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Investigation Into the Microbiological Causes of Epizootics Of Investigation into the microbiological causes of epizootics of Pacific oyster larvae (Crassostrea gigas) in commercial production by Christopher Chapman B. Ag. Sc (Hons) University of Tasmania Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy University of Tasmania Hobart Tasmania Australia February 2012 Declaration This thesis contains no material that has been accepted for the award of any other degree or diploma in any tertiary institution. To the best of my knowledge this thesis does not contain material written or published by another person, except where due reference is made. Christopher Chapman University of Tasmania Hobart February 2012 This thesis may be made available for loan and limited copying in accordance with the Copyright Act 1968 Acknowledgements To my supervisory team, Mark Tamplin, John Bowman, Shane Powell and Michel Bermudes I would like to thank you all. To Mark for his guidance and for keeping me true to my plan; to John (the walking Bergey’s manual) for his fresh ideas and for his encouragement; to Shane, with whom I worked closely on all aspects of this project, thanks especially for helping me survive the laboratory and for being critical on drafts of this thesis; and to Michel for providing the industry perspective. I would like to give particular thanks to the Shellfish Culture team to whom I promised more than I was able to deliver. They made me feel part of the team and gave me free run of their hatchery. Thanks to Tom Spykers and Lee Wilson whose time-tested observations informed the direction of this study. Thanks to Scott Parkinson for initiating this study and providing support throughout. I would like to thank a number of external people for their help and their advice. Thanks to Tom Lewis of Rural Development Services, for pragmatic and strategic advice; to Wayne O’Connor and Stephen O’Connor of NSW fisheries, for introducing me to the industry and for strategic advice in the planning phase of the project; to Graeme Knowles (DPIPWE) for taking an interest and for his collaborative efforts; and to Guy Abell of CSIRO, for technical advice. To members of the university staff who provided assistance I acknowledge their efforts. In particular thanks to Adam Smolenski for trouble shooting problems in the molecular lab; and to David Ratowski for sharing his considerable knowledge and experience in statistical analysis of unbalanced datasets. To Karen Watson - with whom I shared much of this journey from beginning to end - for her support and patience. Finally, to the millions of larvae that died in the making of this thesis, may they be reincarnated as something bigger with sharp teeth, much much higher in the food chain. Table of contents Abbreviations .......................................................................................................................... 1 Abstract ................................................................................................................................... 2 Introduction ............................................................................................................................. 4 1 Chapter 1: Literature review ............................................................................................ 8 1.1 Introduction ............................................................................................................. 8 1.2 Normal development of Oyster larvae .................................................................... 9 1.3 Causes of larvae mortality ..................................................................................... 10 1.4 Larvae immune response ....................................................................................... 17 1.5 Microbiological aspects of oyster hatcheries ........................................................ 20 2 Microbial ecology in a commercial oyster hatchery tank ............................................. 30 2.1 Abstract ................................................................................................................. 30 2.2 Introduction ........................................................................................................... 31 2.3 Methodology ......................................................................................................... 32 2.3.1 Monitoring program design ........................................................................... 32 2.3.2 Hatchery production systems ........................................................................ 33 2.3.3 Analytical procedures .................................................................................... 34 2.4 Results ................................................................................................................... 41 2.4.1 Bacterial communities of the larvae tank ...................................................... 41 2.4.2 Relationships among microbial niches of the larvae tank ............................. 42 2.4.3 Tank type and associated microbial communities ......................................... 44 2.4.4 Temporal variability in microbial communities of larvae and WC ............... 45 2.5 Discussion ............................................................................................................. 45 2.5.1 Bacterial communities of the larvae tank ...................................................... 45 2.5.2 Relationships among microbial niches of the larvae tank ............................. 47 2.5.3 Temporal variability in microbial communities of larvae and WC ............... 52 2.6 Conclusion ............................................................................................................. 53 3 Microbial communities associated with epizootics in a commercial oyster hatchery... 60 3.1 Abstract ................................................................................................................. 60 3.2 Introduction ........................................................................................................... 61 3.3 Methodology ......................................................................................................... 63 3.3.1 Monitoring program design ........................................................................... 63 3.3.2 Analytical procedures .................................................................................... 63 3.4 Results ................................................................................................................... 66 3.4.1 Observation of disease symptoms ................................................................. 66 3.4.2 Microbial communities and disease incidence .............................................. 67 3.4.3 Vibrio abundance and disease incidence ....................................................... 68 3.5 Discussion ............................................................................................................. 69 3.6 Conclusion ............................................................................................................. 73 4 Oyster larvae stress and susceptibility to bacterial disease ........................................... 77 4.1 Abstract ................................................................................................................. 77 4.2 Introduction ........................................................................................................... 77 4.2.1 Interaction between chemical stress and infectious disease .......................... 77 4.2.2 Chemical stressors in the hatchery environment ........................................... 79 4.2.3 Measurement of chemically induced stress in oyster larvae ......................... 81 4.2.4 Research objectives ....................................................................................... 84 4.3 Methodology ......................................................................................................... 85 4.3.1 Copper sulphate exposure followed by bacterial challenge .......................... 85 4.3.2 Copper sulphate exposure and larvae behaviour ........................................... 86 4.4 Results ................................................................................................................... 87 4.4.1 Copper sulphate exposure followed by bacterial challenge .......................... 87 4.4.2 Copper sulphate exposure and larvae behaviour ........................................... 88 4.5 Discussion ............................................................................................................. 89 4.6 Conclusion ............................................................................................................. 93 5 Synopsis ......................................................................................................................... 97 6 Appendix ..................................................................................................................... 101 7 References ................................................................................................................... 115 Abbreviations DSS – days since spawning MVDISP – multivariate dispersion index % CL – percentage of larvae crossing midway line in n – number of samples / replicates cuvette % SL – percentage of larvae swimming in cuvette NSW – natural seawater (above bottom) CFU – colony forming units PCR – polymerase chain reaction CTW – clean tank water ppb – parts per
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