The evolution and ecology of Drosophila sigma viruses Ben Longdon PhD University of Edinburgh 2011 Declaration The work within this thesis is my own, except where clearly stated. Chapters 2-5 are published or submitted manuscripts and so have kept the use of “we”. Chapters 1 and 6 were written purely as thesis chapters and so use “I”. Ben Longdon ii Thesis abstract Insects are host to a diverse range of vertically transmitted micro-organisms, but while their bacterial symbionts are well-studied, little is known about their vertically transmitted viruses. The sigma virus (DMelSV) is currently the only natural host- specific pathogen to be described in Drosophila melanogaster. In this thesis I have examined; the diversity and evolution of sigma viruses in Drosophila, their transmission and population dynamics, and their ability to host shift. I have described six new rhabdoviruses in five Drosophila species — D. affinis, D. obscura, D. tristis, D. immigrans and D. ananassae — and one in a member of the Muscidae, Muscina stabulans (Chapters two and four). These viruses have been tentatively named as DAffSV, DObsSV, DTriSV, DImmSV, DAnaSV and MStaSV respectively. I sequenced the complete genomes of DObsSV and DMelSV, the L gene from DAffSV and partial L gene sequences from the other viruses. Using this new sequence data I created a phylogeny of the rhabdoviruses (Chapter two). The sigma viruses form a distinct clade which is closely related to the Dimarhabdovirus supergroup, and the high levels of divergence between these viruses suggest that they may deserve to be recognised as a new genus. Furthermore, this analysis produced the most robustly supported phylogeny of the Rhabdoviridae to date, allowing me to reconstruct the major transitions that have occurred during the evolution of the family. This data suggests that the bias towards research into plants and vertebrates means that much of the diversity of rhabdoviruses has been missed, and rhabdoviruses may be common pathogens of insects. In Chapter three I examined whether the new sigma viruses in Drosophila affinis and Drosophila obscura are both vertically transmitted. As is the case for DMelSV, both males and females can transmit these viruses to their offspring. Males transmit lower viral titres through sperm than females transmit through eggs, and a lower proportion of their offspring become infected. I then examined natural populations of D. obscura in the UK; 39% of flies were infected and the viral population shows clear evidence of a recent expansion, with extremely low genetic diversity and a large excess of rare polymorphisms. Using sequence data I estimate that the virus has iii swept across the UK within the last ~11 years, during which time the viral population size doubled approximately every 9 months. Using simulations based on lab estimates of transmission rates, I show that the biparental mode of transmission allows the virus to invade and rapidly spread through populations, at rates consistent with those measured in the field. Therefore, as predicted by the simulations, the virus has undergone an extremely rapid and recent increase in population size. In Chapter four I investigated for the first time whether vertically transmitted viruses undergo host shifts or cospeciate with their hosts. Using a phylogenetic approach I show that sigma viruses have switched between hosts during their evolutionary history. These results suggest that sigma virus infections may be short-lived in a given host lineage, so that their long-term persistence relies on rare horizontal transmission events between hosts. In Chapter five I examined the ability of three Drosophila sigma viruses to persist and replicate in 51 hosts sampled across the Drosophilidae phylogeny. I used a phylogenetic mixed model to account for the non-independence of host taxa due to common ancestry, which additionally allows integration over the uncertainty in the host phylogeny. In two out of the three viruses there was a negative correlation between viral titre and genetic distance from the natural host. Additionally the host phylogeny explains an extremely high proportion of the variation (after considering genetic distance from the natural host) in the ability of these viruses to replicate in novel hosts (>0.8 for all viruses). There were strong phylogenetic correlations between all the viruses (>0.65 for all pairs), suggesting a given species’ level of resistance to one virus is strongly correlated with its resistance to other viruses. This suggests the host phylogeny, and genetic distance from the natural host, may be important in determining viruses ability to host switch. This work has aimed to address fundamental questions relating to host-parasite coevolution and pathogen emergence. The data presented suggests that sigma viruses are likely to be widespread vertically transmitted insect viruses, which have dynamic interactions with their hosts. These viruses appear