiled organic baby spinach, TetraFin® floating variety pellets (Tetra Holdings, Blacksburg, VA), algae discs (Wardley, Secaucus, NJ),

SeraMicron® tadpole growth food (Sera, Heinsberg, Germany), Nutrafin® turtle pellets

(Hagen, Montreal, QC), and Bio-Pure® bloodworms (Hikari , Hayward, CA).

Tadpole containers were emptied of geothermal water and replenished every two days, and remnants of food and waste were removed using mesh aquarium fish nets.

Tadpoles were manipulated using the same fish nets, and done minimally so as to reduce stress. Tadpoles were monitored for the appearance of limbs, indicating the beginning of metamorphosis, until both hind and fore limbs emerged, at which point the tadpoles were transferred to glass terraria. Terraria were elevated on one side using a block of wood and filled with enough geothermal water to submerge half the terrarium floor. A plastic ramp was placed between the submerged region and the dry region to facilitate bidirectional

movement. In this way, tadpoles had a constant supply of moisture to prevent drying and dehydration, yet a dry surface to prevent drowning due to the gradual loss of gills.

Metamorphosed green frogs were identified by the retraction of their tails, at which point they were transferred to glass terrariums lined with ExoTerra® tropical terrarium substrate (Hagen, Montreal, QC). Plastic tubs filled with geothermal water were placed on the substrate to provide a source of moisture and hydration, and a plastic bridge was placed over the water and substrate to provide an exit route to prevent drowning.

Halved ice cream containers and artificial greenery were provided for shelter. Water tubs were emptied and replenished every three days, and water levels were monitored and topped up when needed. Every four to six weeks, or depending on accumulated waste build-up and condition of the substrate, terrariums were cleaned and set up anew.

Juvenile frogs were fed crickets (Acheta domestica) and mealworms (Tenerbrio molitor) coated in crushed children’s multivitamins (Loblaws, Toronto, ON) three times a week.

Temperature and light:dark cycles were maintained at 22 C and 16:8, respectively.

Collection and maintenance of green frogs with pre-existing Hepatozoon infections

We sought adult green frogs with pure infections of H. clamatae at 1% parasitaemia (i.e., percentage of erythrocytes infected with parasite gamonts) or pure infections of H. catesbianae at 1% parasitaemia to serve as consistent sources of parasites to subsequently infect clean juveniles frogs via the mosquito vector, C. territans. Adult green frogs were collected from various ponds in Kings County using fish nets. Frogs were transported in screen-covered propylene tubs filled with a small volume of pond water to Acadia University’s Animal Care Facility. Males and females were separated upon capture to prevent any violence resulting from mating competition. Male frogs were

identified based on their large tymphanum-to-eye ratio, bright yellow throat colouration, and thick thumbs used for amplexus. Females were identified based on the absence of these traits. Frogs were left undisturbed for 24 hr to reduce stress following capture.

Frogs were bled from the maxillary vein with a sterile, 27-gauge needle according to the method illustrated in Forzán et al. (2012). A heparinized micro-hematocrit capillary tube was used to collect blood from the surface of the skin between the upper jaw and the anterior side of the tympanum. The collected blood was ejected onto a glass slide and smeared into a monolayer using another clean, glass slide. The resulting puncture wound on the targeted frog was sprayed with Bactine® antiseptic spray, and the frog returned to its original propylene tub. The blood smears were fixed and stained using

Hema 3® (Fisher Scientific, Ottawa, ON) and analyzed using bright field microscopy to determine the presence of Hepatozoon infection. Hepatozoon clamatae and H. catesbianae were identified based on their distinct cytopathological effects, as described by Kim et al. (1998). Hepatozoon clamatae was identified by its ability to fragment the host erythrocyte nucleus, whereas H. catesbianae leaves the host erythrocyte nucleus oval and intact. Parasitaemia was determined by counting the number of parasites per

10,000 erythrocytes.

Wild-caught frogs with appropriate levels of Hepatozoon infection were transferred to flow-through aquaria at Acadia University’s Animal Care Facility, while the remaining frogs were released by 48 hr after capture. Adult frogs were maintained on a diet of crickets and mealworms, at a temperature of 22 C, and on a light:dark cycle of

14:10. Wild-caught frogs were distinguished according to sex, pattern of spots and leg bands, shape of dorsolateral ridges, and any other distinguishing features.

