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POTENTIAL USE OF THE DIGENEAN PARASITE, PLAGIORCHiS

ELEGANS, AS A BIOLOGICAL CONTROL AGENT OF BIOMPHALARIA

GLABRATA (PULMONATA: PLANORBIDAE) AND SCHISTOSOMA

MANSONI (DIGENEA: SCHISTOSOMATIDAE)

By

Simon Daoust

Department of Natural Resource Sciences McGiII University, Montreal, Quebec

June 2008

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science

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1+1 Canada Short Running Title: P. elegans as a biological control agent of Schistosomiasis ABSTRACT

The impact of a primary infection with the incompatible digenean Plagiorchis elegans to groups composed of young, juvenile and adult Biomphalaria glabrata and a challenge infection with the compatible parasite Schistosoma mansoni was examined. Egg production of young B. glabrata was significantly reduced by P. elegans at the higher exposures of 16 eggs per snail, while egg production of adult B. glabrata was reduced at exposures of 8 and 16 eggs per snail. Egg production of juvenile B. glabrata snails was not significantly affected by any of the levels of P. elegans exposures. The survivorship of juvenile B. glabrata snails was significantly lowered by an exposure to 16 P. elegans eggs per snail. The survivorship of adult snails was significantly reduced by exposures to both 8 and 16 P. elegans eggs per snail. Interestingly enough, the survivorship of young B. glabrata snails was not significantly affected by P elegans exposure. Snails harboring small P. elegans infections acquired a resistance to Schistosoma mansoni infection. Infection with P. elegans did not have any significant effect on the egg production of the young and adult sympatric snails, Helisoma trivolvis trivolvis. The survivorship of adult H. trivolvis trivolvis was not significantly affected by exposure to P elegans. This being said, young H. t. trivolvis that were exposed to P. elegans had a higher survivorship than did control snails. Exposed Bulinus truncatus snails laid up to 50% fewer eggs for the first five weeks due to P. elegans infection. There was however no effect on the survivorship of the snails.

iii RESUME

L'effet de l'exposition à des doses graduées d'une infection primaire avec le digénien incompatible Plagiorchis elegans ainsi qu'une infection secondaire avec le parasite compatible Schistosoma mansoni, sur des groupes comprenant des jeunes, juvéniles et adultes Biomphalaria glabrata. À 16 œufs par escargot, la production d'œufs de jeunes B. glabrata a été considérablement réduite par P. elegans, alors que la production d'œufs d'escargots B. glabrata adultes a diminué suite à des niveaux d'exposition de 8 et 16 œufs par escargot. P. elegans n'a eu aucun effet significatif sur la production d'œufs des B. glabrata juvéniles. À 16 œufs de P.elegans par escargot, la survie des B. glabrata juvéniles a diminué de façon considérable. La survie des B. glabrata a diminué de façon significative suite à des niveaux d'exposition de 8 et 16 œufs P.elegans par escargot. Les escargots porteurs de faibles infections de P.elegans développent une résistance aux infections de Schistosoma mansoni. L'exposition des escargots sympatriques Helisoma trivolvis trivolvis jeunes et adultes à l'infection P.elegans n'a eu aucun impact significatif sur leur production d'œufs. L'exposition des adultes H. t. trivolvis au P.elegans n'a eu aucun effet significatif sur leur survie. Notons que les jeunes H.t. trivolvis exposés au P.elegans ont eu un % moyen de survie de 89.4 ±1.6%, alors que les escargots du groupe témoin avaient un % moyen de survie considérablement inférieur, de 74.4 ±3.6%. La production d'œufs des escargots Bulinus truncatus exposés à l'infection P.elegans a diminué de 50% durant les cinq premières semaines. Par contre, aucun impact sur la survie des escargots n'a été observé.

iv ACKNOWLEDGEMENTS

First and foremost I would sincerely like to express my thanks to my supervisor, Dr Manfred E. Rau. His patience, his guidance, his wisdom and his love of biology have been instrumental through the course of my studies. Thanks to Dr. J. D. McLaughlin, Concordia University, for helping me design my project and for reading my thesis; your input has always been greatly appreciated. I would like to thank Dr Paul J. Albert, Concordia University, for initiating me to the wonderful world of insects, parasites and scientific research. His mentorship, his guidance and his dedication to his students have inspired me to follow in his footsteps. To Jeremy, Christian and Krista, your support, love and understanding through the last two years has been paramount to my completing this work. Thank you so very much. With a PhD coming up, I am going to need you all in the years to come. A special thanks goes to Brian Mader for always being there for me when times got rough, for proof reading my work and for assisting me in the setup of my biggest experiment. I seriously could not have done this without you. Thank you my friend. To my parents Michelle and Sylvain, thank you for nurturing the scientist in me (not only allowing me to keep insects in the freezer but also taking care of my giant snails when I was away). Your support, your love and your encouragement have made this work possible. I would also like to thank my friends: Anne, Raphael, Nadia, Michael, Kari, Mariana, Isabelle, Jessie, Virginia and Sue for their love and support.

? CONTRIBUTIONS OF THE AUTHORS

As the author of this work and first author of the manuscripts, I was responsible for the conception, design, setup, data gathering and data analysis of the experiments. My responsibilities also included ordering and maintaining the snails, parasites and other intermediate hosts used in the experiments.

Chapter 3 and Chapter 4 were coauthored by Dr M. E. Rau (McGiII University), Dr J. D. McLaughlin (Concordia University) and M. Brian Mader (Concordia University). Dr M. E. Rau served as mentor and supervisor from conception to submission of this thesis. He helped in designing the experiments as well as was responsible for the maintenance of the parasitic life cycle. Dr J. D. McLaughlin also served as supervisor, helping in the design and creation of the experimental protocol. M. Brian Mader helped in the set up of some of the larger experiments as well as the weekly monitoring of the snail's reproductive output. He also gave valuable input and feedback with regards to the manuscripts.

Vl TABLE OF CONTENTS

TITLEPAGE i

SHORTRUNNINGTIITLE ii

ABSTRACT iii

RÉSUMÉ iv

ACKNOWLEDGEMENTS ?

CONTRIBUTIONS OF THE AUTHORS vi

TABLEOFCONTENTS ?

LIST OF FIGURES x

LISTOFTABLES xiv

CHAPTER 1: INTRODUCTION 1

Literature Cited 4

CHAPTER 2: LITERATURE REVIEW 6

Schistosomiasis a review 6

Biomphalaria glabrata 13

Bulinus truncatus 15

Helisoma trivolvis trivolvis 15

Stagnicolaelodes 16 Plagiorchis elegans 16 Immune system of the snail Biomphalaria glabrata 17 Interactions between digeneans and their snail hosts 19

Interactions between digeneans within their snail host. 21

Control of schistosomiasis 23

vii Infection of P. elegans in the incompatible snail host B. glabrata,

a Review 26

Digeneans and their Impact on Aquatic Communities 27

Literature Cited 29

CHAPTER 3: THE EFFECTS OF A PRIMARY INFECTION WITH

PLAGiORCHIS ELEGANS (DIGENEA: PLAGIORCHMDAE) AND A CHALLENGE INFECTION OF SCHISTOSOMA MANSONI(DiGEUEA: SCHISTOSOMATIDAE) ON A COMMUNITY OF THE INCOMPATIBLE HOST, BIOMPHALARIA GLABRATA (PULMONATA: PLANORBIDAE), COMPOSED

OF DIFFERENT AGE CLASSES 44

Abstract 45

Introduction 46

Materials and Methods 47

Results 54

Discussion 57

Acknowledgments 60

Literature Cited 61

Figures 65

Tables 89

CONNECTING STATEMENT 1 97

Vili CHAPTER 4: THE EFFECTS OF PLAGiORCHIS ELEGANS (DIGENEA: PLAGIORCHIIDAE) ON THE REPRODUCTION AND SURVIVORSHIP OF THE INCOMPATIBLE HOSTS BULINUS TRUNCATUS (PULMONATA: PLANORBIDAE) AND HELISOMA TRIVOLVIS TRIVOLVIS (PULMONATA: PLANORBIDAE) 98

Abstract 99

Introduction 100

Materials and Methods 101

Results 106

Discussion 107

Acknowledgments 110

Literature Cited 111

Figures 114

CHAPTER 5: GENERAL DISCUSSION 126

Literature Cited 129

APPENDICES 130

COAUTHERWAIVERS 131

ANIMAL CARE FORM 132

IX LIST OF FIGURES

CHAPTER 3

Figure 1 A Mean number (±S.E.) of eggs produced by Biomphalaria glabrata exposed as young to 0, 1or 4 P. elegans eggs 65 Figure 1 B Mean number (±S.E.) of eggs produced by Biomphalaria glabrata exposed as young to 0, 8 or 16 P. elegans eggs..65 Figure 2A Mean number (±S.E.) of eggs produced by Biomphalaria glabrata exposed as juvenile to 0, 1 or 4 P. elegans eggs..67 Figure 2B Mean number (±S.E.) of eggs produced by Biomphalaria glabrata exposed as juvenile to 0, 8 or 16 P. elegans egg..67 Figure 3A Mean number (±S.E.) of eggs produced by Biomphalaria glabrata exposed as adult to 0, 1 or 4 P. elegans eggs 69 Figure 3B Mean number (±S.E.) of eggs produced by Biomphalaria glabrata exposed as adult to 0, 8 or 16 P. elegans eggs.. ..69 Figure 4A Log % survivorship curve of young Biomphalaria glabrata exposed to 0, 1 or 4 P. elegans eggs 71 Figure 4B Log % survivorship curve of young Biomphalaria glabrata exposed to 0, 8 or 16 P. elegans eggs 71 Figure 5A Log % survivorship curve of juvenile Biomphalaria glabrata exposed to 0, 1 or 4 P. elegans eggs 73 Figure 5B Log % survivorship curve of juvenile Biomphalaria glabrata exposed to 0, 8 or 16 P. elegans eggs 73 Figure 6A Log % survivorship curve of adult Biomphalaria glabrata exposed to 0, 1 or 4 P. elegans eggs 75 Figure 6B Log % survivorship curve of adult Biomphalaria glabrata exposed to 0, 8 or 16 P. elegans eggs 75 Figure 7A Log % survivorship curve of young Biomphalaria glabrata exposed to 0, 1 or 4 P. elegans eggs with a challenge infection with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans exposure 77 Figure 7B Log % survivorship curve of young Biomphalaria glabrata exposed to 0, 8 or 16 P. elegans eggs with a challenge infection with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans exposure 77 Figure 8A Log % survivorship curve of juvenile Biomphalaria glabrata exposed to 0, 1 or 4 P. elegans eggs with a challenge infection with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans exposure 79 Figure 8B Log % survivorship curve of juvenile Biomphalaria glabrata exposed to 0, 8 or 16 P. elegans eggs with a challenge infection with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans exposure 79

Xl Figure 9A Log % survivorship curve of adult Biomphalaria glabrata exposed to 0, 1 or 4 P. elegans eggs with a challenge

infection with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans exposure 81 Figure 9B Log % survivorship curve of adult Biomphalaria glabrata exposed to 0, 8 or 16 P. elegans eggs with a challenge

infection with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans exposure 81 Figure 1 0 The probability of death for the snail Biomphalaria glabrata within the first two weeks post P elegans

exposure 83 Figure 1 1 The probability of death for Biomphalaria glabrata between weeks three and sixteen after P. elegans infection 85 Figure 1 2 The probability of death for Biomphalaria glabrata three weeks after P elegans and one week after S. mansoni

infection 87

CHAPTER 4

Figure 1 Mean egg production (± S. E.) of adult Helisoma trivolvis

trivolvis exposed to 0 and 16 P. elegans eggs. The snails were paired at week 14 postexposure 115

XIl Figure 2 Mean egg production (± S. E.) of young Helisoma trivolvis

trivolvis exposed to 0 or 16 P. elegans eggs. The snails were paired at week 14 after exposure 117 Figure 3 Log % survivorship of adult Helisoma trivolvis trivolvis exposed to 0 or 16 P. elegans eggs 119 Figure 4 Log % survivorship of young Helisoma trivolvis trivolvis exposed to 0 or 16 P. elegans eggs 121 Figure 5 Mean egg (± S. E.) production of adult Bulinus truncatus

infected with Oor 16 P. elegans eggs 123 Figure 6 Log % survivorship of adult Bulinus truncatus exposed to 16 P. elegans eggs 125

xiii LIST OF TABLES

CHAPTER 3

Table I Tuckey multiple comparisons test between the mean egg

production of young Biomphalaria glabrata exposed to 0, 1 , 4, 8 or 16 P. elegans eggs 89

Table Il Tuckey multiple comparisons test between the mean egg production of adult Biomphalaria glabrata exposed to 0,1,

4, 8 or 16 P. elegans eggs 90 Table Kaplan-Meier survivorship analysis of the % survivorship curve of young Biomphalaria glabrata exposed to 0, 1 ,

4, 8 or 16 P. elegans eggs 91 Table IV Kaplan-Meier log ranked survivorship analysis comparing

the % survivorship curves of young Biomphalaria glabrata exposed to 1 , 4, 8 or 16 P. elegans eggs to the control

snails 91

Table V Kaplan-Meier survivorship analysis of the % Survivorship curve of juvenile Biomphalaria glabrataexposeä to 0, 1 , 4,

8 or 16 P. elegans eggs 92 Table Vl Kaplan-Meier log ranked survivorship analysis comparing

the % survivorship curves of juvenile Biomphalaria glabrata exposed to 1 , 4, 8 or 16 P. elegans eggs to the control

snails 92

XlV Table VII Kaplan-Meier survivorship analysis of the % Survivorship

curve of adult Biomphalaria glabrata exposed to 0, 1 , 4,8 or 16 P. elegans eggs 93 Table Vili Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of adult Biomphalaria glabrata exposed to 1 , 4, 8 or 16 P. elegans eggs to the control

snails 93

Table IX Kaplan-Meier survivorship analysis of the % Survivorship curve of young Biomphalaria glabrata exposed to 0, 1 , 4, 8 or16 P. elegans eggs with a challenge infection with 7 S.

mansoni miracidia, three weeks after P. elegans exposure 94 Table X Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of young Biomphalaria glabrata exposed to 1 , 4, 8 or 16 P. elegans eggs to the control

snails with a challenge infection with 7 S. mansoni miracidial, three weeks after P. elegans exposure 94 Table Xl Kaplan-Meier survivorship analysis of the % Survivorship curve of juvenile Biomphalaria glabrata exposed to 0, 1 , 4, 8 or 16 P. elegans eggs with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans

exposure 95

xv Table XII Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of juvenile Biomphalaria glabrata exposed to 1 , 4, 8 or 16 P. elegans eggs to the control snails with a challenge infection with 7 S. mansoni miracidia,

three weeks after P. elegans exposure 95 Table XIII Kaplan-Meier survivorship analysis of the % Survivorship

curve of adult Biomphalaria glabrata exposed to 0, 1 , 4, 8 or 16 P. elegans eggs with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans exposure 96 Table XIV Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of adult Biomphalaria glabrata exposed to 1 , 4, 8 or16 P. elegans eggs to the control snails with a challenge infection with 7 S. mansoni miracidia, three weeks after P. elegans exposure 96

XVl CHAPTER 1

INTRODUCTION

The present study deals with the relationship between the digenean parasite Plagiorchis elegans (Rudolphi, 1802) and three incompatible snail hosts, two of which are vectors of parasites causing human schistosomiasis. I proposed to investigate the possibility of utilizing P. elegans as an agent in the biological control of human schistosomiasis.

