THE MOLECULAR BIOLOGY OF INSECT DISEASE VECTORS JOIN US ON THE INTERNET VIA WWW, GOPHER, FTP OR EMAIL: WWW: http://www.thomson.com GOPHER: gopher.thomson.com A service of ICDP® FTP: ftp.thomson.com EMAIL: [email protected] THE MOLECULAR BIOLOGY OF INSECT DISEASE VECTORS

A METHODS MANUAL

Edited by J.M. CRAMPTON Division of Molecular Biology and Immunology, Liverpool School of Tropical Medicine, UK C.B. BEARD Centers for Disease Control and Prevention, Atlanta, USA

and C. LOUIS Department of Biology, University of Crete m CHAPMAN & HALL London· Weinheim . New York· Tokyo· Melbourne· Madras Published by Chapman & Ball, 2-6 Boundary Row, London SEt 8HN, UK

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First edition 1997

© 1997 Chapman & Hall Softcover reprint of the hardcover I st edition 1997 Typeset by Saxon Graphics Ltd .• Derby ISBN-13: 978-94-010-7185-7 e-ISBN-13: 978-94-009-1535-0 DOl: 10.1007/978-94-009-1535-0 Apart from any fair dealing for the purposes of research or private study. or criticism or review. as permitted under the UK Copyright Designs and Patents Act. 1988. this publication may not be reproduced. stored, or transmitted, in any form or by any means, without the prior permission in writing of the publishers, or in the case of reprographic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK. or in accordance with the terms of licences issued by the appropriate Reproduction Rights Organization outside the UK. Enquiries concerning reproduction outside the terms stated here should be sent to the publishers at the London address printed on this page. The publisher makes no representation, express or implied. with regard to the accuracy of the information contained in this book and cannot accept any legal responsibility or liability for any errors or omissions that may be made.

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Library of Congress Catalog Card Number: 96-70874

<§ Printed on pennanent acid-free text paper, manufactured in accordance with ANSIINISO Z39.48-1992 and ANSIJNISO Z39.48-1984 (Pennanence of Paper). Contents

A colour plate appears between pages 286 and 287

List of contributors xi Foreword xvii Preface xix Acknowledgements xxi List of abbreviations xxiii

Part One Care and Maintenance of Insect Colonies

1 Care and maintenance of anopheline mosquito colonies 3 Mark Q. Benedict

2 Care and maintenance of Aedes mosquito colonies 13 Leonard E. Munstermann

3 Care and maintenance of phlebotomine sandfly colonies 21 Govind B. Modi

4 Care and maintenance of blackfly colonies 31 Eddie W. Cupp and Frank B. Ramberg

5 Care and maintenance of tsetse colonies 41 Ronald H. Gooding, Udo Feldmann and Alan S. Robinson

6 Care and maintenance of triatomine colonies 56 Patricia de Azambuja and Eloi S. Garcia

Part Two Experimental Infection of Insect Vectors

7 Infection of mosquitoes with rodent malaria 67 Robert E. Sinden

8 Infection of mosquitoes with primate malaria 92 William E. Collins vi Contents

9 Infection of mosquitoes with filaria 101 Harold Townson

10 Infection of phlebotomine sandflies with Leishmania 112 Paul A. Bates

11 Infection of blackflies with Onchocerca 121 Albert E. Bianco and Peter J. Ham

12 Infection of tsetse with Trypanosoma brucei rhodesiense and T. b. gambiense 136 Ian Maudlin

13 Infection of triatomines with Trypanosoma cruzi 146 Eloi S. Garcia and Patricia de Azambuja

Part Three Basic Methods in Isolating, Cloning and Characterizing Nucleic Acids and their Products

14 Isolation of genomic DNA 159 Christos Louis

15 Isolation and analysis of RNA from insect vectors 164 Margaret A. Hughes

16 Determination of insect genome size and complexity by 172 reassociation kinetics Melanie J. Palmer and William C. Black N

