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Signature: __________________________________________________ ____________ Amanda Jo Williams-Newkirk Date Bacterial Community Ecology of the Lone Star Tick (Amblyomma americanum) By Amanda Jo Williams-Newkirk Doctor of Philosophy Graduate Division of Biological and Biomedical Science Population Biology, Ecology and Evolution _________________________________ _________________________________ Gregory A. Dasch, Ph.D. Thomas R. Gillespie, Ph.D. Advisor Advisor _________________________________ _________________________________ Nicole M. Gerardo, Ph.D. Uriel D. Kitron, Ph.D., M.P.H. Committee Member Committee Member _________________________________ _________________________________ Timothy D. Read, Ph.D. James Taylor, Ph.D. Committee Member Committee Member Accepted: ______________________________________ Lisa A. Tedesco, Ph.D. Dean of the James T. Laney School of Graduate Studies ________________ Date Bacterial Community Ecology of the Lone Star Tick (Amblyomma americanum) By Amanda Jo Williams-Newkirk A.B., Bryn Mawr College, 2004 M.S., The University of Southern Mississippi, 2008 Advisor: Gregory A. Dasch, Ph.D. Advisor: Thomas R. Gillespie, Ph.D. An abstract of A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Division of Biological and Biomedical Science Population Biology, Ecology and Evolution 2015 ABSTRACT Bacterial Community Ecology of the Lone Star Tick (Amblyomma americanum) By Amanda Jo Williams-Newkirk Vector-borne diseases dominate the list of emerging infections from the last century. Among arthropod vectors, ticks (Acari, Ixodida) transmit a greater diversity of disease causing organisms than any other known order. In the southeastern United States, the lone star tick (Amblyomma americanum) is the most abundant and aggressive human- biting tick and a vector of multiple known or suspected pathogens, including Rickettsia, Ehrlichia, Borrelia, and Heartland virus. The long life cycle of A. americanum and its broad host range complicate studies aimed at understanding the ecology of these diseases. Furthermore, abundant symbiotic bacteria, many of which are closely related to known pathogens, are a part of the tick’s microbial community. Studies in this and other systems have robustly demonstrated that the non-pathogenic component of a vector’s microbiome is capable of affecting the ability of the arthropod to acquire, maintain, and transmit pathogens to vertebrates. As a result, it is now recognized that pathogen ecology must be described in the context of the vector and the vector’s microbiome. The aim of my dissertation was to provide the necessary context for further studies of A. americanum disease ecology. In my survey of A. americanum bacterial communities, I characterized the bacteria from 131 ticks collected from five sites in three states distributed throughout the species’ eastern range. I then compared A. americanum’s bacterial community to that of sympatric tick species of varying relatedness to determine if species with the opportunity to share hosts also shared similar microbiota and to look for evidence of the evolutionary history of the ticks in their bacterial communities. During these studies I detected multiple genotypes of a novel symbiont, Candidatus ‘Midichloria mitochondrii’, in two Amblyomma species. In addition, I provided the first reference mitochondrial genome sequence for A. americanum, a tool that will be very important to future population genetics studies of this tick. My work provides a foundation for future studies in A. americanum by quantifying both intra and interspecific differences in bacterial communities, detecting new members of the bacterial community, and describing interactions between members which may now be targeted for characterization, metabolic evaluation, and functional studies. Bacterial Community Ecology of the Lone Star Tick (Amblyomma americanum) By Amanda Jo Williams-Newkirk A.B., Bryn Mawr College, 2004 M.S., The University of Southern Mississippi, 2008 Advisor: Gregory A. Dasch, Ph.D. Advisor: Thomas R. Gillespie, Ph.D. A dissertation submitted to the Faculty of the James T. Laney School of Graduate Studies of Emory University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate Division of Biological and Biomedical Science Population Biology, Ecology and Evolution 2015 ACKNOWLEDGEMENTS I was extremely fortunate to have had the support of two advisors while at Emory University. Dr. Gregory Dasch of the Rickettsial Zoonoses Branch (RZB) at the Centers for Disease Control and Prevention (CDC) provided me with an