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

Agrochemical occurrence on colocated wildflowers and wild bees collected near beef cattle feed yards and row crops. (Peterson, E.M., et al. 2021) It is well established that agrochemicals can pose significant threats to native pollinators; however, relatively little is known about pollinator risks associated with agrochemicals that are used on beef cattle feed yards. Recently, feed yard-derived agrochemicals and those from row crop agriculture were quantified on wildflowers growing on the High Plains, USA. To better characterize pollinator risks on the High Plains, we collected colocated wildflowers and foraging bees across three field seasons for analytical determination of residual agrochemicals. Agrochemicals were detected and quantified on the majority of wildflowers (85%) and nearly half of bees (49%). Permethrin was the most frequently detected analyte on wildflowers (32%) and bees (17%). Flower hazard quotients and flower hazard indices were calculated to deterministically evaluate risk to foraging pollinators. Mean flower hazard quotients exceeded one for 5/16 analytes (31%), and flower hazard quotients calculated for 30% of wildflowers were greater than 50. Flower hazard quotients for clothianidin exceeded 400 for 14% of wildflowers, which portends conditions conducive to frequent bee mortalities. Flower hazard indices were greater on wildflowers from mid-July to mid-September as compared with wildflowers collected earlier in the summer, which coincides with row crop planting and increased prevalence of feed yard flies. Hazard quotients and hazard index values calculated from agrochemical residue data suggest that pollinators frequenting wildflowers near beef cattle feed yards and row crops on the High Plains are at risk from both individual sources, and more so when considered in combination.

Impact of Chronic Exposure to Sublethal Doses of Glyphosate on Honey Bee Immunity, Gut Microbiota and Infection by Pathogens. (Castelli, L., et al. 2021) Glyphosate is the most used pesticide around the world. Although different studies have evidenced its negative effect on honey bees, including detrimental impacts on behavior, cognitive, sensory and developmental abilities, its use continues to grow. Recent studies have shown that it also alters the composition of the honey bee gut microbiota. In this study we explored the impact of chronic exposure to sublethal doses of glyphosate on the honey bee gut microbiota and its effects on the immune response, infection by Nosema ceranae and Deformed wing virus (DWV) and honey bee survival. Glyphosate combined with N. ceranae infection altered the structure and composition of the honey bee gut microbiota, for example by decreasing the relative abundance of the core members Snodgrassella alvi and Lactobacillus apis. Glyphosate increased the expression of some immune genes, possibly representing a physiological response to mitigate its negative effects. However, this response was not sufficient to maintain honey bee health, as glyphosate promoted the replication of DWV and decreased the expression of vitellogenin, which were accompanied by a reduced life span. Infection by N. ceranae also alters honey bee immunity although no synergistic effect with glyphosate was observed. These results corroborate previous findings suggesting deleterious effects of widespread use of glyphosate on honey bee health, and they contribute to elucidate the physiological mechanisms underlying a global decline of pollination services.

Metabolomics unveils the influence of dietary phytochemicals on residual pesticide concentrations in honey bees. (Ardalani, H., et al. 2021) The losses of honey bee colonies and declines of other insect pollinators have been associated with negative effects of pesticides. Honey bees as well as other pollinators are nectar and pollen foragers and thus are exposed to an extensive range of phytochemicals. Understanding the synergistic, additive, and antagonistic effects of plant secondary metabolites and pesticides in honey bees may help to protect honey bee colonies against agrochemicals. In this study, we used untargeted metabolomics to investigate the impact of dietary phytochemical composition on the residual concentration of three pesticides: imidacloprid, tau-fluvalinate and tebuconazole in exposed honey bees. Honey bees were given different diets based on pollen or nectar from four plants: Reseda odorata, Borago officinalis, Phacelia tanacetifolia, and Trifolium repens for two days. Thereafter, they were orally exposed to 10 ng/bee imidacloprid or contact-exposed to 0.9 μg/bee tau-fluvalinate or 5 μg/bee tebuconazole. After 1 h of oral exposure or 24 h of contact exposure, the honey bees were anaesthetised with CO2, sacrificed by freezing, extracted with a validated QuEChERS method, and residual pesticide concentrations were determined by LC-QTRAP-MS/MS. The phytochemical composition in the given diets were profiled with an UHPLC-Q Exactive-MS/MS. The results revealed that the dietary phytochemical composition has a noteworthy influence on the concentration of residual pesticides in honey bees. The correlation coefficient analysis demonstrated that flavonoids have a reducing effect on the residual concentration of imidacloprid and tau-fluvalinate in honey bees. The results also highlighted that exposure to imidacloprid impaired the metabolism of sugars in honey bees. Exploiting flavonoid-rich plants may protect honey bees against pesticides and hold promise as forage plants in future beekeeping.

Short-term lab assessments and microcolonies are insufficient for the risk assessment of insecticides for bees. (Van Oystaeyen, A., et al. 2021.) Risk assessment studies addressing effects of agrochemicals on bumblebees frequently use microcolonies. These are queenless colonies consisting of workers only in which typically one worker will lay unfertilized male-destined eggs. In the first tier of risk assessment for bees, short-term laboratory experiments (e.g. microcolonies) are used, the results of which will determine whether higher tier (semi-)field experiments are needed. To evaluate the suitability of microcolonies for risk assessment, a direct comparison between different assessment methods for the neonicotinoid pesticides acetamiprid and thiacloprid was made: microcolonies and queenright colonies under short-term laboratory conditions, queenright colonies under long-term laboratory conditions, and queenright colonies under field conditions. Here, we demonstrate that results from microcolonies contradict results from queenright colonies. While thiacloprid negatively impacted gyne production in queenright colonies, it had a positive effect on microcolony size. By contrast, thiacloprid had no significant effect on fitness parameters of queenright colonies under short-term laboratory conditions when mostly workers are produced. These results thus highlight both the need for long term assessments, allowing evaluation of gyne production, and the risk of reaching erroneous conclusions when using microcolonies. The negative effect of thiacloprid on colony fitness was confirmed under field conditions, where thiacloprid affected the production of reproductives, colony weight gain, worker weight, and foraging behaviour. For acetamiprid, a negative trend on colony fitness could only be shown in a field setup. Therefore, field-realistic setups, which allow colonies to forage freely, are most appropriate to assess sublethal effects of pesticides affecting behaviour and learning.

The development of the solitary bee Osmia bicornis is affected by some insecticide agrochemicals at environmentally relevant concentrations. (Mokkapati, J.S., et al. 2021.) Solitary bees provide essential pollination services for many arable crops, but are prone to global decline. Agricultural intensification, which is connected with pesticide usage, is among major threats to bees and, thus, to the food security and ecosystem stability. As it may not be possible to cease pesticide usage currently because of the growing demand for food, it is crucial to understand the pesticide toxicities to bees for better protection of pollinator populations. The majority of studies have focused on social bees, and those on solitary bees studied effects of adult exposure, whereas these bees are also likely to be exposed as larvae via the consumption of contaminated pollen. Here, the effects of three commonly used insecticide-based plant protection products on the development of the solitary bee, Osmia bicornis (red mason bee), were studied by exposing larvae to insecticide-contaminated multifloral pollen. The tested insecticides were: Dursban480EC, containing the organophosphate chlorpyrifos (CHP), Sherpa100EC, containing the pyrethroid cypermethrin (CYP), and Mospilan20SP with the neonicotinoid acetamiprid (ACT). When compared to the control larvae fed with uncontaminated-pollen, both CHP and CYP significantly reduced the O. bicornis larval survival and their body mass at all tested concentrations. In contrast, ACT did not affect either larval survival or body mass, but the length of larval stage to cocoon formation was significantly shortened compared to controls. None of studied insecticides affected the mass of cocooned individuals. However, at least 80% of individuals exposed to any of the tested insecticides died before reaching the adult stage, whereas 43% of the controls emerged successfully after overwintering. Although no clear monotonic dose-response relationships were found, our study showed that at least some insecticide formulations affect the development of O. bicornis even at concentrations actually found in pollen in the field, indicating an urgent need for revising current pesticide usage recommendations.

The countryside or the city: Which environment is better for the honeybee?. (Mahé, C., et al. 2021.) For a number of years, the decline of honeybee (Apis mellifera) in North America and Europe has been the subject of much debate. Among the many factors proposed by hundreds of studies to explain this phenomenon is the hypothesis that agricultural activities using pesticides contribute to the weakness of bee colonies. Moreover, while urban beekeeping is presently booming in several cities, we do not know if this environment is more beneficial for bees than the typical, rural area. In the summer of 2018, we sampled honeybees (foragers and larvae) in rural (Laurentians) and urban (city of Montreal) areas and compared them using the following biomarkers: carotenoids, retinoids, α-tocopherol, metallothionein-like proteins (MTLPs), lipid peroxidation, triglycerides, acetylcholinesterase activity (AChE) and proteins. Pesticides, pharmaceuticals and personal care products (PPCPs) and metals were also quantified in honeybees' tissues. Our result revealed that, globally, urban foragers had higher levels of insecticides and PPCPs and that metals were in greater concentrations in urban larvae. Compared to rural foragers, urban foragers had higher concentrations of MTLPs, triglycerides, protein and AChE activity. The multifactorial analysis confirmed that insecticides, some metals and PPCPs were the most influential components in the contaminant‒biomarker relationships for both foragers and larvae.

Field-Level Exposure of Bumble Bees to Fungicides Applied to a Commercial Cherry Orchard. 

(Kuivila, K.M., et al. 2021) Bumble bees, Bombus spp. (Apidae), are important native pollinators; however, populations of some species are declining in North America and agricultural chemicals are a potential cause. Fungicides are generally not highly toxic to bees, but little is known about sublethal or synergistic effects. This study evaluates bumble bee exposure to fungicides by quantifying concentrations of boscalid and pyraclostrobin in nectar and pollen collected by colonies of Bombus huntii Greene, 1860 (Hunt bumble bee) deployed in a commercial cherry Prunus avium L. orchard in the spring of 2016. Seven colonies were placed adjacent to an orchard block that was sprayed with a fungicide mixture of boscalid and pyraclostrobin and a control group of seven colonies was placed next to an unsprayed block of orchard 400 m away from the treated block. Nectar and pollen were collected daily, beginning 1 d before spray application and continuing for a total of 12 d, and analyzed for both fungicides. Fungicide concentrations varied spatially by colony and temporally by day. The highest concentrations in nectar occurred 1 and 3 d after spraying: up to 440 ng/g boscalid and 240 ng/g pyraclostrobin. Six days after application, pollen from cherry flowers contained the highest concentrations of the fungicides: up to 60,500 ng/g boscalid and 32,000 ng/g pyraclostrobin. These data can help to determine field-level fungicide concentrations in nectar and pollen and direct future work on understanding the effects of these compounds, including their interactions with important bumble bee pathogenic and beneficial symbionts.

Impacts of a glyphosate-based herbicide on the gut microbiome of three earthworm species (Alma millsoni, Eudrilus eugeniae and Libyodrilus violaceus): A pilot study. (Owagboriaye, F., et al. 2021) While the impact of glyphosate-based herbicides (GBHs) on earthworms has been studied, little is known about their effects on the earthworm gut microbiome. This study investigated the impact of a GBH on the gut microbial communities of three earthworm species (Alma millsoni, Eudrilus eugeniae and Libyodrilus violaceus). Earthworm species accommodated in soil were sprayed with 115.49 mL/m² of Roundup® Alphée or water. Gut microbiome composition was analysed using 16S rRNA Bacterial Tag-Encoded FLX Amplicon Pyrosequencing (bTEFAP) at the 8th week post-herbicide application. A profound shift in bacterial populationswas observed in all exposed earthworms with Proteobacteria becoming the dominant phylum. Affected bacteria were mostly from the genus EnterobacterPantoea and Pseudomonas, which together represented approximately 80 % of the total abundance assigned at the genus level in exposed earthworms, while they were present at a minor abundance (∼1%) in unexposed earthworms. Although consistent results were observed between the three groups of worm species, it is not possible to generalize these outcomes due to a lack of biological replicates, which does not allow for inferential statistical analysis. Nevertheless, our study is the first to report the effects of a GBH on the earthworm gut microbiome and paves the way for future more comprehensive investigations.

Pesticides in honey bee colonies: Establishing a baseline for real world exposure over seven years in the USA. (Traynor, K.S.,  et al. 2021) Honey bees Apis mellifera forage in a wide radius around their colony, bringing back contaminated food resources that can function as terrestrial bioindicators of environmental pesticide exposure. Evaluating pesticide exposure risk to pollinators is an ongoing problem. Here we apply five metrics for pesticide exposure risk (prevalence, diversity, concentration, significant pesticide prevalence, and hazard quotient (HQ)) to a nation-wide field study of honey bees, Apis mellifera in the United States. We examined samples from 1055 apiaries over seven years for 218 different pesticide residues and metabolites, determining that bees were exposed to 120 different pesticide products with a mean of 2.78 per sample. Pesticides in pollen were highly prevalent and variable across states. While pesticide diversity increased over time, most detections occurred at levels predicted to be of low risk to colonies. Varroacides contributed most to concentration, followed by fungicides, while insecticides contributed most to diversity above a toxicity threshold. High risk samples contained one of 12 different insecticides or varroacides. Exposures predicted to be low-risk were nevertheless associated with colony morbidity, and low-level fungicide exposures were tied to queen loss, Nosema infection, and brood diseases.

The sublethal effects of ethiprole on the development, defense mechanisms, and immune pathways of honeybees (Apis mellifera L.). (Liu, Y., et al. 2021) Ethiprole has been widely used in agriculture, but there have been few studies on the adverse effects of ethiprole on nontarget organisms. This study focused on the mechanism of the sublethal effects of ethiprole on the development, antioxidation mechanisms, detoxification mechanisms and immune-related gene expression of honeybees (Apis mellifera L.). Honeybee larvae were found to be more sensitive than pupae to ethiprole. It was found that ethiprole inhibited the pupation and eclosion of bee larvae in a dose-dependent manner, with ethiprole doses of 1 × 10-3 mg/L decreasing pupation and eclosion rates to 50.00 ± 8.84% and 20.83 ± 10.62%, respectively. The activities of antioxidative enzymes (superoxide dismutase and catalase) and detoxification factors (glutathione and glutathione S-transferase) were also significantly increased in ethiprole-exposed honeybees, indicating that a sublethal dose of ethiprole also induced oxidative stress in honeybees. In the 1 × 10-3 mg/L ethiprole-exposure group, the expression of pathogen recognition-related gene PGRP-4300 was upregulated 11.10 ± 0.45-fold, and that of detoxification-related gene CYP4G11 was upregulated 8.84 ± 0.11-fold, indicating that ethiprole induced an immune reaction in honeybees. To the best our knowledge, this study represents the first demonstration that sublethal concentrations of ethiprole inhibit honeybee development and activate honeybee defense and immune systems.

Effects of the Neonicotinoid Acetamiprid in Pollen on Bombus impatiens Microcolony Development.  (Camp, A.A., et al. 2020) Honey bees and other wild bee species including bumble bees have experienced population declines in recent decades. Although many stressors are implicated in bee population declines, much attention has focused on neonicotinoid pesticides, which are widely used and known to be toxic to pollinators. One neonicotinoid, acetamiprid, has been studied very little in bumble bees, despite its use on bumble bee-pollinated crops. We assessed the impacts of acetamiprid to the North American bumble bee Bombus impatiens using the microcolony model. We examined nest growth, development, and subsequent nest productivity as measured by drone production. We found that high concentrations of acetamiprid in pollen (4520 µg/kg) significantly impacted nest growth, development, and, ultimately, reproduction (drone production). We found the no-observable-adverse effect level to be 45.2 µg/kg. Overall, acetamiprid has the potential to negatively impact reproductive endpoints for B. impatiens. However, effects occurred at concentrations substantially higher than expected environmental concentrations that would be achieved when following label rates. Further work is required to assess the effects of this pesticide on B. impatiens via alternate routes of exposure and on queenright colonies.

Assessing field‐scale risks of foliar insecticide applications to monarch butterfly (Danaus plexippus) larvae. (Krishnan, N., et al. 2020) Establishment and maintenance of milkweed plants (Asclepias spp.) in agricultural landscapes of the north central United States are needed to reverse the decline of North America's eastern monarch butterfly (Danaus plexippus) population. Because of a lack of toxicity data, it is unclear how insecticide use may reduce monarch productivity when milkweed habitat is placed near maize and soybean fields. To assess the potential effects of foliar insecticides, acute cuticular and dietary toxicity of 5 representative active ingredients were determined: beta-cyfluthrin (pyrethroid), chlorantraniliprole (anthranilic diamide), chlorpyrifos (organophosphate), and imidacloprid and thiamethoxam (neonicotinoids). Cuticular median lethal dose values for first instars ranged from 9.2 × 10-3 to 79 μg/g larvae for beta-cyfluthrin and chlorpyrifos, respectively. Dietary median lethal concentration values for second instars ranged from 8.3 × 10-3 to 8.4 μg/g milkweed leaf for chlorantraniliprole and chlorpyrifos, respectively. To estimate larval mortality rates downwind from treated fields, modeled insecticide exposures to larvae and milkweed leaves were compared to dose-response curves obtained from bioassays with first-, second-, third-, and fifth-instar larvae. For aerial applications to manage soybean aphids, mortality rates at 60 m downwind were highest for beta-cyfluthrin and chlorantraniliprole following cuticular and dietary exposure, respectively, and lowest for thiamethoxam. To estimate landscape-scale risks, field-scale mortality rates must be considered in the context of spatial and temporal patterns of insecticide use.  

