Host-Seeking Behaviour of Culex Territans Mosquitoes

HOST-SEEKING BEHAVIOUR OF CULEX TERRITANS MOSQUITOES

PARASITIZED WITH HEPATOZOON CLAMATAE

Laura V. Ferguson

Thesis

submitted in fulfillment of the

requirements for the

Degree of Bachelor of Science

with Honours in Biology

Acadia University

April, 2010

This thesis by Laura V. Ferguson

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 Supervisors

______Todd G. Smith Date

______N. Kirk Hillier Date

Approved by the Head of the Department

______Soren Bondrup-Nielsen Date

Approved by the Honours Committee

______Date

iii

I, Laura V. Ferguson, 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 iv

TABLE OF CONTENTS

List of figures...... vi List of tables...... vii List of appendices...... viii Abstract...... ix Introduction...... 1

Culex mosquitoes...... 1 The genus Hepatozoon...... 3 Rana clamitans...... 10 Mosquito-host relationship and general feeding behaviour...... 11 Behavioural modification by parasites...... 13 Objectives...... 16 Materials and methods...... 18

Animal collection and care...... 18 Mosquito infection and dissection procedures...... 19 Data collection...... 22 Statistical analyses...... 23 Results...... 24

General mosquito feeding behaviour...... 24 Mosquito host-seeking behaviour at 15 and 30 days PF...... 25 Mosquito mortality...... 26

Egg retention...... 27 Confirmation of infection...... 27 Discussion...... 31

Mosquito host-seeking behaviour at 15 and 30 days...... 31 Problems posed by egg retention...... 33 v

Areas for further research...... 36 Conclusion...... 39 Appendices...... 41 References...... 43 vi

LIST OF FIGURES

Figure 1. Illustration of a typical Apicomplexan parasite and characterisitc apical complex...... 6

Figure 2. Illustration of the life cycle of Hepatozoon clamatae...... 8 Figure 3. Transmissible stages of Hepatozoon clamatae...... 9

vii

LIST OF TABLES

Table 1. Proportion of mosquitoes fed on infected and uninfected frogs at 0 days...... 28 Table 2. Proportion of infected and control mosquitoes fed on uninfected frogs at 15 days...... 29 Table 3. Proportion of infected and control mosquitoes fed on uninfected

frogs at 30 days...... 30

viii

LIST OF APPENDICES

Appendix 1. Data for mosquitoes, initially fed on infected frogs, that survived until dissection at 30 days...... 41 Appendix 2. Data for uninfected (control) mosquitoes that survived until dissection at 30 days...... 42

ACKNOWLEDGEMENTS

I would first like to extend thanks to my lab mates, Emma McIntyre and

Chris Ogbuah, for their help in the collection of frogs and mosquito larvae; their dedication in the summer heat was greatly appreciated. I would also like to thank them for all of their support and comic relief throughout the duration of this project. I would like to thank Dave Shutler for his timely, much appreciated help in the statistical analyses, as well as Dawn Miner and Tanya Morse-

Outhouse in the Animal Care Facility of Acadia University. I also thank NSERC for the funding provided for this project.

Second, I wish to thank my co-supervisor, Kirk Hillier. The ideas generated from his sharp mind were extremely valuable and his humour and encouragement are greatly appreciated.

Finally, I wish to thank my primary supervisor, Todd Smith, whose support and encouragement helped fuel this project. His enthusiasm was contagious, and instrumental in sparking my interest in the field of parasitology.

For the intellectual input, the laughter, and the opportunity: Thank you. x

ABSTRACT

Animals infected with parasites often display abnormal behaviour that may be a product of the influence of the parasite over the host. Modifying host behaviour may benefit the parasite by increasing transmission success. Trophically transmitted parasites may benefit from reducing host activity during parasite development so as to avoid being consumed prematurely. Conversely, it may be beneficial for the parasite to induce an increase in host activity to attract predators once the parasite is transmissible. Hepatozoon clamatae is a trophically transmitted, apicomplexan blood parasite that passes between the definitive host, the mosquito, Culex territans, and the intermediate host, the green frog,

Rana clamitans. We tested whether, during the 30 day development period, H. clamatae inhibits mosquito host-seeking behaviour, presumably so that mosquitoes avoid being eaten before parasites are mature. We also tested whether, once the parasite has matured, H. clamatae modifies the host-seeking behaviour of the mosquito, presumably so that infected mosquitoes become more conspicuous to the frog. Observations of the behaviour of infected mosquitoes in the presence of uninfected green frogs during parasite development and at parasite maturity suggest parasite manipulation of behaviour is not occurring. However, inhibition of host-seeking behaviour due to egg retention in both infected and uninfected mosquitoes may not allow a true interpretation of this host-parasite relationship. 1

INTRODUCTION

Culex mosquitoes

Mosquitoes are true flies with characteristic blood-sucking females, towards which great animosity is directed for their role in irritation and disease transmission in vertebrates. Mosquitoes of the genus Culex (Diptera: Culicidae) pass through the typical mosquito life cycle consisting of four aquatic larval instars, an aquatic pupal stage and a final moult into an aerial adult. Both males and females feed primarily on the nectar of flowers to obtain sugar, a necessary energy substrate for flight (Rivero and Ferguson, 2003); however females must also imbibe blood. Females in tropical regions will breed continuously (White,

2008) whereas females in temperate regions are inseminated in the fall and will pass the winter in hibernation until spring (Crans, 2004). When females emerge they require a blood meal to produce vitellogen, a protein necessary for egg development (Takken, 1999).

After using several cues, including olfactory, auditory and visual signals, to track an appropriate host, the process of obtaining a blood meal begins by insertion of the fascicle into the vertebrate host. Blood from a pierced blood vessel or damaged capillary is collected and travels to the midgut. Segmental stretch receptors in the abdomen send signals to abdominal ganglia, which relay information through the ventral nerve cord to the brain (Gwadz, 1969) as the gut fills, and feeding is terminated at a threshold level (Friend and Smith, 1977).

Depending on the temperature, in approximately two to six days (Bentley and 2

Day, 1989), the female deposits her eggs in rafts (Crans, 2004) and a new gonotrophic cycle of host searching, blood meal acquisition, ovary maturation and egg laying commences (Klowden and Briegel, 1994).