Collection of mosquito larvae and pupae

Larvae and pupae of the mosquito, Culex territans, were collected from ponds by

Deep Hollow Road and the Wolfville dykes in Kings County, Nova Scotia from June to

September 2014. Larvae and pupae were collected using plastic pipettes from pond water collected in dipping water samplers and transferred into small plastic bags (Whirl-Pak

Nasco®, USA) three-quarters filled with pond water to allow larvae and pupae to reach the air pocket, and transported to the insectary in the Weston Animal Care Facility at

Acadia University. Larvae were transferred using plastic pipettes to uncovered polypropylene tubs filled with 8 cm of geothermal water. Pupae were transferred using plastic pipettes to 50 mL beakers filled with geothermal water, then placed inside a

30 cm3 Plexiglas® cage with mesh windows and a cylindrical opening covered with nylon stocking. A 50 mL Erlenmeyer flask containing geothermal water, and a 50 mL

Erlenmeyer flask containing 10% w/v sucrose solution was placed inside the Plexiglas® cage, and a folded lint-free tissue was inserted into each flask to act as a landing site.

Larvae were maintained on a diet of Nutrafin® baby fish food and yeast, a temperature of

23 C, and a light:dark cycle of 14:10. The propylene tubs containing larvae were monitored every day for newly metamorphosed pupae, which were subsequently transferred to beakers in the Plexiglas® stock cage.

Inoculation of mosquitoes and frogs

Ideally, inoculations would be determined by feeding an exact number of mosquitoes, previously fed on source green frogs with pure H. clamatae infections of 1% parasitaemia or pure H. catesbianae infections of 1% parasitaemia, to lab-raised green frogs. For example, to inoculate with a high intensity of H. clamatae, we would feed four

mosquitoes, that had fed on a source frog with 1% parasitaemia of H. clamatae, to a lab- raised frog. To inoculate with a moderate intensity of both species, we would feed two mosquitoes that had fed on a source frog with 1% parasitaemia of H. clamatae and two mosquitoes that had fed on a source frog with 1% parasitaemia of H. catesbianae. Table

1 describes the ideal scenario for infecting frogs of each of the five cohorts. Therefore, obtaining source green frogs with pure H. clamatae infections of 1% parasitaemia or pure

H. catesbianae infections of 1% parasitaemia, was essential. However, as most infected frogs were naturally infected with both species, finding frogs with pure infections of either species was difficult, and finding frogs with pure infections at 1% parasitaemia was even more challenging. At the end of the summer, three wild-caught frogs with pure H. clamatae infections of 1% parasitaemia and one wild-caught frog with exactly 1% parasitaemia of each of the two parasite species were collected. The frog with the mixed infection was substituted for a pure 1 % H. catesbianae-infected frog, which rendered only cohorts 1, 4 and 5 attainable, with altered inoculation ratios as detailed in Table 2.

To obtain mosquitoes with pure H. clamatae infections and mixed H. clamatae and H. catesbianae infections as described above, two 30 cm3 Plexiglas® feeding cages were set up for each source frog. A 50 mL Erlenmeyer flask containing geothermal water and a 50 mL Erlenmeyer flask containing 10% w/v sucrose solution was placed inside each Plexiglas® cage, and a folded lint-free tissue was inserted into each as a landing site. Approximately 25 female and 5 male mosquitoes were transferred from the stock cage to each feeding cage and allowed 24 hr to adjust to the new environment.

Mosquitoes were deprived of sucrose solution for 12 hr prior to feeding, and deprived of water for 3 hr prior to feeding. A wild-caught green frog with the desired infection level

was placed into its respective feeding cage (e.g., frogs with 1% pure H. clamatae infections were placed inside the feeding cage intended to infect mosquitoes with only H. clamatae) and returned to the Animal Care Facility after 2 hr so that frogs did not become dehydrated. Female mosquitoes that had taken a blood meal were identified by their dark red, distended abdomens, and were collected and placed in plastic holding cages.

Mosquitoes were segregated based on their acquired infection and allowed to incubate for

28 days to allow complete parasite development.

