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Control of the Intestinal Parasite Nosema Ceranae in Apis Mellifera Using Nutraceuticals, Prebiotics and Probiotics

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Control of the Intestinal Parasite Nosema Ceranae in Apis Mellifera Using Nutraceuticals, Prebiotics and Probiotics Control of the intestinal parasite Nosema ceranae in Apis mellifera using nutraceuticals, prebiotics and probiotics by Daniel Borges A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Master of Science in Environmental Sciences Guelph, Ontario, Canada © Daniel Borges, September, 2015 ABSTRACT CONTROL OF THE INTESTINAL PARASITE NOSEMA CERANAE IN APIS MELLIFERA USING NUTRACEUTICALS, PREBIOTICS AND PROBIOTICS Daniel Borges Advisors: University of Guelph, 2015 Dr. Ernesto Guzman Dr. Paul H. Goodwin Nosema ceranae is a damaging parasite of the honey bee, Apis mellifera. The only registered treatment is the antibiotic fumagillin. Thirteen nutraceuticals, three prebiotics and three probiotics were examined as novel alternative compounds for controlling N. ceranae. Caged bees were inoculated with N. ceranae spores, and treatments were administered in sugar syrup. The most effective nutraceuticals were sulforaphane (from cruciferous vegetables), completely eliminating N. ceranae spores but causing 100% bee mortality at higher doses, and naringenin (from citrus), reducing spores by 70% but only causing 10% bee mortality. The most effective prebiotic was acacia gum, reducing spores by 50% but causing over 60% bee mortality. The most effective probiotic was Protexin Concentrate© single-strain (ProtexinC1), reducing spores by 50% and significantly increasing bee survival above uninfected, Nosema-free control bees. Future research on reducing sulforaphane and acacia gum toxicity is needed. Naringenin and ProtexinC1, however, are promising new controls for N. ceranae. ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisors, Dr. Ernesto Guzman and Dr. Paul H. Goodwin, for all of their knowledge, support and guidance. I would also like to thank Dr. Shayan Sharif for his guidance as part of my advisory committee. I would like to thank everyone at the Honey Bee Research Centre for all of their help. Paul Kelly, Nancy Bradbury, Dave Stotesbury and all of the volunteers – their help with everything bee-related, especially the countless hours of feeding bees, was greatly appreciated. Thank you as well to Dr. Mollah Md. Hamiduzzaman for his patience, help and guidance with everything in the lab. I would like to thank Esther Uribe Ortega and Brenda Adriana Ríos Rodríguez for all of their help with my project. This project would not have been possible without their dedication and hard work and I am happy to have met them and to call them my friends. Thank you as well to Mariana Reyes for her help, friendship and guidance, as well as for her company in the lab into the wee hours of the morning running PCRs and counting spores. Finally, I would like to thank my family, especially my husband Jeff Shepstone, for all of their love and support and for putting up with my love of bees and insects. iii TABLE OF CONTENTS TITLE .............................................................................................................................................. i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ....................................................................................................................... ix LIST OF SUPPLEMENTARY MATERIAL ............................................................................... xii LIST OF ABBREVIATIONS ...................................................................................................... xiii CHAPTER ONE LITERATURE REVIEW ............................................................................................................... 1 1.1 The western honey bee, Apis mellifera ................................................................................. 1 1.2 Immunity in A. mellifera ....................................................................................................... 3 1.2.1 Insect humoral immune system ...................................................................................... 4 1.2.2 Insect cellular immune system ....................................................................................... 7 1.2.3 Honey bee social immunity .......................................................................................... 10 1.3 The microbiota of A. mellifera ............................................................................................ 12 1.4 Diseases and parasites of A. mellifera ................................................................................. 17 1.5 Nosema ceranae .................................................................................................................. 21 1.5.1 Biology and lifecycle of N. ceranae ............................................................................. 21 1.5.2 Genome of N. ceranae .................................................................................................. 26 1.5.3 Effects of N. ceranae infection on A. mellifera ............................................................ 27 1.5.4 Differences between N. apis and N. ceranae................................................................ 34 1.5.5 Current treatment of Nosema infection ........................................................................ 37 1.6 Alternative treatment of N. ceranae .................................................................................... 40 1.6.1 Nutraceuticals ............................................................................................................... 40 1.6.2 Prebiotics and probiotics .............................................................................................. 43 1.7 Conclusions ......................................................................................................................... 47 1.8 Hypotheses .......................................................................................................................... 47 1.9 Objectives ............................................................................................................................ 48 iv CHAPTER TWO CONTROL OF NOSEMA CERANAE INFECTION IN APIS MELLIFERA USING NUTRACEUTICALS...................................................................................................... 52 2.1 Introduction ......................................................................................................................... 52 2.2 Materials and methods ........................................................................................................ 58 2.2.1 Nosema ceranae spore extraction ................................................................................. 58 2.2.2 Nosema species identification ...................................................................................... 59 2.2.3 Inoculation with N. ceranae spores .............................................................................. 62 2.2.4 Nutraceutical compounds ............................................................................................. 63 2.2.5 Nutraceutical screening ................................................................................................ 65 2.2.6 Dose response with selected nutraceutical compounds ................................................ 66 2.2.7 Bee mortality with selected nutraceutical compounds ................................................. 68 2.2.8 Statistical analysis......................................................................................................... 69 2.3 Results ................................................................................................................................. 72 2.3.1 Nosema species determination by PCR ........................................................................ 72 2.3.2 Nutraceutical screening ................................................................................................ 72 2.3.3 Dose response with selected nutraceutical compounds ................................................ 73 2.3.4 Bee mortality with selected nutraceutical compounds ................................................. 78 2.4 Discussion ........................................................................................................................... 79 CHAPTER THREE CONTROL OF NOSEMA CERANAE INFECTION IN APIS MELLIFERA USING PREBIOTICS AND PROBIOTICS .............................................................................. 113 3.1 Introduction ....................................................................................................................... 113 3.2 Materials and methods ...................................................................................................... 119 3.2.1 Nosema ceranae spore extraction ............................................................................... 119 3.2.2 Nosema species identification .................................................................................... 119 3.2.3 Inoculation with N. ceranae spores ...........................................................................
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