Pesticide Exposure and Effects on Non-Apis Bees.

Bees are essential pollinators of many crops and wild plants, and pesticide exposure is one of the key environmental stressors affecting their health in anthropogenically modified landscapes. Until recently, almost all information on routes and impacts of pesticide exposure came from honey bees, at least partially because they were the only model species required for environmental risk assessments (ERAs) for insect pollinators. Recently, there has been a surge in research activity focusing on pesticide exposure and effects for non-Apis bees, including other social bees (bumble bees and stingless bees) and solitary bees. These taxa vary substantially from honey bees and one another in several important ecological traits, including spatial and temporal activity patterns, foraging and nesting requirements, and degree of sociality. In this article, we review the current evidence base about pesticide exposure pathways and the consequences of exposure for non-Apis bees. We find that the insights into non-Apis bee pesticide exposure and resulting impacts across biological organizations, landscapes, mixtures, and multiple stressors are still in their infancy. The good news is that there are many promising approaches that could be used to advance our understanding, with priority given to informing exposure pathways, extrapolating effects, and determining how well our current insights (limited to very few species and mostly neonicotinoid insecticides under unrealistic conditions) can be generalized to the diversity of species and lifestyles in the global bee community. We conclude that future research to expand our knowledge would also be beneficial for ERAs and wider policy decisions concerning pollinator conservation and pesticide regulation.

Single and combined exposure to 'bee safe' pesticides alter behaviour and offspring production in a ground-nesting solitary bee (Xenoglossa pruinosa).

Mounting evidence supporting the negative impacts of exposure to neonicotinoids on bees has prompted the registration of novel 'bee-friendly' insecticides for agricultural use. Flupyradifurone (FPF) is a butenolide insecticide that shares the same mode of action as neonicotinoids and has been assessed to be 'practically non-toxic to adult honeybees' using current risk assessment procedures. However, these assessments overlook some routes of exposure specific to wild bees, such as contact with residues in soil for ground-nesters. Co-exposure with other pesticides may also lead to detrimental synergistic effects. In a fully crossed experiment, we assessed the possible lethal and sublethal effects of chronic exposure to two pesticides used on Cucurbita crops, the insecticide Sivanto Prime (FPF) and the fungicide Quadris Top (azoxystrobin and difenoconazole), alone or combined, on solitary ground-nesting squash bees (Xenoglossa pruinosa). Squash bees exposed to Quadris Top collected less pollen per flower visit, while Sivanto-exposed bees produced larger offspring. Pesticide co-exposure induced hyperactivity in female squash bees relative to both the control and single pesticide exposure, and reduced the number of emerging offspring per nest compared to individual pesticide treatments. This study demonstrates that 'low-toxicity' pesticides can adversely affect squash bees under field-realistic exposure, alone or in combination.

Unveiling the submerged secrets: bumblebee queens' resilience to flooding.

In a previous study, an experimental oversight led to the accumulation of water filling a container housing diapausing bumblebee queens. Surprisingly, after draining the water, queens were found to be alive. This observation raises a compelling question: can bumblebee queens endure periods of inundation while overwintering underground? To address this question, we conducted an experiment using 143 common eastern bumblebee (Bombus impatiens) queens placed in soil-filled tubes and subjected to artificially induced diapause in a refrigerated unit for 7 days. Tap water was then added to the tubes and queens (n = 21 per treatment) were either maintained underwater using a plunger-like apparatus or left to float naturally on the water's surface for varying durations (8 h, 24 h or 7 days) while remaining in overwintering conditions. Seventeen queens served as controls. After the submersion period, queens were removed from water, transferred to new tubes with soil and kept in cold storage for eight weeks. Overall, queen survival remained consistently high (89.5 ± 6.4%) across all treatments and did not differ among submersion regimes and durations. These results demonstrate the remarkable ability of diapausing B. impatiens queens to withstand submersion under water for up to one week, indicating their adaptations to survive periods of flooding in the wild.

Effects of early-life exposure to sublethal levels of a common neonicotinoid insecticide on the orientation and migration of monarch butterflies (Danaus plexippus).