Human schistosomiasis remains one of the world's most prevalent parasitic infections and has significant economic and public health consequences (Chitsulo et al., 2000). Schistosomiasis is endemic to 76 countries and territories, primarily in the developing world (Engels et al., 2002). It is estimated that 200 million people are infected, of whom 120 million are symptomatic and 20 million people manifest severe disease (Chitsulo et al., 2000). Three species of schistosomes have been identified as the primary causative agents of human schistosomiasis: Schistosoma haematobium, Schistosoma mansoni and Schistosoma japonicum (Roberts and Janovy, 2005). Like all digenean parasites, schistosomes must complete a first round of asexual reproduction within a snail intermediate host prior to becoming infective to their human host

(Wright, 1973).

1 It is for this reason that much of the work dedicated to the prevention of schistosomiasis has dealt with the control of the intermediate snail host, primarily by means of chemical molluscicides (WHO, 1965). Because of the high production cost of such molluscicides, their adverse environmental impact, and the development of resistance by the snail hosts has made it imperative to develop alternative control strategies, among them the use of competing digenean parasites (Thomas, 1973; Madsen, 1990; Yi et al., 2005). Not only are digenean larvae capable of castrating their snail hosts (Nassi, 1979; de Jong- Brink, 1995), they have also been shown to be mutually antagonistic when they are present in the same snail host (Lie et al., 1968a). In this context, several digeneans have shown significant potential as agents in the biological control of the parasites and the snail vectors of human schistosomiasis. Plagiorchis elegans will establish patent infections in both Stagnicola elodes and Lymnaea stagnalis. Further investigation by Zakikhani and Rau (1998) revealed that the parasite not only castrates the above compatible snail hosts, but also establishes an infection in the incompatible snail Biomphalaria glabrata. However, it is unable to complete its development within this host. Although minute, mother sporocysts were found in the tissues of B. glabrata, there was no evidence of daughter sporocyts or cercariae (Zakikhani and Rau, 1998). Nevertheless, all infected snails developed symptoms of parasitic castration (Zakikhani and Rau, 1998). Perhaps more importantly, there was a concomitant reduction in the number of cercariae shed by a compatible challenge infection with S. mansoni (Zakikhani et al., 2003).

2 Platero (2004) studied the effects of various exposure concentrations of P.elegans on individual B. glabarata of different age classes. Her study revealed that snails responded in a graded fashion to the different levels of P. elegans. Furthermore, young, juvenile and adult B. glabrata were shown to differ in their susceptibility to P. elegans infections. These results may not however accurately reflect what would happen under natural conditions. Snails live in communities and therefore would not be exposed to P. elegans individually. It is likely that snails of different sizes have different probabilities of ingesting the eggs of P. elegans, thereby protecting sympatric conspecifics from infection. What's more, no other incompatible snail species has been exposed to P. elegans infection. This study thus expands our knowledge of host-parasite associations by exposing the snail vectors of another human schistosome. Chapter 1 introduces the conceptual framework and the objectives of this study. Chapter 2 provides a review of the pertinent literature. Chapter 3 examines the impacts of P. elegans and S. mansoni on a community of the incompatible snail host Biomphalaria glabrata composed of different size classes. Chapter 4 asseses the impact of P. elegans on the reproduction and survivorship of the incompatible snail hosts Bulinus truncatus the snail host of S. haematobium, as well as Helisoma trivolvis trivolvis, a snail species occurring sympatrically with S. elodes and P. elegans. Chapter 5 is a general discussion and summary of the findings presented in previous chapters.

3 LITERATURE CITED

Chitsulo, L., Engels, D., Montresor, ?., Savioli, L. (2000). The global status of schistosomiasis and its control. Acta Tropica 77: 41-51. de Jong-Brink, M. (1995). How schistosomes profit from the stress responses they elicit in their hosts. Advances in Parasitology 35: 177-256.

Engels, D., Chitsulo, L., Montresor, A., Savioli, L. (2002). The global epidemiological situation of schistosomiasis and new approaches to

control and research. Acta Tropica 82(2): 139-146.

Lie, J. K., Basch, P. F, Heyneman, D. 1968a. Antagonism between two species of echinostomes (Paryphostomum segregatum and Echinostoma lindoense) in snail Biomphalaria glabrata. Zeitschrift Fur Parasitenkunde 30: 1 17-125.

Madsen, H. (1990). Biological methods for the control of freshwater snails. Parasitology Today 6: 237-241 .

4 Nassi, H. (1979). Coincidence entre le blocage precoce de la ponte de Biomphalaria glabrata (Gastropada: Pulmonata) et la localization cerebrale des jeunes redies mères de Ribeiroia marini guadeloupensis (Trematoda: Cathaemasidae). Comptes Rendus de l'Académie des

Sciences Paris 298D: 165-168.

Platero, I.A. (2004). The effects of parasites dose, host size and the method of exposure on the reproductive capacity and survival of Biomphalaria glabrata infected with the incompatible digenean, Plagiorchis elegans. M. Sc. Thesis. McGiII University, Montreal, Canada.

Roberts, L. S., Janovy, J. (2005). Foundations of Parasitology. Seventh Edition. McGraw Hill. New York. USA. p. 248-262.

Thomas, J. D. (1973). Schistosomiasis and the control of molluscan hosts of human schistosomes with particular reference to possible self-regulatory mechanisms. Advances in Parasitology 1 1 : 307-394.

World Health Organization WHO. (1965). Snail control in the prevention of

bilharziasis. Geneva.

Wright, CA., (1973). Flukes and snails. Macmillan, New York, New York, 168 p.

5 Yi, Y., Xing-Jian, X., Hui-fen, D., Ming-Sen, J., Hui-Go, Z. (2005). Transmission control of schistosomiasis japónica: implementation and evaluation of different snail control interventions. Acta Tropica 96: 191-197.

Zakikhani, M., Rau, M. E. (1998a). Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection on the reproduction of Biomphalaria glabrata (Pulmonata: Planorbidae). Journal of Parasitology 84 (5): 927-930.

Zakikhani, M., Smith, J. M., Rau, M. E. (2003). Effects oí Plagiorchis elegans (Digenea: Plagiorchiidae) infection of Biomphalaria glabrata (Pulmonata: Planorbidae) on a challenge infection with Schistosoma mansoni (Digenea: Schistosomatidae). Journal of Parasitology 89 (1): 70-75.

6 CHAPTER 2

LITERATURE REVIEW

This chapter reviews the relevant background information on the history, biology and treatment of human schistosomiasis. This is followed by a brief outline of the life history of the snails Biomphalaria glabrata (Say, 1818), Bulinus truncatus, Helisoma trivolvis trivolvis (Say, 1816) and Stagnicola elodes (Say, 1821), as well as the life history of the digenean parasite Plagiorchis elegans (Rudolphi, 1802). Chapter 2 provides a review of the molluscan immune response, a review of the interactions between digeneans and their hosts, as well as the interactions of digeneans within their hosts. I will then provide a brief description of the methods previously used to control schistosomiasis, including previous work done on the interactions between the incompatible parasite P. elegans and the snail Biomphalaria glabrata. Lastly I will review the impact of parasites on community structure.

Schistosomiasis, a Review Human schistosomiasis remains one of the world's most prevalent parasitic infections and has significant economic and public health consequences (Chitsulo et al., 2000). Schistosomiasis is endemic to 76 countries and territories, and continues to be a global public health concern in the developing world

7 (Engels et al., 2002). It is estimated that 200 million people are infected, of whom 120 million are symptomatic and 20 million people have severe disease (Chitsulo et al., 2000). For thousands of years a chronic endemic disease, characterized by blood in the urine and by various bladder troubles, has been known to exist in Egypt and elsewhere (WHO, 1959). The eggs of the parasite which cause the disease have been discovered on the kidneys of mummies of the Twentieth Dynasty (1250-1000 B.C.) (WHO, 1959). The first Europeans to record contact with schistosomiasis were surgeons with Napoleon's army in Egypt (1799-1801) (Roberts and Janovy, 2005). They remarked on an elevated presence of blood in the urine of the soldiers (hematuria), although the causal agent was unknown to them at the time. Nothing further was learned about the causative agent of this disease until fifty years later, when a young German parasitologist, Theodor Bilharz, discovered the worm that was responsible (Roberts and Janovy, 2005). It is in honour of Bilharz that schistosomiasis is often referred to as Bilharziasis.

Schistosomiasis is caused by digenean parasites of the family Schistosomatidae (Roberts and Janovy, 2005). Three species of schistosomes have been shown to be the primary causative agents of human schistosomiasis and therefore are of great medical importance: Schistosoma haematobium, Schistosoma mansoni and Schistosoma japonicum (Roberts and Janovy, 2005). For the purpose of this work, I will focus our attention on Schistosoma mansoni. Which has the largest geographic range of all schistosomes infecting humans (Morgan et al., 2001). It is present throughout most of the African continent,

8 Indonesia, Thailand, China and Japan. It is also the only schistosome to be found in the New World, with foci located in Antigua, Brazil, the Dominican Republic, Guadeloupe, Martinique, Montserrat, Puerto Rico, Suriname and Venezuela (Chitsulo et al., 2000). Files (1951) suggested that the presence of S. mansoni in the New World is due to extensive slave trade in the 16th and 19th centuries. Further studies using enzyme-linked electrophoresis supported this theory as researchers detected very little variation between South American and African isolates (Fletcher et al., 1981). There exists considerable sexual dimorphism within the genus. Males are shorter and stouter than females, possessing a ventral, longitudinal groove, the gynecophoral canal. The female resides within this canal and the couple remains paired for life (see Roberts and Janovy, 2005). Adult S. mansoni live in the portal veins draining the large intestine of their human definitive hosts. Here the females deposit their eggs. The eggs must then traverse the wall of the portal veins and the surrounding connective tissue before they can be expelled by way of the feces of their host (Roberts and Janovy, 2005). The mechanism by which the eggs cross through tissue is not fully understood. It has been suggested that endothelial cells lining the venule actively move over, away from the schistosome eggs, excluding them from the lumen (File 1995). Doenhoff et al. (1985) postulated that the worm then exploits the host's immune response to transport its eggs to the lumen of the gut. The egg stimulates the formation of a granuloma of motile immune cells to enclose itself and allow passage through the tissues of the gut (Doenhoff et al. 1985). This

9 being said, only about one third of eggs produced are passed out with the feces. The remaining two-thirds either build up in the gut wall or are swept up by the blood stream to be trapped and lodged in the liver or capillary beds of other organs (Roberts and Janovy, 2005). During their tissue migration the miracidia within the eggs develop and become ready to hatch (Wright, 1967). Hatching is stimulated by the low osmolarity of fresh water (Wright, 1967). The ciliated miracidium leaves the egg and swims ceaselessly during its short life. Miracidia have one to two hours to find and penetrate their intermediate snail host before they die. S. mansoni has several snails hosts in which they can successfully develop, all of which are of the genus Biomphalaria (see Roberts and Janovy, 2005). Host snail populations constitute a fragmented and heterogeneous environment for the free-swimming miracidia (Théron et al., 1998). Individual snails within a population are morphologically (size), physiologically (sexually mature/immature, infected/non-infected) and genetically different (susceptible / resistant) (Theron et al., 1998). As a consequence, the miracidia face a number of critical choices. In the majority of cases, the survival success of the miracidia is negatively correlated with host size, due to an increase in host resistance (Anderson et al., 1982; McKindsey et al., 1995, Théron et al., 1998;). Interestingly enough, Théron et al. (1998) have demonstrated that the miracidia of S. mansoni preferentially selected subadult B. glabrata snails over young, juvenile or adult snails. Furthermore, the prevalence of infection within a snail population is characteristically low in areas where schistosomiasis is endemic,

10 wherein 1% to 5% of compatible snails are usually infected (Anderson and May,

1979). Within the snail, the miracidium develops into a mother sporocyst, not far from the point of penetration (Wright, 1967). The mother sporocyst produces a large number of germ-balls asexually from its body wall; developing into daughter sporocysts (Wright, 1967). The daughter sporocysts typically migrate to other organs of the snail, particularly the digestive gland. Furcocercous cercariae start to emerge from the daughter sporocysts and the snail host about four weeks after initial penetration by the miracidium (Roberts and Janovy, 2005). The emergence of the cercariae is a highly controlled event. Depending on the chronobiology of the strain of schistosome; they can emerge from the snail at dusk or at dawn (Theron et al., 1997). Schistosomes do not require a second intermediate host in order to complete their life-cycle. The cercariae may swim for up to 3 days on stored glycogen reserves, but only remain infective for the first 2 days. During this time they may find and penetrate their human host. The cercariae penetrate human skin using histolytic enzymes and by vigorous body and tail action. It takes about half an hour for complete penetration of the epidermis. Once within the epidermis, cercariae shed their tails and transform into schistosomules. The schistosomules migrate through the circulatory system and reach the liver by way of the hepatoportal system where they continue their development. Three-weeks thereafter the worms pair and migrate up the hepatoportal vein to the venules of the gut (see Roberts and Janovy, 2005).

11 Schistosomiasis is unusual among parasitic infections in that pathogenesis is almost entirely attributable to the eggs and not to adult worms (Elsdon-Dew, 1967). As previously mentioned, two-thirds of the eggs produced are trapped in the gut wall or are swept up by the blood stream to capillary beds of the liver and other organs. The eggs, surrounded by granuloma of immune cells, continuously leak antigens and do this over a considerable length of time (Elsdon-Dew, 1967). Therefore, the primary lesion in schistosomiasis is a delayed type hypersensitivity reaction in the tissues surrounding the eggs (Elsdon-Dew, 1967). Human schistosomiasis is often divided into three phases: migratory, acute and chronic (Roberts and Janovy, 2005). The migratory phase is asymptomatic; it encompasses the time from penetration of the cercariae until the mature flukes begin to lay their eggs (Roberts and Janovy, 2005). The acute phase occurs when the schistosomes begin to produce eggs, approximately 4 to 10 weeks post initial infection (Roberts and Janovy, 2005). This phase is marked by chills, fever, fatigue, headache, malaise, muscle aches, lymphadenopathy and gastrointestinal discomfort (Elsdon-Dew, 1967). Chronic phase patients usually live in endemic areas and tend to be asymptomatic (Roberts and Janovy, 2005).

However, in about 8% of cases, the development of egg granulomas and accompanying fibrosis of the liver seriously impedes portal blood flow (Roberts and Janovy, 2005). Furthermore, the presence of eggs trapped in the capillaries of the spleen, coupled with the chronic passive congestion of the liver, induces splenomegaly (Mousa et al., 1967; Roberts and Janovy, 2005). Although

12 Schistosomiasis is not usually associated with high levels of mortality, American and British soldiers stationed in the Far East and in West Africa during the Second World War had a significantly high number of mortality due to the acute phase of schistosomiasis (Mostofi, 1967). Indeed, schistosomiasis can be lethal to individuals who have not previously been exposed to the parasite and are brought into an endemic area (Mostofi, 1967). The drug of choice for the treatment of schistosomiasis is praziquantel, an anti-helminthic drug effective against all species of schistosomes (Chandiwana et al. ,1991; Gabrielli et al., 2006). Praziquantel works by disrupting the tegument of the fluke; which may facilitate the immune recognition of adult worms (Roberts and Janovy, 2005). The drug also disrupts the ion flow across the membrane of the parasite, resulting in muscular tetany (Roberts and Janovy, 2005). As a consequence, adult worms are dislodged from the gut and swept back to the liver.

