Eco-immunology: Thermal Variation and Parasitology of the Three-Spined Stickleback Alexander Thomas Stewart July 2016 A thesis submitted to Cardiff University for the degree of Doctor of Philosophy (PhD) in the School of Biosciences Declaration Declaration This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed………………………………………… (candidate) Date…………………… STATEMENT 1 This thesis is being submitted in partial fulfillment of the requirements for the degree of PhD Signed………………………………………… (candidate) Date…………………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated. Other sources are acknowledged by explicit references. The views expressed are my own. Signed………………………………………… (candidate) Date…………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed………………………………………… (candidate) Date…………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available for photocopying and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed………………………………………… (candidate) Date…………………… i Collaborators Collaborators The Leverhulme Trust funded this PhD and some of the key experiments presented in this thesis (Chapter 3 and 4) were outlined in this original grant held by my supervisors. Chapters 5 and 6 arose as extensions to the original grant with the experiments presented in Chapters 7 and 8 designed outside the original grant. Chapter 2 arose from the information gap identified during preparation of the other chapters, producing a single methods article that will hopefully be useful for furthering stickleback and associated parasite studies. Initial Argulus foliaceus specimens and the Saprolegnia parasitica cultures were provided by Chris Williams and Amy Reading, and Pieter van West, respectively. Chapter 2: AS wrote this chapter, with additional input and suggestions from Jo Cable. Experts commented on individuals sections: Chris Williams, the argulid section; Rachel Paterson the Diplostomum section; Pieter van West the Saprolegnia section; and Iain Barber the Schistocephalus section. Other authors may provide additional information before submission of this manuscript for publication. Chapter 3: Fish capture and mesocom care was performed at Aberystwyth University by Joseph Jackson, Martha Brown and Pascal Hablützel. Infections and other experimental procedures, data analysis and writing were conducted by AS with additional suggestions from Jo Cable and Joseph Jackson on statistics and writing. Chapter 4: AS conducted fish capture, care, infections and other experimental procedures. RNA extractions and qPCR were conducted at Aberystwyth University by Sophie Parker-Normal and Pascal Hablützel. All data analysis and writing was conducted by AS with suggestions from Joseph Jackson and Jo Cable. Chapter 5: Fish capture, care, infection and dissection were conducted by AS, with additional help from Anya Tober. RNA extractions and qPCR were conducted at Aberystwyth University by Hayley Watson and Pascal Hablützel. AS conducted all data analysis and with suggestions from Joseph Jackson and Jo Cable. Chapter 6: AS conducted fish collection and other experimental procedures with the exception of RNA extractions and qPCR by Hayley Watson, Sophie Parker-Norman and Pascal Hablützel. AS preformed all data analysis and writing with suggestions from Joseph Jackson and Jo Cable. Chapter 7: AS performed majority of experimental procedures with additional help from Sophie Tuppen and Karan Gupta. AS performed all data analysis with writing suggestions from Jo Cable. Chapter 8: Experimental procedures were conducted by AS and Robby Mitchel with additional help from Valentine Muhawenimana. The flumes were kindly loaned by Catherine Wilson and Cardiff University’s School of Engineering. Video analyses were performed by AS and Rhi Hunt. AS performed all data analysis with writing suggestions from Jo Cable and Catherine Wilson. ii Acknowledgements Acknowledgements First I would like to thank my two supervisors Jo Cable and Joseph Jackson, without their support and encouragement this thesis would not have been possible. In particular, Jo was an immense help with writing this thesis and stressed and worried on my behalf, even if it was not always necessary and Joe a constant pillar of statistical support. I would also like to thank them both for giving me free reign with a number of experiments, allowing me to develop and produce my own ideas. My thanks also go to my collaborators in Cardiff, Aberystwyth and elsewhere. Pascal Hablützel, Sophie Parker-Norman and Hayley Watson, who conducted the RNA extractions and qPCR runs at Aberystwyth on my behalf. Catherine Wilson, who let me run riot in the Cardiff’s hydrology department for some months and put up with some of my more unusual ideas. Chris Williams and Amy Reading who supplied me with the first individuals of my now much utilised Argulus culture. Pieter van West, who supplied me with the Saprolegnia cultures. Ida Jackson, who made the long journey from Aber to Cardiff with crates of sticklebacks at her husband’s behest. To my small army of undergraduates and postgraduates, Robby Mitchell, Anya Tober, Rhi Hunt, Karan Gupta and Sophie Tuppen, without whom such a depth and breadth of data might not have been achieved, thank you. My apologies also go to these five people who had to put up with some of the more tedious chores involved in this project and attempt to keep up with my often rapid pace. Thanks also to my research support group: Jo James, Rhidian Thomas, Katie Dunkley, Becky Pawluk, Willow Smallbone, Amy Ellison, Mike Reynolds, Alberto Maceda, Linda Erlandsson, Don A’Bear, Ifan Jâms, Fran Hockley, Emma Gillingham, Jess Stephenson, Valentine Muhawenimana, Ben Harvey, Alley Evan, Lizzy Donkin and Nathan King who supplied much needed friendship, coffee and, as frequent as was necessary, beer or gin. To my family without whose support and encouragement I would not be where I am today. Particularly, my long suffering parents who continue to provide a solid foundation of love and financial support. And lastly, to my loving partner Hannah, who has kept me constantly grounded in the real world and reasonably sane. Let’s go exploring. “It is well known that a vital ingredient of success is not knowing that what you're attempting can't be done.” Terry Pratchett iii Contents Table of Contents Declaration ........................................................................................................................ i