Trypanosomes of the Australian brush-tailed bettong (Bettongia penicillata)- the parasites, the host and their potential vectors Craig K Thompson BSc (Hons) A dissertation submitted to Murdoch University in fulfilment of the requirements for a Doctor of Philosophy Supervisors Professor R. C. Andrew Thompson, Murdoch University Dr. Adrian F. Wayne, Department of Parks and Wildlife Dr. Stephanie S. Godfrey, Murdoch University June 2014 P a g e | i Prelude Science may set limits to knowledge, but should not set limits to imagination Bertrand Russell Nature composes some of the loveliest poems for the microscope and the telescope Theodore Roszak P a g e | ii Declaration I declare that this thesis is my own account of my research and contains as its main content, work that has not been previously submitted for a degree at any other tertiary education institution Craig K Thompson BSc (Hons) June 2014 P a g e | iii Acknowledgements This thesis has been a result of extensive field and laboratory work that would have not been achievable without the guidance and support of the following people. I would like to thank Manda Page, Jo Kuiper and Alexander Kabat at Karakamia Wildlife Sanctuary, Marika Maxwell, Chris Vellios and Colin Ward at DPaW Science and Nature Conservation Division, Lizzie Aravidis and Kelli Ellemor at NAR for their expertise, assistance and understanding during field work, especially in the early stages of woylie handling when things did not always go to plan- “Have you got the woylie?”....”yep” “Are you sure?” ... “yep” “Where did it go?” .... “opps, my bad!!”. I am also grateful to the numerous volunteers who helped with the collection of samples during the monthly trapping at NAR. I am in debt to both Kerry and Kerry Littleton, who graciously allowed the transfer of eight woylies from their private colony to NAR for the intensive observational study; thank you. In the laboratory, a huge thanks goes to Adrian Botero-Gomez and Louise Pallant; the results that I present here are due, in part to their molecular super-powers. When I started this investigation, I did not understand what PCR stood for, the importance of pipettes, or why Taq is required in each and every reaction; thank-you, thank-you, thank-you. I would also like to thank Andrew Li and Peter Adams for their assistance with developing the FISH P a g e | iv protocol and Wan Hon for her continuous guidance throughout, in particular her comments- “you’re doing it all wrong!” Another huge thanks goes to Judy Dunlop; you made the four years of my candidature very enjoyable. The therapy sessions at the Tav (which became more and more regular as time went by) helped to blow away the cobwebs, to vent frustrations and to talk about something else other than the thesis. I am very much grateful; thank you. To my supervisors, Prof. Andy Thompson, Dr Adrian Wayne and Dr Stephanie Godfrey, again I am very much grateful for your time, assistance, guidance, support and quick turnaround with earlier drafts. Thank you. And finally to my wife, Eileen; thank you for your unconditional support and understanding, allowing me the time and freedom to complete this mammoth task. Now that it is over......Never Again! In completing this thesis, I dedicate it to my son, Ash Nicholas (17.05.2013). You have changed my life for the better, and I am so very grateful for the opportunity to be at home and watch you grow during the last six months. PRICELESS And now that this thesis has been submitted........Papa’s back!! P a g e | v Abstract The brush-tailed bettong (Bettongia penicillata) is known locally as the woylie and is one of two critically endangered potoroids in Australia. At a species level, they have declined by 90% since 1999, with their current distribution occupying a small fraction of their former Australian range. The predation of individuals made more vulnerable by disease is thought to be the primary cause of this decline; however, there may be other, as yet unidentified, stressors. This thesis details research that investigated whether trypanosomes are the causative agent that has reduced the fitness of the woylie and made them more vulnerable to predation. Woylies and haematophagous insects were sampled from five locations in southern Western Australia. Woylie health checks included the measurement of weight and skeletal morphometrics from adults, sub-adults and pouch young; reproductive observations on adult females; the collection of blood and ectoparasites from adults and sub-adults; and the single collection of tissues from a deceased adult woylie. During reproductive examinations, the maximum annual breeding potential was observed for the adult female, as to the pouch life, rate of growth, age of independence, sexual maturity and mating of the pouch young. Crown-rump and skeletal measurements of the developing pouch young were adequate predictors of age, with resulting growth curves being incorporated by the Department of Parks and Wildlife during conservation field-work. During the morphological investigation of trypanosomes from the woylie, a