Understanding Aphids: Transcriptomics, Molecular Evolution and Pest Control Submitted By Amol Bharat Ghodke Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy JUNE 2018 School of Biosciences, Faculty of Science The University of Melbourne Melbourne, Australia Abstract Aphids exhibit fascinating biological features including parthenogenesis, symbiosis, altruism and host-plant preference; all of which would be better understood if genetic tools and molecular biological techniques were applied to them. Aphids are also agricultural pests that vector plant viruses and new approaches to control them are required. This thesis addresses questions motivated by an interest in the biology of aphids and a desire to improve the agricultural impact of aphids. It does so through transcriptomic analyses and RNA interference (RNAi) technology. I examined the ways in which the transcriptome of aphid changes with host-plant, between tissues, within species and between species. The three-aphid species studied (the green peach aphid: Myzus persicae, the mustard aphid: Lipaphis erysimi, and the cabbage aphid: Brevicoryne brassicae) are all pests of economically important brassica crops (such as cabbage, cauliflower, mustard and canola). These data may provide insights into the way different aphid species deal with plant secondary compounds such as glucosinolates. These data also allowed me to examine the structure, function and evolution of myrosinase enzymes that have allowed some aphid species to develop an anti-predator ‘mustard bomb’. RNAi has been suggested as a way to specifically target pest that would be more ‘environmentally friendly’ than conventional insecticides. I experimentally assessed the feasibility of orally-delivered RNAi to control aphids and the potential of this technology to be developed as a functional genomic tool. RNAi was fed to aphids via artificial diets at various concentrations and with various delivery agents and via transgenic Arabidopsis thaliana plants that I created that produced dsRNA’s corresponding to aphid genes. These studies lead me to suggest that more work needs to be done to limit the effects of RNase enzymes of the aphid gut digesting orally delivered RNAi and to more carefully characterize factors that may affect within-species variation in RNAi efficacy. II | P a g e Declaration This is to certify that: 1. The thesis comprises only my original work towards the PhD except where indicated. 2. Due acknowledgement has been made in the text to all other material used 3. The thesis is fewer than 100,000 words in length, exclusive of tables, amps, bibliographies, and appendices as approved by the Research Higher Degrees Committee. SIGNATURE (Amol Bharat Ghodke) III | P a g e Preface The tackling of large problems using science often requires team efforts and so funding agencies often support large international collaborations. The so called ‘Australia: India Grand- Challenge’ scheme funded a research program within which this PhD was nested. It was entitled Caterpillar and Aphid Resistance in Brassica (CARiB). This was a joint project among six major institutes, three from Australia and three from India. It included The University of Melbourne, the University of Queensland and the CSIRO from Australia, and the International Centre for Genetic Engineering and Biotechnology, the National Research Centre of Plant Biotechnology and the Indian Agriculture Research Institute from India. The CARiB projects main focus was to develop transgenic cultivars of cabbage, canola, cauliflower and mustard that will be resistant for aphid as well as caterpillars. The project had three main parts, 1) development of new Bt (Bacillus thuringenesis) gene-based control strategy for caterpillars, 2) development of the transgenic plant lines with specific landing sites for targeted gene insertion and 3) the development of RNAi (RNA interference) technology to control aphid species. This thesis falls with the 3rd objective and focussed on aphids and the development of RNAi directed against them. The specific aims of the third objective was to find the superior gene targets for RNAi technology that would greatly affect the fitness of the pest aphid species. The technology development was aimed to achieve universal effectiveness with applicability against different aphid species. Thus, there were several criteria that needed to be considered. The targeted gene sequences needed to be conserved among the pest aphids, but ideally would minimize the chance of affecting other organisms in the environment. They were to be designed so minimize the chance of RNAi resistance arising and therefore polymorphism within species needed to be considered. Target genes also needed to affect aphid fecundity or viability IV | P a g e