Queen honey bee (Apis mellifera) pheromone and reproductive behavior are affected by pesticide exposure during development. (Walsh, E.M. et al. 2020) Pollinator diversity and abundance in North America have been at a steep decline over the last two decades due to the combinatorial effects of several environmental and anthropogenic stressors. In particular, managed honey bees (Apis mellifera) face multiple health risks including nutritional stress, exposure to pests and pathogens, poor queen quality, and pesticide contamination, which cause problems at the individual and colony levels. One of the gravest problems faced by honey bees is parasitization by the mite Varroa destructor, which is typically controlled through the application of miticides such as tau-fluvalinate, coumaphos, and amitraz. In addition to miticides, colonies are also exposed to pesticides brought by foragers from agricultural settings, including the fungicide chlorothalonil and the insecticide chlorpyrifos. Here, we explored whether exposure of wax to combinations of these pesticides during development affects honey bee queen physiology and worker behavior. To do this, we reared queens in plastic cups coated with molten beeswax that was either pesticide-free or containing field-relevant concentrations of tau-fluvalinate and coumaphos, amitraz, or chlorothalonil and chlorpyrifos. Once queens mated naturally, we placed them in observation hives to measure egg-laying rate and worker retinue size. We then dissected the queens and used the contents of their mandibular glands to measure worker attractiveness in caged bioassays and to analyze their chemical components using GC-MS. Exposure of wax to field-relevant concentrations of the tested pesticides during queen development significantly lowered the adult queens’ egg-laying rate and worker retinue size. Miticide exposure during development also lowered the attractiveness of queen mandibular gland contents to workers and affected the relative amounts of the glands’ chemical components. Our results support the ideas that mandibular gland pheromones act as honest indicators of queen reproductive fitness and that pesticide exposure of wax during bee development is an important and concerning factor impairing honey bee health.

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 (LD50/100). 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 alone pesticides, toxic to bees, and are also present in drug and personal care products, particularly shampoos, and thus represent an important component of the chemical landscape to which pollinators and humans are exposed. This mini review is the first to possibly link spray adjuvant use with declining health of honey bee populations.

A neonicotinoid impairs olfactory learning in Asian honey bees (Apis cerana) exposed as larvae or as adults
Xenobiotics such as the neonicotinoid pesticide, imidacloprid, are used globally, but their effects on native bee species are poorly understood. We studied the effects of sublethal doses of imidacloprid on olfactory learning in the native honey bee species, Apis cerana, an important pollinator of agricultural and native plants throughout Asia. We provide the first evidence that imidacloprid can impair learning in A. cerana workers exposed as adults or as larvae. Adults that ingested a single imidacloprid dose as low as 0.1 ng/bee had significantly reduced olfactory learning acquisition, which was 1.6-fold higher in control bees. Longer-term learning (1-17 h after the last learning trial) was also impaired. Bees exposed as larvae to a total dose of 0.24 ng/bee did not have reduced survival to adulthood. However, these larval-treated bees had significantly impaired olfactory learning when tested as adults: control bees exhibited up to 4.8-fold better short-term learning acquisition, though longer-term learning was not affected. Thus, sublethal cognitive deficits elicited by neonicotinoids on a broad range of native bee species deserve further study.

Neonicotinoid-Coated Zea mays Seeds Indirectly Affect Honeybee Performance and Pathogen Susceptibility in Field Trials
Thirty-two honeybee (Apis mellifera) colonies were studied in order to detect and measure potential in vivo effects of neonicotinoid pesticides used in cornfields (Zea mays spp) on honeybee health. Honeybee colonies were randomly split on four different agricultural cornfield areas located near Quebec City, Canada. Two locations contained cornfields treated with a seed-coated systemic neonicotinoid insecticide while the two others were organic cornfields used as control treatments. Hives were extensively monitored for their performance and health traits over a period of two years. Honeybee viruses (brood queen cell virus BQCV, deformed wing virus DWV, and Israeli acute paralysis virus IAPV) and the brain specific expression of a biomarker of host physiological stress, the Acetylcholinesterase gene AChE, were investigated using RT-qPCR. Liquid chromatography-mass spectrometry (LC-MS) was performed to detect pesticide residues in adult bees, honey, pollen, and corn flowers collected from the studied hives in each location. In addition, general hive conditions were assessed by monitoring colony weight and brood development. Neonicotinoids were only identified in corn flowers at low concentrations. However, honeybee colonies located in neonicotinoid treated cornfields expressed significantly higher pathogen infection than those located in untreated cornfields. AChE levels showed elevated levels among honeybees that collected corn pollen from treated fields. Positive correlations were recorded between pathogens and the treated locations. Our data suggests that neonicotinoids indirectly weaken honeybee health by inducing physiological stress and increasing pathogen load.

Wings as a new route of exposure to pesticides in the honey bee
In pesticide risk assessment, estimating the routes and levels of exposure is critical. For honey bees subjected to pesticide spray, toxicity is assessed by thorax contact to account for all possible contact exposures. In the present study, the authors tested 6 active substances with different hydrophobicity. For the first time, the authors demonstrated that it is possible to induce mortality by pesticide contact with only the wings of the honey bee. The toxicities induced by contact with the wings and thorax were similar, with the wing median lethal dose (LD50) being 0.99 to 2.23 times higher than that of the thorax. This finding demonstrates that the wings represent a relevant route of exposure in the honey bee. In a second approach, the authors estimated the air volume displaced by the wings during 1 beating cycle to be 0.51 ± 0.03 cm(3), which corresponds to a volume of 116.8 ± 5.8 cm(3) s(-1) at a wing beat frequency of 230 Hz. The authors then tested realistic scenarios of exposure for bees flying through a pesticide cloud at different concentrations. In the worst-case scenario, the dose accumulated during the flight reached 525 ng bee(-1) s(-1). These results show that the procedure used to assess the risk posed by contact with pesticides could be improved by accounting for wing exposure.

Chronic exposure to a neonicotinoid pesticide alters the interactions between bumblebees and wild plants
Researchers of this study, published in the journal Functional Ecology, observed the behavior of individual bumblebees from colonies chronically exposed to a neonicotinoid pesticide (10 ppb thiamethoxam) or control solutions foraging for the first time on an array of morphologically complex wildflowers (Lotus corniculatus and Trifolium repens) in an outdoor flight arena. They found that more bees released from pesticide-treated colonies became foragers, and that they visited more L. corniculatus flowers than controls. Interestingly, bees exposed to pesticide collected more pollen than controls, but control bees learnt to handle flowers efficiently after fewer learning visits than bees exposed to pesticide. There were also different initial flower preferences of our treatment groups; control bees visited a higher proportion of T. repens flowers, and bees exposed to pesticide were more likely to choose L. corniculatus on their first visit. The results suggest that the foraging behavior of bumblebees on real flowers can be altered by sublethal exposure to field-realistic levels of pesticide. This has implications for the foraging success and persistence of bumblebee colonies, but perhaps more importantly for the interactions between wild plants and flower-visiting insects and ability of bees to deliver the crucial pollination services to plants necessary for ecosystem functioning. 

Neonicotinoid pesticide exposure impairs crop pollination services provided by bumblebees
In this study, published in Nature, researchers show the first evidence that pesticide exposure can reduce the pollination services bumblebees deliver to apples, a crop of global economic importance. Bumblebee colonies exposed to a neonicotinoid pesticide provided lower visitation rates to apple trees and collected pollen less often. Most importantly, these pesticide-exposed colonies produced apples containing fewer seeds, demonstrating a reduced delivery of pollination services. These results also indicated that reduced pollination service delivery is not due to pesticide-induced changes in individual bee behavior, but most likely due to effects at the colony level. These findings show that pesticide exposure can impair the ability of bees to provide pollination services, with important implications for both the sustained delivery of stable crop yields and the functioning of natural ecosystems.

Reconciling laboratory and field assessments of neonicotinoid toxicity to honeybees
European governments have banned the use of three common neonicotinoid pesticides due to insufficiently identified risks to bees. This policy decision is controversial given the absence of clear consistency between toxicity assessments of those substances in the laboratory and in the field. Although laboratory trials report deleterious effects in honeybees at trace levels, field surveys reveal no decrease in the performance of honeybee colonies in the vicinity of treated fields. In this study, published in the journal Proceeding of the Royal Society B, researchers provide the missing link, showing that individual honeybees near thiamethoxam-treated fields do indeed disappear at a faster rate, but the impact of this is buffered by the colonies' demographic regulation response. Although researchers could ascertain the exposure pathway of thiamethoxam residues from treated flowers to honeybee dietary nectar, they uncovered an unexpected pervasive co-occurrence of similar concentrations of imidacloprid, another neonicotinoid normally restricted to non-entomophilous crops in the study country. Thus, its origin and transfer pathways through the succession of annual crops need be elucidated to conveniently appraise the risks of combined neonicotinoid exposures. This study reconciles the conflicting laboratory and field toxicity assessments of neonicotinoids on honeybees and further highlights the difficulty in actually detecting non-intentional effects on the field through conventional risk assessment methods.

Exposure of native bees foraging in an agricultural landscape to current-use pesticides
In this study conducted by the U.S. Geological Survey and published in the journal Science of The Total Environment, researchers examine for the first time pesticide residues on native bee populations and find that they are exposed to neonicotinoids, as well as other pesticides, at significant rates. Compounds detected in > 2% of the samples included: insecticides thiamethoxam (46%), bifenthrin (28%), clothianidin (24%), chlorpyrifos (17%), imidacloprid (13%), fipronil desulfinyl (7%; degradate); fungicides azoxystrobin (17%), pyraclostrobin (11%), fluxapyroxad (9%), and propiconazole (9%); herbicides atrazine (19%) and metolachlor (9%). Concentrations ranged from 1 to 310 ng/g for individual pesticides. Pesticides were detected in samples collected from both grasslands and wheat fields; the location of the sample and the surrounding land cover at the 1000 m radius influenced the pesticides detected but because of a small number of temporally comparable samples, correlations between pesticide concentration and land cover were not significant. The results show native bees collected in an agricultural landscape are exposed to multiple pesticides, these results can direct future research on routes/timing of pesticide exposure and the design of future conservation efforts for pollinators.

Are bee diseases linked to pesticides? - A brief review
The negative impacts of pesticides, in particular insecticides, on bees and other pollinators have never been disputed. Insecticides can directly kill these vital insects, whereas herbicides reduce the diversity of their food resources, thus indirectly affecting their survival and reproduction. At sub-lethal level (<LD50), neurotoxic insecticide molecules are known to influence the cognitive abilities of bees, impairing their performance and ultimately impacting on the viability of the colonies. In addition, widespread systemic insecticides appear to have introduced indirect side effects on both honey bees and wild bumblebees, by deeply affecting their health. Immune suppression of the natural defences by neonicotinoid and phenyl-pyrazole (fipronil) insecticides opens the way to parasite infections and viral diseases, fostering their spread among individuals and among bee colonies at higher rates than under conditions of no exposure to such insecticides. This causal link between diseases and/or parasites in bees and neonicotinoids and other pesticides has eluded researchers for years because both factors are concurrent: while the former are the immediate cause of colony collapses and bee declines, the latter are a key factor contributing to the increasing negative impact of parasitic infections observed in bees in recent decades.

The neonicotinoids thiacloprid, imidacloprid, and clothianidin affect the immunocompetence of honey bees (Apis mellifera L.).
A strong immune defense is vital for honey bee health and colony survival. This defense can be weakened by environmental factors that may render honey bees more vulnerable to parasites and pathogens. Honey bees are frequently exposed to neonicotinoid pesticides, which are being discussed as one of the stress factors that may lead to colony failure. Authors investigated the sublethal effects of the neonicotinoids thiacloprid, imidacloprid, and clothianidin on individual immunity, by studying three major aspects of immunocompetence in worker bees: total hemocyte number, encapsulation response, and antimicrobial activity of the hemolymph. In laboratory experiments, we found a strong impact of all three neonicotinoids. Thiacloprid (24h oral exposure, 200μg/l or 2000μg/l) and imidacloprid (1μg/l or 10μg/l) reduced hemocyte density, encapsulation response, and antimicrobial activity even at field realistic concentrations. Clothianidin had an effect on these immune parameters only at higher than field realistic concentrations (50-200μg/l). These results suggest that neonicotinoids affect the individual immunocompetence of honey bees, possibly leading to an impaired disease resistance capacity.

Multi-Drug Resistance Transporters and a Mechanism-Based Strategy for Assessing Risks of Pesticide Combinations to Honey Bees.
Dangerous combinations of pesticides, plant-produced compounds and antibiotics added to hives may cause or contribute to losses, but it is very difficult to test the many combinations of those compounds that bees encounter. Authors propose a mechanism-based strategy for simplifying the assessment of combinations of compounds, focusing here on compounds that interact with xenobiotic handling ABC transporters. We evaluate the use of ivermectin as a model substrate for these transporters. Compounds that increase sensitivity of bees to ivermectin may be inhibiting key transporters. Authors show that several compounds commonly encountered by honey bees (fumagillin, Pristine, quercetin) significantly increased honey bee mortality due to ivermectin and significantly reduced the LC50 of ivermectin suggesting that they may interfere with transporter function. These inhibitors also significantly increased honey bees sensitivity to the neonicotinoid insecticide acetamiprid. Mechanism-based strategies for simplifying the assessment of adverse chemical interactions such as described here could improve our ability to identify those combinations that pose significantly greater risk to bees and perhaps improve the risk assessment protocols for honey bees and similar sensitive species.

Survey and Risk Assessment of Apis mellifera (Hymenoptera: Apidae) Exposure to Neonicotinoid Pesticides in Urban, Rural, and Agricultural Settings.
A comparative assessment of apiaries in urban, rural, and agricultural areas was undertaken in 2013 and 2014 to examine potential honey bee colony exposure to neonicotinoid insecticides from pollen foraging. Apiaries ranged in size from one to hundreds of honey bee colonies, and included those operated by commercial, sideline (semicommercial), and hobbyist beekeepers. Residues in and on wax and beebread (stored pollen in the hive) were evaluated for the nitro-substituted neonicotinoid insecticides imidacloprid and its olefin metabolite and the active ingredients clothianidin, thiamethoxam, and dinotefuran. Beebread and comb wax collected from hives in agricultural landscapes were more likely to have detectable residues of thiamethoxam and clothianidin than that collected from hives in rural or urban areas (∼50% of samples vs. <10%). The maximum neonicotinoid residue detected in either wax or beebread was 3.9 ppb imidacloprid. A probabilistic risk assessment was conducted on the residues recovered from beebread in apiaries located in commercial, urban, and rural landscapes. The calculated risk quotient based on a dietary no observable adverse effect concentration (NOAEC) suggested low potential for negative effects on bee behavior or colony health.

Sublethal Dosage of Imidacloprid Reduces the Microglomerular Density of Honey Bee Mushroom Bodies.
Previous studies have indicated that neonicotinoid insecticides cause behavioural abnormalities and have proven that exposure to sublethal doses of imidacloprid during the larval stage decreases the olfactory learning ability of adults. The present study shows the effect of sublethal doses of imidacloprid on the neural development of the honey bee brain by immunolabelling synaptic units in the calyces of mushroom bodies. Authors found that the density of the synaptic units in the region of the calyces, which are responsible for olfactory and visual functions, decreased after being exposed to a sublethal dose of imidacloprid. This not only links a decrease in olfactory learning ability to abnormal neural connectivity but also provides evidence that imidacloprid damages the development of the nervous system in regions responsible for both olfaction and vision during the larval stage of the honey bee.

Widespread contamination of wildflower and bee-collected pollen with complex mixtures of neonicotinoids and fungicides commonly applied to crops
There is considerable and ongoing debate as to the harm inflicted on bees by exposure to agricultural pesticides. Authors quantify concentrations of neonicotinoid insecticides and fungicides in the pollen of oilseed rape, and in pollen of wildflowers growing near arable fields. They compare this to concentrations of these pesticides found in pollen collected by honey bees and in pollen and adult bees sampled from bumble bee colonies placed on arable farms. Study also compared this with levels found in bumble bee colonies placed in urban areas. Pollen of oilseed rape was heavily contaminated with a broad range of pesticides, as was the pollen of wildflowers growing nearby. Consequently, pollen collected by both bee species also contained a wide range of pesticides, notably including the fungicides carbendazim, boscalid, flusilazole, metconazole, tebuconazole and trifloxystrobin and the neonicotinoids thiamethoxam, thiacloprid and imidacloprid. In bumble bees, the fungicides carbendazim, boscalid, tebuconazole, flusilazole and metconazole were present at concentrations up to 73nanogram/gram (ng/g). It is notable that pollen collected by bumble bees in rural areas contained high levels of the neonicotinoids thiamethoxam (mean 18ng/g) and thiacloprid (mean 2.9ng/g), along with a range of fungicides, some of which are known to act synergistically with neonicotinoids. Pesticide exposure of bumble bee colonies in urban areas was much lower than in rural areas. Understanding the effects of simultaneous exposure of bees to complex mixtures of pesticides remains a major challenge.

Neonicotinoid Residues in Wildflowers, a Potential Route of Chronic Exposure for Bees
In recent years, an intense debate about the environmental risks posed by neonicotinoids, a group of widely used, neurotoxic insecticides, has been joined. When these systemic compounds are applied to seeds, low concentrations are subsequently found in the nectar and pollen of the crop, which are then collected and consumed by bees. Study demonstrate that the current focus on exposure to pesticides via the crop overlooks an important factor: throughout spring and summer, mixtures of neonicotinoids are also found in the pollen and nectar of wildflowers growing in arable field margins, at concentrations that are sometimes even higher than those found in the crop. Indeed, the large majority (97%) of neonicotinoids brought back in pollen to honey bee hives in arable landscapes was from wildflowers, not crops. Both previous and ongoing field studies have been based on the premise that exposure to neonicotinoids would occur only during the blooming period of flowering crops and that it may be diluted by bees also foraging on untreated wildflowers. Results show that exposure is likely to be higher and more prolonged than currently recognized because of widespread contamination of wild plants growing near treated crops.

Neonicotinoid pesticides severely affect honey bee queens
Queen health is crucial to colony survival of social bees. Recently, queen failure has been proposed to be a major driver of managed honey bee colony losses, yet few data exist concerning effects of environmental stressors on queens. Here authors demonstrate for the first time that exposure to field-realistic concentrations of neonicotinoid pesticides during development can severely affect queens of western honey bees (Apis mellifera). In pesticide-exposed queens, reproductive anatomy (ovaries) and physiology (spermathecal-stored sperm quality and quantity), rather than flight behaviour, were compromised and likely corresponded to reduced queen success (alive and producing worker offspring). This study highlights the detriments of neonicotinoids to queens of environmentally and economically important social bees, and further strengthens the need for stringent risk assessments to safeguard biodiversity and ecosystem services that are vulnerable to these substances.

