A novel insecticide impairs bumblebee memory and sucrose responsiveness across high and low nutrition.

Wild bees are important pollinators of crops and wildflowers but are exposed to a myriad of different anthropogenic stressors, such as pesticides and poor nutrition, as a consequence of intensive agriculture. These stressors do not act in isolation, but interact, and may exacerbate one another. Here, we assessed whether a field-realistic concentration of flupyradifurone, a novel pesticide that has been labelled as 'bee safe' by regulators, influenced bumblebee sucrose responsiveness and long-term memory. In a fully crossed experimental design, we exposed individual bumblebees (Bombus impatiens) to flupyradifurone at high (50% (w/w)) or low (15% (w/w)) sucrose concentrations, replicating diets that are either carbohydrate rich or poor, respectively. We found that flupyradifurone impaired sucrose responsiveness and long-term memory at both sucrose concentrations, indicating that better nutrition did not buffer the negative impact of flupyradifurone. We found no individual impact of sugar deficiency on bee behaviour and no significant interactions between pesticide exposure and poor nutrition. Our results add to a growing body of evidence demonstrating that flupyradifurone has significant negative impacts on pollinators, indicating that this pesticide is not 'bee safe'. This suggests that agrochemical risk assessments are not protecting pollinators from the unintended consequences of pesticide use.

Breaking the cycle: Reforming pesticide regulation to protect pollinators.

Over decades, pesticide regulations have cycled between approval and implementation, followed by the discovery of negative effects on nontarget organisms that result in new regulations, pesticides, and harmful effects. This relentless pattern undermines the capacity to protect the environment from pesticide hazards and frustrates end users that need pest management tools. Wild pollinating insects are in decline, and managed pollinators such as honey bees are experiencing excessive losses, which threatens sustainable food security and ecosystem function. An increasing number of studies demonstrate the negative effects of field-realistic exposure to pesticides on pollinator health and fitness, which contribute to pollinator declines. Current pesticide approval processes, although they are superior to past practices, clearly continue to fail to protect pollinator health. In the present article, we provide a conceptual framework to reform cyclical pesticide approval processes and better protect pollinators.

Pesticide licensing in the EU and protecting pollinators.

Intensive agriculture is reliant on pesticides to control crop pests, but these chemicals can have negative environmental consequences. This has resulted in repeated calls for pesticide risk assessments to be modified to better protect ecosystem services such as pollination. However, the pesticide licensing process is complex, and consequently there is often confusion between risk assessments where the environmental impact of pesticide use is considered, and risk management where licensing decisions are made. Using bees as a case study, we provide a roadmap for how pesticides are licensed for use in the European Union. By outlining the regulatory process, we highlight key data gaps that need to be addressed to generate a holistic approach to environmental risk assessment. Such an approach is vital to protect pollinators and wildlife more broadly from the unintended consequences of pesticide use.

Wild bees are exposed to low levels of pesticides in urban grasslands and community gardens.

Globally documented wild bee declines threaten sustainable food production and natural ecosystem functioning. Urban environments are often florally abundant, and consequently can contain high levels of pollinator diversity compared with agricultural environments. This has led to the suggestion that urban environments are an increasingly important habitat for pollinators. However, pesticides, such as commercial bug sprays, have a range of lethal and sub-lethal impacts on bees and are widely available for public use, with past work indicating that managed bees (honeybees and bumblebees) are exposed to a range of pesticides in urban environments. Despite this, we still have a poor understanding of (i) whether wild bees foraging in urban environments are exposed to pesticides and (ii) if exposure differs between genera. Here we assessed pesticide exposure in 8 bee genera foraging across multiple urban landscapes. We detected 13 different pesticides, some at concentrations known to have sub-lethal impacts on pollinators. Both the likelihood of pesticides being detected, and the concentrations observed, were higher for larger bees, likely due to their greater foraging ranges. Our results suggest that restricting agrochemical use in urban environments, where the economic benefits are limited, is a simple way to reduce anthropogenic stress on wild bees.

Exposure to the novel insecticide flupyradifurone impairs bumblebee feeding motivation, learning, and memory retention.

