Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps THESIS SUBMITTED TO THE COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY In partial fulfillment of the degree of DOCTOR OF PHILOSOPHY IN MARINE BIOTECHNOLOGY UNDER THE FACULTY OF MARINE SCIENCES BY SWAPNA P. ANTONY DEPT. OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY SCHOOL OF MARINE SCIENCES COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY KOCHI-682 016, INDIA March, 2011 Dedicated to My Family, My Guide & God Almighty Declaration I hereby do declare that the thesis entitled “Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps” is an authentic record of research work done by me under the supervision and guidance of Dr. Rosamma Philip, Assistant Professor, Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, for the degree of Doctor of Philosophy in Marine Biotechnology and that no part thereof has been presented before for the award of any other degree, diploma or associateship in any University. Kochi-682 016 Swapna P. Antony March 2011 Dr. Rosamma Philip Assistant Professor Department of Marine Biology, Microbiology and Biochemistry School of Marine Sciences Cochin University of Science and Technology Kochi-682 016 Certificate This is to certify that the thesis entitled “Molecular Characterization and Gene Expression Profiling of Antimicrobial Peptides in Penaeid Shrimps” is an authentic record of research work carried out by Ms. Swapna P. Antony under my supervision and guidance in the Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, Cochin University of Science and Technology, in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Marine Biotechnology and that no part thereof has been presented before for the award of any other degree, diploma or associateship in any University. Kochi-682 016 Dr. Rosamma Philip March 2011 (Supervising Guide) Acknowledgements This work was carried out at the Department of Marine Biology, Microbiology and Biochemistry, School of Marine Sciences, CUSAT. I have received generous support of numerous people from the beginning till the end of the present thesis work.I would like to thank the people that made this work possible. I thank: Dr. Rosamma Philip, my supervisor, for accepting me as a Ph.D student, for introducing me to an amazing world of Molecular Biology, for sharing your vast knowledge, for never ending encouragement, interesting discussions and being patient for all aspects. Mam, I am so appreciated with your generousness and kindness. Thank you indeed for your constant support and for being there for questions big and small. I am very grateful for your thorough revision and your invaluable comments of the manuscripts that remarkably improved the quality of our papers and made it acceptable for publication. Most of all I thank you for the trust, for believing in me and for your invaluable friendship. Mam, you only taught me how to focus on a problem, approach it carefully and present it precisely. Without your support, this work would never have been possible. I will always be honored to be called as your student. Dr. I. S. Bright Singh, for being generous enough inviting me to join your group and for providing a lab full of equipments. For always being extremely kind whenever I need help; your support, interesting discussions and making it possible for me to stay in the field of antimicrobial compounds and shrimp immunity. Especially I thank you for taking interest in my research and for sharing your vast knowledge. Sir, I am proud and encouraged with your devotion to science. I will always be honored to be called as your student. The Head, Department of Marine Biology, Microbiology and Biochemistry, for all the help and support extended to me throughout the period. Dr. Ram Mohan, Dean and Director, School of Marine Sciences, CUSAT for all the help rendered and facilities provided for research. The faculty members of the Department, Dr. A.V. Saramma, Dr. Babu Philip, Dr. Aneykutty Joseph, Dr. Mohammed Hatha, and Dr. Bijoy Nandan for your help, concern and encouragement. Dr. C. K. Radhakrishnan for all the help rendered. Dr. A. Mohandas, Dr. Rajesh Ramachandran, Dr. Valsamma Joseph, Dr. Somnath Pai, and Dr. Nandini Menon for your valuable suggestions and all help rendered during my research. My teachers at St. Teresa’s College, Ernakulum, who moulded me. Special thanks are due to Miss. Gladys Francis, the Head, Dept. of Zoology for directing me to the research field, for your kind concern and encouragement. Rev. Sr. Francis Ann, Ms. Reema Kuriakose, Ms. Meera and Ms. Mini of St. Teresa’s College