Wolbachia in Eurema butterflies

268 Pages·2016·3.24 MB·English

by  Peter Kern

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.
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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

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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
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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.
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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
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1.7 Research scope and aims ................................................... 47

Chapter 2 Double trouble: combined action of meiotic drive and
Wolbachia feminisation in Eurema butterflies ................................. 52
2.1. Abstract ............................................................................. 53
2.2. Introduction ....................................................................... 54
2.3. Material and Methods ......................................................... 56
2.3.1. Sampling and Wolbachia screening ................................ 56
2.3.2. W chromatin body assays .............................................. 56
2.3.3. Real-time quantitative PCR (qPCR) ................................. 56
2.3.4. Tpi sequence analysis .................................................... 57
2.4. Results ............................................................................... 57
2.4.1. Wolbachia infection status ............................................. 57
2.4.2. W chromatin body assays .............................................. 57
2.4.3. GDR of Z-linked genes in males and females ................. 58
2.4.4. Inheritance of the Z chromosome .................................. 60
2.5. Discussion ......................................................................... 62

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