to have switched between hosts iv during their evolutionary history and it is likely the host phylogeny is a determinant of such host shifts. v Acknowledgments “That’s a fool’s experiment. But I love fools' experiments. I am always making them” Charles Darwin. “One becomes a gourmet chef for the diptera” Terri Markow It is traditional to start a thesis with a quote (or two) and I thought these summed up my PhD rather nicely. I have tried to be succinct in writing these acknowledgements, but realised that this thesis would not have been possible without the help and support of a great number of people. Specific acknowledgments with regards to experimental work are made in each chapter. I owe a huge thanks to Frank Jiggins for his help, constant advice and reassurance (even if he did leave me and run away to Cambridge!). I am also greatly indebted to Darren Obbard who has always been friendly, keen to help, and has shown constant enthusiasm (coupled with extreme pessimism and whinging). I could not have asked for a better pair of supervisors. I would like to thank the following to whom I am hugely grateful: Lena Wilfert for her help, advice and occasional accommodation in Cambridge. Jarrod Hadfield for being the source of knowledge for all things statistical, his musings over a pint, and unrepeatable quotes. Tom Little for being my official supervisor, and for offering useful advice. Mark Blaxter for being my post-graduate carer and also providing money towards my external examiner travel expenses. Claire Webster who has provided continuous supplies of gossip and helped with fly work. Mike Magwire for going out of his way to be helpful. Andrew Rambaut for numerous informal discussions on the correct phylogenetic practices. Fergal Waldrin, Jewelna Osei- Poku, Floh Bayer and Jenny Carpenter for being great lab mates. The Edinburgh coevolution group for numerous interesting, and often heated, discussions. The French sigma virus researchers of days gone by for providing many snippets of useful information. I would also like to thank various IEB folk for offering words of vi advice or kind words (in particular Sarah Reece, Dan Nussey, Penny Haddrill and Desiree Allen). Thanks to everyone who put me up on fieldwork (Chapter 2 and 3) and supplied flies at various points during my PhD. Thank you to Nigel Franks for his help and encouragement in my undergraduate degree. I would like to thank the folk at the CIE at Deakin University for allowing me to work in the department whilst writing up. Thanks to Helen Borthwick and Helen Cowan for making me laugh and helping with fly food preparation. Thanks to the other IEB PhD students for putting things into perspective (and making me realise flies are relatively good compared to daphnia, plasmodium or some long term “wild” dataset). I received funding from the BBSRC, Royal Entomological Society, Genetics Society UK and the James Rennie bequest. On a personal note I would like to thank my parents, for their enduring support, even when I was forced to abandon them on their visit to Edinburgh for lab work. Thanks to Matt Boyce and Saskia Pedersen for providing a very welcome distraction from work. Finally I want to thank Laura Kelley for her kindness and unwavering faith in me, for putting up with my grumpiness and for generally being brilliant. Lastly thank you to Andrew Rambaut and John Jaenike for taking the time to read this thesis. vii Table of Contents Declaration ii Thesis Abstract iii Acknowledgments vi 1. General Introduction 1.1 General background 1 1.2 Questions 2 1.3 Sigma virus general biology 12 2. Sigma virus discovery and phylogeny 25 2.1 Introduction 26 2.2 Methods 28 2.3 Results 33 2.4 Discussion 41 3. Vertical transmission and a recent sweep 46 3.1 Introduction 47 3.2 Methods 48 3.3 Results 55 3.4 Discussion 65 4. Host-switching 70 4.1 Introduction 71 4.2 Methods 72 4.3 Results 73 4.4 Discussion 75 5. Phylogenetic determinants of host shifts 78 5.1 Introduction 79 5.2 Methods 82 5.3 Results 89 5.4 Discussion 93 6. General discussion 6.1 Summary of field 98 6.2 Overview 99 6.3 Future directions 104 6.4 Conclusions 109 References 110 Appendix/Supplementary material 130 viii Chapter 1: General Introduction 1. General Introduction I wrote this chapter with comments on drafts from Darren Obbard and Frank Jiggins. 1.1 Background Parasites are a major cause of mortality, being responsible for 13 million deaths in humans each year (WHO 1999). Similarly, parasites are responsible for diseases affecting crops, livestock and natural populations of plants and animals (Altizer and Pedersen 2008). However, parasites are also of interest to evolutionary biologists, as antagonistic reciprocal interactions between hosts and parasites drive rapid coevolutionary dynamics (Woolhouse et al. 2002). In turn, understanding the coevolutionary processes that occur between hosts and parasites may have implications for the control and prediction of infectious disease.