Deciphering the genomic tool-kit underlying animal- bacteria interactions Insights through the demosponge Amphimedon queenslandica Benedict Yuen Jinghao B.MarSt. (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2016 School of Biological Sciences Deciphering the genomic tool-kit underlying animal-bacteria interactions Abstract All animals are inhabited by bacteria, and maintaining homeostasis in the multicellular environment of the host involves the complex balancing act of promoting the survival of symbionts while defending against intruders. Sponges (Porifera), in addition to housing diverse bacterial symbiont assemblages, also rely on bacteria filtered from the water column for nutrition. My research uses the genome-enabled demosponge, Amphimedon queenslandica, a member of one of the earliest-diverging animal phyletic lineages, as an experimental platform to investigate the genomic toolkit underpinning animal-bacteria interactions. Using comparative bioinformatics tools, I characterised a surprisingly large and complex repertoire of innate immune receptors from the NLR family of genes encoded in the A. queenslandica genome. I then used a high throughput RNAseq approach to profile the sponge’s global transcriptomic response to foreign versus its own native bacteria. Conserved metazoan innate immune pathways were activated in response to both foreign and native bacteria. However, only the native bacteria elicited the expression of a more extensive suite of signalling pathways, involving TGF-β signalling and the transcription factors NF-κB and FoxO. Upregulation of the nutrient sensor AMPK in all treatments along with immune signalling genes, which all regulate FoxO activity, further suggests an interplay between metabolic homeostasis and immunity. Finally, I used microscopy to track the cellular-level processing of the different bacteria by the sponge. Consistent with the observed transcriptional response, the native bacteria were ingested by archaeocytes more rapidly than the foreign bacteria. The slower processing of foreign bacteria also correlated with the expression of numerous Nrf2-associated detoxification genes, indicative of cellular stress, only in response to foreign bacteria. ii Deciphering the genomic tool-kit underlying animal-bacteria interactions Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. iii Deciphering the genomic tool-kit underlying animal-bacteria interactions Publications during candidature Peer-reviewed paper Yuen B, Bayes JM, Degnan SM 2015. The characterization of sponge NLRs provides insight into the origin and evolution of this innate immune gene family in animals. Mol. Biol. Evol. 31(1):106. Conference abstracts Yuen B, Degnan SM 2013. Why does the genome of the sponge, Amphimedon queenslandica encode highly complex and diversified innate immune receptor systems? In: 9th World Sponge Conference: 2013; Fremantle, Western Australia. Yuen B, Degnan SM 2015. Why does the genome of the humble marine sponge, Amphimedon queenslandica, encode the potential for a complex innate immune response? In: The 2nd Annual EMBL Australia PhD Symposium – Completing the pipeline: from biology to bioinformatics and back again: 2015; Melbourne, Victoria. Publications included in this thesis Publication citation – incorporated as Chapter 2. Yuen B, Bayes JM, Degnan SM 2015. The characterization of sponge NLRs provides insight into the origin and evolution of this innate immune gene family in animals. Mol. Biol. Evol. 31(1):106. Contributor Statement of contribution Benedict Yuen (Candidate) Designed experiments (50%) Data analysis and interpretation (80%) Wrote the paper (90%) Joanne Marie Bayes Designed experiments (20%) Data analysis and interpretation (10%) Sandra M Degnan Designed experiments (30%) Data analysis and interpretation (10%) Wrote and edited paper (10%) iv Deciphering the genomic tool-kit underlying animal-bacteria interactions Contributions by others to the thesis Sandra M. Degnan contributed to the conception and design of this research, advised on methods and analyses, and provided critical comments on the thesis and on the associated publication in Chapter 2 Joanne M. Bayes was also involved in the of Chapter 2 of this thesis. conception and the analysis of 10% of the data – specifically the identification of the clade AqNLRsC . Selene Fernandez Valverde, William Hatleberg and Shunsuke Sogabe produced several of the datasets analysed across this thesis (these contributions are specifically acknowledged in the relevant chapters). Transcriptome sequencing was conducted by the Ramaciotti Centre for Genomics, University of New South Wales, NSW, Australia Statement of parts of the thesis submitted to qualify for the award of another degree None. v Deciphering the genomic tool-kit underlying animal-bacteria interactions Acknowledgements I would like to begin by expressing my heartfelt gratitude to my supervisor, Sandie Degnan, for all the guidance you have given me since I first joined the lab as an Honours student in 2011. Thank you for patiently keeping me on track with encouragement and tough love when it was necessary. My career in science is only just beginning and I could not have picked a better mentor to guide me through the transition from an undergraduate to a post-graduate student. I also wish to thank Bernie Degnan, because I believe this journey began when he sparked my interest in evolution as an undergraduate student in his Marine Invertebrates course back in 2010. I am also grateful for having been given the opportunity to tutor in that same course years later. Thank you both for all the wisdom you have imparted in aspects of both work and life, and for teaching me how to science with rigour and integrity. I wish to thank my co-supervisor Andy Barnes for his advice and also for taking me on as a tutor on the MARS2005 field trips. I’ve had so much fun working with you and the other tutors on Heron, and look forward to one final round this September. I also want to thank my panel of readers Sassan Asgari and Kate Stacey. The frank and honest advice you gave me at each milestone is something I’ve come to greatly appreciate. This project was funded by an Australian Research Council Discovery grant to Sandie Degnan (DP110104601). I also wish to acknowledge the University of Queensland for awarding me the UQ International scholarship that covered my tuition fees and provided me with a stipend so that I would not starve in my quest to further our understanding of the world around us. I would also like to thank Bernie and Sandie for financing my trip to the 9th World Sponge Conference in Fremantle. Thank you also for funding my fieldwork in one of the most magnificently beautiful places on this planet. Doing fieldwork on a remote island is challenging on a good day and nightmarish when the weather is not in your favour. I wish to acknowledge the staff at the Heron Island Research Station for all the support they have provided over the multiple field trips I have undertaken over the years. I have been surrounded by truly intelligent and wonderful colleagues who have made my time learning to science in the Degnan lab immensely enjoyable. I must express my appreciation to the lab members vi Deciphering the genomic tool-kit underlying animal-bacteria interactions who have been so generous with the time they have spent training me and providing technical advice. Special thanks goes out to Kerry, Carmel, Laura, Daniel, Selene, Bec and Will. I wish to thank to wonder woman Kerry for the amazing job that she does keeping the day to day business of the lab running so smoothly. I am grateful to Carmel, the all-knowing oracle, for allowing me to badger her constantly with all manner of science-y questions. Thank you both for helping me get through the CEL-Seq protocol. I am grateful to Laura for her insight and advice over the years, the chats about food and other random things. I’m always going to remember the 90’s dance party on Heron Island. Thank you to Selene and Will for help with the computational stuff and for the precious insight into dealing with large datasets. I am grateful to Daniel who taught me the fundamentals of microscopy and imaging techniques. Thank you Bec for the conversations about microbes. Both of you have helped me put what would otherwise have been abstract data into a meaningful spongy context. A big thank you goes out to all other Degnan Labsters, past and present: Jo, Jaret, Andrew, Jabin, Felipe, Shun, Katia, Fede, Maely, Yasu, Kevin, Claire, Gemma, Markus, Arun, Tahsha, Simone, Eunice, Jake, Mel, Betty, Aude.