Antiviral immune responses in abalone and influence of potential abiotic and biotic factors A thesis submitted to the Flinders University By Vinh Dang In candidature for the degree Doctor of Philosophy School of Biological Sciences Flinders University August 2012 1 Contents Contents Contents ........................................................................................................................ 2 List of tables ................................................................................................................. 6 List of figures ............................................................................................................... 7 Abbreviations ............................................................................................................... 8 Declaration ................................................................................................................... 9 Authority of access ..................................................................................................... 10 Abstract ...................................................................................................................... 11 Acknowledgement ...................................................................................................... 14 Thesis Structure .......................................................................................................... 15 CHAPTER 1: General introduction ........................................................................... 17 1.1. Major economic abalone species in Australia ................................................. 17 1.2. Effects of microbial infection and environmental stress on abalone production ................................................................................................................................ 19 1.2.1. Virus infections ......................................................................................... 19 1.2.2. Bacterial infections ................................................................................... 20 1.2.3. Environmental stress factors ..................................................................... 23 1.3. Immune responses against microbial infections .............................................. 25 1.3.1. Cell-mediated immunity ........................................................................... 26 1.3.2. Humoral immunity defense ....................................................................... 27 1.3.3. Dietary derived anti-microbial factors ...................................................... 29 1.4. Research Aims ................................................................................................. 30 CHAPTER 2: In vitro antiviral activity against herpes simplex virus in abalone Haliotis laevigata ....................................................................................................... 32 Abstract ................................................................................................................... 33 2.1. Introduction ...................................................................................................... 34 2.2. Materials and methods ..................................................................................... 37 2.2.1. Abalone ..................................................................................................... 37 2.2.2. Cell culture and virus ................................................................................ 37 2.2.3. Tissue and haemolymph collection ........................................................... 38 2.2.4. Lipophilic and non-lipophilic extraction ................................................... 38 2.2.5. Peptide extraction ...................................................................................... 39 2.2.6. Protein determination ................................................................................ 39 2 Contents 2.2.7. Cytotoxicity assays ................................................................................... 40 2.2.8. Anti-HSV assay ......................................................................................... 40 2.2.9. Timing of antiviral activity ....................................................................... 41 2.2.10. Attachment assay .................................................................................... 42 2.2.11. Entry assay .............................................................................................. 43 2.2.12. Virucidal assay ........................................................................................ 43 2.2.13. Statistical analysis ................................................................................... 44 2.3. Results.............................................................................................................. 44 2.4. Discussion ........................................................................................................ 53 Acknowledgments .................................................................................................. 58 CHAPTER 3: Variation in the antiviral and antibacterial activity of abalone Haliotis laevigata, H. rubra and their hybrid in South Australia ............................................ 59 Abstract ................................................................................................................... 60 3.1. Introduction ...................................................................................................... 62 3.2. Materials and methods ..................................................................................... 65 3.2.1. Abalone ..................................................................................................... 65 3.2.2. Spawning induction ................................................................................... 66 3.2.3. Haemolymph collection ............................................................................ 66 3.2.4. Cell culture and virus ................................................................................ 67 3.2.5. Anti-HSV assay ......................................................................................... 67 3.2.6. Bacteria ..................................................................................................... 68 3.2.7. Antibacterial assay .................................................................................... 68 3.2.8. Statistical analysis ..................................................................................... 69 3.3. Results.............................................................................................................. 70 3.4. Discussion ........................................................................................................ 76 Acknowledgments .................................................................................................. 83 Appendix 3.1. Map of abalone collecting locations ............................................... 84 CHAPTER 4: Effects of micro and macroalgal diet supplementations on growth and immunity of greenlip abalone, Haliotis laevigata ...................................................... 85 Abstract ................................................................................................................... 86 4.1. Introduction ...................................................................................................... 88 4.2. Materials and methods ..................................................................................... 91 4.2.1. Experimental abalone and diets ................................................................ 91 4.2.2. Macro-and microalgae feeding trials ........................................................ 91 3 Contents 4.2.3. Haemolymph analyses .............................................................................. 93 4.2.4. Lipophilic extraction and antiviral assay .................................................. 96 4.2.5. Statistical analysis ..................................................................................... 96 4.3. Results.............................................................................................................. 97 4.3.1. Growth rates .............................................................................................. 97 4.3.2. Haemolymph immune parameters ............................................................ 99 4.3.3. Antiviral activity of lipophilic extracts ................................................... 103 4.4. Discussion ...................................................................................................... 105 Acknowledgments ................................................................................................ 110 CHAPTER 5: Influence of elevated temperatures on the immune response of abalone, Haliotis rubra ............................................................................................. 111 Abstract ................................................................................................................. 112 5.1. Introduction .................................................................................................... 114 5.2. Materials and methods ................................................................................... 117 5.2.1. Field-sampled abalone ............................................................................ 117 5.2.2. Laboratory