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, 223ng/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.

Genetics, Synergists, and Age Affect Insecticide Sensitivity of the Honey Bee, Apis mellifera
The number of honey bee colonies in the United States has declined to half of its peak level in the 1940s, and colonies lost over the winter have reached levels that are becoming economically unstable. While the causes of these losses are numerous and the interaction between them is very complex, the role of insecticides has garnered much attention. As a result, there is a need to better understand the risk of insecticides to bees, leading to more studies on both toxicity and exposure. While much research has been conducted on insecticides and bees, there have been very limited studies to elucidate the role that bee genotype and age has on the toxicity of these insecticides. The goal of this study was to determine if there are differences in insecticide sensitivity between honey bees of different genetic backgrounds (Carniolan, Italian, and Russian stocks) and assess if insecticide sensitivity varies with age. Authors found that Italian bees were the most sensitive of these stocks to insecticides, but variation was largely dependent on the class of insecticide tested. There were almost no differences in organophosphate bioassays between honey bee stocks (<1-fold), moderate differences in pyrethroid bioassays (1.5 to 3-fold), and dramatic differences in neonicotinoid bioassays (3.4 to 33.3-fold). Synergism bioassays with piperonyl butoxide, amitraz, and coumaphos showed increased phenothrin sensitivity in all stocks and also demonstrated further physiological differences between stocks. In addition, as bees aged, the sensitivity to phenothrin significantly decreased, but the sensitivity to naled significantly increased. These results demonstrate the variation arising from the genetic background and physiological transitions in honey bees as they age. This information can be used to determine risk assessment, as well as establishing baseline data for future comparisons to explain the variation in toxicity differences for honey bees reported in the literature.

Similar Comparative Low and High Doses of Deltamethrin and Acetamiprid Differently Impair the Retrieval of the Proboscis Extension Reflex in the Forager Honey Bee (Apis mellifera)
In the present study, the effects of low (10 ng/bee) and high (100 ng/bee) doses of acetamiprid and deltamethrin insecticides on multi-trial learning and retrieval were evaluated in the honey bee Apis mellifera. After oral application, acetamiprid and deltamethrin at the concentrations used were not able to impair learning sessions. When the retention tests were performed 1 h, 6 h, and 24 h after learning, we found a significant difference between bees after learning sessions when drugs were applied 24 h before learning. Deltamethrin-treated bees were found to be more sensitive at 10 ng/bee and 100 ng/bee doses compared to acetamiprid-treated bees, only with amounts of 100 ng/bee and at 6 h and 24 h delays. When insecticides were applied during learning sessions, none of the tested insecticides was able to impair learning performance at 10 ng/bee or 100 ng/bee but retention performance was altered 24 h after learning sessions. Acetamiprid was the only one to impair retrieval at 10 ng/bee, whereas at 100 ng/bee an impairment of retrieval was found with both insecticides. The present results therefore suggest that acetamiprid and deltamethrin are able to impair retrieval performance in the honey bee Apis mellifera.

Neonicotinoid-contaminated puddles of water represent a risk of intoxication for honey bees
While multiple routes of exposure to systemic insecticides have been documented for honey bees, contamination from puddle water has not been investigated. In this study, authors used a multi-residue method based on LC-MS/MS to analyze samples of puddle water taken in the field during the planting of treated corn and one month later. If honey bees were to collect and drink water from these puddles, our results showed that they would be exposed to various agricultural pesticides. All water samples collected from corn fields were contaminated with at least one neonicotinoid compound, although most contained more than one systemic insecticide. Concentrations of neonicotinoids were higher in early spring, indicating that emission and drifting of contaminated dust during sowing raises contamination levels of puddles. Although the overall average acute risk of drinking water from puddles was relatively low, concentrations of neonicotinoids ranged from 0.01 to 63 µg/L and were sufficient to potentially elicit a wide array of sublethal effects in individuals and colony alike. Results also suggest that risk assessment of honey bee water resources underestimates the foragers' exposure and consequently miscalculates the risk. In fact, data shows that honey bees and native pollinators are facing unprecedented cumulative exposure to these insecticides from combined residues in pollen, nectar and water. These findings not only document the impact of this route of exposure for honey bees, they also have implications for the cultivation of a wide variety of crops for which the extensive use of neonicotinoids is currently promoted.

Programmed Cell Death in the Honey Bee (Apis mellifera) (Hymenoptera: Apidae) Worker Brain Induced by Imidacloprid
Honey bees are at an unavoidable risk of exposure to neonicotinoid pesticides, which are used worldwide. Compared with the well-studied roles of these pesticides in nontarget site (including midgut, ovary, or salivary glands), little has been reported in the target sites, the brain. In the current study, laboratory-reared adult worker honey bees (Apis mellifera L.) were treated with sublethal doses of imidacloprid. Neuronal apoptosis was detected using the TUNEL technique for DNA labeling. Authors observed significantly increased apoptotic markers in dose- and time-dependent manners in brains of bees exposed to imidacloprid. Neuronal activated caspase-3 and mRNA levels of caspase-1, as detected by immunofluorescence and real-time quantitative PCR, respectively, were significantly increased, suggesting that sublethal doses of imidacloprid may induce the caspase-dependent apoptotic pathway. Additionally, the overlap of apoptosis and autophagy in neurons was confirmed by transmission electron microscopy. It further suggests that a relationship exists between neurotoxicity and behavioral changes induced by sublethal doses of imidacloprid, and that there is a need to determine reasonable limits for imidacloprid application in the field to protect pollinators.

Assessing Honey Bee (Hymenoptera: Apidae) Foraging Populations and the Potential Impact of Pesticides on Eight U.S. Crops
In this study, published in the Journal of Economic Entomology, researchers assessed changes, from 2009-2010, in the field force populations of 9–10 colonies at one location per crop on each of the eight crops by counting departing foragers leaving colonies at regular intervals during the respective crop blooming periods. The number of frames of adult bees was counted before and after bloom period. For pesticide analysis, researchers collected dead and dying bees near the hives, returning foragers, crop flowers, trapped pollen, and corn-flowers associated with the cotton crop. The number of departing foragers changed over time in all crops except almonds; general patterns in foraging activity included declines (cotton), noticeable peaks and declines (alfalfa, blueberries, cotton, corn, and pumpkins), and increases (apples and cantaloupes). The number of adult bee frames increased or remained stable in all crops except alfalfa and cotton. A total of 53 different pesticide residues were identified in samples collected across eight crops. Hazard quotients (HQ) were calculated for the combined residues for all crop-associated samples and separately for samples of dead and dying bees. A decrease in the number of departing foragers in cotton was one of the most substantial crop-associated impacts and presented the highest pesticide risk estimated by a summed pesticide residue HQ.

Sublethal imidacloprid effects on honey bee flower choices when foraging
Authors examined one neonicotinoid pesticide, imidacloprid, for its effects on the foraging behavior of free-flying honey bees (Apis mellifera anatoliaca) visiting artificial blue and white flowers. Imidacloprid doses, ranging from 1/5 to 1/50 of the reported LD50, were fed to bees orally. The study consisted of three experimental parts performed sequentially without interruption. In Part 1, both flower colors contained a 4 μL 1 M sucrose solution reward. Part 2 offered bees 4 μL of 1.5 M sucrose solution in blue flowers and a 4 μL 0.5 M sucrose solution reward in white flowers. In Part 3 we reversed the sugar solution rewards, while keeping the flower color consistent. Each experiment began 30 min after administration of the pesticide. We recorded the percentage of experimental bees that returned to forage after treatment. We also recorded the visitation rate, number of flowers visited, and floral reward choices of the bees that foraged after treatment. The forager return rate declined linearly with increasing imidacloprid dose. The number of foraging trips by returning bees was also affected adversely. However, flower fidelity was not affected by imidacloprid dose. Foragers visited both blue and white flowers extensively in Part 1, and showed greater fidelity for the flower color offering the higher sugar solution reward in Parts 2 and 3. Although larger samples sizes are needed, study suggests that imidacloprid may not affect the ability to select the higher nectar reward when rewards were reversed. We observed acute, mild effects on foraging by honey bees, so mild that storage of imidacloprid tainted-honey is very plausible and likely to be found in honey bee colonies.

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, authors 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.

The neonicotinoid imidacloprid impairs honey bee aversive learning of simulated predation
Neonicotinoid insecticides can impair bee learning and memory--cognitive features that play a key role in colony fitness because they facilitate foraging. For example, the commonly used neonicotinoid imidacloprid reduces honey bee olfactory learning. However, no studies have previously determined whether imidacloprid can impair aversive associative learning, although such learning should enhance bee survival by allowing bees to avoid dangerous foraging sites. To mimic attempted predation of foragers, authors developed an electro-mechanical predator that consistently attacked foragers with a pinching bite at a fixed force and elicited aversive olfactory learning in a sting extension response (SER) assay. Study shows that chronic exposure to a sublethal concentration of imidacloprid (25.6 µg l(-1)=20.8 ppb) over 4 days (mean of 1.5 µg per bee day(-1)), significantly impaired aversive short-term learning and memory retention. Imidacloprid treatment reduced short-term learning by 87% and memory retention by 85% in comparison with control bees. Imidacloprid therefore impairs the ability of honey bees to associate a naturalistic predation stimulus--biting--with floral odor compounds. Such learning should enhance bee survival, suggesting that xenobiotics could alter more complex ecological interactions such as predator-prey relationships.

Neonicotinoid Insecticides and Their Impacts on Bees: A Systematic Review of Research Approaches and Identification of Knowledge Gaps
In order to synthesize which research approaches have been used to examine the effect of neonicotinoids on bees and to identify knowledge gaps, the researchers systematically reviewed research on this subject that was available on the Web of Science and PubMed in June 2015. Most of the 216 primary research studies were conducted in Europe or North America (82%), involved the neonicotinoid imidacloprid (78%), and concerned the western honey bee Apis mellifera (75%). Thus, little seems to be known about neonicotinoids and bees in areas outside Europe and North America. Furthermore, because there is considerable variation in ecological traits among bee taxa, studies on honey bees are not likely to fully predict impacts of neonicotinoids on other species. Studies on crops were dominated by seed-treated maize, oilseed rape (canola) and sunflower, whereas less is known about potential side effects on bees from the use of other application methods on insect pollinated fruit and vegetable crops, or on lawns and ornamental plants. Laboratory approaches were most common, and the authors suggest that their capability to infer real-world consequences are improved when combined with information from field studies about realistic exposures to neonicotinoids. Studies using field approaches often examined only bee exposure to neonicotinoids and more field studies are needed that measure impacts of exposure. Most studies measured effects on individual bees. Authors suggest that effects on the individual bee should be linked to both mechanisms at the sub-individual level and also to the consequences for the colony and wider bee populations. As bees are increasingly facing multiple interacting pressures future research needs to clarify the role of neonicotinoids in relative to other drivers of bee declines.

Differential responses of Apis mellifera heat shock protein genes to heat shock, flower-thinning formulations, and imidacloprid
In this study, published in the Journal of Asia-Pacific Entomology, researchers used mRNA levels of heat shock protein (HSP) genes as molecular markers of response to three types of external stress: thermal shock, flower-thinning agents, and pesticides. When worker bees were exposed to temperatures of 4, 27, 40, 45 and 50°C for 1 h, decreased survival occurred only at 50°C. Further, increased levels of hsp70, grp78, and hsp90, but not hsp40, were detected, and reached a maximum at 45C, particularly in the hypopharyngeal glands and fat bodies. Artificial ingestion of two flower-thinning agents containing either 0.1% boron and zinc, or 1% sulfur increased hsp70 and grp78 levels at different rates without affecting hsp40 and hsp90 levels, and had no effect on workers’ mortality. However, ingestion of imidacloprid solution (0.5–50 ppm) increased mortality in workers and decreased the levels of hsp70, grp78, and hsp90 in a dose-dependent manner. The results showed that the responses of honey bees to each hsp are differential and highly specific to different stresses. This study suggests that the unique expression profiles of hsps can be used as valuable tools for monitoring the susceptibility of honey bees to various environmental impacts.

A neonicotinoid impairs olfactory learning in Asian honey bees (Apis cerana) exposed as larvae or as adults
Authors studied the effects of sublethal doses of imidacloprid on olfactory learning in the native honey bee species, Apis cerana, an important pollinator of agricultural and native plants throughout Asia. Study provides the first evidence that imidacloprid can impair learning in A. cerana workers exposed as adults or as larvae. Adults that ingested a single imidacloprid dose as low as 0.1 ng/bee had significantly reduced olfactory learning acquisition, which was 1.6-fold higher in control bees. Longer-term learning (1-17 h after the last learning trial) was also impaired. Bees exposed as larvae to a total dose of 0.24 ng/bee did not have reduced survival to adulthood. However, these larval-treated bees had significantly impaired olfactory learning when tested as adults: control bees exhibited up to 4.8-fold better short-term learning acquisition, though longer-term learning was not affected. Thus, sublethal cognitive deficits elicited by neonicotinoids on a broad range of native bee species deserve further study.

Bee declines driven by combined stress from parasites, pesticides, and lack of flowers
In this review, published in Science, the authors recognize that bees are subject to numerous pressures in the modern world. The abundance and diversity of flowers has declined, bees are chronically exposed to cocktails of agrochemicals, and they are simultaneously exposed to novel parasites accidentally spread by humans. Climate change is likely to exacerbate these problems in the future. Stressors do not act in isolation; for example pesticide exposure can impair both detoxification mechanisms and immune responses, rendering bees more susceptible to parasites. It seems certain that chronic exposure to multiple, interacting stressors is driving honey bee colony losses and declines of wild pollinators, but such interactions are not addressed by current regulatory procedures and studying these interactions experimentally poses a major challenge. In the meantime, taking steps to reduce stress on bees would seem prudent; incorporating flower-rich habitat into farmland, reducing pesticide use through adopting more sustainable farming methods, and enforcing effective quarantine measures on bee movements are all practical measures that should be adopted. Effective monitoring of wild pollinator populations is urgently needed to inform management strategies into the future.

Honeybee colony disorder in crop areas: the role of pesticides and viruses
As in many other locations in the world, honeybee colony losses and disorders have increased in Belgium. Some of the symptoms observed rest unspecific and their causes remain unknown. The present study aims to determine the role of both pesticide exposure and virus load on the appraisal of unexplained honeybee colony disorders in field conditions. From July 2011 to May 2012, 330 colonies were monitored. Honeybees, wax, beebread and honey samples were collected. Morbidity and mortality information provided by beekeepers, colony clinical visits and availability of analytical matrix were used to form 2 groups: healthy colonies and colonies with disorders (n = 29, n = 25, respectively). Disorders included: (1) dead colonies or colonies in which part of the colony appeared dead, or had disappeared; (2) weak colonies; (3) queen loss; (4) problems linked to brood and not related to any known disease. Five common viruses and 99 pesticides (41 fungicides, 39 insecticides and synergist, 14 herbicides, 5 acaricides and metabolites) were quantified in the samples.The main symptoms observed in the group with disorders are linked to brood and queens. The viruses most frequently found are Black Queen Cell Virus, Sac Brood Virus, Deformed Wing Virus. No significant difference in virus load was observed between the two groups. Three acaricides, 5 insecticides and 13 fungicides were detected in the analysed samples. A significant correlation was found between the presence of fungicide residues and honeybee colony disorders. A significant positive link could also be established between the observation of disorder and the abundance of crop surface around the beehive. According to our results, the role of fungicides as a potential stressor for honeybee colonies should be further studied, either by their direct and/or indirect impacts on bees and bee colonies.

Evidence for pollinator cost and farming benefits of neonicotinoid seed coatings on oilseed rape
Chronic exposure to neonicotinoid insecticides has been linked to reduced survival of pollinating insects at both the individual and colony level, but so far only experimentally. Here, study combines large-scale pesticide usage and yield observations from oilseed rape with those detailing honey bee colony losses over an 11 year period, and reveal a correlation between honey bee colony losses and national-scale imidacloprid (a neonicotinoid) usage patterns across England and Wales. Study also provides the first evidence that farmers who use neonicotinoid seed coatings reduce the number of subsequent applications of foliar insecticide sprays and may derive an economic return. Results inform the societal discussion on the pollinator costs and farming benefits of prophylactic neonicotinoid usage on a mass flowering crop.

In vitro effects of thiamethoxam on larvae of Africanized honey bee Apis mellifera (Hymenoptera: Apidae)
Several investigations have revealed the toxic effects that neonicotinoids can have on Apis mellifera, while few studies have evaluated the impact of these insecticides can have on the larval stage of the honeybee. From the lethal concentration (LC50) of thiamethoxam for the larvae of the Africanized honeybee, authors evaluated the sublethal effects of this insecticide on morphology of the brain. After determine the LC50 (14.34 ng/μL of diet) of thiamethoxam, larvae were exposed to a sublethal concentration of thiamethoxam equivalent to 1.43 ng/μL by acute and subchronic exposure. Morphological and immunocytochemistry analysis of the brains of the exposed bees, showed condensed cells and early cell death in the optic lobes. Additional dose-related effects were observed on larval development. Results show that the sublethal concentrations of thiamethoxam tested are toxic to Africanized honeybees larvae and can modulate the development and consequently could affect the maintenance and survival of the colony. These results represent the first assessment of the effects of thiamethoxam in Africanized honeybee larvae and should contribute to studies on honey bee colony decline.

