Lethal Effects of High Intensities of Hepatozoon Species on The

LETHAL EFFECTS OF HIGH INTENSITIES OF HEPATOZOON SPECIES ON THE

MOSQUITO, CULEX TERRITANS (DIPTERA: CULICIDAE)

Caoimhe A. McParland

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 Caoimhe McParland

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, Caoimhe McParland, 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

ACKNLOWLEDGEMENTS

First and foremost, I would like to thank my supervisor, Dr. Todd Smith. I could not have asked for a better, more understanding supervisor and I am so grateful. Thank you for always believing things would get finished on time, even when I didn’t, for the countless hours helping me put everything together and for always having time for a laugh. I have gained so much from this experience and cannot thank you enough for guiding me through it.

To my co-supervisor Dr. Glenys Gibson, thank you for taking me on and helping me learn so many new skills that were completely unknown to me less than a year ago.

Thank you for everything you have taught me, and thank you for trusting me with your diamond knife.

I would also like to thank my honours lab mate Meghan Kerr for keeping me company on endless frog- and mosquito-catching trips, and for helping keep me sane. To my other lab mates Francine Heelan and Marie-Catherine French: thank you for all of your continued help with maintaining the mosquito colonies, it is very much appreciated.

Thank you to Haixin Xu for teaching me how to use the microtome, and thank you to Tanya Morse-Outhouse and Dawn Miner in the Animal Care Facility for taking such wonderful care of our frogs. Thank you to NSERC, the Atlantic Canada Society for

Microbial Ecology and Acadia University for providing the funding necessary to complete this research project.

Finally, thank you to my friends and family for all of their support throughout this process. The moral support, encouragement and late night coffee runs have been invaluable and I cannot thank any of you enough.

TABLE OF CONTENTS

Page

List of figures ...... vii

List of appendices ...... viii

Abstract ...... ix

Introduction ...... 1

Digestion and excretion in mosquitoes ...... 1

Culex territans ...... 3

Phylum Apicomplexa and genus Hepatozoon ...... 4

Mosquito immunity ...... 7

Objectives ...... 11

Materials and methods ...... 15

Collection and care of mosquitoes ...... 15

Collection and care of green frogs ...... 16

Infection of mosquitoes ...... 17

Processing of infected mosquitoes ...... 17

Results ...... 19

Development of Hepatozoon species in the midgut of Culex territans ...... 19

Development of Hepatozoon species in the Malpighian tubules of

Culex territans ...... 20

Discussion ...... 29

Effect of Hepatozoon species on the midgut of Culex territans ...... 29

Effect of Hepatozoon on the Malpighian tubules of Culex territans ...... 32

Pathology of other mosquito-infecting parasites ...... 34

Experimental challenges ...... 36

Further research and implications ...... 37

References ...... 39

Appendices ...... 47

LIST OF FIGURES

Page

Figure 1. Life cycle of Hepatozoon species in ranid frogs and in the

mosquito, Culex territans ...... 13

Figure 2. Malpighian tubules of Culex territans infected with oocysts of

Hepatozoon species ...... 14

Figures 3-4. Midgut of Culex territans 12 hr post-feeding ...... 21

Figures 5-8. Midgut of Culex territans 36 hr post-feeding ...... 22

Figures 9-12. Midgut of Culex territans 48 hr post-feeding ...... 23

Figures 13-14. Midgut of Culex territans 72 hr post-feeding ...... 24

Figures 15-16. Malpighian tubules of Culex territans 12 hr post-feeding ...... 25

Figures 17-18. Malpighian tubules of Culex territans 36 hr post-feeding ...... 26

Figures 19-22. Malpighian tubules of Culex territans 48 hr post-feeding ...... 27

Figures 23-26. Malpighian tubules of Culex territans 72 hr post-feeding ...... 28

vii

LIST OF APPENDICES

Page

Appendix 1. Fixation protocol ...... 47

Appendix 2. Embedding protocol ...... 48

Appendix 3. Staining protocol ...... 49

viii

ABSTRACT

Two sympatric species of Hepatozoon, H. clamatae and H. catesbianae, have a high prevalence in frogs of Nova Scotia. Although the vertebrate hosts appear not to be adversely affected by these parasites, high parasitaemia values (i.e., where more than 5% of erythrocytes are infected) that occur naturally in some frogs have been observed to cause death in mosquito vectors. The first objective of this study was to investigate the cause of death in mosquitoes, Culex territans, that have fed on frogs with heavy infections of Hepatozoon species. The second objective was to determine the route travelled by these parasites through the body of C. territans from the blood meal in the midgut to the Malpighian tubules, where parasite development occurs. Culex territans mosquitoes were allowed to feed on green frogs with parasitaemia values of 5%. Blood- fed mosquitoes were fixed in 2.5% gluteraldehyde at specific times (24 hr, 36 hr, 48 hr, and 72 hr) post-feeding (PF). Fixed mosquitoes were embedded in TAAB resin, sectioned at 1 µm sections, stained and observed using bright-field microscopy. At 12 hr PF, the midgut was healthy and parasites had not invaded Malpighian tubules. At 36 hr PF, there was extensive damage to the midgut, but parasites were not yet present in Malpighian tubules. At 48 hr PF, there was extensive trauma to the midgut, and Malpighian tubules were riddled with parasite zygotes. At 72 hr PF, Malpighian tubules were infected with growing oocysts, but there was negligible damage to the midgut. The results support the hypothesis that mosquito death is caused by morphological trauma to the midgut, and is not likely a result of septicaemia. This further supports the hypothesis that Hepatozoon species penetrate the midgut and enter Malpighian tubules through the haemocoel, rather than entering the tubules at their origin near the junction of the midgut and the hindgut.

INTRODUCTION

The family Culicidae, comprising 3500 species of insects known as mosquitoes, are a family of true flies, distributed worldwide, that have been the subject of intense entomological research (Becker et al., 2010). As important vectors for a wide variety of disease-causing organisms, mosquitoes have been more debilitating to humans than any other animal (Stout, 2008). Mosquitoes are able to survive and adapt to a broad range of habitats, which has allowed species of the group to become incredibly successful. They are able to lay eggs in virtually any aquatic habitat, in many cases regardless of size, level of salinity, or level of pollution of the body of water. More than half of the world’s population is at risk of infections caused by organisms or viruses that are transmitted by mosquitoes (Becker et al., 2010; Centers for Disease Control and Prevention, 2012), and they are responsible for the death of hundreds of thousands of people every year (Centers for Disease Control and Prevention, 2012).

Digestion and excretion in mosquitoes

Mosquitoes feed on the flowers of plants for the sugars found in their nectar.

Anautogenous females must also feed on blood, as protein from a blood meal is essential for the development and maturation of their eggs (Becker et al., 2010). Female mosquitoes have highly specialized mouthparts that allow them to pierce the skin of their host, whereas the lack of blood-feeding habits in males has resulted in reduced mouthparts (Wahid et al., 2003).