for your love and concern. It is only with deepest respect and appreciation; I thank all my teachers for your help, concern and encouragement. Dr. Sunil Kumar Mohammed, Principal Scientist, Molluscan Fisheries Division, CMFRI, for your valuable suggestions and critical assessment at various stages of my work. Kerala State Council for Science, Technology and Environment for the fellowship awarded as Junior and Senior Research Fellowships. Department of Biotechnology and Ministry of Earth Sciences, Govt. of India, for financial assistance. Office staff and technical staff of the Department, for the support, timely help and assistance. Library staff of the School of Marine Sciences, CUSAT, for extending the necessary facilities. Mr. Balan, the laboratory assistant, for the support and assistance rendered. Mr. Soman, Mr. Aneeshmon, Mr. Jaison and Mr. Biju at the NCAAH, for your timely help in providing the experimental animals. Mr. Sajeevan TK, Mr. Abdul Nazar and Mr. S. Santhosh (Electrical Staff) and Security Staff for your timely help extended to me throughout. Mr. T. Rejil for the administrative help extended. My friends Naveen Sathyan, Anil Kumar P.R. and Chaithanya E.R, (the AMPs) for always being there with help. Thanks a lot for your friendship, support, nice discussions and always having fun. Ann Rose Bright and Jeeshma Jolly for your love and affection. My Seniors, Dr. Annies Joseph, Dr. Lakshmi G. Nair, Dr. Selvan S, Dr. Sajeevan TP, Neil Scolastin Correya, Dr. Sanil Kumar, Dr. Simi Joseph P, Dr. Sreedevi N Kutty, Smitha SL, Dr. Soja Louis, Dr. Sincy Joseph, and Dr. Maya Paul…. for teaching me the basics of research and for taking care of me when I started my research… Thanks a lot for all the help rendered. Vrinda S, Prabha Pillai, Priyaja P, Manjusha K, Sudheer NS, Jayesh P, Rose Mary Jose, Sunitha Paulose, Gigi Poulose, Seena Jose, Sreedharan K, Rajesh VJ, Surya. Thank you for sharing your vast knowledge, for all the help, nice discussions and your invaluable friendship. Anil Kumar PR, Harishankar HS, Naveen Sathyan, Rahul KK and Ajith Kumar for the help rendered in setting the aquarium tanks and for taking care of my shrimps during days of bus strike and hartals. My fellow colleagues and friends, Jisha VK, Sini PJ, Jini Jacob, Deepu AV, Abhilash KR, Anit M Thomas, Manoj P, Remya KD, Jimly C Jacob, Sudha S, Smitha CK, Chitra Som, Deepthi Augustine, Nifty John, Shubankar Ghosh, Neethu, Debora, Divya PS, Jisha Jose, Remya Varadarajan, Anu Pavithran, Sreedevi OK, Shameeda CH, Shyam Kumar, Renjith Kanjur, Sreelakshmi, Vijay, Divya T. Babu, Neelima, Akhilesh, Jayachandran, Dr. Subhash Babu, Dr. Prabhakaran, Dr. Mujeeb Rahman, Dr. Jasmin, Dr. Anupama Nair, Dr. Smitha Bhanu, Dr. Radhika Gopinath, Prajith, Bineesh, Abhilash KS, Abdul Jaleel, Nousher Khan…………..too many friends to be listed here. I am thankful to all of you for your friendship, joyful time and social atmosphere at the lab. My late grandfathers and grandmother for their affection and heavenly blessing that helped me in fulfilling the apparent venture. I humbly offer you with prayers, my admiration and gratitude. My parents-in-law, Veeravunni Varaparambil, and Mariyumbi for your love, care, support and concern. Without your understanding and encouragement it would have been impossible for me to finish this work. Thank you so much for providing a loving environment for me. Perhaps this thesis would not have survived some of the more tiresome times during the process of writing up if it had not been for your unquestioning support. My sister–in-law, Niloufer and my brother, Shiraz Kareem for your friendliness, care, encouragement and concern. My uncles, aunts, cousins and family friends for their love, thoughtfulness, care and support rendered throughout my life. My parents Antony PO and Molly Antony, for being the best…..My grandmother Elsy Easy Kattipparambil, My sister Soumya… Thank you so much for your endless love, encouragement and support … You have been there for me every step of the way, have always loved me unconditionally, and have aided me through all of my tough decisions. You have always been the motivating force behind my morale for higher achievements. The present venture would not have been fulfilled without your genuine interest, love and sacrifice. Words are not enough to express my appreciations for your love and support. My husband Afsal VV, who has always been my pillar, my joy and inspired me to be optimistic … Thank you so much for all your love, care, patience and support. Thanks a lot for being supportive throughout my thesis writing, for