School of Pharmacy Characterisation of the Innate Immune Responses of Marron (Cherax cainii) Bambang Widyo Prastowo This thesis is presented for the Degree of Doctor of Philosophy of Curtin University March 2017 Declaration To the best of my knowledge and belief this thesis contains no material previously published by any other person except where due acknowledgment has been made. This thesis contains no material which has been accepted for the award of any other degree or diploma in any university. Signature: Date: 24/03/2017 ACKNOWLEDGMENTS I would like to thank the following organizations for the support to this thesis: Australia Award Scholarship (AAS) and School of Pharmacy, Curtin University Australia. I would also like to express my deep gratitude to my present and former supervisors: Dr. Ricardo Lareu, my main supervisor, for sharing knowledge, research experience and opinions in my research and for his patience for correcting my thesis writing. I also be grateful for his continuous support, kindness and understanding during the final stage of my study. Dr. Rima Caccetta, my co-supervisor, for all the constructive criticism, interesting discussion during our meeting, fruitful assistance and her kindness during my research journey. Prof. Ravi Fotedar, my associate supervisor, for introducing me to the marron aquaculture in Western Australia and also in the field of innate immunity. Dr. Andrew McWilliams, my previous main supervisor, for informing me about the world of innate immunity. For his moral support and guidance during my PhD study. I would like to extend my sincere gratitude to the person below: Dr. Jeanne LeMasurier, core facility staff at Curtin Health Innovation Research Institute (CHIRI), for helping me to operate flow cytometer and sorting my cell using fluorescence- activated cell sorting during the early morning of my research. Also for his consideration regarding my data analysis using the FlowJo software. Peter Fallon, core facility staff at School of Veterinary and Biomedical Sciences Murdoch University, for teaching me the preparation process and using transmission electron microscope, as well as interpretation of my results. Dr. Richard Parson, statistician at School of Pharmacy, for significant assistance and valuable contribution to the statistical analysis. Julie Craig and Staff at International Sponsored Student Unit (ISSU) Curtin University for the continuous support and a chance to finish my PhD at Curtin University. Angela Rodgers and Staffs at Counselling and Disability Services Curtin University for helping me through to the situation that beyond my control. Associate Professor Lynne Emmerton and all staff at School of Pharmacy for kind understanding and support. And also giving us the best workstation to finish my thesis. I would also like to thank to the Director and Staffs in the CHIRI Biosciences for their kind and generous assistance during my research. To all my colleagues in the CHIRI Biosciences laboratory: Dr. Simon Fox, Wolfgang Wimmer, Vanathi Perumal, Martha Mungkaje, Alex Richards, Hean Teik Humphrey Ko and Adnan Mannan for their friendship and joyful surroundings. Simon Longbottom and Rowan Kleindienst, core facility staffs at Curtin Aquatic Research Laboratory (CARL), for giving me a hand during my preparation for my marron acclimatisation tanks. And everyone at CARL, for the technical support, warm hospitality and the most important thing for helping me taking care of my marrons for many years. And also for all the marrons that have already devoted their lives during my research. Indonesian government, Ministry of Marine Affairs and Fisheries (Directorate General of Aquaculture) and Station of Investigation for Fish Health and Environment Serang for giving me the chance to continue my PhD. It is a privilege and an honour to be entrusted with such an opportunity. I also would like to thank to all my friends in Perth and Indonesia for being my source of inspiration, happiness and encouragement since I arrived at Perth until returned home. I also thank to everyone that have important part in my life, but I might have their name forgotten. I wish to express my deepest appreciation to my whole family and my late parents for their inspiration, thoughtfulness and patience during my PhD journey. Especially for the most important person in my life, my lovely wife Ella Yustanti and my children Shafira Ayuditha Kirana and Dimas Diandra Audiansyah for their endless love, trust, patience, support and source of strength. Without your help, I could not finish my thesis. Finally all thanks are due to Allah for keeping me healthy and for giving me the strength and perseverance to finish my work. TABLE OF CONTENTS TABLE OF CONTENTS …………………………………………………………………………………..…………..