A holobiont characterization of reproduction in a live-bearing cockroach, Diploptera punctata A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biological Sciences of the McMicken College of Arts and Sciences By Emily C. Jennings B.S. Neurobiology, University of Cincinnati, April 2013 Committee Chair: Joshua B. Benoit, Ph.D. Abstract Viviparous reproduction is characterized by maternal retention of developing offspring within the reproductive tract during gestation, culminating in live birth. In some cases, a mother will provide nutrition beyond that present in the yolk; this is known as matrotrophic viviparity. While this phenomenon is best associated with mammals, it is observed in insects such as the viviparous cockroach, Diploptera punctata. Female D. punctata carry developing embryos in the brood sac, a reproductive organ that acts as both a uterus and placenta by protecting and providing a nutritive secretion to the intrauterine developing progeny. While the basic physiology and hormonal changes of D. punctata pregnancy have been characterized, little else is known about this phenomenon. This study attempts to broaden the understanding of D. punctata reproduction by using a multi-omics approach to characterize multiple aspects of the holobiome. First, I utilized RNA-seq analysis to characterize molecular changes associated with D. punctata reproduction and provides the most complete gene set to date for this species. A comparison of four stages of the female reproductive cycle revealed unique gene expression profiles corresponding to each stage. Differentially regulated transcripts of interest include the previously identified family of milk proteins, transcripts associated with juvenile hormone metabolism, and other reproduction-associated transcripts. I next utilized 16S rRNA gene sequencing to characterize the microbiome of Diploptera punctata during development, from embryonic development to female adulthood. We identified 50 phyla and 121 classes overall and found that mothers and their developing embryos had significantly different microbial communities, with embryos harboring only a single endosymbiont known as Blattabacteria. Our analysis of postnatal development reveals i that significant amounts of non-Blattabacteria species are not able to colonize newborn D. punctata until melanization in the first instar, after which the microbial community rapidly and dynamically diversifies. This rapid change of the microbial community appears to stabilize after the second juvenile stage with no major changes even through the last molt to adulthood for females. Lastly, I sought to better understand the role of the cockroach specific endosymbiont Blattabacteria as the sole member of the D. punctata embryonic microbial community. We leveraged next generation genome sequencing to computationally characterize the metabolic capacity of Blattabacteria isolated from D. punctata. Our analysis produced a 670,273 base pair chromosome with a high degree of similarity to previously sequenced genomes of Blattabacteria isolated from other cockroach species. Our genome assembly contained 593 putatively protein coding DNA sequences, which included proteins required for nearly all key metabolic processes including DNA replication, transcription, and translation. Additionally, the D. punctata strain of Blattabacteria also holds the ability to synthesize nearly all 10 essential amino acids and process nitrogenous waste to do so. This suggests that Blattabacteria are likely providing nutritional supplementation to intrauterine developing embryos to accommodate the previously reported lack of methionine and tryptophan in maternally provided nutrition. Together, the research presented in this dissertation contributes to a more holistic understanding of this human-like reproductive mode in the cockroach Diploptera punctata, broadening our understanding of viviparity throughout the animal kingdom. ii © Copyright Emily C Jennings 2019 All Rights Reserved iii Acknowledgements First and foremost, I want to thank my advisor, Dr. Joshua Benoit, for going out on a limb and taking me on as one of two graduate students the same year he started as a new faculty member at UC. He provided constant support and exhibited unfathomable patience as I stumbled through the last six years in his lab. He encouraged me to think not only critically as a scientist but creatively, encouraging me to take risks on wild experiments like modeling fetal alcohol syndrome in cockroaches. When some of those risks resulted in failed experiments Josh was always there to help me bounce back, just as he was always there to casually offer kudos when something did manage to work out. Josh helped keep me on track when I started to lose focus and dealt with my constant questions. He never questioned my decisions to enroll in courses outside of traditional biology curriculum, whether it was a course about birth defects, or an undergraduate level python programming course. When I realized that I was interested in at least trying a career outside of academia after graduating, he never wavered in his support even when I felt like a quitter. Josh was always open to and encouraged discussions about my project, which sometimes turned into fun debates about where the experiment was going or needed to go. What wasn’t always fun was when one of us had to admit we were wrong, but Josh was a good sport and never gloated too much when I was wrong even though I wasn’t always so kind when he was wrong. It’s been a wonderful six years in the lab, and I know I’ll miss it when I’m gone. I’d also like to acknowledge and thank Dr. Elke Buschbeck, Dr. Joshua Gross, Dr. Mike Polak, and Dr. Matthew Weirauch, for being part of this crazy journey as members of my research advisory committee. They have offered invaluable guidance and support, helping me iv to navigate and focus my disjointed project ideas, overcome the roadblocks of failed experiments and helping to know when it was time to let go of a stalled-out project. Through it all, I’ve always been able to count on them to be in my corner of the ring. In addition to serving on my advisory committee, Dr. Elke Buschbeck has played an especially important role in this journey. I’d like to thank her for gambling on hiring me, a weird bug fanatic in her neuroscience course, as a work study back in 2011. She has consistently served as a mentor and role model, helping me grow as a scientist. I cannot begin to describe the value of her constant encouragement and support, letting me know when I was too hard on myself but also making sure to ask if I had something better to do when she noticed I was slacking. I want to extend my gratitude to all of the current and former members of the Benoit lab for their constant support, listening to me whine when I needed to, dealing with me talking to myself at the lab bench or in our shared office space, and keeping things fun in and out of lab. This work would not be possible without the undergraduate students who have been part of our lab throughout the last 6 years. LaVeta Burke, Luke Bernhardt, John Cavanaugh, Jake Hendershot, Matthew Korthauer, Erica McDonald, and Sophie Shemas have all made contributions to my research, running countless experiments. Working with them has taught me so much about mentoring, communication, and leadership. It has been an honor to have worked with them. Christopher Holmes and Elise Didion deserve special recognition. I cannot thank them enough for being wonderful lab mates and two of my best friends. Without them this process would have been much harder and very lonely. v I am so grateful for the patience and support from my family over the last six years. They never complained when I missed countless birthdays and holidays because I was working or studying, instead welcoming me home with open arms every chance they got. My parents and step-parents have been supportive, kind, and forgiving beyond any reasonable expectation. Without them, my education would have been impossible. They believed in me when I couldn’t believe in myself, pushed me to constantly be better and strive for more than I thought I could. I would like to recognize the immense support of my running family. Not only have they been there for me when I needed a friend, but they have listened to me talk about all aspects of graduate school ad nauseum (sometimes for 26.2 miles). I am grateful for the unique perspective and advice that each one of them has provided me over the last two years. In addition to coaching me through three full marathons, countless training runs, and smaller races, they have also coached me throughout the last two years of my PhD, a very different kind of marathon. Samantha Isler deserves more recognition than I can give her here, meeting her dancing in the rain back in high school might be one of the greatest things that has ever happened to me. I cannot imagine my life without her, and I cannot thank her enough for being my best friend for all of these years. She has been there for me through every high and every low, my light in the dark. Lastly, I need to thank my husband, Justin. He has loved me when I was a nightmare, always understood how much my research means to me, and forgiven me when I put him second to science. He has always supported all of my wildest dreams, even when I changed my mind constantly about what those were. Justin is constantly lifting me up, assuring me that I am vi enough, and that I deserve to be where I am. I’m fairly certain that I wouldn’t be able to accomplish anything without him, including this degree.