Pesticides and reduced-risk insecticides, native bees and pantropical stingless bees: pitfalls and perspectives
Although invertebrates generally have a low public profile, the honey bee, Apis mellifera L., is a flagship species whose popularity likely derives from the products it provides and its perceived ecological services. Therefore, the raging debate regarding honey bee decline has surpassed the realm of beekeepers, academia, industry and regulatory agencies and now also encompasses non-governmental agencies, media, fiction writers and the general public. The early interest and concern about honey bee colony collapse disorder (CCD) soon shifted to the bigger issue of pollinator decline, with a focus on the potential involvement of pesticides in such a phenomenon. Pesticides were previously recognised as the potential culprits of the reported declines, particularly the neonicotinoid insecticides owing to their widespread and peculiar use in agriculture. However, the evidence for the potential pivotal role of these neonicotinoids in honey bee decline remains a matter of debate, with an increased recognition of the multifactorial nature of the problem and the lack of a direct association between the noted decline and neonicotinoid use. The focus on the decline of honey bee populations subsequently spread to other species, and bumblebees became another matter of concern, particularly in Europe and the United States. Other bee species, ones that are particularly important in other regions of the world, remain the object of little concern (unjustifiably so). Furthermore, the continuous focus on neonicotinoids is also in need of revision, as the current evidence suggests that a broad spectrum of compounds deserve attention.

Chronic exposure to neonicotinoids increases neuronal vulnerability to mitochondrial dysfunction in the bumblebee (Bombus terrestris)
In this 2015 study, published in The Journal of the Federation of American Studies for Experimental Biology, researchers show that bumblebees (Bombus terrestris audax) fed field levels [10 nM, 2.1 ppb (w/w)] of neonicotinoid accumulate between 4 and 10 nM in their brains within 3 days. Acute (minutes) exposure of cultured neurons to 10 nM clothianidin, but not imidacloprid, causes a nicotinic acetylcholine receptor-dependent rapid mitochondrial depolarization. However, a chronic (2 days) exposure to 1 nM imidacloprid leads to a receptor-dependent increased sensitivity to a normally innocuous level of acetylcholine, which now also causes rapid mitochondrial depolarization in neurons. Finally, colonies exposed to this level of imidacloprid show deficits in colony growth and nest condition compared with untreated colonies. These findings provide a mechanistic explanation for the poor navigation and foraging observed in neonicotinoid treated bumblebee colonies.

Field realistic doses of pesticide imidacloprid reduce bumblebee pollen foraging efficiency
This 2014 study, published in the journal Ecotoxicology, reveals that near infinitesimal exposure to neonicotinoids reduces bees ability to gather food by 57%. “Whilst the nectar foraging efficiency of bees treated with imidacloprid was not significantly different than that of control bees, treated bees brought back pollen less often than control bees (40% of trips vs 63% trips, respectively) and, where pollen was collected, treated bees brought back 31% less pollen per hour than controls. This study demonstrates that field-realistic doses of these pesticides substantially impacts on foraging ability of bumblebee workers when collecting pollen, and we suggest that this provides a causal mechanism behind reduced queen production in imidacloprid exposed colonies.” Read more onThe Daily News.

Four Common Pesticides, Their Mixtures and a Formulation Solvent in the Hive Environment Have High Oral Toxicity to Honey Bee Larvae
This 2014 study, published in PLOS ONE, demonstrates the chronic oral and mixture toxicity of common pesticides at hive levels to honey bees at the larval stage. Most notable are the chronic larval toxicities of the fungicide chlorothalonil and its synergistic combinations with frequently used in-hive miticides, and the unexpected high toxicity of the formulation ingredient N-methyl-2-pyrrolidone. Considering the extensive detection of chlorothalonil and its coexistence with other pesticides in diverse combinations especially in hive pollen and wax, and its substantial larval toxicity alone and in mixtures shown here, the application of this and other fungicides during crop bloom cannot be presumed innocuous to pollinating honey bees. The study scientists stress that given the critical sensitivity of larvae to chlorothalonil and its complex interactions with other pesticides, the potential impacts of fungicides on colony survival and development need further investigation. They add that in the more complex milieu of this social insect and its aging hive environment, pesticides, formulation additives and their resulting mixtures may have greater long-term impacts on colony health than previously considered. Consequently, the scope of pesticide risk assessment for non-target honey bees should be expanded from the present emphasis on acute toxicity of individual pesticides to a priority for assessment of chronic and mixture toxicities that incorporate fungicides, other pesticide pollutants and their ‘inert’ ingredients.

Impacts of a neonicotinoid, neonicotinoid–pyrethroid premix, and anthranilic diamide insecticide on four species of turf-inhabiting beneficial insects
In this 2014 study, published in Ecotoxicology, researchers compared the impact of a neonicotinoid (clothianidin), a premix (clothianidin + bifenthrin), and an anthranilic diamide (chlorantraniliprole), the main insecticide classes used for multiple targeting, on four species of beneficial insects: Harpalus pennsylvanicus, an omnivorous ground beetle, Tiphia vernalis, an ectoparasitoid of scarab grubs, Copidosoma bakeri, a polyembryonic endoparasitoid of black cutworms, and Bombus impatiens, a native bumble bee. Ground beetles that ingested food treated with clothianidin or the premix suffered high mortality, as did C. bakeri wasps exposed to dry residues of those insecticides. Exposure to those insecticides on potted turf cores reduced parasitism by T. vernalis. Bumble bee colonies confined to forage on white clover (Trifolium repens L.) in weedy turf that had been treated with clothianidin or the premix had reduced numbers of workers, honey pots, and immature bees. Premix residues incapacitated H. pennsylvanicus and C. bakeri slightly faster than clothianidin alone, but otherwise we detected no synergistic or additive effects. Chlorantraniliprole had no apparent adverse effects on any of the beneficial species. Implications for controlling turf pests with least disruption of non-target invertebrates are discussed.

Cytotoxic effects of thiamethoxam in the midgut and malpighian tubules of Africanized Apis mellifera (Hymenoptera: Apidae)
In this 2014 study, published in Microscopy Research and Technique, researchers aimed to analyze the effects of thiamethoxam in the midgut and Malpighian tubule cells of Africanized Apis mellifera. Newly emerged workers were exposed until 8 days to a diet containing a sublethal dose of thiamethoxam. The bees were dissected and the organs were processed for transmission electron microscopy. The results showed that thiamethoxam is cytotoxic to midgut and Malpighian tubules. In the midgut, the damage was more evident in bees exposed to the insecticide on the first day. On the eighth day, the cells were ultrastructurally intact suggesting a recovery of this organ. The Malpighian tubules showed pronounced alterations on the eighth day of exposure of bees to the insecticide. This study demonstrates that the continuous exposure to a sublethal dose of thiamethoxam can impair organs that are used during the metabolism of the insecticide.

Chronic impairment of bumblebee natural foraging behaviour induced by sublethal pesticide exposure
In this 2014 study, published in Functional Ecology, researchers examined how the day-to-day foraging patterns of bumblebees (Bombus terrestris) were affected when exposed to either a neonicotinoid (imidacloprid) and/or a pyrethroid (λ-cyhalothrin) independently and in combination over a four-week period. This is the first study to provide data on the impacts of combined and individual pesticide exposure on the temporal dynamics of foraging behaviour in the field over a prolonged period of time. Their results show that neonicotinoid exposure has both acute and chronic effects on overall foraging activity. Whilst foragers from control colonies improved their pollen foraging performance as they gained experience, the performance of bees exposed to imidacloprid became worse: chronic behavioural impairment. They also found evidence suggesting that pesticide exposure can change forager preferences for the flower types from which they collect pollen. These findings highlight the importance of considering prolonged exposure (which happens in the field) when assessing the risk that pesticides pose to bees. The effects of chronic pesticide exposure could have serious detrimental consequences for both colony survival and also the pollination services provided by these essential insect pollinators.

Pesticide residues and bees--a risk assessment
Bees are essential pollinators of many plants in natural ecosystems and agricultural crops alike. In recent years the decline and disappearance of bee species in the wild and the collapse of honey bee colonies have concerned ecologists and apiculturalists, who search for causes and solutions to this problem. Whilst biological factors such as viral diseases, mite and parasite infections are undoubtedly involved, it is also evident that pesticides applied to agricultural crops have a negative impact on bees. Most risk assessments have focused on direct acute exposure of bees to agrochemicals from spray drift. However, the large number of pesticide residues found in pollen and honey demand a thorough evaluation of all residual compounds so as to identify those of highest risk to bees. Using data from recent residue surveys and toxicity of pesticides to honey and bumble bees, a comprehensive evaluation of risks under current exposure conditions is presented here. Standard risk assessments are complemented with new approaches that take into account time-cumulative effects over time, especially with dietary exposures. Whilst overall risks appear to be low, this analysis indicates that residues of pyrethroid and neonicotinoid insecticides pose the highest risk by contact exposure of bees with contaminated pollen. However, the synergism of ergosterol inhibiting fungicides with those two classes of insecticides results in much higher risks in spite of the low prevalence of their combined residues. Risks by ingestion of contaminated pollen and honey are of some concern for systemic insecticides, particularly imidacloprid and thiamethoxam, chlorpyrifos and the mixtures of cyhalothrin and ergosterol inhibiting fungicides. More attention should be paid to specific residue mixtures that may result in synergistic toxicity to bees.

Chronic Exposure of Imidacloprid and Clothianidin Reduce Queen Survival, Foraging, and Nectar Storing in Colonies of Bombus impatiens
In this 2014 study, published in PLOS ONE, caged queenright colonies of Bombus impatiens Cresson, were fed treatments imidacloprid and clothianidin that overlapped the residue levels found in pollen and nectar of many crops and landscape plants, which have higher residue levels than seed-treated crops. Researchers ultimately found that feeding on imidacloprid and clothianidin can cause changes in behavior (reduced worker movement, consumption, wax pot production, and nectar storage) that result in detrimental effects on colonies (queen survival and colony weight). Wild bumblebees depending on foraging workers can be negatively impacted by chronic neonicotinyl exposure at 20 ppb.

Sub-lethal exposure to neonicotinoids impaired honey bees winterization before proceeding to colony collapse disorder
This 2014 study, published in the Bulletin of Insectology, undercuts chemical industry arguments that neonicotinoids are not the primary contribute factor in Colony Collapse Disorder (CCD).The results find that hives exposed to low doses of two neonicotinoid pesticides—imidacloprid and clothianidin—do not recover from over winter losses from which control hives quickly rebound. “It is imperative to emphasize that while pathogen infections are common and serious diseases found in honey bees that often lead to colony death, the post-mortem examinations of the pathogen- caused dead colonies are vastly different to those suffered from CCD.One of the defining symptomatic observations of CCD colonies is the emptiness of hives…. [Thus] the absence of dead bees in the neonicotinoid-treated colonies is remarkable and consistent with CCD symptoms.”

Pesticide residue and bees - a risk assessment
This 2014 study, published in the journal PLoS One provides a comprehensive evaluation of risks under current exposure conditions using data from recent residue surveys and toxicity of pesticides to honey and bumble bees. "Whilst overall risks appear to be low, our analysis indicates that residues of pyrethroid and neonicotinoid insecticides pose the highest risk by contact exposure of bees with contaminated pollen. However, the synergism of ergosterol inhibiting fungicides with those two classes of insecticides results in much higher risks in spite of the low prevalence of their combined residues. Risks by ingestion of contaminated pollen and honey are of some concern for systemic insecticides, particularly imidacloprid and thiamethoxam, chlorpyrifos and the mixtures of cyhalothrin and ergosterol inhibiting fungicides. More attention should be paid to specific residue mixtures that may result in synergistic toxicity to bees."

Honey bees, neonicotinoids and bee incident reports: the Canadian situation
In this 2013 study, published in Pest Management Science, researchers summarize honey bee incident report data obtained from the Canadian Pest Management Regulatory Agency (PMRA). In Canada, there were very few honey bee incidents reported in 2007–2011 and data were not collected prior to 2007. In 2012, a significant number of incidents were reported in the province of Ontario, where exposure to neonicotinoid dust during planting of corn was suspected to have caused the incident in up to 70% of cases. Most of these incidents were classified as ‘minor’ by the PMRA, and only six cases were considered ‘moderate’ or ‘major’. In that same year, there were over three times as many moderate or major incidents due to older non-neonicotinoid pesticides, involving numbers of hives or bees far greater than the number of moderate or major incidents suspected to be due to neonicotinoid poisoning. These data emphasize that, while exposure of honey bees to neonicotinoid-contaminated dust during corn planting needs to be mitigated, other pesticides also pose a risk.

Brain morphophysiology of Africanized Bee Apis mellifera exposed to sublethal doses of imidacloprid
This 2013 study, published in Archives of Environmental Contamination and Toxicology, demonstrated that imidacloprid causes changes to the brain in Africanized bees, particularly to the optic lobes of the brain, disrupting their visual system and impairing their learning capacity. "The organs of both control bees and bees exposed to insecticide were subjected to morphological, histochemical and immunocytochemical analysis after exposure to imidacloprid, respectively, for 1, 3, 5, 7, and 10 days. In mushroom bodies of bees exposed to imidacloprid concentrations of LD50/10 and in optic lobes of bees exposed to imidacloprid concentrations of LD50/10, LD50/100, and LD50/50, we observed the presence of condensed cells. The Feulgen reaction revealed the presence of some cells with pyknotic nuclei, whereas Xylidine Ponceau stain revealed strongly stained cells. These characteristics can indicate the occurrence of cell death. Furthermore, cells in mushroom bodies of bees exposed to imidacloprid concentrations of LD50/10 appeared to be swollen. Cell death was confirmed by immunocytochemical technique. Therefore, it was concluded that sublethal doses of imidacloprid have cytotoxic effects on exposed bee brains and that optic lobes are more sensitive to the insecticide than other regions of the brain."

Fatal powdering of bees in flight with particulates of neonicotinoids seed coating and humidity implication
This 2012 study, published in the Journal of Applied Entomology, examined the effect of direct aerial powdering on foragers in free flight near the drilling machine. Bees were conditioned to visit a dispenser of sugar solution whilst a drilling machine was sowing corn along the flight path. Samples of bees were captured on the dispenser, caged and held in the laboratory. Chemical analysis showed some hundred nanograms of insecticide per bee. Nevertheless, caged bees, previously contaminated in flight, died only if kept in conditions of high humidity. After the sowing, an increase in bee mortality in front of the hives was also observed. Spring bee losses, which corresponded with the sowing of corn-coated seed, seemed to be related to the casual encountering of drilling machine during foraging flight across the ploughed fields.

Multiple routes of pesticide exposure for honey bees living near agricultural fields
Populations of honey bees and other pollinators have declined worldwide in recent years. A variety of stressors have been implicated as potential causes, including agricultural pesticides. Neonicotinoid insecticides, which are widely used and highly toxic to honey bees, have been found in previous analyses of honey bee pollen and comb material. However, the routes of exposure have remained largely undefined. We used LC/MS-MS to analyze samples of honey bees, pollen stored in the hive and several potential exposure routes associated with plantings of neonicotinoid treated maize. Results demonstrate that bees are exposed to these compounds and several other agricultural pesticides in several ways throughout the foraging period. During spring, extremely high levels of clothianidin and thiamethoxam were found in planter exhaust material produced during the planting of treated maize seed. Study also found neonicotinoids in the soil of each field sampled, including unplanted fields. Plants visited by foraging bees (dandelions) growing near these fields were found to contain neonicotinoids as well. This indicates deposition of neonicotinoids on the flowers, uptake by the root system, or both. Dead bees collected near hive entrances during the spring sampling period were found to contain clothianidin as well, although whether exposure was oral (consuming pollen) or by contact (soil/planter dust) is unclear. Authors detected the insecticide clothianidin in pollen collected by bees and stored in the hive. When maize plants in our field reached anthesis, maize pollen from treated seed was found to contain clothianidin and other pesticides; and honey bees in our study readily collected maize pollen. These findings clarify some of the mechanisms by which honey bees may be exposed to agricultural pesticides throughout the growing season. These results have implications for a wide range of large-scale annual cropping systems that utilize neonicotinoid seed treatments.

Neonicotinoid pesticide reduces bumble bee colony growth and queen production
This 2012 study, published in Science, "exposed colonies of the bumble bee Bombus terrestris in the laboratory to field-realistic levels of the neonicotinoid imidacloprid, then allowed them to develop naturally under field conditions. Treated colonies had a significantly reduced growth rate and suffered an 85% reduction in production of new queens compared with control colonies. Given the scale of use of neonicotinoids, we suggest that they may be having a considerable negative impact on wild bumble bee populations across the developed world."

Neonicotinoids in bees: a review on concentrations, side-effects and risk assessment
This review summarizes, for the first time, 15 years of research on the hazards of neonicotinoids to bees including honey bees, bumble bees and solitary bees. The focus of the paper is on three different key aspects determining the risks of neonicotinoid field concentrations for bee populations: (1) the environmental neonicotinoid residue levels in plants, bees and bee products in relation to pesticide application, (2) the reported side-effects with special attention for sublethal effects, and (3) the usefulness for the evaluation of neonicotinoids of an already existing risk assessment scheme for systemic compounds. Although environmental residue levels of neonicotinoids were found to be lower than acute/chronic toxicity levels, there is still a lack of reliable data as most analyses were conducted near the detection limit and for only few crops. Many laboratory studies described lethal and sublethal effects of neonicotinoids on the foraging behavior, and learning and memory abilities of bees, while no effects were observed in field studies at field-realistic dosages. The proposed risk assessment scheme for systemic compounds was shown to be applicable to assess the risk for side-effects of neonicotinoids as it considers the effect on different life stages and different levels of biological organization (organism versus colony). Future research studies should be conducted with field-realistic concentrations, relevant exposure and evaluation durations. Molecular markers may be used to improve risk assessment by a better understanding of the mode of action (interaction with receptors) of neonicotinoids in bees leading to the identification of environmentally safer compounds.

