Nova Southeastern University NSUWorks HCNSO Student Theses and Dissertations HCNSO Student Work 5-1-2013 De novo Transcriptome Analysis of the Marine Sponge Cinachyrella spp: A Potential Model Organism for Oil and Dispersant Ecotoxicology Emily Smith Nova Southeastern University, [email protected] Follow this and additional works at: https://nsuworks.nova.edu/occ_stuetd Part of the Marine Biology Commons, and the Oceanography and Atmospheric Sciences and Meteorology Commons Share Feedback About This Item NSUWorks Citation Emily Smith. 2013. De novo Transcriptome Analysis of the Marine Sponge Cinachyrella spp: A Potential Model Organism for Oil and Dispersant Ecotoxicology. Master's thesis. Nova Southeastern University. Retrieved from NSUWorks, Oceanographic Center. (141) https://nsuworks.nova.edu/occ_stuetd/141. This Thesis is brought to you by the HCNSO Student Work at NSUWorks. 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NOVA SOUTHEASTERN UNIVERSITY OCEANOGRAPHIC CENTER De novo transcriptome analysis of the marine sponge Cinachyrella spp: A potential model organism for oil and dispersant ecotoxicology By Emily Smith Submitted to the Faculty of Nova Southeastern University Oceanographic Center in partial fulfillment of the requirements for the degree of Master of Science with a specialty in: Marine Biology Nova Southeastern University May, 2013 1 Thesis of Emily Smith Submitted in Partial Fulfillment of the Requirements for the Degree of Masters of Science: Marine Biology Nova Southeastern University Oceanographic Center May 2013 Approved: Thesis Committee Major Professor: _____________________________ Dr. Jose Lopez, Ph.D. Committee Member: ___________________________ Dr. Patricia Blackwelder, Ph.D. Committee Member: ___________________________ Dr. David Willoughby, Ph.D. Committee Member: ___________________________ Dr. Scott Nichols, Ph.D. 2 TABLE OF CONTENTS ACKNOWLEDGEMENTS....................................................................................... 5 ABSTRACT............................................................................................................... 7 List of Figures............................................................................................................ 8 List of Tables............................................................................................................. 10 List of Abbreviations................................................................................................. 11 INTRODUCTION..................................................................................................... 13 Marine Sponges as Bioindicators.................................................................. 13 Cinachyrella spp............................................................................................ 16 The Harmful Effects of Crude Oil in the Marine Environment..................... 20 Dispersant Toxicity in the Aquatic Environment................................……... 24 The Toxic Effects of Oil and Dispersant Mixtures on Marine Organisms.... 26 Illumina RNA Sequencing and Transcriptome Analysis................................ 28 Bioinformatics……………………………………………………………… 34 HYPOTHESES and OBJECTIVES........................................................................... 38 MATERIALS and METHODS..................................................................................39 Collection and Preparation of Sponges……………………………………. 39 Preparation of WAF, CE-WAF and Dispersant-Only Solution……………. 41 Sponge Exposure to Macondo Crude Oil and Corexit 9500………………. 44 Total RNA Isolation, Purification and RNA Quality Scoring ……………... 45 Illumina cDNA Library Construction and RNA-Sequencing………………. 49 De novo Transcriptome Assembly…………………………………………. 51 Bioinformatics……………………………………………………………… 53 RESULTS.................................................................................................................. 55 RNA Quality Scoring………………………………………………………..55 Illumina RNA Sequences and De Novo Transcriptome Assembly…………. 59 Hierarchical Gene Clustering using Cluster 3.0…………………………... 62 Principal Component Analysis using NIA Array Analysis Tool…………… 67 KEGG Orthology System, BRITE Hierarchies, and Pathway Assignments...69 DISCUSSION............................................................................................................ 98 Transcriptome Sequences………………………………………………….. 98 Gene Cluster and Expression Analysis ……………………………………. 99 Principal Component Analysis ……………………………………………. 107 KEGG BRITE Hierarchies ………………………………………………… 108 Metabolism ………………………………………………………… 109 Genetic Information Processing…………………………………... 111 Environmental Information Processing……………………………. 