regardless of environmental variation such as host plants. This justified substantial transcriptomic analyses of the pest aphid species. This thesis describes my contribution to the CARIB project. I performed many RNAi assays and generated two major transcriptome datasets. It also describes how I utilized these data to address some specific questions about the aphid biology. This included characterizing differences between generalist and specialist aphids, and in particular the ways they handled plant-generated secondary metabolite toxicity. I hope, the reader will enjoy this short scientific tour in the world of aphids and appreciate the need to further improve our understanding about the aphids with greater depth. V | P a g e Acknowledgements This thesis is a result of an arduous, yet a momentous journey of some considerable years of my life. As it happens with every journey, there are a number of people who have been instrumental in shaping the story of this journey. My supervisors, Charles Robin and John Golz along with my committee members, Philip Batterham (Chair), Derek Russell, Owain Edwards have been the biggest constructors of my PhD’s journey. Charlie, you not only guided me as a supervisor but nourished me as a researcher and helped me develop a necessary attitude towards science. John, you introduced me to the world of Arabidopsis and granted me all the freedom in your lab. I am also grateful to Phil Batterham and Derek Russell for their invaluable support and guidance within and beyond the research. Owain Edward, even though we had long distance relationship, it only flourished with time and I had wonderful collaboration experience with you and your group. Dr. Keshav Kranthi and Dr. Sandhya Kranthi, without your encouragement and support I wouldn’t be doing PhD at The University of Melbourne. Thank you all for timely advice and all the support that you have provided during this period. You are the people who have still kept my hunger for research and knowledge alive. I cannot thank you enough gentlemen! I would also like to thank my colleagues at School of Biosciences, Bio21 Institute and CSIRO. You made this journey more enjoyable for me with your fun loving but supportive attitude. Alex Fournier-level, I feel blessed for your charismatic company during my PhD that helped me explore some unknown territories of science. I am grateful to Nancy Endersby, Jill Williams and Venessa White, three Wonder-Women who pulled me out from several different apocalyptic situations and helped me without any expectations. I would like to thank to Ronald Lee, Heng-lip Yeap, Mike Murry, Tom Walsh and Andrew Warden for their generous advice during my PhD. During my short experience at VI | P a g e CSIRO, Canberra, I met my dear colleague Chris Coppin, I am thankful to you Chris for teaching me some of the cool molecular biology methods and tricks. Huge thanks to my fellow lab members, especially Rob, Sue, Caitlyn, Llewellyn, Paul, Rebecca S, Pontus, Jack and Kat as well as Hoffman lab Rahul, Rebecca J, who shared my journey with me not just intellectually but emotionally as well. I will forever be thankful to you all for those hilarious moments that made us laugh making this ride a lot more fun. I would like to mention the special contribution of Rob, Rahul, Sue, Alex and Rebecca J who helped me tackle the completely unknown territory, bioinformatics. All the anger, joy, sorrows, I felt during this bioinformatics adventure you all made it worthwhile. This thesis wouldn’t have been possible without you guys. The story of this journey will especially remain incomplete without mentioning a vast number of friends who always made this tough period of my life a lot happier. The gang of ‘Breakers’ in India, Snehal-Rohit, Tejas-Super, Mona-Gaurav, Rohan-Nikita, Avinash-Rohini, Sachin, Shweta, you guys pulled me out of my sadness every now and then and helped me stand strong in difficult time. The remote encouragement provided by Dipak, Sachin, Bala sir, Sandip sir, Varsha. My PhD travel would not have been possible without you guys. Thanks! Finally, big thanks to my grandfather (Baba), Aai-Dada, Bhau-Mothi aai, Raju kaka – Kaki, lovely Mama’s and mami’s and all uncles and aunts, Manisha, Kavita Rajshree (Sisters), Swapnil, Suhas, Sangram and Sanchit (Brothers) and The Ghodke’s for unconditional love, unshakable belief and very long patience. Just thank you is not enough and I will always feel blessed to have you in my life. I hope, I have lived up to your expectations and hope to make this life more fruitful with your support and blessings. Sincerely Amol Bharat Ghodke VII | P a g e Contents Abstract ....................................................................................................................................