Effects of imidacloprid, a neonicotinoid pesticide, on reproduction in worker bumble bees  
One conspicuous threat to bumble bees is their unintended exposure to trace residues of systemic neonicotinoid pesticides, such as imidacloprid, which are ingested when bees forage on the nectar and pollen of treated crops. To determine whether environmentally realistic levels of imidacloprid are capable of making a demographic impact on bumble bees, study exposed queenless microcolonies of worker bumble bees, Bombus terrestris, to a range of dosages of dietary imidacloprid between zero and 125 μg L(-1) and examined the effects on ovary development and fecundity. Microcolonies showed a dose-dependent decline in fecundity, with environmentally realistic dosages in the range of 1 μg L(-1) capable of reducing brood production by one third. In contrast, ovary development was unimpaired by dietary imidacloprid except at the highest dosage. Imidacloprid reduced feeding on both syrup and pollen but, after controlling statistically for dosage, microcolonies that consumed more syrup and pollen produced more brood. Authors speculate that the detrimental effects of imidacloprid on fecundity emerge principally from nutrient limitation imposed by the failure of individuals to feed. Findings raise concern about the impact of neonicotinoids on wild bumble bee populations. However, we recognize that to fully evaluate impacts on wild colonies it will be necessary to establish the effect of dietary neonicotinoids on the fecundity of bumble bee queens.

Exposure to sublethal doses of fipronil and thiacloprid highly increases mortality of honeybees previously infected by Nosema ceranae
This 2011 study, published in the journal PLoS ONE, explores the effect of Nosema ceranae infection on honeybee sensitivity to sublethal doses of the insecticides fipronil and thiacloprid. Honeybee mortality and insecticide consumption were analyzed daily and the intestinal spore content was evaluated 20 days after infection. A significant increase in honeybee mortality was observed when N. ceranae-infected honeybees were exposed to sublethal doses of insecticides. The synergistic effect of N. ceranae and insecticide on honeybee mortality, however, did not appear strongly linked to a decrease of the insect detoxification system. These data support the hypothesis that the combination of the increasing prevalence of N. ceranae with high pesticide content in beehives may contribute to colony depopulation.

Neonicotinoid insecticides translocated in guttated droplets of seed-treated maize and wheat: a threat to honeybees?
This study, published in Apidologie, demonstrates that guttated water of plants germinated from seeds dressed with neonicotinoids contains neonicotinoids. Maize seeds treated with clothianidin (Poncho® 0.5mg/seed and Poncho® Pro 1.25mg/seed) resulted in neonicotinoid concentrations up to 8,000ngmL−1 in the guttated fluid. This concentration decreases rapidly, but remained detectable over several weeks. Seeds treated with Poncho® Pro did not result in higher concentrations in guttated droplets in the first stages of plant development, but the concentration decreased more slowly. Triticale seed treated with imidacloprid contained small quantities of this active agent (up to 13ngmL−1) in the guttated fluid the following spring after overwintering.

Sub-lethal effects of pesticide residues in brood comb on worker honey nee (Apis mellifera) development and longevity
This 2011 study, published in the journal PLoS ONE, examines the "possible direct and indirect effects of pesticide exposure from contaminated brood comb on developing worker bees and adult worker lifespan. Results demonstrate sub-lethal effects on worker honey bees from pesticide residue exposure from contaminated brood comb. Sub-lethal effects, including delayed larval development and adult emergence or shortened adult longevity, can have indirect effects on the colony such as premature shifts in hive roles and foraging activity."

Rapid analysis of neonicotinoid insecticides in guttation drops of corn seedlings obtained from coated seeds
Published in the Journal of Environmental Monitoring, ths study examined guttation drops of corn plants obtained from commercial seeds coated with thiamethoxam, clothianidin, imidacloprid and fipronil have been analyzed. "The young plants grown both in pots – in greenhouse – and in open field from coated seeds, produced guttation solutions containing high levels of the neonicotinoid insecticides (up to 346 mg L-1 for imidacloprid, 102 mg L-1 for clothianidin and 146 mg L-1 for thiamethoxam). These concentration levels may represent lethal doses for bees that use guttation drops as a source of water. The neonicotinoid concentrations in guttation drops progressively decrease during the first 10–15 days after the emergence of the plant from the soil. Otherwise fipronil, which is a non-systemic phenylpyrazole insecticide, was never detected into guttation drops. Current results confirm that the physiological fluids of the corn plant can effectively transfer neonicotinoid insecticides from the seed onto the surface of the leaves, where guttation drops may expose bees and other insects to elevated doses of neurotoxic insecticides."

Overview of pesticide residues in stored pollen and their potential effect on bee colony (Apis mellifera) losses in Spain
In the last decade, an increase in honey bee (Apis mellifera L.) colony losses has been reported in several countries. The causes of this decline are still not clear. This study was set out to evaluate the pesticide residues in stored pollen from honey bee colonies and their possible impact on honey bee losses in Spain. In total, 1,021 professional apiaries were randomly selected. All pollen samples were subjected to multiresidue analysis by gas chromatography-mass spectrometry (MS) and liquid chromatography-MS; moreover, specific methods were applied for neonicotinoids and fipronil. A palynological analysis also was carried out to confirm the type of foraging crop. Pesticide residues were detected in 42% of samples collected in spring, and only in 31% of samples collected in autumn. Fluvalinate and chlorfenvinphos were the most frequently detected pesticides in the analyzed samples. Fipronil was detected in 3.7% of all the spring samples but never in autumn samples, and neonicotinoid residues were not detected. More than 47.8% of stored pollen samples belonged to wild vegetation, and sunflower (Heliantus spp.) pollen was only detected in 10.4% of the samples. A direct relation between pesticide residues found in stored pollen samples and colony losses was not evident accordingly to the obtained results. Further studies are necessary to determine the possible role of the most frequent and abundant pesticides (such as acaricides) and the synergism among them and with other pathogens more prevalent in Spain.

Pesticides and honey bee toxicity – USA
This 2010 study, published in Apidologie, "examines pesticides applied to crops, pesticides used in apiculture and pesticide residues in hive products. Authors discuss the role that pesticides and their residues in hive products may play in colony collapse disorder and other colony problems. Although no single pesticide has been shown to cause colony collapse disorder, the additive and synergistic effects of multiple pesticide exposures may contribute to declining honey bee health."

High levels of miticides and agrochemicals in North American apiaries: Implications for honey bee health
This 2010 study, published in the journal PLoS ONE, conducted "A broad survey of pesticide residues... on samples from migratory and other beekeepers across 23 states, one Canadian province and several agricultural cropping systems during the 2007–08 growing seasons. The 98 pesticides and metabolites detected in mixtures up to 214 ppm in bee pollen alone represents a remarkably high level for toxicants in the brood and adult food of this primary pollinator. This represents over half of the maximum individual pesticide incidences ever reported for apiaries. While exposure to many of these neurotoxicants elicits acute and sublethal reductions in honey bee fitness, the effects of these materials in combinations and their direct association with CCD or declining bee health remains to be determined." See: Daily News Blog.

Interactions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera)
This 2010 study, published in Environmental Microbiology, examines "Global pollinators, like honeybees, are declining in abundance and diversity, which can adversely affect natural ecosystems and agriculture. The authors tested the current hypotheses describing honeybee losses as a multifactorial syndrome, by investigating integrative effects of an infectious organism and an insecticide on honeybee health. Study demonstrated that the interaction between the microsporidia Nosema and a neonicotinoid (imidacloprid) significantly weakened honeybees. In the short term, the combination of both agents caused the highest individual mortality rates and energetic stress. This provides the first evidences that interaction between an infectious organism and a chemical can also threaten pollinators, interactions that are widely used to eliminate insect pests in integrative pest management."

Toxicities of fipronil enantiomers to the honeybee Apis mellifera L. and enantiomeric compositions of fipronil in honey plant flowers
Published in Environmental Contamination and Toxicology, this study examined fipronil, a "chiral phenylpyrazole insecticide that is effective for control of a wide range of agricultural and domestic pests at low application rates. Wide application of fipronil also causes poisoning of some nontarget insects, such as honeybees. In the present study, toxicities of fipronil enantiomers and racemate to the honeybee Apis mellifera L. were determined to examine whether using formulations of single or enriched fipronil enantiomer is a possible option to reduce risks to bees. The results indicate that it is unlikely that use of formulations with single or enriched fipronil enantiomer would reduce the risk that fipronil poses to honeybees. Improved fipronil application practices (based on safest timing and bloom conditions) and reduction of overall fipronil usage seem to be more realistic options."

Is Apis mellifera more sensitive to insecticides than other insects?
This 2010 study, published in Pest Management Science, summarized "insecticide toxicity data between A. mellifera and other insects to determine the relative sensitivity of honey bees to insecticides. It was found that, in general, honey bees were no more sensitive than other insect species across the 62 insecticides examined. In addition, honey bees were not more sensitive to any of the six classes of insecticides (carbamates, nicotinoids, organochlorines, organophosphates, pyrethroids and miscellaneous) examined. While honey bees can be sensitive to individual insecticides, they are not a highly sensitive species to insecticides overall, or even to specific classes of insecticides. However, all pesticides should be used in a way that minimizes honey bee exposure, so as to minimize possible declines in the number of bees and/or honey contamination."

Exposure to pesticides at sublethal level and their distribution within a honey bee (Apis mellifera) colony
This 2010 study, published in the Bulletin of Environmental Contamination and Toxicology, exposed honey bee colonies "to pesticides used in agriculture or within bee hives by beekeepers: coumaphos; diazinon; amitraz or fluvalinate. Samples of bee workers, larvae and royal jelly were analysed. Amitraz residues in all sampled material were below the level of detection of 10 ng/g. Diazinon was not detected in any of the analysed samples. The large quantities of fluvalinate found in bee heads and larvae, the coumaphos residues in royal jelly, and additional potential sub-lethal effects on individual honey bees or brood are discussed."

Residues of pesticides in honeybee (Apis mellifera carnica) bee bread and in pollen loads from treated apple orchards
This 2010 study, published in Bulletin of Environmental Contamination and Toxicology, placed honey bee (Apis mellifera carnica) colonies in two apple orchards treated with the insecticides diazinon and thiacloprid and the fungicide difenoconazole in accordance with a Protection Treatment Plan in the spring of 2007. "The residue of diazinon in pollen loads 10 days after orchard treatment was 0.09 mg/kg, and the same amount of residue was found in bee bread 16 days after treatment. In pollen loads 6 days after application 0.03 mg/kg of thiacloprid residues and 0.01 mg/kg of difenoconazole were found on the first day after application. Possible sub-lethal effects on individual honey bees and brood are discussed."

A meta-analysis of experiments testing the effects of a neonicotinoid insecticide (imidacloprid) on honey bees
Published in Ecotoxicology, this study provides a meta-analysis of "fourteen published studies of the effects of imidacloprid on honey bees under laboratory and semi-field conditions that comprised measurements on 7073 adult individuals and 36 colonies, fitted dose–response relationships [which] estimate that trace dietary imidacloprid at field-realistic levels in nectar will have no lethal effects, but will reduce expected performance in honey bees by between 6 and 20%. Statistical power analysis showed that published field trials that have reported no effects on honey bees from neonicotinoids were incapable of detecting these predicted sublethal effects with conventionally accepted levels of certainty. These findings raise renewed concern about the impact on honey bees of dietary imidacloprid, but because questions remain over the environmental relevance of predominantly laboratory-based results, I identify targets for research and provide procedural recommendations for future studies"

The impact of neonicotinoid insecticides on bumblebees, Honey bees and other non-target invertebrates
This 2009 report, published by BugLife,, reviews existing approvals research and independent research on the effects of neonicotinoid pesticides on Honey bees, bumblebees and other non-target invertebrates, and investigates the current approvals mechanism and its standards.

Subchronic exposure of honeybees to sublethal doses of pesticides: effects on behavior
This 2009 study, published in Environmental Toxicology and Chemistry conducted laboratory bioassays "to evaluate the effects on honeybee behavior of sublethal doses of insecticides chronically administered orally or by contact. After exposure to fipronil, acetamiprid or thiamethoxam, behavioral functions of honeybees were tested on day 12. Fipronil, used at the dose of 0.1 ng/bee, induced mortality of all honeybees after one week of treatment. In the olfactory conditioning paradigm, fipronil-treated honeybees failed to discriminate between a known and an unknown odorant. Thiamethoxam by contact induced either a significant decrease of olfactory memory 24 h after learning at 0.1 ng/bee or a significant impairment of learning performance with no effect on memory at 1 ng/bee. The only significant effect of acetamiprid (administered orally, 0.1 microg/bee) was an increase in responsiveness to water. Data on the intrinsic toxicity of the compounds after chronic exposure have to be taken into account for evaluation of risk to honeybees in field conditions."

Translocation of Neonicotinoid Insecticides From Coated Seeds to Seedling Guttation Drops: A Novel Way of Intoxication for Bees
This 2009 study, published in Ecotoxicology, examine the contamination of guttation droplets which bees are regularly exposed to: "Although neonicotinoid systemic insecticides used for seed coating of agricultural crops were suspected as possible reason, studies so far have not shown the existence of unquestionable sources capable of delivering directly intoxicating doses in the Þelds. Guttation is a natural plant phenomenon causing the excretion of xylem ßuid at leaf margins. Here, we show that leaf guttation drops of all the corn plants germinated from neonicotinoid-coated seeds contained amounts of insecticide constantly higher than 10 mg/l, with maxima up to 100 mg/l for thiamethoxam and clothianidin, and up to 200 mg/l for imidacloprid. The concentration of neonicotinoids in guttation drops can be near those of active ingredients commonly applied in Þeld sprays for pest control, or even higher. When bees consume guttation drops, collected from plants grown from neonicotinoid-coated seeds, they encounter death within few minutes."

Effects of sublethal concentrations of bifenthrin and deltamethrin on fecundity, growth, and development of the honeybee Apis mellifera ligustica
This 2009 study, published in Environmental Contamination and Toxicology, examined bifenthrin and deltamethrin whichhave been widely used as pesticides in agriculture and forestry and are becoming an increasing risk to honeybees. "The honeybee, Apis mellifera ligustica, is widely recognized as a beneficial insect of agronomic, ecological, and scientific importance. It is important to understand what effects these chemicals have on bees. Effects of two pesticides at sublethal concentrations on fecundity, growth, and development of honeybees were examined with the feeding method for a three-year period (2006–2008). It was shown that both bifenthrin and deltamethrin significantly reduced bee fecundity, decreased the rate at which bees develop to adulthood, and increased their immature periods. The toxicity of bifenthrin and deltamethrin on workers of Apis mellifera ligustica was also assessed, and the results from the present study showed that the median lethal effects of bifenthrin and deltamethrin were 16.7 and 62.8?mg/L, respectively." See Daily News Blog.

Synergistic interactions between in-hive miticides in Apis mellifera
This 2009 study published in the Journal of Economic Entomology, examined the varroa mite, Varroa destructor Anderson & Trueman, " a devastating pest of honey bees, Apis mellifera L., that has been primarily controlled over the last 15 yr with two in-hive miticides: the organophosphate coumaphos (Checkmite+), and the pyrethroid tau-fluvalinate (Apistan). In this laboratory study, the authors observed a large increase in the toxicity of tau-fluvalinate to 3-day-old bees that had been treated previously with coumaphos, and a moderate increase in the toxicity of coumpahos in bees treated previously with tau-fluvalinate. The observed synergism may result from competition between miticides. These results suggest that honey bee mortality may occur with the application of otherwise sublethal doses of miticide when tau-fluvalinate and coumaphos are simultaneously present in the hive."

Translocation of neonicotinoid insecticides from coated seeds to seedling guttation drops: A novel way of intoxication for bees
This 2009 study, published in the Journal of Economic Entomology, examines "The death of honey bees, Apis mellifera L., and the consequent colony collapse disorder causes major losses in agriculture and plant pollination worldwide. The phenomenon showed increasing rates in the past years, although its causes are still awaiting a clear answer. Here, we show that leaf guttation drops of all the corn plants germinated from neonicotinoid-coated seeds contained amounts of insecticide constantly higher than 10 mg/l, with maxima up to 100 mg/l for thiamethoxam and clothianidin, and up to 200 mg/l for imidacloprid. The concentration of neonicotinoids in guttation drops can be near those of active ingredients commonly applied in field sprays for pest control, or even higher. When bees consume guttation drops, collected from plants grown from neonicotinoid-coated seeds, they encounter death within few minutes."

Abnormal foraging behavior induced by sublethal dosage of imidacloprid in the honey bee (Hymenoptera: Apidae)
This 2008 study, published in the Journal of Economic Entomology, demonstrates that low dosages of the neonicotinoid insecticide imidacloprid may affect honey bee, Apis mellifera L., behavior. "In this article, the foraging behavior of the honey bee workers was investigated to show the effects of imidacloprid. Results demonstrated that sublethal dosages of imidacloprid were able to affect foraging behavior of honey bees. By measuring the time interval between two visits at the same feeding site, we found that the normal foraging interval of honey bee workers was within 300 s. However, these honey bee workers delayed their return visit for >300 s when they were treated orally with sugar water containing imidacloprid."

The relevance of sublethal effects in honey bee testing for pesticide risk assessment
This 2007 study, published in Pest Management Science, considers whether and if sublethal effects should be incorporated into risk assessment, by addressing a number of questions: The authors conclude that sublethal studies may be helpful as an optional test to address particular, compound-specific concerns, as a lower-tier alternative to semi-field or field testing, if the effects are shown to be ecologically relevant. However, available higher-tier data (semi-field, field tests) should make any additional sublethal testing unnecessary, and higher-tier data should always override data of lower-tier trials on sublethal effects.

Comparative sublethal toxicity of nine pesticides on olfactory learning performances of the honeybee Apis mellifera
Published in Archives of Environmental Contamination and Toxicology, this study used "a conditioned proboscis extension response (PER) assay, honeybees (Apis mellifera L.) can be trained to associate an odor stimulus with a sucrose reward. In the present study, the effects of sublethal concentrations of nine pesticides on learning performances of worker bees subjected to the PER assay were estimated and compared. Reduced learning performances were observed for bees surviving treatment with fipronil, deltamethrin, endosulfan, and prochloraz. A lack of behavioral effects after treatment with lambda-cyalothrin, cypermethrin, tau-fluvalinate, triazamate, and dimethoate was recorded."

