An investigation into the stranding of Australian sea snakes. Amber Kristen Gillett BVSc (Hon I) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 School of Veterinary Science Abstract Strandings of marine animals are a regular occurrence both on Australian and international beaches and provide an opportunity for gathering data on the anatomy, health, and ecology of many ocean- dwelling species that might be unattainable otherwise. Analysis of stranding data has revealed many important diseases, anthropogenic injuries (primarily as a result of commercial or recreational fishing and boating) and other potential threats in a number of marine species. As such, stranding information is important to include when evaluating the conservation status of marine species whilst developing processes to mitigate declines. Stranding analyses and scientific reports are readily available for a range of marine species, including cetaceans, pinnipeds, dugong and marine turtles, but have not been previously undertaken in sea snakes. True sea snakes (subfamily Hydrophiinae: Hydrophiini) spend their entire life in the marine environment, as opposed to their amphibious counterparts the sea kraits (genus Laticauda). Sea snake species inhabiting Australia’s waters are almost exclusively Hydrophiinae, yet are regularly found beach-washed on Australia’s coastline, particularly in Queensland. As such, stranded sea snakes in Australia are likely ill or injured individuals. Despite frequent sea snake strandings, demonstrated population declines in some species and recognition that sea snakes are likely to be bio-indicators of the health of the marine ecosystem, an epidemiological investigation of sea snake strandings has not been conducted. The main objective of this thesis was to help address gaps in the literature about sea snake health, disease and stranding events, with particular focus in south east Queensland, where stranding reports occur most frequently. A comprehensive analysis of information from multiple stranding databases was undertaken to assess: (i) species occurrence, (ii) the cause of strandings (where known), (iii) sex predisposition (where identified), (iv) biometrics, (v) stranding locations, and (vi) times of year when strandings occur. This information was from stranding reports, and was collated to provide a comprehensive assessment of reported sea snake stranding events in Queensland, identifying key ‘hot-spots’ for stranding observations, seasonal occurrence of strandings, and factors that are likely to influence stranding events. Abnormally high numbers of stranded animals were reported in 1996 and 2009 and this appeared to coincide with times of higher than average rainfall, as well as increased onshore winds and wind speed (Chapter 2). To address the paucity of information in the literature about normal clinical health parameters for sea snakes an antemortem assessment guide was developed, detailing procedures for the clinical i assessment and diagnostic investigation of sea snakes by veterinarians and researchers (Chapter 3). In addition, the first comprehensive set of haematological and biochemical reference ranges for three species of Australian sea snakes were developed from blood collected during an intensive field survey of wild caught sea snakes (Chapter 4). A comprehensive examination of sea snakes found stranded on beaches in south east Queensland and presented to the Australia Zoo Wildlife Hospital, was conducted from 2009 to 2015. Gathered information was used to develop the first sea snake postmortem guide, illustrating normal anatomy and outlining common diseases and pathology found in stranded animals. Traumatic injuries, including eye injuries, spinal fractures and jaw amputations, were prevalent in examined snakes and neoplasia (predominantly pancreatic ductal adenocarcinoma) was identified as one of the most common disease conditions (Chapter 5). In addition to the presence of trauma and neoplasia, granulomas were also found in the hypaxial and epaxial musculature of a small number (4) of necropsied snakes and contained dense aggregations of microsporidial organisms. Ultrastructural examination using transmission electromicroscopy suggested these organisms were most similar to the genus Pleistophora affecting the muscle of many species, including marine and freshwater fish, and terrestrial reptiles. However, Bayesian and maximum likelihood analysis of ssrRNA placed the sea snake microsporidia within the Heterosporis clade, known most notably to affect the muscle of freshwater eels. Although molecular analysis appeared to place the sea snake microsporidia within this clade, our specimens lacked a key defining morphological feature of this group, a sporophorocyst. Microsporidial infection was previously undescribed in sea snakes and the identified organism may represent a novel species. Due to the disparity between morphological and molecular identification methods complete classification was not possible with the current sample set (Chapter 6). The investigation on sea snakes undertaken for this thesis is the first of its kind anywhere in the world, and has made a significant contribution towards closing the gaps in our knowledge of this group. This body of work revealed that stranded sea snakes are often suffering from injuries and diseases that predispose them to stranding. Anthropogenic, environmental, infectious and natural causes are all implicated. Stranded individuals are likely to only represent a very small proportion of overall morbidity and mortality in these animals. In order to determine the true impact of these conditions on sea snake populations both in Australia, and globally, further research is required. ii 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 Publications during candidature Peer reviewed publications Chapter 3: Gillett AK, Flint M, Mills PC. An antemortem guide for the assessment of stranded Australian sea snakes (Hydrophiinae). J Zoo Wildl Med 2014;45:755-765. Chapter 4: Gillett AK, Flint M, Hulse L, Hanger J, Mills PC. Haematological and biochemical reference intervals for three species of hydrophiine sea snakes (Hydrophis curtus, Hydrophis elegans and Hydrophis peronii) in Australia. Vet J 2015;204(3): 275-281. Chapter 5: Gillett AK, Ploeg R, Flint M, Mills PC. Postmortem examination of Australian sea snakes (Hydrophiinae): anatomy and common pathologic conditions. J Vet Diagn Invest. 2017, May. doi: 10.1177/1040638717710056 Chapter 6: Gillett AK, Ploeg R, O’Donoghue PJ, Chapman PA, Webb RI, Flint M, Mills PC. Ultrastructural and molecular characterisation of an Heterosporis-Like microsporidian in Australian sea snakes (Hydrophiinae). PLoS ONE 2016;11(3) e0150724. doi:10.1371/journal.pone.0150724 Book Chapters Haberfield J, Martelli P, Johnson R, Barten S, Gillett A, Lock B, Jones R, Simpson S, Jackson TNW, Cochran C, Dunstan N, Sunagar K, Fry BG. Veterinary care of venomous reptiles. In: Fry BG, editor. Venomous Reptiles and Their Toxins. Oxford University Press, New York, 2015: 133-152. Conference abstracts Gillett AK, Mills PC, Flint M, Ploeg R, Limpus C. An assessment of disease in stranded sea snakes in South East Queensland. In: Wildlife Disease Association Australasian Section Conference Proceedings, The Coorong, South Australia, 25-30 September 2011: 32. iv Gillett AK, Flint M, Hulse L, Mills PC. The sea snake rodeo: madness, mayhem and blood. In: The Wildlife Disease Association Australasia Annual Conference Proceedings, North Stradbroke Island, Queensland, 23-28 September 2012: 32-33. Gillett AK, Flint M, Ploeg R, Mills PC. Investigation into mortality and morbidity of stranded Australian sea snakes (Hydrophiinae) on the south Queensland coast. In: International Association for Aquatic Animal Medicine Conference Proceedings, SeaWorld, Gold Coast, Queensland, 17-21 May 2014. General interest publications – magazine articles Crikey magazine (Australia Zoo) 2013 – ‘The struggle of stranded sea snakes’ by Dr Amber Gillett