Biological and cultural control tactics are available for many agricultural pests but insecticides still play an important role in the rapid reduction of pest incidence when damage reaches economic levels. Laboratory and field toxicities of the reduced-risk products spinetoram and flubendiamide to Itame argillacearia (blueberry spanworm) was compared to deltamethrin, a conventional synthetic pyrethroid insecticide. In laboratory experiments, I. argillacearia larvae were highly susceptible to spinetoram and flubendiamide, and efficacy in the field was comparable to that of deltamethrin. Lethal and sublethal effects of the biopesticide formulations of Beauveria bassiana and Bacillus subtilis, and spirotetramat, a new tetramic acid insecticide, to bumble bees, Bombus impatiens, were also assessed. When ingested, field rates of spirotetramat caused high mortality after a week, and B. subtilis significantly reduced drone production. Field rates of spirotetramat, when applied topically, reduced drone production, but drone production varied following topical treatments of either biopesticide.

Pollinator-driven selection is thought to drive much of the extraordinary diversity of flowering plants. Plants that produce floral traits preferred by particular pollinators are more likely to receive conspecific pollen and to evolve further adaptations to those pollinators that enhance pollination and ultimately generate floral diversity. Two mechanisms in particular, sensory bias and learning, are thought to explain how pollinator preference can contribute to divergence and speciation in flowering plants. While the preferences of pollinators, such as bees, flies, and birds, are frequently implicated in patterns of floral trait evolution, the role of learning in generating reproductive isolation and trait divergence for different floral types within plant populations is not well understood. Floral color polymorphism in particular provides an excellent opportunity to examine how pollinator behavior and learning might maintain the different floral morphs. In this study we asked if bumble bees showed innate preferences for different color morphs of the pollen-only plant Solanum tridynamum, whether bees formed preferences for the morphs with which they had experience collecting pollen from, and the strength of those learned preferences. Using an absolute conditioning protocol, we gave bees experience collecting pollen from a color polymorphic plant species that offered only pollen rewards. Despite initially-naïve bees showing no apparent innate bias toward human-white versus human-purple flower morphs, we did find evidence of a bias in learning. Specifically, bees learned strong preferences for purple corollas, but learned only weak preferences for hypochromic (human-white) corollas. We discuss how our results might explain patterns of floral display evolution, particularly as they relate to color polymorphisms. Additionally, we propose that the ease with which floral visual traits are learned—i.e., biases in learning—can influence the evolution of floral color as a signal to pollinators.

The astonishing diversity of floral form in angiosperm plants is driven in large part by preferences of pollinators for various floral traits, including learned preferences. Remarkably, almost all of a vast literature on learning and memory in pollinators relates to nectar as a reward, even though bees and many flies, beetles, and butterflies must collect pollen. In this study, we asked if bees formed preferences for plant species from which pollen had been collected successfully. Using absolute conditioning, we gave pollen foraging bees experience with plant species that offered only pollen rewards. Naive bees generally showed modest preferences, whereas experienced bees adopted strong preferences for those species over alternative species not previously experienced. Learned preferences were retained for at least 24 h, consistent with preferences learned with nectar rewards. These experience-mediated changes in preference raised the possibility that bees formed associations between particular floral features and pollen rewards. We therefore asked if learned preferences required that bees successfully collect pollen. Using differential conditioning, we determined that learned preferences were strongly influenced by receipt of a pollen reward. In a final experiment, we characterized the importance of 2 floral features, the corolla and the anther, in the expression of learned preferences. Although experience altered responses to both floral parts, responses to anthers were influenced more strongly. We discuss recent evidence in the literature for associative learning with pollen rewards and propose that learned preferences in the context of pollen collection have played an important role in floral display evolution.

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 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.

