ResearchOnline@JCU This file is part of the following reference: Bastos Gomes, Giana (2017) Ecology and detection of harmful freshwater fish ciliate parasites Chilodonella spp. in aquaculture. PhD thesis, James Cook University. Access to this file is available from: http://dx.doi.org/10.4225/28/5a399ef03d55d The author has certified to JCU that they have made a reasonable effort to gain permission and acknowledge the owner of any third party copyright material included in this document. If you believe that this is not the case, please contact [email protected] Ecology and detection of harmful freshwater fish ciliate parasites Chilodonella spp. in aquaculture Thesis submitted by Giana Bastos Gomes August, 2017 For the degree of Doctor of Philosophy in the School of Marine and Aquaculture Sciences James Cook University 1 Statement of animal ethics: Experiments within this research were performed in conformity to JCU Animal Ethics Approval Number A2065. 2 Statement on the contributions of others: At the time of the submission of this thesis Chapters 2, 3 and 4 were published in peer-reviewed international journals. Chapter 5 is in preparation for publication in peer-reviewed international journal. I am the lead author of all three articles and the unpublished Chapter derived from this PhD thesis. I acknowledge the specific contribution of co-authorship to these articles: Nature and extent of the intellectual input of each author and their Chapter Details of publication(s) on which chapter is based affiliations Bastos Gomes, G.1, Miller, T.L. 1,2, Jerry, D.R.1, Hutson K.S.1, Bastos Gomes, G.1 Project conception, writing and editing. 2016. Impacts and current status of parasitic Chilodonella spp. Miller, T.L.1,2, Jerry, D.R.1, Hutson K.S.1 Research supervision, editing and 2 (Phyllopharyngea: Chilodonellidae) in freshwater fish aquaculture. funding. Journal of Fish Diseases, 40(5): 703-715. DOI: 10.1111/jfd.12523 Bastos Gomes, G.1, Miller, T.L.1,2, Vaughan, D.B. 1, Jerry, D.R.1, Bastos Gomes, G.1 Execution of project, writing and editing. McCowan, C.3, Bradley, T.L.4, Hutson K.S.1, 2017. Evidence of Miller, T.L.1,2 Research supervision, data analysis, editing and funding. multiple species of Chilodonella (Protozoa, Ciliophora) infecting Vaughan, D.B.1 Execution of project, editing. 3 Australian farmed freshwater fishes. Jerry, D.R.1 Research supervision and editing. Veterinary Parasitology, 237, 8-16. DOI: McCowan, C. 3, Bradley, T.L. 4 Assistance providing samples and editing. http://doi.org/10.1016/j.vetpar.2017.03.004 Hutson K.S.1 Project conception, research supervision, editing and funding. Bastos Gomes, G.1, Hutson K.S.1, Domingos, J.A.1,5, Chung, C.6, Bastos Gomes, G.1 Project conception, execution of project, writing and editing. Hayward, S.6, Miller, T.L.1,2, Jerry, D.R.1, 2017. Use of Hutson K.S.1 Research supervision, editing and funding. environmental DNA (eDNA) and water quality data as a Domingos, J.A.1 Data analysis, editing. 4 management tool to predict protozoan parasites outbreaks in fish Chung, C.6, Hayward, S.6 Assistance providing samples and editing. farms. Aquaculture. Miller, T.L.1,2 Research supervision, editing and funding. DOI: https://doi.org/10.1016/j.aquaculture.2017.06.021 Jerry D.R.1 Research supervision, editing. 3 Bastos Gomes, G.1, Hutson K.S.1, Domingos, J.A.1,5, Bastos Gomes, G.1 Project conception, execution of project, writing and editing. 1 Huerlimann, R.1, Miller, T.L.1,2, Jerry, D.R.1. Interactions between Hutson K.S. Research supervision, editing and funding. 1,5 parasitic protozoan Chilodonella spp. and bacterial communities Domingos, J.A. Data analysis, editing. 5 within a tropical aquaculture system. (In preparation) Aquaculture Huerlimann, R.1 Data analysis, editing. Environment Interactions. Miller, T.L.1,2 Research supervision, editing and funding. Jerry D.R.1 Research supervision, editing. Author’s affiliations: 1 Marine and Aquaculture Sciences, College of Science and Engineering and Centre for Sustainable Tropical Fisheries and Aquaculture, James Cook University, Townsville, 4811, QLD, Australia 2 Fish Health Laboratory, Department of Fisheries Western Australia, South Perth 6151, WA, Australia 3 Department of Economic Development, Jobs, Transport and Resources, 5 Ring Road Bundoora, 3083, VIC Australia 4 Department of Economic Development, Jobs, Transport and Resources, Chief Veterinary Officer's Unit, 475 Mickleham Road, Attwood, 3049, VIC Australia 5 Department of Agriculture and Fisheries, Agri-Science Queensland, Bribie Island Research Centre, Woorim 4507, QLD, Australia. 6 Sealord King Reef, 188 Cowley Creek Road Cowley, 4871, QLD, Australia. 