UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE GENOMIC INVESTIGATION OF CNIDARIAN TOXIN EVOLUTION A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By ANUJ GURUACHARYA Norman, Oklahoma 2017 GENOMIC INVESTIGATION OF CNIDARIAN TOXIN EVOLUTION A DISSERTATION APPROVED FOR THE DEPARTMENT OF BIOLOGY BY ______________________________ Dr. Richard Broughton, Chair ______________________________ Dr. Lawrence Weider ______________________________ Dr. JP Masly ______________________________ Dr. Michael Markham ______________________________ Dr. Cecil Lewis © Copyright by ANUJ GURUACHARYA 2017 All Rights Reserved. Dedication To my family and friends. Acknowledgement I would like to thank Richard Broughton for his helpful advice throughout my education. He has also helped me through one year of research assistantship and provided a space and motivation to work. I would like to thank Christian Lemon, who has been my friend and helped me better understand statistics in a biological concept. I would also like to thank David Durica, who has listened to some of my work and been supportive. I would like to thank Larry Weider for his very helpful comments on the early draft of the first two chapters and his moral support. I would also like to thank JP Masly, who has provided me some useful advice, which I unfortunately, forgot to heed. I would also like to thank my advisors Michael Markham and Cecil Lewis, who agreed to serve in my committee, despite their busy schedule. I would also like to thank Dahang Yu and Si Wu, who helped me a lot of the proteomics portions of the project and also helped me in writing portions of the manuscript for proteomics. I would like to thank my friend Binita Rajbanshi for discussing most of chapter 3 with me and helping me in the formatting of the document. I would like to thank Sharmistha Shyamal for listening to me create my initial hypothesis and showing me wet lab procedures. I have benefitted from conversations with her as equally as she has benefitted from me. I would like to thank Krithi Sankanarayan for discussing some of the procedures for my initial studies. iv Table of Contents Acknowledgement ........................................................................................................... iv List of Tables ................................................................................................................... ix List of Figures ................................................................................................................... x Abstract ............................................................................................................................ xi Introduction ...................................................................................................................... 1 Chapter 1: Co-evolution of ion channels and neurotoxins in cnidarians .......................... 3 Abstract ....................................................................................................................... 3 Introduction ................................................................................................................ 3 Methods ...................................................................................................................... 8 Data collection ...................................................................................................... 8 Species tree ........................................................................................................... 9 Gene tree-species tree reconciliation .................................................................... 9 Gene presence/absence analysis ......................................................................... 10 Selection analysis ............................................................................................... 10 Analysis based on evolutionary rates of gene trees ............................................ 11 Data access ......................................................................................................... 11 Results ...................................................................................................................... 12 Species tree ......................................................................................................... 12 Occurrence of toxin and ion channel genes among species ............................... 13 Gene tree-species tree reconciliation .................................................................. 13 Selection analysis ............................................................................................... 14 v Correlation analysis of gene presence/absence .................................................. 15 Analysis of evolutionary rates ............................................................................ 17 Discussion ................................................................................................................. 17 Rapid evolution of parasitic cnidarians .............................................................. 19 Ion channels and neurotoxins may have co-evolved in an evolutionary arms race ................................................................................................................ 20 Conclusion ................................................................................................................ 22 References ................................................................................................................ 23 List of Tables ............................................................................................................ 32 List of Figures ........................................................................................................... 32 List of Supplementary Information .......................................................................... 32 Chapter 2: Transcriptomic and proteomic analyses of toxin composition in clownfish- host and non-host sea anemones ......................................................................... 33 Abstract ..................................................................................................................... 33 Introduction .............................................................................................................. 34 Methods .................................................................................................................... 37 Sample collection and dissection ........................................................................ 37 DNA extraction and COX-1 sequencing ............................................................ 38 mRNA extraction and sequencing ...................................................................... 38 Transcriptome assembly and annotation ............................................................ 39 Protein extraction from sea anemone tissue samples ......................................... 40 Bottom-up MS/MS analysis ............................................................................... 40 Results ...................................................................................................................... 42 vi COX1 barcode sequencing ................................................................................. 42 Transcriptome analysis ....................................................................................... 42 Identification of Toxin proteins .......................................................................... 44 Proteome analysis ............................................................................................... 47 Discussion ................................................................................................................. 49 References ................................................................................................................ 54 List of Tables ............................................................................................................ 62 List of Figures ........................................................................................................... 63 List of Supplementary Information .......................................................................... 63 Chapter 3: Tissue-specific differential toxin gene expression analysis of toxins in two sea anemones ...................................................................................................... 64 Abstract ..................................................................................................................... 64 Introduction .............................................................................................................. 65 Methods .................................................................................................................... 67 Sample collection and dissection ........................................................................ 67 Transcriptome assembly and annotation ............................................................ 68 Differential gene expression analysis of toxins .................................................. 69 Results ...................................................................................................................... 70 Sequencing, assembly, and annotation ............................................................... 70 Mapping, abundance, and Gene Ontology (GO) analysis .................................