Biomphalaria glabrata (Say, 1 81 8) Biomphalaria glabrata (Say, 1818) is a pulmonate snail of the family Planorbidae and is the most important intermediate host of S. mansoni, the causative agent of schistosomiasis (WHO, 1968). This species is found in many islands of the West Indies (Hispaniola, Puerto Rico, Vieques, St. Kitts, Antigua, St. Philip, Guadeloupe, Marie-Glante, Martique, St. Lucia), Venezuela, Surinam, French Guiana and Brazil (WHO, 1968). They are usually found in standing water: pools, marshes, permanent and temporary ponds, irrigation and drainage

13 ditches as well as water reservoirs (WHO, 1968). They are adapted to a wide range of water temperatures, having an optimal oviposition temperature of 26°C and an optimal growth temperature of 28°C (WHO, 1968; El Emam and Madsen, 1982). Laboratory studies have demonstrated that oviposition behaviour of B. glabrata is influenced by water quality: clean, fresh water will stimulate whereas dirty water will inhibit egg laying (Boyle and Yoshino, 2000). Studies documenting the behaviour of the snail under different light regimes have demonstrated that they are significantly more active in the dark (Rotenberg et al., 1989). B. glabrata can both self- and cross-fertilize (Vianey-Liaud and Dussart, 2002). In most cases, copulation involves two partners both acting simultaneously as a male and as a female. However copulation may involve more than two different partners (Vianey-Liaud and Dussart, 2002). Snails have significantly higher reproductive success in terms of egg production, development and hatching success when cross-fertilization occurs. Cross- fertilization results in a higher number of eggs produced and a greater hatching success (Vianey-Liaud, 2002J. The eggs are spherical and are contained within a yellow, translucent gelatinous case. Hatching occurs approximately 10 days after being laid, varying with water temperature. The shell diameter of newly emerged neonates measures between 0.3 and 0.8 mm. Adult shell diameter ranges from 15 to 30 mm. Adults can survive for up to 24 months under laboratory conditions (WHO, 1968).

14 Bulinus truncatus

The genus Bulinus comprises some 30 nominal species (Preston and Southgate, 1994). Bulinus truncatus is a pulmonate snail of the family Planorbidae (Preston and Southgate, 1994). They are pan-African, extending from Malawi into much of West and North Africa, onto some of the Mediterranean islands, the Iberian Peninsula, and into the Middle East as far as Iran (Preston and Southgate, 1994). They live in shallow ponds, slow running rivers and lakes. Contrary to other Bulinus species, Bulinus truncatus are polyploid snails, with diploid, tetraploid, hexaploid and octoploid species. Adults have an average shell length between 0.8 - 1 .0 cm (Preston and Southgate, 1994). The eggs are laid in yellowish, circular bands, approximately 3-6 mm in diameter on the surface of stones, plants or containers (Daoust 2008, personal observations).

HeI¡soma trivolvis trivolvis (Say, 1816) Helisoma trivolvis trivolvis, also known as the Larger Eastern Ramshorn snail, is the most abundant of the large eastern helisomas (Clark, 1981). In Canada, this pulmonate occurs throughout the boreal and deciduous forest regions from eastern Quebec and Nova Scotia to southeastern Manitoba with small foci in central Saskatchewan (Clark, 1981). This species is characteristically found in well-vegetated perennial watersheds, ponds and slow moving streams (Clark, 1981). Many species of pulmonate snails are hermaphroditic, capable of self-fertilization as well as cross-fertilization. This being said, there is very little information available in the literature describing the

15 reproductive capability of this particular species of snail. The eggs are laid in yellowish, circular bands, approximately 5-7 mm in diameter on the surface of stones, plants or containers (Daoust, personal observations).

Stagnicola elodes (Say, 1821) Stagnicola elodes (Say, 1821) is a member of the Lymnaeidae family (Clark, 1981). It possesses a dextral, lymnaeiform shell measuring up to 32 mm in height and 14 mm in width (Clark, 1981). It is a herbivorous, pulmonate, freshwater snail (Clark, 1981). This species is ubiquitous, found in many aquatic habitats. It is especially numerous in thick vegetation as well as on muddy substrates (Clark, 1981). S. elodes is monoecious, and capable of self- fertilization as well as cross- fertilization, producing 20-28 eggs per week.

Plagiorchis elegans (Rudolphi, 1802) Plagiorchis elegans is a digenean trematode of the family Plagiorchiidae (Styczynska-Jurewicz, 1962). The lymnaeid snails Stagnicola elodes and Lymnaea stagnalis serve as its first intermediate hosts (Styczynska-Jurewicz, 1962; Leflore, 1978). Emergence of the cercariae from the snail is governed primarily by photoperiod (Webber et al., 1986; Lowenberger and Rau, 1994). Most cercariae emerge within the first hour of darkness (Webber et al. 1986). Aquatic insect larvae such as Coenagrion hastulatum, Aedes aegypti, Anopheles maculipennis, Culex pipes, Corethra sp., Cloeon sp., Lestes sponsa, serve as second intermediate hosts (Styczynska-Jurewicz, 1962). P. elegans lacks

16 specificity for its final host, and completes development in many small mammals such as muskrats, mice, voles as well as in many species of bird (Styczynska-

Jurewicz, 1962). The mature flukes are usually located in the upper region of the gastrointestinal tract of their final host. Under experimental conditions, the parasites require between eight and ten days to initiate egg production. Eggs are expelled in the final host's feces (Zakikhani and Raul 998b). The eggs are small, measuring approximately 43 X 21 urn (Genov and Samnaliev, 1984) and are ingested by the first intermediate (there is no free-swimming miracidial stage). Once ingested by the snail host, the miracidium breaks out of the egg, penetrates the gut wall and forms the mother sporocyst, which in turn produces many daughter sporocysts. The daughter sporocysts migrate to the tissues of the snail hepatopancreas where they generate a large number of cercariae (Cort and Ameel, 1944).

Immune System of Snail Biomphalaria glabrata Snails possess two types of defense mechanisms against foreign objects; a cell mediated immune response and a humoral response (Lackie, 1980). The main effector cells of the gastropod's cell mediated immune response are hemocytes. These mobile haemolymph cells are responsible for the recognition and destruction of a large variety of pathogens, including parasites (lakovleva et al., 2006). Hemocytes destroy or sequester pathogens or foreign materials within the snail host by phagocytosis and encapsulation (Lackie, 1980). Both

17 mechanisms are accompanied by the release of lysosomal enzymes such as ß - glucuronidase and acid phosphatase, which break down the foreign materials (Lackie, 1980). Foreign material larger than approximately 10 µ?? in diameter is encapsulated rather than phagocytosed (Lackie, 1980). Hemocytes of gastropods were usually considered to be of a single cell type, but recent studies have demonstrated subpopulations, classified according to age, enzyme content, and surface determinants. Three major types of hemocytes have been discovered to date; the almost equally numerous large and medium size hemocytes and comparatively fewer, small hemocytes. The larger hemocytes (8 by 12 µ?t?) have a small nucleoplasms ratio and are asymmetrical with prominent cytoplasmic extensions. The cells' centers are comprised of a centriole, a few dyctosomes and a few dense secretory granules. There are numerous mitochondria in the cytoplasm and large aggregates of glycogen particles. The medium-sized hemocytes (~ 8 µ?t? in diameter) have a higher nucleoplasm^ ratio. They are more symmetrical and have a homogenous cytoplasm with few organelles. The smaller cells (5 - 6 µ?t? in diameter) appear to be a heterogeneous population. The mature small hemocytes possess a high nucleoplasm^ ratio, thin cortex and the cytoplasm is scarce with few dense secretory granules and rich in organelles. Quiescent circulating hemocytes can be found in the lumina of the heart cavities, in the main vessels and in the sinuses of the middle part of the kidney or in those draining the hemocyte- producing organ (see Matricon-Gondran and Letocart., 1999a).

18 Molluscs also rely on several humoral factors to protect themselves from foreign invaders. One group of humoral factors which has been of great interest are the haemaglutinins. It is now recognized that most agglutinins are lectin-like, in that they have specific receptors for certain specific carbohydrate antigens. These molecules appear to be involved in several immunological processes. They seem to be involved in the recognition of foreign antigens, binding to the latter and serving as "recognition molecules" for the cell mediated immune system (see Lackie, 1980). They also have been shown to opsonize bacteria and enhance phagocytosis in vitro (Hardy et al., 1977).

Interactions Between Digeneans and their Snail Hosts One of the characteristic features of digenean-mollusk associations is their high specificity (Cort, 1941; Llewellyn, 1965; Wright, 1973; Shoop, 1988). The majority of digenean species solely develop successfully within members of a single molluscan family, genus or even species and strain (Wright, 1973; Llewellyn 1965; Shoop 1988). This specificity is expressed at the level of the miracidia, which may not be able to penetrate an incompatible host and has been shown to select compatible hosts over others (Haas et al., 1991). In some cases the incompatible host may lack certain biochemical attributes rendering it unsuitable for the parasite (Sullivan and Richards, 1981). However, in most cases it is more likely that immunological phenomena are ultimately responsible for the success or failure of the digenean larva in the snail host (Adema and Loken, 1997; Sapp and Loken, 2000).

19 Parasites draw energy from their hosts: their growth and rapid multiplication progressively debilitate the snail host (Platero, 2004). Snails, however, are not helpless against such attacks. As previously mentioned, the main effectors of the gastropod internal defense system are hemocytes. Depending on the degree of immunological compatibility, the hemocyte responses to the miracidium to mother sporocyst transformation varies from none to benign association of hemocytes with the parasite surfaces, to complete encapsulation and elimination of the parasite (Amen et al., 1992). This being said, parasites have had to develop ways of coping with their specific snail host's immune systems (Amen et al., 1992). One way by which compatible parasites evade the immune response is to suppress its activity (Amen et al., 1992). For example, in order for Schistosoma mansoni to successfully establish itself in the tissues of its compatible host snail, the parasite must produce "secretory factors" that suppresse the activation of the hemocytes (Nunez et al., 1994). The digenean parasite Echinostoma paraensi evades the immune response of the snail by diminishing the capacity of its host snail's hemocytes to encapsulate and destroy the invading parasites (Loker et al., 1992) Digenean parasites not only modulate the snail host's immune system, they also have a significant impact on the snail's reproductive output (Zakikhani and Raul 998a). This is accomplished in two ways. Echinostome rediae, have been shown to feed actively on the gonadal tissues of the snail host causing direct parasitic castration (Nassi, 1979). Indirect parasitic castration of the snail host is induced by the sporocyst by chemically manipulating the snail's

20 neuroendocrine system to divert resources toward the development and reproduction of the parasite (de Jong-Brink, 1995). This type of castration has been thoroughly documented in the L. stagnalis- Trichobilharzia ocellata system (Amen et al., 1992; de Jong-Brink et al., 2001). In the early stage of infection, T. ocellata not only has immunomodulatory effects but also inhibits the development of the reproductive organs of the host snail (Amen et al., 1992; de Jong-Brink et al., 2001). If the snails are infected as neonates, the development of the reproductive tract is almost completely inhibited (Amen et al., 1992; de Jong- Brink et al., 2001). In contrast, when subadult snails, which are not yet laying egg masses but may already have a well developed reproductive tract, become infected, they start producing egg masses before the non-infected controls. This accelerated burst in egg production is of short duration, and the infected snails soon completely cease egg production (Amen et al., 1992; de Jong-Brink 2001).

Interactions Between Digeneans Within their Snail Host Organisms living in the same community may interact with each other so that one organism or population is negatively affected by the other (Halvòrsen, 1976). In the field, an individual snail may be host to more than one species of digenean parasite (Farley, 1967). In such concurrent infections, the larval parasites may act antagonistically. The primary result of such antagonism is the inhibition or destruction of one of the competing trematode species. Antagonistic interaction may occur between rediae of 2 species or between rediae and a sporocyst (Lie et al., 1968a). Antagonistic interaction may be either direct or

21 indirect (Lie et al., 1968a; Lie et al., 1968b). Direct antagonism consists of prédation by rediae of one parasite species upon the sporocysts, rediae or cercariae of the other species. Indirect antagonism between larval digenean parasites leads to developmental retardation or degeneration of the competing larval trematode infection (Lie et al., 1968a; Lie et al., 1968b). Indirect antagonism between trematodes within the snail host may be affected chiefly by: the liberation of inhibitory substances by rediae or sporocysts, by production of inhibitory substances or cellular reaction by the snail host and lastly by competition for limited resources within the snail (Lie et al., 1968b). Lie et al. (1968a) studied the antagonistic relationship between the trematodes Paryphostomum segregatum and Echinostoma lindoense, in their compatible snail host Biomphalaria glabrata. The rediae of P. segregatum were shown to actively consume the rediae of E. lindoense, whereas the reverse was never demonstrated (Lie et al., 1968a). A curious feature is that prédation always seemed to act in one direction, even though the prey itself may be an active predator in other combinations (Lie and al., 1968b). Much of the work dealing with trematode antagonism has been conducted using the Schistosoma mansoni I Echinostome model. The negative effects of the aggressor trematodes on the development of S. mansoni infection has formed the basis of a new approach to the biological control of this human pathogen by broadcasting mass-produced eggs of the echinostomes into schistosome infected habitats (Basch et al., 1970; Lie et al. ,1970; Lie et al., 1971; Page and Huizinga, 1976).