Pesticide Impacts on Other Pollinators

Larval exposure to the neonicotinoid imidacloprid impacts adult size in the farmland butterfly Pieris brassicae.
Populations of farmland butterflies have been suffering from substantial population declines in recent decades. These declines have been correlated with neonicotinoid usage both in Europe and North America but experimental evidence linking these correlations is lacking. The potential for non-target butterflies to be exposed to trace levels of neonicotinoids is high, due to the widespread contamination of agricultural soils and wild plants in field margins. Here we provide experimental evidence that field realistic, sub-lethal exposure to the neonicotinoid imidacloprid negatively impacts the development of the common farmland butterfly Pieris brassicae. Cabbage plants were watered with either 0, 1, 10, 100 or 200 parts per billion imidacloprid, to represent field margin plants growing in contaminated agricultural soils and these were fed to P. brassicae larvae. The approximate digestibility (AD) of the cabbage as well as behavioural responses by the larvae to simulated predator attacks were measured but neither were affected by neonicotinoid treatment. However, the duration of pupation and the size of the adult butterflies were both significantly reduced in the exposed butterflies compared to the controls, suggesting that adult fitness is compromised through exposure to this neonicotinoid.

Effects of Field-Relevant Concentrations of Clothianidin on Larval Development of the Butterfly Polyommatus icarus (Lepidoptera, Lycaenidae).
Arable field margins are often sown with wildflowers to encourage pollinators and other beneficial or desirable insects such as bees and butterflies. Concern has been raised that these margins may be contaminated with systemic pesticides such as neonicotinoids used on the adjacent crop, and that this may negatively impact beneficial insects. The use of neonicotinoids has been linked to butterfly declines, and species such as the common blue butterfly ( Polyommatus icarus) that feed upon legumes commonly sown in arable field margins, may be exposed to such toxins. Here, we demonstrate that the larval food plants of P. icarus growing in an arable field margin adjacent to a wheat crop treated with the neonicotinoid clothianidin not only contain the pesticide at concentrations comparable to and sometimes higher than those found in foliage of treated crops (range 0.2-48 ppb) but also remain detectable at these levels for up to 21 months after sowing of the crop. Overall, our study demonstrates that nontarget herbivorous organisms in arable field margins are likely to be chronically exposed to neonicotinoids. Under laboratory conditions, exposure to clothianidin at 15 ppb (a field-realistic dose) or above reduced larval growth for the first 9 days of the experiment. Although there was evidence of clothianidin inducing mortality in larvae, with highest survival in control groups, the dose-response relationship was unclear. Our study suggests that larvae of this butterfly exhibit some deleterious sublethal and sometimes lethal impacts of exposure to clothianidin, but many larvae survive to adulthood even when exposed to high doses.

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.

The reduced-risk insecticide azadirachtin poses a toxicological hazard to stingless bee Partamona helleri (Friese, 1900) queens.
Large-scale pesticide application poses a major threat to bee biodiversity by causing a decline in bee populations that, in turn, compromises ecosystem maintenance and agricultural productivity. Biopesticides are considered an alternative to synthetic pesticides with a focus on reducing potential detrimental effects to beneficial organisms such as bees. The production of healthy queen stingless bees is essential for the survival and reproduction of hives, although it remains unknown whether biopesticides influence stingless bee reproduction. In the present study, we investigated the effects of the biopesticide azadirachtin on the survival, behavior, morphology, development, and reproduction of queens of the stingless bee Partamona helleri (Friese, 1900). The neonicotinoid imidacloprid was used as a toxic reference standard. Queens were orally exposed in vitro to a contaminated diet (containing azadirachtin and imidacloprid) during development. Azadirachtin resulted in reduced survival, similarly to imidacloprid, altered development time, caused deformations, and reduced the size of the queens' reproductive organs. All of these factors could potentially compromise colony survival. Results from the present study showed azadirachtin posed a toxicological hazard to P. helleri queens.

Field evidence of bird poisonings by imidacloprid-treated seeds: a review of incidents reported by the French SAGIR network from 1995 to 2014.
The large-scale use of neonicotinoid insecticides has raised growing concerns about their potential adverse effects on farmland birds, and more generally on biodiversity. Imidacloprid, the first neonicotinoid commercialized, has been identified as posing a risk for seed-eating birds when it is used as seed treatment of some crops since the consumption of a few dressed seeds could cause mortality. But evidence of direct effects in the field is lacking. Here, we reviewed the 103 wildlife mortality incidents reported by the French SAGIR Network from 1995 to 2014, for which toxicological analyses detected imidacloprid residues. One hundred and one incidents totalling at least 734 dead animals were consistent with an agricultural use as seed treatment. Grey partridges (Perdix perdix) and "pigeons" (Columba palumbus, Columba livia and Columba oenas) were the main species found. More than 70% of incidents occurred during autumn cereal sowings. Furthermore, since there is no biomarker for diagnosing neonicotinoid poisonings, we developed a diagnostic approach to estimate the degree of certainty that these mortalities were due to imidacloprid poisoning. By this way, the probability that mortality was due to poisoning by imidacloprid-treated seeds was ranked as at least "likely" in 70% of incidents. As a result, this work provides clear evidence to risk managers that lethal effects due to the consumption by birds of imidacloprid-treated seeds regularly occur in the field. This in turn raises the question of the effectiveness of the two main factors (seed burying and imidacloprid-treated seeds avoidance) that are supposed to make the risk to birds negligible. Risk factors and the relevance of mitigation measures are discussed.

Are neonicotinoid insecticides driving declines of widespread butterflies?
There has been widespread concern that neonicotinoid pesticides may be adversely impacting wild and managed bees for some years, but recently attention has shifted to examining broader effects they may be having on biodiversity. For example in the Netherlands, declines in insectivorous birds are positively associated with levels of neonicotinoid pollution in surface water. In England, the total abundance of widespread butterfly species declined by 58% on farmed land between 2000 and 2009 despite both a doubling in conservation spending in the UK, and predictions that climate change should benefit most species. Here we build models of the UK population indices from 1985 to 2012 for 17 widespread butterfly species that commonly occur at farmland sites. Of the factors we tested, three correlated significantly with butterfly populations. Summer temperature and the index for a species the previous year are both positively associated with butterfly indices. By contrast, the number of hectares of farmland where neonicotinoid pesticides are used is negatively associated with butterfly indices. Indices for 15 of the 17 species show negative associations with neonicotinoid usage. The declines in butterflies have largely occurred in England, where neonicotinoid usage is at its highest. In Scotland, where neonicotinoid usage is comparatively low, butterfly numbers are stable. Further research is needed urgently to show whether there is a causal link between neonicotinoid usage and the decline of widespread butterflies or whether it simply represents a proxy for other environmental factors associated with intensive agriculture.

Imidacloprid-treated seed ingestion has lethal effect on adult partridges and reduces both breeding investment and offspring immunity.
The ingestion of imidacloprid treated seeds by farmland birds may result in exposure to toxic amounts of this insecticide. Here we report on the effects that the exposure to the recommended application rate and to 20% of that rate may produce on birds feeding on treated seeds. Experimental exposure to imidacloprid treated seeds was performed on red-legged partridges (Alectoris rufa) (n=15 pairs per treatment group: control, 20% or 100% of the recommended application rate) during two periods that corresponded to the autumn (duration of exposure: 25 days) and late winter (10 days) cereal sowing times in Spanish farmlands. We studied effects on the survival, body condition, oxidative stress biomarkers, plasma biochemistry, carotenoid-based coloration, T-cell mediated immune response and reproduction of exposed adult partridges, and on the survival and T-cell immune response of their chicks. The high dose (recommended application rate) killed all partridges, with mortality occurring faster in females than in males. The low dose (20% the recommended application rate) had no effect on mortality, but reduced levels of plasma biochemistry parameters (glucose, magnesium and lactate dehydrogenase), increased blood superoxide dismutase activity, produced changes in carotenoid-based integument coloration, reduced the clutch size, delayed the first egg lay date, increased egg yolk vitamins and carotenoids and depressed T-cell immune response of chicks. Moreover, the analysis of the livers of dead partridges revealed an accumulation of imidacloprid during exposure time. Despite the moratorium on the use of neonicotinoids in the European Union, birds may still be at high risk of poisoning by these pesticides through direct sources of exposure to coated seeds in autumn and winter.

Increasing neonicotinoid use and the declining butterfly fauna of lowland California.
The butterfly fauna of lowland Northern California has exhibited a marked decline in recent years that previous studies have attributed in part to altered climatic conditions and changes in land use. Here, we ask if a shift in insecticide use towards neonicotinoids is associated with butterfly declines at four sites in the region that have been monitored for four decades. A negative association between butterfly populations and increasing neonicotinoid application is detectable while controlling for land use and other factors, and appears to be more severe for smaller-bodied species. These results suggest that neonicotinoids could influence non-target insect populations occurring in proximity to application locations, and highlights the need for mechanistic work to complement long-term observational data.

Insight into the mechanism of reproductive dysfunction caused by neonicotinoid pesticides.
Neonicotinoids, which were developed in the 1990 s as an insecticide having selective toxicity, were later found to cause reproductive abnormalities in experimental animals. In Japan there is an attempt to preserve endangered animals, including the Japanese crested ibis, and there is a question of whether neonicotinoids affect the reproduction of this bird, since they are used in its habitat. Hence, we investigated whether the daily oral administration of the neonicotinoid clothianidin (CTD) has any deleterious effects on the reproductive function of mature male only or both young male and female quails as experimental animals. Vacuolization and the number of germ cells having fragmented DNA in seminiferous tubules, as well as the number and size of vacuoles in hepatocytes, increased dose-dependently. The ovaries showed abnormal histology in the granulosa cells, which produce progesterone. There were significant differences in egg-laying rates and embryo weights between the groups. Glutathione Peroxidase 4 (GPx4) and Manganese Superoxide Dismutase (Mn-SOD), which protect the organism from oxidative damage, showed a dose-dependent decrease. Thus, it is possible neonicotinoids affect the bird's reproductive system through oxidative stress, reflecting an imbalance between the production of reactive oxygen species (ROS) and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Responding to our study, Sado Island has since succeeded in breeding Japanese crested ibis in the wild without the use of neonicotinoids.

Ecology: Pesticides linked to bird declines.
The debate over the environmental risks posed by neonicotinoid insecticides has raged since the late 1990s, when French beekeepers began blaming the chemicals for losses of honeybee colonies. The discussion has focused closely on bees, particularly the risks posed by the use of neonicotinoid treatments on flowering crops that bees visit. But on page 341 of this issue, Hallmann et al.1 provide strong evidence that this debate may have missed the bigger picture. Analysing long-term data sets on bird populations in the Netherlands, the authors demonstrate that regional patterns of population decline in insect-eating birds are neatly predicted by levels of neonicotinoids detected in environmental samples. In other words, birds have declined faster in places with more neonicotinoid pollution.

Soil-applied imidacloprid translocates to ornamental flowers and reduces survival of adult Coleomegilla maculata, Harmonia axyridis, and Hippodamia convergens lady beetles, and larval Danaus plexippus and Vanessa cardui butterflies

Integrated Pest Management (IPM) is a decision making process used to manage pests that relies on many tactics, including cultural and biological control, which are practices that conserve beneficial insects and mites, and when needed, the use of conventional insecticides. However, systemic, soil-applied neonicotinoid insecticides are translocated to pollen and nectar of flowers, often for months, and may reduce survival of flower-feeding beneficial insects. Imidacloprid seed-treated crops (0.05 mg AI (active ingredient) /canola seed and 1.2 mg AI/corn seed) translocate less than 10 ppb to pollen and nectar. However, higher rates of soil-applied imidacloprid are used in nurseries and urban landscapes, such as 300 mg AI/10 L (3 gallon) pot and 69 g AI applied to the soil under a 61 (24 in) cm diam. tree. Translocation of imidacloprid from soil (300 mg AI) to flowers of Asclepias curassavica resulted in 6,030 ppb in 1X and 10,400 ppb in 2X treatments, which are similar to imidacloprid residues found in another plant species we studied. A second imidacloprid soil application 7 months later resulted in 21,000 ppb in 1X and 45,000 ppb in 2X treatments. Consequently, greenhouse/nursery use of imidacloprid applied to flowering plants can result in 793 to 1,368 times higher concentration compared to an imidacloprid seed treatment (7.6 ppb pollen in seed- treated canola), where most research has focused. These higher imidacloprid levels caused significant mortality in both 1X and 2X treatments in 3 lady beetle species, Coleomegilla maculata, Harmonia axyridis, and Hippodamia convergens, but not a fourth species, Coccinella septempunctata. Adult survival were not reduced for monarch, Danaus plexippus and painted lady, Vanessa cardui, butterflies, but larval survival was significantly reduced. The use of the neonicotinoid imidacloprid at greenhouse/nursery rates reduced survival of beneficial insects feeding on pollen and nectar and is incompatible with the principles of IPM.

Fipronil application on rice paddy fields reduces densities of common skimmer and scarlet skimmer

In this study, published in the journal Scientific Reports, researchers conducted paddy mesocosm experiments to investigate the effect of the systemic insecticides clothianidin, fipronil and chlorantraniliprole on rice paddy field biological communities. Concentrations of all insecticides in the paddy water were reduced to the limit of detection within 3 months after application. However, residuals of these insecticides in the paddy soil were detected throughout the experimental period. Plankton species were affected by clothianidin and chlorantraniliprole right after the applications, but they recovered after the concentrations decreased. On the other hand, the effects of fipronil treatment, especially on Odonata, were larger than those of any other treatment. The number of adult dragonflies completing eclosion was severely decreased in the fipronil treatment. These results suggest that the accumulation of these insecticides in paddy soil reduces biodiversity by eliminating dragonfly nymphs, which occupy a high trophic level in paddy fields.

Increased Acetylcholinesterase Expression in Bumble Bees During Neonicotinoid-Coated Corn Sowing

While honey bee exposure to systemic insecticides has received much attention, impacts on wild pollinators have not been as widely studied. Neonicotinoids have been shown to increase acetylcholinesterase (AChE) activity in honey bees at sublethal doses. High AChE levels may therefore act as a biomarker of exposure to neonicotinoids. This two-year study focused on establishing whether bumble bees living and foraging in agricultural areas using neonicotinoid crop protection show early biochemical signs of intoxication. Bumble bee colonies (Bombus impatiens) were placed in two different agricultural cropping areas: 1) control (≥ 3 km from fields planted with neonicotinoid-treated seeds) or 2) exposed (within 500 m of fields planted with neonicotinoid-treated seeds), and maintained for the duration of corn sowing. As determined by Real Time qPCR, AChE mRNA expression was initially significantly higher in bumble bees from exposed sites, then decreased throughout the planting season to reach a similar endpoint to that of bumble bees from control sites. These findings suggest that exposure to neonicotinoid seed coating particles during the planting season can alter bumble bee neuronal activity. This is the first study to report in situ that bumble bees living in agricultural areas exhibit signs of neonicotinoid intoxication.

Are neonicotinoid insecticides driving declines of widespread butterflies?
There has been widespread concern that neonicotinoid pesticides may be adversely impacting wild and managed bees for some years, but recently attention has shifted to examining broader effects they may be having on biodiversity. For example in the Netherlands, declines in insectivorous birds are positively associated with levels of neonicotinoid pollution in surface water. In England, the total abundance of widespread butterfly species declined by 58% on farmed land between 2000 and 2009 despite both a doubling in conservation spending in the UK, and predictions that climate change should benefit most species. Here authors build models of the UK population indices from 1985 to 2012 for 17 widespread butterfly species that commonly occur at farmland sites. Of the factors we tested, three correlated significantly with butterfly populations. Summer temperature and the index for a species the previous year are both positively associated with butterfly indices. By contrast, the number of hectares of farmland where neonicotinoid pesticides are used is negatively associated with butterfly indices. Indices for 15 of the 17 species show negative associations with neonicotinoid usage. The declines in butterflies have largely occurred in England, where neonicotinoid usage is at its highest. In Scotland, where neonicotinoid usage is comparatively low, butterfly numbers are stable. Further research is needed urgently to show whether there is a causal link between neonicotinoid usage and the decline of widespread butterflies or whether it simply represents a proxy for other environmental factors associated with intensive agriculture.

Non-target effects of clothianidin on monarch butterflies.

Monarch butterflies (Danaus plexippus) frequently consume milkweed in and near agroecosystems and consequently may be exposed to pesticides like neonicotinoids. Study conducted a dose response study to determine lethal and sublethal doses of clothianidin using a 36-h exposure scenario. Study then quantified clothianidin levels found in milkweed leaves adjacent to maize fields. Toxicity assays revealed LC10, LC50, and LC90 values of 7.72, 15.63, and 30.70 ppb, respectively. Sublethal effects (larval size) were observed at 1 ppb. Contaminated milkweed plants had an average of 1.14±0.10 ppb clothianidin, with a maximum of 4 ppb in a single plant. This research suggests that clothianidin could function as a stressor to monarch populations.

Effects of the neonicotinoid pesticide thiamethoxam at field-realistic levels on microcolonies of Bombus terrestris worker bumble bees.

Neonicotinoid pesticides are currently implicated in the decline of wild bee populations. Bumble bees, Bombus spp., are important wild pollinators that are detrimentally affected by ingestion of neonicotinoid residues. To date, imidacloprid has been the major focus of study into the effects of neonicotinoids on bumble bee health, but wild populations are increasingly exposed to alternative neonicotinoids such as thiamethoxam. To investigate whether environmentally realistic levels of thiamethoxam affect bumble bee performance over a realistic exposure period, authors exposed queenless microcolonies of Bombus terrestris L. workers to a wide range of dosages up to 98 μgkg(-1) in dietary syrup for 17 days. Results showed that bumble bee workers survived fewer days when presented with syrup dosed at 98 μg thiamethoxamkg(-1), while production of brood (eggs and larvae) and consumption of syrup and pollen in microcolonies were significantly reduced by thiamethoxam only at the two highest concentrations (39, 98 μgkg(-1)). In contrast, study found no detectable effect of thiamethoxam at levels typically found in the nectars of treated crops (between 1 and 11 μgkg(-1)). By comparison with published data, study demonstrates that during an exposure to field-realistic concentrations lasting approximately two weeks, brood production in worker bumble bees is more sensitive to imidacloprid than thiamethoxam. Authors speculate that differential sensitivity arises because imidacloprid produces a stronger repression of feeding in bumble bees than thiamethoxam, which imposes a greater nutrient limitation on production of brood.