Mosquitoes have a closed digestive system that is composed of a long alimentary canal divided into three sections, namely the foregut, the midgut, and the hindgut,

distinguished by their location and the digestive processes that occur within them

(Chapman, 2013). As the meal travels through the alimentary canal, it enters the first section of the alimentary canal, the foregut. The foregut consists of the pharynx, the oesophagus, the crop and the proventriculus. Food can be stored in the crop before passage to the midgut, a junction that is controlled by the proventriculus (Terra and

Ferreira, 2009). The midgut is the main site of digestion; the epithelial cells lining the organ produce and secrete digestive enzymes, and microvilli that line the walls absorb the resulting nutrients (Chapman, 2013). Any indigestible food continues down the alimentary canal and into the hindgut, where waste food is combined with uric acid formed in the Malpighian tubules to form faeces. The majority of the water from the faeces is reabsorbed as it passes through the rectum as waste (Terra and Ferreira, 2009).

Malpighian tubules are a series of blind-ending, convoluted tubules that are composed of a single layer of cells. Comparable to the kidney of vertebrates, Malpighian tubules are the excretory organs of nearly all insects, including mosquitoes (Terra and

Ferreira, 2009). They are attached to the alimentary canal at the junction between the midgut and the hindgut, and project into the haemocoel (Chapman, 2013). Malpighian tubules are surrounded by haemolymph, from which solutes, water and metabolic waste, such as uric acid, are absorbed. On the inner surface of the tubules, microvilli project from principal cells into the lumen. The microvilli are responsible for the uptake of potassium, sodium and hydrogen ions via active transport, as well as the absorption of fluids into the tubule lumen (Klowden, 2008). In the Malpighian tubules of mosquitoes, stellate cells are distributed among the principal cells, and are responsible for exporting bicarbonate produced during the active transport by principal cells (Klowden, 2008;

Chapman, 2013). Cells in the distal region of the tubules (i.e., near the blind end) secrete ions and organic solutes into the lumen of the tubules, while the cells in the proximal region of the tubules (i.e., near the junction with the hindgut) have absorptive functions

(Klowden, 2008). The filtration of the haemolymph that occurs in the Malpighian tubules is relatively unselective, and results in the formation of primary urine. The primary urine undergoes further modifications in the rectum before it is expelled (Chapman, 2013).

Malpighian tubules and the rectum are able to filter their entire extracellular volume up to

200 times a day (Klowden, 2008).

Culex territans

One of the most medically important genera of mosquitoes is Culex, which contains the species responsible for the transmission of many diseases, including West

Nile virus, Japanese encephalitis virus, and bancroftian filariasis caused by the nematode

Wuchereria bancrofti (Service, 2012). Culex is a diverse genus, with over 1200 known species (Burkett-Cadena, 2013). Most of the species in the genus feed on blood from humans and other mammals, whereas others are known to feed on birds, reptiles and amphibians.

One species that feeds on amphibians is Culex territans, a member of the subgenus Neoculex (Carpenter and LaCasse, 1974). Culex territans is able to survive in a wide range of environments, which enables a widespread global distribution (Service,

2012). Inseminated adult females overwinter until early spring, at which time they take a blood meal before laying their first eggs (Bartlett-Healy et al., 2008b). Their egg rafts are laid in non-polluted freshwater bodies, such as swamps, marshes and ponds, which are

often co-inhabited by frogs and other amphibians (Joy and Clay, 2002; Bartlett-Healy et al, 2008a). While Cx. territans have been observed to feed on reptiles, birds and mammals, they exhibit a strong preference for amphibian blood (Savage et al., 2007).

The proximity of their own larval habitats with that of their prey is beneficial for adult females that require blood meals (Savage et al., 2007; Bartlett-Healy et al., 2008a).

Culex territans has been found to carry West Nile virus in the United States, but has not been known to transmit the virus, or any other pathogens, to humans (Centers for

Disease Control and Prevention, 2013). However, it has been found to carry and transmit a number of parasites to amphibians, including frog erythrocytic virus, Foleyella flexicauda, Hepatozoon catesbianae and Hepatozoon clamatae (Benach and Crans, 1973;

Gruia-Gray and Desser, 1992; Desser et al., 1995).

Phylum Apicomplexa and genus Hepatozoon

Apicomplexa is a phylum containing over 4600 known species of parasitic protists (Wise and Hoober, 2007). Unique to the species in the phylum Apicomplexa is the apical complex, a structure involved in the invasion of host cells during the infective stage of these parasites (Sleigh, 1991; Black and Boothroyd, 2000). The apical complex is a group of organelles composed of cytoskeleton and secretory elements (Morrissette and Sibley, 2002). The apicoplast, another structural feature unique to apicomplexans, is a chloroplast-derived plastid that is essential to the survival of the parasite, as it is the site for important metabolic pathways such as fatty acid synthesis, heme biosynthesis, and carbohydrate metabolism (Wise and Hoober, 2007; Becker and Selzer, 2011; Lemgruber and Lupetti, 2012).

Apicomplexans are endoparasitic, and a number of species within the phylum are harmful to the health of humans and other animals. The most important genus in the phylum Apicomplexa, both medically and economically, is Plasmodium, the malaria parasites. Four species of Plasmodium cause human malaria, with P. falciparum the most lethal and one of the most widespread (World Health Organization, 2014). Carried by mosquitoes of the genus Anopheles, Plasmodium species are responsible for between 500

000 and one million deaths per year (Murray et al., 2012; World Health Organization,

2014).

Related to Plasmodium species are member of the genus Hepatozoon, a group of haemogregarines with two-host life cycles. Hepatozoon are found in a wide range of vertebrates, including mammals, birds, lizards and anurans, which serve as intermediate hosts, and haematophagous arthropods, including ticks and mosquitoes, which serve as definitive hosts and vectors (Smith, 1996; Boulianne et al., 2007). There are 42 known species of Hepatozoon that infect frogs and toads worldwide (Smith, 1996; Boulianne et al., 2007). The two species of interest in this study, Hepatozoon clamatae and

Hepatozoon catesbianae, are the only two Hepatozoon species of frogs for which complete life cycles have been elucidated (Fig. 1) (Boulianne et al., 2007).

When Cx. territans, the definitive host of both H. catesbianae and H. clamatae, feeds on an infected frog, it ingests erythrocytes infected with gamonts of the parasite. As the blood meal is digested, the gamonts are released into the midgut. Gamonts migrate to the Malpighian tubules of the mosquito, where they invade the epithelial cells of the tubule wall (Smith, 1996). Parasitophorous vacuoles form in the cytoplasm of the

Malpighian tubule cells, within which micro (male) gamonts and macro (female) gamonts

associate in syzygy (Desser et al., 1995). Gamonts undergo gametogenesis in the parasitophorous vacuole; microgamonts differentiate into two biflagellate micro (male) gametes, whereas macrogamonts differentiate into a single macro (female) gamete. One of the two microgametes fertilizes the macrogamete, producing a zygote that immediately undergoes meiosis to begin the process of sporogony, a type of multiple fission.