After this incubation period, mosquitoes were fed to laboratory-reared green frogs according to the inoculation ratios outlined in Table 2. Feeding was achieved by gently opening the frog’s mouth using small pieces of cardboard and then inserting, using tweezers, infected mosquitoes into the frog’s mouth. Saline solution was gently introduced into the frog’s mouth to induce swallowing, and the frog was monitored in a sealed Ziplock® sandwich container, with breathing holes punctured into the lid, to ensure that mosquitoes were not expelled. Inoculated frogs were bled approximately 10,

20, 30, 40, 50, 60, 80, 100 and 120 days post inoculation (dPI), using the blood-drawing procedure detailed above. Nine frogs were successfully inoculated with a pure 4:0

H. clamatae: H. catesbianae infection (cohort 1), two with a 2:2 infection (cohort 4), and five with a 3:1 infection (cohort 5).

Determining parasitaemia in frogs

Blood smears were analyzed using bright field microscopy, and Hepatozoon species were identified based on their distinct cytopathological effects as detailed by Kim et al. (1998). Parasitaemia was calculated from the number of Hepatozoon species per

10,000 host erythrocytes. Due to the unexpected occurrence of erythrocytic merogony

and ensuing challenges in identifying species, species-specific Hepatozoon counts began at 80 dPI in cohorts 4 and 5 and included a third “unknown” group consisting of merozoites and immature gamonts. Merozoites were identified by their small, stout, and rounded appearance (Fig. 6).

Statistical analysis

Due to the presence of repeated measurements (i.e., bleeding the same frogs over time) and incomplete data set (i.e., resulting from bleedings of cohort 1 that were missed) a linear mixed-effects model was employed. This model accounted for the existence of random effects, which took the form of green frogs in this study. Due to the nominal nature of the data expressed as percent parasitaemia or percent intensity, the data set was arcsine transformed to meet the assumption of normality in the linear mixed-effects model. The linear mixed-effects model was run with mean total parasitaemia over time, mean merozoite intensity over time, and the ratio of H. catesbianae to H. clamatae in cohorts 4 and 5 over time. To achieve the most statistically accurate results, statistical tests were run with data starting at 30 dPI. To compare parasitaemia and merozoite intensity among cohorts at a given time point, the bleeding days of cohort 1 were shifted to match the bleeding days of cohorts 4 and 5.

Table 1. The five experimental cohorts with ideal inoculation ratios of H. clamatae to H. catesbianae. Ideal inoculation ratios of each species of parasite would be achieved by feeding mosquitoes fed on source frogs with pure H. clamatae infections at 1% parasitaemia and mosquitoes fed on source frogs with pure H. catesbianae infections at

1% parasitaemia, to lab-raised frogs.

Cohort Number of mosquitoes infected Number of mosquitoes infected

with H. clamatae with H. catesbianae

(1% parasitaemia) (1% parasitaemia)

1 4 0

2 0 4

3 1 3

4 2 2

5 3 1

Table 2. The three experimental cohorts with actual inoculation ratios of H. clamatae to H. catesbianae. Actual inoculation ratios of each species were achieved by feeding mosquitoes fed on source frogs with pure H. clamatae infections at 1% parasitaemia and mosquitoes fed on source frogs with mixed H. clamatae to

H.catesbianae infections at 1% for each parasite species.

Cohort Number of mosquitoes infected Number of mosquitoes infected with

with H. clamatae H. clamatae and H. catesbianae

(1% parasitaemia) (1% parasitaemia for each species)

1 4 0

4 0 2

5 2 1

A B C D

Figure 6. Cytopathological features of various stages in the life cycle of Hepatozoon species. A. Seven erythrocytic merozoites of Hepatozoon species in a host erythrocyte, species unknown. B. An immature Hepatozoon gamont, species unknown. C. Mature gamont of Hepatozoon clamatae and the resulting host cell nucleus fragmentation, a hallmark of this Hepatozoon species. D. Mature gamont of Hepatozoon catesbianae and the laterally displaced, oval, darkly-stained, and intact host cell nucleus.

RESULTS

Hepatozoon species first invaded host cell erythrocytes at 30 dPI in all cohorts.