22 Control of Schistosomiasis

Theoretically, control of schistosomiasis can be achieved by breaking the lifecycle of the parasite at any one of a number of points (WHO, 1959). For instance; adult worms can be killed with anthelminthic therapy in their human host, eggs can be prevented from reaching water, snail vectors may be eliminated, and lastly the human population in endemic regions may be protected from contact with cercaria-contaminated water (WHO, 1959). In practice, anthelminthic treatment and snail control are the most commonly used methods. The two methods are complementary; chemotherapy will rid the human host of infection, and snail control prevents reinfection. Over the past four decades, various methods have been applied in order to control the intermediate snail host. The earliest procedures involved physical modification of snail habitat (WHO, 1965). The most effective habitat modification against snail populations is the drainage of wetlands. Where this is not possible, alternative methods such as weed clearance, straightening the water banks, and deepening of the marginal areas have also shown to be effective in reducing snail habitat (WHO, 1965). Environmental control is likely to be more permanent in its effects than are chemical methods. This being said, the cost of making permanent habitat alterations and the difficulty of obtaining the co-operation of local people may preclude the immediate adoption of such measures (WHO, 1965). The second and most widely used method of controlling intermediate snail host populations is by adding chemical molluscicides to their aquatic habitats (Levine, 1970). Initially, copper sulfates and other copper salts were used with

23 great success to control the snails (Levine, 1970). Although the chemicals were very effective in killing intermediate snail host species, they were also shown to kill non-target snail species as well as some fish species (Levine, 1970). Due to such serious ecological implications, their use was terminated and they were replaced by the molluscicide niclosamide (Bayluscide, Bayer 73, 5,2'-dichloro-4'- nitroethanolamine salicylanilide) (Levine, 1970). This compound kills snails at 0.5 ppm and snail eggs at 1 ppm in 24 hours and remains effective in the field for two days (Levine, 1970). Niclosamide has not been shown to be as harmful to fish or aquatic plants at the low concentrations required for snail control (Levine, 1970; Takouag et al., 2007). It is important to note that molluscicides cannot be used effectively without a thorough understanding of the bionomics of the snail and the associated parasite stages (Webbe, 1962a). One such successful campaign was conducted on the West Indian Island of St-Lucia (Sturrock, 1973). Scientists successfully controlled the spread of schistosomiasis on St-Lucia, with reasonably small controlled amounts of molluscicides (Sturrock, 1973). However, the high production cost of molluscicides, their adverse environmental impact, and the development of resistance by the snail hosts has made it imperative to develop alternative control strategies (Thomas, 1973; Madsen, 1990; Yi et al.,

2005). Biological agents have been considered for the last few decades as an alternative approach to chemical molluscicides (WHO, 1984). A great number of organisms have been considered for the biological control of snails. They may be briefly categorized as micropathogens, predators, parasites and competitors

24 (Pointier and Jourdane, 2000). Predators and competitors of freshwater snails have been studied more extensively. Notably, fieldwork introducing molluscivorous fish including cichlids (Haplochromis xenognathus, Haplochromis sauvagei and Haplochromis ishmaeli), tilapia {Talapia rendalli) and American crayfish (Procambarus clarkii) has been shown to have great potential as biological control agents of the snail vectors of human schistosomaisis (Graber et al., 1981; Slootweg, 1987; Hofkinseta., 1991). A competitor snail species, Marisa cornuarietis, is a voracious herbivore, which in the course of feeding not only ingests B. glabrata egg masses and newly hatched young, but also out- competes B. glabrata for food resources (Chemin et al., 1956). As previously mentioned not only are trematode larvae capable of castrating their snail hosts (Nassi, 1979; de Jong-Brink, 1995), they have also been shown to be mutually antagonistic when they are present in the same host snail (Lie et al., 1968a). For these reasons, digeneans have some potential as biological control agents of the parasites and their snail vectors causing human schistosomiasis. Fieldwork done by Lie et ai. (1970) demonstrated that the eggs of the digenean Echinostoma malayanum when deployed in small bodies of water in Kuala Lumpur, Malaysia, successfully eliminated the subordinate parasite Schistosoma spindale from snail hosts. Nassi et al. in their 1979 field study, applied eggs of the trematode Ribeiroia guadeloupensis to a small pond on Grande-Terre Island, Guadeloupe, which contained a large number of B. glabrata snails. Despite the low rate of hatching among the introduced parasite

25 eggs, due particularly to high turbidity of the water, this trial resulted in near total elimination of the snails (Nassi et al., 1979).

Infection of P. elegans in the Incompatible Snail Host B. glabrata - A

Review

As previously mentioned, P. elegans can establish patent infections in both S. elodes and L. stagnalis. Studies by Zakikhani and Rau (1998a) revealed that the parasite did not only castrate these two compatible hosts, it also established an infection in B. glabrata but was shown to be unable to complete its development. Although minute, the mother sporocysts were found in the tissues of this snail and here was no evidence of daughter sporocyts or cercariae. Nevertheless, all infected snails developed symptoms of parasitic castration, and the total number of eggs produced was reduced by 90% (see Zakikhani and Rau 1998). More importantly, there was a concomitant reduction in the number of cercariae shed by a challenge infection with S. mansoni (Zakikhani et al., 2003). There is evidence that plagiorchiid digeneans may have a rather unique interaction with the immune responses of their compatible snail host (Zakikhani et al., 2003). Unlike S. mansoni sporocysts, which suppress the activity of host haemocytes in terms of reduced mobility and phagocytic activity, certain plagiorchiids immunostimulate the host and incorporate responding hemocytes which provide nourishment and protection (Monteil et al., 1991). Although it is not known whether P. elegans can establish this kind of relationship in the

26 incompatible snail host, it is conceivable that the minute mother sporocyts may stimulate the defense system of their incompatible snail hosts (Zakikhani et al., 2003). It is hypothesized that in the incompatible host, where P. elegans sporocysts remain small, free haemocytes may theoretically remain very high and may therefore diminish the success of a subsequent challenge infection with S. mansoni (Zakikhani et al., 2003). Recent laboratory studies have demonstrated that ß. glabrata can ingest large numbers of P. elegans eggs by ad libitum browsing on contaminated substrates with eggs. The number of eggs acquired in this manner is a function of the amount of substrate ingested (Platero, 2004). The age at which B. glabrata are infected also has a significant effect on their subsequent reproductive success (Platero, 2004). In a twenty-four hour period, young snails were shown to consume a mean of 6.2 cm2 ± S. E. 0.8 of substrate, whereas juveniles consumed a mean of 29.9 cm2 ± S. E. 5.7 and adults 91 .8 cm2 ± S. E. 1 2.6

(Platero, 2004). The younger the snail is at the time of exposure, the smaller the number of eggs it will produce when it reaches maturity (Platero, 2004).

Digeneans and their Impact on Aquatic Communities Digeneans play a crucial role in the biology of gastropods and their communities. Parasitic infection leads to a substantial reduction in the reproductive output of their snail hosts, as well as having a negative impact on survivorship of their snail hosts. It is important to note that not all infected snails die due to parasitic infection, but almost all become castrated. Because castrated snails remain in the population and consume resources that may or may not be

27 limited, they compete not only with other infected snails but with uninfected snails as well (Lafferty, 1997). These factors reduce the fitness of the snails within the population, eventually resulting in a general decrease in the population.

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43 CHAPTER 3

THE EFFECTS OF A PRIMARY INFECTION WITH PLAGIORCHIS ELEGANS

(DIGENEA: PLAGIORCHIIDAE) AND A CHALLENGE INFECTION OF SCHISTOSOMA MANSONI (DIGENEA: SCHISTOSOMATIDAE) ON A

COMMUNITY OF THE INCOMPATIBLE HOST BIOMPHALARIA GLABRATA

(PULMONATA: PLANORBIDAE) COMPOSED OF DIFFERENT AGE

CLASSES.

*Daoust, S., **Mader, B., **McLaughlin, J.D., and M. E. *Rau

* Department of Natural Resource Sciences, McGiII University (Macdonald Campus), 21111 Lakeshore Road, Ste-Anne-de-Bellevue, Qc, Canada, H9X 3V9

** Department of Biology, Concordia University (Loyola Campus), 7141 Sherbrooke St W.

Montreal, Qc, Canada, H4B 1R6

Running Title: Plagiorchis elegans and Schistosoma mansoni in Biomphalaria glabrata: effect of snail age/size.

44 ABSTRACT

The impact of a primary infection with the incompatible digenean Plagiorchis elegans on groups composed of young, juvenile and adult Biomphalaria glabrata and a challenge infection with the compatible parasite Schistosoma mansoni was assessed. Egg production of young Biomphalaria glabrata was only significantly reduced by P. elegans at the higher exposures of 16 eggs per snail and that of adult B. glabrata was reduced at exposures of 8 and 16 eggs per snail. Juvenile B. glabrata were not significantly affected. The survivorship of juvenile and adult B. glabrata was significantly reduced at an exposure of 16 P. elegans eggs per snail and an exposure of 8 and 16 P. elegans eggs per snail, respectively. Interestingly enough, the survivorship of young B. glabrata snails was not significantly affected by P. elegans exposure. Furthermore, snails harboring small P. elegans infections seem to acquire resistance to Schistosoma mansoni infection. In communities where snails of different age groups are present, the larger snails have a higher probability of ingesting the P. elegans eggs and therefore serve as parasitic sinks, preventing or protecting the smaller snails in the community from being exposed to digenean eggs, to a certain extent.

45 INTRODUCTION

Schistosomiasis is endemic to 76 countries and territories, and continues to be a global public health concern in the developing world (Engels et al., 2002). It is estimated that 200 million people are infected, of whom 120 million are symptomatic and 20 million people manifest severe disease. Furthermore, a total of 600 million people are at risk of infection (Chitsulo et al., 2000). Control of the snail intermediate host remains an essential component of integrated schistosomiasis control programs (Pointier & Jourdane, 2000). The preferred method of control involves the use of chemical molluscicides (Levine, 1970). Among those, niclosamide (Bayluscide, Bayer 73, 5,2'-dichloro-4'- nitroethanolamine salicylanilide) is the most widely used, killing snails at 0.5 ppm and snail eggs at 1 ppm within 24 hours. Residual effects in the field persist for two days (Levine, 1970; Takouagang et al., 2007). However, high production cost, their adverse environmental impact, and development of resistance by the snail hosts have underscored the need for alternative approaches of control (Thomas, 1973; Madsen, 1990; Yi et al., 2005). Biological agents have been considered for the last few decades as an alternative to chemical molluscicides (WHO, 1984). They can be loosely categorized as micropathogens, predators, parasites and competitors of snail hosts (Pointier & Jourdane, 2000). Parasites, especially larval digeneans, have been the subject of investigation in the last two decades. The ¡ntramolluscan development stages of these parasites are able to castrate and potentially kill

46 their snail hosts and as well as have antagonistic effects on other trematodes within the same snail host (Nassi, 1979; de Jong-Brink, 1995). Until recently, research conducted on the use of digeneans as biological control agents has focused on digenean species compatible with the target snail host. However, studies by Zakikhani and Rau (1998a) found that eggs of Plagiorchis elegans, ingested by Biomphalaria glabrata, elicited the same rapid, severe, and permanent suppression of reproductive output in this incompatible host snail as in the compatible Stagnicola elodes (Zakikhani and Rau, 1998a). Interestingly, P. elegans development did not progress beyond the early embryological stages in this incompatible snail host (Zakikhani and Rau, 1998a). This study work consists of two related investigations. The first examines the impact of exposure of P. elegans to the adult, juvenile and young incompatible B. glabrata host. Since these snails commonly occur in mixed size/age classes, they were exposed jointly to the incompatible parasite. The second study assesses the impact of a subsequent challenge infection with a compatible digenean Schistosoma mansoni on these same parameters.

Materials and Methods

Snail Hosts

Biomphalaria glabrata Young (2-3 mm), juvenile (4 - 7mm) and adult (8-10 mm) of the Puerto Rican strain of Biomphalaria glabrata were obtained from the Biomedical

47 Research Institute, Rockville, Maryland. The colony was maintained in an incubator at 26.5 0C, under a L12:D12 photoperiod with ~ 50% relative humidity (El-Emam and Madsen, 1982). Snails were initially kept in 26 ? 18 ? 8 cm aquaria filled with 1 .5 L of aerated tap water; and fed washed Romaine lettuce and powdered calcium carbonate ad libitum.

Stagnicola elodes A colony of laboratory-reared S. elodes has been maintained in our laboratory for more than ten years. The snails were derived from a wild stock with a high natural prevalence of infection with the digenean P. elegans. Snails were reared under a L16:D8 photoperiod, in 473 ml containers (Solo Cup Company, Urbana, Illinois) filled with aerated tap water at 22 0C. The water was changed monthly.

Parasites

Plagiorchis elegans Plagiorchis elegans has been maintained in our laboratory for more than a decade, using Mesocricetus auratus as the definitive host. Labortaory-reared S. elodes serve as the first intermediate host and were exposed to P. elegans eggs in a small volume of water. The addition of small amounts of ground Tetramin® fish food to the exposure chambers helped stimulate feeding of the snail and the consumption of parasite eggs (Zakikhani and Rau, 1998a). Infections reach patency in five to six weeks, and cercariae were induced to emerge from the

48 snail host by an environmental change in light intensity from light to dark (Lowenberger and Rau, 1994). Laboratory-reared fourth instar Aedes aegypti larvae were used as a second intermediate host. Larvae were exposed to cercariae, which penetrate their tissues where they form metacercariae. When metacercariae reached ¡nfectivity three to seven days later, the host mosquito larvae were lightly crushed. The numbers of metacercariae were estimated on the basis of ten random samples, and approximately one hundred metacercariae were fed to each hamster. Metacercariae require eight days to develop into ovipositing adult worms (Zakikhani and Rau, 1998a). Eggs were passed with the feces of the hamsters and collected overnight as needed. The eggs were collected in a tray of water and separated from the fecal debris by washing through a series of four brass sieves (mesh sizes 200, 65,37 and 20 urn). The eggs were then collected on the 20 urn mesh, and re-suspended in deionized water. Eggs were allowed to embryonate at 20 ± 4°C, for 4 days before they were fed to snails (Zakikhani and

Rau, 1998b).

Schistosoma mansoni

Adult S. mansoni were obtained from the Biomedical Research Institute, Rockville, Maryland, in CD-1 outbread mice, serving as their final hosts. The mice had been given 80-100 S. mansoni cercariae by skin penetration. The infected mice were kept following level 2 biological quarantine procedures. Mice were euthanized with carbon dioxide followed by cervical dislocation.

49 Schistosome mirac¡d¡a were collected following the procedure developed by Rau et al. (1972). The homogenized mouse livers were placed in a modified Erlenmeyer flask, possessing a side arm, filled with distilled water. The Erlenmeyer flask provides a large bottom surface area so that the liver homogenate is spread out in a thin layer to facilitate the escape of miracidia from the eggs. Free-swimming miracidia are brought to the top of the masked flask by negative geotactic responses and migrate into the unmasked side arm by positive phototaxis where they can be collected.

Experimental Methods Experiment 1 - The effects of P. elegans on a community of the incompatible host, Biomphalaria glabrata, composed of different size classes.

Eggs of P. elegans were obtained from the feces of experimentally infected hamsters as described above. The mean density of eggs in suspension was determined on the basis of ten 0.025 ml samples drawn by pipette (Zakikhani and Rau, 1998b). The suspension was diluted serially to obtain the desired number of 1 , 4, 8 and 16 embryonated eggs per sample. The young (2-3 mm), juvenile (4-7 mm) and pre-reproductive adult (8-10 mm) Biomphalaria glabrata were kept separately in 26 ? 18 ? 8 cm aquaria for one week after arrival from the Biological Research Institute, allowing them to acclimatize to laboratory conditions. All experiments took place in a locked, walk-

50 in, incubator kept at 26.5 0C, under a L12:D12 photoperiod with ~ 50% relative humidity. One week after arrival, three snails, one of each size class were placed into 473 ml containers (Solo Cup company, Urbana, Illinois), with a bottom surface area of 70.88 cm2, filled with 200 ml of aerated tap water. The P. elegans egg suspensions, along with tetramin ® and lettuce (to promote feeding) were added to the containers and stirred. Ten groups of three snails were used for each Plagiorchis egg concentration (0, 1, 4, 8 or 16 per snail or 0,;(2,J12, 24, 48 per container) (N=10 per size class per treatment). The snails exposed to 0 parasite eggs served as our control groups. A total of 150 snails were used for this experiment. The groups, each composed of the three snails of three different size classes were exposed to P. elegans for one week before they were separated and placed into new individual containers. This provided the snails with ample time to encounter and ingest the parasite eggs. Snail survival and egg production were monitored weekly for a period of four months. After every egg count, the egg bands were removed from the containers. Snails were fed small pieces of Romaine lettuce ad libitum and the water was changed every two weeks to stimulate egg production (Boyle and Yoshino, 2000). A group of 10 S. elodes were simultaneously infected with P. elegans eggs of the same batch to assure infectivity. These snails were exposed individually and kept in 473 ml containers (Solo Cup company, Urbana, Illinois), with a bottom surface area of 70.88 cm2 . They were monitored weekly for cercarial production.

