INSECT PATHOGENS Molecular Approaches and Techniques This page intentionally left blank INSECT PATHOGENS Molecular Approaches and Techniques Edited by S. Patricia Stock Department of Entomology University of Arizona USA John Vanderberg USDA-ARS US Plant, Soil and Nutrition Laboratory USA Noël Boemare Institut National de la Recherche Agronomique (INRA) Université Montpellier France and Itamar Glazer Agricultural Research Organization The Volcani Centre Israel CABI is a trading name of CAB International CABI Head Offi ce CABI North American Offi ce Nosworthy Way 875 Massachusetts Avenue Wallingford 7th Floor Oxfordshire OX10 8DE Cambridge, MA 02139 UK USA Tel: +44 (0)1491 832111 Tel: +1 617 395 4056 Fax: +44 (0)1491 833508 Fax: +1 617 354 6875 E-mail: [email protected] E-mail: [email protected] Website: www.cabi.org ©CAB International 2009. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. Library of Congress Cataloging-in-Publication Data Insect pathogens : molecular approaches and techniques/edited by S. Patricia Stock . [et al.]. p. cm. Includes bibliographical references and index. ISBN 978-1-84593-478-1 (alk. paper) 1. Insects--Pathogens. 2. Insects--Molecular aspects. I. Stock, S. Patricia. II. Title. SB942.I57 2009 632'.7--dc22 2008031290 ISBN-13: 978 1 84593 478 1 Typeset by SPi, Pondicherry, India. Printed and bound in the UK by MPG Books Group. The paper used for the text pages in this book is FSC certified. The FSC (Forest Stewardship Council) is an international network to promote responsible management of the world’s forests. Contents Contributors xi Preface xiii Glossary of Terms xv PART I IDENTIFICATION AND DIAGNOSTICS 1 1 Molecular Approaches to Virus Characterization 3 and Detection M.A. Erlandson and D.A. Theilmann 1.1. Introduction 4 1.2. Current Virus Taxonomy and Classification 4 1.3. Preliminary Approaches to Virus Identification 13 1.4. Methodology for Virus Isolation and Fractionation 13 (Nucleic Acid and Protein Purification Techniques) 1.5. Biochemical/Molecular Approaches to Virus 14 Identification and Diagnosis 1.6. PCR and Virus-specific Primer Development 21 1.7. Conclusions 26 2 Molecular Approaches and Techniques for the 32 Study of Entomopathogenic Bacteria N. Boemare and P. Tailliez 2.1. Introduction 32 2.2. Classification of Entomopathogenic Bacteria 33 2.3. Conclusions 44 v vi Contents 3 Molecular Methods for Identification and Diagnosis 50 of Fungi L.A. Castrillo and R.A. Humber 3.1. Introduction 50 3.2. General Considerations 51 3.3. Genetic Fingerprinting 54 3.4. DNA Sequencing 64 3.5. Diagnosis and Detection 65 3.6. Conclusions and Future Prospects 66 4 Molecular Approaches and the Taxonomy of 71 Insect-parasitic and Pathogenic Nematodes S.P. Stock 4.1. Introduction 71 4.2. Nematode Diagnosis and the Barcode System 72 4.3. A Review of Molecular Approaches Considered for 73 Insect-parasitic and Pathogenic Nematode Taxonomy 4.4. Techniques Considered for Obtaining DNA Sequences 75 4.5. Sequencing 93 4.6. Sequence Manipulation and Analysis 93 4.7. Conclusions 94 5 Identification and Diagnostics of Entomopathogenic 101 Protozoa M. Oborník 5.1. Introduction 101 5.2. Molecular Identification of Species and Strains 102 5.3. Conclusion and Future Perspectives 123 PART II EVOLUTIONARY RELATIONSHIPS AND 129 POPULATION