Graduate Theses, Dissertations, and Problem Reports 2016 Microbial Dynamics within Shed Mucosal Secretions of Hirudo verbana, the European Medicinal Leech Brittany Maree Ott Follow this and additional works at: https://researchrepository.wvu.edu/etd Recommended Citation Ott, Brittany Maree, "Microbial Dynamics within Shed Mucosal Secretions of Hirudo verbana, the European Medicinal Leech" (2016). Graduate Theses, Dissertations, and Problem Reports. 6361. https://researchrepository.wvu.edu/etd/6361 This Dissertation is protected by copyright and/or related rights. It has been brought to you by the The Research Repository @ WVU with permission from the rights-holder(s). You are free to use this Dissertation in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you must obtain permission from the rights-holder(s) directly, unless additional rights are indicated by a Creative Commons license in the record and/ or on the work itself. This Dissertation has been accepted for inclusion in WVU Graduate Theses, Dissertations, and Problem Reports collection by an authorized administrator of The Research Repository @ WVU. For more information, please contact [email protected]. Microbial Dynamics within Shed Mucosal Secretions of Hirudo verbana, the European Medicinal Leech by Brittany Maree Ott Dissertation submitted to the Eberly College of Arts and Sciences at West Virginia University in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology Rita V. M. Rio, Ph.D., Chair Nyles Charon, Ph.D. Andrew Dacks, Ph.D. Stephen DiFazio, Ph.D. Jennifer Hawkins, Ph.D. Department of Biology Morgantown, West Virginia 2016 Keywords: symbiosis, transmission, leech, Aeromonas veronii, mucus, Illumina sequencing, RNA-seq © 2016 Brittany M. Ott ABSTRACT Microbial Dynamics within Shed Mucosal Secretions of Hirudo verbana, the European Medicinal Leech Brittany Ott Microbes are widespread throughout our planet, residing in soils, oceans, and even beneath the arctic glaciers. Most interact with each other and other life forms, and there is no known complex organism that lacks an associated microbiome. These microbial symbionts are critical to the survival and proliferation of their host by assisting in nutrient provisioning, physiological development, immunological priming, providing protection from pathogens, contributing to predator evasion, etc. As such, the transmission of these symbionts is a crucial aspect of host biology. Despite a growing appreciation for the prevalence of mixed transmission (incorporating vertical, or from a parental route, and horizontal, or environmental, mechanisms) for establishing microbial symbioses, features that enable these infections are not well understood. My work investigated the mechanistic basis of symbiont acquisition by the European medicinal leech, Hirudo verbana. In the leech cocoon, only a portion of the albumenotrophic larvae obtain their beneficial gut symbiont, the Gammaproteobacterium Aeromonas veronii. However, by early adulthood, all leeches harbor this bacterium, indicating the complementation of incomplete vertical transmission by an unknown horizontal mechanism. Insight from a number of other organisms, aquatic and terrestrial, suggests that host-secreted mucus may provide a transmission vehicle. Through the use of genetically- tractable A. veronii, I demonstrated that cyclical host mucosal secretions are seeded by digestive tract symbionts. Using quantitative PCR, I verified that A. veronii are not only viable, but also proliferate within mucosal casts at a frequency synchronous to host shedding. Subsequent experiments using mucus inoculated with a gfp-expressing A. veronii demonstrates that mucosal contact is sufficient for symbiont transmission to novel hosts. Importantly, behavior assays show that leeches are attracted to these castings, providing an efficient mechanism for transmission of A. veronii between conspecifics. Additional behavioral assays show that host symbiont-state does not influence attraction towards mucus and that symbiont content of the mucus does not alter attraction, suggesting that A. veronii exploits a preexisting host physiological process. Lastly, Illumina-based RNA-seq was used to identify how the A. veronii transcriptome, with regards to metabolism and information processing, responds to the lifestyle shift from mutualistic within the leech digestive tract to free-living within shed mucus. This dual mode of symbiont transmission may prove evolutionarily advantageous, as it not only ensures the infection of leeches by beneficial symbionts, but also provides accessibility to a higher genetic diversity of symbionts and biome lifestyle options to A. veronii beyond that of mutualism. Additionally, examination