The mosquito alimentary canal consists of the mouth, oesophagus, crop, midgut, Malpighian tubules, hindgut, rectum and anus. Carbohydrate meals contain sugars that trigger receptors to release a signal to open the crop sphincter where the sugar meal will be stored, whereas a blood meal travels directly to the midgut (Billingsley and Lehane, 1996). The Malpighian tubules lie in proximity to the fat body and consist of a single cell layer surrounding a lumen that forms a junction to the hindgut and the rectum (Coast, 2000). Water, ions and organic solutes cross an epithelial barrier by active transport from the haemocoel to the lumen of the Malpighian tubules; most of the water and ions are reabsorbed and wastes are excreted through the anus (Klaus and Piermarini, 2008). Fluid consisting of plasma from a blood meal is excreted as a female mosquito feeds.

Initial diuresis of NaCl and water follows this primary urine to rid the mosquito of an excess of these components from the blood meal. Excess nitrogen and haemoglobin are excreted as uric acid and hematin (Klaus and Piermarini, 2008).

The biology of the mosquito gut is important to explore since the Malpighian tubules serve as the site for the development of the protozoan parasite

Hepatozoon clamatae.

Culex territans is a diurnal, zoophilic mosquito that prefers the blood of amphibians (Bartlett-Healy et al., 2008). Adult integument is brown in colour, as 3 with all Culex species, but C. territans is distinguishable by a band of white scales on the apical end of the dorsal side of each abdominal segment (Wood et al.,

1979). Larvae are distinguished by a specific arrangement of hairs on the head and siphon. Single hairs, with occasional pairs, are found on the head, and four or five tufts of hairs following a row of pecten teeth are found on the siphon

(Wood et al., 1979). Larvae are found in unpolluted, freshwater ponds and bogs that contains vegetation (Crans, 2004), a type of habitat also preferred by the green frog, Rana clamitans (Bartlett-Healy et al., 2008). Culex territans, unusual among other species of mosquitoes, can complete a gonotrophic cycle at the low temperature of 3˚C when R. clamitans is exiting diapause in the spring (Bartlett-

Healy et al., 2008), which suggests that there is a preferential relationship established between C. territans and R. clamitans. It is not unlikely, then, that this sympatric relationship has been exploited by the parasite Hepatozoon clamatae, which passes trophically between the two hosts.

The Genus Hepatozoon

The genus Hepatozoon (Apicomplexa: Adeleina) comprises a group of blood-dwelling, protozoan parasites that are heteroxenous, residing in the blood cells of intermediate tetrapod vertebrate hosts and various definitive hematophagous arthropod hosts (Boulianne et al., 1997). Unlike other blood- borne parasites, such as human malaria parasites, Plasmodium spp., transmission of these haemogregarines from the arthropod host to the vertebrate host does not 4 occur during the blood meal. Rather, the parasite is passed by ingestion of the arthropod vector by the vertebrate host, in a reciprocal trophic fashion. When ingested by the vertebrate host, sporozoites, or infective stages of the parasite, may infect various internal organs before reaching the bloodstream to complete further rounds of merogony, or multiple fission. Ingestion of infected blood cells by a hematophagous arthropod allows passage of intraerythrocytic gamonts into the arthropod host. Gametogony, or the formation of male and female gametes, fertilisation and sporogony, or the asexual divison in the zygote to form haploid sporozoites in a thick-walled oocyst occurs either in the arthropod haemocoel or cells of the gut wall (Smith, 1996). In species such as Hepatozoon sipedon, dizoic cysts are formed in the liver of a frog to await ingestion by a snake where further merogony, followed by gamogony, will occur. In other species, such as H. catesbianae and H. clamatae, only one vertebrate host is required; merogony will occur once in the liver before merozoites migrate to the bloodstream to infect red blood cells. In all species of Hepatozoon, large multipsorocystic oocysts are observed in the arthropod vector; the number of sporozoites in each sporocyst may be different from species to species (Desser, 1995).

Sporozoites of Hepatozoon species share the unique feature of the apicomplexans: the apical complex (Desser 1995) (Fig. 1). The apical complex is machinery designed for host cell invasion which makes members of the apicomplexan phylum well suited for an intracellular parasitic lifestyle. The apical complex is based around the conoid, a fibrous structure that becomes 5 motile during invasion. The polar ring serves as a microtubule organizing centre from which structural subpellicular microtubules arise (Blackman and Bannister,

2001). The rhoptries and micronemes are secretory organelles that discharge components necessary for adherence to the host cell, invasion, and formation of a parasitophorous vesicle in which the parasite resides in the host cell (Hu et al.,

2006).

Hepatozoon species of interest in this study are the anuran parasites H. clamatae and H. catesbianae. Although there is low host specificity within these species (Smith, 1996), each exhibits an apparent preference for Rana clamitans, the green frog, and Rana catesbeiana, the bullfrog, respectively (Boulianne et al., 2007).

Hepatozoon clamatae and H. catesbianae are recognizable from other species of

Hepatozoon in that these species have sporocysts containing four sporozoites.

Although nuclear distortion is observed in infections by both species, H. clamatae fragments the erythrocytic nucleus whereas H. catesbianae leaves the nucleus intact (Kim et al., 1998). Further distinction of the two species lies in differences in ITS-1 sequence (Boulianne et al., 2007). Both species cause severe swelling of

Malpighian tubules of mosquitoes in a heavy infection (Kim et al., 1998).

Although H. catesbianae was present in double infections of wild caught green frogs, H. clamatae is the parasite of primary interest in this study.

Figure 1. Illustration of a typical Apicomplexan parasite and characteristic apical complex. This represents the infective stages of an apicomplexan parasite

(sporozoites, merozoites and gamonts). The organelles comprising the ampical complex are concentrated at the apical end of the cell (Hickman et al., 2006). 7

The life cycles of H. clamatae and H. catesbianae (Fig. 2) follow the general format of the Hepatozoon life cycle outlined above. Specifically, sporozoites from an ingested mosquito (Fig. 2g) penetrate hepatic parenchymal cells of the frog and undergo one round of multiple fission to form a meront (Fig. 2h). The meront ruptures, releasing merozoites that travel in the bloodstream and invade erythrocytes (Fig. 2i). Once in an erythrocyte, the merozoite transforms into a gamont (Fig. 2a), the stage easily and often observed under a light microscope as a sausage-shaped entity within the erythrocyte (Fig. 3A) (Boulianne et al., 2007).

