Influence of metal exposure history on metal tolerance in the Sydney rock oyster (Saccostrea glomerata) Thanvapon Senee Yingprasertchai A thesis submitted to the Discipline of Biological Sciences, University of Newcastle, in fulfillment of the requirements of the Degree of Doctor of Philosophy Supervised by: Dr Geoffrey MacFarlane and Dr Richard Yu Declaration The thesis contains no material which has been accepted for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made in the text. I give consent to the final version of my thesis being made available worldwide when deposited in the University’s Digital Repository, subject to the provisions of the Copyright Act 1968. I hereby certify that the work embodied in this thesis has been done in collaboration with other researchers, or carried out in other institutions (delete if not applicable). I have included as part of the thesis a statement clearly outlining the extent of collaboration, with whom and under what auspices. Thanvapon Senee Yingprasertchai ii Acknowledgements Firstly, I wish to acknowledge the untiring efforts of my supervisors; Dr. Geoff MacFarlane and Dr. Richard Yu, in providing logistical support, critical appraisal, encouragement and friendship over the many years that it has taken for this thesis to come to fruition. Secondary, I am indebted to Dr Wayne O‘Connor at Port Stephens Fisheries Institute, Taylors Beach (PSFI) who works as an unofficial supervisor and has provided great support in field oyster sampling and laboratory facilities for exposure experiments. Thanks to all PSFI staff, Dr. Michael Dove, Kyle Johnston, Stephen O‘Connor, Lynne Foulkes, Brandt Archer and Andrew Parnell, for their kind support and friendship. I extend thanks to Dr Megan Priestley for teaching techniques at the beginning of the project in ecotoxicology laboratory and her friendship. I am greatly indebted to Mr.Tony Rothkirch, Professional Officer in research services of Analytical and Biomolecular Research Facility (ABRF) for technical advice in metals analysis using Inductively Coupled Plasma Mass Spectrometer (ICP-MS). In particular, I wish to thank the Dunstan’s lab; Dr. Anthony Martin, Marcus Crompton and members for support equipment for enzyme activity and for metal analysis sample preparation. I would also thankthe School of Environmental and Life Sciences office; Marina Besseny, Vicki Purcell, Anna Honeysett and Amy Glumbeton for their kind support. I also thank Mr.Brett Hill, technical Officer in Faculty of Health for allowing me to use facilities such as the cold room, safety cabinet etc. Thanks to friends in Ecotoxicology group, Thi Kim Anh Tran, Grace Bourke and Motsawadi Maswabi. Thirdly, I am grateful to Professor Barney Glover who initiated a bilateral agreement between University of Newcastle and Kanchanaburi Rajabhat University, Thailand. The agreement provided a great opportunity for my life to study at University of Newcastle, live and learn so many things in Australia. I would also like to thank the University of Newcastle Research Scholarship Central (UNRSC) and Kanchanaburi Rajabhat University (KRU) for Financial support thoughout my PhD study. Finally I would extend an enormous thanks to my mother and family Peach, Jessy & Grace for their love, patience, encouragement and support during this long and arduous process. iii Table of contents Page Declaration .................................................................................................. ii Acknowledgements .................................................................................... iii Table of contents ........................................................................................ iv List of figures ............................................................................................ viii List of tables ............................................................................................. xiii Abbreviations ......................................................................................... xviii Abstract ........................................................................................................ 1 Chapter 1 Introduction and Literature review ......................................... 4 1.1 Overall thesis aims ................................................................................... 4 1.2 Metals ........................................................................................................ 4 1.3 Metals in aquatic environment ............................................................... 7 1.4 NSW estuaries .......................................................................................... 9 1.5 Effects of metals on aquatic organisms ................................................ 10 1.6 Accumulation versus regulation of metals ........................................... 11 1.7 Molluscs as monitors .............................................................................. 12 1.8 Sydney rock oysters ............................................................................... 15 1.9 Metal tolerance development - acclimation versus adaptation .......... 18 1.10 Adaptation studies ............................................................................... 20 1.11 Acclimation due to prior metals exposure history ............................ 22 1.12 Metallothionein .................................................................................... 23 1.13 Metal induced oxidative stress ............................................................ 25 1.14 Response of antioxidant enzymes GPx and SOD to metal contamination .................................................................................................. 26 1.15 Chapter aims ........................................................................................ 29 Chapter 2 : Molecular cloning, characterization and expression of metallothionein (MT) gene from S. glomerata ........................................ 31 2.1. Summary ................................................................................................... 31 2.2. Introduction .............................................................................................. 32 2.3. Materials and methods ............................................................................. 37 2.3.1. Collection of Sydney Rock Oysters .................................................... 37 2.3.2. Total RNA extraction .......................................................................... 38 2.3.3. Agarose gel electrophoresis ................................................................ 39 2.3.4. First stand cDNA synthesis ................................................................. 39 2.3.5. Amplification of S. glomerata MT (sgMT) cDNA by degenerate PCR ....................................................................................................................... 40 iv 2.3.6. Purification of sgMT gene fragment from agarose gel ....................... 42 2.3.7. Cloning of MT gene fragments ........................................................... 42 2.3.8. Genomic DNA isolation ...................................................................... 44 2.3.9. Genome walking ................................................................................. 44 2.3.10. Direct PCR amplification and sequencing of sgMT ......................... 47 2.3.11. Computer analyses ............................................................................ 48 2.3.12. Phylogenetic analysis ........................................................................ 49 2.3.13. Real-time quantitative PCR (qPCR) ................................................. 50 2.3.14. Tissue specific sgMT gene expression studies .................................. 51 2.3.15. Statistical analysis ............................................................................. 52 2.4. Results ....................................................................................................... 53 2.4.1. Amplified MT-gene in S.glomarata .................................................... 53 2.4.2. Cloning of metallothionein .................................................................. 54 2.4.3. Genome walking ................................................................................. 56 2.4.4. Direct PCR amplification and sequencing of genomic DNA and cDNA of gill and digestive gland ............................................................................. 58 2.4.5. Analysis of the sgMT amino acid sequence ........................................ 61 2.4.6. Phylogenetic analysis .......................................................................... 63 2.4.7. Real-time quantitative PCR (RT-qPCR) development ....................... 65 2.4.8. Tissue specific sgMT gene expression studies .................................... 70 2.5. Discussion .................................................................................................. 73 Chapter 3 : Multivariate analysis approach for the determination of metal contamination history in S. glomerata from New South Wales, Australia ..................................................................................................... 77 3.1. Summary ................................................................................................... 77 3.2.