What the Science Shows

There is widespread consensus in the scientific community that pesticides are having a devastating effect on the sustainability of pollinator populations. A recent systematic review of insect declines worldwide finds pollinators, and insects as a whole, in dire trouble. Research findings across several studies confirm that agricultural intensification, pesticide use, and in particular, the spread of systemic insecticides, are the main drivers for ongoing mass pollinator declines.

Multiple studies have confirmed that the levels of neonicotinoid pesticides that bees encounter in the environment are toxic enough to impair foraging, navigational, and learning behaviors, as well as suppress immune responses. These individual impacts are compounded at the level of social colonies, weakening collective resistance to common parasites, pathogens other pesticides, and thus leading to colony losses and mass population declines. In 2018, more than two hundred scientists co-authored a “Call to restrict neonicotinoids” on the basis of the bulk of evidence implicating neonicotinoids in mass pollinator and beneficial insect declines.

In the early 2000s, Colony Collapse Disorder (CCD) brought national attention to increased honey bee colony losses. During the same period that CCD and colony losses spiked, neonicotinoid prevalence skyrocketed, in large part due to the introduction of seed-delivered technologies. As of 2011, 34-44% of soybeans and 79-100% of maize hectares were preemptively treated with neonicotinoids. While CCD prevalence has decreased, colony loss rates (and systemic insecticide use) remain high. A 2018 national survey indicates that U.S. beekeepers currently experience an average annual colony mortality rate of 30.7%, double the pre-CCD baseline of 15% losses.

Native pollinators are similarly threatened by increased use of systemic insecticides. Recent studies of wild and managed pollinators in the field have shown significant colony and population declines as a direct result of neonicotinoid crop treatment and intensified pesticide use. A 2008-2013 study of wild bee populations across various land types in the US found the greatest declines in regions of concentrated corn production, concomitant with the tripling of neonicotinoid use in maize. Wild bee populations are declining by more than 30% in the US corn-belt, where neonicotinoid use is by now ubiquitous. A 23% decline in California butterfly species documented over the last few decades began sharply following the introduction of neonicotinoids to the state in 1995.

The research article abstracts quoted below highlight the impact of pesticides on wild and managed bees, other pollinators like butterflies and birds, and other organisms that are beneficial to the environment.



Pesticide impacts on bees

Pesticide impacts on other pollinators

Pesticide impacts on beneficial organisms

Parasites and Viruses

Pesticide Impacts on Bees

Insecticide exposure during brood or early-adult development reduces brain growth and impairs adult learning in bumblebees. (Smith, D. B. et al. 2020) For social bees, an understudied step in evaluating pesticide risk is how contaminated food entering colonies affects residing offspring development and maturation. For instance, neurotoxic insecticide compounds in food could affect central nervous system development predisposing individuals to become poorer task performers later-in-life. Studying bumblebee colonies provisioned with neonicotinoid spiked nectar substitute, we measured brain volume and learning behaviour of 3 or 12-day old adults that had experienced in-hive exposure during brood and/or early-stage adult development. Micro-computed tomography scanning and segmentation of multiple brain neuropils showed exposure during either of the developmental stages caused reduced mushroom body calycal growth relative to unexposed workers. Associated with this was a lower probability of responding to a sucrose reward and lower learning performance in an olfactory conditioning test. While calycal volume of control workers positively correlated with learning score, this relationship was absent for exposed workers indicating neuropil functional impairment. Comparison of 3- and 12-day adults exposed during brood development showed a similar degree of reduced calycal volume and impaired behaviour highlighting lasting and irrecoverable effects from exposure despite no adult exposure. Our findings help explain how the onset of pesticide exposure to whole colonies can lead to lag-effects on growth and resultant dysfunction.

County-level analysis reveals a rapidly shifting landscape of insecticide hazard to honey bees (Apis mellifera) on US farmland. (Douglas, M. R. et al. 2020) Each year, millions of kilograms of insecticides are applied to crops in the US. While insecticide use supports food, fuel, and fiber production, it can also threaten non-target organisms, a concern underscored by mounting evidence of widespread decline of pollinator populations. Here, we integrate several public datasets to generate county-level annual estimates of total 'bee toxic load' (honey bee lethal doses) for insecticides applied in the US between 1997–2012, calculated separately for oral and contact toxicity. To explore the underlying components of the observed changes, we divide bee toxic load into extent (area treated) and intensity (application rate x potency). We show that while contact-based bee toxic load remained relatively steady, oral-based bee toxic load increased roughly 9-fold, with reductions in application rate outweighed by disproportionate increases in potency (toxicity/kg) and extent. This pattern varied markedly by region, with the greatest increase seen in Heartland (121-fold increase), likely driven by use of neonicotinoid seed treatments in corn and soybean. In this "potency paradox", farmland in the central US has become more hazardous to bees despite lower volumes of insecticides applied, raising concerns about insect conservation and highlighting the importance of integrative approaches to pesticide use monitoring.

Neonicotinoids and ectoparasitic mites synergistically impact honeybees. (Straub, L. et al. 2019) The Western honeybee, Apis mellifera, is the most important managed pollinator globally and has recently experienced unsustainably high colony losses. Synergistic interactions among stressors are believed to be primarily responsible. However, despite clear evidence of strong effect on honeybee longevity of widely-employed neonicotinoid insecticides and of the ubiquitous ectoparasitic mite Varroa destructor, no data exist to show synergistic effects between these two stressors. Even though neonicotinoids had no significant impact by themselves, we here show for the first time a synergistic time-lag interaction between mites and neonicotinoids that resulted in significantly reduced survival of long-lived winter honeybees. Even though these mites are potent vectors of viruses, the virus-insecticide interaction had no significant impact. The data suggest a previously overlooked mechanism possibly explaining recent unsustainably high losses of managed A. mellifera honeybee colonies in many regions of the world. Future mitigation efforts should concentrate on developing sustainable agro-ecosystem management schemes that incorporate reduced use of neonicotinoids and sustainable solutions for V. destructor mites.

Pesticide exposure affects flight dynamics and reduces flight endurance in bumblebees. (Kenna, D. et al. 2019) The emergence of agricultural land use change creates a number of challenges that insect pollinators, such as eusocial bees, must overcome. Resultant fragmentation and loss of suitable foraging habitats, combined with pesticide exposure, may increase demands on foraging, specifically the ability to collect or reach sufficient resources under such stress. Understanding effects that pesticides have on flight performance is therefore vital if we are to assess colony success in these changing landscapes. Neonicotinoids are one of the most widely used classes of pesticide across the globe, and exposure to bees has been associated with reduced foraging efficiency and homing ability. One explanation for these effects could be that elements of flight are being affected, but apart from a couple of studies on the honeybee (Apis mellifera), this has scarcely been tested. Here, we used flight mills to investigate how exposure to a field realistic (10 ppb) acute dose of imidacloprid affected flight performance of a wild insect pollinator—the bumblebee, Bombus terrestris audax. Intriguingly, observations showed exposed workers flew at a significantly higher velocity over the first ¾ km of flight. This apparent hyperactivity, however, may have a cost because exposed workers showed reduced flight distance and duration to around a third of what control workers were capable of achieving. Given that bumblebees are central place foragers, impairment to flight endurance could translate to a decline in potential forage area, decreasing the abundance, diversity, and nutritional quality of available food, while potentially diminishing pollination service capabilities.

Neural conduction, visual motion detection, and insect flight behaviour are disrupted by low doses of imidacloprid and its metabolites. (Parkinson, R.H. and Gray, J.R., 2019) While neonicotinoid insecticides impair visually guided behaviours, the effects of their metabolites are unknown and measurements of environmental concentrations of neonicotinoids, typically lower than those required to elicit toxic effects, tend to exclude metabolites. Here we examined the contributions of imidacloprid and two of its metabolites, imidacloprid-olefin and 5-hydroxy-imidacloprid, on neural conduction velocity, visual motion detection and flight in the locust (Locusta migratoria) using a combination of electrophysiological and behavioural assays. We show reduced visual motion detection and impaired flight behaviour following treatment of metabolite concentrations equal to sublethal doses of the parent compound. Additionally, we show for the first time that imidacloprid and its metabolites result in a decrease in conduction velocity along an unmyelinated axon. We suggest that secondary effects of the insecticide on the biophysical properties of the axon may result in decreased neural conduction. As these metabolites display neurotoxicity similar to the parent compound they should be considered when quantifying environmental concentrations.

Chronic contact with realistic soil concentrations of imidacloprid affects the mass, immature development speed, and adult longevity of solitary bees. (Anderson, N.L. and Harmon-Threatt, A.N., 2019) The non-target effects of pesticides are an area of growing concern, particularly for ecologically and economically important organisms such as bees. Much of the previous research on the effects of neonicotinoids, a class of insecticide that has gained attention for non-target effects, on bees focused on the consumption of contaminated food resources by a limited number of eusocial species. However, neonicotinoids are known to accumulate and persist in soils at concentrations 2 to 60 times greater than in food resources, and may represent an important route of exposure for diverse and ecologically important ground-nesting bees. This study aimed to assess the effect of chronic contact exposure to realistic soil concentrations of imidacloprid, the most widely used neonicotinoid pesticide, on bee longevity, development speed, and body mass. Cohorts of Osmia lignaria and Megachile rotundata were used as proxies for ground-nesting species. We observed species- and sex-specific changes to adult longevity, development speed, and mass in response to increasing concentrations of imidacloprid. These results suggest that chronic exposure to nesting substrates contaminated with neonicotinoids may represent an important route of exposure that could have considerable physiological and ecological consequences for bees and plant-pollinator interactions.