51 Experiment 2 - The effects of P. elegans on a community of the incompatible host, Biomphalaria glabrata, composed of different size classes followed by a challenge infection of Schistosoma mansoni. Experimental exposure of Biomphalaria glabrata to Plagiorchis elegans eggs follows the procedure outlined in Experiment 1 . Three weeks post exposure to P. elegans eggs, the B. glabrata snails of the three size classes were also individually exposed to seven miracidia of S. mansoni. The miracidia were pipetted from the side arm of the modified Erlenmeyer flask and placed in small drops on a clean plastic slide, where they were counted under a dissecting scope. Seven miracidia were washed in the 473 ml containers (Solo Cup company, Urbana, Illinois) harboring the snails. Survivorship, egg production and cercarial production were monitored for four months. Work with the schistosome cercariae followed level 2 biological safety procedures. Because S. mansoni cercariae tend to leave the snail in response to environmental changes from dark to light, cercariae were always counted in the morning. The containers with the shedding snails were gently shaken, to make sure that the cercariae were evenly distributed. Five samples of 20 ml of cercarial suspension (10%) were taken from each container. The samples were placed on a filter paper, water was removed over a vacuum flask, cercariae were killed and dyed using 10% iodine. The cercariae were counted and a mean was calculated for the total volume of water. After every cercarial count, the water was placed into a container and sterilized using high concentrations of chlorine bleach.

52 Statistical Analysis All experiments followed a complete randomized design. Data were analyzed using SPSS (SPSS inc. 2008, Chicago Illinois). The weekly number of eggs produced from young, juvenile and adult B. glabrata were rank transformed to satisfy the conditions of normality (Sokal and Rohlf, 1995). A repeated measures ANCOVA as well as a Tukey multiple comparison post-hoc test was used to analyze these data. Possible variation due to the initial container in which the snail groups were exposed to the P. elegans eggs was included into the model as a covariate. Standard error and confidence intervals for the B. glabrata survivorship curves were calculated using a Kaplan-Meier survivorship analysis. Kaplan-Meier log-ranked survivorship analyses were used to compare the survivorship curves of the control snails with those of snails exposed to P. elegans. Because four multiple comparisons were made, Bonferroni corrections were used, which adjusted the alpha to 0.0125. Probabilities were calculated from the slopes of the linears derived from the log survivorship curves. Linear regressions were used to verify if there was a significant effect of treatments on the probability of dying.

53 RESULTS

Experiment 1 - The effects of P. elegans on a community of the incompatible host, Biomphalaria glabrata, composed of three different size classes.

There was no significant attributable effect to the initial container in which the snails were exposed to the P. elegans eggs (Repeated Measures ANCOVA,

DF= 1, 48, F= 0.272, P= 0.604, a = 0.05). There was, however, a significant difference between the mean number of eggs produced by young ß. glabrata exposed to 0,1 4, 8 or 16 Plagiorchis elegans eggs (Repeated Measures ANCOVA, DF= 4, 45, F= 2.882, P = 0.033, a = 0.05) (Figures 1A, 1B). Young snails exposed to 8 or 16 eggs laid significantly fewer eggs than snails exposed to 0, 1 or 4 eggs (Table I). No significant difference was found between the mean number of eggs produced by juvenile B. glabrata at the four levels of exposure to P. elegans eggs (Repeated Measures ANCOVA, DF= 4, 45, F= 0.517, P = 0.725 a = 0.05) (Figure 2A, 2B). The large spikes at weeks 10 and 13 are due to the presence of two snails laying over 100 eggs each, which is very uncommon. There is, however, a significant difference between the mean number of eggs produced by adult B. glabrata exposed to 0,1 , 4, 8 or 16 P. elegans eggs (Repeated Measures ANCOVA, DF= 4, 45, F= 2.782, P = 0.0378, a = 0.05) (Figure 3A, 3B). Adult snails exposed to 8 or 16 P. elegans eggs produced significantly fewer eggs than snails exposed to 0, 1 or 4 eggs

(Table II).

54 There was no significant difference between the % survivorship of young B. glabrata exposed to 1 , 4, 8 or 16 P. elegans eggs and the control snails (Figure 4A, 4B) (Table III, Table IV). There was no significant difference between the mean % survivorship of juvenile B. glabrata exposed to 1,4 or 8 P. elegans eggs and control snails (Figure 5A, 5B) (Table V, Table Vl). However, the mean survivorship of juvenile B. glabrata exposed to 16 P. elegans significantly differed from that of the control snails (DF = 1, 18, statistic = 4.33, P = 0.0119, a = 0.0125) (Figure 5A, 5B) (Table V, Table Vl). There was no significant difference between the mean % survivorship of adult B. glabrata exposed to 1 or 4 P. elegans eggs and adult control snails (Figure 6A) (Table VII, Table VIII). However, the mean survivorship of adult B. glabrata exposed to 8 or 16 P. elegans eggs (43.5 % and 27.1 %) differed significantly from that of control snails (DF = 1, 18, statistic = 6.26, P = 0.0100, a = 0.0125) (DF = 1, 18, statistic = 7.25, P = 0.0071 , a = 0.0125) (Figure 6B) (Table VII, Table VIII).

Experiment 2 - The effects of P. elegans on a community of the incompatible host, Biomphalaria glabrata, composed of different size classes followed by a challenge infection with Schistosoma mansoni. Only a very small minority of snails from the adult control group (only infected with 7 S. mansoni miracidia) laid eggs. The majority of the young, juvenile and adult snails died before they were able to lay eggs. It is for this reason that egg production data are not part of the study. The majority of the mortality of young, juvenile and adult R glabrata occurred primarily within the first

55 week post S. mansoni infection. There was no significant difference between the mean % survivorship of young and juvenile B. glabrata exposed to the 1,4,8 and 16 P. elegans eggs / 7 S. mansoni miracidia treatments and control snails (Figure 7A, 7B) (Table IX, Table X) (Figure 8A, 8B) (Table Xl, Table XII). There was no significant difference between the % survivorships of adult snails exposed to the 1 and 4 P. elegans eggs / 7 S. mansoni miracidia treatments and the control snails, however, there was a significant difference between the mean % survivorship of adult snails exposed to 8 and 16 P. elegans eggs / 7 S. mansoni miracidia treatments and the control snails exposed only to S. mansoni (Figure 9A, 9B) (Table XIII, Table XIV). There was no significant effect of the concentration of P. elegans eggs given to the snails on the probability of death of young or juvenile B. glabrata within the first week post infection (Linear Regression D. F = 1, 4, F = 3.079, P = 0.178) (Linear Regression D. F = 1, 4, F = 10.068, P = 0.05) (Figure 10). There was however, a significant positive linear relationship for adult snails (Linear Regression D.F = 1, 4, F = 35.974, P = 0.009) (Figure 10). There was no significant effect of the concentration of P. elegans eggs given to the snails on the probability of death of young, juvenile or adult B. glabrata between weeks three and sixteen post P. elegans infection (Linear Regression D.F = 1 , 4, F = 3.068, P = 0.178) (Linear Regression D.F = 1, 4, F = 0.984, P = 0.394) (Linear Regression D.F = 1 , 4, F = 1 .21 8, P = 0.350) (Figure 1 1 ).

56 DISCUSSION

This study supports previous work demonstrating that P. elegans has a direct impact on the reproductive output and survivorship of the incompatible snail intermediate host, B. glabrata (Zakikhani and Rau, 1998a). It furthermore supports the hypothesis that the relative intensity of these effects within a snail population heterogeneous in terms of size/age is skewed. We demonstrated that larger individuals bear the brunt of the effects whereas smaller individuals escape relatively unscathed particularly at the lower levels of exposure to the incompatible parasite. Large individuals appear to "outcompete" smaller individuals for available parasite eggs. Platero (2004) demonstrated that the impact of the parasite on individually exposed, incompatible hosts, are a function of the amount of substrate, and therefore the number of parasite eggs these snails could ingest in a given time. Large snails can ingest almost 15 times as much substrate than can small individuals. When exposed in a group heterogeneous in size/age, as in the present study, large individuals will acquire a disproportionally large number of parasites, and small snails relatively few. This is supported by observations that the reproductive loss and host mortality were more severe among large individuals in the group at all intensities of exposure but appeared in the small snails only at high levels of exposure. Our study also reveals that mortality due to infection with Plagiorchis manifests itself early, within the first three weeks after exposure. This suggests

57 that a significant part of the pathology rests with the initial stages of infection, perhaps the penetration process of the miracidia through the gut wall, rather than the mother spororocysts in the hemocoel. Challenge of B. glabrata with S. mansoni miracidia three weeks after P. elegans exposure caused even greater mortality, virtually all among smaller and younger snails. This confirms the work of Zakikhani et al. (2003) that P. elegans infections protect these snails from S. mansoni. Pre-challenge mortality and reproductive deficits among snails exposed to P. elegans were particularly severe among older snails, suggesting that survivors may be harboring many P. elegans sporocysts and be therefore protected from the adverse effects of schistosome invasion. Few younger snails may have picked up sufficiently large numbers of eggs to benefit from their protection. Plagiorchis elegans infections may stimulate hemocyte production in the incompatible host and provide protection, particularly among the older/larger snails. Indeed, unpublished work (Daoust) has indicated that hemocyte number rise precipitously following exposure to P. elegans. In sum, adult B. glabrata appear to serve as parasitic sinks but only at the lower and intermediate P elegans egg exposure levels. At the higher exposure levels, the young, juvenile and adult snails' reproductive output and survivorship are significantly reduced. Furthermore, B. glabrata surmised to be harboring a light P. elegans infection were shown to be resistant to S. mansoni infection. By killing, castrating and preventing S. mansoni infections, P. elegans is of potential use in controlling B. glabrata. To date, little work has been done on the

58 relationship between digenean parasites and incompatible snail hosts, there is much research that needs to be done before any field trials are to be conducted. It would be interesting to investigate, at the cellular and molecular level, what happens inside B. glabrata when infected with P. elengans. It would also be worthwhile to investigate the effects of P. elegans on other intermediate snail hosts of Human Schistosomiasis such as Bulinus truncatus and Oncomelania hupensis.

59 ACKNOWLEDGEMENTS

We would like to thank Professor P. J. Albert, Concordia University for his guidance and help. This work was supported by an NSERC grant to M. E. Rau.

60 LITERATURE CITED

Boyle, J. P., Yoshino, TP. (2000). The effects of water quality on oviposition in Biomphalaria glabrata (Say, 1818) (Planorbidae) and a description of the stages of the egg-laying process. Journal of Molluscan Studies 66: 83-93.

Chitsulo, L., Engels, D., Montresor, ?., Savioli, L. (2000). The global status of schistosomiasis and its control. Acta Tropica 77: 41-51. de Jong-Brink, M. (1995). How schistosomes profit from the stress responses they elicit in their hosts. Advances in Parasitology 35: 177-256.

El-Emam, M.A., Madsen, H. (1982). The effect of temperature, darkness, starvation and various food types on growth and reproduction of Helisoma duryi, Biomphalaria alexandria and Bulinus truncates (Gastropoda: Planorbidae). Hydrobiologia 88: 265-275.

Engels, D., Chitsulo, L., Montresor, A., Savioli, L. (2002). The global epidemiological situation of schistosomiasis and new approaches to control and research. World Health Organization WHO.

Levine, N. D. (1970). Integrated control of snails. American Zoologist 10: 579-582.

61 Lowenberger, CA. Rau, M. E. (1994). Plagiorchis elegans: emergence, longevity and infectivity of cercariae, and host behavioural modifications during cercarial emergence. Parasitology 109: 65-72.

Madsen, H. (1990). Biological methods for the control of freshwater snails. Parasitology Today 6: 237-241 .

Monteil, J.-F., Matricon-Gondran, M. (1991). Interactions between the snail Lymnaea truncata and the plagiorchiid trematode Haplometra cylindracae. Journal of Invertebrate Pathology 58: 127-135

Nassi, H., Pointier, J. P., Golvan, Y.P. (1979). Bilan d'un essai de contrôle de Biomphalaria glabrata en Guadeloupe a l'aide d'un trématode stérilisant.

Journal Annal de Parasitologic 54: 185-192.

Platero, I. A. (2004). The effects of parasite dose, host size and the method of exposure on the reproductive capacity and survival of Biomphalaria glabrata infected with the incompatible digenean, Plagiorchis elegans. M. Sc. Thesis. McGiII University, Montreal, Canada.

Pointier, J. P., Jourdane, J. (2000). Biological control of the snail hosts of schistosomiasis in areas of low transmission: the example of Caribbbean

area. Acta Tropica 77: 53-60.

62 Sokal, R. R. and F.J. Rholf . (1995). Biometry: The Principles and Practice of Statistics in Biological Research. W.H. Freeman and Company, New York,

882 pp.

Takouagang, I., Meli, J., Pone, J.W., Angwafo, F. III. (2007). Community acceptability of the use of low-dose niclosamide (Bayluscide®), as a

molluscicide in the control of human schistosomiasis in Sahelian

Cameroon. Annals of Tropical Medicine & Parasitology 101(6): 479-486.

Thomas, J. D. (1973). Schistosomiasis and the control of molluscan hosts of human schistosomes with particular reference to possible self-regulatory

mechanisms. Advances in Parasitology 1 1 : 307-394.

World Health Organization WHO. (1984). Report of an informal consultation on research on the biological control of snail intermediate hosts. TDR/VBC- SCH.SI 1984-3, pp.1-39.

Yi, Y., Xing-Jian, X., Hui-fen, D., Ming-Sen, J., Hui-Go, Z. (2005). Transmission control of schistosomiasis japónica: implementation and evaluation of different snail control interventions. Acta Tropica 96: 191-197.

63 Zakikhani, M., Rau, M. E. (1998a). Effects of Plagiorchis elegans (Digenea:

Plagiorchiidae) infection on the reproduction of Biomphalaria glabrata (Pulmonata: Planorbidae). Journal of Parasitology 84 (5): 927-930.

Zakikhani, M., Rau, M. E. (1998b). The effects of temperature and light regimens on the survival, development, and infectivity of Plagiorchis elegans eggs.

Journal of Parasitology 84: 1 1 70-1 1 73.

Zakikhani, M., Smith, J. M., Rau, M. E. (2003). Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection of Biomphalaria glabrata (Pulmonata: Planorbidae) on a challenge infection with Schistosoma mansoni (Digenea: Schistosomatidae). Journal of Parasitology 89 (1): 70-75.

64 •

Figure 1 A: Mean Number (±S.E.) of eggs produced by B. glabrata exposed as young to 0, 1 or 4 P. elegans eggs.

Figure 1B: Mean number (±S.E.) of eggs produced by B. glabrata exposed as young to 0, 1, 8 or 16 P. elegans eggs.

