Phylogenetic Diversity, Functional Convergence, and Stress Response of the Symbiotic System Between Sponges and Microorganisms Lu Fan A thesis submitted to the University of New South Wales for the degree of Doctor of Philosophy April 2012 Abstract All living multicellular organisms contain associated microorganisms, which often make substantial symbiotic contributions to host physiology, behaviors and evolution. Marine sponges host dense and highly diverse communities of symbiotic microorganisms. These symbionts are assembled in stable and host-specific community structures and are often inherited through the sponge’s generations. Previous investigations have provided a good understanding of the phylogenetic diversity of microbial symbionts in marine sponges. However the functional features that underpin their symbiotic interactions are largely unknown. This thesis uses sponges as models to address fundamental concepts of microbial symbiosis, including community diversity and assembly, metabolic interactions with the host and other symbionts, as well as symbiosis stability under perturbations. State-of-the-art techniques including 16S rRNA gene pyro-tag-sequencing, metagenomics and metaproteomics were applied. Specifically, a pipeline was developed to reconstruct full-length ribosomal rRNA genes from pyrosequencing metagenomic shotgun data. This work showed that a substantial proportion of microbial diversity has been typically missed by the PCR-based approaches. Detailed metagenomic analyses then identified the functional core in symbiont communities from taxonomically distinct sponge species. The result indicated that common functions were provided by distinct but functionally equivalent symbionts and enzymes in different sponge hosts. Moreover, the abundant elements involved in horizontal gene transfer suggested their key roles in distributing core functions between co-evolutionary symbionts and in facilitating functional convergence on the community scale. To investigate the expression profile of sponge symbionts, metaproteogenomic analysis was conducted on the sponge Cymbastela concentrica. The analysis detected abundant protein expression for the functions of substrate transport, aerobic and anaerobic metabolisms, stress response, and host-symbiont interactions. To study the stability and dynamics of the sponge holobiont during perturbation, the sponge Rhopaloeides odorabile was examined under controlled thermal stress. Dramatic changes in community structure, functional composition and gene expression were observed along with the host stress response suggesting that a decline in symbiotic interactions was likely to be the key factor in the loss of holobiont function. i These discoveries combined with a theoretical framework advanced our knowledge in sponge microbiology and provided new insights into the ecology and evolution of microbial symbiosis. ii Acknowledgements First, I would like to thank Torsten Thomas for being such a cool supervisor and helpful friend to me. I admire his great knowledge in research, detailed research insights, and creative thinking. His constant demand for ‘German precision’ towards data analysis trained me in the ability to examine every experimental result or conclusion independently and critically with requirement for definative results. Besides the training to be a hard-nosed scientist, I also feel grateful for the freedom and trust he gave me to fulfill my science aspirations. Torsten’s wonderful personality, his positive thinking towards life and great sense of humor has had a great influence on me. I still remember the words he gave me before I started my PhD study that, ‘The first thing to be a good scientist is to be a good person’. It has been my great honour to have had the chance to work with Torsten for the last four years. The remarks on my research from my co-supervisor Staffan Kjelleberg have always been insightful. I appreciate his guidance throughout my PhD study including project design, progress review and manuscript writing. Staffan’s views in the big picture of science and his endeavours in combining science and industry have been of great influence to me. I would also like to show my gratitude to Nicole Webster, my supervisor during my visit to AIMS as well as my wonderful collaborator. Nicole was excellent in coordinating the heat- stress experiment described in this thesis and provided important data for the GeXP analysis. As a colleague, I am very impressed by her passion towards her research and her love to the sponges. I thank her for her wonderful arrangement for my sampling at AIMS. I really enjoyed my stay there, especially the exclusive ship trip and I deeply fell in love with the fascinating nature of the GBR. I hope to have the chance to re-visit AIMS one day and get seasick there again. I thank Michael Liu as my wonderful colleague and good friend. Michael contributed to the proteomic sample analyses described in this thesis. Working with him has been great fun. I owe sincere and earnest thankfulness for all his help in life over the last four years, especially when I first came to Australia. I would like to congratulate him on his recent marriage and I wish him a bright future in his career. I also want to thank David Reynolds, iii who did a wonderful analysis for the ELPs described in this thesis. David is a quick learner and very interesting person. It has been my pleasure to supervise him and I wish him joy for whatever he decides to do in the future. My thanks also go to other colleagues and collaborators who have had a direct contribution to the analyses in this thesis, including Manuel Stark, Rachel Simister, and Ling Zhong. Special thanks to the smart super girl Kerensa McElroy, who contributed a number of neat scripts used in my bioinformatic analyses and to Bill O’Sullivan and Susan Cooke for proofreading of this thesis. Thank you to everyone from AIMS for their support in sample collection and shipment; including Rose, Rochelle, Chris B, Raffaella, Florita, Heidi, and Shawn. I thank Martin from UNSW for his help in server operation and Patricia and Merrick from the Queensland Museum for sponge identification. I thank my former and present colleagues at CMB, especially Kirsty and Adam for their grateful support with my scholarship, reagent ordering, sample shipment and conference travel arrangements. My experiments would not have been successful without their assistance. I cherish the memory I had in Lab 304 especially at the beginning of my PhD study. Thanks to Neil, Shaun, Cathy, Maria, Gee, Vickey, Sharon, Melani, Raymond, Carla, and Alex for their great help and advice during the time when I was establishing my experiments. I am also thankful for the 'golden old days' I had with Francesco, Kathrine, Adrian, Sylvain and others whilst enjoying camping, surfing, soccer and badminton. Lastly, I am exceedingly grateful to my parents and my grandmothers. Thanks for supporting my decision to study in Australia. Thanks for your countless sacrifices that you made on my part over the years and the education you gave me to be honest, responsible and grateful. I am really indebted to the endless love and encouragement I receive from you. Last but not the least, I would like to thank my friends in China and Australia who appreciated me for my work and made my PhD life more enjoyable. iv Table of Contents Abstract ..................................................................................................................................... i Acknowledgements ............................................................................................................... iii Table of Contents .................................................................................................................... v List of Figures .......................................................................................................................... x List of Tables ....................................................................................................................... xiii List of Abbreviations ........................................................................................................... xiv Chapter One General Introduction ....................................................................................... 1 1.1 Common features of eukaryota-associated microorganisms ................................. 1 1.1.1 Diversity, distribution and functional significance of the micro-biosphere .......... 1 1.1.2 Significance of eukaryote-associated microbial community and the holobiont concept .............................................................................................................................. 3 1.1.3 Loosely associated microbiota and host-restricted symbionts .............................. 4 1.1.4 Microbial community assembly mechanisms ....................................................... 5 1.1.5 Evolution of symbiont genomes ............................................................................ 7 1.2 Sponge-microbiota holobionts ................................................................................... 8 1.2.1 General background and basic biology of sponges ............................................... 8 1.2.2 Diversity and distribution of sponge microorganisms ........................................ 10 1.2.3 Evolution of sponge holobiont ............................................................................ 10 1.2.4 Functional features in the sponge holobiont .......................................................