A mechanistic framework to explain the immunosuppressive effects of neurotoxic pesticides on bees. (Pamminger, T. et al. 2018). There is growing concern that declines in some managed and wild bee pollinator populations threaten biodiversity, the functioning of vital ecological processes and sustainable food production on a global scale. In recent years, there has been increasing evidence that sublethal exposure to the neurotoxic class of insecticides (neonicotinoids) can harm the immune systems of pollinators. This then results in them being more susceptible to the effects of disease, which is one of the main causes of pollinator declines. However, exactly how neonicotinoid insecticides might inhibit pollinator immunity has remained something of a mystery. In this review, we propose that the close connection during development between the immune and nervous systems of insects makes them inherently susceptible to neurotoxins such as neonicotinoids interfering with the proper function of their immune systems. Investigation of this physiological connection is urgently needed to develop a clear understanding of the interplay between neonicotinoids and disease ecology in pollinators. This in turn may enable us to develop strategies to mitigate the impacts of neurotoxins on pollinators and help better manage their populations in the future.

Quantifying the impact of pesticides on learning and memory in bees. (Siviter, H. et al. 2018). 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.

Neonicotinoid exposure disrupts bumblebee nest behavior, social networks, and thermoregulation. (Crall, JD et al. 2018). Neonicotinoid pesticides cause mortality and decline in insect pollinators. One repeatedly noted effect is a reduction in bee colony size. However, the mechanism behind this reduction is unclear. Crall et al. performed complex real-time monitoring of bumblebee behavior within their nests (see the Perspective by Raine). Neonicotinoid exposure reduced nurse and caretaking behaviors, which affected productivity and harmed colony thermoregulation. These changes in behavior acted together to decrease colony viability, even when ex

Impacts of neonicotinoid use on long-term population changes in wild bees in England. (Woodcok et al. 2016). Wild bee declines have been ascribed in part to neonicotinoid insecticides. While short-term laboratory studies on commercially bred species (principally honeybees and bumblebees) have identified sub-lethal effects, there is no strong evidence linking these insecticides to losses of the majority of wild bee species. We relate 18 years of UK national wild bee distribution data for 62 species to amounts of neonicotinoid use in oilseed rape. Using a multi-species dynamic Bayesian occupancy analysis, we find evidence of increased population extinction rates in response to neonicotinoid seed treatment use on oilseed rape. Species foraging on oilseed rape benefit from the cover of this crop, but were on average three times more negatively affected by exposure to neonicotinoids than non-crop foragers. Our results suggest that sub-lethal effects of neonicotinoids could scale up to cause losses of bee biodiversity. Restrictions on neonicotinoid use may reduce population declines.

The Neonicotinoid Insecticide Thiacloprid Impacts upon Bumblebee Colony Development under Field Conditions. (Ciaran, E et al. 2017). The impacts of pesticides, and in particular of neonicotinoids, on bee health remain much debated. Many studies describing negative effects have been criticized as the experimental protocol did not perfectly simulate real-life field scenarios. Here, we placed free-flying bumblebee colonies next to raspberry crops that were either untreated or treated with the neonicotinoid thiacloprid as part of normal farming practice. Colonies were exposed to the raspberry crops for a two week period before being relocated to either a flower-rich or flower-poor site. Overall, exposed colonies were more likely to die prematurely, and those that survived reached a lower final weight and produced 46% fewer reproductives than colonies placed at control farms. The impact was more marked at the flower-rich site (all colonies performed poorly at the flower poor site). Analysis of nectar and pollen stores from bumblebee colonies placed at the same raspberry farms revealed thiacloprid residues of up to 771 ppb in pollen and up to 561 ppb in nectar. The image of thiacloprid as a relatively benign neonicotinoid should now be questioned.

Sulfoxaflor exposure reduces bumblebee reproductive success. (Siviter, H. et al. 2018). Intensive agriculture currently relies on pesticides to maximize crop yield. Neonicotinoids are the most widely used insecticides globally, but increasing evidence of negative impacts on important pollinators and other non-target organisms has led to legislative reassessment and created demand for the development of alternative products. Sulfoximine-based insecticides are the most likely successor, and are either licensed for use or under consideration for licensing in several worldwide markets, including within the European Union, 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 pollinators, because such effects are rarely detected by standard ecotoxicological assessments, but can have major impacts at larger ecological scales. 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.

Ecotoxiclogical effects of the insecticide fipronil in Brazilian native stingless bees Melipona scutellaris (Apidae: Meliponini). (de Morais, C et al. 2018). Melipona scutellaris Latreille, 1811 (Hymenoptera, Apidae) is a pollinator of various native and cultivated plants. Because of the expansion of agriculture and the need to ensure pest control, the use of insecticides such as fipronil (FP) has increased. This study aimed to evaluate the effects of sublethal doses of FP insecticide on M. scutellaris at different time intervals (6, 12, and 24 h) after exposure, via individually analyzed behavioral biomarkers (locomotor activity, behavioral change) as well as the effect of FP on different brain structures of bees (mushroom bodies, antennal cells, and optic cells), using sub-individual cell biomarkers (heterochromatin dispersion, total nuclear and heterochromatic volume). Forager bees were collected when they were returning to the nest and were exposed to three different concentrations of FP (0.40, 0.040, and 0.0040 ng a.i/bee) by topical application. The results revealed a reduction in the mean velocity, lethargy, motor difficulty, paralysis, and hyperexcitation in all groups of bees treated with FP. A modification of the heterochromatic dispersion pattern and changes in the total volume of the nucleus and heterochromatin were also observed in the mushroom bodies (6, 12, and 24 h of exposure) and antennal lobes (6 and 12 h) of bees exposed to 0.0040 ng a.i/bee (LD 50/100 ). FP is toxic to M. scutellaris and impairs the essential functions required for the foraging activity.

Sub-lethal effects of dietary neonicotinoid insecticide exposure on honey bee queen fecundity and colony development. (Wu-Smart, J. and Spivak, M., 2016). Many factors can negatively affect honey bee (Apis mellifera L.) health including the pervasive use of systemic neonicotinoid insecticides. Through direct consumption of contaminated nectar and pollen from treated plants, neonicotinoids can affect foraging, learning, and memory in worker bees. Less well studied are the potential effects of neonicotinoids on queen bees, which may be exposed indirectly through trophallaxis, or food-sharing. To assess effects on queen productivity, small colonies of different sizes (1500, 3000, and 7000 bees) were fed imidacloprid (0, 10, 20, 50, and 100 ppb) in syrup for three weeks. We found adverse effects of imidacloprid on queens (egg-laying and locomotor activity), worker bees (foraging and hygienic activities), and colony development (brood production and pollen stores) in all treated colonies. Some effects were less evident as colony size increased, suggesting that larger colony populations may act as a buffer to pesticide exposure. This study is the first to show adverse effects of imidacloprid on queen bee fecundity and behavior and improves our understanding of how neonicotinoids may impair short-term colony functioning. These data indicate that risk-mitigation efforts should focus on reducing neonicotinoid exposure in the early spring when colonies are smallest and queens are most vulnerable to exposure.

Impaired associative learning after chronic exposure to pesticides in young adult honey bees. Neonicotinoids are the most widespread insecticides in agriculture, preferred for their low toxicity to mammals and their systemic nature. Nevertheless, there have been increasing concerns regarding their impact on non-target organisms. Glyphosate is also widely used in crops and, therefore, traces of this pesticide are likely to be found together with neonicotinoids. Although glyphosate is considered a herbicide, adverse effects have been found on animal species, including honey bees. Apis mellifera is one of the most important pollinators in agroecosystems and is exposed to both these pesticides. Traces can be found in nectar and pollen of flowers that honey bees visit, but also in honey stores inside the hive. Young workers, which perform in-hive tasks that are crucial for colony maintenance, are potentially exposed to both these contaminated resources. These workers present high plasticity and are susceptible to stimuli that can modulate their behaviour and impact on colony state. Therefore, by performing standardised assays to study sublethal effects of these pesticides, these bees can be used as bioindicators. We studied the effect of chronic joint exposure to field-realistic concentrations of the neonicotinoid imidacloprid and glyphosate on gustatory perception and olfactory learning. Both pesticides reduced sucrose responsiveness and had a negative effect on olfactory learning. Glyphosate also reduced food uptake during rearing. The results indicate differential susceptibility according to honey bee age. The two agrochemicals had adverse effects on different aspects of honey bee appetitive behaviour, which could have repercussions for food distribution, propagation of olfactory information and task coordination within the nest.

The pesticide flupyradifurone impairs olfactory learning in Asian honey bees (Apis cerana) exposed as larvae or as adults.

Relatively little attention has focused on how pesticides may affect Asian honey bees, which provide vital crop pollination services and are key native pollinators. We therefore studied the effects of a relatively new pesticide, flupyradifurone (FLU), which has been developed, in part, because it appears safer for honey bees than neonicotinoids. We tested the effects of FLU on Apis cerana olfactory learning in larvae (lower dose of 0.033 µg/larvae/day over 6 days) and, in a separate experiment, adults (lower dose of 0.066 µg/adult bee/day) at sublethal, field-realistic doses given over 3 days. A worst-case field-realistic dose is 0.44 µg/bee/day. Learning was tested in adult bees. The lower larval dose did not increase mortality, but the lower adult dose resulted in 20% mortality. The lower FLU doses decreased average olfactory learning by 74% (larval treatment) and 48% (adult treatment) and reduced average memory by 48% (larval treatment) and 22% (adult treatment) as compared to controls. FLU at higher doses resulted in similar learning impairments. The effects of FLU, a pesticide that is reported to be safer than neonicotinoids for honey bees, thus deserve greater attention.

Toxicity and motor changes in Africanized honey bees (Apis mellifera L.) exposed to fipronil and imidacloprid.