indescribable amount of professional and personal support. I will always be grateful to him for providing me with an amazing environment in which to develop as a scientist. Dr. Thomas Gillespie gave me a space at Emory to call home, and I thank him for the many opportunities to work with the incredible undergraduate students in his laboratory. I am also very grateful to my dissertation committee members: Drs. Nicole Gerardo, Uriel Kitron, Timothy Read, and James Taylor. They were all exceedingly generous with their time and knowledge, and it would have been extremely difficult to design and execute such an interdisciplinary dissertation without their support. Funding for this work was provided by many generous organizations. The RZB at CDC supported me from 2010 – 2014 through an Oak Ridge Institute for Science and Education Fellowship. In 2013 – 2014, I was supported by a National Institutes of Health Training Grant awarded to the Emory Graduate Program in Population Biology, Ecology and Evolution. I was supported in 2014 – 2015 by the Association of Public Health Laboratories Bioinformatics in Public Health Practical Fellowship. Additionally, in 2013 I was awarded a National Science Foundation Doctoral Dissertation Improvement Grant to support a portion of my tick ecology research. I was also fortunate in 2009 to receive funding from the Emory Global Health Institute to support tick collections in Uganda. I thank all of these organizations for their support of my research and my education. I was assisted during my dissertation work by many undergraduate students to whom I am grateful for their hard work and endless enthusiasm for all things tick-related. I thank Ellen Dugan, Deema Elchoufi, Ian Fried, Dana Greenlee, Jasmine Hensley, Ho Joon Hwang, Benjamin Kramer-Roach, Gwen Parker, Karen Wu, Audrey Zeis, and Gene Zhou. I made many friends at both Emory and CDC that brought much joy and many laughs to my studies, in addition to contributing their substantial intellectual might to my work. Many thanks to Johanna Salzer, Maria Zambrano, Ryan Lash, Shari Lydy, Sandor Karpathy, Marina Eremeeva, Lori Rowe, and Seth Sims. I also thank all of the members of the RZB for their constant support and encouragement throughout this process. It would have been impossible for me to reach this goal without the support, guidance, and example of my parents, Kim and Jimmie Williams. I will always be thankful for their love and belief in me. Finally, I thank my husband, Rob Newkirk, for always saying “I want you to be whatever you want to be” and going above and beyond every day to make that promise a reality. TABLE of CONTENTS CHAPTER 1 pg. 1 Introduction: The evolution of tick microbiome research 1.1 Known effects of the tick microbiome on disease ecology pg. 2 1.2 Next generation sequencing as a tool for descriptive and experimental microbiome studies pg. 6 1.3 Figures pg. 10 1.4 References pg. 11 CHAPTER 2 pg. 19 Presence, genetic variability, and potential significance of "Candidatus Midichloria mitochondrii" in the lone star tick Amblyomma americanum 2.1 Summary pg. 19 2.2 Introduction pg. 20 2.3 Materials and Methods pg. 22 2.4 Results pg. 25 2.5 Discussion pg. 26 2.6 Figures pg. 30 2.7 Tables pg. 33 2.8 References pg. 35 CHAPTER 3 pg. 40 Characterization of the bacterial communities of life stages of free living lone star ticks (Amblyomma americanum) 3.1 Summary pg. 40 3.2 Introduction pg. 41 3.3 Materials and Methods pg. 44 3.4 Results pg. 52 3.5 Discussion pg. 57 3.6 Figures pg. 66 3.7 Tables pg. 84 3.8 References pg. 134 CHAPTER 4 pg. 151 Sex and species phylogenies influence the bacterial communities of four sympatric North American tick species 4.1 Summary pg. 151 4.2 Introduction pg. 152 4.3 Materials and Methods pg. 155 4.4 Results pg. 161 4.5 Discussion pg. 165 4.6 Figures pg. 176 4.7 Tables pg. 193 4.8 References pg. 231 CHAPTER 5 pg. 241 The mitochondrial genome of Amblyomma americanum 5.1 Summary pg. 241 5.2 Introduction pg. 241 5.3 Materials and Methods pg. 244 5.4 Results and Discussion pg. 250 5.5 Conclusions pg. 256 5.6 Figures pg. 257 5.7 Tables pg. 283 5.8 References pg. 285 CHAPTER 6 pg. 293 Strengths, limitations, and conclusions 6.1 Key Findings and Strengths pg. 293 6.2 Limitations pg. 298 6.3 Future Directions pg. 301 6.4 Figures pg. 303 6.5 References pg. 304 TABLE of FIGURES Figure 1.1: Generalized ixodid tick three host life cycle and natural transmission of pathogens. pg. 10 Figure 2.1: Phylogenetic tree of Midichloria mitochondrii 16S rRNA gene fragments obtained from Amblyomma americanum and related sequences from GenBank.