Distinct Bacterial Composition Associated with Different Laboratory-cultured Aiptasia Strains Across Two Thermal Conditions Thesis by Hanin Ibrahim Ahmed In Partial Fulfillment of the Requirements For the Degree of Master of Science King Abdullah University of Science and Technology Thuwal, Kingdom of Saudi Arabia May, 2018 1 EXAMINATION COMMITTEE PAGE The thesis of Hanin Ibrahim Ahmed is approved by the examination committee. Committee Chairperson: Manuel Aranda Committee Members: Christian R Voolstra, Xosé Anxelu G. Morán 2 © May, 2018 Hanin Ibrahim Ahmed All Rights Reserved 3 ABSTRACT Distinct bacterial composition associated with different laboratory-cultured Aiptasia strains across two thermal conditions Hanin Ibrahim Ahmed Coral reefs are crucial for the ecological sustainability of the oceans, yet, increasing sea surface temperature is threatening these ecosystems globally. Microbial communities associated with corals have become a recent research focus, as the associated microbiome may contribute to coral resilience to environmental stressors, e.g., heat stress. However, research in this area is hampered by the difficulty of working with corals. This study aims to use Aiptasia, a sea anemone, as a tractable laboratory model system to study the role of the coral microbiome. Analyses of the bacterial compositions associated with different Aiptasia strains across two temperatures (25 °C and 32 °C), based on 16S rRNA gene sequencing. This study aims also to identify a “core” microbiome associated with heat stress acclimation, as well as host-specific differences. In general, results showed that bacterial composition associated with Aiptasia strains differs significantly with temperature. Higher bacterial diversity and richness were observed when all Aiptasia strains were placed under heat stress. Moreover, results showed an increase in beta diversity and dispersion of bacterial communities in response to heat stress. These changes in the bacterial composition are in line with the recently described “Anna Karenina principle” for animal microbiomes, which suggests that the microbiomes of unhealthy individuals vary more than healthy and stable individuals. This study further shows that while temperature had the greatest effect on structuring the bacterial compositions, there were some variations better attributed to batch and host effects. This suggests that technical aspects have to be carefully addressed in the framework of microbiome studies. Members of a putative “core” microbiome associated with 32 °C Aiptasia have been identified as indicator species of heat stress (i.e., Francisella sp.,). Previous reports have shown that these indicator taxa are associated with saline environments and can tolerate high temperatures. Putative functional profiles based on taxonomic inference of associated bacterial taxa (i.e., enrichment and depletion of various metabolic processes) were also identified, implying functional differences of the microbiomes associated with Aiptasia strains in response to heat stress. Future studies 4 should more specifically examine how the microbiome influences the animal ability to respond to environmental changes. 5 ACKNOWLEDGEMENTS First and foremost, I would like to offer my sincere gratitude to my supervisor Prof. Manuel Aranda who has supported me throughout my research. I am very grateful for the continuous support of my thesis, for his patience, motivation and knowledge. I attribute the level of my master degree to his assistance and effort. His office was always open whenever I had questions about my research or even any unrelated questions. Beside my supervisor, I would like to express my gratitude to the members of examination committee; Prof. Christian Voolstra who gave useful advice that helped in addressing some of the results, and Prof. Xelu Moran for taking the time to examine the thesis. Special thanks to Marcela Herrera for her guidance in all the time of the research and help in writing this thesis. She spent a lot of time to teach me and encourage me. I am very thankful to Dr. Yi Jin Liew for his support, help and assistance. My thanks also go to all my fellow labmates, for their friendly smiles and a hello every time we met. I also want to extend my gratitude to all my friends who had a good influence on my life. I also would like to thank my colleagues and the department faculty for making my time great at KAUST. The bioscience core lab has provided the support and equipment I have needed to produce and complete my research. Finally, I want to send my deepest gratitude to my parents and to my sisters for providing me with continuous support and encouragement throughout my higher studies. 6 TABLE OF CONTENTS EXAMINATION COMMITTEE PAGE ............................................................................................. 2 ABSTRACT ................................................................................................................................... 3 ACKNOWLEDGEMENTS .............................................................................................................. 6 TABLE OF CONTENTS.................................................................................................................. 7 LIST OF ABBREVIATIONS ............................................................................................................ 8 LIST OF FIGURES ......................................................................................................................... 9 LIST OF TABLES ......................................................................................................................... 10 1. INTRODUCTION ................................................................................................................ 11 1.1 Coral reefs in a changing environment ....................................................................................... 11 1.2 The coral holobiont ..................................................................................................................... 11 1.3 Coral-associated microbial communities and environmental stressors .................................... 15 1.4 Aiptasia sp. as model organism to study the coral holobiont .................................................... 16 2. PROJECT OBJECTIVES, RATIONALE AND SIGNIFICANCE ....................................................... 19 3. MATERIALS AND METHODS ................................................................................................. 20 3.1 Aiptasia Rearing .......................................................................................................................... 20 3.2 DNA extraction ........................................................................................................................... 20 3.3 16S rRNA gene amplification and sequencing ............................................................................ 21 3.4 Sequencing data processing ....................................................................................................... 21 3.5 Bacterial community analysis ..................................................................................................... 23 4. RESULTS ................................................................................................................................ 25 4.1 Distinct bacterial microbiomes of Aiptasia and rearing water ................................................... 25 4.2 Temperature has the largest effect on the microbiome ............................................................ 28 4.3 Aiptasia reared at higher temperatures display greater microbiome variability ....................... 33 4.4 Aiptasia “core” microbiome responds to heat stress ................................................................ 36 4.5 Host-specific microbiomes ......................................................................................................... 42 4.5.1. Red Sea Aiptasia microbiome ................................................................................................. 42 5. DISCUSSION .......................................................................................................................... 46 5.1 The microbiome of Aiptasia and rearing water .......................................................................... 46 5.2 Temperature has an effect on the microbiome compositions ................................................... 47 5.3 Treatment has the highest effect on structuring the bacterial community ............................... 49 5.4 “Core” microbiome and indicator taxa ....................................................................................... 50 5.5 Taxonomy-based functional profiling of bacterial communities change with temperature ..... 52 6. CONCLUSION ........................................................................................................................ 54 7. REFERENCES ......................................................................................................................... 55 8. APPENDICES ......................................................................................................................... 64 7 LIST OF ABBREVIATIONS AMOVA Analysis of Molecular Variance CaCO3 Calcium carbonate DMSP Dimethylsulfoniopropionate DMSO Dimethyl sulfoxide DSCs Dimethyl Sulfate Compounds OTU Operational taxonomic unit PCoA Principal Coordinate Analysis ppt Parts per thousand PSU Practical salinity unit ROS Reactive Oxygen Species