Initial parasitaemia greatly varied within each cohort, ranging from 0.47% to 7.83% in cohort 1, 0.4% to 8.13% in cohort 4, and 0.12% to7.91% in cohort 5. Mean total parasitaemia of gamonts (i.e., the percentage of erythrocytes infected with gamonts of either species) decreased drastically in all cohorts during the subsequent 20 days, at which point mean parasitaemia of cohort 5 increased until levelling off at 80 dPI. Mean total parasitaemia of cohort 4 appeared to level off at 80 dPI, whereas mean total parasitaemia of cohort 1 continued to decline throughout the 98 days (Fig. 7). Despite these trends, the linear mixed-effects model did not detect any statistically significant differences between the development of Hepatozoon infections in cohort 4 and cohorts 1 and 5, although there was a trend toward a significant difference between the parasitaemia of cohorts 1 and 5 over time (p=0.0837). The statistical analysis detected a significant difference within cohorts over time (p=0.0000), however, indicating that day has a significant effect on parasitaemia (Table 3).

Mean total merozoite intensity within cohorts revealed relatively steady, decreasing trends over time (Fig. 8). Mean intensity remained highest in cohort 4 over

121 days, and was lowest in cohort 1 over 98 days (Fig. 8). A statistically significant difference was not detected in mean merozoite intensity among cohorts over time, but the effect of day on merozoite intensity within all cohorts was determined to be statistically significant (p=0.0207) (Table 4).

By 80 days PI in the pure H. clamatae infection (i.e., cohort 1), the distinctive cytopathological effect of gamonts on host erythrocytes started to appear, resulting in the

disturbance or fragmentation of host nuclei in the majority of parasitized cells. Thus,

H. clamatae did not fragment the host cell nucleus until it reached maturity. In both mixed infections (cohorts 4 and 5), the persistence of erythrocytic merogony and the subsequent abundance of immature gamonts rendered species distinction difficult by light microscopy. The identification of H. clamatae and H. catesbianae in mixed infections

(i.e., cohorts 4 and 5) began at 80 dPI until the final bleeding at 121 dPI, and resulted in a third group consisting of merozoites and immature gamonts that could not be identified to the species level. Over the 41 days, as more H. clamatae and H. catesbianae gamonts matured and revealed their cytopathological effects in infected frogs of both mixed cohorts, the parasitaemia of identifiable Hepatozoon species increased as the parasitaemia of unknown Hepatozoon decreased (Figs. 9, 10). In both mixed cohorts, mean parasitaemia of H. clamatae remained higher than H. catesbianae (Fig. 11); however, the linear mixed-effects model did not detect any significant difference between parasitaemia of H. catesbianae and H. clamatae within or between cohorts 4 and 5 over time

(Table 5).

Green frogs in all cohorts increased in mass over time (Fig. 12). However, due to a lack of data on growth of frogs before infection, the absence of a control group for comparison, and because frogs were randomly selected for each cohort without regard to mass, statistical analysis was not performed on these data.

Table 3. Statistical output for the linear mixed-effects model investigating differences in mean total parasitaemia of Hepatozoon species within and among cohorts 1, 4 and 5 over time. Percent parasitaemia values for each frog at each time point were arcsine transformed to meet the assumption of normality. Statistical analysis revealed that day of bleeding had a significant effect on parasitaemia across all cohorts

(p=0.0000). In addition, the analysis detected that there is at trend toward a significant difference between the mean parasitaemia of cohort 1 and 5 over time (p=0.0837).

DF t-value p-value

Cohort 4 13 -0.821708 0.4261

Cohort 5 13 -1.873351 0.0837

Day 111 -4.636522 0.0000

Cohort 4: Day 111 0.725451 0.4697

Cohort 5: Day 111 1.425298 0.1569

Table 4. Statistical output for the linear mixed-effects model investigating differences in mean merozoite intensity of Hepatozoon species within and among cohorts 1, 4 and 5 over time. Percent merozoite intensity values for each frog at each time point were arcsine transformed to meet the assumption of normality. Statistical analysis revealed that day of bleeding had a significant effect on intensity across all cohorts (p=0.0207).

