Effects of used brood comb and propolis on honey bees (Apis mellifera L.) and their associated bacterium, Melissococcus plutonius Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Stephanie K. Murray, B.S. Graduate Program in Entomology The Ohio State University 2019 Thesis Committee Dr. Reed M. Johnson, Advisor Dr. P. Larry Phelan Dr. Rachelle M. M. Adams 1 Copyrighted by Stephanie K. Murray 2019 2 Abstract Wax brood comb is the place where larval honey bees are reared, making it the physical heart of a honey bee colony. Generations of larval rearing darken the color of the wax and leave behind layers of excrement, silken cocoons and sometimes bacterial or fungal spores that are harmful to developing or adult honey bees. Additionally, the chemical nature of beeswax creates a sink for many compounds to be absorbed—these include pesticides and beekeeper-applied acaricides, as well as pheromones produced by larval honey bees. Finally, propolis—a dark brown antimicrobial substance collected by bees from plant buds—can also become concentrated around the rims of wax comb cells. As beekeepers become more aware of the risks caused by potential pesticide and microbe build-up, a practice known as brood comb replacement is becoming more popular in the United States. With this practice, beekeepers are systematically removing and replacing old brood combs after several years in use. However, there is little research on the effects of old brood comb on honey bee survival or the frequency with which beekeepers should replace brood comb. Additionally, some beekeepers prefer to save old brood combs, as they are preferred by honey bee swarms that are settling into a new nest space. This thesis aims to elucidate some of the potential benefits of old darkened brood comb by recording differences in colony preference for, larval survival on, and antimicrobial activity of wax combs that have or have not been used for larval rearing. Overall, we observed no differences in colony preference for or larval survival on comb ii treatments that were or were not previously used for larval rearing. However, there were antimicrobial effects of extracts made from both darkened brood comb and light honey comb against a honey bee pathogen, Melisococcus plutonius—the bacterial agent responsible for a larval bee disease called European Foulbrood. Extracts of propolis were also found to inhibit M. plutonius growth, suggesting that the antimicrobial effects of wax may derive from the propolis incorporated into wax. However, the antibacterial components of wax combs are still unknown and should be studied further. iii Acknowledgments I would first like to acknowledge the University and the CFAES Research Enhancement Competitive Grants Program for funding the research included in Chapter 3 of this thesis. Next, I would like to thank my graduate advisor, Dr. Reed Johnson for his kindness, support and guidance throughout my graduate career at OSU. Reed has given me room to explore avenues of research not typically covered in his lab and has made great efforts to connect me with professionals who share similar interests. He has given me time to learn from my mistakes, but also pushed me in the right direction when needed. Finally, Reed is always supportive and encouraging when I want to explore new opportunities, or when circumstances feel discouraging. I would also like to thank my committee members Dr. Rachelle Adams and Dr. Larry Phelan for their guidance and support. Rachelle has not only provided lab space and equipment to make my research possible, but she has also helped guide me through experimental design and push me through the writing process. Larry has not only provided expertise on chemical ecology but has also greatly developed my critical thinking skills through one-on-one conversation and lecture activities. Both Rachelle and Larry have always had open doors when I was feeling overwhelmed through this process. I would also like to acknowledge the many lab members and colleagues who made my research possible through equipment preparation, data collection, expert guidance, donation of space and equipment and being a listening ear. Colleagues outside iv of our lab include Dr. Thaddeus Ezeji, Dr. Christopher Okonkwo, Dr. Vanessa Hale, Dr. Christopher Madden, Andrew Mularo and Robert Filbrun. Lab members include Jacob Shuman, Jessica Lyons, Colin Kurkul, Tyler Eaton and Sreelakshmi Suresh. I would like to thank many beekeepers throughout the state of Ohio that have encouraged my research through their genuine interest and engaging conversation. Beekeepers from the Tri-County Beekeepers Association and the Greater Cleveland Beekeepers Association have welcomed me to participate and present at meetings. Conversation with local beekeepers has provided me with a great deal of joy, inspiration and encouragement. Finally, I would like the thank my friends and family who have been immensely supportive throughout this journey. I would be nowhere without the unconditional love, support and encouragement that my parents and siblings have always given me. They helped remind me to smile and laugh through the mentally trying times of a earning a graduate degree. My grandparents and extended family have also been involved and encouraging throughout all my years in school. Lastly, I would like to acknowledge my dear cat, Lulu, for the companionship and joy that she has brought me during my time in Ohio. v Vita June 2013 .................................................... Ringgold High School May 2017 .................................................... B.S. Biology, California University of Pennsylvania (Cal U of PA) Aug 2016 – May 2017 ................................. Lab Assistant, Cal U of PA Aug 2018 – May 2019 ................................. Graduate Teaching Assistant, The Ohio State University (OSU) Aug 2019 – Present ..................................... Instructor, OSU’s Agricultural Technical Institute February 2019 ............................................. OSU SEEDS Grant March 2019 ................................................. Entomological Society of America, North Central Branch 1st Place M.S. Poster Expected December 2019 ............................ M.S. Entomology, The Ohio State University Fields of Study Major Field: Entomology vi Table of Contents Abstract .......................................................................................................................... ii Acknowledgments ......................................................................................................... iv Vita ............................................................................................................................... vi List of Tables................................................................................................................. ix List of Figures .................................................................................................................x Chapter 1. Literature Review ...........................................................................................1 1.1 Introduction ...........................................................................................................1 1.2 Brood Comb as a Composite Material ....................................................................3 1.3 Pesticide Residues and Microbial Contaminants .....................................................5 1.4 Benefits of Old Brood Comb: Pheromones, Propolis and Social Immunity .............9 1.5 Brood Comb Replacement in the United States .................................................... 12 1.6 Research Objectives ............................................................................................. 15 1.7 References ........................................................................................................... 15 Chapter 2. Effects of comb used for brood rearing on honey bee (Apis mellifera L.) preference and survival .................................................................................................. 20 2.1 Abstract ............................................................................................................... 20 2.2 Introduction ......................................................................................................... 21 2.3 Materials and Methods ......................................................................................... 23 2.3.1 Brood Comb Treatments ............................................................................... 23 2.3.2 Choice Arena and Honey Bee Package Installation ........................................ 24 2.3.3 Larval Survival Estimation ............................................................................ 26 2.3.4 Statistical Analysis ........................................................................................ 27 2.4 Results ................................................................................................................. 28 2.4.1 Choice Experiment ........................................................................................ 28 2.4.2 Larval Survival.............................................................................................. 28 2.5 Discussion ........................................................................................................... 31 vii 2.6 Acknowledgements .............................................................................................