Sporogony results in the formation of a polysporocystic oocyst in the Malpighian tubule wall (Fig. 2). Every oocyst contains dozens of sporocysts, each of which houses four sporozoites. Oocysts remain in the Malpighian tubules, and the parasite is only transmitted to the frog host upon ingestion of an infected mosquito (Smith, 1996).

When a frog ingests an infected mosquito, sporozoites within oocysts are released into the frog gut. Sporozoites enter parenchymal cells of the liver, where they divide asexually by merogony, another type of multiple fission, to produce meronts (Desser et al., 1995). Merozoites emerge from meronts, enter the bloodstream of the frog, and invade erythrocytes. Inside these blood cells, merozoites undergo gamogony to become gamonts. When a mosquito feeds on the newly infected frog, the gamonts in erythrocytes migrate to the Malpighian tubules and perpetuate the parasitic cycle (Smith, 1996).

Hepatozoon clamatae and Hepatozoon catesbianae are similar parasites, and although DNA sequence data from the internal transcribed spacer regions, located between the 18S rRNA and 28 rRNA genes, have revealed several nucleotide differences among isolates of the two species (Boulianne et al., 2007), there are only a few distinguishing characteristics of a phenotypic nature. The primary phenotypic characteristic that distinguishes the two species is the cytological effects of gamonts in host erythrocytes. The invasion of erythrocytes by gamonts of H. clamatae causes the

erythrocyte nucleus to fragment, whereas invasion of erythrocytes by gamonts of H. catesbianae leaves the erythrocyte nucleus intact (Kim et al., 1998). Another distinguishing characteristic between the two species is their host preferences. Although both species use Cx. territans as their definitive host and vector, H. clamatae prefers green frogs (Rana clamitans) as their intermediate host, whereas H. catesbianae prefers bullfrogs (Rana catesbeiana) (Kim et al., 1998).

Parasites ingested by mosquitoes in a blood meal must be able to survive the initial barriers of the digestive system. In mosquitoes, sclerotized teeth protrude into the foregut lumen, destroying larger pathogens. However, bacteria, viruses and protozoans such as Plasmodium and Hepatozoon species are small enough to evade elimination

(Hillyer, 2010). In the midgut, parasites must penetrate the thick, acellular peritrophic matrix encasing the lumen that is produced as a result of blood feeding (Hillyer, 2010;

Vega and Kaya, 2012). The mechanism by which the midgut itself is penetrated by

Hepatozoon species, as an essential part of their life cycle, is not well understood, although Hepatozoon muris has been observed to penetrate the gut wall of its intermediate host Laelaps echidninus into the haemocoel (Smith, 1996; Hillyer, 2010).

Mosquito immunity

Insects, like all invertebrates, lack the adaptive immune system of vertebrate animals, and must rely solely on innate immunity to protect them against infection

(Jiravanichpaisal et al., 2006). The innate immune system is divided into two branches; the humoral response and the cellular response. The humoral response is acellular, and consists of various molecules in the body fluids. The production of antimicrobial

peptides, reactive oxygen and nitrogen intermediates, as well as the processes of melanisation and coagulation of haemolymph, are the principal forms of humoral defense

(Lavine and Strand, 2002). The cellular response is haemocyte-mediated, relying on specialized cells to get rid of foreign invaders. Microorganisms are killed by phagocytosis, nodulation or encapsulation, all of which are dependent on haemocytes

(Jiravanichpaisal et al., 2006). Despite their different mechanisms of defense, the humoral and cellular immunities are not independent of each other. A number of humoral molecules affect the function of haemocytes, and haemocytes produce a number of important humoral molecules (Lavine and Strand, 2002).

Melanisation is the production and deposition of the pigment melanin, and is a humoral immune response to invaders (Christensen et al., 2005). Among other functions, melanisation can help prevent the spread of microorganisms through the haemolymph

(Jiravanichpaisal et al., 2006; Hillyer, 2010). The production of antimicrobial proteins is another humoral defense, and occurs both in the midgut and in the fat body of the haemocoel upon detection of a pathogen (Hillyer, 2010).

Haemocytes are immunosurveillance cells found in the haemocoel (Lavine and

Strand, 2002; Hillyer, 2010). In mosquitoes, the most common types of haemocytes are granular cells, plasmatocytes, spherule cells and oenocytoids (Lavine and Strand, 2002).

Granular cells, or granulocytes, are highly phagocytic haemocytes that exhibit strong adhesive capabilities and produce proteins involved in humoral immunity (Hillyer, 2010).

Plasmatocytes are the main cells responsible for phagocytosis (Jiravanichpaisal et al.,

2006), spherule cells are believed to transport components that make up the cuticle, and

oenocytoids produce phenoloxidase, a precursor involved in melanisation

(Jiravanichpaisal et al., 2006).

Soluble immune molecules act as pattern recognition receptors (PRRs) that bind to pathogen-associated molecular patterns (PAMPs) on the surface of invaders and subsequently surround, or osponise, them. The bound PRRs, acting as opsonins, are then recognized by receptors on haemocytes and enhance the phagocytic or nodulation responses (Lavine and Strand, 2002). Thioester-containing proteins (TEPs) are one type of PRR that is involved in killing the motile zygotes, or ookinetes, of apicomplexans such as Plasmodium species in mosquitoes (Hillyer, 2010). PRRs on haemocytes also have the ability to recognize and bind PAMPs directly, but much less are known about these receptors (Lavine and Strand, 2002). Once an invader has been recognized, the haemocytes can mediate the processes of phagocytosis, nodulation or encapsulation to destroy it (Jiravanichpaisal et al., 2006).

PRRs on haemocytes recognize bacteria and fungi via the molecularly distinct proteins found on surfaces of these invaders. However, haemocytes are also capable of recognizing less obvious foreign invaders like protozoans, which are not as distinguishable as ‘non-self’ on a molecular level. Despite lacking acquired immunity, insects are able to recognize a remarkably wide range of invaders. Approximately 150

PRR genes have been discovered in the mosquito Anopheles gambiae. These genes encode proteins with pattern-recognition domains that can interact with PAMPs on foreign invaders and thus recognize them as non-self (Das et al., 2009). It was recently discovered that insects use alternative splicing to arrange binding domains into proteins

that are specific to invading pathogens, providing them with the ability to respond with specificity to a wide range of invaders (Dong et al., 2012).