This study evaluated the in vitro toxicity and motor activity changes in African-derived adult honey bees (Apis mellifera L.) exposed to lethal or sublethal doses of the insecticides fipronil and imidacloprid. Mortality of bees was assessed to determine the ingestion and contact lethal dose for 24 h using probit analysis. Motor activities in bees exposed to lethal (LD50) and sublethal doses (1/500th of the lethal dose) of both insecticides were evaluated in a behavioral observation box at 1 and 4 h. Ingestion and contact lethal doses of fipronil were 0.2316 ? 0.0626 and 0.0080 ? 0.0021 μg/bee, respectively. Ingestion and contact lethal doses of imidacloprid were 0.1079 ? 0.0375 and 0.0308 ? 0.0218 μg/bee, respectively. Motor function of bees exposed to lethal doses of fipronil and imidacloprid was impaired; exposure to sublethal doses of fipronil but not imidacloprid impaired motor function. The insecticides evaluated in this study were highly toxic to African-derived A. mellifera and caused impaired motor function in these pollinators.

Environmental Risks and Challenges Associated with Neonicotinoid Insecticides.

Neonicotinoid use has increased rapidly in recent years, with a global shift toward insecticide applications as seed coatings rather than aerial spraying. While the use of seed coatings can lessen the amount of overspray and drift, the near universal and prophylactic use of neonicotinoid seed coatings on major agricultural crops has led to widespread detections in the environment (pollen, soil, water, honey). Pollinators and aquatic insects appear to be especially susceptible to the effects of neonicotinoids with current research suggesting that chronic sublethal effects are more prevalent than acute toxicity. Meanwhile, evidence of clear and consistent yield benefits from the use of neonicotinoids remains elusive for most crops. Future decisions on neonicotinoid use will benefit from weighing crop yield benefits versus environmental impacts to nontarget organisms and considering whether there are more environmentally benign alternatives.

Detrimental interactions of neonicotinoid pesticide exposure and bumblebee immunity.

Pesticides are well known to have a number of ecological effects. However, it is only now becoming understood that sublethal exposures may have effects on nontarget insects of conservation concern through interactions with immunity, thus increasing detrimental impacts in the presence of pathogens. Pesticides and pathogens are suggested to have played a role in recent declines of several wild bee pollinators. Compromised immunity from exposure to widely used neonicotinoids has been demonstrated in honeybees, but further research on interactions between neonicotinoids and immunity in other important bees is lacking. In this study, adult workers of the bumblebee Bombus impatiens received 6-day pulses of either low (0.7 ppb) or high (7 ppb) field realistic doses of the neonicotinoid imidacloprid prior to assaying immunity and survival following a nonpathogenic immune challenge. High-dose imidacloprid exposure reduces constitutive levels of phenoloxidase, an enzyme involved in melanization. Hemolymph antimicrobial activity initially increases in all groups following an immune challenge, but while heightened activity is maintained in unexposed and low imidacloprid dose groups, it is not maintained in the high exposure dose bees, even though exposure had ceased 6 days prior. Additionally, imidacloprid exposure followed by an immune challenge significantly decreased survival probability relative to control bees and those only immune challenged or imidacloprid exposed. A temporal lag for immune modulation and combinatorial effects on survival suggest that resource-based trade-offs may, in part, contribute to the detrimental interactions. These interactions could have health consequences for pollinators facing multiple stresses of sublethal neonicotinoid exposure and pathogens.

Analysis and evaluation of (neuro)peptides in honey bees exposed to pesticides in field conditions.

There is a big absence of studies with quantitative results correlating the effect of pesticide exposure with changes on neuropeptides insects, and most of them are conducted under laboratory conditions, typically with individual active ingredients. In this study, we present an analytical workflow to evaluate pesticide effects on honey bees through the analysis of (neuro)peptides. The workflow consists of a rapid extraction method and liquid chromatography with triple quadrupole for preselected neuropeptides. For non-target analysis, high resolution mass spectrometry, multivariate analysis and automatic identification of discriminated peptides using a specific software and protein sequence databases. The analytical method was applied to the analysis of target and non-target (neuro)peptides in honey bees with low and high content of a wide range of pesticides to which have been exposed in field conditions. Our findings show that the identification frequency of target neuropeptides decreases significantly in honey bees with high concentration of pesticides (pesticide concentrations ≥ 500 μg kg-1) in comparison with the honey bees with low content of pesticides (pesticide concentrations ≤ 20 μg kg-1). Moreover, the principal component analysis in non-target search shows a clear distinction between peptide concentration in honey bees with high level of pesticides and honey bees with low level. The use of high resolution mass spectrometry has allowed the identification of 25 non-redundant peptides responsible for discrimination between the two groups, derived from 18 precursor proteins.

Neonicotinoid pesticide limits improvement in buzz pollination by bumblebees.

Neonicotinoid pesticides have been linked to global declines of beneficial insects such as bumblebees. Exposure to trace levels of these chemicals causes sub-lethal effects, such as reduced learning and foraging efficiency. Complex behaviours may be particularly vulnerable to the neurotoxic effects of neonicotinoids. Such behaviours may include buzz pollination (sonication), in which pollinators, usually bees, use innate and learned behaviours to generate high-frequency vibrations to release pollen from flowers with specialised anther morphologies. This study assesses the effect of field-realistic, chronic exposure to the widely-used neonicotinoid thiamethoxam on the development of sonication buzz characteristics over time, as well as the collection of pollen from buzz-pollinated flowers. We found that the pollen collection of exposed bees improved less with increasing experience than that of unexposed bees, with exposed bees collecting between 47% and 56% less pollen by the end of 10 trials. We also found evidence of two distinct strategies for maximising pollen collection: (1) extensions to the duration of individual buzzes and (2) extensions of the overall time spent buzzing. We find new complexities in buzz pollination, and conclude that the impacts of field-realistic exposure to a neonicotinoid pesticide may seriously compromise this important ecosystem service.

Neonicotinoid pesticides can reduce honeybee colony genetic diversity.

Neonicotinoid insecticides can cause a variety of adverse sub-lethal effects in bees. In social species such as the honeybee, Apis mellifera, queens are essential for reproduction and colony functioning. Therefore, any negative effect of these agricultural chemicals on the mating success of queens may have serious consequences for the fitness of the entire colony. Queens were exposed to the common neonicotinoid pesticides thiamethoxam and clothianidin during their developmental stage. After mating, their spermathecae were dissected to count the number of stored spermatozoa. Furthermore, their worker offspring were genotyped with DNA microsatellites to determine the number of matings and the genotypic composition of the colony. Colonies providing the male mating partners were also inferred. Both neonicotinoid and control queens mated with drones originating from the same drone source colonies, and stored similar number of spermatozoa. However, queens reared in colonies exposed to both neonicotinoids experienced fewer matings. This resulted in a reduction of the genetic diversity in their colonies (i.e. higher intracolonial relatedness). As decreased genetic diversity among worker bees is known to negatively affect colony vitality, neonicotinoids may have a cryptic effect on colony health by reducing the mating frequency of queens.

Honey bee-collected pollen in agro-ecosystems reveals diet diversity, diet quality, and pesticide exposure.

European honey bees Apis mellifera are important commercial pollinators that have suffered greater than normal overwintering losses since 2007 in North America and Europe. Contributing factors likely include a combination of parasites, pesticides, and poor nutrition. We examined diet diversity, diet nutritional quality, and pesticides in honey bee-collected pollen from commercial colonies in the Canadian Maritime Provinces in spring and summer 2011. We sampled pollen collected by honey bees at colonies in four site types: apple orchards, blueberry fields, cranberry bogs, and fallow fields. Proportion of honey bee-collected pollen from crop versus noncrop flowers was high in apple, very low in blueberry, and low in cranberry sites. Pollen nutritional value tended to be relatively good from apple and cranberry sites and poor from blueberry and fallow sites. Floral surveys ranked, from highest to lowest in diversity, fallow, cranberry, apple, and blueberry sites. Pesticide diversity in honey bee-collected pollen was high from apple and blueberry sites and low from cranberry and fallow sites. Four different neonicotinoid pesticides were detected, but neither these nor any other pesticides were at or above LD50 levels. Pollen hazard quotients were highest in apple and blueberry sites and lowest in fallow sites. Pollen hazard quotients were also negatively correlated with the number of flower taxa detected in surveys. Results reveal differences among site types in diet diversity, diet quality, and pesticide exposure that are informative for improving honey bee and land agro-ecosystem management.

Neonicotinoids act like endocrine disrupting chemicals in newly-emerged bees and winter bees.

Accumulating evidence suggests that neonicotinoids may have long-term adverse effects on bee health, yet our understanding of how this could occur is incomplete. Pesticides can act as endocrine disrupting chemicals (EDCs) in animals providing characteristic multiphasic dose-response curves and non-lethal endpoints in toxicity studies. However, it is not known if neonicotinoids act as EDCs in bees. To address this issue, we performed oral acute and chronic toxicity studies including concentrations recorded in nectar and pollen, applying acetamiprid, clothianidin, imidacloprid, and thiamethoxam to bumble bees, honey bees and leafcutter bees, the three most common bee species managed for pollination. In acute toxicity studies, late-onset symptoms, such as ataxia, were recorded as non-lethal endpoints for all three bee species. Clothianidin and thiamethoxam produced biphasic dose-response curves for all three bee species. Clothianidin and thiamethoxam were extremely toxic to winter worker honey bees prior to brood production in spring, making this the most sensitive bee stage identified to date. Chronic exposure to field-realistic levels of neonicotinoids reduced bee survival and caused significant late-onset symptoms for all three bee species. Given these findings, neonicotinoid risk should be reevaluated to address the EDC-like behavior and the sensitivity of winter worker honey bees.

Concentration and movement of neonicotinoids as particulate matter downwind during agricultural practices using air samplers in southwestern Ontario, Canada.