Bees are vital pollinators of crops and wildflowers and as such, wild bee declines threaten food security and functioning ecosystems. One driver of bee declines is the use of systemic insecticides, such as commonly used neonicotinoids. However, rising pest resistance to neonicotinoids, and restrictions on their use in the EU, has increased the demand for replacement insecticides to control crop pests. Flupyradifurone is a novel systemic insecticide that is thought to be relatively 'bee safe' although it can be present in the nectar and pollen of bee-attractive crops. Bumblebees rely on learning to forage efficiently, and thus detriments to learning performance may have downstream consequences on their ability to forage. While neonicotinoids negatively influence bumblebee learning and memory, whether this is also the case for their replacements is unclear. Here, we exposed bumblebees (Bombus impatiens) to an acute, field-realistic dose of flupyradifurone before training them to learn either an olfactory or colour association. We found that flupyradifurone impaired bumblebees' learning and memory performance in both olfactory and visual modalities. Flupyradifurone-treated bees were also less motivated to feed. Given the similarity between the detriments to cognition found here and those previously reported for neonicotinoids, this implies that these insecticides may have similar sub-lethal effects on bees. Restrictions on neonicotinoid use are therefore unlikely to benefit bees if novel insecticides like flupyradifurone are used as an alternative, highlighting that current agrochemical risk assessments are not protecting bees from the unwanted consequences of pesticide use. Sub-lethal assessments on non-Apis bees should be made mandatory in agrochemical regulation to ensure that novel insecticides are indeed 'bee safe'.

A Combined LD50 for Agrochemicals and Pathogens in Bumblebees (Bombus terrestris [Hymenoptera: Apidae]).

Neonicotinoid insecticides are the most commonly used insecticide in the world and can have significant sub-lethal impacts on beneficial insects, including bumblebees, which are important pollinators of agricultural crops and wild-flowers. This has led to bans on neonicotinoid use in the EU and has resulted in repeated calls for the agrochemical regulatory process to be modified. For example, there is increasing concern about 1) the underrepresentation of wild bees, such as bumblebees, in the regulatory process, and 2) the failure to determine how agrochemicals, such as neonicotinoids, interact with other commonly occurring environmental stressors, such as parasites. Here, we modify an OECD approved lethal dose (LD50) experimental design and coexpose bumblebees (Bombus terrestris) to the neonicotinoid thiamethoxam and the highly prevalent trypanosome parasite Crithidia bombi, in a fully crossed design. We found no difference in the LD50 of thiamethoxam on bumblebees that had or had not been inoculated with the parasite (Crithidia bombi). Furthermore, thiamethoxam dosage did not appear to influence the parasite intensity of surviving bumblebees, and there was no effect of either parasite or insecticide on sucrose consumption. The methodology used demonstrates how existing ring-tested experimental designs can be effectively modified to include other environmental stressors such as parasites. Moving forward, the regulatory process should implement methodologies that assess the interactions between agrochemicals and parasites on non-Apis bees and, in cases when this is not practical, should implement post-regulatory monitoring to better understand the real-world consequences of agrochemical use.

Bumblebees Exposed to a Neonicotinoid Pesticide Make Suboptimal Foraging Decisions.

Bumblebees are important pollinators of agricultural crops and wildflowers, but many species are in decline. Neonicotinoid insecticides are the most commonly used insecticide globally and can have negative sublethal effects on bumblebee colony growth and reproduction. Individual bumblebees can visit hundreds to thousands of flowers a day to forage for their colony. As such, they are a model species for studying optimal foraging, and small impairments to an individual's foraging decisions may have compounding effects on the colony's nutritional intake. We exposed bumblebees (Bombus impatiens) to an acute, field-realistic dose of the neonicotinoid insecticide imidacloprid, before allowing them to forage on an artificial floral array. We found that neonicotinoid-exposed bumblebees made suboptimal foraging decisions, as they were more likely to visit flowers located further apart than control bees. This indicates that for a given flower patch, individual bees exposed to a neonicotinoid will likely use more energy and forage less efficiency than unexposed bees, although further studies that directly measure energetic cost are required to confirm this. Given the robust and growing body of evidence demonstrating negative sublethal effects of neonicotinoids on bees, sublethal assessments on non-Apis bees should be made mandatory within the regulatory process.

Field-realistic neonicotinoid exposure has sub-lethal effects on non-Apis bees: A meta-analysis.

Neonicotinoid insecticides can have sub-lethal effects on bees which has led to calls from conservationists for a global ban. In contrast, agrochemical companies argue that neonicotinoids do not harm honeybees at field-realistic levels. However, the focus on honeybees neglects the potential impact on other bee species. We conducted a meta-analysis to assess whether field-realistic neonicotinoid exposure has sub-lethal effects on non-Apis bees. We extracted data from 53 papers (212 effects sizes) and found that it largely consisted of two genera: bumblebees (Bombus) and mason bees (Osmia), highlighting a substantial taxonomic knowledge gap. Neonicotinoid exposure negatively affected reproductive output across all bees and impaired bumblebee colony growth and foraging. Neonicotinoids also reduced Bombus, but not Osmia, individual development (growth and body size). Our results suggest that restrictions on neonicotinoids should benefit bee populations and highlight that the current regulatory process does not safeguard pollinators from the unwanted consequences of insecticide use.

