Environmental influences on the Pacific oyster (Crassostrea gigas) microbiome and disease associated with Ostreid herpesvirus-1 (OsHV-1) Bhagini Erandi PATHIRANA A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy THE UNIVERSITY OF SYDNEY Farm Animal Health Sydney School of Veterinary Science Faculty of Science February 2020 Declaration of Authorship Apart from the assistance stated in the acknowledgements section, this thesis represents the original work of the author. To the best of my knowledge the results from this study have not been presented for award for any other degree or diploma at this or any other university. Bhagini Erandi PATHIRANA MSc, BVSc (Hons) February 2020 i Acknowledgements First, I would like to extend my sincere thanks to Dr. Paul Hick for his expert supervision as the primary supervisor of my doctoral research study. His patience and guidance helped me to develop skills and competence in research and scientific communication. I would like to take this opportunity to thank him for supervising and guiding me to achieve valuable outcomes from my PhD research and for providing me with great opportunities to reach new horizons in molecular diagnostics, bioinformatics and aquatic animal health, through professional training. The role of Emeritus Professor Richard Whittington as my auxiliary supervisor is no way smaller. I would like to take this opportunity to thank him again for accepting me to the diverse and amazing Farm Animal Health Research group of the University of Sydney, as a PhD candidate. Without his acceptance, I would not have been able to pursue a doctoral research at the University of Sydney. Further, I would like to extend my gratitude for his expert supervision in helping me to develop my research and scientific communication skills. The financial assistance provided by the Australian Government through the Endeavour Leadership Program, supporting my doctoral studies and my stay in Australia as an international student is greatly appreciated. Securing an Australian Government Scholarship to pursue doctoral studies at the University of Sydney is one of the greatest achievements of my academic career and a turning point of my career as a university academic and as a researcher. Further, I would like to acknowledge the financial assistance provided by the University of Sydney to support my doctoral studies. It has been a wonderful privilege to work with a highly qualified, friendly and a supportive team of people in the Farm Animal Health Research group. In particular, I would like to extend my sincere appreciation to Associate Professor Jenny-Ann Toribio, Dr. Om Dhungyel, Dr. Karren Plain, Dr. Auriol Purdie and Dr. Kumudika de Silva, for their support and expert views provided during research group meetings and research presentations which positively contributed to improve my doctoral research. My sincere thanks also go to Associate Professor Joy Becker for providing me with the opportunity to continue my teaching career and to widen my teaching experience as a graduate teaching assistant. Further, I would like to thank Anna Waldron, Alison Tweedie, Ann-Michele Whittington, Slavicka Patten, Natalie Schiller, Nicole ii Carter, Rebecca Maurer, Stuart Glover and Craig Kristo for their technical expertise, support and guidance in carrying out various laboratory procedures and for creating a pleasant laboratory environment. The support from late Pabitra Dhungyel in the laboratory, during the early years of my candidature is fondly remembered. The laboratory skills and competence that I developed by working in NATA-accredited laboratories will undoubtedly be a great asset in my future career. The support and thoughtful assistance extended by Marion Saddington, was an invaluable strength during my entire PhD candidature. I would also like to take this opportunity to thank Associate Professor Navneet Dhand and Dr. Kathrin Schemann for their expert statistical advice in refining the statistical analyses of my research. I am very much thankful for Dr. Olivia Evans and Dr. Marine Fuhrmann for their great support in carrying out various research activities during my doctoral programme. My sincere thanks also go to Dr. Andrew McPherson for helping me to get started with microbiome analysis using the QIIME software and for his invaluable contribution for the first research publication during my PhD candidature. It has been a pleasure to work alongside friendly and competent PhD candidates, Dr. Matt Johansen, Dr. Kamal Aacharya and Dr. Hannah Pooley (as former PhD students) and Maximillian de Kantzow, Karen Smith, Anna Ly, Katherine Wright, Cahya Fusianto and Ed Annand. I would also like to thank the bioinformatics staff at the Sydney Informatics Hub, especially