4 Acknowledgements I am sincerely thankful to my supervisor Dr Kate Hutson for the immense support and encouragement during my candidature. Each and every piece of advice given to me by Kate during my struggles were an incentive to continue my journey. Kate provided me scientific guidance and writing skills that were crucial for the completion of this thesis. I also learned with Kate the importance of professional ethics in the workplace and the skills necessary to be a great team leader. Kate has been an important role model for my professional career. But most importantly, thank you Kate for being a good friend. I am also grateful to my co-supervisor Prof. Dean Jerry for his teachings on career development and the importance of having an open mind to innovation. Dean gave me many work opportunities before my PhD which were essential to develop my self-confidence to embark into this journey. Thank you Dean for every single opportunity you offered me and for the friendship we developed during the last eight years. I am also very grateful to my co- supervisor Dr Terry Miller for his scientific guidance, field trip support and friendship during my PhD. I thank all my colleagues in the Marine Parasitology Laboratory, especially David B. Vaughan, Alejandro Trujillo Gonzalez and Alex Brazenor for their help and support in different aspects of my PhD. My gratitude goes also to my colleagues in the Molecular Ecology and Evolution Laboratory, especially Tansyn Noble and Julie Goldsbury for their support in different phases of my PhD. I’m very grateful to Scott Hayward and Catherine Chung from Sealord King Reef barramundi farm for the research partnership which enabled the experiments of this thesis to be performed using real data from a large-scale commercial fish farm. I am very thankful to James Cook University for offering me all the resources necessary to develop my 5 research. I am eternally grateful to James Cook University for supporting my PhD stipend scholarship and my candidature. I am immensely grateful to my dog Maya and my cat Baby, who stood by my side, without judgement, during each difficulty I encountered. Finally, I would like to express my deepest gratitude to my husband, Jose, who always believed in me more than myself. Jose, thank you for your immense patience and respect. Ze, thank you, with all my love, for giving me unconditional emotional, financial and technical support. 6 Abstract ___________________________________________________________________________ Freshwater fish farming contributes to more than two thirds of global aquaculture production. This rapidly growing agribusiness can be seriously compromised by diseases which can constrain industry development and profitability. In fact, disease problems account for forty percent of global aquaculture production loss. Parasitic ciliates are one of the largest causes of production loss in aquaculture and evidence suggests that they serve as the first portal of entry for many secondary infections in farmed fishes. Ciliate protozoan species of the genus Chilodonella are problematic and ubiquitous in global freshwater aquaculture. While Chilodonella spp. are important components of the 'free-living' fauna of freshwater ecosystems, some species have been implicated in outbreaks resulting in fish mortalities, particularly in high density aquaculture. A comprehensive review on the impacts and current status of parasitic Chilodonella spp. in freshwater fish aquaculture is presented in Chapter 2. Limited information exists on the ecology, infection dynamics and potential triggers leading to outbreaks of Chilodonella spp. in fish production. To assist with preventative management practices and sustainability of freshwater fish aquaculture, this research addressed key knowledge gaps related to the biology, ecology, geographic distribution, and identity of harmful Chilodonella spp. in Australia and examined new non-invasive diagnostic methods. Collectively, the research reported in this thesis provides fundamental baseline understanding of Chilodonella spp. ecology and their detection in fish aquaculture. While Chilodonella piscicola ((Zacharias, 1894; syn. C. cyprini (see Moroff, 1902)) and Chilodonella hexasticha (Kiernik, 1909) have been reported to globally infect wild and farmed fishes, the diversity of Chilodonella spp. parasitising Australian freshwater farmed fishes was 7 unknown prior to this study. Chilodonella cells were isolated from infected barramundi, Lates calcarifer (Bloch, 1790) and Murray cod, Maccullochella peelii (Mitchell, 1838) from fish farms in tropical north Queensland (QLD), temperate Victoria (VIC) and New South Wales (NSW) for genetic and morphological analysis (Chapter 3). Parasites were stained and measured for morphological description and comparative phylogenetic analyses were performed using three markers: ITS1-5.8S-ITS2, small