Agrochemicals interact synergistically to increase bee mortality.

Global concern over widely documented declines in pollinators1-3 has led to the identification of anthropogenic stressors that, individually, are detrimental to bee populations4-7. Synergistic interactions between these stressors could substantially amplify the environmental effect of these stressors and could therefore have important implications for policy decisions that aim to improve the health of pollinators3,8,9. Here, to quantitatively assess the scale of this threat, we conducted a meta-analysis of 356 interaction effect sizes from 90 studies in which bees were exposed to combinations of agrochemicals, nutritional stressors and/or parasites. We found an overall synergistic effect between multiple stressors on bee mortality. Subgroup analysis of bee mortality revealed strong evidence for synergy when bees were exposed to multiple agrochemicals at field-realistic levels, but interactions were not greater than additive expectations when bees were exposed to parasites and/or nutritional stressors. All interactive effects on proxies of fitness, behaviour, parasite load and immune responses were either additive or antagonistic; therefore, the potential mechanisms that drive the observed synergistic interactions for bee mortality remain unclear. Environmental risk assessment schemes that assume additive effects of the risk of agrochemical exposure may underestimate the interactive effect of anthropogenic stressors on bee mortality and will fail to protect the pollinators that provide a key ecosystem service that underpins sustainable agriculture.

Do novel insecticides pose a threat to beneficial insects?

Systemic insecticides, such as neonicotinoids, are a major contributor towards beneficial insect declines. This has led to bans and restrictions on neonicotinoid use globally, most noticeably in the European Union, where four commonly used neonicotinoids (imidacloprid, thiamethoxam, clothianidin and thiacloprid) are banned from outside agricultural use. While this might seem like a victory for conservation, restrictions on neonicotinoid use will only benefit insect populations if newly emerging insecticides do not have similar negative impacts on beneficial insects. Flupyradifurone and sulfoxaflor are two novel insecticides that have been registered for use globally, including within the European Union. These novel insecticides differ in their chemical class, but share the same mode of action as neonicotinoids, raising the question as to whether they have similar sub-lethal impacts on beneficial insects. Here, we conducted a systematic literature search of the potential sub-lethal impacts of these novel insecticides on beneficial insects, quantifying these effects with a meta-analysis. We demonstrate that both flupyradifurone and sulfoxaflor have significant sub-lethal impacts on beneficial insects at field-realistic levels of exposure. These results confirm that bans on neonicotinoid use will only protect beneficial insects if paired with significant changes to the agrochemical regulatory process. A failure to modify the regulatory process will result in a continued decline of beneficial insects and the ecosystem services on which global food production relies.

Individual and combined impacts of sulfoxaflor and Nosema bombi on bumblebee (Bombus terrestris) larval growth.

Sulfoxaflor is a globally important novel insecticide that can have negative impacts on the reproductive output of bumblebee (Bombus terrestris) colonies. However, it remains unclear as to which life-history stage is critically affected by exposure. One hypothesis is that sulfoxaflor exposure early in the colony's life cycle can impair larval development, reducing the number of workers produced and ultimately lowering colony reproductive output. Here we assess the influence of sulfoxaflor exposure on bumblebee larval mortality and growth both when tested in insolation and when in combination with the common fungal parasite Nosema bombi, following a pre-registered design. We found no significant impact of sulfoxaflor (5 ppb) or N. bombi exposure (50 000 spores) on larval mortality when tested in isolation but found an additive, negative effect when larvae received both stressors in combination. Individually, sulfoxaflor and N. bombi exposure each impaired larval growth, although the impact of combined exposure fell significantly short of the predicted sum of the individual effects (i.e. they interacted antagonistically). Ultimately, our results suggest that colony-level consequences of sulfoxaflor exposure for bumblebees may be mediated through direct effects on larvae. As sulfoxaflor is licensed for use globally, our findings highlight the need to understand how novel insecticides impact non-target insects at various stages of their development.

Sulfoxaflor exposure reduces egg laying in bumblebees Bombus terrestris.

