A PROTEOMIC INVESTIGATION OF THE HEAT STRESS RESPONSE OF THE SOUTH AFRICAN ABALONE HALIOTIS MIDAE By Bridget Calder Thesis Presented for the Degree of DOCTOR OF PHILOSOPHY in the Department of Molecular and Cell Biology, Faculty of Science University of Cape Town, South Africa. July 2014 Supervised by Prof. V. Coyne and Dr. S. Rafudeen Declaration I know the meaning of plagiarism and declare that all of the work in this thesis, save for that which is properly acknowledged, is my own. Signed: B. Calder __________________ Acknowledgements I would like to acknowledge the support and assistance offered to me by my supervisor, Professor Vernon. E. Coyne, and co-supervisor Dr Suhail Rafudeen during my time at the University of Cape Town. The marine biotechnology laboratory has been a source of constant comfort and much needed advice during my time in the molecular and cell biology department. Thank you also to the department as a whole for providing me with so much stimulating tea-time conversation and helpful guidance. I would also like to thank Dr Zac McDonald for his assitance with mass spectrometry in the molecular and cell biology department, without which this work would not have been possible. Then, I would like to thank my friends and loved ones who have been so patient with me while I completed this work. This would not have been possible without a loving and understanding support structure. I would also like to formally acknowledge the financial assistance offered to me by the postrgraduate funding office at the University of Cape Town, and the National Research foundation for providing bursaries during my postgraduate studies. TABLE OF CONTENTS ABSTRACT ........................................................................................................................ 2 Chapter 1 LITERATURE REVIEW ............................................................................. 4 Chapter 2 TWO DIMENSIONAL POLYACRYLAMIDE GEL ELECTROPHORESIS ANALYSIS OF THE H. MIDAE HEAT STRESS RESPONSE ..................................................................................................................... 27 Chapter 3 AN iTRAQ BASED PROTEOMIC PROFILING OF THE H. MIDAE HAEMOCYTE RESPONSE TO ACUTE THERMAL STRESS .......... 50 Chapter 4 CHARACTERISATION OF THE RESPONSE OF A MOLECULAR CHAPERONE TO ACUTE THERMAL STRESS IN H. MIDAE HAEMOCYTES ...........................................................................................105 Chapter 5 GENERAL DISCUSSION ......................................................................134 Appendix A Solutions ..................................................................................................143 Appendix B Supplementary data ................................................................................147 Appendix C Literature cited ........................................................................................185 1 A PROTEOMIC INVESTIGATION OF THE HEAT STRESS RESPONSE OF THE SOUTH AFRICAN ABALONE HALIOTIS MIDAE By Bridget Calder 2014 ABSTRACT The abalone Haliotis midae has been fished to near-extinction on the South African coastline, primarily to satisfy a growing international market. In order to meet demands, H. midae has been produced in South Africa by aquaculture for several decades, and the South African abalone aquaculture industry continues to expand. Internationally, abalone aquaculture has been negatively affected by the outbreak of bacterial and viral diseases, which spread rapidly and lead to high abalone mortality. There is evidence that environmental stresses on abalone farms may lead to immunosuppression, and thereby increase the severity of disease outbreaks. The water temperature on abalone farms fluctuates seasonally, and increased abalone mortality has been associated with warmer water during the summer months. However, the molecular mechanisms affecting the abalone during exposure to stress remain unclear. With advances in proteomics technology, it is possible to identify and quantify the expression of several hundred proteins simultaneously. This study therefore aimed to gain insight into the H. midae stress response by using proteomic tools to identify proteins that are differentially regulated in haemocytes during exposure to acute heat stress. Identifying which biochemical pathways are involved in the abalone stress response will give some insight into the molecular mechanism by which H. midae responds to heat stress. Proteins that were significantly regulated under heat shock conditions were identified using two different proteomic techniques: 2D PAGE analysis and iTRAQ. Several proteins with cytoskeletal function were identified by 2D PAGE, indicating that cytoskeletal remodelling may be induced by acute heat stress. As the abalone immune response is predicated on the ability of the haemocytes to engulf foreign particles by phagocytosis, it was therefore hypothesised that damage to the cytoskeleton could lead to immunosuppression. iTRAQ analysis of heat stressed abalone haemocyte total protein was then carried out and additional heat-regulated proteins were identified. A statistically stringent method was employed, and 11 proteins with significant heat-induced differential expression were identified. Additional differentially regulated proteins were identified using statistical seriation to cluster proteins with similar heat-induced expression profiles. As many of the identified proteins lack functional characterisation in abalone, Blast2GO® analysis was performed to associate gene ontology keywords with the protein identities that had the most consistent response to heat stress. The GO terms were then simplified using the tool 2 REViGO to identify biochemical pathways that were implicated by these protein identities. Based on these data, the cellular functions that appear to be affected by heat stress in H. midae are cell signalling, response to stress and metabolic regulation. As several important proteins identified by iTRAQ remain uncharacterised in abalone, one was selected for further characterisation in order to determine its suitability as a potential molecular heat stress indicator in H. midae. Thus, further expression analysis of the molecular chaperone calreticulin (CRT) was carried out using alternate molecular techniques. The transcriptional regulation of CRT in H. midae during exposure to heat stress was measured using qPCR, which detected a significant early decrease in the amount of CRT mRNA followed by a return to basal levels. The translational regulation of CRT was then measured by western blot analysis, which detected a significant increase in the amount of CRT in the haemocytes after 120 minutes of exposure to 23 °C. This data also confirmed the expression profile determined by iTRAQ for CRT. The findings of this study indicate that the H. midae molecular heat stress response is complex and interconnected, and capable of rapidly responding to acute thermal stress. However, the metabolic cost to the abalone and the potential damage caused to the haemocytes should be of concern to abalone farmers. This information is also of particular concern when considering the effects of anthropogenic climate change on the survival in the wild of this already threatened species. This study presents a set of molecular tools that may be of use in future molluscan studies, and with the growing popularity of proteomics as a diagnostic tool in aquaculture may serve as a platform for the investigation of the effect of environmental stress on other molluscan species. 3 Chapter 1 LITERATURE REVIEW 1.1. Introduction .................................................................................................................... 5 1.2. The abalone ..................................................................................................................... 5 1.3. Naturally occurring H. midae populations in South Africa ....................................... 6 1.4. The South African abalone aquaculture industry ...................................................... 7 1.5. The molluscan immune system .................................................................................... 8 1.6. The link between stress and the immune response ................................................... 9 1.7. The heat-shock response ............................................................................................. 11 1.8. The molluscan response to thermal stress ................................................................ 12 1.8.1. Mussel ........................................................................................................................ 13 1.8.2. Oysters ....................................................................................................................... 14 1.8.3. Abalone ..................................................................................................................... 15 1.9. Proteomics and the characterisation of the stress response ................................... 17 1.9.1. An introduction to proteomics technology .......................................................... 18 1.9.1.1. The mass spectrometer ....................................................................................... 18 1.9.1.2. Reduction in proteome complexity .................................................................. 21 1.9.1.3. Software for proteomic investigations ............................................................. 22 1.10. Aims of this study ........................................................................................................