Feasibility of bacterial probiotics for mitigating coral bleaching Ashley M. Dungan ORCID: 0000-0003-0958-2177 Thesis submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy September 2020 School of BioSciences The University of Melbourne Declaration This is to certify that: 1. This thesis comprises only of my original work towards the PhD, except where indicated in the preface. 2. Due acknowledgements have been made in the text to all other material used. 3. The thesis is under 100,000 words, exclusive of tables, bibliographies, and appendices. Signed: Date: 11 September 2020 ii General abstract Given the increasing frequency of climate change driven coral mass bleaching and mass mortality events, intervention strategies aimed at enhancing coral thermal tolerance (assisted evolution) are urgently needed in addition to strong action to reduce carbon emissions. Without such interventions, coral reefs will not survive. The seven chapters in my thesis explore the feasibility of using a host-sourced bacterial probiotic to mitigate bleaching starting with a history of reactive oxygen species (ROS) as a biological explanation for bleaching (Chapter 1). In part because of the difficulty to experimentally manipulate corals post-bleaching, I use Great Barrier Reef (GBR)-sourced Exaiptasia diaphana as a model organism for this system, which I describe in Chapter 2. The comparatively high levels of physiological and genetic variability among GBR anemone genotypes make these animals representatives of global E. diaphana diversity and thus excellent model organisms. The ‘oxidative stress theory for coral bleaching’ provides rationale for the development of a probiotic with a high free radical scavenging ability. In Chapter 3, I construct a probiotic comprised of E. diaphana-associated bacteria able to reduce oxidative stress by neutralizing free radicals such as ROS. I identified six strains with high free radical scavenging ability belonging to the families Alteromonadaceae, Rhodobacteraceae, Flavobacteriaceae, and Micrococcaceae. In parallel, I established a “negative” probiotic consisting of closely related strains with poor free radical scavenging capacities. The application of this probiotic to mitigate the negative impacts of exposure to a simulated heat wave was tested in Chapter 4. There was no evidence for improved thermal tolerance in E. diaphana. Changes in the relative abundance of anemone-sourced Labrenzia provided evidence for its integration in the E. diaphana microbiome. Uptake of other probiotic members was inconsistent and probiotic members did not persist in the anemone microbiome over time. Consequently, the failure of the probiotic inoculation to confer improved thermal tolerance may have been due to the absence of probiotic bacteria for the full duration of the experiment. Importantly, there were no apparent physiological impacts on the holobiont following inoculation, thus showing that shifting the abundance of native anemone microbiome members was not detrimental to holobiont iii health. Further, I found no evidence for an increase in ROS in the E. diaphana holobiont when it was exposed to heat. Some of the most compelling evidence in support of the ‘oxidative stress theory of coral bleaching’ comes from three published studies that expose corals, cultures of their algal endosymbiont, or E. diaphana to exogenous antioxidants during thermal stress. To confirm that ROS is the main driver behind thermal bleaching in E. diaphana, I replicated these previous experiments with novel methods that allowed a more accurate quantitation of ROS, and found that dosing with exogenous antioxidants (mannitol and ascorbate plus catalase) mitigates bleaching in E. diaphana, with no correlation between bleaching and increased ROS (Chapter 5). A serendipitous finding was that the E. diaphana bacterial community diversity can be rapidly reduced when anemones are reared in sterile seawater, making this model suitable for testing the efficacy of microbial restructuring strategies (Chapter 6). Taken together, the work from my PhD has shown that ROS scavenging varies among anemone-associated bacteria and that a high ROS-scavenging probiotic can be developed. Further, my findings have unveiled several main knowledge gaps that need to be filled before probiotics can be implemented, including administration strategies and choice of probiotic bacteria that maximise the maintenance of probiotic communities over time and a direct measurements of ROS in bleaching corals (Chapter 7). iv Preface Three of the seven chapters of this thesis (Chapters 2, 3, and 6) have been submitted to peer-review journals and are either published or under review. Chapters 4-5 have been written in publication format for future submission. Details of contribution for each manuscript are outlined below. Chapter 2 (published in Symbiosis on 29 January 2020) Dungan A., Hartman L., Tortorelli G., Belderok R., Lamb A., Pisan L., McFadden G., Blackall L., van Oppen M. (2020), Exaiptasia diaphana from the Great Barrier