The blood meal ingested by a mosquito will include the intraerythrocytic gamonts; the motile gamonts escape from the red blood cells in the mosquito gut and travel by an as yet unknown path to penetrate epithelial cells of the

Malpighian tubules (Fig. 2b). Here a microgamont and a macrogamont will lie together within a parsitophorous vacuole, created around the parasites after cell penetration, and gametogenesis will occur. Two biflagellated microgametes develop from a microgamont and one macrogamete forms from the macrogamont (Fig. 2c). Fertilisation occurs as the macrogamete and one microgamete fuse to form a zygote (Fig. 2d), the only diploid stage of the parasite. The zygote will then immediately undergo meiosis to form an oocyst, which forms sporoblasts that transform into sporocysts in the Malpighian tubules of a mosquito (Fig. 2e, 3B). Within each sporocyst lie four sporozoites

(Fig. 2f) capable of transmitting infection to the next host, the green frog (Fig. 2g). 8

Figure 2. Life cycle of Hepatozoon clamatae. a. Mosquito feeds upon frog infected with intraerythrocytic gamonts. b. Gamonts emerge from erythrocytes in mosquito gut, travel to Malpighian tubules and associate in syzygy in a vacuole. c. Gametogony occurs to form a microgamete and a macrogamete. d. Fertilisation occurs to form a diploid zygote. e. Zygote undergoes multiple fission to form an oocyst, which contains several sporocysts. f. Each sporocyst contains four sporozoites. g. Mosquito containing infective sporozoites is ingested by a frog. h. sporozoites travel to parenchymal cells of the liver and undergo one round of merogony to form merozoites. i. Merozoites travel in bloodstream, penetrate erythrocytes, and becomes gamonts. (Smith, 1996). 9

A B

Figure 3. Transmissible stages of Hepatozoon clamatae. A. extracellular gamont next to a red blood cell of a frog. B. mature sporocysts in mosquito

Malpighian tubule. (Harkness et al. 2010)

Rana clamitans

The green frog, Rana clamitans, is an abundant species found throughout eastern North America. Although primarily green, integument may also be brown, yellowish or olive coloured. Males display a yellow throat, distinguishable from the white throat of the female. Males are also distinct from females by the size of the tympanum; a male tympanum is larger than the eye whereas a female tympanum is approximately the same size as the eye. Lateral folds of skin extend from the eye to the posterior portion of the body on green frogs and allow distinction from the physically similar bullfrog (Dickerson,

1969).

Green Frogs are generally aquatic and are found in many freshwater settings such as ponds, bogs, lakes, swamps and slowing moving streams. They are active both during the day and at night until the temperatures become cold, signalling a change in the season. During the winter they will hibernate, burying themselves under mud and moss until emerging in the early spring. (Dickerson,

1969).

The diet of the green frog is primarily carnivorous, consisting of insects, including mosquitoes, and various other invertebrates; they may also eat small snakes and other small frogs. They are passive hunters, remaining stationary until prey comes close enough to snap up. Predators of the adult green frog include snakes, birds, and small mammals (Dickerson, 1969).

Mosquito-host relationship and general feeding behaviour

A female mosquito must obtain a blood meal from a vertebrate host to gain protein necessary for egg production (Takken, 1999). However, mosquitoes prompt defensive behaviour such as swatting, tail swishing, and even consumption if the intended host is insectivorous (Anderson and Roitberg, 1999).

As such, the mosquito must be aware of the loss-gain ratio in its search for blood

(Anderson and Roitberg, 1999). Fecundity of a female mosquito is positively correlated with an increase in blood meal size (Briegel, 1990) and thus the mosquito will attempt to gain the largest blood meal that she can. If a mosquito is interrupted in her feeding and a full blood meal cannot be attained, she will not lay as many eggs as others in her cohort; this serves as motivation to feed to repletion (Briegel, 1990). The tsetse fly (Glossinidae) feeds infrequently but aims to feed to repletion in one session; this is an attempt to decrease the amount of times the fly comes in contact with a defensive host and possible mortality

(Randolph et al., 1992). Anautogenous mosquitoes, those requiring blood for oogenesis, must employ this strategy of persistence as it feeds on defensive or insectivorous hosts to maximize the amount of blood imbibed and minimize the risk of death (Anderson and Roitberg, 1999).

The defensive behaviour of a host is a factor in host selection for a mosquito. Diurnal mosquitoes are more likely to feed on vertebrates that are less active during the day, including animals that exhibit “freeze” behaviour as a defensive strategy against predators during the day (Day and Edman, 1984). 12

Crepuscular and nocturnal feeders are more likely to feed on animals that are less active and preferably sleeping at night, as defensive behaviour will be minimal (Day and Edman, 1984). Some species of mosquitoes will “host shift” if a host becomes too defensive and will choose to feed on a new species (Day and

Edman, 1984). It has also been observed that female mosquitoes become increasingly antsy over time as they feed; they are most likely to abandon the host if they have only just begun to probe or if they are nearing repletion

(Roitberg, 2008). As mass increases or as a host become disturbed, a mosquito should be more willing to leave the host (Roitberg et al., 2003). Presumably the signal to disengage from the host and fly away originates from stretch receptors in the gut which signal to the mosquito that the gut is full (Friend and Smith,

1977). Unfortunately for the mosquito, flight from a host is impeded by the increase in body mass; anopheline mosquitoes can increase mass by 200% after receiving a full blood meal. This decreases the speed and agility of the mosquito thereby increasing the risk of mortality by defensive behaviour or predation,

(Roitberg et al., 2003). The risk involved in blood feeding is an important consideration for parasites developing within hematophagous hosts as death to the mosquito from defensive behaviour signals death for the parasite as well.

The attraction of a female mosquito to a host is variable. When feeding is terminated by a signal from abdominal stretch receptors, this also serves as an inhibitory cue for the female and she will not host seek so long as the distension remains (Klowden, 1989). If the blood meal is sufficient to trigger oogenesis, a 13 humoral factor released by the fat body will inhibit host seeking until a nervous signal indicates that eggs have passed through the ovipositor. This “oocyte induced inhibition” overlaps the “distention induced inhibition”, barring any host-seeking in a fully fed female (Klowden, 1989). Half or less of a full blood meal may be insufficient to trigger oogenesis (Edman et al., 1975) and host seeking will resume if the distension threshold is not met (Klowden, 1989). The host-seeking behaviour of the mosquito is an important behaviour that may affect a developing or mature parasite as it brings the symbiotic pair in contact with potential harm or potential hosts for the parasite.

Behavioural modification by parasites

There are cases of host behaviour manipulation by parasites to increase the likelihood of their transmission to the next host. A rather dramatic example is that of the Gordian worm (Phylum Nematomorpha) infecting a cricket. Inside the cricket, the worm produces various compounds that alter the development of the cricket‟s nervous system. These changes force suicidal behaviour in the cricket, making it seek out water and drown itself. This allows the parasite to emerge and continue to the next stage of development in the water (Lefèvre et al., 2008). Similarly, the trematode parasite Microphallus sp. which infects the snail Potamopygrus antipodarum, induces changes in feeding behaviour. Normally the snails remain hidden during the morning to avoid predation by birds; however, once infected, the snails move to the tops of rocks during morning 14 hours, becoming conspicuous to birds and provoking their own death (Levri,

1999). This example of a trophically transmitted parasite highlights the main goal in parasite manipulation which is to increase the contact between definitive and intermediate hosts so that the parasite may pass between the two and continue its life cycle.