Atmospheric emissions of neonicotinoid seed treatment insecticides as particulate matter in field crops occur mainly for two reasons: 1) due to abraded dust of treated seed generated during planting using vacuum planters, and 2) as a result of disturbances (tillage or wind events) in the surface of parental soils which release wind erodible soil-bound residues. In the present study, concentration and movement of neonicotinoids as particulate matter were quantified under real conditions using passive and active air samplers. Average neonicotinoid concentrations in Total Suspended Particulate (TSP) using passive samplers were 0.48 ng/cm2, trace, trace (LOD 0.80 and 0.04 ng/cm2 for clothianidin and thiamethoxam, respectively), and using active samplers 16.22, 1.91 and 0.61 ng/m3 during planting, tillage and wind events, respectively. There was a difference between events on total neonicotinoid concentration collected in particulate matter using either passive or active sampling. Distance of sampling from the source field during planting of treated seed had an effect on total neonicotinoid air concentration. However, during tillage distance did not present an effect on measured concentrations. Using hypothetical scenarios, values of contact exposure for a honey bee were estimated to be in the range from 1.1% to 36.4% of the reference contact LD50 value of clothianidin of 44 ng/bee.

Assessment of acute sublethal effects of clothianidin on motor function of honeybee workers using video-tracking analysis.

Sublethal impacts of pesticides on the locomotor activity might occur to different degrees and could escape visual observation. Therefore, our objective is the utilization of video-tracking to quantify how the acute oral exposure to different doses (0.1-2ng/bee) of the neonicotinoid "clothianidin" influences the locomotor activity of honeybees in a time course experiment. The total distance moved, resting time as well as the duration and frequency of bouts of laying upside down are measured. Our results show that bees exposed to acute sublethal doses of clothianidin exhibit a significant increase in the total distance moved after 30 and 60min of the treatment at the highest dose (2ng/bee). Nevertheless, a reduction of the total distance is observed at this dose 90min post-treatment compared to the distance of the same group after 30min, where the treated bees show an arched abdomen and start to lose their postural control. The treated bees with 1ng clothianidin show a significant increase in total distance moved over the experimental period. Moreover, a reduction in the resting time and increase of the duration and frequency of bouts of laying upside down at these doses are found. Furthermore, significant effects on the tested parameters are observed at the dose (0.5ng/bee) first at 60min post-treatment compared to untreated bees. The lowest dose (0.1ng/bee) has non-significant effects on the motor activity of honeybees compared to untreated bees over the experimental period.

Landscape Scale Study of the Net Effect of Proximity to a Neonicotinoid-Treated Crop on Bee Colony Health.

Since 2013, the European Commission has restricted the use of three neonicotinoid insecticides as seed dressings on bee-attractive crops. Such crops represent an important source of forage for bees, which is often scarce in agro-ecosystems. However, this benefit has often been overlooked in the design of previous field studies, leaving the net impact of neonicotinoid treated crops on bees relatively unknown. Here, we determine the combined benefit (forage) and cost (insecticide) of oilseed rape grown from thiamethoxam-treated seeds on Bombus terrestris and Apis mellifera colonies. In April 2014, 36 colonies per species were located adjacent to three large oilseed rape fields (12 colonies per field). Another 36 were in three nearby locations in the same agro-ecosystem, but several kilometers distant from any oilseed rape fields. We found that Bombus colony growth and reproduction were unaffected by location (distant versus adjacent) following the two month flowering period. Apis colony and queen survival were unaffected. However, there was a small, but significant, negative relationship between honey and pollen neonicotinoid contamination and Apis colony weight gain. We hypothesize that any sublethal effects of neonicotinoid seed dressings on Bombus colonies are potentially offset by the additional foraging resources provided. A better understanding of the ecological and agronomic factors underlying neonicotinoid residues is needed to inform evidence-based policy.

Lethal and sublethal effects, and incomplete clearance of ingested imidacloprid in honey bees (Apis mellifera).

A previous study claimed a differential behavioural resilience between spring or summer honey bees (Apis mellifera) and bumble bees (Bombus terrestris) after exposure to syrup contaminated with 125 µg L-1 imidacloprid for 8 days. The authors of that study based their assertion on the lack of body residues and toxic effects in honey bees, whereas bumble bees showed body residues of imidacloprid and impaired locomotion during the exposure. We have reproduced their experiment using winter honey bees subject to the same protocol. After exposure to syrup contaminated with 125 µg L-1 imidacloprid, honey bees experienced high mortality rates (up to 45%), had body residues of imidacloprid in the range 2.7-5.7 ng g-1 and exhibited abnormal behaviours (restless, apathetic, trembling and falling over) that were significantly different from the controls. There was incomplete clearance of the insecticide during the 10-day exposure period. Our results contrast with the findings reported in the previous study for spring or summer honey bees, but are consistent with the results reported for the other bee species.

Planting of neonicotinoid-coated corn raises honey bee mortality and sets back colony development.

Worldwide occurrences of honey bee colony losses have raised concerns about bee health and the sustainability of pollination-dependent crops. While multiple causal factors have been identified, seed coating with insecticides of the neonicotinoid family has been the focus of much discussion and research. Nonetheless, few studies have investigated the impacts of these insecticides under field conditions or in commercial beekeeping operations. Given that corn-seed coating constitutes the largest single use of neonicotinoid, our study compared honey bee mortality from commercial apiaries located in two different agricultural settings, i.e. corn-dominated areas and corn-free environments, during the corn planting season. Data was collected in 2012 and 2013 from 26 bee yards. Dead honey bees from five hives in each apiary were counted and collected, and samples were analyzed using a multi-residue LC-MS/MS method. Long-term effects on colony development were simulated based on a honey bee population dynamic model. Mortality survey showed that colonies located in a corn-dominated area had daily mortality counts 3.51 times those of colonies from corn crop-free sites. Chemical analyses revealed that honey bees were exposed to various agricultural pesticides during the corn planting season, but were primarily subjected to neonicotinoid compounds (54% of analysed samples contained clothianidin, and 31% contained both clothianidin and thiamethoxam). Performance development simulations performed on hive populations' show that increased mortality during the corn planting season sets back colony development and bears contributions to collapse risk but, most of all, reduces the effectiveness and value of colonies for pollination services. Our results also have implications for the numerous large-scale and worldwide-cultivated crops that currently rely on pre-emptive use of neonicotinoid seed treatments.

Bumblebee colony development following chronic exposure to field-realistic levels of the neonicotinoid pesticide thiamethoxam under laboratory conditions.

Neonicotinoid pesticides are used in agriculture to reduce damage from crop pests. However, beneficial insects such as bees can come into contact with these pesticides when foraging in treated areas, with potential consequences for bee declines and pollination service delivery. Honeybees are typically used as a model organism to investigate insecticide impacts on bees, but relatively little is known about impacts on other taxa such as bumblebees. In this experiment, we chronically exposed whole mature bumblebee (Bombus terrestris) colonies to field-realistic levels of the neonicotinoid thiamethoxam (2.4ppb & 10ppb) over four weeks, and compared colony growth under laboratory conditions. We found no impact of insecticide exposure on colony weight gain, or the number or mass of sexuals produced, although colonies exposed to 2.4ppb produced larger males. As previous studies have reported pesticide effects on bumblebee colony growth, this may suggest that impacts on bumblebee colonies are more pronounced for colonies at an earlier stage in the reproductive cycle. Alternatively, it may also indicate that thiamethoxam differs in toxicity compared to previously tested neonicotinoids in terms of reproductive effects. In either case, assessing bumblebee colony development under field conditions is likely more informative for real world scenarios than tests conducted in laboratory conditions.

Differential physiological effects of neonicotinoid insecticides on honey bees: A comparison between Apis mellifera and Apis cerana.

Acute toxicities (LD50s) of imidacloprid and clothianidin to Apis mellifera and A. cerana were investigated. Changing patterns of immune-related gene expressions and the activities of four enzymes between the two bee species were compared and analyzed after exposure to sublethal doses of insecticides. Results indicated that A. cerana was more sensitive to imidacloprid and clothianidin than A. mellifera. The acute oral LD50 values of imidacloprid and clothianidin for A. mellifera were 8.6 and 2.0ng/bee, respectively, whereas the corresponding values for A. cerana were 2.7 and 0.5ng/bee. The two bee species possessed distinct abilities to mount innate immune response against neonicotinoids. After 48h of imidacloprid treatment, carboxylesterase (CCE), prophenol oxidase (PPO), and acetylcholinesterase (AChE) activities were significantly downregulated in A. mellifera but were upregulated in A. cerana. Glutathione-S-transferase (GST) activity was significantly elevated in A. mellifera at 48h after exposure to imidacloprid, but no significant change was observed in A. cerana. AChE was downregulated in both bee species at three different time points during clothianidin exposure, and GST activities were upregulated in both species exposed to clothianidin. Different patterns of immune-related gene expression and enzymatic activities implied distinct detoxification and immune responses of A. cerana and A. mellifera to imidacloprid and clothianidin.

Impact of Thiamethoxam on Honey Bee Queen (Apis mellifera carnica) Reproductive Morphology and Physiology.

High honey bee losses around the world have been linked in part by the regular use of neonicotinoids in agriculture. In light of the current situation, the aim of this study was to investigate the effects of thiamethoxam on the development of the reproductive system and physiology in the honey bee queen. Two experimental groups of honey bee queen larvae were treated with thiamethoxam during artificial rearing, applied via artificial feed in two cycles. In the first rearing cycle, honey bee larvae received a single treatment dose (4.28 ng thiamethoxam/queen larva on the 4th day after larvae grafting in artificial queen cells), while the second honey bee queen rearing cycle received a double treatment dose (total of 8.56 ng thiamethoxam/queen larva on the 4th and 5th day after larvae grafting in artificial queen cells). After emerging, queens were anesthetized and weighed, and after mating with drones were anesthetized, weighed, and sectioned. Ovary mass and number of stored sperm were determined. Body weight differed between untreated and treated honey bee queens. The results also show a decrease in the number of sperm within honey bee queen spermathecae that received the double thiamethoxam dose.