new species was identified and described: Trypanosoma vegrandis sp. nov. Morphological polymorphism was also identified for Trypanosoma copemani, with two different phenotypes described. P a g e | vi Spatially, the prevalence of parasitic infections varied among the five study sites, with contrasting trypanosome prevalence observed from the two declining indigenous populations within the Upper Warren region in southern Western Australia. Parasitaemia associated with trypanosome infection in the peripheral blood of the woylie exhibited a temporal decline as the infection progressed, being indicative of the infection transitioning between the acute and chronic phase. This thesis addresses host reproductive biology, trypanosome identification, spatial, temporal and transmission dynamics of infections, with relation to acute and chronic health of the woylie. It appears that the chronic intracellular association of trypanosomes with the internal organs of the woylie may be potentially pathogenic and adversely affect the fitness and coordination of the woylie, making them more susceptible to predation. As evident from this thesis, the chronic effect of trypanosome infections requires consideration during future conservation efforts to protect the woylie from extinction. P a g e | vii Publications Publications arising from this and associated research: Thompson, C.K., Botero, A., Wayne, A.F., Godfrey, S.S., Lymbery, A.J., Thompson, R.C.A. 2013. Morphological polymorphism of Trypanosoma copemani and description of the genetically diverse T. vegrandis sp. nov. from the critically endangered Australian potoroid, the brush-tailed bettong (Bettongia penicillata (Gray, 1837)). Parasites and Vectors: 6, 121. Botero, A., Thompson, C.K., Peacock, C.S., Clode, P.L., Nicholls, P.K., Wayne, A.F., Lymbery, A.J., Thompson, R.C.A. 2013. Trypanosomes genetic diversity, polyparasitism and the population decline of the critically endangered Australian marsupial, the brush- tailed bettong or woylie (Bettongia penicillata). International Journal for Parasitology: Parasites and Wildlife: 2, 77–89. Thompson, C.K., Godfrey, S.S., Thompson, R.C.A. 2013. Trypanosomes of Australian mammals: A review. International Journal for Parasitology: Parasites and Wildlife: 3, 57– 66. Thompson, C.K., Wayne, A.F., Godfrey, S.S., Thompson, R.C.A. 2013. Temporal and spatial dynamics of trypanosomes infecting the brush-tailed bettong (Bettongia penicillata); a cautionary note of disease-induced population decline. Parasites and Vectors: 7, 169. Thompson, C.K., Wayne, A.F., Godfrey, S.S., Thompson, R.C.A. 2014. Survival, age estimation and sexual maturity for pouch young of the brush-tailed bettong (Bettongia penicillata) in captivity. Australian Mammalogy: in press. P a g e | viii Dunlop, J., Thompson C. K., Godfrey, S. S., Thompson, R. C. A. 2014. Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods. Journal of Experimental Parasitology: 146, 20-24. Conference and Invited presentations Thompson, C.K., Smith, A., Botero, A., Wayne, A.F., Thompson, R.C.A. 2010. Trypanosomes of woylies from Western Australia; vector Identification and phylogenetic analysis. XIIth International Congress of Parasitology (ICOPA). Melbourne, Victoria: 16 – 20 August 2010. Thompson, C.K., Smith, A., Lymbery, A. J., Wayne, A.F., Thompson, R.C.A. 2010. Trypanosomes of woylies from Western Australia; vector Identification and phylogenetic analysis. Royal Society of Western Australian (RAWA).Murdoch, Western Australia: 18 September 2010. Thompson, C.K., Smith, A., Lymbery, A. J., Wayne, A.F., Thompson, R.C.A. 2010. Who’s biting the woylie and what are they transmitting. Australian Wildlife Conservancy (AWC) symposium. Karakamia, Western Australia: 01 October 2010. Thompson, C.K., Smith, A., Lymbery, A. J., Wayne, A.F., Thompson, R.C.A. 2010. Who’s biting the woylie and what are they transmitting. Ecological Society of Australia (ESA). Canberra, Australian Capital Territory: 06 – 10 December 2010. Thompson, C.K., Smith, A., Wayne, A.F., Thompson, R.C.A. 2011. Transmission dynamics of trypanosomes in declining, stable and enclosed populations of brush-tailed bettongs (Bettongia penicillata). Australian Society of Parasitology (ASP). Cairns, Queensland: 11 - 15 July 2011. P a g e | ix Thompson, C.K., Smith, A., Lymbery, A. J., Wayne, A.F., Thompson, R.C.A. 2010. Seeing is believing; the first visualisation of a trypanosome from woylies and its potential impact. Australian Wildlife Conservancy (AWC) symposium. Karakamia, Western Australia: 22 June 2012. Thompson, C.K., Botero, A., Smith, A., Wayne, A.F., Thompson, R.C.A. 2012. Morphological heterogeneity of mixed trypanosome infections; an explanation for the decline of the brush-tailed bettong (Bettongia penicillata) in Australia. Wildlife Disease Association (WDA). Lyon, France: 22 – 27 July 2012. Thompson, C.K., Botero, A., Smith, A., Wayne, A.F., Thompson, R.C.A. 2012. Trypanosome polyparasitism and the decline of the critically endangered Australian potoroid, the brush-tailed bettong (Bettongia penicillata (Gray, 1837)).