Collaborators ................................................................................................................... ii Acknowledgements ......................................................................................................... iii Table of Contents ........................................................................................................... iv Definitions of Terms ..................................................................................................... viii Biological terms ......................................................................................................... viii Immunological Terms .................................................................................................. ix List of Figure and Tables ................................................................................................ x Figures ........................................................................................................................... x Tables ........................................................................................................................... xi Thesis Abstract .............................................................................................................. xii Thesis Layout ................................................................................................................ xiii Chapter 1 - General Introduction .................................................................................. 1 Climate change .............................................................................................................. 1 Study Species ................................................................................................................ 4 Teleost Immunology ...................................................................................................... 5 Thesis Aims ................................................................................................................... 8 References ..................................................................................................................... 9 Chapter 2 - Hook, line and infection: culture methods for common parasites of the three-spined stickleback and associated immunological responses .......................... 18 Abstract ....................................................................................................................... 18 Introduction ................................................................................................................. 18 Stickleback Husbandry ................................................................................................ 21 Ethics ....................................................................................................................... 21 Collection ................................................................................................................. 21 Maintenance ............................................................................................................. 22 Breeding ................................................................................................................... 23 Hatchery ................................................................................................................... 25 Common Stickleback Parasite Cultures ...................................................................... 26 Gyrodactylus spp. ........................................................................................................ 26 Introduction .............................................................................................................. 26 iv Contents Source, culture and infection ................................................................................... 27 Immunology ............................................................................................................. 28 Diplostomum spp. ........................................................................................................ 28 Introduction .............................................................................................................. 28 Source, culture and infection ................................................................................... 29 Immunology ............................................................................................................. 30 Schistocephalus solidus ............................................................................................... 31 Introduction .............................................................................................................. 31 Source, culture and infection ................................................................................... 31 Immunology ............................................................................................................. 34 Camallanus lacustris ................................................................................................... 34 Introduction .............................................................................................................. 34 Source, culture and infection ................................................................................... 35 Immunology ............................................................................................................. 36 Saprolegnia parasitica ................................................................................................ 37 Introduction .............................................................................................................. 37 Source, culture and infection ................................................................................... 38 Immunology ............................................................................................................. 39 Glugea anomala .......................................................................................................... 39 Introduction .............................................................................................................. 39 Source, culture and infection ................................................................................... 39 Immunology ............................................................................................................. 