Chronic exposure to neonicotinoids reduces honey bee health near corn crops.

Experiments linking neonicotinoids and declining bee health have been criticized for not simulating realistic exposure. Here we quantified the duration and magnitude of neonicotinoid exposure in Canada's corn-growing regions and used these data to design realistic experiments to investigate the effect of such insecticides on honey bees. Colonies near corn were naturally exposed to neonicotinoids for up to 4 months-the majority of the honey bee's active season. Realistic experiments showed that neonicotinoids increased worker mortality and were associated with declines in social immunity and increased queenlessness over time. We also discovered that the acute toxicity of neonicotinoids to honey bees doubles in the presence of a commonly encountered fungicide. Our work demonstrates that field-realistic exposure to neonicotinoids can reduce honey bee health in corn-growing regions.

Country-specific effects of neonicotinoid pesticides on honey bees and wild bees.

Neonicotinoid seed dressings have caused concern world-wide. We use large field experiments to assess the effects of neonicotinoid-treated crops on three bee species across three countries (Hungary, Germany, and the United Kingdom). Winter-sown oilseed rape was grown commercially with either seed coatings containing neonicotinoids (clothianidin or thiamethoxam) or no seed treatment (control). For honey bees, we found both negative (Hungary and United Kingdom) and positive (Germany) effects during crop flowering. In Hungary, negative effects on honey bees (associated with clothianidin) persisted over winter and resulted in smaller colonies in the following spring (24% declines). In wild bees (Bombus terrestris and Osmia bicornis), reproduction was negatively correlated with neonicotinoid residues. These findings point to neonicotinoids causing a reduced capacity of bee species to establish new populations in the year following exposure.

The challenges of predicting pesticide exposure of honey bees at landscape level.

To evaluate the risks of pesticides for pollinators, we must not only evaluate their toxicity but also understand how pollinators are exposed to these xenobiotics in the field. We focused on this last point and modeled honey bee exposure to pesticides at the landscape level. Pollen pellet samples (n = 60) from 40 Belgian apiaries were collected from late July to October 2011 and underwent palynological and pesticide residue analyses. Areas of various crops around each apiary were measured at 4 spatial scales. The most frequently detected pesticides were the fungicides boscalid (n = 19, 31.7%) and pyrimethanil (n = 10, 16.7%) and the insecticide dimethoate (n = 10, 16.7%). We were able to predict exposure probability for boscalid and dimethoate by using broad indicators of cropping intensity, but it remained difficult to identify the precise source of contamination (e.g. specific crops in which the use of the pesticide is authorized). For pyrimethanil, we were not able to build any convincing landscape model that could explain the contamination. Our results, combined with the late sampling period, strongly suggest that pesticides applied to crops unattractive to pollinators, and therefore considered of no risk for them, may be sources of exposure through weeds, drift to neighboring plants, or succeeding crops.

Exposure of larvae to thiamethoxam affects the survival and physiology of the honey bee at post-embryonic stages.

Under laboratory conditions, the effects of thiamethoxam were investigated in larvae, pupae and emerging honey bees after exposure at larval stages with different concentrations in the food (0.00001 ng/μL, 0.001 ng/μL and 1.44 ng/μL). Thiamethoxam reduced the survival of larvae and pupae and consequently decreased the percentage of emerging honey bees. Thiamethoxam induced important physiological disturbances. It increased acetylcholinesterase (AChE) activity at all developmental stages and increased glutathione-S-transferase (GST) and carboxylesterase para (CaEp) activities at the pupal stages. For midgut alkaline phosphatase (ALP), no activity was detected in pupae stages, and no effect was observed in larvae and emerging bees. We assume that the effects of thiamethoxam on the survival, emergence and physiology of honey bees may affect the development of the colony. These results showed that attention should be paid to the exposure to pesticides during the developmental stages of the honey bee. This study represents the first investigation of the effects of thiamethoxam on the development of A. mellifera following larval exposure.

The environmental risks of neonicotinoid pesticides: a review of the evidence post 2013.

Neonicotinoid pesticides were first introduced in the mid-1990s, and since then, their use has grown rapidly. They are now the most widely used class of insecticides in the world, with the majority of applications coming from seed dressings. Neonicotinoids are water-soluble, and so can be taken up by a developing plant and can be found inside vascular tissues and foliage, providing protection against herbivorous insects. However, only approximately 5% of the neonicotinoid active ingredient is taken up by crop plants and most instead disperses into the wider environment. Since the mid-2000s, several studies raised concerns that neonicotinoids may be having a negative effect on non-target organisms, in particular on honeybees and bumblebees. In response to these studies, the European Food Safety Authority (EFSA) was commissioned to produce risk assessments for the use of clothianidin, imidacloprid and thiamethoxam and their impact on bees. These risk assessments concluded that the use of these compounds on certain flowering crops poses a high risk to bees. On the basis of these findings, the European Union adopted a partial ban on these substances in May 2013. The purpose of the present paper is to collate and summarise scientific evidence published since 2013 that investigates the impact of neonicotinoids on non-target organisms. Whilst much of the recent work has focused on the impact of neonicotinoids on bees, a growing body of evidence demonstrates that persistent, low levels of neonicotinoids can have negative impacts on a wide range of free-living organisms.

Ornamental plants on sale to the public are a significant source of pesticide residues with implications for the health of pollinating insects.

Garden centres frequently market nectar- and pollen-rich ornamental plants as "pollinator-friendly", however these plants are often treated with pesticides during their production. There is little information on the nature of pesticide residues present at the point of purchase and whether these plants may actually pose a threat to, rather than benefit, the health of pollinating insects. Using mass spectrometry analyses, this study screened leaves from 29 different 'bee-friendly' plants for 8 insecticides and 16 fungicides commonly used in ornamental production. Only two plants (a Narcissus and a Salvia variety) did not contain any pesticide and 23 plants contained more than one pesticide, with some species containing mixtures of 7 (Ageratum houstonianum) and 10 (Erica carnea) different agrochemicals. Neonicotinoid insecticides were detected in more than 70% of the analysed plants, and chlorpyrifos and pyrethroid insecticides were found in 10% and 7% of plants respectively. Boscalid, spiroxamine and DMI-fungicides were detected in 40% of plants. Pollen samples collected from 18 different plants contained a total of 13 different pesticides. Systemic compounds were detected in pollen samples at similar concentrations to those in leaves. However, some contact (chlorpyrifos) and localised penetrant pesticides (iprodione, pyroclastrobin and prochloraz) were also detected in pollen, likely arising from direct contamination during spraying. The neonicotinoids thiamethoxam, clothianidin and imidacloprid and the organophosphate chlorpyrifos were present in pollen at concentrations between 6.9 and 81 ng/g and at levels that overlap with those known to cause harm to bees. The net effect on pollinators of buying plants that are a rich source of forage for them but simultaneously risk exposing them to a cocktail of pesticides is not clear. Gardeners who wish to gain the benefits without the risks should seek uncontaminated plants by growing their own from seed, plant-swapping or by buying plants from an organic nursery.

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.

Neonicotinoid pesticides and nutritional stress synergistically reduce survival in honey bees.

The honey bee is a major pollinator whose health is of global concern. Declines in bee health are related to multiple factors, including resource quality and pesticide contamination. Intensive agricultural areas with crop monocultures potentially reduce the quality and quantity of available nutrients and expose bee foragers to pesticides. However, there is, to date, no evidence for synergistic effects between pesticides and nutritional stress in animals. The neonicotinoids clothianidin (CLO) and thiamethoxam (TMX) are common systemic pesticides that are used worldwide and found in nectar and pollen. We therefore tested if nutritional stress (limited access to nectar and access to nectar with low-sugar concentrations) and sublethal, field-realistic acute exposures to two neonicotinoids (CLO and TMX at 1/5 and 1/25 of LD50) could alter bee survival, food consumption andhaemolymph sugar levels. Bee survival was synergistically reduced by the combination of poor nutrition and pesticide exposure (-50%). Nutritional and pesticide stressors reduced also food consumption (-48%) and haemolymph levels of glucose (-60%) and trehalose (-27%). Our results provide the first demonstration that field-realistic nutritional stress and pesticide exposure can synergistically interact and cause significant harm to animal survival. These findings have implications for current pesticide risk assessment and pollinator protection.

Disruption of quercetin metabolism by fungicide affects energy production in honey bees (Apis mellifera)

Cytochrome P450 monooxygenases (P450) in the honey bee, Apis mellifera, detoxify phytochemicals in honey and pollen. The flavonol quercetin is found ubiquitously and abundantly in pollen and frequently at lower concentrations in honey. Worker jelly consumed during the first 3 d of larval development typically contains flavonols at very low levels, however, RNA-Seq analysis of gene expression in neonates reared for three days on diets with and without quercetin revealed that, in addition to up-regulating multiple detoxifying P450 genes, quercetin is a negative transcriptional regulator of mitochondrion-related nuclear genes and genes encoding subunits of complexes I, III, IV, and V in the oxidative phosphorylation pathway. Thus, a consequence of inefficient metabolism of this phytochemical may be compromised energy production. Several P450s metabolize quercetin in adult workers. Docking in silico of 121 pesticide contaminants of American hives into the active pocket of CYP9Q1, a broadly substrate-specific P450 with high quercetin-metabolizing activity, identified six triazole fungicides, all fungal P450 inhibitors, that dock in the catalytic site. In adults fed combinations of quercetin and the triazole myclobutanil, the expression of five of six mitochondrion-related nuclear genes was down-regulated. Midgut metabolism assays verified that adult bees consuming quercetin with myclobutanil metabolized less quercetin and produced less thoracic ATP, the energy source for flight muscles. Although fungicides lack acute toxicity, they may influence bee health by interfering with quercetin detoxification, thereby compromising mitochondrial regeneration and ATP production. Thus, agricultural use of triazole fungicides may put bees at risk of being unable to extract sufficient energy from their natural food.