17 Constructing and screening lambda libraries 195 Margaret A. Hughes

18 Constructing and screening cosmid libraries 207 Kostas D. Mathiopoulos

19 Constructing and screening cDNA libraries 218 Kostas D. Mathiopoulos

20 Southern/northern blotting and hybridization techniques 230 Inga Siden-Kiamos Contents vii

21 The polymerase cMin reaction (PCR) and RT-PCR 244 Bruno Arca and Charalambos Savakis

22 Differential display of mRNA 261 George Dimopoulos and Christos Louis

23 Localization of RNA transcripts by in situ hybridization 268 Philip W. Ingham and Trevor Jowett

24 In situ localization of proteins in whole mounted tissue 283 Marcos A. Gonzalez-Gaitan and Herbert Jackle

25 DNA sequencing and primer extension mapping 295 Hans-Michael Muller

Part Four Genome Mapping Techniques

26 RFLP analysis of insect genomes 309 David W. Severson

27 Microsatellite mapping of insect genomes 321 Liangbiao Zheng

28 Polytene chromosome preparation from anopheline mosquitoes 329 Alessandra della Torre

29 In situ hybridization to Anopheles polytene chromosomes 337 Vasantha Kumar, Anthony J. Cornel and Odette Mukabayire

30 Microdissection and microamplification of dipteran polytene 346 chromosomes Robert D.C. Saunders

Part Five Insect Identification Techniques

31 RAPD-PCR and SSCP analysis for insect population genetic studies 361 William C. Black IV and Nancy M. DuTeau

32 PCR amplification of insect ribosomal DNA 374 Susan M. Paskewitz and Frank H. Collins viii Contents

33 Generation and use of species-specific DNA probes for insect vector 384 iden tification Julian M. Crampton and Susannah M. Hill

34 Cloning and detection of insecticide resistance genes 399 Nicole Pasteur, Michel Raymond, Fran~ois Rousset, Jean-Baptiste Berge, Marcel Amichot, David Pauron, Andre Cuany and Didier Fournier

Part Six Transformation Techniques and Viral Systems

35 Microinjection of mosquito embryos 423 Alison C. Morris

36 Transfection of cultured mosquito cells 430 Ann Marie Fallon

37 Viral transducing vectors for mosquitoes 444 Jon O. Carlson, Boris N. Afanasiev, Stephen Higgs, Tomoyo Matsubara and Jane c. Bums

38 Viral expression systems and viral infections in insects 459 Stephen Higgs, Ken E. Olson, Kurt 1. Kamrud, Ann M. Powers and Barry J. Beaty

Part Seven Cell and Organ Culture

39 Establishing primary cell cultures from disease vectors and 487 maintenance of continuous cell lines Colin J. Leake

40 Culture and analysis of insect salivary glands 500 Alison C. Morris and Anthony A. James

41 Culture and analysis of the insect fat body 507 Alexander S. Raikhel, Kirk W. Deitsch and Thomas W. Sappington

42 Culture and analysis of insect ovaries 523 Thomas W. Sappington, Mark R. Brown and Alexander S. Raikhel Contents ix

43 Culture and analysis of insect midgut 536 Hans-Michael Muller and Andrea Crisanti

Part Eight Insect Symbionts

44 Isolation and culture of tsetse secondary endosymbionts 547 Susan C. Welburn and Colin Dale

45 Genetic manipulation of insect symbionts 555 C. Ben Beard and Serap Aksoy

46 PCR-based detection and identification of insect symbionts 561 Scott L. O'Neill

Index 567 Contributors

Boris N. Afanasiev Arthropod Borne Animal Diseases Research Laboratory, Department of Microbiology, Colorado State University, Fort Collins CO 80523, USA Serap Aksoy Yale-MacArthur Center for Molecular Parasitology, Department of Epidemiology and Public Health, Yale University School of Medicine, Room 700 LEPH, 333 Cedar Street, New Haven CT 06510, USA Marcel Amichot Genetique MoIeculaire, INRA, 06606 Antibes, France BrunoArd Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Hellas, GR-71110 Heraklion, Greece Patricia de Azambuja Funda~ao Oswaldo Cruz, Departamento de Bioquimica e Biologia Molecular, Rio de Janeiro RJ, Brazil 21045-900 Paul A. Bates Division of Molecular Biology and Immunology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 SQA , UK C. Ben Beard Centers for Disease Control and Prevention, Division of Parasitic Diseases, Mail Stop F-12, 4770 Buford Hwy, Atlanta GA 30341-3724, USA Barry J. Beaty Department of Microbiology, Colorado State University, College of Veterinary Medicine & Biomedical Science, Fort Collins CO 80523, USA Mark Q. Benedict Centers for Disease Control and Prevention, Division of Parasitic Diseases, Mail Stop F-22, 4770 Buford Hwy, Atlanta GA 30341-3724, USA Jean-Baptiste Berge Genetique Moleculaire, INRA, 06606 Antibes, France Albert E. Bianco Division of Molecular Biology and Immunology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK William C. Black IV Department of Microbiology, Colorado State University, Fort Collins CO 80523, USA Mark R. Brown Department of Entomology, University of Georgia, Athens GA 30602-2603, USA Jane c. Burns University of California San Diego, School of Medicine, Department of Pediatrics/0830, 9500 Gilman Drive, La Jolla, CA 92093-0830, USA. xii Contributors