Migratory insects use a variety of innate mechanisms to determine their orientation and maintain correct bearing. For long-distance migrants, such as the monarch butterfly (Danaus plexippus), these journeys could be affected by exposure to environmental contaminants. Neonicotinoids are synthetic insecticides that work by affecting the nervous system of insects, resulting in impairment of their mobility, cognitive performance, and other physiological and behavioural functions. To examine how neonicotinoids might affect the ability of monarch butterflies to maintain a proper directional orientation on their ∼4000 km migration, we grew swamp milkweed (Asclepias incarnata) in soil that was either untreated (0 ng g-1: control) or mixed with low (15 ng g-1 of soil) or high (25 ng g-1 of soil) levels of the neonicotinoid clothianidin. Monarch caterpillars were raised on control or clothianidin-treated milkweed and, after pupation, either tested for orientation in a static flight simulator or radio-tracked in the wild during the autumn migration period. Despite clothianidin being detectable in milkweed tissue consumed by caterpillars, there was no evidence that clothianidin influenced the orientation, vector strength (i.e. concentration of direction data around the mean) or rate of travel of adult butterflies, nor was there evidence that morphological traits (i.e. mass and forewing length), testing time, wind speed or temperature impacted directionality. Although sample sizes for both flight simulator and radio-tracking tests were limited, our preliminary results suggest that clothianidin exposure during early caterpillar development does not affect the directed flight of adult migratory monarch butterflies or influence their orientation at the beginning of migration.

Neonicotinoid pesticide exposure impairs crop pollination services provided by bumblebees.

Recent concern over global pollinator declines has led to considerable research on the effects of pesticides on bees. Although pesticides are typically not encountered at lethal levels in the field, there is growing evidence indicating that exposure to field-realistic levels can have sublethal effects on bees, affecting their foraging behaviour, homing ability and reproductive success. Bees are essential for the pollination of a wide variety of crops and the majority of wild flowering plants, but until now research on pesticide effects has been limited to direct effects on bees themselves and not on the pollination services they provide. Here we show the first evidence to our knowledge that pesticide exposure can reduce the pollination services bumblebees deliver to apples, a crop of global economic importance. Bumblebee colonies exposed to a neonicotinoid pesticide provided lower visitation rates to apple trees and collected pollen less often. Most importantly, these pesticide-exposed colonies produced apples containing fewer seeds, demonstrating a reduced delivery of pollination services. Our results also indicate that reduced pollination service delivery is not due to pesticide-induced changes in individual bee behaviour, but most likely due to effects at the colony level. These findings show that pesticide exposure can impair the ability of bees to provide pollination services, with important implications for both the sustained delivery of stable crop yields and the functioning of natural ecosystems.

Combined pesticide exposure severely affects individual- and colony-level traits in bees.

Reported widespread declines of wild and managed insect pollinators have serious consequences for global ecosystem services and agricultural production. Bees contribute approximately 80% of insect pollination, so it is important to understand and mitigate the causes of current declines in bee populations . Recent studies have implicated the role of pesticides in these declines, as exposure to these chemicals has been associated with changes in bee behaviour and reductions in colony queen production. However, the key link between changes in individual behaviour and the consequent impact at the colony level has not been shown. Social bee colonies depend on the collective performance of many individual workers. Thus, although field-level pesticide concentrations can have subtle or sublethal effects at the individual level, it is not known whether bee societies can buffer such effects or whether it results in a severe cumulative effect at the colony level. Furthermore, widespread agricultural intensification means that bees are exposed to numerous pesticides when foraging, yet the possible combinatorial effects of pesticide exposure have rarely been investigated. Here we show that chronic exposure of bumblebees to two pesticides (neonicotinoid and pyrethroid) at concentrations that could approximate field-level exposure impairs natural foraging behaviour and increases worker mortality leading to significant reductions in brood development and colony success. We found that worker foraging performance, particularly pollen collecting efficiency, was significantly reduced with observed knock-on effects for forager recruitment, worker losses and overall worker productivity. Moreover, we provide evidence that combinatorial exposure to pesticides increases the propensity of colonies to fail.

General and species-specific impacts of a neonicotinoid insecticide on the ovary development and feeding of wild bumblebee queens.

Bumblebees are essential pollinators of crops and wild plants, but are in decline across the globe. Neonicotinoid pesticides have been implicated as a potential driver of these declines, but most of our evidence base comes from studies of a single species. There is an urgent need to understand whether such results can be generalized across a range of species. Here, we present results of a laboratory experiment testing the impacts of field-relevant doses (1.87-5.32 ppb) of the neonicotinoid thiamethoxam on spring-caught wild queens of four bumblebee species: Bombus terrestris, B. lucorum, B. pratorum and B. pascuorum. Two weeks of exposure to the higher concentration of thiamethoxam caused a reduction in feeding in two out of four species, suggesting species-specific anti-feedant, repellency or toxicity effects. The higher level of thiamethoxam exposure resulted in a reduction in the average length of terminal oocytes in queens of all four species. In addition to providing the first evidence for general effects of neonicotinoids on ovary development in multiple species of wild bumblebee queens, the discovery of species-specific effects on feeding has significant implications for current practices and policy for pesticide risk assessment and use.