Trophic transmission of a parasite requires an increase in predation risk by the prey to the predator provided that the parasite has reached a transmissible stage (Lefèvre, 2008). If the parasite is in a non-transmissible stage, then activity of the vector should be suppressed, thereby decreasing the risk of predation and allowing the parasite to develop (Anderson et al., 1999; Parker et al., 2008). For example, in mosquitoes infected with Plasmodium species, increased sugar intake is observed during the developing oocyst stage of the parasite. This may be an attempt by the parasite to reduce blood feeding by the mosquito by filling the gut with sugar and reducing host seeking (Rivero and Ferguson, 2003). Sugar meals suppress host seeking by the mosquito (Roitberg and Friend, 1992; Foster and Eischen, 1987), which would consequently allow the mosquito, and parasite, to avoid death by predation or defensive behaviour and thereby allow the oocyst to release the transmissible sporozoite stage (Rivero and Ferguson, 2003).

Similarly, by an unknown mechanism, host-seeking behaviour of the mosquito,

Aedes sierrensis, is inhibited when infected with the ciliate Lambornella clarki

(Egerter and Anderson 1989). Inhibition of host-seeking behaviour in mosquitoes during parasite infection may be a strategy used by parasites to reduce mortality 15 during development and increase transmission success. Due to the incubation period of 30 d in the mosquito host, H.clamatae could benefit by employing this strategy during development.

In infected hematophagous vectors, the ability to obtain a full blood meal is often impaired which prompts increased probing time and persistent feeding behaviour. Leishmania parasites are flagellated protozoans that block the foregut of the sandfly host, diminishing the amount of blood that the fly can ingest. In turn, the fly will increase probing attempts and thus increase the transmission of the parasites residing in the salivary glands (Koella, 1996). Infections by

Plasmodium species reduce the amount of apyrase, a compound that impeded the formation of blood clots, secreted by the salivary glands in the mosquito (Wekesa et al., 1992; Hurd, 1990). By inhibiting blood clotting, the mosquito may continue to feed unhindered. When this advantage is lost, the mosquito spends more time probing the host to find blood instead of being able to gain a blood meal from a single site (Wekesa et al. 1992). Although increased probing on one host may not increase transmission of the parasite, increased probing over several hosts is still highly advantageous to the parasite. Field experiments have shown that the mosquito Anopheles gambiae infected with sporozoites of P. falciparum fed on more hosts than uninfected mosquitoes, effectively spreading the parasite (Koella,

1998). If a feeding attempt by an infected mosquito is thwarted by a host, the infected mosquito is also more likely to seek out a new host than is an uninfected mosquito (Hurd et al. 1995). 16

In addition to increased probing, infected mosquitoes are also more persistent in their feeding attempts. Koella et al. (1998) showed that Anopheles gambiae infected with Plasmodium falciparum were relatively undisturbed by host defensive movement and were more willing to return to a host after being dislodged than uninfected mosquitoes. In another study by Koella (1996)

Anopheles punctulatus infected with P. falciparum or P. vivax fed maximally throughout the night whereas uninfected mosquitoes fed progressively as time passed, feeding maximally during the time when hosts were sleeping and less defensive. Inefficient blood feeding and persistent feeding behaviour by a haematophagous vector act to increase the contact between the host and the vector, giving the parasite a greater chance of being transmitted (Wekesa et al.

1992). Should H. clamatae be exerting influence over the behaviour of its mosquito host, an increase in feeding time or more persistent feeding behaviour could increase the conspicuousness of the mosquito to the frog and consequently increase the chances of the mosquito being eaten by the frog.

Objectives

This study explored the feeding behaviour of the mosquito Culex territans infected with the parasite Hepatozoon clamatae. The general feeding behaviour of uninfected mosquitoes was also studied to observe the interaction between mosquito and frog without the influence of parasitism. The behaviour of mosquitoes parasitized with Hepatozoon clamatae was observed during parasite 17 development to determine if mosquito host-seeking behaviour was inhibited, thus reducing the chance of mortality for both mosquito and parasite. The behaviour of infected mosquitoes in the presence of an uninfected green frog was also observed at parasite maturity to determine if mosquito host-seeking behaviour was modified from that of an uninfected mosquito. We investigated these observations to determine whether Culex territans mosquitoes infected with

H. clamatae become less inclined to feed during development and, conversely, more conspicuous once the parasite is mature, in order to increase transmission success. 18

MATERIALS AND METHODS

Collection and care of mosquitoes and green frogs

Culex territans larvae were collected from the Hillcrest Brook in Chipman‟s

Corner, Nova Scotia. A 2 L ice cream bucket was used to scoop into the water and larvae were transferred into a Rubbermaid container using a disposable plastic pipette. De-ionized water was added to pond water in the Rubbermaid container upon return to the lab. Temperature in the lab was maintained at 20˚C

± 2˚C and a light: dark cycle of 14: 10. Larvae were supplied a diet of Wardley®

Goldfish Flakes. Upon pupation, pupae were transferred into a 50 mL beaker filled with de-ionized water and placed in a 30 cm x 30 cm x 30 cm Plexiglas cage where adults were also maintained. The cage had a cylindrical opening covered by a stocking cut at both ends to allow passage of materials in and out of the cage. Large mesh windows covered each wall of the cage. De-ionized water and

5% (w/v) sugar solution, prepared from table sugar and de-ionized water, were kept in separate 50 mL Erlenmeyer flasks and a folded Kimwipe was placed in each flask to supply a landing place for adult mosquitoes.

Green frogs, Rana clamitans, were collected from the Hillcrest Brook in

Chipman‟s Corner, Nova Scotia. Frogs were transported to the laboratory in

Rubbermaid containers with mesh screening covering a hole cut into the lid.

Upon return to the lab, blood samples were taken from each frog to determine the presence and intensity of Hepatozoon clamatae infection. A 27 gauge 0.5 inch needle was used to pierce the maxillary vein along the jaw anterior to the 19 tympanum. Blood was collected in a Fisherbrand® heparinized micro-hematocrit capillary tube. Bactine® (Bayer Healthcare, Toronto, ON) antiseptic spray was sprayed on the puncture site of the frog before the frog was returned to a holding cage. Blood was smeared on a glass slide and fixed and stained with Hema 3®

(Fisher Scientific, Ottawa, ON). Prepared blood smears were examined for gamonts of H. clamatae in red blood cells using bright field microscopy.

