The diversity and pathogenicity of amoebae on the gills of Atlantic salmon (Salmo salar) with amoebic gill disease (AGD) Chloe Jessica English BMarSt (Hons I) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2019 i Abstract Amoebic gill disease (AGD) is an ectoparasitic condition affecting many teleost fish globally, and it is one of the main health issues impacting farmed Atlantic salmon, Salmo salar in Tasmania’s expanding aquaculture industry. To date, Neoparamoeba perurans is considered the only aetiological agent of AGD, based on laboratory trials that confirmed its pathogenicity, and its frequent presence on the gills of farmed Atlantic salmon with branchitis. However, the development of gill disease in salmonid aquaculture is complex and multifactorial and is not always closely associated with the presence and abundance of N. perurans. Moreover, multiple other amoeba species colonise the gills and their role in AGD is unknown. In this study we profiled the Amoebozoa community on the gills of AGD-affected and healthy farmed Atlantic salmon and performed in vivo challenge trials to investigate the possible role these accompanying amoebae play alongside N. perurans in AGD onset and severity. Significantly, it was shown that despite N. perurans being the primary aetiological agent, it is possible AGD has a multi-amoeba aetiology. Specifically, the diversity of amoebae colonising the gills of AGD-affected farmed Atlantic salmon was documented by culturing the gill community in vitro, then identifying amoebae using a combination of morphological and sequence-based taxonomic methods. In addition to N. perurans, 11 other Amoebozoa were isolated from the gills, and were classified within the genera Neoparamoeba, Paramoeba, Vexillifera, Pseudoparamoeba, Vannella and Nolandella. This work highlighted that there is a far greater diversity of amoebae colonising AGD-affected gills than previously established. Drawing on this culture-based study, five new TaqMan quantitative PCR (qPCR) assays were developed and applied to more accurately determine the prevalence and abundance of multiple amoeba species colonising the gills of Atlantic salmon held at two Tasmanian farm sites over a one- year period. The presence of N. perurans was also assessed in parallel using a previously established qPCR method. N. perurans was the dominant species in the Amoebozoa community on gills, and its abundance positively correlated with the progression of gross gill pathology. Only sporadic detections of Pseudoparamoeba sp. and Vannellida species were observed across the sampling period at either farm site. Nolandella spp., however, were highly prevalent at one site at ii one sample time when N. perurans were not detected on gills presenting low levels of gross gill pathology. To investigate the pathogenic potential of Nolandella sp. and a more closely related amoeba to N. perurans, Pseudoparamoeba sp., in vivo challenges of naïve Atlantic salmon were performed. Additionally, to elucidate how Nolandella sp. and Pseudoparamoeba sp. influence the onset or severity of N. perurans-induced AGD, the gill condition of fish challenged with N. perurans alone was compared to fish challenged with a mix of all three amoeba strains. Immersion challenge of all three species resulted in minor gill lesions, with the most severe epithelial hyperplasia documented in the N. perurans treatments, while lesions with infiltrating lymphocytes were the predominate pathology observed in fish challenged by Nolandella sp. and Pseudoparamoeba sp. The presence of individual Nolandella or Pseudoparamoeba cells were not linked with lesion sites, so the precise cause of pathology remains inconclusive. Moreover, the presence of these non-N. perurans species did not significantly increase the severity of N. perurans-induced branchitis. Overall this investigation supports N. perurans being the primary agent of AGD, yet also shows that other species of amoebae which colonise the gills of Tasmanian Atlantic salmon can dominate the gill community and may be capable of causing some gill pathology under specific conditions. Thus, the involvement of non-N. perurans amoebae in AGD should not yet be discounted. The increasing list of putative gill pathogens, and the complexity of disease expression, provides supportive rationale to the consideration of gill disease in the context of dysbiosis of microbial