Phagocytosis is the predominant cellular immune response to small foreign particles (Jiravanichpaisal et al., 2006). Phagocytosis is an essential process in which individual cells internalize small, potentially damaging substrates for destruction. The process begins with recognition and binding of the substrate by receptors on the haemocytes. The target is then engulfed, and a phagosome forms. The phagosome develops into a phagolysosome upon fusion with endosomes and lysosomes, and the target is isolated or destroyed (Beckage, 2008). Haemocytes can phagocytize a range of foreign entities, both biotic and abiotic, including bacteria, fungi and protozoans

(Jiravanichpaisal et al., 2006; Beckage, 2008).

Encapsulation and nodulation are forms of cellular defense in which multiple haemocytes surround the targets, and occur when invaders are too large to be internalised by an individual cell. Both encapsulation and nodulation ultimately result in the target being surrounded by layers of haemocytes, typically granular cells and plasmatocytes, to form an enveloping capsule. The two processes are fundamentally the same, but are employed against different targets. Encapsulation is the process in which multiple haemocytes bind to the surface of large targets like parasitoids and nematodes

(Jiravanichpaisal et al., 2006). Granular cells or humoral PRRs bind to the surface of the target and cause the release of cytokines, including plasmatocyte-spreading peptide, which induce plasmatocytes to become strongly adhesive (Clark et al., 1997; Lavine and

Strand, 2002). Plasmatocytes form the majority of the capsule, but granular cells make an outer layer, completing the process of encapsulation (Lavine and Strand, 2002).

Nodulation occurs when large quantities of bacteria are surrounded and trapped in a mesh of haemocytes (Jiravanichpaisal et al., 2006; Vega and Kaya, 2012).

The nodules and capsules formed during nodulation and encapsulation are often melanised, which decreases the flexibility and penetrability of the capsule around the pathogen (Vega and Kaya, 2012). Phenoloxidase and the prophenoloxidase activating system (proPO system) are essential to melanogenesis. The proPO system is activated upon detection of the presence of foreign microbial components (Jiravanichpaisal et al.,

2006). Prophenoloxidase, an inactive zymogen, is activated to phenoloxidase through a series of enzymatic reactions (Christensen et al., 2005). Phenoloxidase catalyzes the oxygenation of mono-phenols, resulting in cytotoxic quinones and reactive oxygen species that are believed to have a function in killing the target within the capsule

(Jiravanichpaisal et al., 2006; Vega and Kaya, 2012). The mechanism by which the sequestered pathogen is killed is unclear, but may also be a result of starvation, oxygen depravation or reactive oxygen and nitrogen species that lyse the target (Jiravanichpaisal et al., 2006; Hillyer, 2010). Cross-links formed between quinones and haemolymph proteins ultimately form the melanin pigment (Hillyer, 2010).

Objectives

This study will investigate the relationship between development of the parasites

Hepatozoon clamatae and H. catesbianae in their mosquito vector and definitive host,

Culex territans. It has been observed that most Cx. territans that feed on the blood of frogs with Hepatozoon infections of parasitaemia values higher than 5% (i.e., 5% of the erythrocytes of the frogs are parasitised by gamonts of Hepatozoon species) will die

within four days of feeding. The objectives of this study are to ascertain the route taken by parasites to arrive at the Malpighian tubules, the cells of which are required for parasitic development, and to determine the mechanism(s) by which the mosquito is killed by such heavy infections of the parasite. To pursue these objectives, Culex territans were allowed to feed on green frogs with different parasitaemia values. The abdomens of mosquitoes at various time points after feeding were sectioned and observed with bright-field microscopy to determine the path of the parasite through the mosquito, and how this movement and subsequent development ultimately kills its host. We hypothesise that parasites penetrate the midgut wall of the mosquito, travel through the haemocoel, and enter Malpighian tubules. In blood meals with high intensities of parasites, trauma caused by the large numbers of parasites penetrating the midgut wall may be the cause of death. Alternatively, the natural microbiota in the gut may infiltrate the haemocoel through the holes in the midgut wall formed by the parasites, resulting in a fatal septicaemia. The results of this study will further knowledge of the pathology of parasites that utilise mosquitoes as vectors and hosts, and may provide useful information on mosquito immunity.

FIGURE 1. Life cycle of Hepatozoon species 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 microgametes fertilizes female gamete to form a diploid zygote. d. Zygote undergoes meiosis and develops into an oocyst, which undergoes sporogony. 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 merogony to produce meronts. i. Merozoites emerge from hepatic meronts and enter bloodstream of the frog to invade the erythrocytes, where they will mature into gamonts capable of infecting subsequent mosquitoes (Smith, 1996).

FIGURE 2. Malpighian tubules of Culex territans infected with oocysts of

Hepatozoon clamatae. Two oocysts (arrows), containing numerous sporocysts, of H. clamatae are visible in Malpighian tubules at 30 days post-feeding (PF) (150x) (Kim et al., 1998).

MATERIALS AND METHODS

Collection and care of mosquitoes

Larvae and pupae of Culex territans were collected at various ponds and ditches off Deep Hollow Road in Greenwich, Nova Scotia (45.0839, -64.3999) and off Oak

Avenue in Wolfville, Nova Scotia (45.0833, -64.3667). Water samples were collected using a small plastic container attached to a long wooden pole. Larvae and pupae of

Culex territans were removed from water samples using a plastic pipette and transferred to Whirl-Pak® bags containing pond water from the collection site. The Whirl-Pak® bags were transported back to the lab, where larvae were transferred using a plastic pipette to

Rubbermaid® tubs half filled with geothermal water. The tubs were kept in the insectary of the Acadia Weston Animal Care Facility at 24°C ± 2°C under a 14:10 light: dark cycle. Larvae were fed a pinch of yeast and crushed Nutrafin® fish food every two days.

Pupae were transferred with a plastic pipette to a small plastic cup containing geothermal water, and the cup was placed in a stock mosquito cage. Adults emerged from pupae after about 48 hr. The mosquito cages were 30 x 30 x 30 cm Plexiglas® cubes with a circular hole cut in the top. The four sides contained windows covered with mesh screening. The hole at the top was covered with a nylon stocking to keep the mosquitoes inside, while allowing easy access to the inside of the cage. Adult mosquitoes were provided with a constant supply of geothermal water and a 10% weight by volume sucrose solution in 100 mL Erlenmeyer flasks. Each solution contained a Kimwipe® half submerged in the liquid to serve as a landing pad for mosquitoes. Both the water and sucrose solutions were refilled every second day.

