Myxosporean Parasites in Australian Frogs and Tadpoles Ashlie Hartigan B Sc. (Zoology), M App Sci (Wildlife Health and Pop. Mgmt) Thesis by published works This thesis is submitted in full satisfaction of the requirements for the degree of Doctor of Philosophy at the University of Sydney. Faculty of Veterinary Science University of Sydney March 2012 Statement of authentication This thesis is submitted to the University of Sydney in fulfilment of the requirement for the degree of Doctor of Philosophy. The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institution. Signed: Date: ii Abstract The investigation of new threats to amphibian conservation is a priority of researchers and wildlife managers. Emerging infectious diseases are one of the most threatening processes to wildlife around the world including amphibians. Australian frogs have suffered large scale declines and extinctions from pathogens such as chytrid fungus (Batrochochytrium dendrobatidis). The once overly abundant Green and golden bell frog (Litoria aurea) has declined over 90% of its range with disease listed as a key threat. A routine pathogen screen of tadpoles from a captive breeding population of Green and golden bell frogs found an unknown parasitic infection in the brains, bile ducts and gallbladders of tadpoles (later confirmed as Myxosporea). It was this preliminary identification that was the impetus for my thesis. Myxosporean parasites found in Australian frog gallbladders were thought to be Cystodiscus immersus from Central and South America. The parasite was assumed to have been introduced to Australia with the Cane toad (Rhinella marina, syn. Bufo marinus) in 1935. Cystodiscus immersus was supposed to have translocated with the Cane toad from native Brazil and was infecting a broad range of Australian frog species with no apparent host impact. The aim of this thesis was to challenge all aspects of this assumption, to establish the true myxosporean species present in Australian frogs, if it was an exotic infection, if it was an emerging pathogen and what threat did it pose to hosts. The parasite found in Australian frogs and tadpoles was examined using histological and ultrastructural morphology as well as molecular identification. This revealed there was in fact two myxosporean species in multiple species of frogs and tadpoles. Sequencing of partial small iii subunit and large subunit as well as the internal transcribed spacers (ITS1 and ITS2) of ribosomal DNA regions confirmed these were distinct species (differing 9%, 7%, 34% and 37% at each region respectively). They were in fact separate species infecting multiple Australian host species, samples of C. immersus from Brazil were compared to establish if either of these was the exotic C. immersus. We found instead that these were completely unrelated despite sharing similar spore morphology. In addition the Brazil material revealed the cryptic diversity of myxosporea in frog gallbladders around the world and the ambiguous identity of Cystodiscus immersus. To determine if the parasites were emerging, the presence of these parasites in 130 years was established using voucher specimens. Four native species and the exotic Cane toad were examined for gallbladder myxospores from 1879 to 2009, the first positive was in 1966 in Green tree frog outside of Sydney. The prevalence of myxosporea increased in both native and exotic species over the last 40 years including the threatened Green and golden bell frog. The emergence of these parasites is a cause for concern, especially when found in endangered species. Close scrutiny of these parasites prompted the formal description of Cystodiscus axonis and Cystodiscus australis, resurrecting the original genus name proposed by Lutz in 1889. The description revealed diagnostic features previously unknown for frog gallbladder myxosporea species. Scanning electron microscopy revealed filiform polar appendages on the myxospores of C. axonis, absent in C. australis. In addition, histological examination showed unique developmental stages in the nervous tissue of tadpoles and frog hosts infected with C. axonis, these stages are only found in hosts infected with this parasite. The use of development stages as a species diagnostic character has not been previously reported for any amphibian myxosporea as iv yet. The genetic and morphological differences between C. axonis and C. australis prompted the development of a species specific multiplex PCR using ITS1, 5.8S and ITS2 ribosomal DNA. Multifaceted diagnostic tools (morphology and species specific PCR) demonstrated significant disease associated with these pathogens in native and exotic frog species. Seven host species were morphologically examined for Cystodiscus infection showing endangered species to have significant disease associated with infection. Cystodiscus axonis was linked to neurological signs, haemorrhage, necrosis and