DF t-value p-value

Cohort 4 12 1.429378 0.1784

Cohort 5 12 0.344861 0.7362

Day 33 -2.429676 0.0207

Cohort 4: day 33 -1.131086 0.2662

Cohort 5: day 33 -0.135037 0.8934

Table 5. Statistical output for the linear mixed-effects model investigating differences in mean parasitaemia of H. clamatae and H. catesbianae within and among cohorts 4 and 5 over time. Percent parasitaemia values for each frog at each time point were arcsine transformed to meet the assumption of normality. Significant differences in parasitaemia were not detected between H. clamatae and H. catesbianae within or between cohorts of frogs over time.

DF t-value p-value

Cohort 5 5 0.304747 0.7728

Day 12 -1.379027 0.1930

Cohort 5: day 12 0.508245 0.6205

Figure 7. Mean total parasitaemia of Hepatozoon species in three cohorts of infected frogs over time. Cohort 1 (n=9) was followed over 98 days, whereas cohort 4 (n=2) and cohort 5 (n=5) were monitored over 121 days. Parasitaemia is expressed as a percentage, which reflects the number of infected cells per 10,000 erythrocytes. The statistical analysis revealed that day of bleeding had a significant effect on parasitaemia across all cohorts (p=0.0000). In addition, the analysis detected that there is at trend toward a significant difference between the mean parasitaemia of cohort 1 and 5 over time

(p=0.0837). Error bars represent ± 1 standard error of the mean.

Figure 8. Mean total merozoite intensity of Hepatozoon species in three cohorts of infected frogs over time. Cohort 1 (n=9) was followed from 61 dPI to 98 dPI, whereas cohort 4 (n=2) and cohort 5 (n=5) were monitored from 52 dPI to 121 dPI. Intensity is expressed as a percentage, which reflects the total number of merozoites per 10,000 erythrocytes. The statistical analysis revealed that day of bleeding had a significant effect on intensity across all cohorts (p=0.0207). Error bars represent ± 1 standard error of the mean.

Figure 9. Mean parasitaemia of mixed 2:2 (H. clamatae to H. catesbianae) infections in cohort 4 (n=2) over 121 days. Due to the unexpected occurrence of erythrocytic merogony and ensuing challenges differentiating species, species-specific Hepatozoon counts began at 80 dPI, and consisted of a third “unknown” group consisting of merozoites and immature gamonts. Error bars represent ± 1 standard error of the mean.

Figure 10. Mean parasitaemia of mixed 3:1 (H. clamatae to H. catesbianae) infections in cohort 5 (n=5) over 121 days. Due to the unexpected occurrence of erythrocytic merogony and ensuing challenges differentiating species, species-specific

Hepatozoon counts began at 80 dPI, and consisted of a third “unknown” group consisting of merozoites and immature gamonts. Error bars represent ± 1 standard error of the mean.

Figure 11. Proportion of gamonts of H. catesbianae to H. clamatae in cohorts 4 and 5 over time. Proportions were calculated from mean parasitaemia values recorded from 80 dPI to 121 dPI. Statistically significant differences were not detected within or between cohorts over time. Error bars represent ± 1 standard error of the mean.

Figure 12. Mean mass of green frogs in three cohorts over time. Cohort 1 (n=9) was measured over 98 days and cohorts 4 (n=2) and 5 (n=5) over 121 days. Error bars represent ± 1 standard error of the mean.

DISCUSSION

Infection dynamics of Hepatozoon species

Across all cohorts, merozoites were first detected in the blood at 30 dPI at relatively high mean parasitaemia values (Fig. 7). The initial mean parasitaemia in every cohort, however, was calculated from highly variable initial parasitaemias of independent infections of individual frogs within each cohort. Hepatozoon species were identified based on the cytopathological effects of mature gamonts, but species distinction could not be determined at the merozoite stage (Kim et al., 1998). Although significant differences were not detected among cohorts over time (Table 3) the trend toward a significant difference between cohort 1 (n=9) and cohort 5 (n=5) (p=0.0837) suggests that, with complete cohorts of 10 green frogs, a significant difference among cohorts may emerge.

Such a result would indicate that infection dynamics of pure infections differ from infection dynamics of 3:1 mixed infections. A trend toward significance was not detected in cohort 4 (p=0.4261), perhaps due to small sample size (n=2), and again, a complete cohort of 10 green frogs may reveal significant differences between pure infections and

2:2 mixed infections.