Uninfected frogs were returned to the Hillcrest Marsh within 48 h of capture.

Frogs with low (0.2% of RBCs infected), moderate (0.5% of RBCs infected) and high infections (5% of RBCs infected) were placed in the Animal Care Facility.

Guidelines of the Animal Care Committee of Acadia University were followed during handling and maintenance of all frogs.

Six captured green frogs, two each of low, moderate and high parastitaemia, as well as uninfected, lab-raised green frogs, were housed in the

Animal Care Facility in semi-aquatic aquarium conditions. Five frogs, all male, were housed in one aquarium; one female frog was housed in the neighbouring aquarium to reduce competition between males. Captive frogs were maintained on a diet of mealworms (Tenebrio molitor).

Mosquito infection and dissection procedures

For feeding experiments, five to ten female mosquitoes were removed, using a plastic aspirator, from the main Plexiglas holding cage, into a separate 30 cm x 30 cm x 30 cm Plexiglas feeding cage. Males were added to the feeding cage 20 in equal numbers to the females to allow mating to occur. A frog with moderate parasitaemia was placed in the feeding cage for 2 h. After 2 h the frog was removed and returned to the Animal Care Facility. Female mosquitoes with red, distended abdomens indicative of a blood meal were collected and placed in holding containers; an equal number of males were added to encourage mating in case mating had not occurred before the blood meal was taken. Holding containers were prepared using 2 L plastic pop bottles; the top half of the pop bottle was removed using a sharp knife. A stocking was placed over the top of the bottle and the closed end of the stocking was cut off to create a passageway.

A hole large enough to allow the aspirator to enter was cut in the side of the pop bottle and a square of latex cut from a latex glove was taped over the opening. A small incision was made in the latex and another square of latex was taped on only three sides over the opening to form a flap. Two 10 mL centrifuge tubes were attached to the sides of the holding cages using UHU® Tac; one tube was filled with water and the other was filled with 5% (w/v) sugar solution, prepared in the same manner detailed above. A folded Kimwipe was placed in each tube.

In an attempt to provide a suitable oviposition site, a wet cotton makeup sponge inside a small Petri dish was placed in each holding cage. A sprig of plastic leaves was fastened to the inside of the cage using UHU® Tac. The sprig of leaves extended over the Petri dish as a supplementary oviposition site. 21

At 15 d post-infection (PI), female mosquitoes were transferred from holding cages to the larger, Plexiglas feeding cage using the aspirator. An uninfected green frog was introduced into the cage. Female mosquito behaviour was observed to note host-seeking and blood feeding attempts. The trial was complete after 1 h and the frog was returned to the Animal Care Facility. Female mosquitoes that blood fed were transferred into separate holding cages from the original group and labelled accordingly. All other mosquitoes from the trial were replaced in original holding cage.

At 30 d PI, the presence of an uninfected frog introduced in the same method as above was used to observe the behaviour of female mosquitoes carrying mature H. clamatae oocysts. Following observations, all females from the trial were dissected. The head of the mosquito was quickly removed by pinching at the base of the head with forceps; subsequently, legs and wings were removed with forceps. The mosquito was placed in a small pool of Frog Ringer solution contained in a glass Petri dish. Under a dissecting microscope, one pair of forceps was used to grasp the mesothorax and another pair was used to grasp the tip of the abdomen. Slowly and gently the forceps were pulled in opposite directions and the digestive tract, with Malpighian tubules attached, was extracted. The presence or absence of eggs in the abdomen was noted. Using phase contrast microscopy, the Malpighian tubules were examined to confirm the presence of mature oocysts. Observations were noted for each mosquito. 22

Control trials were also performed by in the manner detailed above; however, an uninfected frog was used in the initial feeding trial. Uninfected mosquitoes were maintained in the same conditions as infected mosquitoes and all experiments conducted in the same style. Dissections were also performed to confirm the presence or absence of eggs.

Data collection

Mosquito behaviour was observed and recorded during initial feeding experiments with an infected frog as well as subsequent feeding experiments with uninfected frogs. Host-seeking behaviour was recorded as the occurrence of landing on a host and initiating a blood meal. During initial feedings (0 d), the number of mosquitoes that fed during the course of each trial was recorded, as well as the extent to which each mosquito fed (partially fed versus fully engorged, based on visual estimation of abdominal distension). A record was also made if the mosquito was forced to leave the frog due to defensive movements made by the frog. The area of the frog from which each mosquito fed was noted.

During feeding trials at 15 and 30 d, the number of female mosquitoes that approached and fed on the uninfected frog, as well as female mosquitoes that remained stationary or avoided the frog, was recorded. The area of the frog from which each mosquito fed was also noted. All data were compiled into contingency tables for 0, 15 and 30 d. 23

Statistical analyses

A chi-square test was used to determine whether or not the proportions of mosquitoes that fed on an uninfected frog compared to those that fed on an infected frog, were statistically significant. Due to values less than 5 in the contingency tables, a Fischer exact test was used to determine significance between proportions of uninfected or infected mosquitoes that did or did not feed at 15 and 30 d. A Fischer exact test was also used to determine if significance existed between the numbers of infected mosquitoes that did, or did not, feed at 15 and 30 d. All statistical analyses were performed in Minitab. 24

RESULTS

General mosquito feeding behaviour

Mosquitoes approaching a green frog were observed to hover in the air and feign landings several times before actually landing on the frog. On several occasions the mosquito would land on the floor of the cage, walk slowly towards the frog and crawl onto the body. Probing time varied; some mosquitoes were able to take a blood meal from the first site probed while others tested several sites by exploring the surface of the frog, inserting and withdrawing the fascicle before receiving blood. Mosquitoes appeared to be more apt to fly away when the frog moved if the proboscis had not yet been inserted. Once the proboscis was inserted and blood was visible in the abdomen, mosquitoes were observed to remain in place even if the frog exhibited movements such as walking, jumping, climbing the sides of the cage, twitching and leg kicking. Mosquitoes appeared to prefer the back and hind legs of the frog; only two attempts of feeding on the head were observed. Feeding time was also variable and often lasted upwards of 40 min. Mosquitoes fed until the abdomen was red and appeared fully distended unless knocked off by movements made by the frog.

Those that did not feed to repletion were observed to either return to the frog and attempt to blood feed again, or remain at rest on the side of the cage. After the proboscis was withdrawn, a fully fed, undisturbed mosquito often rested upon the frog for several minutes before flying away. Fully fed mosquitoes were also observed to remain on the floor of the cage for several minutes after leaving 25 the frog before walking or flying to a wall. It should be noted that not all mosquitoes initially exposed to a frog at 0 d exhibited host-seeking behaviour.