Reef: a valuable resource for coral symbiosis research, Symbiosis 80: 195-206. DOI: 10.1007/s13199- 020-00665-0 All authors contributed to conceptual development and the final edited version of the manuscript. AD, GT, RB, and LP collected data, AD, LH, and AL performed data analysis, and AD and LH wrote the first draft of the manuscript. AD contributed to approximately 60% of this work. Chapter 3 (published as a preprint on bioRxiv on 3 July 2020; submitted to Microbial Biotechnology on 21 August 2020) Dungan A., Bulach D., Lin H., van Oppen M., Blackall L. (2020), Development of a free radical scavenging probiotic to mitigate oxidative stress in cnidarians, bioRxiv. DOI: 10.1101/2020.07.02.185645 (Submitted to Microb. Biotechnol.) AD, MvO, and LB conceived and designed the study. AD performed the sampling and sample processing. AD, DB, and HL completed bioinformatic analyses. AD wrote the first draft. All authors edited and approved the final manuscript. AD contributed with ~75% of the work. Chapter 4 (in progress for Journal of Applied Microbiology) Dungan A., Hartman L., Blackall L., van Oppen M., (2020), Assessment of a ROS-targeted bacterial probiotic aimed at improving thermal tolerance in the coral model, the sea anemone Exaiptasia diaphana. (in prog. J. Appl. Microbiol.) v AD, LH, MvO and LB conceived and designed the study. AD and LH performed the sampling, sample processing and bioinformatic analyses. AD and LH wrote the first draft. All authors edited and approved the final manuscript. AD completed 51% of this work. Chapter 5 (in progress for Global Change Biology) Dungan A., Maire J., Perez-Gonzalez A., Blackall L., van Oppen M., (2020), Lack of evidence for the oxidative stress theory of bleaching in the sea anemone, Exaiptasia diaphana. (in prog. Glob. Chang. Biol.) AD, LB, and MvO conceived and designed the study. AD and JM performed the sampling, sample processing and analyses. APG assisted with flow cytometry. AD wrote the first draft. All authors edited and approved the final manuscript. AD contributed with ~70% of the work. Chapter 6 (submitted to Frontiers in Marine Science on 25 August 2020) Dungan A., van Oppen M., Blackall L., (2020), Short-term exposure to sterile seawater reduces bacterial community diversity in the sea anemone, Exaiptasia diaphana. (Submitted to Front. Mar. Sci.) AD, MvO, and LB designed and conceived the experiments. AD conducted the experiment, collected, analysed, and interpreted the data. MvO and LLB contributed reagents, materials, and analysis tools. AD wrote the first draft of the manuscript. All authors read and approved the final manuscript. AD completed 70% of the work. Other relevant publications during candidature: Blackall L., Dungan A., Hartman L., van Oppen M., (2020), Probiotics for corals. Microbiol. Aust., 41(2), 100-104. Ng E-L., Silk Y.L., Dungan A., Colwell J., Ede S., Lwanga E.H., Geissen V., Blackall L., Chen D., (2020), Forest soil microbiome altered by microplastic pollution. (Submitted to J. Hazard. Mater.) vi Acknowledgements Like many tasks in life, it really does take a village to complete a PhD. My experience has been no exception and there are several people I would like to acknowledge for their role in my growth and development as a scientist, colleague, and friend. When I reached out to Madeleine and Linda at the 2016 ICRS, I could not have anticipated how enjoyable they would make my experience at The University of Melbourne. Thank you both for supporting my ideas, giving me confidence, and being excellent role models. I could not have picked better advisors to lead me on this incredible journey. Leon, you have been there for me from the beginning – from teaching me fundamental lab and analytical skills to listening to me ramble in our office. You have been a great “Uncle” to Kelly and an even better friend to Avery and me. You are a true mate! I am incredibly thankful to Justin for both his unwavering mateship and enthusiasm for science. To the students (Giulia Holland, Louis Dorison, Shona Elliot-Kerr, Xavier Smith, Emily Derrick, Sarah Jane Tsang Min Ching) that helped along the way and allowed me to be your teacher, thank you. I am indebted especially to Giulia and Louis who spent months working on my projects and made significant contributions to my research. Much of the bioinformatics work in this thesis would not have been possible without Dieter and Gayle. Thank you for your patience when teaching me. I would be remiss not to mention by other lab mates and colleagues who participated in group discussions and asked difficult questions leading to more scientifically sound research. For this contribution I would like to thank my friends and colleagues Dr. Kat Damjankovic, Giada Tortorelli, Dr. Pat Buerger, Dr. Wing Chan, Talisa Doering, Annie Lamb, Ruby Vanstone, Dr. Adrian Lutz, Sam Girvan, and Sarah Jane Tsang Min Ching. I would like to thank Seeing Eye Dogs Vision Australia. This organization really made me vii feel like a valued member of the community and provided respite during my PhD.