Repeated Measures ANCOVA;

DF= 4, 45 F= 2.882 P = 0.033 a = 0.05

65 1A. Control 1 P.e. egg ¦+—4 P.e. eggs

» 40

UJ 35

9 10 11 12 13 14 15 16 17 Time (weeks)

1B. -?— Control 8 P.e. eggs -*— 16 P.e. eggs

W 40 O» S 35

10 11 12 13 14 15 16 17 Time (weeks)

66 Figure 2A: Mean number (±S.E.) of eggs produced by B. glabrata

exposed as juveniles to 0, 1 or 4 P. elegans eggs.

Figure 2B: Mean number (±S.E.) of eggs produced by ß. glabrata

exposed as juveniles to 0, 8 or 16 P. elegans

eggs.

Repeated Measures ANCOVA;

DF= 4, 44 F= 0.517 P = 0.725 a = 0.05

67 2A. -?— Control 1 P.e. egg 4 P.e. eggs

«J 10

9 10 11 12 13 14 15 16 Time (weeks)

2B.

-?— Control 8 P.e. eggs -*— 16 P.e. eggs

35 -?

°> 25

ra 10

9 10 11 12 Time (weeks)

68 Mean number (±S.E.) of eggs produced by B. glabrata exposed as adults to 0, 1 or 4 P. elegans eggs.

Mean number (±S.E.) of eggs produced by B. glabrata exposed as adults to 0, 8 or 16 P. elegans eggs.

Repeated Measures ANCOVA;

DF= 4, 45 F= 2.782 P = 0.0378 a = 0.05

69 3A.

-?- Control 1 P.e. egg ¦*— 4 P.e. eggs

70

5 6 7 8 9 10 11 12 13 14 15 16 17 Time (Weeks)

3B.

-?—Control -·-8 P.e. eggs -*— 16 P.e. eggs

70

7 8 9 10 11 12 13 14 15 16 17 Time (Weeks)

70 Figure 4A: Log % survivorship curve of young ß. glabrata exposed to 0, 1 or 4 P. elegans eggs.

Figure 4B: Log % survivorship curve of young B. glabrata exposed to 0, 8 or 16 P. elegans eggs.

71 4A.

? Control ¦ 1 P.e. egg a 4 P.e. eggs

-0.1 ? 4 6 8 10 12 14 ¦ A a. -0.2 Í HSf IBJ IH? B? ?? S^ UH I -0.3 i ¦ i ¦ I -0.4 I AAA è *> L. -0.5 3 ? W -0.6 # as -0.7 J -0.8 -0.9

-1 Time (Weeks)

4B.

? Control • 8 P.e. eggs * 16 P.e. eggs op- -0.1 2 4 6 8 10 12 14 16j § § § f f § • · ·

warna -0.2 XL % -0.3 rP' ^^ f^ ^p ^F ^^ • t · ê ?. i t > -0.4 -0.5 3 fitti! -0.6 # a -0.7 * ? O -> -0.8

-0.9

-1 Time (Weeks)

72 Figure 5A: Log % survivorship curve of juvenile B. glabrata exposed to 0, 1 or 4 P. elegans eggs.

Figure 5B: Log % survivorship curve of juvenile ß. glabrata exposed to 0, 8 or 16 P. elegans eggs.

73 5A.

? Control 1 P.e. egg a 4 P.e.eggs

•0,1 o I ¦ il ì 10 12 14 16 t II I SP ^P ? t 5· -0.2 1 AAA A A I -0.3 t t| > -0.4 : ¦ A â| -0.5 3 ¦ il « -0.6 «? 1-0.7 S -0.8 -0.9 -1 Time (Weeks)

5B.

? Control • 8 P.e. eggs * 16 P.e. eggs

op -o.i ö ! t 14 16! t I ? t B -0.2 h % WWWW » -0.3 ¦; X <^? ?\ ^f* #? I t X % X i! KM*> -0.4 i > 3 « -0.6

°0Ì -OJ ! 3 -0.8 -0.9 -1 Time (Weeks)

74 Figure 6A: Log % survivorship curve of adult B. glabrata exposed to 0, 1 or 4 P. elegans eggs.

Figure 6B: Log % survivorship curve of adult B. glabrata exposed to 0, 8 or 16 P. elegans eggs.

75 6A.

? Control 1 Re. egg a 4 P.e. eggs

0 ¦ ¦ ?- W ¦ -0.1 T ? I I m 12 14 16; a-0.2 A WE ?«ß -0.3n „ ? im ? tí > -0.4 I -0.5 «-0.6 œ-0.7 J -0.8 -0.9 -I Time (Weeks)

6B.

? Control • 8 P.e. eggs * 16 P.e. eggs

- ? ? - f~ ? 12 14 -0.1 2 t f 16 ? ? a -0.2 • · · · ·

«?. -0.3 * ? * ? # O > -0.4 ?> w w w y w· ? ^^ ^\ ypy -/»^ ?? y^- • · ·· · *-0,6

o* <^t «j/ >^' 1S^ >y S^ a-0.7 <^K ^y ^k ^t <¿K ?% o J -0.8 -0.9 -1 Time (Weeks)

76 Figure 7A: Log % survivorship curve of young B. glabrata exposed to 0, 1 or 4 P. elegans eggs with a challenge infection of 7 S. mansoni miracidia, three weeks after P. elegans

exposure.

Figure 7B: Log % survivorship curve of young B. glabrata exposed to 0, 8 or 16 P. elegans eggs with a challenge infection of 7 S. mansoni miracidia, three weeks after P. elegans

exposure.

77 7A.

? Control ¦ 1 P.e.egg a 4 Re, eggs

-0.10 2 10 12 14 16!

a -0.2 -j è è u 0.3 ! O > -0.4

3 -0.5 W -0.6 $ -0.7 Ol ¦ O •0.8 •0.9 ?

Time (Weeks)

7B.

? Control • 8 P.e. eggs x 16 P.e. eggs

0 1" -0.1 0 2 io 12 14 16! .9· -0.2 JC £ -0.3 O > -0.4 9 I -0.5 m ¦ -0.6 i -0.7 ? * 3 -0.8 -0.9

-1 ? ????? Time (Weeks)

78 Figure 8A: Log % survivorship curve of juvenile B. glabrata exposed to 0, 1 or 4 P. elegans eggs with a challenge infection of 7 S. mansoni miracidial / snail, three weeks

after P. elegans exposure.

Figure 8B: exposedLog % survivorshipto 0, 8 or 16curveP. elegansof juvenileeggsB.withglabrataa challenge

infection of 7 S. mansoni miracidial / snail, three weeks

after P. elegans exposure.

79 8A.

? Control ¦ 1 P.e. egg a 4 P.e. eggs

-0.1 ó 10 12 14 16s a 111 2 -0.2 -! I "»-? \ > -0.4 -; I -0.5 \ ? t m .0,6 \ S" -0.7 -i ¦ ¦ ¦ ¦ ¦ · ? -0.8 I J -0.9 -I -1 A A Time (Weeks)

8B.

? Control • 8 P.e. eggs x 16 P.e. eggs

-0.1 o I S 10 12 14 161 .5* -o 2 ? i -o 3 O > -o 4 > 5 i 6 7 fiftttttt O 8 J 9 -1 ¦ a Time (Weeks)

80 Figure 9A: Log % survivorship curve of adult B. glabrata exposed to 0, 1 or 4 P. elegans eggs with a challenge infection of 7 S. mansoni miracidial / snail, three weeks after P.

elegans exposure.

Figure 9B: Log % survivorship curve of adult ß. glabrata exposed Jp 0, 8jorJJB P. elegans eggs^ of 7 S. mansoni miracidial / snail, three weeks after P.

elegans exposure.

81 9A.

? Control ¦ 1 P.e. egg a 4 P.e. eggs

a -0.1 0*1*4 6 8 10 12 14 16J JE -0.2 * 0.3 A O rM "Ui^r ™- H m * * W ^Jp W >

Mr

9B.

? Control · 8 P.e. eggs x 16 P.e. eggs

Of -0.1 0 * Î f 4 6 8 10 12 14 16j 5- -0.2 • ? 2 -0-3 ¦¦; t > -0.4 i t # t t t f 3 "?·3-0.5 : % * ?????? « -0.6 J # -0.7 i iff 3 -0.8 ? -0.9 i

^K ^? ?? «fc ^t imi ?* ?? ^K ?* ^K ¿? ^K Time (Weeks)

82 Figure 10: The probability of death for the ß. glabrata within the first two weeks after P. elegans exposure.

Effect of treatments on the probability of death one-week post exposure:

Linear Regression

Young = D.F = 1, 4, F = 3.079, P = 0.178 Juvenile = D.F = 1, 4, F = 10.068, P = 0.05

Adult = D.F = 1, 4, F = 35.974, P = 0.009

83 ? Young ¦ Juvenile a Adult — Linear (Young) — Linear (Juvenile) — Linear (Adult)

4 6 8 10 12 14 16 Treatments (Number of P. elegans egg given)

84 Figure 1 1 : The probability of death for the B. glabrata between weeks three and sixteen after P. elegans

infection.

Linear Regression Young = D.F = 1, 4, F = 3.068, P = 0.178 Juvenile = D.F = 1, 4, F = 0.984 P = 0.394

Adult = D.F = 1, 4, F = 1.218, P = 0.350

85 ? YoungLinear (Young) Juvenile a Adult ¦ Linear (Juvenile) — Linear (Adult)

4 6 8 10 12 16 Treatment (Number of P. elegans eggs given)

86 Figure 12: The probability of death for the ß. glabrata three weeks after P. elegans and one-week post S. mansoni

infection.

87 Young •Juvenile Adult

4 6 8 10 12 14 16 Treatment (Number of P, elegans eggs given)

88 Table I. Tu key multiple comparisons test between the mean egg productions of young B. glabrata exposed to 0, 1, 4, 8 or 16 P.

elegans eggs.

#ofP. elegans g 1 4 3 -jß eggs Q - p=0.992 p=1.000 p=0.951 p=0.033

1 . p=0.993 p=0.998 p=0.04

4 _ p=0.962 p=0.797

8 - p=0.359

(a=0.05)

89 Table II. Tuckey multiple comparisons test between the mean egg productions of adult B. glabrata exposed to 0, 1, 4, 8 or 16 P.

elegans eggs.

#ofP. elegans q ^ 4 8 16 eggs

0 - p=0.996 p=0.949 p=0.0116 p=0.034

------1 = p=0:803 ?=0t?47 p=0v027

4 _ p=0.654 p=0.535

8 - p=1.000

(a=0.05)

90 Table III. Kaplan-Meier survivorship analysis of the % survivorship curve of young B. glabrata exposed to 0, 1 , 4, 8 or 16 P. elegans eggs.

Treatment Mean % Surv. Standard Error 0 46.5 % 4.2 1 61.8% 3.1 4 60.0 % 3.8 8 66.5 % 3.1 16 41.8% 5.0

Table IV. Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of young B. glabrata exposed to 1, 4, 8 or 16 P. elegans eggs to the control snails.

Comparison DF Statistic 0 vs 1 1, 18 0.2 0.9021 0 vs 4 1, 18 0.76 0.3831 0 vs 8 1, 18 0.89 0.3454 0vs16 1, 18 0.14 0.7112 a = 0.0125

91 Table V. Kaplan-Meier survivorship analysis of the % survivorship curve of juvenile B. glabrata exposed to 0, 1, 4, 8 or 16 P.

elegans eggs.

Treatments Mean Standard Error 0 70.0 % 3.1 1 68.2 % 4.9 4 61.8% 4.7 8 81.8% 2.0 16 51.2% 3.7

Table Vl. Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of juvenile B. glabrata exposed to 1, 4, 8 or 16 P. elegans eggs to control snails.

Comparisons DF Statistic 0 vs 1 1, 18 0.08 0.7707 0 vs 4 1, 18 0.50 0.4812 0 vs 8 1, 18 0.00 0.9874 0vs16 1, 18 4.33 0.0119 a = 0.0125

92 Table VII. Kaplan-Meier survivorship analysis of the % survivorship curve of adult ß. glabrata exposed to 0, 1 , 4, 8 or 16 P. elegans

eggs.

Treatments Mean Standard Error 0 81.2% 5.0 1 82.4 % 3.4 4 74.1 % 3.2 8 43.5 % 5.3 16 27.1 % 5.9

Table VIII. Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of adult B. glabrata exposed to 1, 4, 8 or 16 P. elegans eggs to control snails.

Comparisons DF Statistic 0 vs 1 1, 18 0.00 0.9480 0 vs 4 1, 18 1.07 0.3001 0 vs 8 1, 18 6.26 0.0100 0vs16 1, 18 7.25 0.0071 a = 0.0125

93 Table IX. Kaplan-Meier survivorship analysis of the % survivorship curve of young B. glabrata exposed to 0, 1, 4, 8 or 16 P. elegans eggs with a challenge infection with 7 S. mansoni miracidial / snail, three weeks after P. elegans exposure.

Treatments Mean Standard Error OP.e. /7S.m. 23.5% 8.1 1P.e./7S.m. 22.9% 8.1 4P.e./7S.m. 24.1% 6.9 8P.e./7S.m. 19.4% 7.6 16P.e./7S.m. 32.9% 5.8

Table X. Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of young B. glabrata exposed to 1, 4, 8 or 16 P. elegans eggs to the control snails with a challenge infection with 7 S. mansoni miracidial / snail, three weeks after

P. elegans exposure.

Comparisons DF Statistic 0vs1 1,18 0.23 0.6338 0vs4 1,18 0.01 0.9318 0vs8 1,18 1.26 0.2621 0vs16 VI8 0?2 0.7316 a =0.0125

94 Table Xl. Kaplan-Meier survivorship analysis of the % survivorship curve of juvenile B. glabrata exposed to 0, 1, 4, 8 or 16 P. elegans eggs with a challenge infection with 7 S. mansoni miracidial / snail, three weeks after P. elegans exposure.

Treatments Mean Standard Error 0 P.e. / 7 S.m. 38.2 % 7.8 1 P.e. / 7 S.m. 38.2 % 7.9 4 P.e. /7 S.m. 21.1% 9.2 8 P.e. / 7 S.m. 36.5 % 8.5 16 P.e. /7 S.m. 32.4% 7.7

Table XII. Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of juvenile B. glabrata exposed to 1, 4, 8 or 16 P. elegans eggs to the control snails with a challenge infection with 7 S. mansoni miracidial / snail, three weeks after

P. elegans exposure.

Comparisons DF Statistic 0vs1 1,18 0.23 0.6338 0vs4 1,18 0.01 0.9318 0vs8 1,18 1.26 0.2621 0vs16 1,18 2.74 0.099 a =0.0125

95 Table XIII. Kaplan-Meier survivorship analysis of the % Survivorship curve of adult B. glabrata exposed to 0, 1, 4, 8 or 16 P. elegans eggs with a challenge infection with 7 S. mansoni miracidial / snail, three weeks after P. elegans exposure.