Dr. Rosemarie Sadsad and Dr. Tracy Chew, for their expert guidance in bioinformatic analyses and for their prompt support whenever needed. Dr. Neil Horadagoda and Kanchana Ekanayake are sincerely acknowledged for their great helping hand whenever needed. They were a great support from the time of our establishment at Camden and throughout the PhD candidature. Last but in no way least, I am very much grateful and indebted to my loving husband Indunil for his continuous encouragement and immense moral and physical support throughout my PhD candidature. Without him this PhD would not be a reality. I would also like to thank my loving son Nisanga for spending long, lonely hours as a little boy and for his support as a teenager to make his mum’s PhD a reality. This thesis is the output of the sacrifice made by all three of us! iii Summary Pacific oyster mortality syndrome (POMS) is a high mortality disease in Pacific oysters (Crassostrea gigas), which has negatively impacted oyster farming. Despite the causal relationship between Ostreid herpesvirus-1 (OsHV-1) and oyster mortality, the incidence and severity of disease is determined by complex interactions between the physiology of oysters, the environmental conditions and secondary pathogens. Understanding the multifactorial nature of this disease is required to develop effective management strategies. Recent investigations revealed a polymicrobial pathogenesis of this disease, with primary infection by OsHV-1 followed by the involvement of opportunistic bacteria present in the microbiome completing the disease expression. Apart from the implications of various environmental risk factors on POMS, the oyster microbiome is also affected by environmental disturbances. It is important to determine if differences in the microbiome such as dysbiosis, are an outcome of the pathogenesis of the disease or whether the features of the microbiome predispose or contribute to the severity of this disease. Although comprehensive studies concerning polymicrobial pathogenesis of POMS were conducted after this thesis was commenced, the influence of environmental factors on the polymicrobial pathogenesis of POMS and how it is mediated by the oyster microbiome remained a knowledge gap in this regard. This thesis aimed at addressing different aspects of this knowledge gap by investigating the impact of different environmental factors on the Pacific oyster microbiome and their subsequent effects on disease associated with OsHV-1. Chapter 3 investigated how the microbiome of genetically related Pacific oysters with a common hatchery origin differed, when grown in different estuaries. Results indicated an influence of farming environment in shaping the microbiome. Bacterial diversity as determined by 16S rRNA gene sequence analysis, indicated that different estuarine environments generated unique microbiomes which were also associated with a differential response to OsHV-1 infection. The quantitative dynamics of total bacteria and Vibrio spp. during an OsHV-1 infection was assessed using qPCR assays. The microbiome changed with the environment and after an experimental OsHV-1 challenge. A strong correlation was observed between the OsHV- 1 and Vibrio quantities in OsHV-1 infected oysters. Different microbiomes prior to infection were associated with altered responses to OsHV-1 challenge and different disease outcomes. iv Optimizing the quality and quantity of bacterial DNA that was purified from Pacific oyster tissues proved critical for accurate characterisation of the Pacific oyster microbiome. This is particularly important in assessing the potential polymicrobial pathogenesis of Pacific oyster mortality diseases. Chapter 4 focused on evaluating methods to sample tissues, extract and appropriately store bacterial nucleic acids from Pacific oyster, to accurately determine the microbiome. Both intrinsic oyster factors and experimental factors influenced the results of oyster microbiome studies with potential biases being introduced due to sampling method and approaches to nucleic acid extraction. A tissue compartmentalization of the Pacific oyster microbiome was confirmed with identification of distinct, tissue-specific microbiomes in the haemolymph, gill and gut. The different experimental procedures substantially impacted the quantity and diversity of bacteria identified. The controlled environment of a laboratory challenge system for OsHV-1 was utilized to investigate the pathogenesis of POMS. A period of acclimation to this environment before an experimental study can induce changes to the microbiome that may confound findings attributed to the disease being studied. Chapter 5 focused on assessing changes in the Pacific oyster microbiome during acclimation to a laboratory