Jon O. Carlson Arthropod-Borne and Infectious Diseases Research Laboratory, Department of Microbiology, Colorado State University, Fort Collins CO 80523, USA Frank H. Collins Centers for Disease Control and Prevention, Division of Parasitic Diseases, Mail Stop F-22, 4770 Buford Hwy, Chamblee GA 30341-3724, USA William E. Collins Centers for Disease Control and Prevention, Division of Parasitic Diseases, Mail Stop F-12, 4770 Buford Hwy, Atlanta GA 30341-3724, USA Anthony J. Cornel Centers for Disease Control and Prevention, Division of Parasitic Diseases, Mail Stop F-22, 4770 Buford Hwy, Atlanta GA 30341-3724, USA Julian M. Crampton Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK Andrea Crisanti Imperial College of Science, Technology and Medicine, Department of Biology, Prince Consort Road, London SW7 2BB, UK Andre Cuany Genetique Moleculaire, INRA, 06606 Antibes, France Eddie w. Cupp Auburn University, Department of Entomology, 301 Funches Hall, Auburn AL 36849-5413, USA Colin Dale Institute for Biological and Life Sciences, Anderson College, University of Glasgow, Glasgow G128QQ, UK Kirk W. Deitsch Department of Entomology and Program in Cell and Molecular Biology, Michigan State University, East Lansing MI 48824-1311, USA Alessandra della Torre Istituto di Parassitologia, Universita di Roma 'La Sapienza', Piazzale AIdo Moro 5, 1-00185 Roma, Italy George Dimopoulos Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Hellas and Department of Biology, University of Crete, GR-7111O Heraklion, Greece Nancy M. DuTeau Department of Microbiology, Colorado State University, Fort Collins CO 80523, USA Ann Marie Fallon Department of Entomology, 219 Hodson Hall, University of Minnesota, 1980 Folwell Ave, St Paul MN 55108-1385, USA Udo Feldmann International Atomic Energy Agency, P.O. Box 100, A-1400, Vienna, Austria Didier Fournier Universite Paul Sabatier, 32062 Toulouse, France Eloi S. Garcia Fundac;ao Oswaldo Cruz, Departamento de Bioquimica e Biologia Molecular, Rio de Janeiro RJ, Brazil 21045-900 Contributors xiii

Marcos A. Gonzalez-Gaitan Max-Plank. Institut fUr Biophysikalische Chemie, Abt. 170, Am Fa~berg, D-37077 Gottingen Germany Ronald H. Gooding Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2EL PeterJ. Ham Department of Biological Sciences, Keele University, Keele, Staffordshire ST5 5BG, UK Stephen Higgs Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Colorado State University, Fort Collins CO 80523, USA Susannah M. Hill Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, Pembroke Place Liverpool L3 5QA, UK Margaret A. Hughes Wolfson Unit of Molecular Genetics, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK Philip W. Ingham Molecular Embryology Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, UK Herbert JackIe Max-Plank. Institut fUr Biophysikalische Chemie, Abt. 170, Am Fa~berg, D-37077 Gottingen, Germany Anthony A. James Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine CA 92697-3900, USA Trevor Jowett Department of Genetics, Catherine Cookson Building, Framlington Place, Newcastle upon Tyne, UK Kurt I. Kamrud USAMRIID, Virology Division, Fort Detrick, Frederick, MD 21702, USA. Vasantha Kumar Brigham and Women's Hospital, Pathology Department, 503 Thorn Building, 20 Shattuck Street, Boston MA 02115, USA Colin J. Leake Vector Biology and Epidemiology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, UK Christos Louis Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Hellas and Department of Biology, University of Crete, GR-711 10 Heraklion, Greece Kostas D. Mathiopoulos Istituto di Parassitologia, Universita di Roma 'La Sapienza', Piazzale Aldo Mora 5, 1-00185, Roma, Italy Tomoyo Matsubara University of California San Diego, School of Medicine, Department of Pediatrics/0830, 9500 Gilman Drive, La Jolla, CA 92093-0830, USA. xiv Contributors