GENETICS 6 Phylogenetic Studies with Entomopathogenic Bacteria 131 with Special Emphasis on Symbionts of Entomopathogenic Nematodes P. Tailliez and N. Boemare 6.1. Introduction 131 6.2. Genes Considered 132 6.3. Conclusions and Perspectives 141 7 Molecular Systematics of Entomopathogenic Fungi 145 S.A. Rehner 7.1. Introduction 145 7.2. Molecular Phylogenies of Fungi and the Origins of 146 Entomopathogens 7.3. Species Recognition 154 7.4. Species-level Phylogenies of Entomopathogenic Fungi 157 7.5. Conclusions 158 Contents vii 8 Phylogenetics and Population Genetics of Entomopathogenic 166 and Insect-parasitic Nematodes S.M. Peat, B.C. Hyman and B.J. Adams 8.1. Introduction 166 8.2. Phylogenetics 167 8.3. Population Genetics 168 8.4. DNA Bar Coding 168 8.5. DNA Markers Considered for Phylogenetic and 169 Population Genetics Studies 8.6. Methodology 175 8.7. Co-phylogenesis and Cospeciation 182 8.8. Population Genetics Methods 184 PART III HOST–PATHOGEN INTERACTIONS 193 9 Host–Virus Interactions 195 J.P. Burand, M. Nakai and I. Smith 9.1. Introduction 195 9.2. Use of Viruses as Insect Pest Control Agents 196 9.3. Development of Baculoviruses for Foreign Gene Expression 197 9.4. Insect Defences Against Viruses 201 9.5. Baculovirus Pathogenesis 202 9.6. Baculovirus Host Range 207 9.7. Unclassified DNA Viruses 210 9.8. Mechanisms of Insect Virus Persistence 212 9.9. Conclusions and Future Prospects 214 10 Insect–Protozoa–Bacteria Associations: a Model System for 223 Investigating Host–Parasite Interactions B.L. Weiss, G.M. Attardo and S. Aksoy 10.1. Introduction 223 10.2. Trypanosomatid Protozoa and Tsetse Flies 224 10.3. Molecular Approaches and Their Application to Study 226 Insect Host Immune Responses 10.4. Tsetse Endosymbionts 228 10.5. Control of Insect-borne Diseases 230 10.6. Current and Future Work 234 10.7. Conclusions 236 11 Methods in Investigating Nematode–Bacterium–Insect 241 Symbiosis H. Goodrich-Blair, D.J. Clarke, P.S. Grewal and T.A. Ciche 11.1. Introduction 242 11.2. Molecular Tools to Study Entomopathogenic Nematodes 244 11.3. Basic Molecular Tools for the Study of 250 Entomopathogenic Bacteria 11.4. Techniques to Investigate Bacteria–Nematode Mutualism 258 viii Contents 11.5. Techniques in Studying EPB Virulence 261 11.6. Conclusions and Future Prospects 265 PART IV GENOMICS AND GENETIC ENGINEERING 273 12 Genetic Engineering of Bacteria to Improve Efficacy Using 275 the Insecticidal Proteins of Bacillus Species H.-W. Park and B.A. Federici 12.1. Introduction 275 12.2. Basic Biology of Bacillus thuringiensis 276 12.3. Insecticidal Proteins of Bacillus thuringiensis 277 12.4. Genetic Factors Regulating Insecticidal Proteins 280 12.5. Construction of Recombinant Bacteria 281 12.6. Conclusions and Future Prospects 298 13 Genomic Analysis of the Symbiotic and Entomopathogenic 306 Photorhabdus Bacteria S. Gaudriault and E. Duchaud 13.1. Introduction 307 13.2. Sequencing and Annotation of Photorhabdus Genomes 308 13.3. Main Features of the P. luminescens Genome 310 13.4. Analogical Post-genomic Analysis 313 13.5. Post-genomic Analysis by a ‘Blind’ Approach 316 13.6. Conclusions and Future Perspectives 325 14 Genomics of Entomopathogenic Viruses 329 J. Slack, Z. Li, S. Escasa, D. Doucet, T. Ladd, G. Quan and B. Arif 14.1. Introduction 329 14.2. General