community structure, rather than a pathological response to a single agent. iii 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, financial support 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 higher degree by research 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 and have sought permission from co- authors for any jointly authored works included in the thesis. Chloe English 08/10/2019 iv Publications included in this thesis English, C., Tyml, T., Botwright, N., Barnes, A., Wynne, J., Lima, P., Cook, M., 2019. A diversity of amoebae colonise the gills of farmed Atlantic salmon (Salmo salar) with amoebic gill disease (AGD). Eur. J. Protistol. 67, 27–45. https://doi.org/10.1016/j.ejop.2018.10.003 English, C., Swords, F., Downes, J., Ruane, N., Botwright, N., Taylor, R., Barnes, A., Wynne, J., Lima, P., Cook, M., 2019. Prevalence of six amoeba species colonising the gills of farmed Atlantic salmon with amoebic gill disease (AGD) using qPCR. Aquac. Environ. Interact. 11, 405–415. https://doi.org/10.3354/aei00325 v Submitted manuscripts included in this thesis English, C., Lima, P., 2018. A review of amoebic disease in aquatic animals: insights into defining aetiological agents. J. Fish Dis. vi Other publications during candidature Conference abstracts English, C.J., Botwright, N.A., Barnes, A.C., Wynne, J.W., Lima, P.C., Cook, M.T (2018). Possible multi-amoeba aetiology of amoebic gill disease (AGD) of farmed Atlantic salmon. (Oral presentation) 8th International Symposium on Aquatic Animal Health, PEI, Canada, 2nd-14th Sep 2018 English, C.J., Botwright, N.A., Barnes, A.C., Wynne, J.W., Lima, P.C., Cook, M.T (2017). The role of non-N. perurans amoeba in amoebic gill disease (AGD) of Atlantic salmon. (Oral presentation) 2017 Australian Society for Parasitology Annual Conference, Leura, Australia, 26th- 29th Jul 2017 English, C.J., Botwright, N.A., Barnes, A.C., Wynne, J.W., Lima, P.C., Cook, M.T (2017). Possible multi-amoeba aetiology of amoebic gill disease (AGD) of farmed Atlantic salmon. (Oral presentation) 4th Australian Scientific Conference on Aquatic Animal Health and Biosecurity, Cairns, Australia, 10th -14th Jul 2017. Awarded Best Student Presentation. English, C.J., Botwright, N.A., Barnes, A.C., Wynne, J.W., Lima, P.C., Cook, M.T (2016). Investigating the role of non-N. perurans amoeba in AGD of Atlantic salmon. (Oral presentation) 4th Gill Health Initiative Meeting, University of Stirling, Scotland, 9th-10th Jun 2016. vii Contributions by others to the thesis Andrew Barnes, Paula Lima, James Wynne and Mat Cook contributed to the conception of this project, advised on methods and analyses, and provided comments on the thesis and the associated publications. Kathryn Green and Erica Lovas provided transmission electron microscopy training and helped extensively during the imaging process for Chapter 3. Tomas Tyml provided training on phylogenetics analysis in Chapter 3. Fiona Sword and Jamie Downes advised on how to design and validate qPCR assays for Chapter 4. Ben Maynard and Richard Taylor sampled 50 % of salmon gill swabs for the field survey in Chapter 4. Natasha Botwright assisted with molecular biology training, resource management and sampling during the in vivo trial in Chapter 5. Russel McCulloch provided microscopy and histology training for Chapter 3 and 5. Joel Slinger and Chris Stratford managed the Atlantic salmon husbandry at Bribie Island Research Centre. Moira Menzies provided molecular biology laboratory training. Mark Adams interpreted the histopathology, scored different gill lesion morphotypes and imaged relevant pathology for Chapter 5. viii Statement of part of the thesis submitted to qualify for award of another degree No works submitted towards another degree have been included in this thesis. ix Research involving animal subjects All Atlantic salmon used for the research in this thesis were approved for sampling by CSIRO Queensland Animal Ethics Committee (AEC). A copy of the ethics approval letter for AEC application A13/2015, A9/2016 and A16/2017 is in Appendix F. x Acknowledgement I would like to acknowledge my supervisors, Mat Cook, Paula Lima, Andrew Barnes and James Wynne. I am grateful for the opportunity to undertake this research project and for their repeated support of my participation in extra activities such as attending many training courses and laboratory visits.