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The Use of a Targeted Omic Approach to Dissect the Nested Symbiosis of the Eastern Subterranean Termite, Reticulitermes Flavipes

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The Use of a Targeted Omic Approach to Dissect the Nested Symbiosis of the Eastern Subterranean Termite, Reticulitermes Flavipes University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 8-28-2019 The seU of a Targeted Omic Approach to Dissect the Nested Symbiosis of the Eastern subterranean termite, Reticulitermes Flavipes Michael Stephens University of Connecticut - Storrs, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Stephens, Michael, "The sU e of a Targeted Omic Approach to Dissect the Nested Symbiosis of the Eastern subterranean termite, Reticulitermes Flavipes" (2019). Doctoral Dissertations. 2304. https://opencommons.uconn.edu/dissertations/2304 The Use of a Targeted Omic Approach to Dissect the Nested Symbiosis of the Eastern subterranean termite, Reticulitermes flavipes Michael Eric Stephens Jr. PhD University of Connecticut 2019 The Eastern subterranean termite, Reticulitermes flavipes is a member of lower wood- feeding termites and feeds on lignocellulose (wood) in nature. The ability to survive on this hard- to-digest, nutrient-poor diet relies on a division of labor between the host termite and its hindgut microbiota. Key players of this symbiotic digestive strategy include a community of various protist species and their associated, prokaryotic, endo- and ectosymbionts. These protists aid the hydrolysis of the various polysaccharides found in wood and it is thought that their bacterial symbionts contribute to a number of processes that are regarded as essential in the termite’s hindgut. The inability to currently culture these organisms has hindered our ability to understand these complex interactions and their contributions to the digestion of wood. By focusing on these protist-associated communities of bacteria and leveraging the sensitivity and coverage of high-throughput sequencing, this complex hindgut community can be dissected and studied to reveal ecological, metabolic, and evolutionary processes that have previously evaded us. This body of work, described in this thesis, aides in our understanding of these communities by first describing the community structures and member transmission trends and then investigating their gene content and expression of both endo- and ectosymbionts. These data have led to precise hypotheses about these protist-associated communities, some of which I was able to test and support. For example, endosymbiotic Endomicrobium species have acquired Michael Eric Stephens Jr.- University of Connecticut 2019 many genes related to their carbon metabolism by horizontal gene transfer, perhaps through a competence pathway which was both conserved and expressed. Due to the complexity of this hindgut community these targeted analyses, and future extensions of similar types, are crucial to our pursuit of understanding of the underlying processes that occur in the termite’s hindgut and how they relate to the efficient digestion of wood. The Use of a Targeted Omic Approach to Dissect the Nested Symbiosis of the Eastern subterranean termite, Reticulitermes flavipes Michael Eric Stephens Jr. B.S., University of North Florida, 2012 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2019 i Copyright by Michael Eric Stephens Jr. 2019 ii APPROVAL PAGE Doctor of Philosophy Dissertation The Use of a Targeted Omic Approach to Dissect the Nested Symbiosis of the Eastern subterranean termite, Reticulitermes flavipes Presented by Michael Eric Stephens Jr., B.S. Major Advisor ___________________________________________________________________ Daniel J. Gage Associate Advisor ___________________________________________________________________ Joerg Graf Associate Advisor ___________________________________________________________________ Johann Peter Gogarten Associate Advisor ___________________________________________________________________ Robertson Thane Papke Associate Advisor ___________________________________________________________________ Spencer Nyholm University of Connecticut 2019 iii Acknowledgements I would first like to thank Dr. Daniel Gage for his mentorship and guidance over these last six years. He taught me how to think critically about my research, as well as dedicated a lot of time towards understanding and troubleshooting the omic approaches that we used in these studies. I could not have done this without him. I would also like to thank all the members of my thesis committee, Dr. Joerg Graf, Dr. Spencer Nyholm, Dr. Peter Gogarten, Dr. Thane Papke, and Dr. Jonathan Klassen who set aside time to meet with me over these last six years and helped refine my research by both challenging me and directing me towards better experimental