Neither sulfoxaflor, Crithidia bombi, nor their combination impact bumble bee colony development or field bean pollination.

Many pollinators, including bumble bees, are in decline. Such declines are known to be driven by a number of interacting factors. Decreases in bee populations may also negatively impact the key ecosystem service, pollination, that they provide. Pesticides and parasites are often cited as two of the drivers of bee declines, particularly as they have previously been found to interact with one another to the detriment of bee health. Here we test the effects of an insecticide, sulfoxaflor, and a highly prevalent bumble bee parasite, Crithidia bombi, on the bumble bee Bombus terrestris. After exposing colonies to realistic doses of either sulfoxaflor and/or Crithidia bombi in a fully crossed experiment, colonies were allowed to forage on field beans in outdoor exclusion cages. Foraging performance was monitored, and the impacts on fruit set were recorded. We found no effect of either stressor, or their interaction, on the pollination services they provide to field beans, either at an individual level or a whole colony level. Further, there was no impact of any treatment, in any metric, on colony development. Our results contrast with prior findings that similar insecticides (neonicotinoids) impact pollination services, and that sulfoxaflor impacts colony development, potentially suggesting that sulfoxaflor is a less harmful compound to bee health than neonicotinoids insecticides.

Weak evidence base for bee protective pesticide mitigation measures.

Pesticides help produce food for humanity's growing population, yet they have negative impacts on the environment. Limiting these impacts, while maintaining food supply, is a crucial challenge for modern agriculture. Mitigation measures are actions taken by pesticide users, which modify the risk of the application to nontarget organisms, such as bees. Through these, the impacts of pesticides can be reduced, with minimal impacts on the efficacy of the pesticide. Here we collate the scientific evidence behind mitigation measures designed to reduce pesticide impacts on bees using a systematic review methodology. We included all publications which tested the effects of any pesticide mitigation measure (using a very loose definition) on bees, at any scale (from individual through to population level), so long as they presented evidence on the efficacy of the measure. We found 34 publications with direct evidence on the topic, covering a range of available mitigation measures. No currently used mitigation measures were thoroughly tested, and some entirely lacked empirical support, showing a weak evidence base for current recommendations and policy. We found mitigation measure research predominantly focuses on managed bees, potentially failing to protect wild bees. We also found that label-recommended mitigation measures, which are the mitigation measures most often applied, specifically are seldom tested empirically. Ultimately, we recommend that more, and stronger, scientific evidence is required to justify existing mitigation measures to help reduce the impacts of pesticides on bees while maintaining crop protection.

No impacts of glyphosate or Crithidia bombi, or their combination, on the bumblebee microbiome.

Pesticides are recognised as a key threat to pollinators, impacting their health in many ways. One route through which pesticides can affect pollinators like bumblebees is through the gut microbiome, with knock-on effects on their immune system and parasite resistance. We tested the impacts of a high acute oral dose of glyphosate on the gut microbiome of the buff tailed bumblebee (Bombus terrestris), and glyphosate's interaction with the gut parasite (Crithidia bombi). We used a fully crossed design measuring bee mortality, parasite intensity and the bacterial composition in the gut microbiome estimated from the relative abundance of 16S rRNA amplicons. We found no impact of either glyphosate, C. bombi, or their combination on any metric, including bacterial composition. This result differs from studies on honeybees, which have consistently found an impact of glyphosate on gut bacterial composition. This is potentially explained by the use of an acute exposure, rather than a chronic exposure, and the difference in test species. Since A. mellifera is used as a model species to represent pollinators more broadly in risk assessment, our results highlight that caution is needed in extrapolating gut microbiome results from A. mellifera to other bee species.

Parasite and Pesticide Impacts on the Bumblebee (Bombus terrestris) Haemolymph Proteome.

Pesticides pose a potential threat to bee health, especially in combination with other stressors, such as parasites. However, pesticide risk assessment tests pesticides in isolation from other stresses, i.e., on otherwise healthy bees. Through molecular analysis, the specific impacts of a pesticide or its interaction with another stressor can be elucidated. Molecular mass profiling by MALDI BeeTyping® was used on bee haemolymph to explore the signature of pesticidal and parasitic stressor impacts. This approach was complemented by bottom-up proteomics to investigate the modulation of the haemoproteome. We tested acute oral doses of three pesticides-glyphosate, Amistar and sulfoxaflor-on the bumblebee Bombus terrestris, alongside the gut parasite Crithidia bombi. We found no impact of any pesticide on parasite intensity and no impact of sulfoxaflor or glyphosate on survival or weight change. Amistar caused weight loss and 19-41% mortality. Haemoproteome analysis showed various protein dysregulations. The major pathways dysregulated were those involved in insect defences and immune responses, with Amistar having the strongest impact on these dysregulated pathways. Our results show that even when no response can be seen at a whole organism level, MALDI BeeTyping® can detect effects. Mass spectrometry analysis of bee haemolymph provides a pertinent tool to evaluate stressor impacts on bee health, even at the level of individuals.