Collection and care of green frogs

Green frogs were collected from a small pond on Prospect Road in Coldbrook,

Nova Scotia (45.0500 N, -64.5833) with the permission of the landowner, Dr. Phyllis

Harvey, and at Gaspereau Lake, south of Kentville, Nova Scotia (44.9692, -64.5296). A permit for frog collection was granted by the Nova Scotia Department of Natural

Resources. Frogs were captured using fishing nets, and transported back to the lab in medium-sized Rubbermaid® tubs containing a small amount of pond water from the collection site. In the lab, blood samples were taken from captured frogs to determine their approximate infection intensity. The facial (maxillary) or musculo-cutaneous vein of the frog was stuck with a 27-gauge syringe needle (Forzán et al., 2012), and a small amount of blood was collected in a Fisherbrand™ microhematocrit capillary tube. The wound was sanitized using Bactine™ antiseptic spray, and the collected blood was used to make a smear on a glass microscope slide, which was then stained with Hema-3™ to differentiate cells. The stained blood smears were viewed under 400x magnification with bright-field microscopy to calculate parasitaemia, a measure of infection intensity, by counting the number of parasites in 10 000 blood cells. Frogs with desired parasitaemia values were maintained in the freshwater room of the Animal Care Facility. They were kept in flow-through tanks at 23°C ± 2°C under a 14:10 light:dark cycle, and were fed crickets (Acheta domesticus) or mealworms (Tenebrio molitor) three times a week. Frogs without desired parasitaemia values were returned to their original site of collection.

Infection of mosquitoes

Female Cx. territans were selected from the main mosquito cage and transferred to a second Plexiglas® cage via an aspirator made of plastic tubing and a nylon stocking.

Geothermal water and 10% weight by volume sucrose solutions in 100 mL Erlenmeyer flasks were placed in the cage, and both were refilled every other day. Female mosquitoes were deprived of sucrose for 12 to 36 hr and water for 3 to 12 hr by removing the respective Erlenmeyer flask from the cage.

Following deprivation, a frog with a desired parasitaemia was transferred from the flow-through tank in the freshwater room to the insectary in a medium-sized

Rubbermaid® tub. The frog was placed in the Plexiglas® feeding cage containing the starved female mosquitoes. The frog was left in the feeding cage in the insect room with the lights at half intensity for 2 hr, and mosquitoes were able to feed during this period.

After the feeding period, the frog was transferred back to the flow-through tank in the freshwater room. Females that had taken a blood meal were easily identified by their red, distended abdomens, and were transferred using the aspirator to a third Plexiglas® cage and maintained until processing for sectioning. The holding cage also had 100 mL

Erlenmeyer flasks containing water and 10% weight by volume sucrose.

Processing of infected mosquitoes

Blood-fed mosquitoes were transferred from the holding cages at pre-determined times post-feeding (12 hr, 36 hr, 48 hr or 72h PF) into a plastic cup closed off with a nylon stocking. The mosquito was brought to the lab, where it was knocked out by flooding the cup with carbon dioxide gas. Under a dissection microscope, the head, legs

and wings were removed using dissecting scissors such that only the thorax and abdomen remained.

The bodies were fixed using 2.5% gluteraldehyde in Sörenson’s phosphate buffer

(Appendix 1). Bodies were then dehydrated through an ascending graded ethanol series from 50 to 70% ethanol. Specimens were stored in 70% ethanol in the refrigerator until the embedding process. Before specimens were embedded, they were continued through the ethanol series from 70% to absolute ethanol. They were then embedded in TAAB resin (Appendix 2).

Glass knives were made using a LKB 7800B KnifeMaker. Embedded mosquitoes were sectioned with these glass knives using a PowerTome X ultramicrotome. Thick (1

µm) sections placed in drops of RO on glass slides, which were then heat-fixed at 60°C ±

2°C on a slide warmer.

Sections were stained using 1% methylene blue and 2% basic fuchsin (Appendix

3). A small amount of Permount™ was used to mount the slides, and a long (50 mm) plastic coverslip was placed over the sections. Slides mounted with Permount™ were left to dry overnight, then observed with a Nikon Eclipse TE-2000-U microscope.

RESULTS

Two green frogs, with parasitaemia values of approximately 5%, were caught for this study; however, one of them died of a bacterial infection, so a single frog was used for feedings. A total of six Culex territans mosquitoes were successfully fed on this green frog, which had a pure infection of Hepatozoon clamatae that varied between 4.8% and

5.0%. Of these six, one mosquito was fixed at 12 hr post-feeding (PF), one was fixed at

36 hr PF, one was fixed at 48 hr PF, and one was fixed at 72 hr PF.

Development of Hepatozoon species in the midgut of Culex territans

At 12 hr PF, the midgut wall of the mosquito did not appear to have sustained any damage from parasite infection. The wall of the gut formed a continuous barrier around the blood meal, and the epithelium was intact and appeared healthy (Figs. 3, 4). At 36 hr

PF, gamonts and bacteria were apparent in the blood meal (Figs. 5, 6) and the midgut wall revealed detectable damage. Gamonts and bacteria were often present in close proximity to the areas of damage in the midgut wall (Fig. 7, 8). In some damaged areas, the epithelium had disappeared, although the basement membrane remained intact (Fig.

8). At 48 hr PF, the midgut wall around the blood meal was difficult to discern (Fig. 9) and epithelial cells were distorted and disorganized (Fig. 10) Parasites had clumped together, forming a few large aggregates within the blood meal (Fig. 11). Damage to the midgut wall was extensive in places, with large sections of the peritrophic matrix, midgut epithelium and basement membrane completely ruptured (Fig. 12). By 72 hr PF, the blood meal in the midgut was barely visible, with only a small amount of matter left in

the lumen (Fig. 13). In this mosquito, the midgut wall was completely intact, and the epithelium cells appeared normal (Fig. 13, 14).

Development of Hepatozoon species in the Malpighian tubules of Culex territans

At 12 hr PF, Malpighian tubules looked healthy (Fig. 15), with a clearly distinguishable lumen surrounded by normal-looking tubule cells containing many uric acid granules (Fig. 16). At 36 hr PF, tubules looked similarly healthy, and did not show any evidence of parasite infection (Figs. 17, 18). Malpighian tubules were in very close proximity to the midgut in the posterior end of the abdomen in mosquitoes at both 36 and

48 hr PF (Figs. 17, 19). By 48 hr PF, Malpighian tubules were riddled with Hepatozoon parasites, with some of the tubule cells infected with multiple zygotes (Figs. 20, 21).

Cells of the tubules were enlarged and pressed up against each other, and the lumen of the tubule was difficult to distinguish (Fig. 20). Gametes undergoing fertilisation, as well as newly formed zygotes, were observed in the Malpighian tubule cells (Fig. 22). At 72 hr PF, Malpighian tubules were infected with zygotes (Fig. 23), around which the parasitophorous vacuole was often apparent (Figs. 24-26). In this mosquito, which had a fewer number of zygotes infecting the tubules compared to the one at 48 hr PF, the lumen of the tubules appeared normal (Fig. 25).

3 4

FIGURES 3-4. Midgut of Culex territans 12 hr post-feeding. (3) Midgut near the posterior end of the abdomen, with epithelial layer (asterisk) and basement membrane

(arrow) (100x). (4) Complete midgut wall (arrow) encompassing the blood meal

(asterisk) (400x).

