View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RMIT Research Repository The characterisation of Australian freshwater fish immune systems and their response to immunomodulators A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Andrew Harford B. Sc. (Hons) Key Centre for Toxicology School of Medical Science Faculty of Life Sciences RMIT-University 11th October 2004 i Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; and any editorial work, paid or unpaid, carried out by a third party is acknowledged. _____________________ _________ Andrew Harford Date Parts of this work have been presented at the following scientific forums; Harford, A.J., O’Halloran, K & Wright P.F.A. (2005). Murray cod (Macullochella peelii) immune function assays and their use in eco/immunotoxicology. Conference of the Australasian Society for Ecotoxicology, Melbourne, Vic, Australia, Sept 25-28, 2005. Harford, A.J., O’Halloran, K & Wright P.F.A. (2004). Immunomodulation of head kidney cell functions in Murray cod by microcystin-LR. Abstracts of the 10th International Congress of Toxicology, Tampere, Finland, July, 2004, Toxicology & Applied Pharmacology, 197 (3): 284. Wright, P.F., Harford, A.J., O’Halloran. K. (2004) Immunotoxic effects of in vivo chlorpyrifos exposure on Murray cod. Oral Platform presentation. Proc. 43rd Ann. Mtg SOT, Baltimore, Maryland, USA, March 2004. The Toxicologist CD, 78 (1-S): 321, abstract no. 1562. Harford, A.J., O’Halloran, K & Wright P.F.A. (2001). Effects of in vitro exposure to environmental pollutants on the phagocytic cells of Golden Perch (Macquaria ambigua). Proceedings of the 9th International Congress of Toxicologists, Brisbane, QLD, July, 2001. Toxicology, 164 (1-3): 212. The following manuscripts have been accepted for publication; Harford, A.J., O’Halloran, K & Wright P.F.A. (2006). Flow cytometric analysis and optimisation for measuring phagocytosis in three Australian freshwater fish. Fish & Shellfish Immunology. (in press; doi: 10.1016/j.fsi.2005.07.005). Harford, A.J., O’Halloran, K & Wright P.F.A. (2006). The effects of in vitro pesticide exposures on the phagocytic function of four native Australian freshwater fish. Aquatic toxicology. (in press). ii Acknowledgements I would like to acknowledge the following people: Paul Wright, my tireless supervisor, who has supported me through the challenging time of this project. My co-supervisor Kathy O’Halloran for her invaluable expertise and critical appraisal of my work. Thanks also to Daphne Cheah, Caroline Teasdale, Carmel Pollino, Phil Smith, Diana Donahue, Jorma Ahokas, Dani Sevior, Simone Yendle and the many other people at the Key Centre for Toxicology and RMIT that have helped me complete this thesis, especially during the move of campus. A special thanks to Caroline Haskard and CSIRO Land and Water in South Australia who also supplied me with cyanobacterial toxins Peter and Susan Coleman for their friendship and editorial help. My fish suppliers especially, Chris Harwood (Beauford Native Fish), Glenwaters native fish, Glenburn and Australian Aquaculture Products, Euroa. Amber for her love, support and patience. iii Table of contents Title..............................................................................................................................................i Declaration.................................................................................................................................ii Acknowledgements ...................................................................................................................iii Table of contents.......................................................................................................................iv List of Figures....................................................................................................................... xiiii List of Tables...........................................................................................................................xix List of Abbreviations..............................................................................................................xxii Summary ...................................................................................................................................1 Aims of this research................................................................................................................4 Chapter 1: General Introduction...........................................................................................5 1.1: The Murray-Darling Basin .............................................................................................5 1.2: Fish species of the Murray-Darling Basin......................................................................6 1.3: Aquaculture in the Murray-Darling basin ......................................................................8 1.3.1: Silver perch aquaculture..........................................................................................8 1.3.2: Murray cod aquaculture...........................................................................................9 1.3.3: Golden perch aquaculture........................................................................................9 1.4: Fish Immunology ...........................................................................................................9 1.4.1: Lymphoid and myeloid tissue ...............................................................................10 1.4.2: Immunocytes .........................................................................................................11 1.4.3: Immunoglobulins...................................................................................................13 1.4.4: Cytokines...............................................................................................................14 1.4.5: Acute Phase Proteins and Non-cellular non-specific mechanisms........................15 1.4.6: Fish immunophysiology........................................................................................16 iv 1.4.7: Sensitivity to stress................................................................................................18 1.4.8: Sensitivity to temperature......................................................................................19 1.5: Aquatic toxicology .......................................................................................................20 1.5.1: Overview ...............................................................................................................20 1.5.2: Aquatic toxicology in Australia ............................................................................21 1.5.3: Biomarkers (Biomonitoring) .................................................................................22 1.6: Immunotoxicology .......................................................................................................23 1.7: Fish immunotoxicology................................................................................................25 1.7.1: Assays used in fish immunotoxicology.................................................................26 1.8: Fish immunomodulators...............................................................................................29 1.8.1: Metals, heavy metals and organometallic compounds..........................................30 1.8.2: Organotins .............................................................................................................31 1.8.3: Pesticides...............................................................................................................31 1.8.4: Aromatic hydrocarbons .........................................................................................32 1.8.5: Antibiotics and immunostimulants........................................................................34 1.8.6: Polluted sites and chemical mixtures ....................................................................35 1.9: Summary of Chapter 1 .................................................................................................39 Chapter 2: Methodology .......................................................................................................41 2.1: Fish procurement, handling and maintenance..............................................................41 2.1.1: Species selection....................................................................................................41 2.1.2: Wet-laboratory water.............................................................................................44 2.1.3: Handling and maintenance ....................................................................................44 2.2: Fish manipulations .......................................................................................................46 2.3: Sample isolation methods.............................................................................................47 2.3.1: Blood and serum collection...................................................................................47 v 2.3.2: Head kidney...........................................................................................................47