Self-reported assessment of compliance with pesticide rules.

How pesticides are used is very important in determining the risk they pose to both the user, and the environment. Given they can have toxic properties, if pesticides are misused they could cause serious harm to the users health as well as a range of environmental damage. Despite this, very little research has quantified whether agricultural use of pesticides is compliant with the legally binding obligations and associated guidance surrounding application. In this survey we used an online, fully anonymous, questionnaire to ask Irish farmers about how they use pesticides. We used a self-reporting methodology, directly asking farmers about their compliance levels. We had a total of 76 unique valid respondents. Our respondents covered the broad range of Irish agriculture, and we quantified how this relates to national demographics. Overall compliance regarding pesticide use was high, with the majority of respondents complying the majority of the time. However, we also found a sizable group who report low compliance levels for certain topics. Respondents reported the highest levels of non-compliance with the use of personal protective equipment, with nearly half of all respondents admitting to not wearing certain required protective equipment on a regular basis. In contrast, for some areas like application rate, very high compliance was reported. Moderate levels of non-compliance with bee protective mitigation measures were found, and some reported practices like not emptying or washing out the spray tank between sprays could have serious impacts on pollinators, soil organisms and other non-targets. Additionally, a minority of respondents admitted to actions which could cause serious water course pollution. As the first survey on a range of pesticide compliance topics within a developed nation, the compliance seen is very high compared to levels in developing nations. Our results demonstrate that the assumption that all legal obligations and guidance surrounding pesticide use are followed is unfounded, but that the majority of the respondents are mostly compliant. Education or enforcement should be targeted to certain areas where compliance is weakest to minimise harm from pesticide use. Reducing the non-compliance we report here could benefit both farmer and environmental health, and ensure that pesticides are used in a manner that risk assessment has deemed safe.

Intra-specific variation in sensitivity of Bombus terrestris and Osmia bicornis to three pesticides.

There is growing evidence that pesticides may be among the causes of worldwide bee declines, which has resulted in repeated calls for their increased scrutiny in regulatory assessments. One recurring concern is that the current frameworks may be biased towards assessing risks to the honey bee. This paradigm requires extrapolating toxicity information across bee species. Most research effort has therefore focused on quantifying differences in sensitivity across species. However, our understanding of how responses to pesticides may vary within a species is still very poor. Here we take the first steps towards filling this knowledge gap by comparing acute, lethal hazards in sexes and castes of the eusocial bee Bombus terrestris and in sexes of the solitary bee Osmia bicornis after oral and contact exposure to the pesticides sulfoxaflor, Amistar (azoxystrobin) and glyphosate. We show that sensitivity towards pesticides varies significantly both within and across species. Bee weight was a meaningful predictor of pesticide susceptibility. However, weight could not fully explain the observed differences, which suggests the existence of unexplored mechanisms regulating pesticide sensitivity across bee sexes and castes. Our data show that intra-specific responses are an overlooked yet important aspect of the risk assessment of pesticides in bees.

No effect of dual exposure to sulfoxaflor and a trypanosome parasite on bumblebee olfactory learning.

Bees are important pollinators in wild and agricultural ecosystems, and understanding the factors driving their global declines is key to maintaining these pollination services. Learning, which has been a focus of previous ecotoxicological studies in bees, may play a key role in driving colony fitness. Here we move beyond the standard single-stressor approach to ask how multiple stressors, an agrochemical (sulfoxaflor, a relatively new insecticide) and a parasite (Crithidia bombi, a prevalent gut parasite of bumblebees), impact learning in the bumblebee Bombus terrestris. We developed a modified version of the classic proboscis extension reflex assay to assess the combined effects of acute oral sulfoxaflor exposure and infection by C. bombi on olfactory learning of bumblebee workers. We found no evidence that either sulfoxaflor, C. bombi, or their combination had any significant effect on bumblebee olfactory learning, despite their known negative impacts on other aspects of bumblebee health. This suggests that losses in cognitive ability, as measured here, are unlikely to explain the impacts of sulfoxaflor and its interactions with other stressors on bumblebees. Our novel methodology provides a model system within which to test interactive effects of other key stressors on bee health.

A Brief Note on Pollinator Exposure to Co-Formulants and Adjuvants.

Bees and other pollinators are exposed to co-formulants and adjuvants at very high levels in agriculture. Thorough, targeted, assessment of the toxicity of co-formulants and adjuvants is urgently required. Created with BioRender.com.

'Inert' ingredients are understudied, potentially dangerous to bees and deserve more research attention.

