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Examining the Roles of Environment, Evolution, and Genetics in Insect Mortality Dana Blackburn Brigham Young University

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Examining the Roles of Environment, Evolution, and Genetics in Insect Mortality Dana Blackburn Brigham Young University Brigham Young University BYU ScholarsArchive All Theses and Dissertations 2015-12-01 Virulence of Photorhabdus spp.: Examining the Roles of Environment, Evolution, and Genetics in Insect Mortality Dana Blackburn Brigham Young University Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Biology Commons BYU ScholarsArchive Citation Blackburn, Dana, "Virulence of Photorhabdus spp.: Examining the Roles of Environment, Evolution, and Genetics in Insect Mortality" (2015). All Theses and Dissertations. 6163. https://scholarsarchive.byu.edu/etd/6163 This Dissertation is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in All Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Virulence of Photorhabdus spp.: Examining the Roles of Environment, Evolution, and Genetics in Insect Mortality Dana Blackburn A dissertation submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Byron J. Adams, Chair David I. Shapiro-Ilan Joel S. Griffitts Michael F. Whiting Joshua A. Udall Department of Biology Brigham Young University December 2015 Copyright © 2015 Dana Blackburn All Rights Reserved ABSTRACT Virulence of Photorhabdus spp.: Examining the Roles of Environment, Evolution, and Genetics in Insect Mortality Dana Blackburn Department of Biology, BYU Doctor of Philosophy Entomopathogenic nematodes (EPNs) (genera Heterorhabditis and Steinernema) kill their invertebrate hosts with the aid of a mutualistic bacterium. The bacteria (Xenorhabdus spp. for steinernematids and Photorhabdus spp. for heterorhabditids) are primarily responsible for killing the host and providing the nematodes with nutrition and defense against secondary invaders. Photorhabdus is a Gram-negative bacterium in the Enterobacteriaceae family with high virulence towards their insect hosts. To achieve high mortality rates Photorhabdus produces a variety of virulence factors such as toxins, lipases, proteases, secretion systems, and fimbriae. EPNs are amenable to laboratory rearing and mass production for biocontrol applications against insects using in vivo or in vitro methods; however, in vitro liquid culture is considered to be the most efficient. In this method the symbiotic bacteria are cultured prior to the addition of their partner EPN. This can leave the bacteria susceptible to a number of problems such as genetic drift and inadvertent selection. Regardless of the culture method the symbiotic bacteria exhibit trait deterioration or changes due to laboratory rearing. This project had three primary aims: 1) investigate the role of nutrition in trait deterioration, 2) examine virulence evolution using a phylogenetic context, and 3) identify genes that are necessary for survival and virulence inside the insect host. Prior to studying these objectives we first determined the optimal conditions for growing and counting viable cells of Photorhabdus. We discovered that growth is enhanced by the addition of pyruvate to growth media. To determine the role of nutrition in trait deterioration we repeatedly sub-cultured Photorhabdus in three different media types. Throughout this study we found that, in contrast to previous studies, trait deterioration does not always happen and the environment influences trait deterioration. Furthermore, based on our phylogenetic studies we found that Photorhabdus spp. are evolving to an increase in insect virulence. Lastly, using Tn- seq we determined a list of 84 genes that are needed for efficient virulence inside the insect host and provide suggestions for ongoing research efforts. Keywords: entomopathogenic nematodes, Photorhabdus, trait deterioration, nutrition, Tn-seq, evolution ACKNOWLEDGEMENTS To my family who has always supported me, listened to me, believed in me, and loved me. I could not have done this without them. I would especially like to thank my mom for teaching me the importance of hard work and a good education. She gave me her everything and I am grateful for her continued presence in my life. I would like to express my sincere gratitude to my committee. They have been invaluable to my graduate work and career. David Shapiro-Ilan and Joel Griffitts have been amazing collaborators that have helped me with ideas, protocols, and troubleshooting. Michael Whiting and Joshua Udall have provided valuable feedback on projects and ideas along the way. To my advisor, Byron Adams, I cannot express enough gratitude. He believed in me from the beginning and gave me the confidence to keep moving forward. This degree would not have been possible without him. Furthermore, I would like to thank John Chaston for teaching me new methods, providing advice, helping with funding, and being my friend. Ed Wilcox has also helped me with all of my sequencing questions. I thank him for always taking the time to answer my questions and helping me with protocols. Additionally, I would like to thank all of the undergraduates that have worked in the Adams’ lab. Many have assisted me with my project and all have shown great patience with me. Lastly, I would like to thank all of my friends that have been there along the way making my life a little happier and a little more fun. TABLE OF CONTENTS TITLE PAGE .............................................................................................................................................................. i ABSTRACT ............................................................................................................................................................... ii ACKNOWLEDGEMENTS .................................................................................................................................... iii TABLE OF CONTENTS ........................................................................................................................................ iv LIST OF TABLES................................................................................................................................................... vii LIST OF FIGURES ............................................................................................................................................... viii Introduction ............................................................................................................................................................ 1 References ............................................................................................................................................................................. 6 Chapter 1 ............................................................................................................................................................... 12 Abstract ................................................................................................................................................................................ 13 Introduction........................................................................................................................................................................ 14 Mass Production .......................................................................................................................................................... 16 Nutritional Effects on Biocontrol Traits ................................................................................................................. 17 Survival/Tolerance..................................................................................................................................................... 17 Infectivity/Virulence ................................................................................................................................................. 20 Reproductive Potential ............................................................................................................................................. 22 Growth and Size ........................................................................................................................................................... 26 Longevity ........................................................................................................................................................................ 26 Conclusions ......................................................................................................................................................................... 27 References ........................................................................................................................................................................... 29 Chapter 2 ............................................................................................................................................................... 42 Abstract ................................................................................................................................................................................ 43 Introduction........................................................................................................................................................................ 44 Materials and Methods ..................................................................................................................................................
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