of the composition of the mucosal microbial community utilizing two separate culture-independent sequencing techniques (i.e. Sanger-sequenced 16S rRNA clone libraries and Illumina deep- sequencing of the V3-V4 hypervariable region of the 16S rRNA gene) revealed that a diverse microbiota resides within the mucus, consisting of both previously-described and potentially novel leech symbionts. Additional transcriptomic analyses indicate that this microbial community actively engages in cell-to-cell communication via quorum sensing and potential DNA transfer. In depth genomic analyses also prove that a novel microbial species, Pedobacter, is a major mucosal microbiota member worthy of future study. Understanding the features that enable mixed transmission, particularly those involving mixed species assemblages, may prove instrumental for the development of strategies to promote beneficial symbioses. Acknowledgements I would first, and foremost, like to thank my incredible Doctoral advisor, Rita Rio, Ph.D., for all of the support and guidance that you have given me over the years, both professionally and personally. Back in 2009, when I completed the summer REU in your lab, I was still debating whether I wanted to continue into research. However, those ten weeks encouraged me to pursue research as my career. When I returned for graduate school, I continued to learn about what it was to be a scientist. I appreciate your pushing me beyond my limits, because it showed me how strong I can be and how much I can accomplish. I also thank you for supporting the decisions I have made for my future; it means more than you can know. I want to also thank my committee members, Nyles, Jen, Steve and Andrew. I appreciate all of your questions, advice and suggestions, as they have strengthened my research and pushed it to meet the high standards that you hold. I would also like to graciously thank all those who helped contribute to my thesis research. I would first like to thank our collaborators at the University of Connecticut in Dr. Joerg Graf’s lab. Your assistance by answering my many questions and providing critical bacterial strains has greatly helped progress my research. I could not have done many of my experiments without your help. I would next like to thank Anna Snyder for all of the late nights we spent in the lab and in our office. I will forever appreciate your opinions and advice that helped me through the tough moments of difficult experiments, as well as difficult times in my life. Additionally, this work would not be possible without the assistance of many past and present lab members, so I would like to thank; Vivian and Adam, as well as many undergraduate students who assisted with so many of my experiments and ideas, specifically Michael, Grace, Noelle, James and Allen. The time spent with you in the lab has helped me become a better researcher and mentor, as well as colleague and friend, and has helped with both my research and peace of mind. While most of my time as a graduate student has been spent in research, I still met with challenges associated with being a graduate student. So I would like to thank Mickey, Diana, Chuck, Judy, Pat and Wendy in the Biology Department for helping make my time here a little bit easier. I would also like to thank the Biology Department graduate students, both past and present, who have helped me make it through each day; I would especially like to thank Micah, Mohna, Dhanu, Kristyn, Jessie Brie, Jen, Tyler and Luke for helping me out with the research- related, graduate student-related, and life-related issues that I have encountered. I would like to thank Katrina Stewart for helping with all of the issues I encountered as a first-time teaching assistant, as I struggled to understand how to teach students not much younger than myself. I would also like to thank Dana Huebert-Lima for helping me go above and beyond as a teacher, and guiding me to find the right balance between strict and lenient, caring and distant, so I could be the best I could be for my students. Finally, I would like to thank my friends Lauren, Alan, Carol, Matt, Samantha, Kate and Kyle for always understanding and remaining my friends as I would occasionally drop off the face of the Earth as my work engulfed me. I want to thank my mum and dad for ALWAYS giving me unwavering support through this whole endeavor. And last, but certainly not least, I want to thank Alex, my rock, my love, and my partner. Your continued support and faith in me have helped me through the good times and the bad. You are my other half, and as long as we have each other, we can make it through anything. iii Table of Contents ABSTRACT II ACKNOWLEDGEMENTS III LIST OF FIGURES VI