Ian Maudlin Institute for Biological and Life Sciences, Anderson College, University of Glasgow, Glasgow GI28QQ, UK Govind B. Modi Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda MD 20892-0425, USA Alison C. Morris Department of Molecular Biology and Biochemistry, School of Biological Sciences, University of California, Irvine CA 92697-3900, USA Odette Mukabayire Centers for Disease Control and Prevention, Division of Parasitic Diseases, Mail Stop F-22, 4770 Buford Hwy, Atlanta GA 30341-3724, USA Hans-Michael Muller European Molecular Biology Laboratory (EMBL), Meyerhofstra~e 1, D-69117 Heidelberg, Germany Leonard Munstermann Division of Infectious Diseases, School of Epidemiology and Public Health, Yale University, 60 College Street, New Haven CT 06520-8034, USA Scott L. O'Neill Department of Epidemiology and Public Health, Yale University School of Medicine, 60 College Street, New Haven CT 06520-8034, USA Ken E. Olson Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Colorado State University, Fort Collins CO 80523, USA Melanie J. Palmer Department of Entomology, Oklahoma State University, Stillwater, Oklahoma 74078, USA Susan M. Paskewitz Department of Entomology, University of Wisconsin, Madison, Wisconsin 53706, USA Nicole Pasteur Laboratoire de Genetique Environnement, Institut des Sciences de l'Evolution, Universite de Montpellier II, 34095 Montpellier 05, France David Pauron Genetique Moleculaire, INRA, 06606 Antibes, France

Ann M. Powers Center for Tropical Diseases, Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609, USA Alexander S. Raikhel Department of Entomology and Program in Cell and Molecular Biology, Michigan State University, East Lansing MI 48824-1311, USA Frank B. Ramberg University of Arizona, Department of Veterinary Science, 204 Veterinary Science and Microbiology Building, Tucson AZ 85721, USA Michel Raymond Laboratoire de Genetique Environnement, Institut des Sciences de l'Evolution, Universite de Montpellier II, 34095 Montpellier 05, France Contributors xv

Alan S. Robinson International Atomic Energy Agency, IAEA Laboratories, A-2444 Siebersdorf, Austria

Fran~ois Rousset Laboratoire de Genetique Environnement, Institut des Sciences de l'Evolution, Universite de Montpellier II, 34095 Montpellier OS, France Thomas W. Sappington Department of Entomology and Program in Cell and Molecular Biology, Michigan State University, East Lansing MI 48824-1311, USA Robert D.C. Saunders Department of Anatomy and Physiology, University of Dundee, Dundee 001 4HN, UK Charalambos Savakis Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Hellas and Department of Medical Sciences, University of Crete Medical School, GR-711 10 Heraklion, Greece David W. Severson Animal Health and Biomedical Sciences, University of Wisconsin-Madison, 1655 Linden Drive, Madison WI 53706-1581, USA Inga Siden-Kiamos Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology, Hellas, GR-71110 Heraklion, Greece Robert E. Sinden Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK Harold Townson Division of Parasite and Vector Biology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK Susan C. Welbum Institute for Biological and Life Sciences, Anderson College, University of Glasgow, Glasgow G128QQ, UK Liangbiao Zheng European Molecular Biology Laboratory (EMBL), Meyerhofstra~e 1, 0-69117 Heidelberg, Germany Foreword