Agrochemical formulations are composed of two broad groups of chemicals: active ingredients, which confer pest control action, and 'inert' ingredients, which facilitate the action of the active ingredient. Most research into the effects of agrochemicals focusses on the effects of active ingredients. This reflects the assumption that 'inert' ingredients are non-toxic. A review of relevant research shows that for bees, this assumption is without empirical foundation. After conducting a systematic literature search, we found just 19 studies that tested the effects of 'inert' ingredients on bee health. In these studies, 'inert' ingredients were found to cause mortality in bees through multiple exposure routes, act synergistically with other stressors and cause colony level effects. This lack of research is compounded by a lack of diversity in study organism used. We argue that 'inert' ingredients have distinct, and poorly understood, ecological persistency profiles and toxicities, making research into their individual effects necessary. We highlight the lack of mitigation in place to protect bees from 'inert' ingredients and argue that research efforts should be redistributed to address the knowledge gap identified here. If so-called 'inert' ingredients are, in fact, detrimental to bee health, their potential role in widespread bee declines needs urgent assessment.

No evidence of effects or interaction between the widely used herbicide, glyphosate, and a common parasite in bumble bees.

BACKGROUND: Glyphosate is the world's most used pesticide and it is used without the mitigation measures that could reduce the exposure of pollinators to it. However, studies are starting to suggest negative impacts of this pesticide on bees, an essential group of pollinators. Accordingly, whether glyphosate, alone or alongside other stressors, is detrimental to bee health is a vital question. Bees are suffering declines across the globe, and pesticides, including glyphosate, have been suggested as being factors in these declines. METHODS: Here we test, across a range of experimental paradigms, whether glyphosate impacts a wild bumble bee species, Bombus terrestris. In addition, we build upon existing work with honey bees testing glyphosate-parasite interactions by conducting fully crossed experiments with glyphosate and a common bumble bee trypanosome gut parasite, Crithidia bombi. We utilised regulatory acute toxicity testing protocols, modified to allow for exposure to multiple stressors. These protocols are expanded upon to test for effects on long term survival (20 days). Microcolony testing, using unmated workers, was employed to measure the impacts of either stressor on a proxy of reproductive success. This microcolony testing was conducted with both acute and chronic exposure to cover a range of exposure scenarios. RESULTS: We found no effects of acute or chronic exposure to glyphosate, over a range of timespans post-exposure, on mortality or a range of sublethal metrics. We also found no interaction between glyphosate and Crithidia bombi in any metric, although there was conflicting evidence of increased parasite intensity after an acute exposure to glyphosate. In contrast to published literature, we found no direct impacts of this parasite on bee health. Our testing focussed on mortality and worker reproduction, so impacts of either or both of these stressors on other sublethal metrics could still exist. CONCLUSIONS: Our results expand the current knowledge on glyphosate by testing a previously untested species, Bombus terrestris, using acute exposure, and by incorporating a parasite never before tested alongside glyphosate. In conclusion our results find that glyphosate, as an active ingredient, is unlikely to be harmful to bumble bees either alone, or alongside Crithidia bombi.

Co-formulant in a commercial fungicide product causes lethal and sub-lethal effects in bumble bees.

Pollinators, particularly wild bees, are suffering declines across the globe, and pesticides are thought to be drivers of these declines. Research into, and regulation of pesticides has focused on the active ingredients, and their impact on bee health. In contrast, the additional components in pesticide formulations have been overlooked as potential threats. By testing an acute oral dose of the fungicide product Amistar, and equivalent doses of each individual co-formulant, we were able to measure the toxicity of the formulation and identify the ingredient responsible. We found that a co-formulant, alcohol ethoxylates, caused a range of damage to bumble bee health. Exposure to alcohol ethoxylates caused 30% mortality and a range of sublethal effects. Alcohol ethoxylates treated bees consumed half as much sucrose as negative control bees over the course of the experiment and lost weight. Alcohol ethoxylates treated bees had significant melanisation of their midguts, evidence of gut damage. We suggest that this gut damage explains the reduction in appetite, weight loss and mortality, with bees dying from energy depletion. Our results demonstrate that sublethal impacts of pesticide formulations need to be considered during regulatory consideration, and that co-formulants can be more toxic than active ingredients.

Rapid Parallel Adaptation to Anthropogenic Heavy Metal Pollution.

The impact of human-mediated environmental change on the evolutionary trajectories of wild organisms is poorly understood. In particular, capacity of species to adapt rapidly (in hundreds of generations or less), reproducibly and predictably to extreme environmental change is unclear. Silene uniflora is predominantly a coastal species, but it has also colonized isolated, disused mines with phytotoxic, zinc-contaminated soils. To test whether rapid, parallel adaptation to anthropogenic pollution has taken place, we used reduced representation sequencing (ddRAD) to reconstruct the evolutionary history of geographically proximate mine and coastal population pairs and found largely independent colonization of mines from different coastal sites. Furthermore, our results show that parallel evolution of zinc tolerance has occurred without gene flow spreading adaptive alleles between mine populations. In genomic regions where signatures of selection were detected across multiple mine-coast pairs, we identified genes with functions linked to physiological differences between the putative ecotypes, although genetic differentiation at specific loci is only partially shared between mine populations. Our results are consistent with a complex, polygenic genetic architecture underpinning rapid adaptation. This shows that even under a scenario of strong selection and rapid adaptation, evolutionary responses to human activities (and other environmental challenges) may be idiosyncratic at the genetic level and, therefore, difficult to predict from genomic data.