An Inert Pesticide Adjuvant Synergizes Viral Pathogenicity and Mortality in Honey Bee Larvae

Honey bees are highly valued for their pollination services in agricultural settings, and recent declines in managed populations have caused concern. Colony losses following a major pollination event in the United States, almond pollination, have been characterized by brood mortality with specific symptoms, followed by eventual colony loss weeks later. In this study, we demonstrate that these symptoms can be produced by chronically exposing brood to both an organosilicone surfactant adjuvant (OSS) commonly used on many agricultural crops including wine grapes, tree nuts and tree fruits and exogenous viral pathogens by simulating a horizontal transmission event. Observed synergistic mortality occurred during the larval-pupal molt. Using q-PCR techniques to measure gene expression and viral levels in larvae taken prior to observed mortality at metamorphosis, we found that exposure to OSS and exogenous virus resulted in significantly heightened Black Queen Cell Virus (BQCV) titers and lower expression of a Toll 7-like-receptor associated with autophagic viral defense (Am18w). These results demonstrate that organosilicone spray adjuvants that are considered biologically inert potentiate viral pathogenicity in honey bee larvae, and guidelines for OSS use may be warranted.

The neonicotinoid pesticide, imidacloprid, affects Bombus impatiens (bumblebee) sonication behavior when consumed at doses below the LD50.

Study investigated changes in sonication (or buzz-pollination) behavior of Bombus impatiens bumblebees, after consumption of the neonicotinoid pesticide, imidacloprid. Authors measured sonication frequency, sonication length, and flight (wing beat) frequency of marked bees collecting pollen from Solanum lycopsersicum (tomato), and then randomly assigned bees to consume 0, 0.0515, 0.515, or 5.15 ng of imidacloprid. The number of bees in each treatment group that resumed sonication behavior after consuming imidacloprid, and re-measured sonication and flight behavior for these bees was recorded. Results did not find evidence that consuming 0.0515 ng imidacloprid affected the sonication length, sonication frequency, or flight frequency for bees that sonicated after consuming imidacloprid. Bumblebees who consumed 0.515 or 5.15 ng of imidacloprid were significantly less likely to sonicate after treatment than bees who consumed no imidacloprid. At the end of the experiment, the study classified bees as dead or alive; our data suggest a trend of increasing mortality with higher doses of imidacloprid. Results show that even modest doses of imidacloprid can significantly affect the likelihood of bumblebees engaging in sonication, a behavior critical for the pollination of a variety of crops and other plants.

The Environmental Risks of neonicotinoid pesticides: a review of the evidence post-2013

The purpose of this review is to collate and summarise scientific evidence published since 2013 that investigates the impact of neonicotinoids on non-target organisms and to bring it into one place to aid informed decision making. Field-realistic laboratory experiments and field trials continue to demonstrate that traces of residual neonicotinoids can have a mixture of lethal and sublethal effects on a wide range of taxa. Susceptibility varies tremendously between different taxa across many orders of magnitude, with some showing a negative response at parts per billion with others show no such effects at many thousands of parts per billion. Relative to the risk assessments produced in 2013 for clothianidin, imidacloprid and thiamethoxam which focussed on their effects on bees, new research strengthens arguments for the imposition of a moratorium, in particular because it has become evident that they pose significant risks to many non-target organisms, not just bees. Given the improvement in scientific knowledge of how neonicotinoids move into the wider environment from all crop types, a discussion of the risks posed by their use on non-flowering crops and in non-agricultural areas is urgently needed.

Impact of controlled neonicotinoid exposure on bumblebees in a realistic field setting

Using 20 bumblebee colonies, authors assess the consequences of exposure to the neonicotinoid clothianidin, provided in sucrose at a concentration of five parts per billion, over 5 weeks. Foraging patterns and pollen collecting performance from 3282 bouts using either a non-invasive photographic assessment, or by extracting the pollen from returning foragers were monitored. Study detected only subtle changes to patterns of foraging activity and pollen foraging during the course of the experiment. However, colony census measures showed a more pronounced effect of exposure, with fewer adult workers and sexuals in treated colonies after 5 weeks. Pesticide-induced impairments on colony development and foraging could impact on the pollination service that bees provide. Therefore, these findings, that bees show subtle changes in foraging behaviour and reductions in colony size after exposure to a common pesticide, have important implications and help to inform the debate over whether the benefits of systemic pesticide application to flowering crops outweigh the costs.

Monitoring of neonicotinoid pesticides in beekeeping

The decline of pollinating species is correlated to the extensive use of neonicotinoids against pest insects for crop protection. In this study, the concentrations of neonicotinoid insecticides were determined in honeybees, honeycomb and honey samples, collected in Spring 2015 (blooming period) from different areas in Sicily (IT), to carry out an evaluation of bees products' safety and an overview of neonicotinoid contamination in beekeeping. The results obtained showed only the presence of clothianidin in bee samples and these concentrations don't represent a risk for bees' vitality and safety. The absence of residue in all honey samples, instead, showed the quality of bee products.

Sub-lethal effects of dietary neonicotinoid insecticide exposure on honey bee queen fecundity and colony development

Many factors can negatively affect honey bee (Apis mellifera L.) health including the pervasive use of systemic neonicotinoid insecticides. Through direct consumption of contaminated nectar and pollen from treated plants, neonicotinoids can affect foraging, learning, and memory in worker bees. Less well studied are the potential effects of neonicotinoids on queen bees, which may be exposed indirectly through trophallaxis, or food-sharing. To assess effects on queen productivity, small colonies of different sizes (1500, 3000, and 7000 bees) were fed imidacloprid (0, 10, 20, 50, and 100 ppb) in syrup for three weeks. We found adverse effects of imidacloprid on queens (egg-laying and locomotor activity), worker bees (foraging and hygienic activities), and colony development (brood production and pollen stores) in all treated colonies. Some effects were less evident as colony size increased, suggesting that larger colony populations may act as a buffer to pesticide exposure. This study is the first to show adverse effects of imidacloprid on queen bee fecundity and behavior and improves our understanding of how neonicotinoids may impair short-term colony functioning. These data indicate that risk-mitigation efforts should focus on reducing neonicotinoid exposure in the early spring when colonies are smallest and queens are most vulnerable to exposure.

Neonicotinoid insecticides can serve as inadvertent insect contraceptives

There is clear evidence for sublethal effects of neonicotinoid insecticides on non-target ecosystem service-providing insects. However, their possible impact on male insect reproduction is currently unknown, despite the key role of sex. Here, we show that two neonicotinoids (4.5 ppb thiamethoxam and 1.5 ppb clothianidin) significantly reduce the reproductive capacity of male honeybees (drones), Apis mellifera Drones were obtained from colonies exposed to the neonicotinoid insecticides or controls, and subsequently maintained in laboratory cages until they reached sexual maturity. While no significant effects were observed for male teneral (newly emerged adult) body mass and sperm quantity, the data clearly showed reduced drone lifespan, as well as reduced sperm viability (percentage living versus dead) and living sperm quantity by 39%. Our results demonstrate for the first time that neonicotinoid insecticides can negatively affect male insect reproductive capacity, and provide a possible mechanistic explanation for managed honeybee queen failure and wild insect pollinator decline. The widespread prophylactic use of neonicotinoids may have previously overlooked inadvertent contraceptive effects on non-target insects, thereby limiting conservation efforts.

Neonicotinoid-contaminated pollinator strips adjacent to cropland reduce honey bee nutritional status

Worldwide pollinator declines are attributed to a number of factors, including pesticide exposures. Neonicotinoid insecticides specifically have been detected in surface waters, non-target vegetation, and bee products, but the risks posed by environmental exposures are still not well understood. Pollinator strips were tested for clothianidin contamination in plant tissues, and the risks to honey bees assessed. An enzyme-linked immunosorbent assay (ELISA) quantified clothianidin in leaf, nectar, honey, and bee bread at organic and seed-treated farms. Total glycogen, lipids, and protein from honey bee workers were quantified. The proportion of plants testing positive for clothianidin were the same between treatments. Leaf tissue and honey had similar concentrations of clothianidin between organic and seed-treated farms. Honey (mean±SE: 6.61 ± 0.88 ppb clothianidin per hive) had seven times greater concentrations than nectar collected by bees (0.94 ± 0.09 ppb). Bee bread collected from organic sites (25.8 ± 3.0 ppb) had significantly less clothianidin than those at seed treated locations (41.6 ± 2.9 ppb). Increasing concentrations of clothianidin in bee bread were correlated with decreased glycogen, lipid, and protein in workers. This study shows that small, isolated areas set aside for conservation do not provide spatial or temporal relief from neonicotinoid exposures in agricultural regions where their use is largely prophylactic.

Non-cultivated plants present a season-long route of pesticide exposure for honey bees.