Concepts 330 14.3. Analyses of DNA and Protein Sequences 335 14.4. Genetic Modification of Baculoviruses 339 14.5. Conclusions 342 15 Genomics and Genetic Improvement of Entomopathogenic 346 Nematodes H. Koltai 15.1. Introduction 346 15.2. Genomic Sequencing and Bioinformatics 347 15.3. Functional Genomics: Towards Deciphering of Genomics 353 and ESTs Sequences 15.4. Genetic Improvement 357 15.5. Conclusion and Future Prospects 358 16 Entomopathonic Fungi and the Genomics Era 365 R.J. St Leger and C. Wang 16.1. Introduction 366 16.2. Procedures for Isolating Pathogen DNA 368 Contents ix 16.3. Procedures for Isolating Pathogen RNA 370 16.4. Pulsed Field Gel Electrophoresis 371 16.5. Construction of Cloning and Expression Vector 372 Components and Markers 16.6. Transformation Systems 376 16.7. Gene Cloning Strategies 379 16.8. Analysing Differential Gene Expression 389 16.9. EST Screening 392 16.10. Microarray Analysis 392 16.11. Targeted Gene Mutagenesis 394 16.12. RNA Interference 395 16.13. Conclusions 395 Index 401 This page intentionally left blank Contributors Byron J. Adams, Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602-5253, USA. Serap Aksoy, Department of Epidemiology and Public Health, Section of Vector Biology, Yale University School of Medicine, LEPH 606, 60 College Street, New Haven, CT 06510, USA. Basil Arif, Great Lakes Forestry Centre, Sault Ste Marie, Ontario, Canada. Geoffrey M. Attardo, Department of Epidemiology and Public Health, Section of Vector Biology, Yale University School of Medicine, LEPH 606, 60 College Street, New Haven, CT 06510, USA. Noël Boemare, Institut National de la Recherche Agronomique (INRA), UMR1133 Laboratoire EMIP, F-34095 Montpellier, France; Université Montpellier II, UMR 1133 Laboratoire EMIP, F-34095 Montpellier, France. John P. Burand, Division of Entomology, Department of Plant Soil and Insect Science, University of Massachusetts, Amherst, Massachusetts, USA. Louela A. Castrillo, Department of Entomology, Cornell University, Ithaca, NY 14853, USA. Todd A. Ciche, Michigan State University, East Lansing, Michigan, USA. David Clarke, University College Cork, Ireland. Daniel Doucet, Great Lakes Forestry Centre, Sault Ste Marie, Ontario, Canada. Eric Duchaud, INRA, UR892, Unité Virologie et Immunologie Moléculaires, F-78350 Jouy-en-Josas, France. Martin A. Erlandson, Agriculture and Agri-Food Canada, Saskatoon Research Centre, 107 Science Place, Saskatoon, SK S7N 0X2, Canada. Shannon Escasa, Great Lakes Forestry Centre, Sault Ste Marie, Ontario, Canada. Brian A. Federici, Department of Entomology and Interdepartmental Graduate Programs in Genetics, Genomics & Bioinformatics and Cell, Molecular & Develop- mental Biology. University of California, Riverside, CA 92521, USA. xi xii Contributors Sophie Gaudriault, INRA and Université Montpellier II, UMR1133 Laboratoire EMIP, F-34000 Montpellier, France. Heidi Goodrich-Blair, University of Wisconsin, Madison, Wisconsin, USA. Parwinder S. Grewal, Ohio State University, Wooster, Ohio, USA. Quan Guoxing, Great Lakes Forestry Centre, Sault Ste Marie, Ontario, Canada. Richard A. Humber, USDA-ARS, Robert W. Holley Center for Agriculture and Health, Ithaca, NY 14853, USA. Bradley C. Hyman, Department of Biology, University of California, Riverside, CA 92521, USA.