As well, frogs were never observed to eat mosquitoes during any of the trials.

Mosquito host-seeking behaviour at 0, 15 and 30 days

The general host-seeking and feeding behaviour of mosquitoes was similar in the presence of an infected frog and an uninfected frog. However, chi- square analysis of the number of mosquitoes fed on an uninfected frog versus an infected frog revealed that significantly more mosquitoes fed on an infected frog than an uninfected frog (p = 0.002, n=80, Table 1). On one occasion, with equal numbers of mosquitoes in each respective cage, 13 mosquitoes fed on an infected frog whereas only two mosquitoes fed on an uninfected frog. This suggests that mosquitoes may prefer to feed on infected frogs.

At 15 d PF, mosquito host-seeking and feeding behaviour resembled that at 0 d. However, most mosquitoes in the experimental group did not feed again and no mosquitoes in the control group fed again at 15 d (Table 2). Analysis with a Fischer exact test revealed that there was not a significant difference in the number of mosquitoes that did, or did not, blood feed at 15 d between the control group of uninfected mosquitoes and the infected mosquitoes (p = 0.28, n=21).

At 30 d, any host-seeking and feeding behaviour was similar to that at 0 and 15 d. As at 15 d, fewer mosquitoes initiated host-seeking behaviour, and at 26

30 d, only one mosquito obtained a blood meal (Table 3). There was no significant difference in the number of mosquitoes that did, or did not, blood feed at 30 d between the uninfected mosquitoes and the infected mosquitoes (p =

1, n=20). Overall, there was a reduction in the number of mosquitoes feeding at both 15 and 30 d after an initial blood meal was taken at 0 d.

There was no statistical significance in a comparison of the proportion of infected mosquitoes that fed at 15 d and the proportion of infected mosquitoes that fed at 30 d (p = 0.17, n=16).

Mosquito mortality

In the first feeding attempt, a frog with high parasitaemia (5% of RBCs infected) was used. Four mosquitoes fed on this frog, but none survived. One mosquito that fed to repletion was found dead approximately four h post feeding (PF); all others were dead within 24 h PF. Rapid death of this nature was not observed in mosquitoes that fed to repletion on uninfected frogs or frogs with a moderate parasitaemia (0.5%). However, mosquito deaths continued to occur in some cases, presumably due to old age of the mosquitoes used in some of the trials. These mosquitoes are included in the row entitled “Other” in Table

2. Mosquitoes that did not complete a full blood meal were not used in further trials and are also included in this row of Table 2.

Egg retention

Of all mosquitoes that gained a blood meal, none were observed to lay eggs. Attempts to provide suitable oviposition sites did not yield positive results. Upon dissection, most mosquitoes contained white, ovoid eggs. Only two mosquitoes did not contain eggs upon dissection; of these two, one re-fed at

30 d (Table 3). All other mosquitoes remained stationary on the sides of the feeding cage unless disturbed by the frog, other mosquitoes, or movements made by the observer.

Confirmation of infection

The presence of H. clamatae sporocysts in the Malpighian tubules confirmed an infection in mosquitoes initially fed from an infected frog. Under a dissecting microscope, heavily infected tubules were distorted and bulged in several areas. Using phase contrast microscopy, numerous ellipsoidal sporocysts were viewed within the Malpighian tubules, similar to those described in Desser et al. (1995). In several mature infections, the oocyst wall itself was no longer visible and it appeared as though sporocysts had dispersed throughout the lumen of the tubule. In several mosquitoes the sporocysts filled the length of the infected tubules whereas in other mosquitoes the tubules contained sporocysts in segregated masses, similar to Figure 3. 28

Table 1. Proportion of mosquitoes fed on infected and uninfected frogs at

0 days. Numbers represent mosquitoes that did, or did not, feed on an

uninfected frog or an infected frog. There was a significant difference

in the proportion of mosquitoes that fed on an infected frog versus the

proportion that fed on an infected frog (p = 0.002)

0 days

Uninfected frog Infected frog

Fed 6 34

Did not feed 19 21

Table 2. Proportion of infected and control mosquitoes fed on uninfected

frogs at 15 d PF. Numbers represent mosquitoes that did and did not

feed on an uninfected frog at 15 d in either the control group (fed on an

uninfected frog) or the infected group (fed on an infected frog).

Mosquitoes in the „Other‟ row represent fatalities or those that did not

feed to repletion. There was no significant difference in the proportion

of mosquitoes that did, or did not, feed at 15 d between the control and

infected groups (p = 0.27)

15 days

Uninfected mosquitoes Infected mosquitoes

Fed at 15 d 0 5

Did not feed at 15 d 5 11

Other 1 18

Table 3. Proportion of infected and control mosquitoes fed on uninfected frog

at 30 d PF. The proportion of mosquitoes that did and did not feed also

includes whether or not eggs were present upon dissection. There was

no significant difference in the proportion of mosquitoes that did, or

did not, feed between the control group (fed on an uninfected frog) and

the infected group (fed on an infected frog) (p = 1).

30 days

Uninfected mosquitoes Infected mosquitoes

Eggs No eggs Eggs No eggs

Fed at 30 d 0 0 0 1

Did not feed 5 0 14 1 at 30 d

DISCUSSION

Host-seeking behaviour at 15 and 30 days

Observed feeding behaviour of Culex territans on green frogs is similar whether the mosquito is infected with Hepatozoon clamatae or not. Mosquitoes infected with H. clamatae and uninfected mosquitoes both experienced a decline in host-seeking behaviour at 15 d and 30 d after an initial blood meal at 0 d. It was expected that infected C. territans would not host-seek at 15 d if the parasite was exerting influence over the behaviour of the mosquito, due to the developmental immaturity of the parasite at this stage. By reducing the host- seeking behaviour of the mosquito, the parasite reduces the chance of being eaten by a frog before reaching a transmissible stage. However, this reduction in host-seeking behaviour was also observed in the control group of mosquitoes and there was no significant difference between the proportion of infected mosquitoes and uninfected mosquitoes that did feed at 15 d. Therefore, it is unlikely that parasite manipulation is acting inhibit mosquito host-seeking behaviour at 15 d post-infection (PI).

At 30 d PI the expectation was that modification of host-seeking behaviour in the infected mosquito would increase the conspicuousness of the mosquito in the presence of the green frog. I expected to see an increase in host-seeking behaviour compared to 15 d, as well as other conspicuous behaviours, such as feeding on the head of the frog, instead of on the hind legs and main body.