Recent efforts to evaluate the contribution of neonicotinoid insecticides to worldwide pollinator declines have focused on honey bees and the chronic levels of exposure experienced when foraging on crops grown from neonicotinoid-treated seeds. However, few studies address non-crop plants as a potential route of pollinator exposure to neonicotinoid and other insecticides. Here we show that pollen collected by honey bee foragers in maize- and soybean-dominated landscapes is contaminated throughout the growing season with multiple agricultural pesticides, including the neonicotinoids used as seed treatments. Notably, however, the highest levels of contamination in pollen are pyrethroid insecticides targeting mosquitoes and other nuisance pests. Furthermore, pollen from crop plants represents only a tiny fraction of the total diversity of pollen resources used by honey bees in these landscapes, with the principle sources of pollen originating from non-cultivated plants. These findings provide fundamental information about the foraging habits of honey bees in these landscapes.

No effect of low-level chronic neonicotinoid exposure on bumblebee learning and fecundity

In recent years, many pollinators have declined in abundance and diversity worldwide, presenting a potential threat to agricultural productivity, biodiversity and the functioning of natural ecosystems. One of the most debated factors proposed to be contributing to pollinator declines is exposure to pesticides, particularly neonicotinoids, a widely used class of systemic insecticide. Also, newly emerging parasites and diseases, thought to be spread via contact with managed honeybees, may pose threats to other pollinators such as bumblebees. Compared to honeybees, bumblebees could be particularly vulnerable to the effects of stressors due to their smaller and more short-lived colonies. Here, we studied the effect of field-realistic, chronic clothianidin exposure and inoculation with the parasite Nosema ceranae on survival, fecundity, sugar water collection and learning using queenless Bombus terrestris audax microcolonies in the laboratory. Chronic exposure to 1 ppb clothianidin had no significant effects on the traits studied. Interestingly, pesticide exposure in combination with additional stress caused by harnessing bees for Proboscis Extension Response (PER) learning assays, led to an increase in mortality. In contrast to previous findings, the bees did not become infected by N. ceranae after experimental inoculation with the parasite spores, suggesting variability in host resistance or parasite virulence. However, this treatment induced a slight, short-term reduction in sugar water collection, potentially through stimulation of the immune system of the bees. Our results suggest that chronic exposure to 1 ppb clothianidin does not have adverse effects on bumblebee fecundity or learning ability.

Influence of pesticide use in fruit orchards during blooming on honeybee mortality in 4 experimental apiaries

Samples of dead honey bees (Apis mellifera L.) were collected periodically from 4 different locations during citrus and stone fruit trees blooming season to evaluate the potential impact of agrochemicals on honey bee death rate. For the determination of mortality, dead honey bee traps were placed in front of the experimental hives entrance located in areas of intensive agriculture in Valencian Community (Spain). A total of 34 bee samples, obtained along the monitoring period, were analyzed by means of QuEChERS extraction method and screened for 58 pesticides or their degradation products by LC-MS/MS. An average of four pesticides per honey bee sample was detected. Coumaphos, an organophosphate acaricide used against varroosis in the experimental hives, was detected in 94% of the samples. However, this acaricide was unlikely to be responsible for honey bee mortality because its constantly low concentration during all the monitoring period, even before and after acute mortality episodes. The organophosphates chlorpyrifos and dimethoate, as well as the neonicotinoid imidacloprid, were the most frequently detected agrochemicals. Almost 80% of the samples had chlorpyrifos, 68% dimethoate, and 32% imidacloprid. Maximum concentrations for these three compounds were 751, 403, 223 ng/g respectively. Influence of these pesticides on acute honey bee mortality was demonstrated by comparing coincidence between death rate and concentrations of chlorpyrifos, dimethoate and imidacloprid.

In-hive Pesticide Exposome: Assessing risks to migratory honey bees from in-hive pesticide contamination in the Eastern United States

This study measured part of the in-hive pesticide exposome by analyzing residues from live in-hive bees, stored pollen, and wax in migratory colonies over time and compared exposure to colony health. We summarized the pesticide burden using three different additive methods: (1) the hazard quotient (HQ), an estimate of pesticide exposure risk, (2) the total number of pesticide residues, and (3) the number of relevant residues. Despite being simplistic, these models attempt to summarize potential risk from multiple contaminations in real-world contexts. Colonies performing pollination services were subject to increased pesticide exposure compared to honey-production and holding yards. We found clear links between an increase in the total number of products in wax and colony mortality. In particular, we found that fungicides with particular modes of action increased disproportionally in wax within colonies that died. The occurrence of queen events, a significant risk factor for colony health and productivity, was positively associated with all three proxies of pesticide exposure. While our exposome summation models do not fully capture the complexities of pesticide exposure, they nonetheless help elucidate their risks to colony health. Implementing and improving such models can help identify potential pesticide risks, permitting preventative actions to improve pollinator health.

Honey Bee Gut Microbiome Is Altered by In-Hive Pesticide Exposures

Honey bees (Apis mellifera) are the primary pollinators of major horticultural crops. Over the last few decades, a substantial decline in honey bees and their colonies have been reported. While a plethora of factors could contribute to the putative decline, pathogens, and pesticides are common concerns that draw attention. In addition to potential direct effects on honey bees, indirect pesticide effects could include alteration of essential gut microbial communities and symbionts that are important to honey bee health (e.g., immune system). The primary objective of this study was to determine the microbiome associated with honey bees exposed to commonly used in-hive pesticides: coumaphos, tau-fluvalinate, and chlorothalonil. Treatments were replicated at three independent locations near Blacksburg Virginia, and included a no-pesticide amended control at each location. The microbiome was characterized through pyrosequencing of V2-V3 regions of the bacterial 16S rRNA gene and fungal ITS region. Pesticide exposure significantly affected the structure of bacterial but not fungal communities. The bee bacteriome, similar to other studies, was dominated by sequences derived from Bacilli, Actinobacteria, α-, β-, γ-proteobacteria. The fungal community sequences were dominated by Ascomycetes and Basidiomycetes. The Multi-response permutation procedures (MRPP) and subsequent Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis indicated that chlorothalonil caused significant change to the structure and functional potential of the honey bee gut bacterial community relative to control. Putative genes for oxidative phosphorylation, for example, increased while sugar metabolism and peptidase potential declined in the microbiome of chlorothalonil exposed bees. The results of this field-based study suggest the potential for pesticide induced changes to the honey bee gut microbiome that warrant further investigation.

Effects of a neonicotinoid pesticide on thermoregulation of African honey bees (Apis mellifera scutellata)

Thiamethoxam is a widely used neonicotinoid pesticide that, as agonist of the nicotinic acetylcholine receptors, has been shown to elicit a variety of sublethal effects in honey bees. However, information concerning neonicotinoid effects on honey bee thermoregulation is lacking. Thermoregulation is an essential ability for the honey bee that guarantees the success of foraging and many in-hive tasks, especially brood rearing. We tested the effects of acute exposure to thiamethoxam (0.2, 1, 2ng/bee) on the thorax temperatures of foragers exposed to low (22°C) and high (33°C) temperature environments. Thiamethoxam significantly altered honey bee thorax temperature at all doses tested; the effects elicited varied depending on the environmental temperature and pesticide dose to which individuals were exposed. When bees were exposed to the high temperature environment, the high dose of thiamethoxam increased their thorax temperature 1-2h after exposure. When bees were exposed to the low temperature, the higher doses of the neonicotinoid reduced bee thorax temperatures 60-90min after treatment. In both experiments, the neonicotinoid decreased the temperature of bees the day following the exposure. After a cold shock (5min at 4°C), the two higher doses elicited a decrease of the thorax temperature, while the lower dose caused an increase, compared to the control. These alterations in thermoregulation caused by thiamethoxam may affect bee foraging activity and a variety of in-hive tasks, likely leading to negative consequences at the colony level. Our results shed light on sublethal effect of pesticides which our bees have to deal with.

The fungicide Pristine® inhibits mitochondrial function in vitro but not flight metabolic rates in honey bees

Honey bees and other pollinators are exposed to fungicides that act by inhibiting fungal mitochondria. Here we test whether a common fungicide (Pristine®) inhibits the function of mitochondria of honeybees, and whether consumption of ecologically-realistic concentrations can cause negative effects on the mitochondria of flight muscles, or the capability for flight, as judged by CO2 emission rates and thorax temperatures during flight. Direct exposure of mitochondria to Pristine® levels above 5 ppm strongly inhibited mitochondrial oxidation rates in vitro. However, bees that consumed pollen containing Pristine® at ecologically-realistic concentrations (≈ 1 ppm) had normal flight CO2 emission rates and thorax temperatures. Mitochondria isolated from the flight muscles of the Pristine®-consuming bees had higher state 3 oxygen consumption rates than control bees, suggesting that possibly Pristine®-consumption caused compensatory changes in mitochondria. It is likely that the lack of a strong functional effect of Pristine®-consumption on flight performance and the in vitro function of flight muscle mitochondria results from maintenance of Pristine® levels in the flight muscles at much lower levels than occur in the food, probably due to metabolism and detoxification. As Pristine® has been shown to negatively affect feeding rates and protein digestion of honey bees, it is plausible that Pristine® consumption negatively affects gut wall function (where mitochondria may be exposed to higher concentrations of Pristine®).

Pollen Contaminated With Field-Relevant Levels of Cyhalothrin Affects Honey Bee Survival, Nutritional Physiology, and Pollen Consumption Behavior

Honey bees are exposed to a variety of environmental factors that impact their health, including nutritional stress, pathogens, and pesticides. In particular, there has been increasing evidence that sublethal exposure to pesticides can cause subtle, yet important effects on honey bee health and behavior. Here, we add to this body of knowledge by presenting data on bee-collected pollen containing sublethal levels of cyhalothrin, a pyrethroid insecticide, which, when fed to young honey bees, resulted in significant changes in lifespan, nutritional physiology,and behavior. For the first time, we show that when young, nest-aged bees are presented with pollen containing field-relevant levels of cyhalothrin, they reduce their consumption of contaminated pollen. This indicates that, at least for some chemicals, young bees are able to detect contamination in pollen and change their behavioral response, even if the contamination levels do not prevent foraging honey bees from collecting the contaminated pollen.