40 Argulus foliaceus ......................................................................................................... 40 Introduction .............................................................................................................. 40 Source, culture and infection ................................................................................... 42 Immunology ............................................................................................................. 44 Treating common infections ........................................................................................ 44 Summary ..................................................................................................................... 47 References ................................................................................................................... 47 Chapter 3 - The impact of global warming and resource availability on three- spined sticklebacks (Gasterosteus aculeatus) and Saprolegnia parasitica and Gyrodactylus gasterostei infections ............................................................................... 58 Abstract ....................................................................................................................... 58 Introduction ................................................................................................................. 58 Materials and Methods ................................................................................................ 61 Results ......................................................................................................................... 66 Discussion ................................................................................................................... 72 References ................................................................................................................... 76 v Contents Chapter 4 – Always winter, never Christmas: Winter length and its influence on sticklebacks, parasites and immunity. ......................................................................... 81 Abstract ....................................................................................................................... 81 Introduction ................................................................................................................. 81 Materials and methods ................................................................................................. 84 Discussion ................................................................................................................... 98 References ................................................................................................................. 100 Chapter 5 - The immunological responses of three-spined sticklebacks (Gasterosteus aculeatus) exposed to constant and shock temperature treatments and Saprolegnia parasitica infection .......................................................................... 105 Abstract ..................................................................................................................... 105 Introduction ............................................................................................................... 105 Materials and Methods .............................................................................................. 107 Results ....................................................................................................................... 110 Discussion ................................................................................................................. 114 References ................................................................................................................. 116 Chapter 6 - I hear the ticking of the clock: the cause of circannual immune rhythms in an ectothermic vertebrate ....................................................................... 120 Abstract ..................................................................................................................... 120 Introduction ............................................................................................................... 120 Materials and Methods .............................................................................................. 121 Results ....................................................................................................................... 124 Discussion ................................................................................................................. 126 References ................................................................................................................. 127 Chapter 7 - Love thy neighbour: co-infection of Argulus foliaceus and Gyrodactylus gasterostei on the three-spined stickleback (Gasterosteus aculeatus) ...................... 130 Abstract ..................................................................................................................... 130 Introduction ............................................................................................................... 130 Materials and Methods .............................................................................................. 132 Results ....................................................................................................................... 135 Discussion ................................................................................................................. 137 References ................................................................................................................. 139 vi Contents Chapter 8 - Argulus foliaceus infection and the consequences for three-spined stickleback (Gasterosteus aculeatus) swimming performance................................. 143 Abstract ..................................................................................................................... 143 Introduction ............................................................................................................... 143 Materials and Methods .............................................................................................. 145 Results ....................................................................................................................... 