Effects of neonicotinoid pesticide exposure on bee health: Molecular, physiological and behavioural investigations Submitted by Elizabeth Jane Collison to the University of Exeter as a thesis for the degree of Doctor of Philosophy in Biological Sciences In June 2015 This thesis is available for Library use on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. I certify that all material in this thesis which is not my own work has been identified and that no material has previously been submitted and approved for the award of a degree by this or any other University. Signature: ………………………………………………………….. 1 2 Abstract Neonicotinoid exposure has been recognised as potentially impacting upon bee health, but whether realistic exposure scenarios are driving declines in bee health is not known. This thesis contributes new insights and perspectives to this research field investigating the use of molecular, physiological and behavioural endpoints as potential ecotoxicological markers for pesticide risk assessment. The thesis presents experimental data for dietary exposures of the European honey bee, Apis mellifera, and the buff-tailed bumble bee, Bombus terrestris, to one of two neonicotinoid pesticides, imidacloprid and thiamethoxam. The first part of this thesis explores impacts of chronic dietary exposures to neonicotinoid pesticides on bee immunocompetence- the ability to mount an immune response- using an artificial challenge to invoke an immune response in adult workers. Levels of phenoloxidase, an enzyme involved in melanisation and part of the bee’s defence system, were largely constitutive and resilient to exposure in honey bees and bumble bees. In honey bees, transient transcriptional changes in antimicrobial effector genes were observed following neonicotinoid exposure, but the physiological antimicrobial response was unaffected. In bumble bees, the induced antimicrobial response was impaired following neonicotinoid exposure, but only when exposed to concentrations likely higher than realistic environmental exposure scenarios. The next phase of this thesis investigates whether transcriptional, physiological and behavioural endpoints associated with the functioning of the honey bee hypopharyngeal gland were altered by imidacloprid exposure. Imidacloprid exposure led to transcriptional changes in foraging genes (associated with the control of temporal polyethism) and major royal jelly proteins (fed to developing larvae by nurse workers) and enzymatic changes in glucose oxidase (an enzyme involved in social immunity), which I hypothesise are linked with hypopharyngeal gland development. Despite these laboratory observations, no behavioural effects were observed in a field setting, monitored using Radio Frequency Identification transponders. Lastly, using RNA-Sequencing to investigate changes across the honey bee transcriptome, this thesis identified a suite of genes that were differentially 3 expressed in adult workers in response to immune challenge and/or dietary neonicotinoid exposure. Wounding and bacterial-like infection led to upregulation of known immune genes, including a peptidoglycan recognition protein and antimicrobial effectors. Chronic exposure to thiamethoxam and imidacloprid led to downregulation of genes associated with several metabolic pathways, such as oxidative phosphorylation, pyruvate- and purine- metabolic pathways, as well as ribosomal activity. Some of these genes identified provide candidates for further study to elucidate functional effects mechanisms and better understand health outcomes, as well as potential new biomarkers for use in pesticide risk assessment. This thesis presents novel findings and offers opportunities for future research that will be of interest to a wide audience, including risk assessors and policy makers, as well as the broader biological community, including ecotoxicologists, insect physiologists and molecular biologists. 4 Table of Contents Abstract .............................................................................................................. 3 Acknowledgements ............................................................................................ 9 List of tables ..................................................................................................... 13 List of figures .................................................................................................... 15 Author’s declaration .......................................................................................... 23 Abbreviations .................................................................................................... 24 1. General Introduction ..................................................................................... 28 1.1. Biology of bees ....................................................................................... 29 1.1.1. The honey bee, Apis mellifera ......................................................... 29 1.1.2. The bumble bee, Bombus terrestris ................................................. 31 1.2. Bees as providers of ecosystem services .............................................. 33 1.3. Risk assessment of plant protection products on bees .......................... 34 1.4. Interactive effects of pesticide exposure and pathogen infection on bee health – a critical analysis ............................................................................. 35 1.4.1. Abstract ............................................................................................ 35 1.4.2. Introduction ...................................................................................... 35 1.4.3. Interactive effects of pesticide exposure and pathogen infection on bee health .................................................................................................. 44 1.4.4. Conclusions ..................................................................................... 60 1.5. Thesis aims ............................................................................................ 64 1.6. References ............................................................................................. 66 2. Methods development and assay validations ............................................... 82 2.1. Method of immune elicitation ................................................................. 83 2.2. Measurement of phenoloxidase activity ................................................. 83 2.2.1. Thorax homogenisation ................................................................... 84 2.2.2. Neat haemolymph collection ............................................................ 85 2.2.3. Perfusion bleed approach ................................................................ 85 2.3. Measurement of antimicrobial activity .................................................... 88 2.3.1. Measurement of antimicrobial activity in honey bees ....................... 89 2.3.2. Measurement of antimicrobial activity in bumble bees ..................... 92 2.4. Measurement of glucose oxidase activity ............................................... 93 2.5. Gene expression assays using qPCR .................................................... 96 2.5.1. Primer design and optimisation ........................................................ 96 2.5.2. Preparation of standard PCR (calibration) curves ............................ 97 2.5.3. Selection of reference genes for normalisation .............................. 102 2.6. References ........................................................................................... 110 5 3. Resilience of honey bee immunocompetence to chronic neonicotinoid exposure......................................................................................................... 115 3.1. Abstract ................................................................................................ 115 3.2. Introduction .......................................................................................... 116 3.3. Methods ............................................................................................... 118 3.3.1. Honey bee provenance and husbandry ......................................... 119 3.3.2. Neonicotinoid exposure ................................................................. 120 3.3.3. Immune challenge .......................................................................... 120 3.3.4. Measurement of gene expression .................................................. 121 3.3.5. Measurement of antimicrobial activity ............................................ 124 3.3.6. Measurement of phenoloxidase activity ......................................... 124 3.3.7. Data analysis ................................................................................. 125 3.4. Results ................................................................................................. 126 3.4.1. Antimicrobial effector gene expression and enzymatic activity ...... 126 3.4.2. Phenoloxidase gene expression and enzymatic activity ................ 130 3.4.3. Sucrose consumption .................................................................... 132 3.5. Discussion ............................................................................................ 132 3.5.1. Conclusions ................................................................................... 136 3.5.2. Implications ...................................................................................