5 6

7 8

FIGURES 5-8. Midgut of Culex territans 36 hr post-feeding. (5) Hepatozoon gamonts

(arrows) in the blood meal (1000x). (6) Unidentified rod-shaped bacteria in the blood meal (1000x). (7) Hepatozoon gamonts (arrows) in an area of broken midgut wall (400x).

(8) Unidentified rod-shaped bacteria in an area of broken midgut wall, with an intact basement membrane (400x).

9 10

11 12

FIGURES 9-12. Midgut of Culex territans 48 hr post-feeding. (9) Midgut containing blood meal (asterisk) (100x). (10) Midgut containing blood meal (asterisk) (100x) (11)

Aggregation of Hepatozoon gamonts in the blood meal (400x). (12) Incomplete midgut wall with multiple breaks (arrows) (100x).

13 14

FIGURES 13-14. Midgut of Culex territans 72 hr post-feeding. (13) Midgut with a largely empty lumen (asterisk) and basement membrane (arrow) (400x). (14) Midgut epithelial cells with nuclei (arrow) (1000x).

15 16

FIGURES 15-16. Malpighian tubules of Culex territans 12 hr post-feeding. (15)

Malpighian tubule in longitudinal section (400x). (16) Malpighian tubule cells (asterisk) containing uric acid granules (arrowhead) surrounding the tubule lumen (arrow) (1000x).

17 18

FIGURES 17-18. Malpighian tubules of Culex territans 36 hr post-feeding. (17 )

Malpighian tubules (arrows) in longitudinal section in close proximity to the midgut wall

(asterisk) (100x). (18) Malpighian tubules in cross-section (400x).

19 20

21 22

FIGURES 19-22. Malpighian tubules of Culex territans 48 hr post-feeding. (19)

Malpighian tubules in longitudinal section (arrow) in close proximity to the midgut wall

(arrowhead) (100x). (20) Malpighian tubule cells infected with Hepatozoon zygotes

(arrow) and an indistinguishable lumen (arrowhead) (1000x). (21) Malpighian tubule cell infected with Hepatozoon zygotes (arrow) (1000x). (22) Fertilization of Hepatozoon macrogametes (arrowhead) by microgametes (arrow) in Malpighian tubule (1000x).

23 24

25 26

FIGURES 23-26. Malpighian tubules of Culex territans 72 hr post-feeding. (23)

Malpighian tubules in cross section (400x). (24) Cross section of a Malpighian tubule infected with a Hepatozoon zygote (arrow) surrounded by a parasitophorous vacuole

(asterisk) (1000x). (25) Malpighian tubule invaded infected with multiple Hepatozoon zygotes (arrow) and an open lumen (asterisk) (1000x). (26) Hepatozoon zygotes (arrow) surrounded by parasitophorous vacuole (asterisk) in a Malpighian tubule (1000x).

DISCUSSION

Effects of Hepatozoon on the midgut of Culex territans

At 12 hr post-feeding (PF), the midgut of the mosquito was fully intact around the entire blood meal (Fig. 3). Both the epithelial layer and the basement membrane were uninterrupted in all sections of the midgut (Figs. 3, 4). The blood meal had clearly expanded the midgut wall, pushing the epithelial layer closer to the cuticle. Mosquitoes, like many haematophagous insects, typically take blood meals that are twice their unfed body weight, since their frequency of feeding is unreliable (Lehane, 1998). The increase in the size of the midgut to accommodate the blood meal results in substantial pressure, and it is plausible that trauma to epithelial cells caused by large (30 x 10 µm) Hepatozoon parasites penetrating the gut wall may cause gut contents to leak or burst out, causing further destruction. At 36 hr PF, the midgut and blood meal had occupied most of the space in the body cavity throughout the length of most of the abdomen, so it is possible that the midgut burst open in this mosquito, potentially from parasite damage. As far as I am aware, accounts of such trauma caused by movement of blood parasites have not been reported; however, individual midgut epithelial cells have been observed to burst as a result of invasion by apicomplexans related to Hepatozoon species, in this case malaria parasites of the genus Plasmodium (Zieler and Dvorak, 2000).

Because of a technical issue, the sections of the midgut at 12 hr PF were approximately 20 µm thick, which caused the contents of the blood meal to be difficult to discern. At 36 hr PF, gamonts of the Hepatozoon parasites were evident throughout the blood meal (Fig. 5) and since both the 12 hr PF and 36 hr PF mosquitoes were fed on the same frog, we can assume that gamonts were also present in the blood meal at 12 hr PF.

High numbers of rod-shaped bacteria were observed in masses within the blood meal at

36 hr PF (Fig. 6). They were often seen in close proximity to many of the areas of the midgut epithelium that was broken; however, the basement membrane usually remained intact, and bacteria were not observed in the haemocoel (Fig. 8). Therefore it seems unlikely that septicaemia is the cause of death in mosquitoes that feed on frogs with heavy Hepatozoon infections.

There were a few regions of the midgut wall at 36 hr PF where Hepatozoon gamonts occurred adjacent to the ruptured regions of the epithelium and basement membrane (Fig. 7). Although parasites were not observed to be moving through the breakages of the midgut into the haemocoel or travelling through the fat body directly adjacent to the midgut, these observations do lend support to the hypothesis that the route that parasites travel through the mosquito includes penetration of the midgut and movement, however briefly, through the haemocoel.

At 48 hr PF, the epithelium and basement membrane of the midgut wall was broken (Fig. 12). At this point in time, gamonts that remained in the blood meal were found in large aggregations (Fig. 11). Aggregation of gamonts and bacteria in the blood meal is suggestive of the cellular immune responses of nodulation and encapsulation, in which pathogens are clumped together by haemocytes. However, immunity in a blood meal is typically a result of reactive oxygen and nitrogen species produced by the midgut epithelial cells (Bryant and Michel, 2014). Haemocytes are the immune cells responsible for the processes of nodulation and encapsulation (Hillyer, 2010), and, although recent studies have shown that blood meals induce an increase in the number of circulating haemocytes (Bryant and Michel, 2014), these immune cells are found primarily in the

haemocoel. Only a few sessile haemocytes, approximately 20 per mosquito, are attached to the basal surface of the midgut epithelium, and none are actually inside the gut (King and Hillyer, 2013). These aggregates of gamonts and bacteria have not been previously reported in blood meals of mosquitoes that have fed on frogs infected with Hepatozoon

(Desser at al., 1995; Kim et al., 1998), so further analysis is required to determine if such a feature is the result of an immune response. It is also possible that parasites are penetrating the midgut wall en masse, which could be the cause of the extensive trauma that was observed.

The midgut of the mosquito fixed at 72 hr PF was intact (Fig. 13), which was surprising considering the trauma observed in the previous two time points. The epithelial cells appeared healthy (Fig. 14), and breaks in the midgut wall were not seen. The blood meal was digested, and only breakdown products remained in the lumen, which is consistent with previous research that by approximately 60 hr PF, the abdomen is no longer distended and contains a minimal level of the breakdown products of digestion

(O’Gower, 1955).