Sulfoximine-based insecticides, such as sulfoxaflor, are of increasing global importance and have been registered for use in 81 countries, offering a potential alternative to neonicotinoid insecticides.Previous studies have demonstrated that sulfoxaflor exposure can have a negative impact on the reproductive output of bumblebee colonies, but the specific life-history variables that underlie these effects remain unknown.Here, we used a microcolony-based protocol to assess the sub-lethal effects of chronic sulfoxaflor exposure on egg laying, larval production, ovary development, sucrose consumption, and mortality in bumblebees. Following a pre-registered design, we exposed colonies to sucrose solutions containing 0, 5, 10 and 250ppb of sulfoxaflor. Exposure at 5 ppb has been previously shown to negatively impact colony reproductive success.Our results showed that sulfoxaflor exposure at 5 ppb (lowest exposure tested) reduced the number of eggs found within the microcolonies (Hedge's d = -0.37), with exposed microcolonies also less likely to produce larvae (Hedge's d = -0.36). Despite this, we found no effect of sulfoxaflor exposure on ovarian development. Sulfoxaflor-exposed bumblebees consumed less sucrose solution, potentially driving the observed reduction in egg laying. Policy implications. Regulatory bodies such as the European Food Safety Authority (EFSA) are under increasing pressure to consider the potential impact of insecticides on wild bees, such as bumblebees, but sublethal effects can go undetected at lower-tier testing. In identifying just such an effect for bumblebees exposed to sulfoxaflor, this study highlights that microcolony-based protocols are a useful tool that could be implemented within an ecotoxicology framework. Furthermore, the results provide evidence for potentially negative consequences of pollinator exposure to an insecticide that is currently undergoing the licensing process in several EU member states.

No evidence for negative impacts of acute sulfoxaflor exposure on bee olfactory conditioning or working memory.

Systemic insecticides such as neonicotinoids and sulfoximines can be present in the nectar and pollen of treated crops, through which foraging bees can become acutely exposed. Research has shown that acute, field realistic dosages of neonicotinoids can negatively influence bee learning and memory, with potential consequences for bee behaviour. As legislative reassessment of neonicotinoid use occurs globally, there is an urgent need to understand the potential risk of other systemic insecticides. Sulfoxaflor, the first branded sulfoximine-based insecticide, has the same mode of action as neonicotinoids, and may potentially replace them over large geographical ranges. Here we assessed the impact of acute sulfoxaflor exposure on performance in two paradigms that have previously been used to illustrate negative impacts of neonicotinoid pesticides on bee learning and memory. We assayed whether acute sulfoxaflor exposure influences (a) olfactory conditioning performance in both bumblebees (Bombus terrestris) and honeybees (Apis mellifera), using a proboscis extension reflex assay, and (b) working memory performance of bumblebees, using a radial-arm maze. We found no evidence to suggest that sulfoxaflor influenced performance in either paradigm. Our results suggest that despite a shared mode of action between sulfoxaflor and neonicotinoid-based insecticides, widely-documented effects of neonicotinoids on bee cognition may not be observed with sulfoxaflor, at least at acute exposure regimes.

Egg incubation temperature influences the growth and foraging behaviour of juvenile lizards.

After laying their eggs, oviparous reptiles are reliant on the external environment to provide the required incubation conditions for successful embryonic development. Egg incubation temperature can impact the behaviour of various species of reptiles, but previous experiments have focused on the impact of incubation environment on hatchlings, with only a limited number of studies focussing on the longer-term behavioural consequences of incubation environment. This study investigated the effects of developmental environment on bearded dragon lizards (Pogona vitticeps) that were incubated at different temperatures within the natural range; half of them were incubated at a 'hot' temperature (30 ±â€¯3 °C) and half at a 'cold' temperature (27 ±â€¯3 °C). The growth and foraging behaviour of the lizards was then compared over 18 weeks of development. Although the lizards incubated at a cool temperatures grew more quickly, those incubated at the hotter temperature completed the foraging task more often and had significantly faster running speeds. These results show that egg incubation temperature impacts the foraging behaviour of juvenile lizards and suggest a potential trade-off between growth and foraging speed, which could influence an animal's life history trajectory.

Quantifying the impact of pesticides on learning and memory in bees.