Spray Toxicity and Risk Potential of 42 Commonly Used Formulations of Row Crop Pesticides to Adult Honey Bees (Hymenoptera: Apidae)

To combat an increasing abundance of sucking insect pests, >40 pesticides are currently recommended and frequently used as foliar sprays on row crops, especially cotton. Foraging honey bees may be killed when they are directly exposed to foliar sprays, or they may take contaminated pollen back to hives that maybe toxic to other adult bees and larvae. To assess acute toxicity against the honey bee, we used a modified spray tower to simulate field spray conditions to include direct whole-body exposure, inhalation, and continuing tarsal contact and oral licking after a field spray. A total of 42 formulated pesticides, including one herbicide and one fungicide, were assayed for acute spray toxicity to 4-6-d-old workers. Results showed significantly variable toxicities among pesticides, with LC50s ranging from 25 to thousands of mg/liter. Further risk assessment using the field application concentration to LC1 or LC99 ratios revealed the risk potential of the 42 pesticides. Three pesticides killed less than 1% of the worker bees, including the herbicide, a miticide, and a neonicotinoid. Twenty-six insecticides killed more than 99% of the bees, including commonly used organophosphates and neonicotinoids. The remainder of the 13 chemicals killed from 1-99% of the bees at field application rates. This study reveals a realistic acute toxicity of 42 commonly used foliar pesticides. The information is valuable for guiding insecticide selection to minimize direct killing of foraging honey bees, while maintaining effective control of field crop pests.

Colonies of Bumble Bees (Bombus impatiens) Produce Fewer Workers, Less Bee Biomass, and Have Smaller Mother Queens Following Fungicide Exposure

Bees provide vital pollination services to the majority of flowering plants in both natural and agricultural systems. Unfortunately, both native and managed bee populations are experiencing declines, threatening the persistence of these plants and crops. Agricultural chemicals are one possible culprit contributing to bee declines. Even fungicides, generally considered safe for bees, have been shown to disrupt honey bee development and impair bumble bee behavior. Little is known, however, how fungicides may affect bumble bee colony growth. We conducted a controlled cage study to determine the effects of fungicide exposure on colonies of a native bumble bee species (Bombus impatiens). Colonies of B. impatiens were exposed to flowers treated with field-relevant levels of the fungicide chlorothalonil over the course of one month. Colony success was assessed by the number and biomass of larvae, pupae, and adult bumble bees. Bumble bee colonies exposed to fungicide produced fewer workers, lower total bee biomass, and had lighter mother queens than control colonies. Our results suggest that fungicides negatively affect the colony success of a native bumble bee species and that the use of fungicides during bloom has the potential to severely impact the success of native bumble bee populations foraging in agroecosystems.

The Status of Honey Bee Health in Italy: Results from the Nationwide Bee Monitoring Network

In Italy a nation-wide monitoring network was established in 2009 in response to significant honey bee colony mortality reported during 2008. The network comprised of approximately 100 apiaries located across Italy. Colonies were sampled four times per year, in order to assess the health status and to collect samples for pathogen, chemical and pollen analyses. The prevalence of Nosema ceranae ranged, on average, from 47-69% in 2009 and from 30-60% in 2010, with strong seasonal variation. Virus prevalence was higher in 2010 than in 2009. The most widespread viruses were BQCV, DWV and SBV. The most frequent pesticides in all hive contents were organophosphates and pyrethroids such as coumaphos and tau-fluvalinate. Beeswax was the most frequently contaminated hive product, with 40% of samples positive and 13% having multiple residues, while 27% of bee-bread and 12% of honey bee samples were contaminated. Colony losses in 2009/10 were on average 19%, with no major differences between regions of Italy. In 2009, the presence of DWV in autumn was positively correlated with colony losses. Similarly, hive mortality was higher in BQCV infected colonies in the first and second visits of the year. In 2010, colony losses were significantly related to the presence of pesticides in honey bees during the second sampling period. Honey bee exposure to poisons in spring could have a negative impact at the colony level, contributing to increase colony mortality during the beekeeping season. In both 2009 and 2010, colony mortality rates were positively related to the percentage of agricultural land surrounding apiaries, supporting the importance of land use for honey bee health.

Sperm viability and gene expression in honey bee queens (Apis mellifera) following exposure to the neonicotinoid insecticide imidacloprid and the organophosphate acaricide coumaphos

Honey bee population declines are of global concern. Numerous factors appear to cause these declines including parasites, pathogens, malnutrition and pesticides. Residues of the organophosphate acaricide coumaphos and the neonicotinoid insecticide imidacloprid, widely used to combat Varroa mites and for crop protection in agriculture, respectively, have been detected in wax, pollen and comb samples. Here, we assess the effects of these compounds at different doses on the viability of sperm stored in the honey bee queens' spermatheca. Our results demonstrate that sub-lethal doses of imidacloprid (0.02ppm) decreased sperm viability by 50%, 7days after treatment. Sperm viability was a downward trend (about 33%) in queens treated with high doses of coumaphos (100ppm), but there was not significant difference. The expression of genes that are involved in development, immune responses and detoxification in honey bee queens and workers exposed to chemicals was measured by qPCR analysis. The data showed that expression levels of specific genes were triggered 1day after treatment. The expression levels of P450 subfamily genes, CYP306A1, CYP4G11 and CYP6AS14 were decreased in honey bee queens treated with low doses of coumaphos (5ppm) and imidacloprid (0.02ppm). Moreover, these two compounds suppressed the expression of genes related to antioxidation, immunity and development in queens at day 1. Up-regulation of antioxidants by these compounds in worker bees was observed at day 1. Coumaphos also caused a repression of CYP306A1 and CYP4G11 in workers. Antioxidants appear to prevent chemical damage to honey bees. We also found that DWV replication increased in workers treated with imidacloprid. This research clearly demonstrates that chemical exposure can affect sperm viability in queen honey bees.

Effects of Imidacloprid and Varroa destructor on survival and health of European honey bees, Apis mellifera.

There has been growing concern over declines in populations of honey bees and other pollinators which are a vital part to our food security. It is imperative to identify factors responsible for accelerated declines in bee populations and develop solutions for reversing bee losses. While exact causes of colony losses remain elusive, risk factors thought to play key roles are ectoparasitic mites Varroa destructor and neonicotinoid pesticides. The present study aims to investigate effects of a neonicotinoid pesticide Imidacloprid and Varroa mites individually on survivorship, growth, physiology, virus dynamics and immunity of honey bee workers. Our study provides clear evidence that the exposure to sublethal doses of Imidacloprid could exert a significantly negative effect on health and survival of honey bees. We observed a significant reduction in the titer of vitellogenin (Vg), an egg yolk precursor that regulates the honey bees development and behavior and often are linked to energy homeostasis, in bees exposed to Imidacloprid. This result indicates that sublethal exposure to neonicotinoid could lead to increased energy usage in honey bees as detoxification is a energy-consuming metabolic process and suggests that Vg could be a useful biomarker for measuring levels of energy stress and sublethal effects of pesticides on honey bees. Measurement of the quantitative effects of different levels of Varroa mite infestation on the replication dynamic of Deformed wing virus (DWV), an RNA virus associated with Varroa infestation, and expression level of immune genes yields unique insights into how honey bees respond to stressors under laboratory conditions.

Drone exposure to the systemic insecticide Fipronil indirectly impairs queen reproductive potential

A species that requires sexual reproduction but cannot reproduce is doomed to extinction. The important increasing loss of species emphasizes the ecological significance of elucidating the effects of environmental stressors, such as pesticides, on reproduction. Despite its special reproductive behavior, the honey bee was selected as a relevant and integrative environmental model because of its constant and diverse exposure to many stressors due to foraging activity. The widely used insecticide Fipronil, the use of which is controversial because of its adverse effects on honey bees, was chosen to expose captive drones in hives via syrup contaminated at 0.1 μg/L and gathered by foragers. Such environmental exposure led to decreased spermatozoa concentration and sperm viability coupled with an increased sperm metabolic rate, resulting in drone fertility impairment. Subsequently, unexposed queens inseminated with such sperm exhibited fewer spermatozoa with lower viability in their spermatheca, leaving no doubt about the detrimental consequences for the reproductive potential of queens, which are key for colony sustainability. These findings suggest that pesticides could contribute to declining honey bee populations through fertility impairment, as exemplified by Fipronil. More broadly, reproductive disorders should be taken into consideration when investigating the decline of other species.

Toxicological Risks of Agrochemical Spray Adjuvants: Organosilicone Surfactants May Not Be Safe

Agrochemical risk assessment that takes into account only pesticide active ingredients without the spray adjuvants commonly used in their application will miss important toxicity outcomes detrimental to non-target species, including humans. Lack of disclosure of adjuvant and formulation ingredients coupled with a lack of adequate analytical methods constrains the assessment of total chemical load on beneficial organisms and the environment. Adjuvants generally enhance the pesticidal efficacy and inadvertently the non-target effects of the active ingredient. Spray adjuvants are largely assumed to be biologically inert and are not registered by the USA EPA, leaving their regulation and monitoring to individual states. Organosilicone surfactants are the most potent adjuvants and super-penetrants available to growers. Based on the data for agrochemical applications to almonds from California Department of Pesticide Regulation, there has been increasing use of adjuvants, particularly organosilicone surfactants, during bloom when two-thirds of USA honey bee colonies are present. Increased tank mixing of these with ergosterol biosynthesis inhibitors and other fungicides and with insect growth regulator insecticides may be associated with recent USA honey bee declines. This database archives every application of a spray tank adjuvant with detail that is unprecedented globally. Organosilicone surfactants are good stand alo