There was obvious, extensive trauma to the midgut at both 36 and 48 hr PF.

While the morphological damage and ensuing physiological trauma are a plausible cause of death in mosquitoes that do die of heavy Hepatozoon infections, we cannot say with certainty that the parasites were the cause of such damage without evidence of gamonts moving through a compromised gut wall. Death as a result of midgut bacteria infiltrating the haemocoel and causing a fatal septicaemia seems less probable, as bacteria were not observed outside of the midgut in any of the four mosquitoes.

Effects of Hepatozoon on the Malpighian tubules of Culex territans

At 12 hr PF, the Malpighian tubules appeared normal (Fig. 15). The lumens of respective tubules were open, and cells did not showed signs of parasite infection (Fig.

16). The Malpighian tubules also appeared healthy at 36 hr PF (Figs. 17, 18), but

Hepatozoon parasites were present at 48 hr PF (Figs. 20, 21). Therefore parasites invade the Malpighian tubules between 36 and 48 hr. At 48 hr, most parasites were observed already as zygotes, however fertilization was also observed in a number of Malpighian tubule cells (Fig. 22), which suggests that invasion was relatively recent (i.e., closer to 48 hr PF). This observation is consistent with the previously reported timeline that by 2 days

PF, gamonts have entered the Malpighian tubules cells (Desser et al., 1995). Unique to this study was that many of the tubule cells had been infected by multiple zygotes (Fig.

20). As Hepatozoon zygotes develop into oocysts, with diameters between 60 and 260

µm (Smith et al., 1994; Desser et al., 1995), they will greatly expand the tubule, which has an outside diameter of only 35 µm (Dow et al. 1994). It is therefore not implausible to suggest that a tubule riddled with parasites would swell to such a degree that the lumen would constrict and tubule function would be greatly compromised.

We originally hypothesised that heavily infected mosquitoes were dying of either trauma to the midgut or of septicaemia caused by gut biota in the haemocoel. However, the high intensity of parasites in Malpighian tubules and concomitant severe constriction of the lumen of these organs observed at 48 hr PF provides another potential hypothesis for the cause of death. Malpighian tubules are necessary for excretion of nitrogenous wastes produced by metabolic processes. If, at high intensity, many of the tubule cells are infected by numerous parasites, causing the cells to expand and collapse upon the lumen

of the tubule, then the ability to filter the haemolymph and remove toxic waste products would be severely diminished, potentially resulting in death. However, nitrogenous waste in mosquitoes is in the form of uric acid, which has a relatively low toxicity in comparison to other forms of nitrogenous wastes (Cochran, 1975). Mosquito death as a result of a heavy infection of Hepatozoon species is typically observed within the first few days after blood feeding, so it is unlikely that the uric acid concentration in the haemolymph would reach a toxic level in that short amount of time. Additionally, the tubule lumens at mosquitoes fixed at 72 hr PF appeared similar to those at fixed at 12 and

36 hr PF. It is possible that the 72 hr PF mosquito was not as heavily infected as the 48 hr

PF mosquito and would not have died. However the lack of pathological effect on the

Malpighian tubules of the 72 hr PF diminishes the plausibility of the hypothesis that

Hepatozoon parasites cause death by reducing the efficacy of the excretory functions of the Malpighian tubules.

In many sections of all four mosquitoes, Malpighian tubules were observed directly opposed to the midgut (Figs. 17, 19), which is of interest in the hypothesis that

Hepatozoon species first penetrate the midgut wall, and then penetrate the Malpighian epithelium for further development. The route for a parasite through the mosquito is dangerous, and each transition between organs results, at least in the case of the malaria parasite Plasmodium berghei, in the death of thousands of parasites (Al-Olayan et al.,

2002). The haemocoel is the most threatening area to the parasite in terms of exposure to the dangers of the mosquito immune system, with between one and five thousand haemocytes on surveillance as they survey for intruders (Lavine and Strand, 2002; Bryant and Michel, 2014). However, if the distance the gamonts must travel through this ‘danger

zone’ to reach the more immune tolerated area of the Malpighian tubules is minimal, then this route is more conceivable.

Pathology of other mosquito-infecting parasites

Hepatozoon sipedon, a closely related species that parasitises the liver of frogs and the blood of snakes (i.e., as opposed to the blood of frogs, as with H. clamatae and H. catesbianae), uses Culex territans or Culex pipiens as its mosquito host. The development in the mosquito differs from H. clamatae and H. catesbianae in that gamonts penetrate the midgut wall and undergoes gametogenesis, fertilization and sporogony in the fat body cells of the haemocoel, rather than in the epithelial cells of the

Malpighian tubules (Smith et al., 1994). Although phylogenetic hypotheses reveal that these three Hepatozoon species form a monophyletic group (Barta et al., 2012), it is not yet known which life cycle is plesiomorphic. If H. clamatae and H. catesbianae evolved from H. sipedon, and given that the haemocoel is the most active region for immunity in a mosquito (Lavine and Strand, 2002), perhaps the route H. clamatae and H. catesbianae take through the mosquito is an adaptation to move from the haemocoel into the

Malpighian tubules as a means of avoiding the constant threat of the immune system.

In Culex mosquitoes that fed on snakes with heavy infections of H. sipedon, death has also been reported anecdotally (Smith et al., 1996; Harkness et al., 2010). This further suggests that the cause of death is not a result of diminished or destroyed

Malpighian tubule function, as H. sipedon does not affect the Malpighian tubules. It does, however, support the hypothesis that morphological trauma to the gut as a result of parasite penetration is the cause of death, as H. sipedon has been shown to penetrate the

midgut wall en route to the fat body (Smith et al., 1994). A number of other Hepatozoon species are also known to penetrate the midgut, including H. mocassini, a parasite of the blood of snakes and H. gracilis, a parasite of the blood of lizards, both species of which are vectored by the mosquito, Aedes aegypti (Bashtar et al, 1987; Lowichik et al., 1993).

Culex pipiens that were allowed to feed on their natural vertebrate host, the bean skink lizard (Trachylepsis quinquetaeniata), with heavy infections (11 to 20% parasitaemia) of

Hepatozoon gravilis, were found to have a 40% reduction in their lifespan compared to those fed a normal, uninfected blood meal (Galal, 2010).

