Wolbachia in Eurema butterflies: endosymbiont effects on host sex ratios and population genetics Peter Kern B.Sc., Dipl.-Ing (Phytomedicine) Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Hawkesbury Institute for the Environment University of Western Sydney, Hawkesbury Campus December 2015 Acknowledgements First I thank my principal supervisor Markus Riegler, who was a great mentor and provided major guidance and encouraged me in my work during the last four years. I learned a lot from him and he always responded to my questions, no matter what time in the morning or night. I am very grateful to my second supervisor James Cook, he always gave helpful advice and helped to discuss ideas and to shape the structure of my thesis. He was especially helpful in drafting paper manuscripts. I am thankful for the top-up scholarship from the Hawkesbury Institute for the Environment which allowed me to extend my candidature and complete this PhD degree. Further, I thank the HIE for granting me a conference fund application to attend the International Wolbachia Conference in June 2014 which gave me the opportunity to present my findings to the Wolbachia research community. I want to thank Daisuke Kageyama for his willingness to share his knowledge, experience and valuable butterfly material as well as for his insightful discussion of some of my research results. Many thanks also go to Darrell Kemp who provided many butterfly samples for my studies and learned a lot about the biology and behaviour of my species and how to identify and find them in the field. I also thank Michael Braby for providing valuable samples for my study from Darwin (Northern Territory) and also for showing me how to I identify the species and for the interesting discussion I had with him at the Australian National Insect Collection in Canberra. I am especially grateful to Jennifer Morrow who was a great and invaluable help in the molecular lab and showed me how things run in the lab and helped me to troubleshoot problems and taught me many new methods and techniques in the lab. To the boys in S8 Aidan Hall, Andrew Gherlenda, Tim Sutton and Adam Frew, many thanks for introducing me to Australia and teaching me some more English. To all my fellow lab members Jenny Shanks, Jocelyn King, Lluvia Flores Renteria, Shannon Smith, Goran Lopaticki, Deane Woruba and Robert Müller – it was great to meet you and it was nice to get to know you a bit better. A heartfelt thank you goes to all my housemates of 2/57 WMS who were always the best housemates and friends you could look for. Special thanks to Christian Stauffer, who first introduced me to the Wolbachia research field during my Masters course and research, and for his valuable advice during my PhD. Finally I want to thank my family who always supported me during the whole PhD and my girlfriend who was a great support, always understanding and helped me to get through the writing phase of this PhD. II Statement of authentication The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. ....................... ....................... Peter Kern December 2015 III Preface This thesis is presented as a series of three experimental chapters. Chapter 2 was accepted and published in Biology Letters (Kern et al. 2015), while Chapter 3 and Chapter 4 have been prepared for future submission to peer-reviewed journals. It is therefore not a thesis by publication but a standard thesis with experimental chapters written in manuscript format (and repetition of concepts in the introductions of individual experimental chapters is kept to a minimum). The thesis also contains an introduction (Chapter 1), including an extensive literature review and an aims and hypothesis section. The thesis finishes with a discussion chapter (Chapter 5) that summarises the findings of the research, discusses its limitations, includes questions that were not answered in experimental chapters and an outlook part with ideas and hypotheses for future research. The appendix of the thesis contains all the supplementary tables and figures. I am the main author of all chapters. I conceptualised and designed all experiments with my supervisory panel, Assoc. Prof Markus Riegler (Principal supervisor) and Prof James Cook (Co-supervisor). With the exception of the execution of the rearing experiment and microscopy analysis, performed by Dr Daisuke Kageyama as part of chapter 2, I performed all the experimental laboratory work and data analysis, and I wrote the individual chapters with contributions and advice of my IV supervisory panel. Field collections were mostly undertaken by myself, the primary supervisor as well as Dr Darrell Kemp (who will be co- author of Chapter 3). The thesis structure is as below: Chapter 1: General Introduction Chapter 2: Double trouble: combined action of meiotic drive and Wolbachia feminisation in Eurema butterflies. Published as Peter Kern, James M. Cook, Daisuke Kageyama, Markus Riegler (2015) Biology Letters, 11 (5) 20150095. Supplemental tables and figures are embedded in Appendix A. The published version of the paper is embedded in Appendix D. Chapter 3: Influence of Wolbachia on the genetic diversity of infected Eurema hecabe in contrast with uninfected Eurema smilax in Australia. Manuscript in preparation for submission to a peer-reviewed journal as Kern, P., Kemp, D. J., Cook, J. M. and Riegler, M. “Influence of Wolbachia on the genetic diversity of the Australian butterflies Eurema hecabe and Eurema smilax”. Supplemental tables and figures are embedded in Appendix B. Chapter 4: Comparison of mitochondrial, nuclear and Wolbachia genes of Australian Eurema butterfly species demonstrates high incidence of horizontal endosymbiont transmission. Manuscript in preparation for submission to a peer-reviewed journal as Kern, P., Cook, J. M. and V Riegler, M. “Comparison of mitochondria, nuclear and Wolbachia genes of Australian Eurema shows high incidence of horizontal endosymbiont transmission”. Supplemental tables and figures are embedded in Appendix C. Chapter 5: General Discussion. VI Table of Contents Acknowledgements ......................................................................... I Statement of authentication ........................................................... III Preface ......................................................................................... IV Table of Contents ......................................................................... VII List of Tables ............................................................................... XV List of Figures ............................................................................ XVII Abbreviations ............................................................................. XXII Abstract .................................................................................... XXIV 1. Chapter 1 General Introduction ................................................. 1 1.1 Selfish genetic elements and genetic conflicts affecting insect sex determination ......................................................................... 2 1.2 Symbiotic bacteria in insects ................................................ 3 1.3 The biology of Wolbachia ..................................................... 4 1.3.1 Cytoplasmic incompatibility (CI) ....................................... 6 1.3.2 Thelytokous parthenogenesis .......................................... 7 1.3.3 Male-killing ..................................................................... 8 1.3.4 Feminisation ................................................................... 9 VII 1.3.5 Effects of Wolbachia on insect sex determination ........... 13 1.3.6 Horizontal Wolbachia transmission ................................ 14 1.3.7 Wolbachia effects other than manipulation of reproduction ............................................................................ 17 1.4 Sex determination of Lepidoptera ....................................... 18 1.5 Eurema butterflies .............................................................. 22 1.5.1 Adaptation strategies of Australian Eurema species to survive tropical and subtropical dry season: Migration and adult diapause .................................................................................. 23 1.5.2 Larval host plant ............................................................ 25 1.5.3 The Large Grass-yellow, Eurema hecabe ........................ 26 1.5.4 The Scalloped Grass-yellow, Eurema alitha .................... 29 1.5.5 The Small Grass-yellow, Eurema smilax ......................... 31 1.5.6 The No-brand Grass-yellow, Eurema brigitta ................. 33 1.5.7 The Lined Grass-yellow, Eurema laeta ............................ 34 1.5.8 The Pink Grass-yellow, Eurema herla ............................. 37 1.5.9 The Broad-margined Grass-yellow, Eurema puella ......... 39 1.5.10 The Grass-yellow, E. mandarina ..................................... 40 1.6 Wolbachia in Eurema hecabe and Eurema mandarina .......... 43 1.6.1 The influence of Wolbachia on mtDNA of Japanese Eurema mandarina and Eurema hecabe ................................................. 46 VIII 1.7 Research scope and aims ................................................... 47 Chapter 2 Double trouble: combined action of meiotic drive and Wolbachia