The initial mean parasitaemia observed in the pure H. clamatae infection (4.5%) was slightly higher than initial mean parasitaemia observed in the mixed infections (4.3% and 3.3%). This may have resulted from previously documented mild host specificity of

H. clamatae in green frogs (Dickson et al., 2013). In experimental infections, Kim and colleagues (1998) revealed that H. clamatae reached a parasitaemia of 4.6% in green frogs, whereas H. catesbianae only reached a parasitaemia 0.6%. The reverse trend was seen in bullfrogs, which developed a 0.4% parasitaemia of H. clamatae and 2.7%

parasitaemia of H. catesbianae. This host specificity was also observed in prevalence data gathered by Boulianne and colleagues (2007) in a study of the distribution of

Hepatozoon species in green frogs. Findings from the present study suggest that cohorts 4 and 5 failed to develop a higher initial total parasitaemia than cohort 1 due to the mild host specificity of H. clamatae for green frogs, giving H. clamatae an advantage over H. catesbianae. Due to the restriction of one round of hepatic merogony in the life cycle of

Hepatozoon species, as well as the previously described host specificity, the higher peak parasitaemia in pure H. clamatae infections suggests that asexual amplification does not occur in hepatocytes in the face of competition.

Exploitation competition and erythrocytic merogony

The progression of the pure H. clamatae infection in cohort 1 revealed a continuously decreasing trend in parasitaemia, without any indication that an equilibrium had been reached by 100 dPI. Parasitaemia in cohort 5 decreased until 52 dPI, at which point the trend reversed and rapidly increased until 80 dPI before levelling off.

Parasitaemia in cohort 4 decreased until 80 dPI, at which point parasitaemia remained fairly constant (Fig. 7). These observations, combined with what initially appeared to be prolonged erythrocytic merogony in mixed infections, led to speculation regarding the occurrence of erythrocytic merogony in the face of competition. The resulting amplification of parasite density would lead to the competitive suppression of one species over the other as the proliferating species acquired free erythrocytes. This form of competition, termed exploitation competition, arises from competitors with overlapping ecological niches with a resulting fight for host resources (Mideo, 2009). However, upon closer examination involving actual counts of merozoites, the progression of mean

merozoite intensity among all three cohorts over time did not reveal any statistically significant differences. There was a statistically significant difference detected within cohorts over time, indicating that merozoite intensity was decreasing across all cohorts.

The species of Hepatozoon that underwent erythrocytic merogony in the mixed infections remains unclear, although the presence of erythrocytic merogony in the pure H. clamatae infection may indicate that H. clamatae was similarly responsible for merogony in the mixed infections. It is possible that both species initiated erythrocytic merogony, for in a study by Smith and colleagues (2000), erythrocytic development in green frogs was identical for both H. clamatae and H. catesbianae. Hepatozoon species are known to undergo one round of asexual division in the liver, yet multiple rounds of asexual division were observed in the blood. Damage caused by immune responses against exoerythrocytic meronts in liver cells is greater than that caused by the destruction of red blood cells housing erythrocytic meronts, which may account for the amplification of parasite numbers in blood stages as opposed to liver stages (Smith et al., 2000).

Relative parasitaemia of Hepatozoon species and apparent competition

In both cohorts of mixed infections, mean parasitaemia of H. clamatae remained higher than H. catesbianae, even as the unknown group matured into identifiable gamonts (Figs. 9, 10). Despite this trend, statistical differences were not found between or within the cohorts of mixed infections over time. Statistical analysis suggests that

Hepatozoon species do not compete within their intermediate host; however, these negative results may have arisen from small sample sizes and large variation. The rapid decline in parasitaemia within all cohorts in the first 10 days after exposure in the blood

(Fig. 7) and ensuing decline in parasite numbers (p=0.0000) suggests that an acquired

immune response was responsible for controlling parasite intensity. Given that apparent competition is the indirect interactions of parasites as a result of host immune activity, the absence of any significant difference between infection dynamics of pure and mixed infection over time, or of the development of significant differences in parasitaemia of H. clamatae and H. catesbianae over time, fails to support the hypothesis of apparent competition among Hepatozoon species.

Mass of green frogs

The gradual increase in mean mass of green frogs within all cohorts over time

(Fig. 12), when compared to the gradual decrease in mean parasitaemia within all cohorts

(Fig. 7), suggests that there may be a negative relationship between Hepatozoon infections and frog mass over time, and that Hepatozoon species do not have a deleterious effect on the health of frogs, at least as measured by weight gain.

