AN ABSTRACT OF THE DISSERTATION OF Ehren J. Bentz for the degree of Doctor of Philosophy in Integrative Biology presented on March 20, 2019. Title: Characterizing the Function of the Harderian Gland and its Interactions with the Vomeronasal Organ in the Red-sided Garter Snake, Thamnophis sirtalis parietalis. Abstract approved: __________________________________________________________________ Robert T. Mason The Harderian gland is a large cephalic gland present in most groups of terrestrial vertebrates. Although the Harderian gland has been the focus of numerous studies for more than 300 years, its physiological function has remained largely unresolved. Harderian gland secretions are diverse among different taxa, and many putative functions have been ascribed to this gland. The Red-sided garter snake (Thamnophis sirtalis parietalis) displays strong seasonal shifts in behavior – mating occurs immediately after emergence from hibernation in the spring and abruptly shifts to feeding several weeks later. These behaviors are mutually exclusive and coincide with sexually dimorphic physical changes to the Harderian gland. Male Harderian glands are hypertrophied upon emergence as male snakes use their vomeronasal chemosensory system to actively search for females expressing sexual attractiveness pheromone, whereas the gland in recently emerged females remains regressed and quiescent. Here, I use garter snakes as a novel model to investigate the function of the Harderian gland, and through the use of modern molecular techniques, describe the mechanisms by which this historically enigmatic gland functions within the vomeronasal chemosensory system. Using high-throughput sequencing and bioinformatic analyses, I examine the functional characteristics of the Harderian gland transcriptome (a collection of all genes which are expressed as mRNA) to describe a general physiological function of this tissue. I describe patterns of variation by sex and season of the genes expressed in the Harderian gland as well as of chemosensory receptor proteins expressed in the vomeronasal organ. Additionally, I use protein mass spectrometry to identify and characterize the functions of proteins present in the secretions of the vomeronasal organ and use an integrated analysis using protein mass spectrometry and RNA-sequencing to infer which proteins within that fluid are likely to be primarily produced in the Harderian gland. The Harderian gland was found to express an abundance of genes associated with lipid-binding proteins and proteins involved antimicrobial defense. Expression within these two categories of genes is significant compared to other tissues. This suggests that gene products produced in this tissue likely function to bind and solubilize lipids and act as component of the immune system within the vomeronasal chemosensory system. The Harderian glands of male snakes were found to be more transcriptionally active in the spring compared to females. Male glands were also found to express more secretory and lipid-binding proteins compared to females throughout the year. Females in the spring were found to express genes involved in stress responses suggesting that they may respond differently to stress than males. These glands in both males and females were found to express genes involved in porphyrin metabolism – a well-described characteristic of the Harderian glands in rodents. This is the first description of porphyrin metabolism in a squamate but was observed only in the summer feeding time period. I found no evidence that the expression of antimicrobial defense proteins varies by either sex or season. Proteins identified in the fluid of the vomeronasal organ were found to contain an abundance of lipocalins (lipid-binding proteins) and extracellular immune proteins. I conducted in-vitro bacteria killing assays demonstrating that this fluid has potent antimicrobial properties. Tissue-specific expression showed that a large proportion of the identified lipocalins and antimicrobial proteins are produced in the Harderian gland and secreted into the vomeronasal organ. Expression of vomeronasal chemosensory receptors showed sexually dimorphic seasonal variation. Males express receptors from early spring and throughout summer. Female snakes expressed very few receptors during the spring mating period, suggesting that their vomeronasal chemosensory system is relatively inactive while mating. The vomeronasal receptor repertoire did not appear to vary greatly by season in either males or females. An important protein of interest was identified which was expressed highly in the male Harderian gland while being nearly absent in those of females. This protein is identified as a lipocalin lipid-binding protein and a likely candidate for a putative pheromone-binding protein facilitating the solubilization and subsequent detection of the female sexual attractiveness pheromone. The findings presented here demonstrate that the Harderian gland is an integral component of the vomeronasal chemosensory system in the Red-sided garter snake functioning both to facilitate the detection of chemical signals and as a component of the extracellular immune system protecting the sensitive vomeronasal sensory epithelium from environmental pathogens. ©Copyright by Ehren J. Bentz March 20, 2019 All Rights Reserved Characterizing the Function of the Harderian Gland and its Interactions with the Vomeronasal Organ in the Red-sided Garter Snake, Thamnophis sirtalis parietalis By Ehren J. Bentz A DISSERTATION submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Presented March 20, 2019 Commencement June 2019 Doctor of Philosophy dissertation of Ehren J. Bentz presented on March 20, 2019. APPROVED: Major Professor, representing Integrative Biology Chair of the Department of Integrative Biology Dean of the Graduate School I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Ehren J. Bentz, Author ACKNOWLEDGEMENTS I would like to thank my daughter Jessica for the incredible amount of support and understanding she has shown me throughout the years. I know it was never easy to be the child of a single father who is in the midst of a doctoral degree program. Jessica, you are an amazing young woman. I grow more proud of you every day. No more puzzles! But, maybe someday we’ll get a chance to conduct some more Y-maze experiments in a sweltering barn. I would also like to extend my deepest and most sincere thanks and appreciation to my parents Gay and Brad Bentz who have supported my every decision and allowed me to become the person I am today. I can say with confidence that I would not have accomplished a fraction of what I have, and I would not be completing this degree without their love and encouragement. I also want to thank my amazing partner Rebecca Amantia who has loved me and supported me through this process. Through long days, and working late into the night, you never once complained, even though some days I didn’t even get around to putting on pants. Thank you! I love you all! I want to thank my doctoral advisor, Dr. Robert T. Mason, for his support and encouragement during my time as a graduate student. Even when experiments devolved into hopeless catastrophes, you continued to provide intellectual and financial support and allowed me freedom to continue pursuing my interests in science. Additionally, I would like to thank my committee members: Dr. Felipe Barreto and Dr. Jeff Anderson for their contributions both to my research and my development as a graduate student. Dr. Jean Hall who performed admirably as my graduate council representative, and Dr. Eli Meyer without whom I would not have been able to perform the various molecular and bioinformatic pursuits on which my dissertation research rests. I would also like to thank my lab partners Dave Hubert and Leslie Blakemore who have been there every step of the way. Whether I wanted to talk research or go on an impromptu snake-catchin’ canoe adventure on the Santiam river. We mutually helped each other retain our sanity (or some of it at least) on cross country drives and crossing international borders in a van filled with hundreds of snakes. Oh, sweet Canada. Last, but certainly not least, I would like to thank the Integrative Biology staff Tara Bevandich, Tresa Bowlin, Traci Durell-Khalife, Trudy Powell, and Torri Givigliano, and Jane Van Order who provided administrative support throughout my degree program. I don’t know how you do everything you do, but you do it extremely well. The degrees of many, many students have been made possible by your efforts. Thank you all! The research presented here was partially funded by the National Science Foundation Graduate Research Fellowship Program (NSF-GRFP), NSF grant 0620125, the J. C. Braly Natural History fund, and the Jack Kent Cooke Foundation. High throughput nucleotide sequencing was performed by the Genomics & Cell Characterization Core Facility at University of Oregon, Eugene, OR; The Massively Parallel Sequencing Shared Resource (MPSSR) at Oregon Health & Science University, Portland, OR; and the Center for Genome Research and Biocomputing (CGRB), Oregon State University, Corvallis, OR. Protein mass spectrometry was performed at the Oregon State University Mass Spectrometry Center, Corvallis, OR on the Orbitrap Fusion Lumos instrument provided by NIH grant # 1S10OD020111-01.