helping me with proofreading and for your valuable suggestions. My research pursuit would not have borne fruit without your sustained interest, support and sacrifice.... I feel extremely blessed to have you in my life and words fail to express my feelings…. All Penaeus monodon and Fenneropenaeus indicus, who sacrificed their lives to fulfill my research leading to Ph.D. God Almighty for my life, knowledge and thoughts. You only give me the strength and energy to face and overcome the hardships and obstacles of my research tenure that paved way to the fulfillment of this much-yearned endeavor. You have made my life more bountiful. May your name be exalted, honored, and glorified. I could not finish without all of you. Thank you all so much… Swapna P. Antony Contents Title Page Nos. 1 General Introduction 1-94 1.1 Introduction 1-2 1.2 Major groups of shrimps cultured worldwide 2-6 1.2.1 Penaeus monodon – Giant tiger shrimp 3-5 1.2.2 Fenneropenaeus indicus– Indian white shrimp 5-6 1.3 Major constraints in aquaculture 6-9 1.4 WSSV 9-18 1.5 Approaches for the control of diseases in shrimp aquaculture 18-22 1.6 Crustacean immune system 22-36 1.6.1 Haemocytes 27 1.6.2 Haematopoiesis 27-29 1.6.3 Cellular immune response 29-30 1.6.4 Humoral immune responses 30-36 1.7 Antimicrobial Peptides (AMPs) 37-92 1.7.1 Background of the AMPs 37-39 1.7.2 Terminology 39 1.7.3 The Nature of AMPs 39-40 1.7.4 Distribution of AMPs 40-44 1.7.5 Biological activity 45-46 1.7.6 Diversity of AMPs 46-58 1.7.7 Induction and regulation of AMP expression 58-60 1.7.8 Regulation of synthesis and release of AMPs 60-61 1.7.9 Localization of AMPs 61-62 1.7.10 Mechanism of action 62-80 1.7.11 Mechanisms of selective toxicity 80-82 1.7.12 Characteristics that affect antimicrobial activity and specificity 82-85 1.7.13 Potential applications of AMPs 85-92 1.8 Relevance of the present study 92-93 1.9 Objectives 94 1.10 Outline of the thesis 94 2 Molecular characterization and phylogenetic analysis of antimicrobial peptides in Penaeus monodon 95-185 2.1 Introduction 95-113 2.1.1 Anti-lipopolysaccharide factor (ALF) 97-99 2.1.2 Crustins 99-105 2.1.3 Penaeidins 105-113 2.2 Materials and Methods 114-120 2.2.1 Experimental animals 114 2.2.2 RNase control 114-115 2.2.3 Haemolymph collection 115 2.2.4 RNA isolation 115-116 2.2.5 Determination of the quantity and quality of RNA 116 2.2.6 Reverse transcription 116 2.2.7 PCR amplification 116-117 2.2.8 Agarose gel electrophoresis 117 2.2.9 Cloning of the PCR product 117-119 2.2.10 Sequencing and sequence analysis 119-120 2.3 Results 120-132 2.3.1 Molecular characterization of AMP genes in P.monodon 120-132 2.3.1.1 Anti-lipopolysaccharide factor -1 (ALF-1) (GU732817) 120-122 2.3.1.2 Anti-lipopolysaccharide factor -2 (ALF-2) (HM588914) 122-123 2.3.1.3 Crustin-1 (GQ334395) 124-126 2.3.1.4 Crustin-2 (FJ535568) 126-128 2.3.1.5 Crustin-3 (GQ334396) 128-130 2.3.1.6 Penaeidin-3 (GU732819) 130-131 2.3.1.7 Penaeidin-5 (GQ334397) 131-132 2.3.2 Control / Reference genes 132 2.4 Discussion 130-140 Tables 140-148 Figures 149-188 3 Molecular characterization and phylogenetic analysis of antimicrobial peptides in Fenneropenaeus indicus 186-237 3.1 Introduction 186-187 3.2 Materials and Methods 187-188 3.2.1 Experimental animals 187 3.2.2 Haemolymph collection 187 3.2.3 Total RNA isolation and Reverse transcription 188 3.2.4 PCR amplification 188 3.2.5 Agarose gel electrophoresis 188 3.2.6 Cloning of the PCR product 188 3.2.7 Sequencing and sequence analysis 188 3.3 Results 188-199 3.3.1 Molecular characterization of AMP genes in F. indicus 188-199 3.3.1.1 Anti-lipopolysaccharide factor (ALF-1) (GU732814) 189-190 3.3.1.2 Anti-lipopolysaccharide factor (ALF-2) (HM366921) 190-192 3.3.1.3 Fi-Crustin (GQ469987) 192-195 3.3.1.4 Fi-penaeidin (HM243617) 195-198 3.3.1.5 Penaeidin-5 (HM243616) 198-199 3.3.2 Control / Reference genes 199 3.4 Discussion 199-205 Tables 205-210 Figures 211-237 4 Time-course of antimicrobial peptide gene expression in response to White spot syndrome virus challenge 238-283 4.1 Introduction 238-248 4.1.1 Antiviral immune response 243-248 4.2 Materials and Methods 249-251 4.2.1 Experimental animals and rearing conditions 249 4.2.2 Sampling 249-250 4.2.3 WSSV challenge 250 4.2.4 Haemolymph collection 250 4.2.5 Total RNA isolation and reverse transcription 250 4.2.6 Semi-quantitative RT-PCR analysis of target gene expression 250-251 4.3 Results 251-255 4.3.1 Expression profile of control genes in the haemocytes of P. monodon in response to WSSV challenge 251 4.3.2 Expression profile of AMP genes in the haemocytes of P. monodon