Host-microbe interactions in brine shrimp Artemia cultured under gnotobiotic and conventional rearing conditions Niu Yufeng Thesis submitted in fulfillment of the requirements for the degree of Doctor (PhD) in Applied Biological Sciences: Environmental Technology Part of the work done during the PhD is being submitted as a patent application. Therefore, non UGent jury members will be asked to sign a confidential disclosure agreement, and all draft versions of the manuscript are to be treated as confidential material. Promoter: Prof. Dr. ir. Peter Bossier (Ghent University); Prof. Dr. ir. Tom Van de Wiele (Ghent University); Prof. Dr. Shuanglin Dong (Ocean University of China) Yufeng Niu. Host-microbe interactions in brine shrimp Artemia cultured under gnotobiotic and conventional rearing conditions. Dissertation, Ghent University, Belgium. Dutch translation of the title: Gastheer-microbe interacties in het pekelkreeftje Artemia in gnotobiotische en conventionele condities ISBN: 978-90-5989-709-0 The author and the promoters give the authorisation to consult and copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. Yufeng Niu was funded by the Chinese Scholarship Council (CSC) and Special Research Fund (BOF) of Ghent University (BOF12/DC1/013; 01DI1312) TABLE OF CONTENTS LIST OF ABBREVIATIONS AND UNITS ................................................................................................... I PART I INTRODUCTION AND LITERATURE REVIEW CHAPTER 1 INTRODUCTION AND THESIS OUTLINE 1. INTRODUCTION .................................................................................................................................... 1 2. THESIS OUTLINE ................................................................................................................................. 23 CHAPTER 2 MICROBIAL MANAGEMENT FOR SUSTAINABLE AQUACULTURE: NEEDS FOR MORE KNOWLEDGE ON HOST-MICROBE INTERACTION 1. INTRODUCTION .................................................................................................................................. 25 2. SUMMARY ........................................................................................................................................ 41 PART II ARTEMIA-MICROBE INTERACTION ASSOCIATED WITH PROBIOTICS CHAPTER 3 A METHOD FOR THE SPECIFIC DETECTION OF RESIDENT BACTERIA IN BRINE SHRIMP LARVAE ARTEMIA FRANCISCANA 1. INTRODUCTION .................................................................................................................................. 44 2. MATERIALS AND METHODS ................................................................................................................... 44 3. RESULTS AND DISCUSSION .................................................................................................................... 49 CHAPTER 4 BACILLUS SP. LT3 IMPROVES THE SURVIVAL OF GNOTOBIOTIC BRINE SHRIMP (ARTEMIA FRANCISCANA) LARVAE CHALLENGED WITH VIBRIO CAMPBELLII BY ENHANCING THE INNATE IMMUNE RESPONSE AND BY DECREASING THE ACTIVITY OF SHRIMP-ASSOCIATED VIBRIOS 1. INTRODUCTION .................................................................................................................................. 57 2. MATERIALS AND METHODS .................................................................................................................. 58 3. RESULTS ........................................................................................................................................... 65 4. DISCUSSION ...................................................................................................................................... 72 PART III ARTEMIA-MICROBE INTERACTION ASSOCIATED WITH HEAT SHOCK PROTEIN INDUCING COMPOUND CHAPTER 5 EFFECT OF HEAT SHOCK PROTEIN INDUCER--TEX-OE® ON BRINE SHRIMP ARTEMIA FRANCISCANA LARVAE UNDER SUB-OPTIMAL REARING CONDITION 1. INTRODUCTION .................................................................................................................................. 81 2. MATERIALS AND METHOD .................................................................................................................... 83 3. RESULT ............................................................................................................................................. 88 4. DISCUSSION ...................................................................................................................................... 96 CHAPTER 6 A PLANT-BASED HEAT SHOCK PROTEIN INDUCING COMPOUND MODULATES HOST-PATHOGEN INTERACTIONS BETWEEN ARTEMIA FRANCISCANA AND VIBRIO CAMPBELLII 1. INTRODUCTION ................................................................................................................................ 