However, both infected and uninfected mosquitoes appeared disinterested in the 32 frog and a comparison of the proportion of infected and uninfected mosquitoes that fed at 30 d did not differ significantly. This suggests that H. clamatae does not exert influence over the host-seeking behaviour of C. territans at 30 d PI.

The lack of interest by previously fed mosquitoes for uninfected frogs at

15 and 30 d necessitated recording mosquito behaviour as simply whether or not the mosquito fed on the host. Ideally, measures such as probing time, time spent near the host, and response to frog defensive behaviour would be described in order to determine if any parasite modification of host behaviour was occurring.

For instance, it is possible that infected mosquitoes ingest a higher volume of sugar during infection, as occurs with infections of Plasmodium species (Hurd et al., 1995; Rivero and Ferguson, 2003). This could also inhibit host-seeking behaviour as large amounts of sugar in the gut would activate stretch receptors and trigger an inhibition of host-seeking behaviour (Foster and Eischen, 1987;

Roitberg and Friend, 1992). Other possible manifestations of host manipulation may involve increased probing time by the mosquito, as occurs with Plasmodium species, although C. territans does not inhabit the salivary glands and reduce production of salivary apyrase as Plasmodium species do (Hurd, 1990; Wekesa et al. 1993; Hurd et al., 1995). As well, C. territans was observed to feed for long periods of time, often upwards of 40 min, therefore an increase in probing time may not be necessary to draw a frog‟s attention to a feeding mosquito, which would increase the chance of consumption by the frog. However, a closer inspection of mosquito behaviour, apart from observing solely whether or not 33 the mosquito lands on the frog and blood feeds during infection, may be necessary to observe modifications of mosquito behaviour by this parasite.

Problems posed by egg retention

Mating was never observed in the laboratory where C. territans were maintained, and although males were added to both feeding cages and holding cages to encourage mating, insemination may have never occurred. Culex territans mate in swarms, as do all mosquitoes; males form large clouds that females dive in and out of in order to find a mate and copulate (Yuval and

Bouskala, 1993). This swarming behaviour was not observed in cages in the lab.

It is possible that a larger area is required for this species to form a mating swarm, or other cues that promote mating in the field are missing in the laboratory.

In most cases, eggs were produced after a blood meal was taken, even if mating was not observed. However, the eggs were never oviposited and were retained until dissection. This may be due to a lack of insemination; in some species of mosquitoes, eggs can be produced without the occurrence of insemination (Bentley and Day, 1983). Chemical factors transferred during insemination also play a role in oviposition. Matrone, a male peptide, is transferred during insemination and stimulates oviposition (Bentley and Day,

1983; Hiss and Fuchs 1972); therefore if insemination did not occur and matrone was not passed to the female, it is possible that oviposition would not occur. 34

Further, Burkett-Cadena et al. (2008) suggest that peak feeding times of Culex territans correspond with breeding times of their anuran hosts. Frog vocalizations are important as a location cue for mosquitoes (Burkett-Cadena et al. 2008) and thus when frogs are calling during mating season, mosquitoes are more likely to find them and feed. This also corresponds with peak breeding times for the mosquitoes (Burkett-Cadena et al. 2008). Therefore, it is possible that C. territans were not receiving the proper cues in the laboratory for mating; thus, the females were not being inseminated and eggs were retained. If insemination did in fact occur, then a lack of suitable oviposition site may be responsible. Female mosquitoes respond to several cues that indicated suitable oviposition sites (Bentely and Day, 1983); if these cues are lacking, it is possible that the eggs will be retained, as was observed in this study. Bentley and Day

(1983) also note that prolonged exposure to sugar may reduce oviposition behaviour or the amount of eggs laid. The mosquitoes in the lab had constant access to sugar which may have conflicted with normal behaviour associated with oviposition.

As previously mentioned, host-seeking behaviour of mosquitoes is inhibited once a blood meal is taken, and this inhibition continues until oviposition occurs. Therefore, host seeking will not occur if the mosquito is carrying eggs. This could explain the general lack of host-seeking behaviour at both 15 and 30 d post-feeding in both infected and uninfected mosquitoes. In hindsight, it would be more accurate to dislodge mosquitoes that host-seek at 35 and insert the fascicle at 15 d instead of allowing them to blood feed again. By allowing them to blood fed at 15 d, the mosquitoes may have then gained enough blood to produce eggs if the initial blood meal at 0 d was insufficient for egg production. This potential for egg production at 15 d interferes with the ideal trajectory of behaviour where the assumption is that a blood meal at 0 d would be sufficient to produce a batch of eggs. Overall, the production and retention of eggs confounds any predictions that parasite manipulation is responsible for changes in host behaviour.

Although it may be beneficial for the parasite to induce egg retention in the mosquito it is unlikely that this is occurring in this host-parasite system.

Culex nigripalpus are recorded to have the ability to retain eggs for long periods of time during unfavourable weather conditions, a characteristic which is taken advantage of by St. Louis Encephalitis Virus (Day and Edman, 1988). The period of egg retention is conducive to the incubation period required by the virus. I considered the possibility that egg retention could be induced by H. clamatae in order to reduce host-seeking activity of the mosquito and allow for a safe parasite incubation period. However, this is unlikely as the control group also retained eggs. As well, one would expect to see oviposition occur at parasite maturity (30 d) and host-seeking behaviour to resume. As this did not occur, it is unlikely that H. clamatae induced the egg retention observed in C. territans.

Areas for further research

Initially, a frog with a high parasitaemia (5% of RBCs infected) was used to feed mosquitoes to repletion. However, mosquitoes that fed on this frog did not survive past 24 h PF. This is an interesting observation which requires further experimentation to elucidate whether or not this occurs every time a mosquito feeds to repletion on a frog with a high infection. If mosquito death is caused by infection with a high intensity of H. clamatae, this suggests that the parasite may require a mechanism for self-regulation of population within the anuran host, so as to not kill its next host. For example, Trypanosoma brucei brucei in the tsetse fly, Glossina morsitans, may possess a mechanism for programmed cell death in order to regulate its population and maintain the life of the host

(Welburn and Maudlin, 1997). Such a strategy would benefit Hepatozoon clamatae if high parasite densities are lethal to the mosquito host.

Additionally, mosquito mortality may prompt research into the unknown path travelled by H. clamatae to reach the Malpighian tubules. It is assumed that the parasite travels through the gut to reach the tubules, thereby avoiding confrontation with immune cells in the haemocoel. However, a related parasite,

H. sipedon, perforates the gut wall and travels through the haemocoel to reach the fat body of the mosquito (Smith, 1996). Therefore, it is possible that H. clamatae travels a similar path, although destined for the Malpighian tubules. Perforation of the gut in large numbers may account for mosquito death for such damage may cause a fatal release of bacteria into the hameocoel, in addition to the costs 37 incurred by overall organ damage. In order to determine if the path travelled by

H. clamatae in the mosquito is causing mortality when a high density of parasites is introduced into the gut, fixing and sectioning of the mosquito at several stages

PI may reveal this yet unknown course of infection.