The inadvertent spread of exotic pests and pathogens has resulted in devastating losses for bees. The vast majority of bee disease research has focused on a single species of managed bee, the European honey bee (Apis mellifera). More recently, pathogen spillover from managed bees is implicated in the decline of several bumble bee species (Bombus spp.) demonstrating a need to better understand the mechanisms driving disease prevalence in bees, transmission routes, and spillover events. RNA viruses, once considered specific to honey bees, are suspected of spilling over from managed honey bees into wild bumble bee populations. To test this, I collected bees and flowers in the field from areas with and without honey bee apiaries nearby. Prevalence of deformed wing virus (DWV) and black queen cell virus (BQCV) as well as replicating DWV infections in Bombus vagans and B. bimaculatus were highest in bumble bees collected near honey bee apiaries (χ 12 < 6.531, P < 0.05). My results suggest that honey bees are significant contributors of viruses to bumble bees. Flowers have been suspected as bridges in virus transmission among bees. I detected bee viruses on 18% of the flowers collected within honey bee apiaries and detected no virus on flowers in areas without apiaries, thus providing evidence that viruses are transmitted at flowers from infected honey bees. In controlled experiments using captive colonies in flight cages, I found that honey bees leave viruses on flowers but not equally across plant species. My results suggest that there are differences in virus ecology mediated by floral morphology and/or pollinator behavior. No bumble bees became infected in controlled experiments, indicating that virus transmission through plants is a rare event that is likely to require repeated exposure. The few studies examining viruses in bumble bees are generally limited to virus detection, resulting in little understanding of the conditions affecting virus titers. In honeybees, infections may remain latent, capable of replicating under certain conditions, such as immunosuppression induced by pesticide exposure. I tested whether exposure to imidacloprid, a neonicotinoid pesticide, affects virus titers in bumble bees. In previous honey bee studies, imidacloprid exposure increased virus titers. In contrast, I found that bumble bee exposure to imidacloprid decreased BQCV and DWV titers (χ42 < 20.873, p < 0.02). My findings suggest that virus-pesticide interactions are species-specific and results from honey bee studies should not be generalized across other bee species. Having found that honey bees are significant contributors of viruses to wild bees and flowers, I investigated how honey bee management practices affect disease spread and developed recommendations and tools to lesson the risk of spillover events. Honey bee disease may be exacerbated by migratory beekeeping which increases stress and opportunities for disease transmission. I experimentally tested whether migratory conditions contribute to disease spread in honey bees and found negative yet varying effects on bees suggesting that the effects of migratory practices may be ameliorated with rest time between pollination events. State apiary inspection programs are critical to controlling disease spread and reducing the risk of spillover. However, these programs are often resource constrained. I developed and deployed a toolkit that enables state programs to prioritize inspections and provide a platform for beekeeper education. Using novel data collected in Vermont, I discovered several promising avenues for future research and provided realistic recommendations to improve bee health.

Unlike other rosaceous fruit crops such as apple and cherry, commercial raspberry cultivars are largely self-fertile and can set fruit in the absence of pollinators. However, their floral morphology often prevents complete self-pollaintion. Incomplete pollination yields unmarketable small or crumbly fruits. Insect visitation is therefore essential to maximizing raspberry yield. Honey bees are typically used to pollinate commercial raspberry; however, escalating prices for hive rentals coupled with increasing acreage encourage evaluation of other manageable pollinators. Bumble bees (Bombus spp.) and several mason bees (Osmia spp.) are promising raspberry pollinators. Five bee species were evaluated and compared for their single-visit pollination efficacies on raspberry. From this a pollinator effectiveness index was created and an estimation of the minimum number of visits required to maximize fruit set was calculated. This estimation was then experimentally verified. Finally, in an attempt to synchronize their brief activity period with raspberry bloom, winter management options aimed at delaying the emergence of the mason bee, O. lignaria, were investigated. All five bee species proved excellent pollinators of raspberry. None of the alternative manageable species greatly outperformed honey bees. For this reason honey bees remain the most economical and practical option for open-field raspberry pollination. The adoption of alternative manageable bees could still be justified in other production systems, such as high-tunnel or greenhouse grown raspberry, which hamper honey bees’ ability to forage effectively. The pollinator effectiveness score for honey bees suggested that as few as two visits can achieve maximum fruit set. This estimate was confirmed through experimentation on three different red raspberry cultivars. For two of these cultivars, just one visit yielded drupelet counts similar to openly-pollinated flowers. This information can be used to help refine stocking density estimates for honey bees on raspberry. Wintering bees at 0° or -3° C rather than 4° C effectively delayed emergence of O. lignaria by more than a month without any impact on post-winter performance. These results suggest winter storage at near freezing temperatures is a viable management option for the use of O. lignaria with later-blooming crops.

Social insect workers can vary in terms of body size, space use, division of labor, and reproductive potential. Here, I begin to 'dissect' a social insect colony, using the bumble bee Bombus impatiens, to determine how this variation affects colony organization. I found that workers are spatially organized inside the nest and they remain at a specific distance from the colony center. Bees that feed larvae tend to remain in the center, whereas foragers are more often found on the periphery when not foraging. Smaller workers are more likely to feed larvae and incubate brood, and larger workers are more likely to fan or guard the nest. Still, workers perform multiple tasks throughout their life. The size of this task repertoire does not depend on body size or age. Furthermore, workers that remain further from the queen while inside the nest and avoid energy-expensive tasks during the ergonomic phase are more likely to reproduce by the end of the colony cycle. Inactive bees are not, however, defensive reserves. Although inactive bees increase their speed inside the nest when the nest was disturbed, they were not more likely to leave the nest (presumably to attack the simulated attacker) or switch to guarding behavior. This suggests that inactive bumble bees that remain farther from the queen may be storing fat reserves to later develop reproductive organs. Finally, I examined how within-group variation affects colony performance. I reduced variation in body size or temperature response thresholds by removing individuals from the colony with extreme phenotypes, and compared colony performance to colonies where random bees were removed. Colonies took longer to cool down the nest after bees were removed, but this effect was most striking when variation in temperature thresholds was reduced. Further, although larger bees are better at carrying items and are more likely to fan, the ability of colonies to perform undertaking behavior or thermoregulation was not affected when size variation was reduced. These studies provide evidence that (1) within-group variation affects colony organization and (2) variation among workers in their inherent tendencies to respond to stimuli positively affects colony performance.