105 2. MATERIALS AND METHODS ................................................................................................................. 107 3. RESULT ........................................................................................................................................... 112 4. DISCUSSION .................................................................................................................................... 119 PART IV DISCUSSION AND CONCLUSION CHAPTER 7 GENERAL DISCUSSION, CONCLUSION AND FUTURE PERSPECTIVES 1. INTRODUCTION AND MAJOR RESULTS .................................................................................................... 125 2. PROBIOTICS ELICIT HOST IMMUNE RESPONSES ........................................................................................ 127 3. FUTHER INVESTIGATION ON PROBIOTICS MODE OF ACTION ........................................................................ 133 4. SUMMARY ...................................................................................................................................... 138 5. POSSIBLE FUTURE WORK .................................................................................................................... 139 PART V APPENDIX APPENDIX A SUPPLEMENTAL TABLES & FIGURES TABLE: CADIDATES FOR HOUSEKEEPING GENE OF ARTEMIA FRANCISCANA ......................................................... 147 FIGURE: LUMINAL AND RESIDENT GUT MICROBIAL COMMUNITY OF ARTEMIA AFTER THE PRETREATMENT OF HSPI (TEX-OE®) ........................................................................................................................................... 148 APPENDIX B REFERENCES REFERENCES ....................................................................................................................................... 149 APPENDIX C SUMMARY/SAMENVATTING SUMMARY .......................................................................................................................................... 181 SAMENVATTING ................................................................................................................................... 184 APPENDIX D CURRICULUM VITAE CURRICULUM VITAE .............................................................................................................................. 187 PART VI ACKNOWLEDGEMENT ACKNOWLEDGEMENT ........................................................................................................................... 191 LIST OF ABBREVIATIONS AND UNITS °C Degree celsius % Percentage μg Microgram μL Microlitre g Relative centrifugal force or G force ± Approximately / Per β-glucan Beta-glucans AID Activation-induced cytidine deaminase ANOVA Analysis of variance BFT Biofloc technology C3 Complement component 3 cDNA Complementary deoxyribonucleic acid cfu Colony forming unit DABCO 1,4-diazobicyclo-2.2.2-octane DCs Dendritic cells DNA Deoxyribonucleic acid FAO Food and Agricultural Organization of the United Nations i FASW Filtered and autoclaved seawater FTS Flow through system g Gram g/L Gram per liter GART Gnotobiotic Artemia test system GenBank Genetic sequence database of the National Institute of Health, USA GF Germ free h Hour Hsps Heat shock proteins Hspi Heat shock protein inducer IL1 Interleukin 1 ILCs Innate lymphoid cells iNKT Invariant natural killer T cells L Litre LAB Lactic Acid Bacteria LB Luria-Bertani LPS Lipopolysaccharides LVS3 Bacterial strain isolated by Laurent Verschuere ii MA Marine agar 2216 MC Microbiota MCC Microbiota composition MCF Mucoid colony forming mRNA ribonucleic acid MHC Major Histocompatibility Complex NMCF Non-mucoid-colony forming NK cells Natural killer cells OD Optical density P Statistical p-value PCR Polymerase chain reaction PD-1 Programmed cell death-1 PO Phenoloxidase proPO Prophenoloxidase PSA Polysaccharide A RAS Recirculating aquaculture system RT-PCR Real time-polymerase chain reaction rpm Rotations per minute SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis iii SFB Segmented filamentous bacteria SPF Specific pathogen-free TLR Toll like receptor TNF Tumor necrosis factor Th17 cells T helper cells 17 Treg Regulatory T cells iv PART I INTRODUCTION AND LITERATURE REVIEW CHAPTER 1 Introduction and thesis outline Introduction and thesis outline 1. Introduction World’s aquaculture status The world population of 7.2 billion in mid-2013 is projected to increase by almost one billion people within the next twelve years, according to official United Nations population estimates. This drastic population expansion is placing higher pressure on the daily demand of