Treatments Mean Standard Error 0 P.e. / 7 S.m. 50.0 % 6.3 1P.e./7S.m. 25.9% 10.5 4 P.e. / 7 S.m. 45.3 % 6.3 8 P.e. /7 S.m. 17.1% 7.1 16 P.e. /7 S.m. 20.6% 6.1

Table XIV. Kaplan-Meier log ranked survivorship analysis comparing the % survivorship curves of adult ß. glabrata exposed to 1, 4, 8 or 16 P. elegans eggs to the control snails with a challenge infection with 7 S. mansoni miracidial / snail, three weeks after

P. elegans exposure.

Comparisons DF Statistic 0vs1 1,18 0.35 0.5540 0vs4 1,18 0.77 0.3815 0vs8 1,18 4.52 0.0109 0vs16 V|8 8/I2 0.0044 a = 0.0125

96 CONNECTING STATEMENT 1

Chapter 3 deals with the effects of P elegans on a community of the incompatible snail host Biomphalaria glabrata composed of different size classes followed by a challenge infection of Schistosoma mansoni. It was demonstrated that, even in a community of snails, P. elegans at high exposure levels does significantly reduce egg production and survivorship of the incompatible snail host Biomphalaria glabrata. The question arises, whether P. elegans has the same negative effect on the survivorship and egg production of other incompatible snails hosts. In Chapter 4, we investigate the effect of P. elegans exposure on a sympatric snail host Helisoma trivolvis trivolvis and on the snail Bulinus truncatus, an intermediate snail host to Schistosoma haematobium.

97 CHAPTER 4

THE EFFECTS OF PLAGIORCHIS ELEGANS (DIGENEA: PLAGIORCHIIDAE)

ON THE REPRODUCTION AND SURVIVORSHIP OF THE INCOMPATIBLE

SNAIL HOSTS BULINUS TRUNCATUS (PULMONATA: PLANORBIDAE) AND HELISOMA TRIVOLVIS TRIVOLVIS (PULMONATA: PLANORBIDAE).

*Daoust, S., **Mader, B., **McLaughlin, J.D., and M. E. *Rau

* Department of Natural Resource Sciences, McGiII University (Macdonald Campus), 21 111 Lakeshore Road, Ste-Anne-de-Bellevue, Qc, Canada, H9X 3V9

** Department of Biology, Concordia University (Loyola Campus), 7141 Sherbrooke St W. Montreal, Qc, Canada, H4B 1R6

Running Title: Effect of P. elegans on B. truncatus and H. trivolvis trivolvis

98 ABSTRACT

Infection with the digenean parasite Plagiorchis elegans does not have a significant effect on egg production of young and adult sympatric snails, Helisoma trivolvis trivolvis. The survivorship of adult H. t. trivolvis was not significantly affected by exposure to P. elegans. This being said, young H. t. trivolvis exposed to P. elegans had a mean % survivorship of 89.4 ± 1 .6 % which was significantly higher than control snails having a mean % survivorship of 74.4 ± 3.6 %. Exposed Bulinus truncatus snails laid up to 50% fewer eggs for the first five weeks following exposure to P. elegans. Although there was no significant effect of P. elegans on the survivorship of B. truncatus, this experiment does demonstrate that the parasite could serve as an agent in the biological control of this snail.

99 INTRODUCTION

One of the characteristic features of digenean - mollusk associations is their high specificity (Cort, 1941; Llewellyn, 1965; Wright, 1973; Shoop, 1988). The majority of digenean species develop successfully solely within members of a single molluscan family, genus or even species or strain (Llewellyn, 1965; Wright, 1973; Shoop, 1988). This specificity is expressed at the level of the miracidia, which may not be able to penetrate an incompatible host (Haas et al., 1991). Plagiorchis elegans lacks a free-swimming miracidial stage that provides behavioral mechanisms for avoiding penetration into incompatible hosts. As a consequence, P. elegans may face a high probability of ingestion by unsuitable hosts.

P. elegans has been shown to establish patent infections in both Stagnicola elodes and Lymnaea stagnalis. Zakikhani and Rau (1998a) demonstrated that this parasite could also establish an infection in the incompatible snail Biomphalaria glabrata (Zakikhani and Rau, 1998a). However, P. elegans was unable to complete its development within this snail host, having its development stopped prior to daughter sporocyst and cercarial production. Nevertheless, all infected snails developed symptoms of parasitic castration, having their total egg production reduced by 90% of control levels. More importantly, there is a concomitant reduction in the number of cercahae shed by a challenge infection with S. mansoni (Zakikhani et al., 2003).

100 The purpose of this work was to extend the investigation by assessing the impact of P. elegans on other incompatible snail host. Here we report results of the effect of P. elegans on reproductive output and survivorship of two incompatible snails; Helisoma trivolvis trivolvis, which occurs sympatrically with Stagnicola elodes, and Bulinus truncatus, the intermediate snail host of the human parasite Schistosoma haematobium.

Materials and Methods

Snail Hosts

Bulinus truncatus

Snails were obtained from the Biomedical Research Institute, Rockville, Maryland. The colony was maintained in an incubator at 26.5 0C, under a L12:D12 photoperiod with ~ 50% relative humidity (El-Emam and Madsen, 1982). Snails were kept in 26 ? 18 ? 8 cm aquaria filled with 1 .5 L of aerated tap water; and fed washed Romaine lettuce and powdered calcium carbonate ad libitum.

Stagnicola elodes A colony of laboratory-reared S. elodes has been maintained in our laboratory for more than ten years. These snails were derived from a wild stock with a high natural prevalence of infection with the digenean P. elegans. Snails were reared under a L16:D8 photoperiod, in 473 ml containers (Solo Cup

101 Company, Urbana, Illinois) filled with aerated tap water at 22 0C. The water was changed monthly.

Helisoma trivolvis trivolvis

Hélisoma t. trivolvis were collected from shores of the Saint-Lawrence

River at Pointe-aux-Cascades, Dorion, Quebec, where they occur sympatrically with S. elodes that were infected with P. elegans. The snails were reared and maintained under the same conditions as Stagnicola elodes.

Parasites

Plagiorchis elegans The life cycle of P. elegans has been maintained in the laboratory for more than a decade, using hamsters, Mesocricetus auratus, as the definitive host. Laboratory-reared S. elodes served as the first intermediate host and were exposed to P. elegans eggs in a small volume of water (Zakikhani and Rau, 1998a; 1998b). The addition of small amounts of ground Tetramin® fish food to the exposure chambers helped stimulate feeding by the snail and the consumption of parasite eggs (Zakikhani and Rau, 1998a; 1998b). Infections reached patency in five to six weeks, and cercariae were induced to emerge from the snail host by an environmental change in light intensity from light to dark (Lowenberger et al., 1994). Laboratory-reared fourth instar Aedes aegypti larvae were used as the second intermediate host. Larvae were exposed to cercariae, which penetrate

102 their tissues where they form metacercariae. When metacercariae reached infectivity three to seven days later, the host mosquito larvae were lightly crushed. Numbers of metacercariae were estimated on the basis often random samples, and approximately one hundred metacercariae were fed to each hamster. Metacercariae require eight days to develop into ovipositing adult worms (Zakikhani and Rau, 1998a; 1999b). Eggs are passed with the feces of the hamsters and collected overnight as needed. The eggs were collected in a tray of water and are separated from the fecal debris by washing through a series of three brass sieves (mesh sizes 200, 65 and 37 urn). The eggs were then CK. collected on the 20 urn mesh, and re-suspended in deionized water. Eggs were allowed to embryonate at 20 ± 4°C, for 4 days before they were fed to snails (Zakikhani and Rau, 1998b).

Experimental Methods Eggs of P. elegans were obtained from the feces of experimentally infected hamsters as described above. The mean density of eggs in suspension was determined on the basis often 0.025 ml samples drawn by automatic pipette (Zakikhani and Rau, 1998b). The suspension was diluted serially to attain the desired number of 16 embryonated eggs per sample.

Juvenile Bulinus truncatus were kept in 26 ? 18 ? 8 cm aquaria for two months after they were received from the Biological Research Institute, allowing them to acclimatize to laboratory conditions and become sexually mature. All

103 experiments took place in a locked, walk-in incubator kept at 26.5 0C, under a L12:D12 photoperiod with a relative humidity of ~ 50%. Once egg masses started appearing on the side of the aquaria, 50 B. truncatus snails were placed in tissue culture wells (3.5 mm diameter, 16 mm deep) (Linbro® Space Saver, Flow Laboratories Inc.) with 4 ml of water and some tetramin ® fish food. Sixteen P. elegans eggs were placed in twenty-five of the tissue culture wells, while the other twenty-five served as controls. A plastic sheet was placed over the tissue culture wells preventing the snails from escaping. The low volume of water and the tetramin ® both serve to facilitate the ingestion of the P. elegans eggs. The B. truncatus were kept in the tissue culture wells for twenty-four hours and then transferred, individually, to 473 ml plastic containers (Solo Cup company, Urbana, Illinois), with a bottom surface area of 70.88 cm2 with 200 ml of aerated tap water. Egg production and survival were monitored for ten weeks. Water was changed monthly and snails were fed ad libitum.

Juvenile pre-reproductive snails (5 mm in shell diameter) and adult reproductive Helisoma trivolvis trivolvis (10 mm in shell diameter) were kept under a L16:D8 photoperiod, and reared in 26 ? 18 ? 8 cm aquaria filled with 1 .5 L of aerated tap water at 22 0C. Twenty four adult and juvenile Helisoma were individually placed in tissue culture wells (3.5 mm diameter, 16 mm deep) (Linbro® Space Saver, Flow Laboratories Inc.), each containing 4 ml of aerated tap water and tetramin ® fish food. Sixteen parasite eggs were added, via micropipette, to twelve of the adult wells and twelve of the juvenile wells. A

104 plastic sheet was then placed over the wells, preventing the snails from escaping. The snails were allowed to forage and ingest the eggs for a period of twenty-four hours. They were then placed into individual plastic containers filled with 200 ml of aerated tap water. Survival and egg production were monitored weekly. Water was changed monthly and snails were fed ad libitum. At week fourteen, the adult and young snails were removed from their individual containers and paired with another snail.

Statistical Analysis All experiments followed a complete randomized design. Data were analyzed using SPSS (SPSS inc. 2008, Chicago Illinois). The data for the weekly number of eggs produced from young and adult Helisoma t. trivolvis and from adult ß. truncatus were ranked transformed to satisfy the conditions of normality (Sokal and Rohlf, 1995). A repeated measures ANOVA was used to analyze these data. Standard error and confidence intervals for the survivorship curves were calculated using a Kaplan-Meier survivorship analysis. Kaplan-Meier log-ranked survivorship analyses were used to compare the survivorship curves of the control snails to the snails exposed to P. elegans.

105 RESULTS

There was no significant difference between the number of eggs produced by adults exposed to 16 P. elegans eggs and adult control H. t. trivolvis (Repeated Measures ANOVA, DF= 1 , 22, F= 1 .247, P = 0.290, a= 0.05) (Figure 1). Interestingly, both the adult infected and control Helisoma ceased egg production fourteen weeks into the experiment. At week fourteen when they were paired with another adult snail, they began to produce eggs again. There was no significant difference between the egg production of young H. t. trivolvis exposed to 0 and 16 P. elegans eggs (Repeated Measures ANOVA, DF= 1, 22, F= 1.161,

P = 0.297, a= 0.05) (Figure 2). The young snails only started laying eggs at week fourteen when they were paired with another snail.

Adult H. t. trivolvis exposed to 16 P. elegans eggs had a mean % survivorship of 86.1 ± 2.9 %. The control snails also had a mean % survivorship of 86.1 ± 3.4 %, there was no significant difference between the latter (Kaplan- Meier log ranked survivorship analysis, DF = 1, 22, Statisitc = 0.88, P = 0.3460) (Figure 3). Young H. t. trivolvis exposed to 16 P. elegans eggs had a mean % survivorship of 89.4 ± 1 .6 % whereas control snails had a significantly lower mean % survivorship of 74.4 ± 3.6 % (Kaplan-Meier log ranked survivorship analysis, DF = 1 , 22, Statisitc = 3.34, P = 0.039) (Figure 4). Adult B. truncatus exposed to 16 P. elegans eggs, laid significantly fewer eggs than did controls (Repeated Measures ANOVA, DF = 1, 49, F = 5.221, P =

106 0.0028) (Figure 5). Adult B. truncatus exposed to 16 P. elegans eggs had a mean % survivorship of 83.6 ± 1 .8 % which did not differ significantly from the control snails having a mean % survivorship of 89.1 ±1.1 % (Kaplan-Meier log ranked survivorship analysis, DF = 1, 22, Statisitc = 0.35, P = 0.5540) (Figure 6).

DISCUSSION

Much of the previous work done studying the effects of Plagiorchis elegans on incompatible snails has primarily focused on the human schistosomiasis snail vector, Biomphalaria glabrata (Zakikhani and Rau, 1998a; Zakikhani et al., 2003; Platero, 2004). These works demonstrated the potential of using P. elegans as a biological control agent of B. glabrata and Schistosoma mansoni.

In the first part of this study, we investigated the effect of P. elegans on a sympatric snail species. The H. t. trivolvis used for this experiment were collected from an area known to have high densities of S. elodes that are also infected with P. elegans. Because the eggs of P. elegans must be ingested by their snail hosts in order to continue its life cycle, there is a high probability that they also be ingested by incompatible snail species. It was therefore of interest to see if sympatric species have developed mechanisms of dealing with this parasite. Egg production of adult and young H. t. trivolvis was not significantly affected at exposure to a high concentration of P. elegans eggs. It is a general assumption that digenean parasites have little to no effect on incompatible snail hosts, but it

107 has been shown that young ß. glabrata when exposed to the same high concentration of P. elegans were permanently castrated (Platero, 2004). Furthermore, when adult B. glabrata where exposed to the same high concentration of P. elegans, their reproductive output was reduced to 13% of the control values (Zakikhani and Rau, 1998b). Interestingly enough, not only are there no significant effects of P. elegans on the survivorship of adult H. t. trivolvis, but the young H. t. trivolvis seem to have a higher survivorship. The ability of P. elegans to castrate or kill competing incompatible snail species may be of selective significance, as would be the ability to resist such effect in the affected sympatric species. Conceivably, H. t. trivolvis has been exposed to such pressures and has adapted. Such adaptive mechanisms, most likely immunological in nature, may enhance the responses of incompatible hosts to extraneous opportunistic bacterial and parasitic infections, enhancing the survival of young, immunologically naive snails. There is very little information available in the literature on the life history of H. t. trivolvis. From our experiment we were able to shine some light on a few interesting facts about this snail. Firstly, unlike most pulmonate snails, H. trivolvis trivolvis does not self fertilize. They can, however, store sperm for up to 14 weeks. Figure 1 clearly shows that egg production of adult snails stops at week fourteen; however, once the snails were paired again at week fourteen and allowed to cross-fertilize their egg production was renewed. These findings are supported by the data gathered from the young Helisoma snails. Young Helisoma snails were segregated before they were able to cross-fertilize and

108 therefore do not have any stored sperm. Figure 2 demonstrates that the young snails only start producing eggs at week fifteen, after being paired off and allowed to cross-fertilize.

