Host-Parasite Interactions: Snails of the Genus Bulinus and Schistosoma Marqrebowiei BARBARA ELIZABETH DANIEL Department of Biol

/ Host-parasite interactions: Snails of the genus Bulinus and Schistosoma marqrebowiei BARBARA ELIZABETH DANIEL Department of Biology (Medawar Building) University College London A Thesis submitted for the degree of Doctor of Philosophy in the University of London December 1989 1 ProQuest Number: 10609762 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609762 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ABSTRACT Shistes c m c a In Africa the schistosomes that belong to the haematobium group are transmitted in a highly species specific manner by snails of the genus Bulinus. Hence the miracidial larvae of a given schistosome will develop in a compatible snail but upe*\. ^entering an incompatible snail an immune response will be elicited which destroys the trematode. 4 The factors governing such interactions were investigated using the following host/parasite combination? Bulinus natalensis and B^_ nasutus with the parasite Spect'e3 marqrebowiei. This schistosome^develops in B^_ natalensis but not in B_;_ nasutus. The immune defence system of snails consists of cells (haemocytes) and haemolymph factors. The haemocytes from the two snail species were compared on both a quantitative and qualitative level. Cell numbers were compared between species befcrcand following infection with S^_ marqrebowiei. The existence of sub-populations of haemocytes was examined using both functional tests and differential binding by lectins. The triggering of the immune response requires that the invasive pathogen be recognised as foreign. Research in other fields of invertebrate immunology indicate that this may be mediated by factors, such as agglutinins, present in the haemolymph. In snails and schistosomes it is postulated that these factors recognise molecules on the surface of the larval schistosomes and the binding of factor to larvae elicits the immune response. The cell-free haemolymph of the snails was 2 investigated for the presence of haemagglutinins. A protein capable of agglutinating vertebrate red blood cells was found in the haemolymph of JL. nasutus snails. The surface carbohydrate and protein profiles of the different larval stages, miracidium, sporocyst and cercaria, were analysed using techniques of lectin binding and biotinylation of proteins followed by subsequent visualisation with labelled avidin. For these purposes the miracidia were transformed into sporocysts in vitro to ensure they were free of acquired snail proteins and sugars. The larvae were also investigated for possible interactions with the snail haemagglutinin. B. nasutus agglutinin was found to bind to the surface of s. marqrebowiei miracidia but not to other larval forms. 3 B ACKNOWLEDGMENTS It never occurred to me, when starting my degree course in 1982, that 7 years later I would be writing the o, acknowledgments page for my doctoral thesis. That this has happened, is due mainly to Mr T.M. Preston, whose constant encouragement and occasional bullying have been invaluable. As a CASE award student, I have had two supervisors and feel fortunate in having had as co-supervisor, Dr V.R. Southgate of the British Museum (Natural History) who also has been ever helpful and encouraging. My thanks go the Experimental taxonomy division BM(NH) and in particular to Mr M. Anderson for the supply of biological materials. I would also like to thank Dr C.A. King and Dr J.E.M. Heaysman of the Biology Department, University College for their support. This thesis would not have been written without the support and help I received from my friends, especially: Ms P. A. Pipkin for proof reading and 'phone calls? Mr F.G. Hickling for lunches, maths and computing; and Dr. K. Hooper for showing me it could be done. I am grateful to Mr J.E. Pipkin who produced figures 1,2 & 11 and Ms H. Wilson for helping me master yet another word processing package. This work was supported by a grant from the SERC which financial support I acknowledge. 4 CONTENTS TITLE PAGE 1 ABSTRACT 2 ACKNOWLEDGEMENTS 4 CONTENTS 5 FIGURES 8 TABLES 13 INTRODUCTION 14 1 Schistosome Life-Cycle 16 2 Schistosomiasis: Human Pathology 24 3 Schistosome Taxonomy 32 4 Snail Immune Response 36 (a) Repertoire of the snails immune system 38 (b) Manifestations of the snail immune response 46 (c) Genetics of snail/schistosome interactions 49 (d) Schistosome immune evasion 50 5 Interactions of Schistosomes and bulinid snails 53 (a) Snail Taxonomy 54 (b) Hybridisation of schistosomes 59 (c) Molluscicides 60 6 Hypothesis 61 MATERIALS AND METHODS 63 1 Snails and Parasites 63 2 Animal Maintenance 64 (a) Snails 64 (b) Schistosome Life-Cycle 67 5 3 Collection of Snail Haemolymph 69 4 Microscopy 70 (a) Phase 70 (b) Scanning Electron Microscopy 70 (c) Fluorescence Microscopy 71 5 Gel Electrophoresis 72 6 Schistosomes 75 (a)'In vitro transformation of miracidia 76 (b) Surface Morphology 80 (c) Surface Chemistry 80 7 Haemocytes 86 (a) Haemocyte Numbers 86 (b) Haemocyte sub-populations 90 (c) Haemocyte cytoskeleton 94 8 Haemagglutinins 96 (a) Haemagglutination Assay 96 (b) Presence of haemagglutinins in bulinid snails 100 (c) Haemagglutinin induction 101 (d) Red cell specificity 101 (e) Requirement for divalent cations 102 (f) Haemagglutination inhibition 102 (g) Absorption by larval schistosomes 104 (h) Fast Protein Liquid Chromatography 105 6 RESULTS 109 1 Schistosomes 109 (a) In vitro transformation of miracidia 109 (b) Surface Morphology 119 (c) Surface Chemistry 120 2 Haemocytes 133 (a) Haemocyte Numbers 13 3 (b) Haemocyte morphology and structure 138 (c) Haemocyte sub-populations 142 3 Haemagglutinins 153 (a) Presence of haemagglutinins in bulinid snails 153 (b) Haemagglutinin induction 156 (c) Red cell specificity 156 (d) Requirement for divalent cations 158 (e) Haemagglutination inhibition 158 (f) Absorption by larval schistosomes 159 (g) Fast Protein Liquid Chromatography 163 DISCUSSION 168 1 Schistosomes 168 2 Haemocytes 181 3 Haemagglutinins 198 REFERENCES 228 7 FIGURES FIGURE 1 The Life-cycle of trematode parasites 15 belonging to the genus Schistosoma FIGURE 2 Stages in the life-cycle of Schistosoma 17 marqrebowiei FIGURE 3 Some Aspects of the vertebrate immune 30 response to infection with S. mansoni FIGURE 4 Snails of the genus Bulinus 58 FIGURE 5 Snails of the genus Bulinus 65 a) Bulinus natalensis b) Bulinus nasutus FIGURE 6 The Sulpho-NHS-Biotin reaction 82 FIGURE 7 The Cytocentrifuge 89 FIGURE 8 Haemagglutination 98 FIGURE 9 Doubling dilution series 99 FIGURE 10 Fast Protein Liquid Chromatography 108 FIGURE 11 Schistosome surface during miracidia/ 110 sporocyst transformation FIGURE 12 Events in the transformation of 112 miracidia to sporocysts FIGURE 13 Photomicrographs of S.marqrebowiei 113 a) Miracidium b) Sporocyst and ciliated plates c) Early mother sporocyst d) Late mother sporocyst FIGURE 14 SEM of S.marqrebowiei miracidium 114 8 15 SEM of S .marqrebowiei miracidium 114 showing apical papilla 16 SEM of S .marqrebowiei sporocyst 115 with ciliated plates 17 SEM of S .marqrebowiei sporocyst 115 18 SEM of S .marqrebowiei sporocyst 116 showing sensory receptor 19 SEM of S .marqrebowiei cercaria 116 showing sensory receptor 20 SEM of S.marqrebowiei cercaria 117 21 SEM of S .marqrebowiei cercaria 117 showing surface spines 22 SEM of S .marqrebowiei cercaria 118 showing receptors on body apex 23 SEM of S .marqrebowiei cercaria 118 showing excretory pore 24 Miracidia stained with FITC-Ricin 121 25 Ciliated plate stained with FITC-PNA 121 26 Transforming miracidium stained with 122 FITC-Ricin 27 Early sporocyst stained with FITC-PNA 122 28 Early sporocyst stained with FITC-WGA 123 29 Mother sporocyst stained with FITC-CON A 123 30 48h sporocyst stained with FITC-Ricin 124 31 48h sporocyst 124 a) stained with FITC-WGA b) stained with FITC-CON A 9 FIGURE 32 Cercaria 125 a) stained with FITC-WGA b) stained with FITC-Asp Pea FIGURE 3 3 Cercaria post biotinylation stained 129 with FITC-Avidin FIGURE 34 Sporocyst post biotinylation stained 130 with FITC-Avidin FIGURE 35 a) Miracidia post biotinylation stained 130 with FITC-Avidin b) Miracidia incubated with B.nasutus130 haemolymph prior to biotinylation FIGURE 3 6 Nitrocellulose blot showing the major 131 proteins of cercariae FIGURE 37 Nitrocellulose blot showing surface 131 proteins of cercariae FIGURE 38 Nitrocellulose blot showing surface 132 proteins of sporocysts and miracidia FIGURE 39 Number of haemocytes in the haemolymph of 134 B. nasutus and B^_ natalensis FIGURE 40 Number of haemocytes in the haemolymph of 136 B. nasutus before and after exposure to S . marqrebowiei miracidia FIGURE 41 Number of haemocytes in the haemolymph of 137 B. natalensis before and after exposure to S . marqrebowiei miracidia FIGURE 42 Phase contrast micrographs of bulinid 139 haemocytes FIGURE 43 SEM of B_i_ natalensis plasmatocyte 140 10 FIGURE 44 SEM of Ek_ natalensis plasmatocyte 140 and granulocyte FIGURE 45 SEM of JL_ natalensis granulocyte 141 FIGURE 46 Plasmatocyte cytoskeleton 143 FIGURE 47 Granulocyte cytoskeleton 143 FIGURE 48 Con A labelling of bulinid haemocytes 144 FIGURE 49 Haemocyte of 'intermediate morphology1 144 (a) stained with FITC-Con A (b) stained with propidium iodide FIGURE 50 WGA labelling of bulinid haemocytes 145 FIGURE 51 Flow cytometry of haemocytes 147 FIGURE 52 In vitro phagocytosis of coli by 148 B. natalensis haemocytes 5 min incubation FIGURE 53 In vitro phagocytosis of EL*. coli by 148 B. natalensis haemocytes 15 min incubation FIGURE 54 In vitro phagocytosis of latex particles 149 by B^.

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