Only one generation ago, entomology was a proudly isolated discipline. In Comstock Hall, the building of the Department of Entomology at Cornell University where I was first introduced to experimental science in the laboratory of Tom Eisner, those of us interested in the chemistry of life felt like interlopers. In the 35 years that have elapsed since then, all of biology has changed, and entomology with it. Arrogant molecular biologists and resentful classical biologists might think that what has happened is a hostile take-over of biology by molecular biology. But they are wrong. More and more we now understand that the events were happier and much more exciting, amounting to a new synthesis. Molecular Biology, which was initially focused on the simplest of organisms, bacteria and viruses, broke out of its confines after the initial fundamental questions were answered - the structure of DNA, the genetic code, the nature of regulatory genes - and, importantly, as its methods became more and more generally applicable. The recombinant DNA revo• lution of the 1970s, the development of techniques for sequencing macromolecules, the polymerase chain reaction, new molecular methods of genetic analysis, all brought molecular biology face to face with the infinite complexity and the exuber• ant diversity of life. Molecular biology itself stopped being an isolated diScipline, pre• occupied with the universal laws of life, and became an approach to addressing fas• cinating specific problems from every field of biology. One example has been the birth of the discipline of molecular entomology, and with it the subdiscipline of mol• ecular insect vector biology. Insects are the most successful and most diverse group of higher animals, in all environments except the sea. Important features of their success are their short life cycle, their ability to evolve rapidly to exploit new niches, and their capacity to sepa• rate the growth and dispersal phases of their life cycle. Thus, they are adept at exploiting the huge food resources of agriculture - concentrated cultivation of plants and animal husbandry by humans - as well as the ready-made source of protein rep• resented by animal or human blood. Agricultural insect pests are ubiquitous and costly exploiters of our efforts to feed and clothe ourselves. Haematophagous insects are serious threats to our health, and that of our animals, by virtue of the viruses and parasites (protozoa or worms) that they happen to carry. To fight insect pests we must know their secrets. For more than a century Entomology has represented the effort of humans to find weak points and disarm our small, but formidable, enemies. We have learned a great deal about them, and it has been very useful. To name only one example, yellow fever was controlled by understanding the habits of the Aedes aegypti mosquito just as much as by the devel• opment of a vaccine against the virus. Much remains to be done, however. Parasitic diseases, and especially malaria, remain among the most important health hazards in much of the world. New or modified agricultural pests continue to erupt, as easy transport brings new insect species in contact with new food sources, and as tradi• tional insect pests stay one step ahead of us by rapidly evolving resistance to pesti- xviii Foreword cides. When coupled with the knowledge accumulated by classical entomology, the new tools of molecular biology promise much help for our side. Again one example: understanding the population biology of malaria mosquitoes is a prerequisite for their control, and molecular genetic markers are now indispensable tools for the study of populations. But the new synthesis works both ways: molecular biology helps entomology, but also vice versa. The best model species for studying the biology of complex organisms is the fruitfly, Drosophila melanogaster. And the enormous amount of accumulated information about Drosophila makes insects especially attractive for those who want to study fundamental biology using the comparative approach. Again one example: in the last 15 years we have understood in outline how genes control the embryonic development of Drosophila, and have discovered to our amazement that many of these same genes are also important for very different animals, including ourselves. More and more molecular developmental biologists are studying the regulatory genes in other insects, to understand the subtleties of how they operate. Why not do it in insects that are pests, thus contributing at the same time to learning about our enemies? Molecular entomology is a rapidly growing and exciting field, whether we are in it for basic biological interests or for reasons of applied biology. The editors of this book have made a wonderful contribution to the progress of this field, by approach• ing it from the synthetic viewpoint. They have addressed the complementary needs both of those entering the field from entomology and those coming from molecular biology. In so doing, they have put together a unique book of methods, each pre• sented at the level of practical detail needed by new practitioners. The first two sec• tions introduce us to the culture of insect vectors of disease, and their experimental infection with parasites. The next three sections present basic methods of molecular biology, genomic mapping and molecular identification techniques as applied to insects of human importance. The final three sections deal with the key special top• ics of DNA transfer to insects (via viruses or direct addition), culture of the most important insect organs and specialized cell types, and the study of insect symbionts. The overall logic and the extent and the quality of coverage of the book are admirable. Whether you are new trainees or researchers in this field, enjoy!