Insecticide Use in Hybrid Onion Seed Production Affects Pre- and Postpollination Processes

In this 2014 study, published in the Journal of Economic Entomology, researchers conducted a field experiment manipulating insecticide use to examine the impacts of insecticides on 1) pollinator attraction, 2) pollen/stigma interactions, and 3) seed set and seed quality. Select insecticides had negative impacts on pollinator attraction and pollen/stigma interactions, with certain products dramatically reducing pollen germination and pollen tube growth. Decreased pollen germination was not associated with reduced seed set; however, reduced pollinator attraction was associated with lower seed set and seed quality, for one of the two female lines examined. These results highlight the importance of pesticide effects on the pollination process. Overuse may lead to yield reductions through impacts on pollinator behavior and postpollination processes. Overall, in hybrid onion seed production, moderation in insecticide use is advised when controlling onion thrips, Thrips tabaci, on commercial fields.

Sublethal neonicotinoid insecticide exposure reduces solitary bee reproductive success

This 2013 study published in Agricultural and Forest Entomology “investigated the influence of field-realistic trace residues of the routinely used neonicotinoid insecticides thiamethoxam and clothianidin in nectar substitutes on the entire life-time fitness performance of the red mason bee Osmia bicornis. We show that chronic, dietary neonicotinoid exposure has severe detrimental effects on solitary bee reproductive output. Neonicotinoids did not affect adult bee mortality; however, monitoring of fully controlled experimental populations revealed that sublethal exposure resulted in almost 50% reduced total offspring production and a significantly male-biased offspring sex ratio.

Single pollinator species losses reduce floral fidelity and plant reproductive function

This 2013 study, published in PNAS, "temporarily removed single pollinator species from study plots in subalpine meadows, to test the hypothesis that interactions between pollinator species can shape individual species’ functional roles via changes in foraging specialization. We show that loss of a single pollinator species reduces floral fidelity (short-term specialization) in the remaining pollinators, with significant implications for ecosystem functioning in terms of reduced plant reproduction, even when potentially effective pollinators remained in the system. Our results suggest that ongoing pollinator declines may have more serious negative implications for plant communities than is currently assumed. More broadly, we show that the individual functional contributions of species can be dynamic and shaped by the community of interspecific competitors, thereby documenting a distinct mechanism for how biodiversity can drive ecosystem functioning, with potential relevance to a wide range of taxa and systems."

Clearance of ingested neonicotinoid pesticide (imidacloprid) in honey bees (Apis mellifera) and bumblebees (Bombus terrestris)

Bees in agricultural landscapes are exposed to dietary pesticides such as imidacloprid when they feed from treated mass-flowering crops. Concern about the consequent impact on bees makes it important to understand their resilience. In the laboratory, the authors therefore fed adult worker bees on dosed syrup (125 μg L(-1) of imidacloprid, or 98 μg kg(-1)) either continuously or as a pulsed exposure and measured their behaviour (feeding and locomotory activity) and whole-body residues.On dosed syrup, honey bees maintained much lower bodily levels of imidacloprid than bumblebees (<0.2 ng versus 2.4 ng of imidacloprid per bee). Dietary imidacloprid did not affect the behaviour of honey bees, but it reduced feeding and locomotory activity in bumblebees. After the pulsed exposure, bumblebees cleared bodily imidacloprid after 48 h and recovered behaviourally.The differential behavioural resilience of the two species can be attributed to the observed differential in bodily residues. The ability of bumblebees to recover may be environmentally relevant in wild populations that face transitory exposures from the pulsed blooming of mass-flowering crops.

Differential sensitivity of honey bees and bumble bees to a dietary insecticide (imidacloprid).

To establish whether imidacloprid, a systemic neonicotinoid and insect neurotoxin, harms individual bees when ingested at environmentally realistic levels, authors exposed adult worker bumble bees, Bombus terrestris L. (Hymenoptera: Apidae), and honey bees, Apis mellifera L. (Hymenoptera: Apidae), to dietary imidacloprid in feeder syrup at dosages between 0.08 and 125μg l(-1). Honey bees showed no response to dietary imidacloprid on any variable measured (feeding, locomotion and longevity). In contrast, bumble bees progressively developed over time a dose-dependent reduction in feeding rate with declines of 10-30% in the environmentally relevant range of up to 10μg l(-1), but neither their locomotory activity nor longevity varied with diet. To explain their differential sensitivity, authors speculate that honey bees are better pre-adapted than bumble bees to feed on nectars containing synthetic alkaloids, such as imidacloprid, by virtue of their ancestral adaptation to tropical nectars in which natural alkaloids are prevalent. Findings do raise new concern about the impact of agricultural neonicotinoids on wild bumble bee populations.

Assessing insecticide hazard to bumble bees foraging on flowering weeds in treated lawns

Maintaining bee-friendly habitats in cities and suburbs can help conserve the vital pollination services of declining bee populations. Despite label precautions not to apply them to blooming plants, neonicotinoids and other residual systemic insecticides may be applied for preventive control of lawn insect pests when spring-flowering weeds are present. Dietary exposure to neonicotinoids adversely affects bees, but the extent of hazard from field usage is controversial. Authors exposed colonies of the bumble bee Bombus impatiens to turf with blooming white clover that had been treated with clothianidin, a neonicotinoid, or with chlorantraniliprole, the first anthranilic diamide labeled for use on lawns. The sprays were applied at label rate and lightly irrigated. After residues had dried, colonies were confined to forage for six days, and then moved to a non-treated rural site to openly forage and develop. Colonies exposed to clothianidin-treated weedy turf had delayed weight gain and produced no new queens whereas those exposed to chlorantraniliprole-treated plots developed normally compared with controls. Neither bumble bees nor honey bees avoided foraging on treated white clover in open plots. Nectar from clover blooms directly contaminated by spray residues contained 171±44 ppb clothianidin. Notably, neither insecticide adversely impacted bee colonies confined on the treated turf after it had been mown to remove clover blooms present at the time of treatment, and new blooms had formed. Results validate EPA label precautionary statements not to apply neonicotinoids to blooming nectar-producing plants if bees may visit the treatment area. Whatever systemic hazard through lawn weeds they may pose appears transitory, however, and direct hazard can be mitigated by adhering to label precautions, or if blooms inadvertently are contaminated, by mowing to remove them. Chlorantraniliprole usage on lawns appears non-hazardous to bumble bees.

Risk assessment for side-effects of neonicotinoids against bumblebees with and without impairing foraging behavior

This 2010 study, published in Ecotoxicology, examined "the development of a new bioassay to assess the impact of sublethal concentrations on the bumblebee foraging behavior under laboratory conditions. In general, the experiments showed that concentrations that may be considered safe for bumblebees can have a negative influence on their foraging behavior. Therefore it is recommended that behavior tests should be included in risk assessment tests for highly toxic pesticides because impairment of the foraging behavior can result in a decreased pollination, lower reproduction and finally in colony mortality due to a lack of food."

Repression and recuperation of brood production in Bombus terrestris bumble bees exposed to a pulse of the neonicotinoid pesticide imidacloprid.

Bumble bees are important wild pollinators that are widely exposed to dietary neonicotinoids by foraging in agricultural environments. In the laboratory, researchers tested the effect of a pulsed exposure (14 days 'on dose' followed by 14 days 'off dose') to a common neonicotinoid, imidacloprid, on the amount of brood (number of eggs and larvae) produced by Bombus terrestris L. bumble bees in small, standardised experimental colonies (a queen and four adult workers). During the initial 'on dose' period they observed a dose-dependent repression of brood production in colonies, with productivity decreasing as dosage increased up to 98 µg kg(-1) dietary imidacloprid. During the following 'off dose' period, colonies showed a dose-dependent recuperation such that total brood production during the 28-day pulsed exposure was not correlated with imidacloprid up to 98 µg kg(-1).Findings raise further concern about the threat to wild bumble bees from neonicotinoids, but they also indicate some resilience to a pulsed exposure, such as that arising from the transient bloom of a treated mass-flowering crop.

Imidacloprid-induced impairment of mushroom bodies and behavior of the native stingless bee Melipona quadrifasciata anthidioides

Native stingless bees (Hymenoptera: Apidae: Meliponinae) are key pollinators in neotropical areas and are threatened with extinction due to deforestation and pesticide use. Few studies have directly investigated the effects of pesticides on these pollinators. Furthermore, the existing impact studies did not address the issue of larval ingestion of contaminated pollen and nectar, which could potentially have dire consequences for the colony. Here, study assessed the effects of imidacloprid ingestion by stingless bee larvae on their survival, development, neuromorphology and adult walking behavior. Increasing doses of imidacloprid were added to the diet provided to individual worker larvae of the stingless bee Melipona quadrifasciata anthidioides throughout their development. Survival rates above 50% were only observed at insecticide doses lower than 0.0056 µg active ingredient (a.i.)/bee. No sublethal effect on body mass or developmental time was observed in the surviving insects, but the pesticide treatment negatively affected the development of mushroom bodies in the brain and impaired the walking behavior of newly emerged adult workers. Therefore, stingless bee larvae are particularly susceptible to imidacloprid, as it caused both high mortality and sublethal effects that impaired brain development and compromised mobility at the young adult stage. These findings demonstrate the lethal effects of imidacloprid on native stingless bees and provide evidence of novel serious sublethal effects that may compromise colony survival.

Pesticide Impacts on Other Beneficial Organisims

Worldwide decline of the entomofauna: A review of its drivers. (Sánchez-Bayo, F. and Wyckhuys, K.A., 2019).     Biodiversity of insects is threatened worldwide. Here, we present a comprehensive review of 73 historical reports of insect declines from across the globe, and systematically assess the underlying drivers. Our work reveals dramatic rates of decline that may lead to the extinction of 40% of the world's insect species over the next few decades. In terrestrial ecosystems, Lepidoptera, Hymenoptera and dung beetles (Coleoptera) appear to be the taxa most affected, whereas four major aquatic taxa (Odonata, Plecoptera, Trichoptera and Ephemeroptera) have already lost a considerable proportion of species. Affected insect groups not only include specialists that occupy particular ecological niches, but also many common and generalist species. Concurrently, the abundance of a small number of species is increasing; these are all adaptable, generalist species that are occupying the vacant niches left by the ones declining. Among aquatic insects, habitat and dietary generalists, and pollutant-tolerant species are replacing the large biodiversity losses experienced in waters within agricultural and urban settings. The main drivers of species declines appear to be in order of importance: i) habitat loss and conversion to intensive agriculture and urbanisation; ii) pollution, mainly that by synthetic pesticides and fertilisers; iii) biological factors, including pathogens and introduced species; and iv) climate change. The latter factor is particularly important in tropical regions, but only affects a minority of species in colder climes and mountain settings of temperate zones. A rethinking of current agricultural practices, in particular a serious reduction in pesticide usage and its substitution with more sustainable, ecologically-based practices, is urgently needed to slow or reverse current trends, allow the recovery of declining insect populations and safeguard the vital ecosystem services they provide. In addition, effective remediation technologies should be applied to clean polluted waters in both agricultural and urban environments.


Call to restrict neonicotinoids. (Goulson, D. et al. 2018). Neonicotinoids are the most widely used insecticides in the world, being applied to a broad range of food, energy and ornamental crops, and used in domestic pest control. They are neurotoxins with very high toxicity to insects, a group of organisms that contains the majority of the described life on Earth, and which includes numerous species of vital importance to humans such as pollinators and predators of pests. Neonicotinoids have proved to be highly persistent in the environment, such that significant residues are commonly found in soils, wildflowers, streams and lakes. For example, a recent study in the journal Science found neonicotinoids in 75% of honey samples collected from around the world. Hundreds of independent scientific studies have been performed to assess their impacts on beneficial organisms such as bees, aquatic insects, butterflies and predatory beetles.   It is the view of the undersigned scientists that the balance of evidence strongly suggests that these chemicals are harming beneficial insects and contributing to the current massive loss of global biodiversity. As such, there is an immediate need for national and international agreements to greatly restrict their use, and to prevent registration of similarly harmful agrochemicals in the future. Failure to respond urgently to this issue risks not only the continued decline in abundance and diversity of many beneficial insects but also the loss of the services they provide and a significant fraction of the biodiversity heritage of future generations.    

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.

Acute toxicity of 6 neonicotinoid insecticides to freshwater invertebrates.

Neonicotinoids are a group of insecticides commonly used in agriculture. Due to their high water solubility, neonicotinoids can be transported to surface waters and have the potential to be toxic to aquatic life. The present study assessed and compared the acute (48- or 96-h) toxicity of 6 neonicotinoids (acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, and thiamethoxam) to 21 laboratory-cultured and field-collected aquatic invertebrates spanning 10 aquatic arthropod orders. Test conditions mimicked species' habitat, with lentic taxa exposed under static conditions, and lotic taxa exposed under recirculating systems. Median lethal concentrations (LC50s) and median effect concentrations (EC50s; immobility) were calculated and used to construct separate lethal- and immobilization-derived species sensitivity distributions for each neonicotinoid, from which 5th percentile hazard concentrations (HC5s) were calculated. The results showed that the most sensitive invertebrates were insects from the orders Ephemeroptera (Neocloeon triangulifer) and Diptera (Chironomus dilutus), whereas cladocerans (Daphnia magna, Ceriodaphnia dubia) were the least sensitive. The HC5s were compared with neonicotinoid environmental concentrations from Ontario (Canada) monitoring studies. For all neonicotinoids except imidacloprid, the resulting hazard quotients indicated little to no hazard in terms of acute toxicity to aquatic communities in Ontario freshwater streams. For the neonicotinoid imidacloprid, a moderate hazard was found when only invertebrate immobilization, and not lethality, data were considered

Effects of chronic exposure to thiamethoxam on larvae of the hoverfly Eristalis tenax (Diptera, Syrphidae).
There is widespread concern over the use of neonicotinoid pesticides in the agro-ecosystem, due in part to their high water solubility which can lead to widespread contamination of non-target areas including standing surface water. Most studies investigating the negative fitness consequences of neonicotinoids have focused on bees, with little research on the impact on other non-target insects. Here we examined the effect of exposure on the aquatic larval stages of the hoverfly Eristalis tenax L. (Diptera: Syrphidae) to a range of concentrations (control, 5, 15, 50, 100 and 500 ppb) of the neonicotinoid thiamethoxam; no published studies have thus far examined the effects of neonicotinoids on hoverflies. Survival was significantly lower when exposed to 500 ppb thiamethoxam, but this concentration exceeds that likely to be found in the field. We observed no effect on survival, development or any latent effects on adult activity budgets resulting from exposure to lower concentrations (up to 100 ppb). Our results suggest that E. tenax exposed as larvae to thiamethoxam are unlikely to be negatively impacted by this neonicotinoid under field conditions.

Community-Level and Phenological Responses of Emerging Aquatic Insects Exposed To Three Neonicotinoid Insecticides: An In Situ Wetland Limnocorral Approach.
Seasonal aquatic insect emergence represents a critical subsidy link between aquatic and terrestrial ecosystems. Early and late instar larvae developing in wetlands near neonicotinoid-treated cropland are at risk of chronic insecticide exposure. An in situ wetland limnocorral experiment compared emergent insect community responses to imidacloprid, clothianidin, and thiamethoxam. Over 15 weeks, 21 limnocorrals were dosed weekly for 9 weeks to target peak nominal doses of 0.0, 0.05 or 0.5 µg/L, followed by a 6-week recovery period. Thirty-nine aquatic insect taxa were recorded but 11 taxa groups made up 97% of the community composition. Principal response curves indicated that during the dosing period, community composition among the treatments resembled the controls. During the 6-week recovery period, significant deviance was observed in the high imidacloprid treatment with similar trends in the clothianidin treatment, suggesting that community effects from neonicotinoid exposure can be delayed. Non-biting midges (Diptera: Chironomidae) and damselflies (Odonata: Zygoptera) also emerged 18 to 25 days earlier in the imidacloprid and clothianidin neonicotinoid treatments, relative to controls. These data suggest that phenology and subtle community effects can occur at measured neonicotinoid concentrations of 0.045 µg/L (imidacloprid) and 0.038 µg/L (clothianidin) under chronic repeated exposure conditions. Synchronization and community dynamics are critical to aquatic insects and consumers; thus, neonicotinoids may have broad implications for wetland ecosystem function.

Lethal and sublethal toxicity of neonicotinoid and butenolide insecticides to the mayfly, Hexagenia spp.
Neonicotinoid insecticides are environmentally persistent and highly water-soluble, and thus are prone to leaching into surface waters where they may negatively affect non-target aquatic insects. Most of the research to date has focused on imidacloprid, and few data are available regarding the effects of other neonicotinoids or their proposed replacements (butenolide insecticides). The objective of this study was to assess the toxicity of six neonicotinoids (imidacloprid, thiamethoxam, acetamiprid, clothianidin, thiacloprid, and dinotefuran) and one butenolide (flupyradifurone) to Hexagenia spp. (mayfly larvae). Acute (96-h), water-only tests were conducted, and survival and behaviour (number of surviving mayflies inhabiting artificial burrows) were assessed. Acute sublethal tests were also conducted with imidacloprid, acetamiprid, and thiacloprid, and in addition to survival and behaviour, mobility (ability to burrow into sediment) and recovery (survival and growth following 21 d in clean sediment) were measured. Sublethal effects occurred at much lower concentrations than survival: 96-h LC50s ranged from 780 μg/L (acetamiprid) to >10,000 μg/L (dinotefuran), whereas 96-h EC50s ranged from 4.0 μg/L (acetamiprid) to 630 μg/L (thiamethoxam). Flupyradifurone was intermediate in toxicity, with a 96-h LC50 of 2000 μg/L and a 96-h EC50 of 81 μg/L. Behaviour and mobility were impaired significantly and to a similar degree in sublethal exposures to 10 μg/L imidacloprid, acetamiprid, and thiacloprid, and survival and growth following the recovery period were significantly lower in mayflies exposed to 10 μg/L acetamiprid and thiacloprid, respectively. A suite of effects on mayfly swimming behaviour/ability and respiration were also observed, but not quantified, following exposures to imidacloprid, acetamiprid, and thiacloprid at 1 μg/L and higher. Imidacloprid concentrations measured in North American surface waters have been found to meet or exceed those causing toxicity to Hexagenia, indicating that environmental concentrations may adversely affect Hexagenia and similarly sensitive non-target aquatic species.