designs. I cannot thank them enough. I would also like to thank the other members of the lab, Charles Bridges, Jamie Micciulla, and Gabrielle Corso, who have been not only great colleges but also amazing friends. I would also like to thank all the past lab members who encouraged me during the beginning of my graduate studies. I would also like to thank all the undergraduate researchers who I had the pleasure of working with during my time here at UConn. I also would like to thank all of my peers who began this journey with me back in 2013, especially Andrea Suria who has been a great friend over this last six years. I couldn’t have done this without her continued support and insights. I would like to thank my family who have always encouraged and supported my childhood dream of becoming a “mad scientist”. They taught me to value education and to pursue my passion, even if they didn’t quite understand what I was passionate about. I also would like to thank my fiancé André Beringhs, who has been by my side over the last several years and has offered constant support and encouragement. iv Table of Contents Chapter One: Introduction……………………………………………………………………1 General Termite Biology and Research Interests……………………………………….............1 Digestion of Wood and Symbiotic Cooperation in Termites…………………………...………2 Protists and Bacterial Symbionts of Termite Hosts…………………………………………......4 An Overview of the Use of Omics to Study Symbioses………………………………...............7 Scope of Dissertation…………………………………………………………………………...10 Chapter Two: Amplicon Sequence Variant (ASV) Inference Reveals Community Structures and Transmission Trends of Protist-Associated Bacteria in the Termite Reticulitermes flavipes…………………………………………………………………………14 Abstract………………………………………………………………………………………...15 Introduction……………………………………………………………………………………16 Materials and Methods………………………………………………………………………..19 Termite Collection, Maintenance, and Identification...……………………………….………...19 Amplification and Sequencing of Protist and Bacterial SSU rRNA Genes…………..….……..21 V4 16S Amplicon Analysis Using ASV Inference……………………………………….….....22 Phylogenetic Analysis of SSU rRNA Genes……………………………………………….…...22 Scanning Electron Microscopy…………………………………………………………..……...22 Live Fluorescent Symbiont Transmission Assays………………………………...…………….23 Results……………………………………………………………………………...………….. 25 Morphological and Phylogenetic Diversity of Hindgut Protists…………………………...……25 ASV Composition of Protist-Associated Bacteria………………………………………………25 Phylogenetic Diversity of Protist-Associated Endomicrobium…………………………………27 Horizontal Transmission of Ectosymbionts………………………………………….………….27 Ectosymbionts in the Free-Living Fraction……………………………………………………..30 Discussion…………………………………………………………………………….…...…….31 Chapter Three: Endosymbionts of Protist Hosts Use Gene Flow to Acquire Niche Specific Traits in the Nested Symbiosis of Termites………………………………....64 Abstract……………………………………………………………………………………...…65 v Introduction……………………………………………………………………………………65 Material and Methods………………………………………………………………………....68 Single Protist Cell Isolation……………………………………………………………………..68 Whole Genome and Transcriptome Amplification……………………………………………...68 Library Preparation and Sequencing…………………………………………………………….69 Genomic Read Processing and Assembly……………………………………………………….69 Genomic Binning, Draft Genome Assessment, and Annotation…………………………….…..70 Analysis of Ribosomal Gene Phylogeny and Average Nucleotide Identities…………….……..71 Detection of Horizontally Acquired Genes……………………………………………….……..72 Analysis of Genes Involved in Competence and Recombination…………………………….....72 Mapping Transcriptome Reads to Draft Genomes………………………………………………73 Verification of comEC Expression by Reverse Transcriptase PCR (RT-PCR)…………………73 Results…………………………………………………………………………………………..74 Phylogeny of Protists Hosts ...…………………………………………………………………..74 Endomicrobium Genome Statistics and Speciation……………………………………………..75 Biosynthesis of Amino Acids, Vitamins, and Peptidoglycan…………………………………...76 Oxidative Stress Response and Aerotolerance………………………………………………….77 Differences in Carbon Metabolism……………………………………………………………..78 Natural Transformation (Competence) as a Putative Mechanism for Acquiring Genes….….…80 Discussion……………………………………………………………………………………....81 Chapter Four: Analysis of Ectosymbiotic Treponema Communities Supports Their Role in Essential Supportive and Hydrolytic Functions in Wood-Feeding Termites……………..106 Abstract………………………………………………………………………………………..107 Introduction…………………………………………………………………………………...107 Materials and Methods……………………………………………………………………….111 Termite Collection and Identification………………………………………………………….111 Protist Isolation and Whole-Genome and Whole-Transcriptome Amplification……………...111 Library Preparations and Sequencing………………………………………………………….112 Metagenome Assembly, Genomic Binning, and Annotation…………………………………..113
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