Variation in parasitaemia within infected frogs

The wide variability in initial parasitaemia may be attributed to the polymorphism of receptor molecules encoded by the multigene major histocompatibility complex, or

MHC (Bernatchez and Landry, 2003). MHC molecules act at the interface between pathogen recognition and the adaptive immune response, and have been conserved as orthologs across all vertebrate species (Richmond et al., 2009). Self peptides, which are typically displayed on MHC class I molecules on the surface of cells, are replaced with foreign peptides when a cell has been invaded. This presentation of non-self peptides by a compromised cell activates circulating CD8+ (cytotoxic) T cells, which clear the infection

(Richmond et al., 2009). In contrast, MHC class II molecules present foreign peptides to

CD4+ (helper) T cells, which then trigger the acquired immune response (Richmond et al., 2009).

Although the general architecture of MHC assembly is relatively conserved across vertebrates, there may be variation in the number of class I and II loci found on the MHC

(Bernatchez and Landry, 2003). MHC class I and II molecules in Xenopus laevis, a model organism for frogs and other amphibians, are not linked on the same gene complex as in birds and humans, but rather contain a large family of non-MHC linked class I genes

(DuPasquier et al., 1989; Cannatella and De Sa, 1993; Flajnik et al., 1993; Carey et al.,

1999). Whether coding for intracellularly derived (MHC class I) or extracellularly derived (MHC class II) peptides, the peptide-binding domain on MHC molecules is the most varied gene-coding region on the MHC, and the entire MHC is the most polymorphic cluster of genes in all vertebrates (Garrigan and Hedrick, 2003; Radwan et al., 2009). The highly polymorphic structure of MHC is of adaptive significance in vertebrates, primarily in the defense against pathogens (Radwan et al., 2009; Savage and

Zamudio, 2011). MHC polymorphism may even be explained by parasite-mediated balancing selection, which is said to maintain several different alleles at gene loci

(Bernatchez and Landry, 2003). The first method by which balancing selection maintains

MHC polymorphism is explained by the theory of overdominance, which predicts that heterozygotes have a fitness advantage over homozygotes. The greater variation in heterozygous organisms results in an extensive repertoire of MHC molecules that confer resistance to a wide range of pathogens (Richmond et al., 2009). The second method, called frequency-dependent selection, predicts that rare host genotypes will have a fitness

advantage over the more common (and more easily exploited) host genotypes, thus promoting MHC variation (Richmond et al., 2009).

Another basis of specificity that was not taken into account in this study was the role of the immune system in the mosquito vector, Culex territans. Mosquitoes possess innate humoral and cellular immune pathways to counter the invasion of pathogens during feeding, resulting in lysis, melanization and a haemocyte-mediated phagocytosis, initiated minutes after exposure (Hillyer, 2010). Plasmodium species are known to face attacks from the mosquito immune system, primarily during ookinete invasion of the midgut epithelium (Cirimotich et al., 2009). Immune defenses against the rodent malaria parasite Plasmodium berghei account for the variation in parasite establishment in the midgut of related, untreated (i.e., immunologically unaltered) mosquitoes, with oocyst numbers ranging from 0 to more than 1000 (Marois, 2011). The role of mosquito immunity in the development of parasitaemia was also demonstrated by manipulating an unsuitable vector, Anopheles quadriannulatus to acquire the ability to support parasitism by P. berghei by knocking down immune factors in the TEP1 pathway that is involved in insect immunity (Habtewold et al., 2008). The variation in initial parasitaemia observed across all cohorts may have paralleled the variation of initial parasitaemia in C. territans.

This variation was similarly observed in a population of Anopheles gambiae mosquitoes, which developed variable infection levels of parasites after taking relatively equal blood meals (Marois, 2011).

The green frogs used in this study were maintained on a uniform diet in environmentally homogenous living conditions, thus environmental factors should not have contributed to the variation in infection dynamics within cohorts. In natural

populations, however, green frogs are distributed over a much larger range of habitats, and thus stress levels, nutritional status, and host condition may play a part in the variation of transmission success and pathogenicity over small spatial scales (Lachish et al., 2011). To understand infection dynamics in wild populations and the ensuing coevolution of hosts and resident parasite populations, the role of biotic and abiotic factors must be taken into account. In an ecological context, the environment occupied by host and parasite may play a role in the strength and specificity of the infection, and therefore accurate infections models would incorporate such spatial variability (Wolinska and

King, 2009).

