Factors Influencing the Gut Microbiota of Antarctic Seals Tiffanie Nelson A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy Evolution & Ecology Research Centre School of Biological, Earth & Environmental Sciences University of New South Wales, Sydney, Australia April 2012 Originality Statement ‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’ Signed …………………………………………….............. Date …………………………………………….............. i Copyright Statement ‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' Signed ……………………………………………........................... Date ……………………………………………........................... Authenticity Statement ‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’ Signed ……………………………………………........................... Date …………………………………………….......................... ii Abstract A mammal’s gastro-intestinal tract houses trillions of bacteria, known as the gut microbiota, which is first acquired during birth. This community provides a number of beneficial functions to the host, including breakdown of food and creation of energy, immunity regulation and cellular development. To date, studies on the mammalian gut microbiota have focused primarily on captive or laboratory-reared terrestrial mammals and there have been a limited number of studies on mammals living under natural conditions or from marine environments. This thesis aimed to address this knowledge gap through a study on the gut microbiota of the southern elephant seal, Mirounga leonina, and the leopard seal, Hydrurga leptonyx, inhabiting Antarctica. The study was designed to understand how time, diet, phylogeny, age, sex, maternal relationship, sewage contamination and captivity influence composition. DNA extracted from faecal samples was analysed via multiple molecular methods to identify an individual’s gut microbiota. The community was dominated by four phyla: Firmicutes, Fusobacteria, Proteobacteria and Bacteroidetes and was identified as stable over a period of months. Diet-driven differences were noted between individuals as a result of species, captivity, sex and age. Specifically, nutrient composition of the diet and bacteria associated with the diet were identified as strong influences on the gut microbiota. Length of the gut, social interactions and antibiotic use were also identified as contributing factors. Despite the observed differences in the gut microbiota between hosts, a ‘core’ microbiota was identified between Antarctic seal species and also between three related species of Arctic seals . This is suggested to be a result of co-evolution which is maintained over generations via transfer from mother to pup. Bacterial isolates possessing virulence factors and resistance to antimicrobial agents were also identified in the gut of these hosts suggesting they may be vulnerable to sewage contamination containing novel bacteria. This research provides a baseline for future work regarding bacterial associations in these hosts. It identifies the complex community inhabiting the gut of mammals and the factors which contribute to its composition. Additionally, it indicates the differences that exist between mammals living in captivity and under natural conditions and has implications for the future health of these hosts. iii Acknowledgements and Thanks There are numerous people who have helped me through this journey. I’d like to thank my partner, Tom Rayner, who has been extremely loving and supportive, particularly during the last two years. Specifically, he has read most of my work and discussed it at length with me, which was extremely helpful in improving my skills. My supervisors, Mark Brown and Tracey Rogers, have been very supportive. Mark has always had patience for my endless questions about microbiology, bacteria, DNA sequencing etc. Tracey together with Michaela Ciaglia facilitated many parts of my research and Tracey always provided me with a practical approach to research. Huge thanks goes to Alejandro Carlini, who taught me so much while literally on the ice and his sudden passing in late 2010 saddened many people. I will never forget his advice on accomplishing tasks in Antarctic conditions, often while holding a cigarette. Funding for field support was provided by the Secretaría de Ciencia y Tecnología with the Dirección Nacional del Antártico (PICTO No. 36054), the Australian Research Council, Winifred Scott Foundation, and the Evolution and Ecology Research Centre, University of New South Wales (UNSW). Research trips involved many transfers between planes, ships, helicopters, and boats and it also included a comfortable stay while at base. In the field, my research would not have been possible without the support provided by Michaela Ciaglia, Javier Negrete, Nigel Edwards, Larry Vogelnest, Nadine Constantinou, David Slip, Matias Baviera, Martin Montes, Karina Smit, Mercedes (Mecha) Santos, Martin Gray, Marie Attard and Mariana Juares. Javier Negrete was an excellent team leader. Nigel, Nadine, Michaela, Mecha and I shared an enjoyable and, sometimes, language-barrier-confusing experience in Primavera. Members of the Argentinean fieldwork team whilst in Jubany were really fun and supportive, especially with my “Spanish”. Many Taronga Zoo staff supported me with the collection of samples from the captive leopard seals. Thanks go to David Slip, Kaye Humphreys, Rebecca Spindler, Larry Vogelnest, Vanessa Di Giglio, Jane Hall, Karrie Rose and Cheryl Sangster who were iv always on hand to answer questions and provide necessary information. Thank you also for assistance from Jose Altunas, Danielle Fox and marine mammal keepers. Laboratory work is never easy when entering a new field and was heavily supported at UNSW by Bill Sherwin and his lab, including Lee Ann Rollins, Emily Miller, Jackie Chan and Jenny Sinclair. Brett Neilan and members of his lab were always helpful in providing access to equipment and answering questions. Special thanks go to Ivan Wong who has been very helpful. The Ramaciotti Centre was very supportive in the processing of samples and particular thanks go to Tonia Russell. Sequencing completed by the Research and Testing laboratories in Lubbock, Texas was made easier with the help of Scot Dowd. Some specific laboratory tests were carried out at the University of Montreal in Saint-Hyacinthe with John Fairbrother, who was warm and supportive. Whilst there, laboratory work was supported by Clarisse Desautels and members of the Reference Laboratory for Escherichia coli. Special thanks for administrative tasks in Canada go to Jacinthe Lachance. At UNSW many people supported me. They included, Jonathan Russell, Matt Hunt, Gemma Smart, Stephen Bonser, Rob Brooks, Alan Wilton, Mark Tanaka, Emma Johnston, Bill Sherwin, Alistair Poore, Richard Kingsford, Firoza Cooper, David Eldridge and Angela Moles. Support from David Cohen has also been exceptional. All research related to animal handling was approved by the University of New South Wales Animal Care and Ethics Committee (08/83B and 03/103B). A number of colleagues provided excellent reviews of chapters and also general advice and support throughout this whole process (emotional and otherwise). These people were Maria Luisa Gutierrez Zamora, Emily Miller, Lee Ann Rollins, Nadine Constantinou, Nigel Edwards, Melanie Sun, Nicole Lima, Jutta Zwielehner, Margo Adler, Alex James, Anna Kopps, James Smith, Jessica Roe, Chris Hellyer, and Patty Zenonos. Lastly but most definitely not least, the Nelson and Rayner families have been very encouraging and loving through this entire process. v Table of Contents Originality Statement ................................................................................................ i Copyright Statement ...............................................................................................