What’s really out there? Investigations into the effects of pesticides and pathogens on bee health NADEJDA TSVETKOV A DISSERTATION SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGY, YORK UNIVERSITY, TORONTO, ONTARIO December 2020 © NADEJDA TSVETKOV, 2020 1 Abstract Bees provide crucial pollination services to both cultivated and wild plants. In recent decades there were large declines in the populations of several native bee species and in the health of managed honey bee colonies. Neonicotinoid pesticides were suspected to negatively impact the health of native and managed bees, although this topic was hotly contested. Unfortunately, we lacked the knowledge regarding the typical duration that bees were exposed to neonicotinoids. Therefore, I first quantified the dose, duration, and types of chemicals honey bee colonies were exposed to near agricultural corn fields (Chapter 2). I found that honey bee colonies were exposed to a cocktail of chemicals, out of which neonicotinoids were the most likely to pose a health risk. I also found that honey bees were exposed to neonicotinoids for up to four months – the majority of the honey bee’s active season. I then performed a controlled experiment, where I exposed honey bee colonies to neonicotinoids in a manner that mimicked the field exposure (Chapter 2 and 3). I found that this field realistic exposure to neonicotinoids reduced worker life span, increased queenlessness, and impacted both social and innate immunity. Then, I studied the genetic underpinnings of neonicotinoid sensitivity in honey bees (Chapter 4). I found that survival after neonicotinoid exposure was heritable and was associated with natural polymorphisms found in two detoxification genes. Although survival after exposure is a convenient trait to study under laboratory conditions, it offers little insight into the plethora of phenotypes sublethal neonicotinoid exposure can affect. Thus, I used transcriptomics to look into the effects of field and field realistic exposures to neonicotinoids on the brain gene expression of forager and nurse honey bees (Chapter 5). I found that neonicotinoids affected the brain states of foragers and nurses in a different manner, possibly reflecting a consequence of developmental alterations. I then applied transcriptomics tools to a declining bumble bee, Bombus terricola (Chapter 6). I discovered that bumble bees near agriculture had signatures of stress due to pesticides and pathogens. Overall, I found that neonicotinoids and agricultural landscapes put undue stress on the health of bees. My research also highlights the importance of conducting season-long studies and quantifying multiple stressors and phenotypes at a time in ecotoxicological research. ii Acknowledgements I want to thank the Zayed lab members, who were always there to listen and offer advice. In particular, I’d like to thank Brock, who helped expend my horizons and who served as a guiding hand in the academic world. I would also like to thank Clement, who was always happy to talk about literature and share in a good cup of coffee. I want to thank my undergraduate field team: Harshil, Keshna, Daniel, Phil, and Pratik. Their enthusiasm transformed the field work into fun excursions. I would also like to thank Tamara Kelly, who had taught me so much about teaching. My thanks goes out to so many in the York Biology community; to all the friendships that were made through social nights, pumpkin carving contests, and just by sharing lunch hours. Of course, I’d like to thank Amro. His trust in me and my work allowed me to push myself (and others) to strive for excellence in research and in the many other skills required to be a good academic. My family has been there for me throughout my academic journey, even when I threatened to move half way across the globe, they were excited for me. I’d like to thank my husband, Egor. His support and understanding of the long hours made this journey easier. iii Table of Contents Abstract ....................................................................................................................................... ii Acknowledgements .................................................................................................................... iii Table of Contents ....................................................................................................................... iv List of Tables ............................................................................................................................... v List of Supplementary Tables ..................................................................................................... v List of Figures ........................................................................................................................... vii List of Supplementary Figures .................................................................................................. vii Chapter 1 Overview ................................................................................................................... 1 Chapter 2 Chronic exposure to neonicotinoids reduces honey bee health near corn crops ....... 6 Chapter 3 Field realistic exposure to neonicotinoids has a subtle impact on the honey bees’ immune system and pathosphere .................................................................................................. 40 Chapter 4 Polymorphisms in CYP9Q genes explain large variation in honey bee sensitivity to neonicotinoid insecticides ............................................................................................................. 65 Chapter 5 Searching beyond the streetlight: neonicotinoid exposure alters the neurogenomic state of worker honey bees ............................................................................................................ 84 Chapter 6 Conservation genomics reveals pesticide and pathogen stress in the declining bumble bee Bombus terricola ..................................................................................................... 108 Chapter 7 Conclusions and future forward ............................................................................ 123 Bibliography ............................................................................................................................... 127 Appendix A: Statement on contributions .................................................................................... 150 iv List of Tables Table 3.1. Results of the repeated measures mixed model for the 2014 field study .................... 60 Table 3.2. Results of the repeated measures mixed model for the 2015 apiary experiment ........ 61 Table 4.1. Percent of deviance explained in survival at different levels of analysis and estimates of broad-sense heritability............................................................................................................. 81 Table 5.1. Summary of previously published papers on the effects of NNIs on the honey bee transcriptomes and the number of differentially expressed genes (DEGs) overlapping with the current study................................................................................................................................ 102 Table 6.1. Number of DEGs overlapping between our study and previously published transcriptomic studies ................................................................................................................. 120 List of Supplementary Tables Table S 2.1. Mean levels of 26 agrochemicals detected in exposed and unexposed sites in 201434 Table S 2.2. The relative contribution of different plant taxa to pollen samples where neonicotinoids were present or absent .......................................................................................... 36 Table S 2.3. Lethal median concentration for the 26 agrochemicals detected in 2014 ................ 37 Table S 2.4. Summary results of SAS mixed models ................................................................... 38 Table S 2.5. Raw toxicology data from bee matrices collected from colonies near or far from Corn in 2014 ................................................................................................................................. 39 Table S 3.1. Primers used for amplification of the pathogen, target and reference control genes as well as their slope and efficiencies ............................................................................................... 62 Table S 3.2. SBV level in each time point in 2014 ....................................................................... 63 Table S 3.3. DWV levels in each time point in 2015 ................................................................... 64 Table S 4.1. The list of primers used to discern the patrilines of the tested worker bees ............. 82 Table S 4.2. The list of primers used to sequence the three CYP9Q genes .................................. 83 Table S 5.1. Differentially expressed transcription factors found in the 2014 field exposed foragers ....................................................................................................................................... 104 Table S 5.2. The overlapping DEGs between the different groups within this study................. 107 v Table S 6.1. The genomes of the common bumble bee pathogens used in this study ................ 121 Table S 6.2. Gene ontology analysis