Bulinus truncatus serves as one the major intermediate snail hosts for the digenean parasite Schistosoma haematobium, the causative agent of Human Schistosomiasis throughout most of the African continent. It was therefore interesting to investigate the effect of P. elegans exposure on the reproductive output and survivorship of this snail. We demonstrated that B. truncatus exposed to a high concentration of P. elegans eggs produced significantly fewer eggs than control snails. As we can see in Figure 5, the infected snail lay about 50% fewer eggs than did control snails for the first five weeks post P. elegans exposure. However, after five weeks, the snails seemed to gradually recover from the parasite induced stress response and the reproduction of infected snails gradually increased back to control levels. Although there was no significant effect of P. elegans on the survivorship of B. truncatus, this experiment does demonstrate that the parasite could serve as a biological control agent of the snail. This being said, several more experiments should be done before field trials are attempted. It would be interesting to see the effect of a second P. elegans exposure, five weeks post initial infection, on the egg production of B. truncatus.

109 ACKNOWLEDGEMENTS

We would like to thank Professor P. J. Albert, Concordia University for his guidance and help. This work was supported by an NSERC grant to M. E. Rau.

110 LITERATURE CITED

Cort, W.W. (1941). Ecological Relations of the Larval Trematodes of Fresh-Water Snails. Symposium on hydrobiology, University of Wisconsin, pp 115-128.

El-Emam, M.A., Madsen, H. (1982). The effect of temperature, darkness, starvation and various food types on growth and reproduction of Helisoma duryi, Biomphalaria alexandria and Bulinus truncates (Gastropoda: Planorbidae). Hydrobiologia 88: 265-275.

Haas, W., Gui, M., Haberl, B., Strobel, M. (1991). Miracidia of Schistosoma japonicum: approach and attachment to the snail host. Journal of

Parasitology 77: 509-513.

Llewellyn, J. (1965). The evolution of parasitic platyhelminths. In: Taylor AER (ed) Evolution of Parasites, pp. 47-78. Blackwell Scientific Publications,

Oxford.

Lowenberger, CA. Rau, M. E. (1994). Plagiorchis elegans: emergence, longevity and infectivity of cercariae, and host behavioural modifications during cercarial emergence. Parasitology 109: 65-72.

Ill Platero, I. A. (2004). The effects of parasite dose, host size and the method of exposure on the reproductive capacity and survival of Biomphalaria glabrata infected with the incompatible digenean, Plagiorchis elegans. M. Sc. Thesis. McGiII University, Montreal, Canada.

Shoop, W.L. (1988). Trematode transmission patterns. Journal of Parasitology

74: 46-49.

Sokal, R. R. and F.J. Rholf. (1995). Biometry: The Principles and Practice of Statistics in Biological Research. W.H. Freeman and Company, New York 882 pp.

Wright, CA., (1973). Flukes and snails. Macmillan, New York, New York, 168 p.

Zakikhani, M., Rau, M. E. (1998a). Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection on the reproduction of Biomphalaria glabrata (Pulmonata: Planorbidae). Journal of Parasitology 84 (5): 927-930.

Zakikhani, M., Rau, M. E. (1998b). The effects of temperature and light regimens on the survival, development, and infectivity of Plagiorchis elegans eggs. Journal of Parasitology 84: 1 1 70-1 1 73.

112 Zakikhani, M., Smith, J. M., Rau, M. E. (2003). Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection of Biomphalaria glabrata (Pulmonata: Planorbidae) on a challenge infection with Schistosoma mansoni (Digenea: Schistosomatidae). Journal of Parasitology 89 (1): 70-75.

113 Figure 1: Mean egg production (± S.E.) of adult Helisoma t. trivolvis exposed to 0 and 16 P. elegans eggs. At

week 14 post exposure, the snails were paired.

Repeated Measures ANOVA;

DF= 1, 22 F= 1.247 P = 0.290 a = 0.05

114 •Adult Infected Adult Control

8 10 12 14 16 18 Time (weeks)

115 Figure 2: Mean egg production (± S.E.) of young Helisoma

t. trivolvis exposed to 0 and 16 P. elegans eggs. At week

14 post exposure, the snails were paired.

Repeated Measures ANOVA;

DF= 1 , 22 F= 1 .161 P = 0.297 a = 0.05

116 ¦?— Young Infected I- «Young Control

120

IUg 100 «?- ? i. » e 3 Z e s (S S

Time (weeks)

117 Figure 3: Log % survivorship of the adult Helisoma t.

trivolvis exposed to 0 and 16 P. elegans eggs.

118 ¦*— Adult Infected ?- Adult Contro!

¿ 12 14 161 -0.05 4 a 2 ¦·—t—H—t" IA -0.1 i. O

•|L -0.15 m -0.2

© -0.25

-0.3

-0.35 Time (weeks)

119 Figure 4: Log % survivorship of the young Helisoma t. trivolvis exposed to 0 and 16 P. elegans eggs.

120 ¦?—Young Infected - ·¦- Young Control

,M5P> j t i I I |. § I tt 12 14 16 i -i -¦ -§ -? > ? g -0,1 ? ?

Iim -0.15 \ 3 m -0,2 % w -0.25 ? w « ß w ß O J -0,3

-0.35 Time (weeks)

121 Figure 5: Mean egg (± S.E.) production of adult ß. truncatus infected with 0 and 16 P. elegans eggs.

Repeated Measures ANOVA;

DF= 1,49 F= 5.221 P = 0.0028 a = 0.05

122 -?-Control Infected

« 30 m m ui 25 O i. & 20

imm 15 Z C 10 ig s S

123456789 10 Time (Weeks)

123 Figure 6: Log % survivorship of adult B. truncatus

exposed to 16 P. elegans eggs.

124 Control Infected

2 io 12!

-t- -« i i · 1 ? S -0.06

# -0.08 m o ¦J -0.1 !·-¦·¦·· ¦

-0.12 Time (Weeks)

125 CHAPTER 5

GENERAL DISCUSSION

Previous research has recognized that the sporocyst stages of the incompatible plagiorchiid digenean Plagiorchis elegans (Rudolphi, 1802), can establish infections within the tissues of the snail Biomphalaria glabrata, intermediate host of Schistosoma mansoni (Zakikhani and Rau, 1998; Zakikhani et al., 2003). Here they elicit the same prompt and severe suppression of reproductive output as they do in their compatible snail host, Stagnicola elodes Say, 1821 (Zakikhani and Rau 1998; Zakikhani et al., 2003). Interestingly enough, not only is B. glabrata castrated by P. elegans but it is also rendered resistant to a concomitant S. mansoni infection. These studies, however, focused on the infection of individual snails, they did not evaluate the effectiveness of P. elegans egg exposures on a community of snails composed of different ages / sizes. Furthermore, these studies only examined the effect of P. elegans on B. glabrata; no other incompatible snail species have been experimentally exposed to P. elegans eggs. In Chapter 3 of this work we found that there was a significant difference after exposure to P. elegans eggs in reproductive output and survivorship of young, juvenile and adult snails whether they were exposed individually or in groups. At lower and intermediate P. elegans exposure levels, adult B. glabrata

126 appear to serve as parasitic sinks, ingesting the majority of the P. elegans eggs preventing smaller snails from acquiring an infection. However, at higher exposure levels, the young and juvenile snails' reproductive output and survivorship was significantly reduced. Our findings also support the work of Zakikhani et al. (2003), demonstrating the ability of P. elegans to render the incompatible snail host B. glabrata resistant to a concomitant infection with

Schistosoma mansoni.

In Chapter 4 we experimentally exposed two incompatible snail hosts: Helisoma t. trivolvis, a snail that occurs sympatrically with P elegans and B. truncatus, the intermediate snail host to Schistosoma haematobium. We demonstrated that egg production of adult and young H. t. trivolvis was not significantly affected by exposure to a high concentration of P. elegans eggs. Interestingly enough, not only were there no significant effects of P elegans on the survivorship of adult H. t. trivolvis, but the young snails seemed to have a higher survivorship when exposed to P elegans. We also demonstrated that B. truncatus, when exposed to a high concentration of P elegans eggs, produces significantly fewer eggs than control snails. However, after five weeks, the snails seemed to gradually recover from the parasite induced stress response and reproduction of infected snails gradually increases back to control levels. Plagiorchis elegans eggs have great potential as a biological agent in the control of human schistosomiasis. Eggs are easily produced in the laboratory; P elegans adults are very prolific, passing more than 30 000 eggs daily in the feces of experimental hamster host, for a period of more than three weeks. At high

127 concentration levels, P. elegans will successfully castrate and reduce the survivorship of young, juvenile and adult B. glabrata within a large community, and rendering them resistant to infection with S. mansoni. Plagiorchis elegans has also demonstrated the potential of being used to control the intermediate snail host of S. haematobium, B. truncatus. This suggests that this one parasite could be used to control the two most common and widely distributed vectors of human schistosomiasis. One of the major concerns of biocontrol are the inevitable negative impact of the introduction of a foreign species into a new ecosystem. P. elegans eggs, however, do not develop in incompatible snail species, and therefore are less likely to successfully reproduce in new ecosystems. Contrarily to molluscicides, the negative effect of P. elegans on snail species might not be generalized (H. t. trivolvis was shown to be unaffected by the parasite), and therefore might not impact all snail species when added to a new ecosystem.

There are several interesting avenues of research left to explore. Firstly, It would be interesting to conduct a controlled field experiment using P. elegans eggs to control the size of B. glabrata communities. It would also be worthwhile to conduct some molecular experiments studying the effects of P. elegans on the immune system and reproductive system of B. glabrata. It would also be instructive to expose B. truncatus to graded concentrations of P. elegans eggs.

128 LITERATURE CITED

Zakikhani, M., Rau, M. E. (1998a). Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection on the reproduction of Biomphalaria glabrata (Pulmonata: Planorbidae). Journal of Parasitology 84 (5): 927-930.

Zakikhani, M., Smith, J. M., Rau, M. E. (2003). Effects of Plagiorchis elegans (Digenea: Plagiorchiidae) infection of Biomphalaria glabrata (Pulmonata: Planorbidae) on a challenge infection with Schistosoma mansoni (Digenea: Schistosomatidae). Journal of Parasitology 89 (1): 70-75.

129 APPENDICES

130 2

Forni version January 2008 3. Summary (in language that will be understood by members ofthe general public) AIMS AND BENEFITS: Describe, in a short paragraph, the overall aim of the study and its potential benefit to human/animal healtit or to the advancement of scientific knowledge (was section Sa in main protocol), We are assessing the role of an entomopathogenic digenean parasite (Plagiorchis eiegans) as a natural enemy of mosquito larvae, with a view to developing a biological method of control ofmosquito vectors of diseases such as yellow fever, malaria, west nile virus and others. The entomopathogemc stage of the parasite is produced asexualîy in the tissues of an aquatic snail intermediate host (Stagnicela elodes), and is released daily in massive numbers into the aquatic environment. The minute parasitic organisms penetrate and kill the larvae of a wide range ofmosquito species. Plagiorchis eiegans and a number of related digenean parasites can also establish themselves in the tissues of snails other than their normal, compatible host species (Stagnicola elodes). In such incompatible hosts, P, eiegans wilinot develop beyond its early embryonic stages, but will nevertheless castrate the incompatible snails and render these unsuitable as hosts oftheir normal guilds of digenean parasites. Thus, P. eiegans infections of the snail Biomphalaria glabrata will stop these snails from reproducing and protects them from infections with Schistosoma mansoni, the aetiological agent of human schistosomiasis (blood fluke disease). Thus, as a group, plagiorchiid digeneans may have profound effects on the reproductive potential of sympatric snail populations and their respective digenean parasite faunas. These attributes may render . plagiorchiid digeneans ofuse in the biological control ofsnails and snail-borne diseases ofmedical and veterinary importance. 4, Has there been any animal care issues? | YES D NO a if yes, supply details:

S. If creating genetically modified animals or new combinations of genetic modifications, complete and attach a Phenotype Disclosureform. If mice expressing new phenotype have been produced, submit a Phenotype Disclosureform. Blank forms at http:/Âmm\mcgillc(i/resem'choffice/complmnce/animal/formsf _ 6. Procedures a) For B attd C level of invasiveness, The procedures are the same as the original protocol: YESJEl NO D IFNQ, complete the following: Detail new procedures that are different from section 10a of the original protocol, including amendments (include a copy ofthe entire revisedprocedure section 10a ofthe originalprotocol with the changes and/or newprocedures in CAPS):

hi For D level of invasiveness, Include here ALL procedures described In the original protocol. New and changed procedures in CAPS (was section 10a in main protocol); Please only attach SOPs related to new and changed procedures to this renewalform.

7. Endpoiiits a) For B and C level of invasiveness. The procedures are the same as the original protocol: YES[Xl NO D IF NO. supply new endpoints that are different from the original protocol: Experimental endpoints:

Clinical endpoints:

b) For D level of invasiveness, . - Include here ALL endpoints, including the ones described in the original protocol as well as new and changed endpoints in CAPS: Experimental endpoints:

Clinical endpoints:

8. Hazards (check here if none are used: f~l) a) Are the hazards different from original protocol? {infectious, radioactive, toxie, carcinogen, tumours) YES D NO _3 if yes, supply details (material, risks, precautions):

b) Have the ceil lines been tested for human and animal pathogens? YES:_3 NO:_] None used:£

9. Description of.Animals to be used to the coming year (only): jQHaUí3tCaSe¿iísSaiS_üS_l To prevent introduction of infectious diseases into animal facilities, a health status report or veterinary inspection certificate may be required prior to receiving animals from all rtoii-commercìal sources or from commerciai sources whose animal health status is unknown or questionable. Quarantine and further testing may be requin»! for these animals. Ifmore than 6 columns are needed, please attach anotherpaga Sp/strain 1 Sp/strain 2 Sp/strain 3 Sp/strain 4 Sp/strain 5 Sp/strain 6 Species Mesocricetus Mus musculus Rana pipiens aitratus Swppler/Seuree Charles Hiver Charles River Boreal Strain LVG CD-I wild Sex female female both Age/Wt 21 days 21 days large # To be purchased 24 "24 # Produced by ia- N.A. NA. N.A. hotise breeding § Other RA. N.A. N.A. (e.g.fMd studies) TOTAlJ /YEAR 24 24 24

10. Explanation of Animal Numbers: BASED ON THE EXPERIMENTAL OBJECTIVES OF THE PROJECT, describe the number of animals required for one year. Include information on experimental and control groups, # per group, and failure rates. For breeding, specify how many adults are used, number of offspring produced, and how many offspring are used in expérimentai procedures. The arithmetic explaining how the total of animals for each colaran in the table above is calculated shoaid be made clear. — Mesocricecus auratus: Production of 200,000 eggs per month requires 2 hamsters. Since P. elegans infections persist for approximately one month, and hamsters develop an immunity to reinfection, they will be infected once only. We will, therefore, need 24 hamsters per annum. The eggs will be used to infect various species of snails . as well as laboratory populations of Biomphaiaria glabrata to assess the protective effects.

Mus museulus; We will infect and kill 2 mice per month to provide a steady supply of Schistosoma mansoni eggs to infect B. glabrata. Rana pipiens: A miriimum order consists of 12 frogs. These will serve as a source of infection for incompatible snail hosts of various species with the eggs of Haplemetrana, Haematoloechus and others, to determine effects on host reproduction.

Submit to your local Facility Animal Care Committee. Please note thai after two renewals, afullprotocol needs to be submitted. This approval does not imply that space, will be made available. Ifa major increase ofspace needs is anticipated, please contact the appropriate animalfacility manager.