Interestingly, the proportion of mosquitoes that fed on an infected frog is significantly higher than the proportion that fed on an uninfected frog. This may indicate that frogs infected with Hepatozoon clamatae are more attractive to female mosquitoes. This is a potential form of manipulation by the parasite in order to increase its own transmission to mosquitoes. It is recognized that infected hosts may emit modified odours (O‟Shea et al. 2002); by somehow provoking the release of an odour from the host, the parasite may be able to attract a higher density of female mosquitoes to infected frogs in order to feed and acquire the parasite. Female mice can detect infected male mice by odour cues and avoid mating with them (Kavaliers et al. 2004), and hamsters infected with Leishmania are more attractive to mosquitoes (O‟Shea 2002). Mosquitoes use a variety of odour cues to find their hosts in order to draw a blood meal (Anton et al. 2003); therefore, it is plausible that an infected frog could be releasing odour cues indicative of its infection which may increase its attractiveness to mosquitoes.

Observations at the collection site, Hillcrest Brook, and evidence from

Bartlett-Healy et al. (2008), suggests that Culex territans and Rana clamitans share a spatial and temporal sympatry. This is beneficial for H. clamatae as the two hosts are already in close association through the reciprocal trophic nature of 38 their relationship. However, Culex territans does not feed exclusively on R. clamitans. Despite the apparent commonality of the green frog in Nova Scotia,

Bullfrogs, R. catesbeiana, and Spring Peepers, Pseudacris crucifer, are also widespread across the mainland. Culex territans prefers Spring Peepers and

Bullfrogs, according to Burkett-Cadena et al. (2008). Bullfrogs can also carry H. clamatae, however Spring Peepers have not yet been shown to host H. clamatae.

Therefore, it is plausible that H. clamatae would increase the attractiveness of the frog host, R. clamitans or R. catesbeiana to the mosquito in order to ensure transmission success. Further, C. territans is a host to other parasites such as H. catesbianae and H. sipedon (Smith, 1996); the low specificity of C. territans as a host to different species of parasites may result in competition between these parasites. Therefore, this may serve as another factor which favours manipulation by the parasite causing female mosquitoes to be more attracted to infected R. clamitans.

It would be beneficial to carry out the same procedures with a mosquito species previously known to lay eggs in the laboratory. Culex pipiens may become infected with Hepatozoon sipedon after feeding on the blood of an infected Eastern garter snake, Thamnophis sirtalis sirtalis or the Northern water snake, Nerodia sipedon sipedon. Hepatozoon sipedon travels through the gut wall to the fat body of the mosquito, via the haemocoel. Fertilization and gametogenesis occur in the fat body and mature oocysts are observed at 28 d PF. The mosquito must be eaten by a frog, Rana pipiens, where sporozoites penetrate hepatic cells and form 39 dizoic cysts after 7 days. The frog is then ingested by a snake where cystozoites invade hepatocytes, undergo asexual division and travel to the red blood cells of the snake and form gamonts, which are the infective stage to the mosquito

(Smith et al. 1994). Infecting C. pipiens with H. sipedon and observing mosquito behaviour around the frog host, R. pipiens at both 15 and 30 d may reveal evidence of host manipulation of behaviour by the parasite.

Initial feeding trials were attempted with Culex pipiens and an Eastern garter snake (Thamnophis siralis siralis), infected with Hepatozoon sipedon, collected in Ontario. Culex pipiens were exposed to the infected garter snake at dusk for 4 h. Three attempts were made with 10 female mosquitoes each time. Males were included in feeding cages, and the snake was exposed to mosquitoes for [how many?] h. Unfortunately, in none of these trials did a mosquito take a blood meal from the snake.

Conclusion

Overall, egg retention in Culex territans confounded any observation of possible manipulation of host-seeking behaviour by Hepatozoon clamatae.

However, the close spatial relationship of the green frog and C. territans and the reciprocal trophic nature of this relationship may be suited for transmission of H. clamatae such that behaviour modification is not necessary. Manipulation of host behaviour requires energy on the part of the parasite and depletes resources that can otherwise be allocated to reproduction (Lefèvre et al. 2008). By taking 40 advantage of an existing relationship between suitable hosts, H. clamatae need not invest in manipulation and thus saves itself the cost associated with this manipulation of host behaviour. However, the relationship between R. clamitans and C. territans is not exclusive and there is room for H. clamatae to be transmitted to an unsuitable host. Therefore, it is feasible to presume that the intermediate host, the definitive host or both are affected by parasitisation by H. clamatae and that modification of host behaviour by this parasite remains a possibility. Further work with this parasite in the frog host may reveal an interesting, and perhaps widely applicable, mechanism for self regulation of parasite density with a host. Investigation into host-parasite life cycles, such as the life cycle of H. clamatae in its anuran and mosquito hosts is beneficial in increasing the breadth of knowledge of the natural world; such knowledge may in turn result in broader applications to medically important avenues of research, such as the amplification of arboviruses in ectothermic hosts (Burkett-Cadena et al. 2008). 41

APPENDICES

Appendix 1. Information table of mosquitoes, initially fed on infected frogs,

that survived until dissection at 30 days. Mosquitoes that did not survive

the duration of the trial are not included.

Mosquito Re-fed at 15 d Re-fed at 30 d Eggs Parasite Date 1 no no yes yes Jun-30 2 no no yes yes Jun-30 3 yes no no yes Jun-30 4 no yes no yes Jul-08 5 yes no yes yes Jul-08 6 yes no yes no Jul-23 7 no no yes yes Jul-23 8 no no yes yes Aug-19 9 no no yes yes Aug-12 10 yes no yes yes Aug-17 11 yes no yes yes Sep-10 12 yes no yes no Sep-10 13 yes no yes yes Sep-10 14 no no yes yes Sep-10 15 no no yes yes Oct-31 16 no no yes yes Sep-10 17 no no yes yes Oct-31 18 no no yes yes Oct-31

Appendix 2. Information table for uninfected control mosquitoes that survived

until dissection at 30 days. Mosquitoes that did not survive the duration

of the trial are not included.

Mosquito Re-fed at 15 d Re-fed at 30 d Eggs Date 1 no no yes Jun-30 2 no no yes Jun-30 3 no no yes Aug-12 4 no no yes Oct-31 5 no no yes Oct-31 43

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