A restatement of recent advances in the natural science evidence base concerning neonicotinoid insecticides and insect pollinators.

A summary is provided of recent advances in the natural science evidence base concerning the effects of neonicotinoid insecticides on insect pollinators in a format (a 'restatement') intended to be accessible to informed but not expert policymakers and stakeholders. Important new studies have been published since our recent review of this field (Godfray et al. 2014 Proc. R. Soc. B 281, 20140558. (doi:10.1098/rspb.2014.0558)) and the subject continues to be an area of very active research and high policy relevance.

Radar tracking and motion-sensitive cameras on flowers reveal the development of pollinator multi-destination routes over large spatial scales.

Central place foragers, such as pollinating bees, typically develop circuits (traplines) to visit multiple foraging sites in a manner that minimizes overall travel distance. Despite being taxonomically widespread, these routing behaviours remain poorly understood due to the difficulty of tracking the foraging history of animals in the wild. Here we examine how bumblebees (Bombus terrestris) develop and optimise traplines over large spatial scales by setting up an array of five artificial flowers arranged in a regular pentagon (50 m side length) and fitted with motion-sensitive video cameras to determine the sequence of visitation. Stable traplines that linked together all the flowers in an optimal sequence were typically established after a bee made 26 foraging bouts, during which time only about 20 of the 120 possible routes were tried. Radar tracking of selected flights revealed a dramatic decrease by 80% (ca. 1500 m) of the total travel distance between the first and the last foraging bout. When a flower was removed and replaced by a more distant one, bees engaged in localised search flights, a strategy that can facilitate the discovery of a new flower and its integration into a novel optimal trapline. Based on these observations, we developed and tested an iterative improvement heuristic to capture how bees could learn and refine their routes each time a shorter route is found. Our findings suggest that complex dynamic routing problems can be solved by small-brained animals using simple learning heuristics, without the need for a cognitive map.

A restatement of the natural science evidence base concerning neonicotinoid insecticides and insect pollinators.

There is evidence that in Europe and North America many species of pollinators are in decline, both in abundance and distribution. Although there is a long list of potential causes of this decline, there is concern that neonicotinoid insecticides, in particular through their use as seed treatments are, at least in part, responsible. This paper describes a project that set out to summarize the natural science evidence base relevant to neonicotinoid insecticides and insect pollinators in as policy-neutral terms as possible. A series of evidence statements are listed and categorized according to the nature of the underlying information. The evidence summary forms the appendix to this paper and an annotated bibliography is provided in the electronic supplementary material.

Foraging errors play a role in resource exploration by bumble bees (Bombus terrrestris).

If the cognitive performance of animals reflects their particular ecological requirements, how can we explain appreciable variation in learning ability amongst closely related individuals (e.g. foraging workers within a bumble bee colony)? One possibility is that apparent 'errors' in a learning task actually represent an alternative foraging strategy. In this study we investigate the potential relationship between foraging 'errors' and foraging success among bumble bee (Bombus terrestris) workers. Individual foragers were trained to choose yellow, rewarded flowers and ignore blue, unrewarded flowers. We recorded the number of errors (visits to unrewarded flowers) each bee made during training, then tested them to determine how quickly they discovered a more profitable food source (either familiar blue flowers, or novel green flowers). We found that error prone bees discovered the novel food source significantly faster than accurate bees. Furthermore, we demonstrate that the time taken to discover the novel, more profitable, food source is positively correlated with foraging success. These results suggest that foraging errors are part of an 'exploration' foraging strategy, which could be advantageous in changeable foraging environments. This could explain the observed variation in learning performance amongst foragers within social insect colonies.

Field agrochemical exposure impacts locomotor activity in wild bumblebees.