Most insecticides are insect neurotoxins. Evidence is emerging that sublethal doses of these neurotoxins are affecting the learning and memory of both wild and managed bee colonies, exacerbating the negative effects of pesticide exposure and reducing individual foraging efficiency.Variation in methodologies and interpretation of results across studies has precluded the quantitative evaluation of these impacts that is needed to make recommendations for policy change. It is not clear whether robust effects occur under acute exposure regimes (often argued to be more field-realistic than the chronic regimes upon which many studies are based), for field-realistic dosages, and for pesticides other than neonicotinoids.Here we use meta-analysis to examine the impact of pesticides on bee performance in proboscis extension-based learning assays, the paradigm most commonly used to assess learning and memory in bees. We draw together 104 (learning) and 167 (memory) estimated effect sizes across a diverse range of studies.We detected significant negative effects of pesticides on learning and memory (i) at field realistic dosages, (ii) under both chronic and acute application, and (iii) for both neonicotinoid and non-neonicotinoid pesticides groups.We also expose key gaps in the literature that include a critical lack of studies on non-Apis bees, on larval exposure (potentially one of the major exposure routes), and on performance in alternative learning paradigms. Policy implications. Procedures for the registration of new pesticides within EU member states now typically require assessment of risks to pollinators if potential target crops are attractive to bees. However, our results provide robust quantitative evidence for subtle, sublethal effects, the consequences of which are unlikely to be detected within small-scale prelicensing laboratory or field trials, but can be critical when pesticides are used at a landscape scale. Our findings highlight the need for long-term postlicensing environmental safety monitoring as a requirement within licensing policy for plant protection products.

Sulfoxaflor exposure reduces bumblebee reproductive success.

Intensive agriculture currently relies on pesticides to maximize crop yield1,2. Neonicotinoids are the most widely used insecticides globally3, but increasing evidence of negative impacts on important pollinators4-9 and other non-target organisms10 has led to legislative reassessment and created demand for the development of alternative products. Sulfoximine-based insecticides are the most likely successor11, and are either licensed for use or under consideration for licensing in several worldwide markets3, including within the European Union12, where certain neonicotinoids (imidacloprid, clothianidin and thiamethoxam) are now banned from agricultural use outside of permanent greenhouse structures. There is an urgent need to pre-emptively evaluate the potential sub-lethal effects of sulfoximine-based pesticides on pollinators11, because such effects are rarely detected by standard ecotoxicological assessments, but can have major impacts at larger ecological scales13-15. Here we show that chronic exposure to the sulfoximine-based insecticide sulfoxaflor, at dosages consistent with potential post-spray field exposure, has severe sub-lethal effects on bumblebee (Bombus terrestris) colonies. Field-based colonies that were exposed to sulfoxaflor during the early growth phase produced significantly fewer workers than unexposed controls, and ultimately produced fewer reproductive offspring. Differences between the life-history trajectories of treated and control colonies first became apparent when individuals exposed as larvae began to emerge, suggesting that direct or indirect effects on a small cohort may have cumulative long-term consequences for colony fitness. Our results caution against the use of sulfoximines as a direct replacement for neonicotinoids. To avoid continuing cycles of novel pesticide release and removal, with concomitant impacts on the environment, a broad evidence base needs to be assessed prior to the development of policy and regulation.

Incubation environment impacts the social cognition of adult lizards.

Recent work exploring the relationship between early environmental conditions and cognition has shown that incubation environment can influence both brain anatomy and performance in simple operant tasks in young lizards. It is currently unknown how it impacts other, potentially more sophisticated, cognitive processes. Social-cognitive abilities, such as gaze following and social learning, are thought to be highly adaptive as they provide a short-cut to acquiring new information. Here, we investigated whether egg incubation temperature influenced two aspects of social cognition, gaze following and social learning in adult reptiles (Pogona vitticeps). Incubation temperature did not influence the gaze following ability of the bearded dragons; however, lizards incubated at colder temperatures were quicker at learning a social task and faster at completing that task. These results are the first to show that egg incubation temperature influences the social cognitive abilities of an oviparous reptile species and that it does so differentially depending on the task. Further, the results show that the effect of incubation environment was not ephemeral but lasted long into adulthood. It could thus have potential long-term effects on fitness.

The impact of egg incubation temperature on the personality of oviparous reptiles.

Personality traits, defined as differences in the behavior of individual animals of the same species that are consistent over time and context, such as 'boldness,' have been shown to be both heritable and be influenced by external factors, such as predation pressure. Currently, we know very little about the role that early environmental factors have upon personality. Thus, we investigated the impact of incubation temperature upon the boldness on an oviparous reptile, the bearded dragon (Pogona vitticeps). Eggs, from one clutch, were incubated at two different average temperatures within the normal range. After hatching the lizards were raised under the same environmental conditions. Novel object and novel environment tests were used to assess personality. Each test was repeated in both the short term and the long term. The results revealed that incubation temperature did impact upon 'boldness' but only in the short term and suggests that, rather than influencing personality, incubation temperature may have an effect on the development of behavioral of oviparous reptiles at different stages across ontogeny.