Fotis C. Kafatos European Molecular Biology Laboratory Heidelberg Preface

This manual aims to provide a source book of detailed protocols covering the impor• tant techniques used in molecular entomology. Although the research literature contains descriptions of practical procedures, extensive practical detail is usually not presented; more particularly, an idea of the problems that can be encountered and how they are identified and overcome is rarely included. Each chapter of this vol• ume concentrates on one specific method, written in such a way that a competent scientist who is unfamiliar with the method can carry out the technique success• fully, either at the first attempt, or with a little practice, by simply following the detailed procedures. The intention was that the methods book would form part of WHOffDRs strategic research initiative in this area and provide a valuable resource to assist in its development. We are particularly grateful to the various experts in these specialized fields who agreed to contribute to the manual. We also greatly appreciate the fact that some of our collaborators 'volunteered' to contribute chap• ters describing general methods! Our target audience includes entomologists with no experience of molecular bio• logy, and molecular biologists interested in entering the field (who have little experience of applying the technology to insect vectors). We also hope that it will prove useful to those organizing or participating in training courses in the molecular biology of dis• ease vectors or molecular entomology in general. Finally, researchers in related fields, such as agricultural entomologists, insect physiologists, etc., may also find some of the methods and techniques applicable, or of interest, to their own field of research.

J.M. Crampton C.B. Beard C. Louis Acknowledgements

This project was initiated during the TOR-sponsored course 'The Molecular Biology of Anopheles' held in Rio de Janeiro in May 1994. We thank Dr Boris Dobrokhotov for his encouragement during the early stages of this project, and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) for financial support during the preparation of this volume. Furthermore, we thank Chapman & Hall for their enthusiasm in wishing to publish this manual. IMC is a Wellcome Trust Senior Research Fellow in Basic Biomedical Sciences. Finally, we thank Jean Gauckwin for providing considerable editorial assistance and compiling the manuscript, and Nektaria Kelaidi for additional secretarial assistance. Abbreviations

A Amps A260 absorbance at a wavelength of 260 nanometre ACD acid/citrate/dextrose APS ammonium persulfate ATP adenosine triphosphate bp base pair(s) BSA bovine serum albumin cDNA complementary DNA CHX cycloheximide cm centimetre(s) cpm counts per minute Da Dalton(s) ddH20 double-distilled deionized water (or equivalent) ddNTP 2',3' -dideoxynucleotide triphosphate(s) DEPC diethylpyrocarbonate diam diameter DMSO dimethyl sulfoxide DNase deoxyribonuclease dNTP deoxynuc1eotide triphosphate(s) WE 2O-hydroxyecdysone EDTA ethylenediaminetetraacetic acid ELISA enzyme linked immunoassay EtBr ethidium bromide EtOH ethanol FCS fetal calf serum g gram(s) g gravitational force G gauge h hour(s) HAP hydroxyapatite HBSS Hank's balanced salt solution HEPES N-2-hydroxyethylpiperazine-N' -2-ethanesulphonic acid !PTG isopropyl-j3-D-thiogalactopyranoside ill International unit(s) kb kilobase pair( s) kDa kilo Dalton(s) L litre(s) L:D light:dark photoperiod L3 third-stage larva(e) m metre(s) M molar xxiv Abbreviations mCi milliCurie(s) MEM modified Eagle's medium mff microfilaria(e) mg milligram(s) min minute(s) mL millilitre(s) mm millimetre(s) mM millimolar mmol millimole(s) moi multiplicity of infection mRNA messenger RNA NBT nitroblue tetrazolium NCS new born calf serum ng nanogram(s) nL nanolitre(s) nm nanometre(s) nM nanomolar nmol nanomole(s) PB phosphate buffer PBS phosphate buffered saline PCR polymerase chain reaction pfu plaque forming units pg picogram(s) PGr positive gland index pM picomolar polyA polyadenylic acid RACE rapid amplification of cDNA ends RAPD random amplified polymorphic DNA RBC red blood cells RFLP restriction fragment length polymorphism RH relative humidity RLO rickettsia-like organism(s) RNase ribonuclease rpm revolutions per minute rRNA ribosomal RNA RT room temperature RT-PCR reverse transcription PCR S Svedburg unit(s) s second(s) SDS sodium dodecyl sulphate SSC saline sodium citrate SSCP saline sodium citrate phosphate TBE Tris/borate/EDTA buffer II transmission index Tris 2-amino-2(hydroxymethyl)-propane-l,3-diol tRNA transfer RNA ~Ci microCurie(s) ~g microgram(s) ~L microlitre(s) Abbreviations xxv

!-Lm micrometre(s) !-LM micromolar !-LMole micromole( s) U unit(s) UV ultra-violet v/v volume per volume V Volts Vg vitellogenin vol volume VSG variant surface glycoprotein w/v weight per volume W Watts X-gal 5-bromo-4-chloro-3-indolyl-I3-D-galactopyranoside xg times gravitational force