112 Cellular Processes…………………………………………………. 112 KEGG Pathway Analysis…………………………………………………... 113 Metabolism Pathways……………………………………………… 113 Genetic Information Processing Pathways………………………… 116 Environmental Information Processing Pathways………………… 117 Cellular Processing Pathways …………………………………….. 120 Organismal Systems Pathways ……………………………………. 122 Human Disease Pathways ………………………………………….125 Challenges…………………………………………………………………..129 3 TABLE OF CONTENTS Improvements and Future Directions……………………………………… 131 CONCLUDING REMARKS..................................................................................... 132 WORKS CITED........................................................................................................ 134 APPENDICES........................................................................................................... 146 4 ACKNOWLEDGEMENTS I would like to thank my major advisor, Dr. Joe Lopez, for giving me the opportunity to work on the BP project as my master’s thesis and as a research assistant in his lab. Thank you for allowing me to dive Florida’s coral reefs as part of my research, funding travel and diving adventures in Central America to learn about marine sponges, and for introducing me to the wonderful people I’ve met along the way. In addition, a special thanks to Dr. Pat Blackwelder, Dr. Scott Nichols, and Dr. David Willoughby for serving on my thesis committee and lending their expertise. Dr. Blackwelder, thank you for your unwavering lab support, technical advice, and always helpful comments and guidance. Dr. Nichols, thank you for your thorough evaluation and insightful suggestions during my proposal and thesis writing process. Dr. Willoughby, sincere thanks for your critical input during the transcriptome assembly and helpful counsel with data analysis. Thank you to The Smithsonian Tropical Research Institute and The Porifera Tree of Life Project for hosting and funding the Taxonomy and Ecology of Caribbean Sponges class and supplying funding for my sponge species taxonomy work for the BP project. Funding for the project was provided by a Florida Institute of Oceanography $200K grant from a $10M block grant awarded from BP and an NSU Chancellor’s Faculty Research and Development grant awarded to Dr. Joe Lopez. I would also like to thank my lab mates, Dawn Formica, Jigu Patel, Andia Chaves-Fonnegra, Rebecca Mulheron, and Alexandra Campbell. Dawn, thank you for the boisterous laughter and hilarity in lab; you made frustrating days fun. Jigu, thank you for your mischief and playful teasing; I will always miss your wit. Andia, thank for your 5 constant advice and support in lab. You are one of the most knowledgeable people I know about marine sponges – thank you for imparting your sponge knowledge! To Rebecca, thank you tremendously for your help with the BP project. You’ve been an asset for both the sponge transcriptome and bacterial metagenomic work. Alexandra, thank you for your help with the aquaculture set up and RNA work . A special thank you to my best friend, Katie Strickland; without your kind and supportive words, late night text messages, and dinner date Skype sessions, I would be overstressed and in tears. To my loving, comical, and sometimes quite irritating boyfriend, Cory Blake, thank you for your support as I worked tirelessly to finish my degree. To my dear friend, Angela Loson, thank you for always cheering me on; I can always count on you! Last, but certainly not least, I would like to thank my family: Dad, Mom, and sister, Kristin. I could not have quit my job and moved almost 800 miles away from home to follow my dream without your love, support, and money. Dad, thank you for always pushing me to be a better person; I owe all of my success to you. To my Mom, thank you for always lending an ear and giving me sound advice; if it weren’t for you, I would be defeated. To my loving sister, Kristin, thank you for teaching me patience and kindness; you taught me to take things day by day. Finally, a warm thank you to my belated MeMa who always believed in the importance of education; I dedicate the completion of this thesis to you. 6 ABSTRACT In order to study the potential effects of an oil spill on coral reef organisms, the marine sponge, Cinachyrella spp. was investigated. In this study, Cinachyrella spp. was placed in a closed aquaculture system and exposed to sub-lethal water-accommodated fractions (WAFs) of Macondo crude oil and chemically-enhanced water accommodated fractions (CE-WAFs) of the dispersant, Corexit 9500, over a 24-hour time course, in order to model the BP Deepwater Horizon oil spill and oil spill sponge response. Illumina RNA sequencing and gene expression analysis utilizing hierarchical