Habitat loss and climate change are major factors implicated in the decline of bumble bees worldwide. These factors may be particularly acute in regions subject to climatic extremes such as seasonal drought. Combining methods from pollinator research and phylogenetic community ecology, I investigated the impacts of seasonal drought on plant phenology and bumble bee community ecology across gradients of disturbance and soil moisture in a semi-arid ecosystem. Seasonal fluctuations in floral resources coincided with significant phylogenetic clustering in plant communities, with decreasing plant diversity observed under conditions of increasing drought stress. In the late season, modified wet areas supported higher floral resource availability and greater bumble bee abundances as compared to dry woodlands, though wetlands were also an important source of late season floral resources. Despite these local effects, however, the areal extent of natural vs modified matrix habitat accounted for the majority of variation in models explaining bumble bee abundances. Modified matrix habitat was negatively associated, and natural matrix habitat positively associated, with the occurrence of bumble bee workers in June and late-flying queens in July and August. Results provide insight into the temporal niche dynamics of entomophilous flowering plants in this system, and emphasize the importance of conserving natural habitat diversity in efforts to promote resilient plant-pollinator communities. This study also provides evidence for the local extinction of Bombus occidentalis Greene, 1858 and Bombus suckleyi Greene, 1860 from Galiano Island, BC, Canada, as well as the island’s recent colonization by Bombus vosnesenskii Radoszkowski, 1862. / Graduate

Bumble bees and other wild pollinators are crucial to the support of both natural and agricultural ecosystems. However, unprecedented declines of pollinator populations have been observed all over the world, raising concerns of a looming threat to both the human food supply, as well as sustainability of the biodiversity in local ecological niches. Though declines are well described, the cause behind these still evades scientists. Proposed contributors include anthropogenic-mediated environmental stress, including application of xenobiotics for pest control, and increase of pathogen diversity and abundance due to the shipment of infection human-managed colonies. This research examined these theories and attempted to quantify the threats they may pose. Through development of a chronic, oral toxicity experiment, susceptibility of all Bombus impatiens castes to clothianidin exposure was examined. This exposed a substantial increase in vulnerability of male bumble bees to realistic concentrations of neonicotinoid pesticides, highlighting the crucial need to examine all members of wild bumble bee life cycles before determining pesticide regulations. Additionally, sublethal effects on fitness-related foraging behaviors in Bombus impatiens were examined through development of a voluntary task switching assay. The results of this experiment suggest humoral immune stimulation, through pathogenic infection, leads to significant impairment of cognitive flexibility. Taken together, this data suggests that pesticides and pathogens are capable of causing severe detrimental effects, both lethally and sublethally, in wild bumble bees. I hope this data will eventually contribute to reassessment of environmental regulations and establishment of effective conservation strategies in order to sustain the critical populations of wild bumble bees.

Det här arbetet handlar om tambins inverkan på naturligt förekommande pollinatörer. Min frågeställning berör: tillgången på näring, hälsotillståndet och populationsnivån hos naturligt förekommande pollinatörer i förhållande till tambins närvaro. Det som framkommit av den här litteraturstudien är att introducerade tambin leder till att naturligt förekommande pollinatörer minskar i antal kring bikuporna, en del arter söker föda på andra blommor eller senare på dygnet än tidigare. Den minskade tillgången på föda som denna konkurrens innebär har en negativ inverkan på humlors kroppsstorlek och därmed deras överlevnad. Samt att tambin kan föra över patogener så som varroakvalster, nosemasjuka och deformed wing virus till humlor vilket leder till minskad livslängd och sämre fortplantning. / This study is about the impact of honeybees on native pollinators. My questions concerns: availability of food, the health and population level of native pollinators in relation to the presence of honeybees. What has emerged from this literature review is that the introduction of honeybees lead to a decline in numbers of bee and bumblebee pollinators in proximity of the hives and also alters their behaviour, some species choose to forage on other flowers, or later in the day than before honeybees where introduced. The reduced availability of food that this competition induces has a negative impact on the size of bumblebees body’s and thus their survival. What was also discovered was that honeybees can transfer pathogens such as varroa mites, the microsporidium Nosema ceranae and deformed wing virus to bumblebees, leading to reduced life expectancy and poor propagation.