Agricultural intensification has been identified as one of the key causes of global insect biodiversity losses. These losses have been further linked to the widespread use of agrochemicals associated with modern agricultural practices. Many of these chemicals are known to have negative sublethal effects on commercial pollinators, such as managed honeybees and bumblebees, but less is known about the impacts on wild bees. Laboratory-based studies with commercial pollinators have consistently shown that pesticide exposure can impact bee behavior, with cascading effects on foraging performance, reproductive success, and pollination services. However, these studies typically assess only one chemical, neglecting the complexity of real-world exposure to multiple agrochemicals and other stressors. In the summer of 2020, we collected wild-foraging workers of the common eastern bumblebee, Bombus impatiens, from five squash (Cucurbita) agricultural sites (organic and conventional farms), selected to represent a range of agrochemical, including neonicotinoid insecticide, use. For each bee, we measured two behaviors relevant to foraging success and previously shown to be impacted by pesticide exposure: sucrose responsiveness and locomotor activity. Following behavioral testing, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) chemical analysis to detect and quantify the presence of 92 agrochemicals in each bumblebee. Bees collected from our sites did not vary in pesticide exposure as expected. While we found a limited occurrence of neonicotinoids, two fungicides (azoxystrobin and difenoconazole) were detected at all sites, and the pesticide synergist piperonyl butoxide (PBO) was present in all 123 bees. We found that bumblebees that contained higher levels of PBO were less active, and this effect was stronger for larger bumblebee workers. While PBO is unlikely to be the direct cause of the reduction in bee activity, it could be an indicator of exposure to pyrethroids and/or other insecticides that we were unable to directly quantify, but which PBO is frequently tank-mixed with during pesticide applications on crops. We did not find a relationship between agrochemical exposure and bumblebee sucrose responsiveness. To our knowledge, this is the first evidence of a sublethal behavioral impact of agrochemical exposure on wild-foraging bees.

Chronic sublethal stress causes bee colony failure.

Current bee population declines and colony failures are well documented yet poorly understood and no single factor has been identified as a leading cause. The evidence is equivocal and puzzling: for instance, many pathogens and parasites can be found in both failing and surviving colonies and field pesticide exposure is typically sublethal. Here, we investigate how these results can be due to sublethal stress impairing colony function. We mathematically modelled stress on individual bees which impairs colony function and found how positive density dependence can cause multiple dynamic outcomes: some colonies fail while others thrive. We then exposed bumblebee colonies to sublethal levels of a neonicotinoid pesticide. The dynamics of colony failure, which we observed, were most accurately described by our model. We argue that our model can explain the enigmatic aspects of bee colony failures, highlighting an important role for sublethal stress in colony declines.

The microsporidian parasites Nosema ceranae and Nosema apis are widespread in honeybee (Apis mellifera) colonies across Scotland.

Nosema ceranae is spreading into areas where Nosema apis already exists. N. ceranae has been reported to cause an asymptomatic infection that may lead, ultimately, to colony collapse. It is thought that there may be a temperature barrier to its infiltration into countries in colder climates. In this study, 71 colonies from Scottish Beekeeper's Association members have been screened for the presence of N. apis and N. ceranae across Scotland. We find that only 11 of the 71 colonies tested positive for spores by microscopy. However, 70.4 % of colonies screened by PCR revealed the presence of both N. ceranae and N. apis, with only 4.2 or 7 % having either strain alone and 18.3 % being Nosema free. A range of geographically separated colonies testing positive for N. ceranae were sequenced to confirm their identity. All nine sequences confirmed the presence of N. ceranae and indicated the presence of a single new variant. Furthermore, two of the spore-containing colonies had only N. ceranae present, and these exhibited the presence of smaller spores that could be distinguished from N. apis by the analysis of average spore size. Differential quantification of the PCR product revealed N. ceranae to be the dominant species in all seven samples tested. In conclusion, N. ceranae is widespread in Scotland where it exists in combination with the endemic N. apis. A single variant, identical to that found in France (DQ374655) except for the addition of a single nucleotide polymorphism, is present in Scotland.

Safeguarding pollinators requires specific habitat prescriptions and substantially more land area than suggested by current policy.

Habitat loss and fragmentation are major drivers of global pollinator declines, yet even after recent unprecedented periods of anthropogenic land-use intensification the amount of habitat needed to support insect pollinators remains unknown. Here we use comprehensive pan trap bee survey datasets from Ontario, Canada, to determine which habitat types are needed and at what spatial scales to support wild bee communities. Safeguarding wild bee communities in a Canadian landscape requires 11.6-16.7% land-cover from a diverse range of habitats (~ 2.6-3.7 times current policy guidelines) to provide targeted habitat prescriptions for different functional guilds over a variety of spatial scales, irrespective of whether conservation aims are enhancing bee species richness or abundance. Sensitive and declining habitats, like tallgrass woodlands and wetlands, were important predictors of bee biodiversity. Conservation strategies that under-estimate the extent of habitat, spatial scale and specific habitat needs of functional guilds are unlikely to protect bee communities and the essential pollination services they provide to both crops and wild plants.