Exploitation competition by coinfection vs superinfection

Although undetected in this study, future replications with a greater sample of green frogs per cohort may detect the presence of exploitation competition between

Hepatozoon species in the form of increased asexual amplification. In cases of simultaneous inoculation, the infectious agent with higher initial densities of asexual forms or more rapid replication would be more successful in colonizing the host than the infectious agent that is fewer in number or is slower to multiply. In a study by Shute

(1946), a simultaneous inoculation of P. falciparum and P. vivax in humans resulted in the suppression and elimination of P. vivax after one week. Plasmodium vivax was only detectable in the blood following treatment for P. falciparum. Despite its low and declining numbers in the face of competition, the prevalence of H. catesbianae in natural green frog populations may be the result of a seasonal pattern evolved to avoid the challenge of super-infecting a host with a previously established H. clamatae infection.

This parallels the adaptation of P. malariae to peak 60 days before P. falciparum, the

more exploitative species, allowing it to achieve and maintain relatively high parasitaemia levels, although incidence rates of P. malariae were rare (Molineaux and

Gramiccia, 1980; Mason et al., 1999).

Experimental challenges

The first challenge emerged during the attempted collection of wild green frogs with pure infections of H. catesbianae with a 1% parasitaemia. The failure to capture and maintain green frogs with pure H. catesbianae infections resulted in the substitution of a frog with a mixed infeciton of 1% parasitaemia of each species, and ensuing possible cohorts with altered inoculation ratios as detailed in Tables 1 and 2.

Another challenge was the collection of sufficient quantities of mosquitoes to support the initial objective of feeding four infected mosquitoes to each of 50 laboratory- raised green frogs. Collected Culex territans larvae suffered a drastic decline in numbers once transferred into laboratory conditions, which may be partly attributed to the presence of damselfly nymphs (Enallagma civile) that were unintentionally transferred alongside Culex larvae into the holding tubs in the insectary. Damselfly nymphs feed on aquatic invertebrates and are difficult to see due to their transparent exoskeleton and quick movements. In previous studies, damselfly nymphs fed on Culex tarsalis in larger amounts as prey density increased, explaining what may have caused the decimation of

C. territans larvae in the holding tubs (Miura and Takahashi, 1988). The inoculation of mosquito vectors by taking equal blood meals proved to be another experimental challenge, as the quantity of inoculated vectors and subsequent size of blood meal is dependent upon their desire to feed.

The last unanticipated experimental setback was the persistence of erythrocytic merogony in all cohorts, causing H. clamatae to delay fragmenting host cell nuclei as immature gamonts predominated early in infections. This rendered visual distinction of

Hepatozoon species in mixed infections to be difficult, as immature H. clamatae and H. catesbianae appeared similar before their cytopathology took effect. Species identification was thus delayed until 80 dPI, and resulted in a creation of a third unknown group that included merozoites and unidentifiable immature gamonts.

Future studies

In future replications of this study, sufficient quantities of infected mosquito vectors must be obtained to infect 10 green frogs per cohort to produce statistically significant findings. Due to the subjective nature of species identification based on cytopathological features alone, future studies may incorporate species distinction based on polymorphisms in the ITS-1 of H. clamatae and H. catesbianae using molecular techniques, and counts should begin beyond 60 dPI. In addition to monitoring infection dynamics and competition between Hepatozoon species in green frogs, this study may be replicated in bullfrogs to determine if there is a parallel or reverse trend in a different anuran host. The effect of superinfection, as opposed to coinfection of Hepatozoon species may also be investigated in green frogs and bullfrogs, as superinfections are known to occur in nature and may alter infection dynamics.

Lastly, future studies may target the role of the mosquito immune system and the potential occurrence of competition within the gut of the mosquito. Due to the complex developmental transitions that Hepatozoon species must achieve during their indirect life

cycle, the role of the immune pathways in both the intermediate and definitive hosts must be considered to investigate the infection dynamics of these parasites.

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