Apicomplexans of the genus Plasmodium also invade mosquitoes when these vectors ingest blood. Following fertilization in the midgut, the motile zygotes, or ookinetes, which are the same size as Hepatozoon gamonts, penetrate the midgut epithelium and form oocysts on the haemocoel side of the midgut (Centers for Disease Control and

Prevention, 2012). Plasmodium falciparum, a human malaria parasite, has been found to travel intercellularly between epithelial cells of the midgut, whereas other species like P. berghei, a parasite of rodents, travels intracellularly through these epithelial cells (Meis et al., 1989). Mosquitoes infected with Plasmodium species that travel intracellularly were found to have 30% higher death rates within the first 3 days PF, whereas those infected with Plasmodium species that travel intercellularly did not sustain any damage to the midgut epithelium and did not show any increase in mortality (Gad et al., 1979; Meis et al., 1989). When the midgut epithelial cells are invaded by Plasmodium, the morphology of the cell is altered, including nuclear swelling, and the cell dies quickly. An apoptosis cascade is initiated upon invasion of the midgut cells, and some of the midgut epithelial cell die is a result of this programmed cell death (Vlachou et al., 2004). However, Zieler

and Dvorak (2000) argue that this cell death occurs too rapidly for it to be attributed to apoptosis, as they observed that invaded cells undergo morphological changes so rapidly that they explode. Baton and Ranford-Cartwright (2004) found that midgut epithelial cells that had been invaded by Plasmodium ookinetes may be ejected from the midgut wall, as many of them were found within the midgut lumen. Hepatozoon moccasini, a parasite of the blood of snakes and the mosquito, Aedes aegypti, has been observed by transmission electron microscopy to penetrate midgut epithelial cells to reach the haemocoel, the site of oocyst development in this species (Lowichik et al., 1993). If

Hepatozoon species cause death to epithelial cells that they penetrate, and heavy infections cause massive gut trauma, then the hypothesis that movement of many gamonts causes death of mosquitos may end up as the correct one.

Experimental challenges

To perform this study, it was necessary to catch green frogs that had naturally occurring high parasitaemias. It was found that, of those infected, the majority of infected frogs had low infections between 1 and 2% parasitaemia. Ultimately, only two green frogs with the desired levels of infection were caught. One of the frogs then died in

Animal Care as a result of a bacterial infection, leaving only one suitable frog.

The most significant challenge encountered in this study was convincing mosquitoes to take a blood meal from green frogs. The feeding cage was placed in various temperatures and under different light levels, from daylight to moonlight to complete darkness, but mosquitoes, which were feeding avidly on other frogs as part of my colleague’s BScH project, seemingly had little interest in the heavily infected frog.

The starvation and dehydration periods were increased in length, up to 36 hr and 12 hr, respectively, with little improvement. Green frog calls were played on speakers in the insectary to stimulate feeding, as phonotaxis to frog calls has been observed (Bartlett-

Healy et al., 2008b), but again very few mosquitoes fed. It has been suspected that mosquitoes may have the ability to sense some factor prevalent in a frog with a heavy parasite infection and therefore avoid feeding, although conclusive evidence was not found for this to occur with Hepatozoon species of frogs (Ferguson et al., 2013).

Culex territans have a limited life span in captivity, and will not lay viable eggs, so the amount of time available to do the feedings was limited to the summer months. In the end, only six mosquitoes were successfully fed on the frog and processed. Originally, thick (1 µm) sections of the mosquito were to be cut for maximum resolution. However, we found that the blood meal did not section well, and caused the knife to slip. Slightly thicker sections of 2 to 3 µm were taken instead, which alleviated this issue, and which did not adversely affect interpretation of results.

Further research and implications

The results suggest that Hepatozoon catesbianae and H. clamatae penetrate the midgut of the mosquito, briefly enter the haemocoel and infect the Malpighian tubules from the exterior of the organ, rather than travelling all the way down the alimentary canal to the mutual junction of the midgut, hindgut and Malpighian tubules, and invading the tubules from the inside. Results also suggest that death of Culex territans as a result of feeding on frogs with high parasitaemias is not a result of septicaemia, but more likely a result of morphological trauma to the gut. The next step in testing the hypothesis that

morphological midgut trauma is the cause of death would be to dissect mosquitoes at various time periods post-feeding to observe any visible trauma to the gut. The entire gut could also be fixed and processed for scanning electron microscopy to view any pathological effects at high resolution.

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APPENDICES

Appendix 1: Fixation Protocol

Making Sörenson’s Buffer

1) Mix 18.89 g dibasic NaP anhydrous in 1 L of distilled water to create 0.133M

Na2HPO4

2) Mix 9.06 g monobasic potassium-phosphate in 500 mL of distilled water to create

0.133M KH2PO4

3) Mix 71.5 mL Na2HPO4 and 28.5 mL KH2PO4

4) Adjust pH to 7.2

a. If pH is too high, add 1M hydrochloric acid drop wise with a micropipette

until the desired pH is reached

b. If pH too low, add 1M sodium hydroxide drop wise with a micropipette until

the desired pH is reached

Fixing

1) Mix 25% gluteraldehyde stock solution with Sörenson’s buffer in a 1:10 ratio to

create a 2.5% solution

2) Soak mosquito in 0.5 mL 2.5% gluteraldehyde in Sörenson’s buffer on ice for 1 hr

3) Rinse in 0.5 mL pure Sörenson’s buffer at room temperature for 15 min

4) Dehydrate in 0.5 mL of 30% ethanol at room temperature for 15 min

5) Dehydrate in 0.5 mL of 50% ethanol at room temperature for 15 min

6) Dehydrate in 0.5 mL of 70% ethanol, store in fridge until ready to embed

Appendix 2: Embedding Protocol

Creating TAAB Resin (makes 20 mL, can multiply to make more)

1) Mix 10 mL TAAB resin, 6.75 mL DDSA and 3.25 mL MNA, stir constantly for 15 to

20 min until mixed well

2) Stir in 0.2 mL DMP-30 and mix well, stirring for minimum 5 min

3) Store in 20 mL syringes in freezer

Embedding

1) Dehydrate specimen again in 0.5 mL of 70% ethanol at room temperature, 15 min

2) Dehydrate in 0.5 mL of 95% ethanol at room temperature, 15 min

3) Dehydrate in two changes of 0.5 mL absolute ethanol at room temperature, 15 min

each

4) Rinse in two changes of 0.5 mL propylene oxide at room temperature, 15 min each

5) Place in a 1:1 propylene oxide to TAAB resin mix (1 mL total) for 1 hr

6) Place in a 1:3 propylene oxide to TAAB resin mix (1 mL total) for 2 hr

7) Place in 1 mL of pure TAAB resin overnight on a rocker table

8) Block up mosquito in a flat embedding mould using fresh resin, with mosquito as

close to the base of the cavity as possible

9) Polymerize in TAAB MK II embedding oven at 60°C ± 1°C oven for exactly 48 hr

10) Remove blocks from oven, let stand at least 1 hr before sectioning

Appendix 3: Staining Protocol

Creating 1% methylene blue in borax

1) Mix 1.0 g methylene blue with 1.0 g sodium tetraborate (borax) in 100mL

distilled water

2) Filter the stain

Creating basic fuchsin

1) Mix 2.0 g basic fuchsin in 100 mL distilled water

2) Filter the stain

Staining sections

1) Flood the slide with 1% methylene blue in borax, place on hot plate for 10 to 20 s