WHAT’S INSIDE? AN EXAMINATION OF THE CHARACTERISTIC MICROBIOME OF PRAIRIE GRASSHOPPERS AND KATYDIDS Thesis Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Master of Science in Biology By Melani K Muratore Dayton, OH May 2020 WHAT’S INSIDE? AN EXPLORATION OF THE MICROBIOME OF PRAIRIE GRASSHOPPERS AND KATYDIDS Name: Muratore, Melani K APPROVED BY: Yvonne Sun, Ph.D. Faculty Advisor Assistant Professor Department of Biology Chelse Prather, Ph.D. Committee Member Associate Professor Chair, Department of Biology Ryan W. McEwan, Ph.D. Committee Member Associate Professor Department of Biology ii © Copyright by Melani K Muratore All rights reserved 2020 iii ABSTRACT WHAT’S INSIDE? AN EXPLORATION OF THE MICROBIOME OF PRAIRIE GRASSHOPPERS AND KATYDIDS Name: Muratore, Melani K University of Dayton Advisor: Dr. Yvonne Sun There are many ways in which microbiomes can influence the health and fitness of their insect hosts. While much research has been done on the microbiomes of economically important species like honeybees, and charismatic groups like butterflies, little work has been done to understand the microbiome of grasshoppers and katydids. Grasshoppers are an important herbivore in grassland ecosystems and provide important ecosystem services like nutrient cycling. Alternatively, grasshoppers can be a pest organism requiring management and control. In relationship to the total ecosystem, the limitation of abiotic factors like essential nutrients can influence the abundance and fitness of insect herbivores such as grasshoppers. However, the effect of these abiotic factors on grasshopper microbiomes is largely unknown. Using culture-independent, high-throughput 16s and ITS sequencing and statistical analysis, we examined the gut microbiome of six species of grasshoppers (n=60) from the site of a fully-factorial fertilization experiment in a coastal tallgrass prairie ecosystem in order to gain a better understanding of the microbial communities present across the orthopteran order in this ecosystem. We also examined the relationship between environmental nutrients and gut microbes of one orthopteran species, Orchelimum vulgare. Our observations support the iv hypothesis that there is a “core” group of bacterial families in these grasshopper species and factors such as trophic behaviors and the evolution of the host may contribute to the shifts in prevalence among these core microbial groups. We also found significantly higher Shannon diversity in the gut bacterial communities of those grasshoppers from plots fertilized with added sodium in contrast to plots without sodium. There is significantly lower diversity in gut fungal communities from plots amended with nitrogen and phosphorus, as well as sodium, suggesting that both nitrogen and phosphorus and sodium are limiting nutrients for the fungal community. There was also a strong positive correlation between bacterial and fungal diversity within each sample. These results support the hypothesis that environmental nutrients will alter the characteristic gut microbiome of O. vulgare, though these alterations will be different for bacterial and fungal communities. v ACKNOWLEDGMENTS I would like to express my appreciation to my advisor, Dr. Yvonne Sun, who first gave me the opportunity to do research in her lab in the Summer of 2017. She has been a supportive mentor, giving me many opportunities to learn and lots of help and direction. She is someone who is enthusiastic about learning new things and collaborating with other scientists. She has a great love of teaching and a desire to share her knowledge and help her students succeed. She is one of the best instructors that I have had. It is her desire to collaborate and explore new ideas to which I owe my association with Dr. Chelse Prather. I am grateful for Dr. Prather’s knowledge, patient guidance, and her enthusiasm for her work. I am also grateful for the good advice and kind assistance of Dr. Ryan McEwan, who served as a member of my committee. I would also like to express my gratitude to the other members of the Biology department past and present who have offered their help to me over the last several years. I am very grateful for the other students who have shared their knowledge and friendship with me, especially Amanda Finke, Michaela Woods, and Julia Chapman who all helped with coding. I am grateful to Erica Rhinehart for her help in the lab. I am most grateful for the support of my family, most of all my husband, Chris. Ever since I decided to go “back to school,” you have all been behind me, encouraging me to persist. This research would not have been possible without funding from the University of Dayton Graduate Student Summer Fellowship, a University of Dayton STEM catalyst grant, and a Keck Environmental Fellowship. vi TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iv ACKNOWLEDGMENTS ................................................................................................. vi LIST OF FIGURES ......................................................................................................... viii LIST OF TABLES ............................................................................................................. ix INTRODUCTION .............................................................................................................. 1 CHAPTER 1 THE GUT BACTERIAL COMMUNITIES ACROSS SIX SPECIES FROM A COASTAL TALLGRASS PRAIRIE ................................................................. 8 Abstract .......................................................................................................................... 8 Introduction ................................................................................................................... 9 Materials and Methods ............................................................................................... 11 Results .......................................................................................................................... 13 Discussion..................................................................................................................... 15 Conclusion ................................................................................................................... 21 Acknowledgements ..................................................................................................... 22 References .................................................................................................................... 22 CHAPTER 2 EFFECTS OF ENVIRONMENTAL NUTRIENTS ON BACTERIAL AND FUNGAL MICROBIOMES IN THE COMMON MEADOW KATYDID, ORCHELIMUM VULGARE ............................................................................................. 32 Abstract ........................................................................................................................ 32 Introduction ................................................................................................................. 33 Methods ........................................................................................................................ 36 Results .......................................................................................................................... 39 Discussion..................................................................................................................... 42 Conclusion ................................................................................................................... 46 References .................................................................................................................... 47 vii LIST OF FIGURES Figure 1. Mean relative abundance of bacterial phyla in the gut of six grasshopper species……………………………………………….……….………………………….54 Figure 2. Mean relative abundance of prevalent bacterial families in the gut of six grasshopper species. ……………………………………………..……………………..55 Figure 3. Beta diversity comparison of bacterial communities…………………………56 Figure 4. Comparison of the gut microbiome in male and female grasshoppers……….57 Figure 5. Average relative abundance of microbial phyla in grasshopper samples from four treatments…………………………………………………………………………..58 Figure 6. Average relative abundance of microbial taxa in grasshopper samples from four treatments……………………………………….…………………………….59 Figure 7. Nonmetric multidimensional scaling of grasshopper gut community assemblages at the family level………………………………………………………….60 Figure 8. Analysis of diversity over four treatments………………………………….…61 Figure 9. An analysis of shared groups among the four treatments……………………..62 viii LIST OF TABLES Table 1. Gut bacterial richness and diversity for six grasshopper species……………....63 Table 2. Gut microbiome richness and diversity for four treatments……………………64 ix INTRODUCTION Over the last decade, advances in culture-independent methods have led to an explosion in microbiome research. These methods include 16S rRNA gene sequencing which uses variable regions (V3-V4) of the 16S rRNA gene to identify bacterial and archaeal taxa from a variety of samples, such as content inside the gut. The discovery that humans have an intricate symbiosis with their commensal microbes from which they derive health and well-being represented a paradigm shift in the study of human physiology. Microbiomes can include both