Middlesex University Research Repository An open access repository of Middlesex University research http://eprints.mdx.ac.uk Landeryou, Toby (2019) Molecular assessment of parasite infection within socioeconomically important UK salmonid populations. PhD thesis, Middlesex University. [Thesis] Final accepted version (with author’s formatting) This version is available at: https://eprints.mdx.ac.uk/26874/ Copyright: Middlesex University Research Repository makes the University’s research available electronically. Copyright and moral rights to this work are retained by the author and/or other copyright owners unless otherwise stated. The work is supplied on the understanding that any use for commercial gain is strictly forbidden. A copy may be downloaded for personal, non-commercial, research or study without prior permission and without charge. 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See also repository copyright: re-use policy: http://eprints.mdx.ac.uk/policies.html#copy MIDDLESEX UNIVERSITY Molecular Assessment of Parasite Infection within Socioeconomically Important UK Salmonid Populations A thesis submitted to Middlesex University in partial fulfilment of the requirements for the degree of Doctor of Philosophy Toby Landeryou M00513028 Faculty of Science and Technology, Department of Natural Science Hendon Campus, London, England 1 Acknowledgements I would like to first and foremost thank my supervisory team of Dr Steve Kett, Dr Dirk Wildeboer and Dr Scott Lawton with helping me out throughout the planning, practical and most importantly writing of my thesis. Also although not part of the official supervisory team, Dr Anne Ropiquet has been a constant guiding hand, taking time out of her own work to aid me throughout the project and for that I am extremely grateful. They have all gone above and beyond in helping me achieve the final completed copy of this work and I am extremely thankful for their due diligence and great attention to detail when helping me through to completion. Alongside the academic supervisory there has been a substantial input to create the extensive archive at Middlesex University, which was vital to this project. For this I would the Wester Ross Fishery Trust, a truly great bunch of people to work with who are dedicated to maintaining a sustainable Gairloch system that can be enjoyed by anglers and naturalists for generations to come. The contributors have been many but to mentions a few I would like to thank the generous list of highly skilled and adept anglers who made the archive possible; Dr Steve Kett, Dr Andy Vick, Vu Dang, Dr James Close, Dr Robin Ade, Prof Barry Blake, Ben Rushbrooke, Prof Dave Barclay, David, Dougal and Florence Foremen, Frank Buckley, Hugh Whittle, Prof Pete Maguire, Pat Wilson, , Dr Carl Forbes, Ray Dingwall, Ronnie Buchan Roger McLachlan, Robin Payne, Radenko Velinov, Calum Button and Adam Choonara. Most importantly I would like to thank my caring and patient wife for putting up with constant late nights and misguided thesis-based tantrums. Also my mother who has always been there to keep me on the straight and narrow and to fill my stomach full of home baked goods. 2 A man who this thesis is dedicated to is my beloved father who passed away on the 16th of August 2018 after a brief but courageous fight against cancer. I will always be indebted to a man that always put the needs of his family before the needs of his own. Like his son he was an average but enthusiastic fly fisherman, whose snoozes in the sunshine will vastly outweigh the time spent with fish on line. Although you won’t see me walk on stage in a “poncy hat” you will be in my mind every step and every day of my journey in the world science and life. Love you Dad and thank you, your son did good. R.I.P Bryan Landeryou 1957-2018 3 Abstract Salmo trutta (Brown trout) is a native salmonid species to the United Kingdom. The economy generated via fish farming and eco-tourism has aided isolated communities in job creation and tourism income. It is particular true in highland communities in Scotland, where parasite infection has threatened the health of Scottish salmonids. The work presented in this thesis assessed parasite infection of wild brown trout populations in the northwest highlands of Scotland. Initial analysis of fish health screened subpopulations within the Gairloch region for infective parasite species. Eye fluke Diplostomum spp. was highly prevalent throughout the system with all lochs populations harboring infection. Through molecular analysis the species was confirmed as D. baeri which, when compared to other European isolates indicated highly diverse species complex infecting freshwater fish across the continent, most likely through definitive bird host. A cestode species that infected 4 trout subpopulations across the system was the medically relevant Diphyllobothrium dendriticum. Using molecular species identification techniques, it was the first finding of the parasite within UK freshwater fish populations. In response to differential parasite infection adaptive immunogenic traits were also observed within trout host subpopulations. The MHC II related gene Satr-DAB variability was higher in populations with differential parasite species infection suggesting the diversity of infection maintains MHC diversity within the population. The evasion of immune recognition to achieve high levels of infection is key to sustained parasite infection. The highly infective parasite in the Gairloch system, D. baeri, utilizes intracellular antigens Tetraspanins and Venom allergen-like antigenic proteins. These antigens were isolated using genomic techniques to highlight potential vaccine targets in aquaculture. The body of work presented here has furthered the knowledge of the highly infective D. baeri and provided molecular methodologies to identify medically relevant D. dendriticum. Genomic analysis of trout population immunogenics and parasite antigenic factors provides key knowledge to further conservation stocking methods and sustainable aquaculture practice. 4 Contents 1 Introduction 12 1.1 Brown trout ecology ........................................................................................................................... 12 1.1.2 Brown trout health .......................................................................................................................... 14 1.1.3 Brown trout-parasite relationship............................................................................................ 14 1.2 Diplostomum 17 1.2.1 Life cycle and pathology ................................................................................................................ 17 1.2.2 Taxonomy of Diplostomum ........................................................................................................... 19 1.3 Evolutionary ecology of the MHC complex 20 1.3.1 MHC structure and function ........................................................................................................ 20 1.3.3 Salmonid MHC ecology .................................................................................................................. 23 1.3.2 MHC ecology and parasite driven selection .......................................................................... 23 1.3.3 Functional variation of amino acid sequence within antigen binding site .............. 25 1.4 Host-parasite infection dynamics 26 1.4.1 Host-parasite molecular interactions ...................................................................................... 26 1.4.2 Parasite tegument and surface antigens ................................................................................ 26 1.5 Rationale and aims 30 1.5.1 Study area............................................................................................................................................ 30 2 Survey of parasites infecting wild brown trout in a highland hill loch system 33 2.1 Introduction 33 2.1.1 Brown trout health in Scotland .................................................................................................. 33 2.1.2 Parasite identification .................................................................................................................... 35 2.2 Study Area ............................................................................................................................................... 36 2.3 Materials and Methods 37 2.3 Sample protocol.................................................................................................................................... 37 2.3.1 Molecular