Bees do not use nearest-neighbour rules for optimization of multi-location routes.

Animals collecting patchily distributed resources are faced with complex multi-location routing problems. Rather than comparing all possible routes, they often find reasonably short solutions by simply moving to the nearest unvisited resources when foraging. Here, we report the travel optimization performance of bumble-bees (Bombus terrestris) foraging in a flight cage containing six artificial flowers arranged such that movements between nearest-neighbour locations would lead to a long suboptimal route. After extensive training (80 foraging bouts and at least 640 flower visits), bees reduced their flight distances and prioritized shortest possible routes, while almost never following nearest-neighbour solutions. We discuss possible strategies used during the establishment of stable multi-location routes (or traplines), and how these could allow bees and other animals to solve complex routing problems through experience, without necessarily requiring a sophisticated cognitive representation of space.

Fungicides and bees: a review of exposure and risk.

Fungicides account for more than 35% of the global pesticide market and their use is predicted to increase in the future. While fungicides are commonly applied during bloom when bees are likely foraging on crops, whether real-world exposure to these chemicals - alone or in combination with other stressors - constitutes a threat to the health of bees is still the subject of great uncertainty. The first step in estimating the risks of exposure to fungicides for bees is to understand how and to what extent bees are exposed to these active ingredients. Here we review the current knowledge that exists about exposure to fungicides that bees experience in the field, and link quantitative data on exposure to acute and chronic risk of lethal endpoints for honey bees (Apis mellifera). From the 702 publications we screened, 76 studies contained quantitative data on residue detections in honey bee matrices, and a further 47 provided qualitative information about exposure for a range of bee taxa through various routes. We compiled data for 90 fungicides and metabolites that have been detected in honey, beebread, pollen, beeswax, and the bodies of honey bees. The risks posed to honey bees by fungicide residues was estimated through the EPA Risk Quotient (RQ) approach. Based on residue concentrations detected in honey and pollen/beebread, none of the reported fungicides exceeded the levels of concern (LOC) set by regulatory agencies for acute risk, while 3 and 12 fungicides exceeded the European Food Safety Authority (EFSA) chronic LOC for honey bees and wild bees, respectively. When considering exposure to all bees, fungicides of most concern include many broad-spectrum systemic fungicides, as well as the widely used broad-spectrum contact fungicide chlorothalonil. In addition to providing a detailed overview of the frequency and extent of fungicide residue detections in the bee environment, we identified important research gaps and suggest future directions to move towards a more comprehensive understanding and mitigation of the risks of exposure to fungicides for bees, including synergistic risks of co-exposure to fungicides and other pesticides or pathogens.

Quantifying exposure of bumblebee (Bombus spp.) queens to pesticide residues when hibernating in agricultural soils.

Exposure to pesticides is a major threat to bumblebee (Bombus spp.) health. In temperate regions, queens of many bumblebee species hibernate underground for several months, putting them at potentially high risk of exposure to soil contaminants. The extent to which bumblebees are exposed to residues in agricultural soils during hibernation is currently unknown, which limits our understanding of the full pesticide exposome for bumblebees throughout their lifecycle. To generate field exposure estimates for overwintering bumblebee queens to pesticide residues, we sampled soils from areas corresponding to suitable likely hibernation sites at six apple orchards and 13 diversified farms throughout Southern Ontario (Canada) in fall 2019-2020. Detectable levels of pesticides were found in 65 of 66 soil samples analysed for multi-pesticide residues (UPLC-MS/MS). A total of 53 active ingredients (AIs) were detected in soils, including 27 fungicides, 13 insecticides, and 13 herbicides. Overall, the frequency of detection, residue levels (median = 37.82 vs. 2.20 ng/g), and number of pesticides per sample (mean = 12 vs. 4 AIs) were highest for orchard soils compared to soils from diversified farms. Ninety-one percent of samples contained multiple residues (up to 29 different AIs per sample), including mixtures of insecticides and fungicides that might lead to synergistic effects. Our results suggest that when hibernating in agricultural areas, bumblebee queens are very likely to be exposed to a wide range of pesticide residues in soil, including potentially harmful levels of insecticides (e.g., cyantraniliprole up to 148.82 ng/g). Our study indicates the importance of empirically testing the potential effects of pesticide residues in soils for hibernating bumblebee queens, using field exposure data such as those generated here. The differences in potential exposure that we detected between cropping systems can also be used to better inform regulations that govern the use of agricultural pesticides, notably in apple orchards.