150 Discussion ................................................................................................................. 152 References ................................................................................................................. 154 Chapter 9 - General Discussion ................................................................................. 158 Temperature, Saprolegnia and Immunity ................................................................. 158 The Foothold Hypothesis .......................................................................................... 159 Body Condition: Fuelling the Fires of Infection ....................................................... 161 The Pathology and Co-infection of Argulids ............................................................ 161 Summary ................................................................................................................... 163 References ................................................................................................................. 163 Appendix 1 ................................................................................................................... 166 Appendix 2 ................................................................................................................... 167 vii Figures and Tables Definitions of Terms Biological terms Term Definition Argulus foliaceus Generalist ectoparasitic crustacean, commonly referred to as the fish louse. AUC Area Under Curve. A measure of parasitic infections with a time component. Calculated by plotting the parasite intensity over time and calculating the area under the curve produced. Camallanus Parasitic gut nematode, paratenic in sticklebacks. Definitive hosts lacustris include perch and pike, intermediate hosts include copepod species. Definitive Host Host in or on which a parasite reaches sexual maturity. Diplostomum spp. Digenean trematode occurs as a metacercaria in the lens or vitreous humour of the fish eye, the 2nd intermediate host. Piscivorous birds are the definitive hosts and snails the 1st intermediate hosts. Ectoparasite Parasite which lives on the exterior of a host e.g. on the skin or fins. Endoparasite Parasite which lives inside the body e.g. in the gut or body cavity. Gasterosteus Three-spined stickleback. Ray fined fish in the Infraclass Teleostei of aculeatus the Order Gasterostiformes. Glugea anomala Microsporidian parasite of the three-spined stickleback. Gyrodactylus Monogenean ectoparasite of the three-spined stickleback. gasterostei Intermediate Host A host required for larval development or growth of a parasite before it is infective to another intermediate host or the definitive host. Parasite Intensity Number of individual parasites of a particular species infecting a host. Peak of Infection The highest intensity of parasites found in/on a host over a period of time. Prevalence Number of individuals of a host species infected with a species of parasite. Expressed as a percentage. Saprolegnia Generalist oomycete freshwater pathogen that is both saprotrophic parasitica and necrotrophic. Causes white/grey cotton-like mould on fish. Schistocephalus Cestode, plerocercoid stage in the 2nd intermediate host the three- solidus spined stickleback. The definitive hosts are piscivorous birds and the 1st intermediate hosts copepods. Sham Infection Exposing an individual to an infection procedure without the inclusion of the infective stage of a parasite. Time until Peak Period of time between date of an infection and Peak of Infection. Infection viii Figures and Tables Immunological Terms Term Definition cd8a Cluster of Differentiation 8a, marker of cytotoxic T-cells. IgM Immunoglobulin M, tetrameric antibody in teleosts. First class of antibody usually secreted during an immune response, receptor of naïve B-cells. When referenced in immune data primer is specifically for the heavy chain. IgZ Immunoglobulin Z, referred to as IgT in some literature. Specialised to gut and mucosal immunity. Present in the serum as a monomer and in gut as a polymer. Anti-bacterial antibody of teleosts thought to be similar to human IgA. foxp3b Transcription factor localised to the nucleus, marker of regulatory T-cells. il1r-like Teleost cytokine receptor gene analogous to human IL-1R, marker of pro- inflammatory response. il-4 Cytokine that is a marker of the Th2 immune phenotype, typically an anti-helminth response. Stimulates growth and differentiation of B-cells. il-17 Cytokine that is a marker of the Th17 immune phenotype, marker of the pro-inflammatory response. In humans also linked to autoimmune diseases. Stimulates other pro-inflammatory cytokines such as IL-1. il-12ba Cytokine important in differentiation of Th1 T-cells. orai1 Calcium release-activation calcium channel protein 1. Calcium ion channel necessary for T-cell proliferation. β-def Β-defensin. Antimicrobial peptide active against bacteria, fungi and enveloped viruses. tbk1 TANK-binding kinase 1. NFκB activator. lyso Lysozyme. Enzyme that digests bacterial cell walls, non-specific antibacterial. tirap Toll-interleukin 1 receptor domain containing adaptor protein. Protein involved in Toll-like receptor signalling and NFκB and JNK activation. gpx4a Glutathione peroxidase, housekeeping gene. Protects cells against lipid peroxidation. NFκB Transcription factor associated with the pro-inflammatory response Innate Non-specific and rapid immunity involving phagocytic, antimicrobial and Immunity inflammatory activity. Adaptive Host defences mediated by T and B-cells. Response is specific, diverse, Immunity has memory and is normally able to discriminate between self and non- self. Takes longer to become active than the innate response. B-cells B-lymphocytes. Humoral immune component of adaptive immunity. Primarily involved in secretion of immunoglobulins (antibodies) and immune memory. T-cells T-lymphocytes. Mediators of immune response separated into different T subtypes, see below. H T 1 Helper type-1 CD4+ cells. Aids immune response against intracellular H pathogens. T 2 Helper type-2 CD4+ cells. Enhance B-cell production and aids H elimination of helminths. T 9 Helper type-9 CD4+ cells. Enhance antibody production, mostly H antihelminthic. T 17 Helper type-17 pro-inflammatory CD4+ cells. Effective against bacteria H and fungi. ix