…………. I LIST OF FIGURES ……………………………………………………………………………………………………………….. VI LIST OF TABLES ……………………………………………………………………………………………..……….…………. IX LIST OF ABBREVIATIONS …………………………………………………………………………………….…………….. X ABSTRACT ………………………………………………………………………………………………………..……………… XII CHAPTER 1. LITERATURE REVIEW 1.1. Marron (Cherax cainii) ……………………………………………………………………………………..……... 1 1.1.1. Overview …………………………………………………………………………..………………………… 1 1.1.2. Taxonomy …………………………………………………………………………..………………………. 2 1.1.3. Biology …………………………………………………………………………..……………….…………… 5 1.1.4. Aquaculture …………………………………………………………………………..………….………… 6 1.1.5. Disease …………………………………………………………………………..………………….……….. 7 1.2. Ecological innate immune responses in invertebrates ……………………………………........... 8 1.2.1. Evolution of innate immune system …………………………………….......................... 8 1.2.2. Innate immune responses in invertebrates ………………………………………..……… 10 1.2.3. Cellular immune responses ………………………………………………………………….……. 12 1.2.4. Humoral immune responses ……………………………………………………………….…….. 13 1.2.5. Immune cell production …………………………………………………………………….………. 14 1.2.6. Effect of environment ………………………………………………………………………….……. 15 1.3. Crayfish haemocytes and their immune functions ……………………………………............... 16 1.3.1. Haemolymph, circulating haemocytes and haemocytes differentiation ….…. 16 1.3.2. Antimicrobial systems …………………………………….............................................. 17 1.3.3. Phagocytic system …………………………………….................................................... 18 CHAPTER 2. MATERIALS AND METHODS 2.1. Acclimatization system ……………………………………......................................................... 22 2.2. Animals ……………………………………................................................................................. 22 2.3. Preparation of haemocytes …………………………………….................................................. 22 2.4. Preparation of Vibrio mimicus stock solution ……………………………………....................... 23 2.5. Transmission electron microscopy ……………………………………....................................... 23 2.6. Flow cytometry ……………………………………..................................................................... 24 2.7. Griess reaction ……………………………………...................................................................... 24 I 2.8. Two-step density gradient centrifugation using Percoll (Chapter 3) ………………..………. 25 2.9. Phagocytosis assay using TEM (Chapter 4) …………………………………………………………….. 25 2.10. Phagocytosis assay using specific fluorophore (Chapter 5) …………………………..…………. 26 2.11. Activation of haemocytes (Chapter 6) …………………………………………………………..………… 26 2.12. Haemocyte responses to activators (Chapter 7) ………………………………………………..……. 26 2.13. Separation of different haemocyte types using FACS (Chapter 7) ………………………….. 27 2.14. Challenge test in vivo (Chapter 8) ……………………………………………………………………….….. 27 CHAPTER 3. DETERMINATION OF CELL TYPES AND MORPHOMETRIC OF MARRON (Cherax cainii) HAEMOCYTES 3.1. Introduction ………………………………………………………………………………………………………….. 29 3.2. Experimental Outline ………………………….…………………………………………………………………. 31 3.2.1. Experimental outline of different temperature effect at C. cainii haemocytes …………………………………………………………………………………….….…….. 31 3.2.2. List of experimental procedures ………………………………………………….…………….. 32 3.3. Results …………………………………………………………………………………………………………….…….. 33 3.3.1. Determination of C. cainii cell types by light microscope (LM) …………….…….. 33 3.3.2. Determination of C. cainii cell types by transmission electron microscope (TEM) ……………………………………………………………………………………………….……….. 33 3.3.3. Determination of C. cainii cell types by flow cytometer (FCM) ……………..……. 36 3.3.4. Morphometric of the C. cainii haemocytes ……………………………………………..… 36 3.3.5. Total and differential haemocytes counts (THC and DHC) ………………………..… 37 3.3.6. Two-step density gradient centrifugation with Percolltm ………………………..….. 38 3.4. Discussion ……………………………………………………………………………………………………………... 41 CHAPTER 4. ULTRASTRUCTURAL AND FUNCTIONAL CHARACTERISATION OF MARRON Cherax cainii HAEMOCYTES TO PHAGOCYTIC ACTIVITY AT DIFFERENT TEMPERATURES IN VITRO 4.1. Introduction …………………………………………………………………………………………………………… 46 4.2. Experimental Outline ……………………………………………………………….……………………………. 48 4.2.1. Experimental outline of phagocytic experiments ……………………………….……. 48 4.2.2. List of experimental procedures ……………………………………………………………… 49 4.3. Results …………………………………………………………………………………………………………….…….. 50 4.3.1. Non-challenged of C. cainii haemocytes …………………………………….…….……… 50 II 4.3.2. Phagocytic activity by C. cainii haemocytes for live V. mimicus ………………………………………………………………………………………………………………… 51 4.3.3. Phagocytic activity by C. cainii haemocytes for heat-killed V. mimicus and E. coli ………………………………………………………………………….………………………………. 51 4.4. Discussion ……………………………………………………………………………………………………………… 55 CHAPTER 5. PHAGOCYTOSIS