AN ABSTRACT OF THE THESIS OF Stephanie Parreira for the degree of Master of Science in Horticulture presented on May 4, 2016. Title: Effects of Pesticide Exposure on Honey Bee Health: Impacts of Pesticide Interactions and Exposure Through Multiple Routes Abstract approved: ______________________________________________________ Ramesh R. Sagili Honey bees (Apis mellifera) are responsible for approximately $17 billion in crop production per year in the United States, and are arguably the most important pollinators in the nation. The future of crop pollination and production is threatened by widespread national honey bee colony losses, which have averaged approximately 30% per year over the past decade. Many factors contribute to colony mortality, but the particular impacts of pesticides are still poorly understood. Here, we investigated the impacts of pesticides under conditions that have not been examined in previous research. Our research focused on the effects of an interaction between the neonicotinoid imidacloprid and the fungicide chlorothalonil and effects of exposure through multiple routes. To understand the potential impacts of pesticide interactions, we exposed whole colonies to imidacloprid, chlorothalonil, or combination of both chemicals through a pollen diet for one month. We found that many of our response variables were unaffected by our treatments, and that outliers influenced the outcome of several analyses. Brood area and prophenoloxidase activity were significantly affected by different treatments when outliers were excluded, although these differences were no longer significant after the multiple comparisons confidence interval adjustment. Similarly, the number of non-pollen foragers returning to the colonies was affected by the interaction between imidacloprid and time, chlorothalonil and time, and both chemicals and time, when outliers were removed. The interactions indicated that seven weeks after the end of the exposure period, both imidacloprid and chlorothalonil reduced the number of non-pollen foragers returning to the colonies. Imidacloprid and chlorothalonil also reduced the number of total foragers returning to the colonies overall. Our results indicate that colonies may be affected by pesticide exposure long after the exposure period, and that bees exposed to pesticides early in life may be detrimentally affected by that exposure at later stages. To determine whether pesticide exposure through multiple routes has a greater effect on bees than single-route exposure, we conducted a laboratory experiment in which we exposed bees to imidacloprid through pollen diet, sugar syrup, or both routes. We found that exposure through sugar syrup increased the midgut proteolytic enzyme activity overall, as well as glucose oxidase activity after four weeks of exposure. Exposure through sugar syrup, as well as exposure through both routes, increased glucose oxidase activity when outliers were included and excluded from the analysis, respectively. Mortality differed significantly between bees exposed to imidacloprid through sugar syrup and those exposed through both matrices, but none of the treatments were significantly different from the control group. We also found that bees in different treatment groups consumed different amounts of sugar syrup and pollen. Our results indicate the importance of conducting laboratory experiments that better reflect field-realistic pesticide exposure by both incorporating effects over a longer period of exposure, and exposure through multiple routes. In summary, our results provide new knowledge and insights on how pesticides impact long-term colony health. Future research must thoroughly examine statistical procedures, outliers, and statistical power, and must also determine interactions between pesticides and pathogens under different conditions, such as different types of pesticide application, honey bee subspecies, nutritional conditions, season, etc. Discerning the variability in results when these conditions vary will provide a fuller understanding of the true impacts of pesticides on colony health. ©Copyright by Stephanie Parreira May 4, 2016 All Rights Reserved Effects of Pesticide Exposure on Honey Bee Health: Impacts of Pesticide Interactions and Exposure Through Multiple Routes by Stephanie Parreira A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Master of Science Presented May 4, 2016 Commencement June 2016 Master of Science thesis of Stephanie Parreira presented on May 4, 2016 APPROVED: Major Professor, representing Horticulture Head of the Department of Horticulture 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. Stephanie Parreira, Author ACKNOWLEDGEMENTS I sincerely thank my advisor, Dr. Ramesh Sagili, for being the cornerstone of my success as a researcher and as graduate student. He has both guided my experimental design, analyses, and writing, and has been an incredibly supportive mentor to me in both the good times and hard times throughout my graduate education. I also thank all of my committee members, who have been instrumental to this research: Dr. Paul Jepson, Dr. Jeff Jenkins, Dr. Jennifer Parke, and Dr. Louisa Hooven; and the professors who provided the support for my statistical analyses: Dr. Lisa Ganio, Dr. Charlotte Wickham, and Dr. Sharmodeep Bhattacharya. Additionally, I thank all of the members of the OSU Honey Bee Lab who assisted me in the field and in the lab: Ciera Wilson, Chloe Donegan, Jon Dempster, Andrew Richards, Wayne Walters, Casey Cruse, Payton Stovall, Ellen Leinhaupel, Elizabeth Renshaw, and Molla Nawsher. Special thanks Carolyn Breece for driving hours with me to pick up my experimental bees and helping me maintain my experimental colonies; Ashrafun Nessa; for being the master of hemolymph extraction and who has been a constant source of moral support; Jared Jorgenson, for making the queen- excluder frame covers and all of the cages for my lab experiment, and Hannah Lucas, for developing the laboratory protocols used in this thesis and providing unending moral support. I would also like to thank Jan Lohman and Vince Vazza, who donated their bees for my experimental colonies. I want to thank my union family, the Coalition of Graduate Employees, for bargaining for a fair contract for graduate employees. Solidarity forever! Finally, I thank everyone who helped me not only complete graduate school, but helped me to get here in the first place: my undergraduate advisors, Dr. Caroline Christian and Dr. J. Hall Cushman; the director of the Sonoma State University McNair Scholars Program, Daniel Smith; my parents, Lisa and Christopher Parreira; my siblings Megan, Amelia, and Zachary Parreira; my friends Monica, Cara, Jill, Natalie, Thomas, Pete, Cathy, Stu, Chris, and Josephine; and my partner and best friend, Matt Zweier. Your support and encouragement have helped me make it this far. CONTRIBUTION OF AUTHORS Conceived and designed the experiments: Stephanie Parreira, Ramesh Sagili Performed the experiments: Stephanie Parreira, Ramesh Sagili, Hannah Lucas Analyzed the data: Stephanie Parreira Wrote the paper: Stephanie Parreira TABLE OF CONTENTS Page Chapter 1: Introduction .................................................................................................. 1 1.1 Insect Pollination and Pollinator Declines ................................................................ 1 1.1.1 Honey Bee Pollination ....................................................................................... 1 1.1.2 Honey Bee Colony Losses ................................................................................ 2 1.2. Factors Contributing to Honey Bee Colony Losses ................................................. 2 1.2.1. The Role of Pesticides ................................................................................ 3 1.2.2. Interactions Between Pesticides, Pests and Pathogens ................................ 3 1.2.2.1. Individual Immunity ........................................................................... 4 1.2.2.2. Social Immunity .................................................................................. 5 1.2.2.3. Nutritional Physiology ........................................................................ 5 1.2.3. Neonicotinoids ............................................................................................. 7 1.2.3.1. Systemic Properties, Persistence and Mobility ................................... 8 1.2.3.2. Potential Role in Colony Declines ...................................................... 9 1.2.3.3. Imidacloprid ........................................................................................ 9 1.2.4. Fungicides ................................................................................................ 10 1.2.4.1. Chlorothalonil ................................................................................... 11 1.3. Research Gaps and Impetus for Thesis Research .................................................. 12 1.3.1. Field-Realistic Exposure to Pesticides ..................................................... 12 1.3.2. Multiple Routes of Pesticide Exposure .................................................... 13 1.3.3. Effects of Pesticides on Whole Honey Bee Colonies ............................... 13 1.3.4. Long-Term Effects of Sublethal