Department of Ecology Neonicotinoids and honeybee health The effect of the neonicotinoid clothianidin, applied as a seed dressing in Brassica napus, on pathogen and parasite prevalence and quantities in free-foraging adult honeybees (Apis mellifera) Julia Goss European Master of Environmental Science (EnvEuro) Uppsala 2014 Independent project/Degree project / SLU, Department of Ecology 2014:4 Neonicotinoids and honeybee health The effect of the neonicotinoid clothianidin, applied as a seed dressing in Brassica napus, on pathogen and parasite prevalence and quantities in free-foraging adult honeybees (Apis mellifera) Supervisor: Dr. Joachim Rodrigues de Miranda, SLU, Department of Ecology Assistant Supervisor: Prof. Dr. Ingemar Fries, SLU, Department of Ecology Co-supervisor: Prof. Dr. Peter Rosenkranz, University of Hohenheim Examiner: Dr. Bengt Olsson, SLU, Department of Ecology Credits: 30 hec Level: A2E Course title: Independent Project in Environmental Science – Master’s thesis Course code: EX0431 Programme/education: European Master in Environmental Science (EnvEuro) Place of publication: Uppsala Year of publication: 2014 Cover picture: Foraging honeybee, Julia Goss 2013 Title of series: Independent project/Degree project / SLU, Department of Ecology Part no: 2014:4 Online publication: http://stud.epsilon.slu.se Keywords: honeybees; pathogens; parasites; virus; Nosema; Varroa; neonicotinoids; clothianidin Sveriges lantbruksuniversitet Swedish University of Agricultural Sciences Faculty of Natural Resources and Agricultural Sciences Department of Ecology Table of content Table of content Figures .................................................................................................................................................... IV Equations ................................................................................................................................................ V Tables ...................................................................................................................................................... V Abbreviations ......................................................................................................................................... VI Abstract ................................................................................................................................................. VII Svensk sammanfattning ....................................................................................................................... VIII Zusammenfassung ................................................................................................................................. IX 1. Introduction .................................................................................................................................... 1 1.1 The importance of bees .......................................................................................................... 2 1.2 Colony losses ........................................................................................................................... 3 1.3 Honeybee immunity................................................................................................................ 5 1.4 Parasites and Pathogens ......................................................................................................... 6 1.4.1 Varroa destructor ............................................................................................................ 6 1.4.2 Nosema ........................................................................................................................... 7 1.4.3 Viruses ............................................................................................................................. 7 1.5 Neonicotinoids ...................................................................................................................... 10 1.5.1 Lethal effects of neonicotinoids on honeybees ............................................................ 12 1.5.2 Sub-lethal effects of neonicotinoids on honeybees ..................................................... 12 1.6 Aim ........................................................................................................................................ 13 1.7 Hypothesis ............................................................................................................................. 13 2. Methods ........................................................................................................................................ 14 2.1 General structure of the study .............................................................................................. 14 2.2 Laboratory work .................................................................................................................... 17 2.2.1 Varroa destructor .......................................................................................................... 17 I Table of content 2.2.2 Preparation of a bee/water solution ............................................................................ 18 2.2.3 Determining the Nosema spp. prevalence and infestation rate ................................... 18 2.2.4 Nucleic acid detection ................................................................................................... 21 2.2.4.1 RNA and DNA extraction ............................................................................................... 21 2.2.4.2 Real time quantitative polymerase chain reaction (qPCR) ........................................... 22 2.2.4.3 DNA analysis for N. apis and N. ceranae ....................................................................... 23 2.2.4.4 RNA analysis for different viruses ................................................................................. 24 2.3 Statistical Analysis ................................................................................................................. 26 3. Results ........................................................................................................................................... 28 3.1 Varroa destructor .................................................................................................................. 28 3.2 Nosema spp. .......................................................................................................................... 29 3.3 Comparison of two methods to quantify Nosema spp. ........................................................ 31 3.4 Viral diseases ......................................................................................................................... 32 3.4.1 Black queen cell virus .................................................................................................... 32 1.1.1 Sacbrood virus ............................................................................................................... 32 1.1.2 Deformed wing virus ..................................................................................................... 34 2. Discussion ...................................................................................................................................... 35 2.1 Methods used ....................................................................................................................... 35 2.1.1 Field experimental setup .............................................................................................. 35 2.1.2 Laboratory work ............................................................................................................ 35 2.2 Results ................................................................................................................................... 36 2.2.1 Varroa destructor .......................................................................................................... 36 2.2.2 Nosema apis and Nosema ceranae ............................................................................... 37 2.2.3 Different methods used for Nosema ............................................................................ 38 2.2.4 Viruses ........................................................................................................................... 38 2.3 Social immunity versus individual immunity ........................................................................ 40 2.4 Sustainable use of pesticides ................................................................................................ 40 3. Conclusion ..................................................................................................................................... 43 II Table of content Acknowledgment .................................................................................................................................. 44 Literature .............................................................................................................................................. 45 Appendices ............................................................................................................................................ 53 Appendix 1: Primers used for RT-qPCR ............................................................................................. 53 Appendix 2: Code for SAS ................................................................................................................. 54 Appendices 3: SAS output for all tests .............................................................................................