gliosis in Southern bell frogs (Litoria raniformis), Green and golden bell frogs (Litoria aurea), Booroolong frogs (Litoria booroolongensis), and the recently re- discovered Yellow spotted bell frogs (Litoria castanea). Cystodiscus spp. infection in the bile ducts of tadpoles was associated with biliary hyperplasia, loss of hepatocytes and inflammation. These lesions were statistically associated with the presence of infection in the Green and golden bell frog. This research disproved an assumption about myxosporean parasites that had held for 25 years. The disease found with Cystodiscus parasite infection in Australian frogs and tadpoles, as well as the previously unknown biodiversity in this cryptic species complex, highlight a number of areas in need of further research. This work has provided insight into the importance of multifaceted approaches to species identification and amphibian pathogen surveillance which uncovered a threat to endangered Australian amphibians, now listed as one of the key disease threats to frogs by the Department of Environment and Health (Australia) in 2011. v Acknowledgements This thesis has taken roughly three years to complete, during that time I had the opportunity to meet and collaborate with a number of fantastic people. My greatest gratitude is to my two supervisors Dr. Jan Šlapeta and Assoc. Prof. David Phalen. The two of you provided me with amazing support, wisdom and patience during the steepest learning curve of my life. Jan, you have been a constant source of inspiration and motivation as my mentor and I count myself lucky for the opportunity to have learnt from you. David, thank you for your endless patience, awful jokes and letting me soak up as much of your wisdom as I could during this time. I’ve appreciated the influence you both have had on my development as a researcher and the skills I’ve learned from you along the way. I’d like to thank Dr. Karrie Rose at the Australian Registry of Wildlife Health, she has been a champion for frog myxosporea since the beginning and her enthusiasm, collaboration and friendship have supported me immensely. My PhD is the culmination of substantial international collaboration with the Institute of Parasitology, Biology Centre, Academy of Sciences of the Czech Republic. I was extremely lucky to work with Dr. Ivan Fiala during this time and I’d like to thank him for his expertise, helpful suggestions and support during my visits to České Budějovice. I am grateful to have had the opportunity to discuss myxozoa and gain valuable training from Dr. Iva Dyková whilst at the Institute. None of this research would be possible without the support and unending patience of Mr. Lance Jurd. He has been a fountain of information and source of help to me during this time and has been absolutely essential to this project. A number of people within the Faculty of Veterinary Science were vital to my sanity in my doctorate. I’d like to thank Mrs Denise McDonell and Dr. Graeme Browne for giving me invaluable advice and learning opportunities. Thanks to Dr. Jessica King and Ms Christie Foster for being great listeners and giving good advice and Ms Elaine Chew for her seemingly endless patience and knowledge on the histopathology. vi I’m especially in debt to my friends and family who had to learn to love parasites and frogs by proxy. I am extremely grateful to Dr. Stephen Hoggard (Macquarie University) for his advice, encouragement and amazing friendship over the years. Thank you to Kristie Flannery who has been a fire under me, a comforting ear and an inspiration to me since I was five. I’d like to thank my sister Amber, Steph Snoyman, Rochanne Barratt, and Sarah Atkinson for understanding when I missed life whilst in the lab, for letting me vent and especially for giving me a good laugh when I needed it most. Thank you to my friend Ms Lauren Walsh (Rouge), who propped me up when I was down, fed me when I was hungry and listened when I needed it. A special mention to my Mum Pauline, I wouldn’t have done any of this without her support, motivation and inexhaustible faith in me. Lastly, thanks to the frogs and tadpoles, without which I would be a parasitologist looking for a host. vii Publications Hartigan. A, Peacock. L, Rosenwax. A, Phalen. D.N and Šlapeta. J, (2012), Emerging myxosporean parasites of Australian frogs take a ride with fresh fruit transport, Parasites & Vectors, 5: 208, 10.1186/1756-3305-5-208 Hartigan. A, Dhand. N, Rose. K, Šlapeta. J and Phalen. D.N, (2012) Comparative pathology and ecological implications of two myxosporean parasites in native Australian frogs and the invasive Cane toad, PLoS One, 7(10): e43780. 10.1371/journal.pone.0043780. Hartigan. A, Sangster. C, Rose. K, Phalen. D.N and Šlapeta. J, (2012), Myxozoan parasite in brain of critically endangered frog [letter]. Emerging Infectious Diseases, 18 (4), 10.3201/eid1804.111606 Hartigan. A, Fiala. I, Dyková.