An update of the Worldwide Integrated Assessment (WIA) on systemic insecticides. Part 2: impacts on organisms and ecosystems.
New information on the lethal and sublethal effects of neonicotinoids and fipronil on organisms is presented in this review, complementing the previous Worldwide Integrated Assessment (WIA) in 2015. The high toxicity of these systemic insecticides to invertebrates has been confirmed and expanded to include more species and compounds. Most of the recent research has focused on bees and the sublethal and ecological impacts these insecticides have on pollinators. Toxic effects on other invertebrate taxa also covered predatory and parasitoid natural enemies and aquatic arthropods. Little new information has been gathered on soil organisms. The impact on marine and coastal ecosystems is still largely uncharted. The chronic lethality of neonicotinoids to insects and crustaceans, and the strengthened evidence that these chemicals also impair the immune system and reproduction, highlights the dangers of this particular insecticidal class (neonicotinoids and fipronil), with the potential to greatly decrease populations of arthropods in both terrestrial and aquatic environments. Sublethal effects on fish, reptiles, frogs, birds, and mammals are also reported, showing a better understanding of the mechanisms of toxicity of these insecticides in vertebrates and their deleterious impacts on growth, reproduction, and neurobehaviour of most of the species tested. This review concludes with a summary of impacts on the ecosystem services and functioning, particularly on pollination, soil biota, and aquatic invertebrate communities, thus reinforcing the previous WIA conclusions (van der Sluijs et al. 2015).

Response of the mayfly (Cloeon dipterum) to chronic exposure to thiamethoxam in outdoor mesocosms.
Thiamethoxam is a widely used neonicotinoid insecticide that has been detected in surface water monitoring programs in North America and Europe. This has led to questions about its toxicity to nontarget insects, specifically those with an aquatic life stage. To address the uncertainty associated with possible impacts from environmental exposures, a chronic (35-d) outdoor mesocosm study with a formulated product containing thiamethoxam was conducted. The specific focus of the study was the response of mayflies (Ephemeroptera), which have been reported to be particularly sensitive in laboratory studies. A range of concentrations (nominally 0.1, 0.3, 1.0, 3.0, and 10.0 µg/L thiamethoxam), plus untreated controls were tested, and the abundance and emergence of mayflies (Cloeon dipterum) were assessed weekly for 35 d. Mean measured time-weighted average exposures were within 6% of nominal over the duration of the study, with the mean half-life of thiamethoxam in each treatment ranging from 7 to 13 d. Statistically significant reductions in both larval abundance and adult emergence were observed at 10.0, 3.0, and 1.0 μg/L following 1, 2, and 3 wk of exposure, respectively. Exposure to 0.1 and 0.3 µg/L thiamethoxam had no statistically significant effect on larval mayfly abundance or adult emergence at any point in the study. These findings support a 35-d no-observed-effect concentration (NOEC) of 0.3 µg thiamethoxam/L for mayflies (C. dipterum) under chronic conditions. Furthermore, because the 95th percentile of environmental concentrations has been reported to be 0.054 µg/L, these results indicate that populations of C. dipterum and similarly sensitive aquatic insects are unlikely to be significantly impacted by thiamethoxam exposure in natural systems represented by the conditions in our study.

Comparative toxicity of imidacloprid and thiacloprid to different species of soil invertebrates
Neonicotinoid insecticides have come under increasing scrutiny for their impact on non-target organisms, especially pollinators. The current scientific literature is mainly focused on the impact of these insecticides on pollinators and some aquatic insects, leaving a knowledge gap concerning soil invertebrates. This study aimed at filling this gap, by determining the toxicity of imidacloprid and thiacloprid to five species of soil invertebrates: earthworms (Eisenia andrei), enchytraeids (Enchytraeus crypticus), Collembola (Folsomia candida), oribatid mites (Oppia nitens) and isopods (Porcellio scaber). Tests focused on survival and reproduction or growth, after 3-5 weeks exposure in natural LUFA 2.2 standard soil. Imidacloprid was more toxic than thiacloprid for all species tested. F. candida and E. andrei were the most sensitive species, with LC50s of 0.20-0.62 and 0.77 mg/kg dry soil for imidacloprid and 2.7-3.9 and 7.1 mg/kg dry soil for thiacloprid. EC50s for effects on the reproduction of F. candida and E. andrei were 0.097-0.30 and 0.39 mg/kg dry soil for imidacloprid and 1.7-2.4 and 0.44 mg/kg dry soil for thiacloprid. The least sensitive species were O. nitens and P. scaber. Enchytraeids were a factor of 5-40 less sensitive than the taxonomically related earthworm, depending on the endpoint considered. Although not all the species showed high sensitivity to the neonicotinoids tested, these results raise awareness about the effects these insecticides can have on non-target soil invertebrates.

Comparative ecotoxicity of imidacloprid and dinotefuran to aquatic insects in rice mesocosms.
There are growing concerns about the impacts of neonicotinoid insecticides on ecosystems worldwide, and yet ecotoxicity of many of these chemicals at community or ecosystem levels have not been evaluated under realistic conditions. In this study, effects of two neonicotinoid insecticides, imidacloprid and dinotefuran, on aquatic insect assemblages were evaluated in experimental rice mesocosms. During the 5-month period of the rice-growing season, residual concentrations of imidacloprid were 5-10 times higher than those of dinotefuran in both soil and water. Imidacloprid treatment (10kg/ha) reduced significantly the populations of, Crocothemis servilia mariannae and Lyriothemis pachygastra nymphs, whereas those of Orthetrum albistylum speciosum increased slightly throughout the experimental period. However, Notonecta triguttata, which numbers were high from the start, later declined, indicating possible delayed chronic toxicity, while Guignotus japonicus disappeared. In contrast, dinotefuran (10kg/ha) did not decrease the populations of any species, but rather increased the abundance of some insects, particularly Chironominae spp. larvae and C. servilia mariannae nymphs, with the latter being 1.7x higher than those of controls. This was an indirect effect resulting from increased prey (e.g., chironomid larvae) and lack of competition with other dragonfly species. The susceptibilities of dragonfly nymphs to neonicotinoids, particularly imidacloprid, were consistent with those reported elsewhere. In general, imidacloprid had higher impacts on aquatic insects compared to dinotefuran.

Effects of clothianidin on aquatic communities: Evaluating the impacts of lethal and sublethal exposure to neonicotinoids.
The widespread usage of neonicotinoid insecticides has sparked concern over their effects on non-target organisms. While research has largely focused on terrestrial systems, the low soil binding and high water solubility of neonicotinoids, paired with their extensive use on the landscape, puts aquatic environments at high risk for contamination via runoff events. We assessed the potential threat of these compounds to wetland communities using a combination of field surveys and experimental exposures including concentrations that are representative of what invertebrates experience in the field. In laboratory toxicity experiments, LC50 values ranged from 0.002 ppm to 1.2 ppm for aquatic invertebrates exposed to clothianidin. However, freshwater snails and amphibian larvae showed high tolerance to the chemical with no mortality observed at the highest dissolvable concentration of the insecticide. We also observed behavioral effects of clothianidin. Water bugs, Belostoma flumineum, displayed a dose-dependent reduction in feeding rate following exposure to clothianidin. Similarly, crayfish, Orconectes propinquus, exhibited reduced responsiveness to stimulus with increasing clothianidin concentration. Using a semi-natural mesocosm experiment, we manipulated clothianidin concentration (0.6, 5, and 352 ppb) and the presence of predatory invertebrates to explore community-level effects. We observed high invertebrate predator mortality with increases in clothianidin concentration. With increased predator mortality, prey survival increased by 50% at the highest clothianidin concentration. Thus, clothianidin contamination can result in a top-down trophic cascade in a community dominated by invertebrate predators. In our Indiana field study, we detected clothianidin (max = 176 ppb), imidacloprid (max = 141 ppb), and acetamiprid (max = 7 ppb) in soil samples. In water samples, we detected clothianidin (max = 0.67 ppb), imidacloprid (max = 0.18 ppb), and thiamethoxam (max = 2,568 ppb). Neonicotinoids were detected in >56% of soil samples and >90% of the water samples, which reflects a growing understanding that neonicotinoids are ubiquitous environmental contaminants. Collectively, our results underscore the need for additional research into the effects of neonicotinoids on aquatic communities and ecosystems.

Neonicotinoids thiamethoxam and clothianidin adversely affect the colonisation of invertebrate populations in aquatic microcosms.
Surface waters are sometimes contaminated with neonicotinoids: a widespread, persistent, systemic class of insecticide with leaching potential. Previous ecotoxicological investigations of this chemical class in aquatic ecosystems have largely focused on the impacts of the neonicotinoid imidacloprid; few empirical, manipulative studies have investigated the effect on invertebrate abundances of two other neonicotinoids which are now more widely used: clothianidin and thiamethoxam. In this study, we employ a simple microcosm semi-field design, incorporating a one-off contamination event, to investigate the effect of these pesticides at field-realistic levels (ranging from 0 to 15 ppb) on invertebrate colonisation and survival in small ephemeral ponds. In line with previous research on neonicotinoid impacts on aquatic invertebrates, significant negative effects of both neonicotinoids were found. There were clear differences between the two chemicals, with thiamethoxam generally producing stronger negative effects than clothianidin. Populations of Chironomids (Diptera) and Ostracoda were negatively affected by both chemicals, while Culicidae appeared to be unaffected by clothianidin at the doses used. Our data demonstrate that field-realistic concentrations of neonicotinoids are likely to reduce populations of invertebrates found in ephemeral ponds, which may have knock on effects up the food chain. We highlight the importance of developing pesticide monitoring schemes for European surface waters.

Comparative toxicity of imidacloprid and thiacloprid to different species of soil invertebrates.
Neonicotinoid insecticides have come under increasing scrutiny for their impact on non-target organisms, especially pollinators. The current scientific literature is mainly focused on the impact of these insecticides on pollinators and some aquatic insects, leaving a knowledge gap concerning soil invertebrates. This study aimed at filling this gap, by determining the toxicity of imidacloprid and thiacloprid to five species of soil invertebrates: earthworms (Eisenia andrei), enchytraeids (Enchytraeus crypticus), Collembola (Folsomia candida), oribatid mites (Oppia nitens) and isopods (Porcellio scaber). Tests focused on survival and reproduction or growth, after 3-5 weeks exposure in natural LUFA 2.2 standard soil. Imidacloprid was more toxic than thiacloprid for all species tested. F. candida and E. andrei were the most sensitive species, with LC50s of 0.20-0.62 and 0.77 mg/kg dry soil for imidacloprid and 2.7-3.9 and 7.1 mg/kg dry soil for thiacloprid. EC50s for effects on the reproduction of F. candida and E. andrei were 0.097-0.30 and 0.39 mg/kg dry soil for imidacloprid and 1.7-2.4 and 0.44 mg/kg dry soil for thiacloprid. The least sensitive species were O. nitens and P. scaber. Enchytraeids were a factor of 5-40 less sensitive than the taxonomically related earthworm, depending on the endpoint considered. Although not all the species showed high sensitivity to the neonicotinoids tested, these results raise awareness about the effects these insecticides can have on non-target soil invertebrates.

Sensitivity of the early-life stages of freshwater mollusks to neonicotinoid and butenolide insecticides.
Neonicotinoid insecticides can be transported from agricultural fields, where they are used as foliar sprays or seed treatments, to surface waters by surface or sub-surface runoff. Few studies have investigated the toxicity of neonicotinoid or the related butenolide insecticides to freshwater mollusk species. The current study examined the effect of neonicotinoid and butenolide exposures to the early-life stages of the ramshorn snail, Planorbella pilsbryi, and the wavy-rayed lampmussel, Lampsilis fasciola. Juvenile P. pilsbryi were exposed to imidacloprid, clothianidin, or thiamethoxam for 7 or 28 d and mortality, growth, and biomass production were measured. The viability of larval (glochidia) L. fasciola was monitored during a 48 h exposure to six neonicotinoids (imidacloprid, thiamethoxam, clothianidin, acetamiprid, thiacloprid, or dinotefuran), or a butenolide (flupyradifurone). The 7-d LC50s of P. pilsbryi for imidacloprid, clothianidin, and thiamethoxam were ≥4000 μg/L and the 28-d LC50s were ≥182 μg/L. Growth and biomass production were considerably more sensitive endpoints than mortality with EC50s ranging from 33.2 to 122.0 μg/L. The 48-h LC50s for the viability of glochidia were ≥456 μg/L for all seven insecticides tested. Our data indicate that neonicotinoid and butenolide insecticides pose less of a hazard with respect to mortality of the two species of mollusk compared to the potential hazard to other non-target aquatic insects.

Pesticide seed dressings can affect the activity of various soil organisms and reduce decomposition of plant material

Seed dressing with pesticides is widely used to protect crop seeds from pest insects and fungal diseases. While there is mounting evidence that especially neonicotinoid seed dressings detrimentally affect insect pollinators, surprisingly little is known on potential side effects on soil biota. We hypothesized that soil organisms would be particularly susceptible to pesticide seed dressings as they get in direct contact with these chemicals. Using microcosms with field soil we investigated, whether seeds treated either with neonicotinoid insecticides or fungicides influence the activity and interaction of earthworms, collembola, protozoa and microorganisms. The full-factorial design consisted of the factor Seed dressing (control vs. insecticide vs. fungicide), Earthworm (no earthworms vs. addition Lumbricus terrestris L.) and collembola (no collembola vs. addition Sinella curviseta Brook). We used commercially available wheat seed material (Triticum aesticum L. cf. Lukullus) at a recommended seeding density of 367 m(-2). Seed dressings (particularly fungicides) increased collembola surface activity, increased the number of protozoa and reduced plant decomposition rate but did not affect earthworm activity. Seed dressings had no influence on wheat growth. Earthworms interactively affected the influence of seed dressings on collembola activity, whereas collembola increased earthworm surface activity but reduced soil basal respiration. Earthworms also decreased wheat growth, reduced soil basal respiration and microbial biomass but increased soil water content and electrical conductivity. The reported non-target effects of seed dressings and their interactions with soil organisms are remarkable because they were observed after a one-time application of only 18 pesticide treated seeds per experimental pot. Because of the increasing use of seed dressing in agriculture and the fundamental role of soil organisms in agroecosystems these ecological interactions should receive more attention.

Contamination of wild plants near neonicotinoid seed-treated crops, and implications for non-target insects

Neonicotinoid insecticides are commonly-used as seed treatments on flowering crops such as oilseed rape. Their persistence and solubility in water increase the chances of environmental contamination via surface-runoff or drainage into areas adjacent to the crops. However, their uptake and fate into non-target vegetation remains poorly understood. In this study, we analysed samples of foliage collected from neonicotinoid seed-treated oilseed rape plants and also compared the levels of neonicotinoid residues in foliage (range: 1.4-11ng/g) with the levels found in pollen collected from the same plants (range: 1.4-22ng/g). We then analysed residue levels in foliage from non-target plants growing in the crop field margins (range: ≤0.02-106ng/g). Finally, in order to assess the possible risk posed by the peak levels of neonicotinoids that we detected in foliage for farmland phytophagous and predatory insects, we compared the maximum concentrations found against the LC50 values reported in the literature for a set of relevant insect species. Our results suggest that neonicotinoid seed-dressings lead to widespread contamination of the foliage of field margin plants with mixtures of neonicotinoid residues, where levels are very variable and discontinuous, but sometimes overlap with lethal concentrations reported for some insect species. Understanding the distribution of pesticides in the environment and their potential effects on biological communities is crucial to properly assess current agricultural management and schemes with biodiversity conservation aims in farmland.

Acute and chronic toxicity of neonicotinoids to nymphs of a mayfly species and some notes on seasonal differences.
Mayfly nymphs are among the most sensitive taxa to neonicotinoids. The present study presents the acute and chronic toxicity of 3 neonicotinoids (imidacloprid, thiacloprid, and thiamethoxam) to a mayfly species (Cloeon dipterum) and some notes on the seasonality of the toxicity of imidacloprid to C. dipterum and 5 other invertebrate species. Imidacloprid and thiamethoxam showed equal acute and chronic toxicity to a winter generation of C. dipterum, whereas thiacloprid was approximately twice as toxic. The acute and chronic toxicity of imidacloprid was much higher for the C. dipterum summer generation than for the winter one. The acute toxicity differs by a factor of 20 for the 96-h 50% effective concentration (EC50) and by a factor of 5.4 for the chronic 28-d EC50. Temperature had only a slight effect on the sensitivity of C. dipterum to imidacloprid because we only found a factor of 1.7 difference in the 96-h EC50 between tests performed at 10 °C and 18 °C. The difference in sensitivity between summer and overwintering generations was also found for 3 other insect species. The results indicate that if the use and environmental fate of the 3 neonicotinoids are comparable, replacing imidacloprid by another neonicotinoid might not reduce the environmental impact on the mayfly nymph C. dipterum. The results also show the importance of reporting which generation is tested because sensitivity values of insects in the summer might be underestimated by the experiments performed with neonicotinoids and an overwintering population.

Neonicotinoids in the Canadian aquatic environment: a literature review on current use products with a focus on fate, exposure, and biological effects.
Developed to replace organophosphate and carbamate insecticides, neonicotinoids are structurally similar to nicotine. The three main neonicotinoid insecticides, imidacloprid, clothianidin, and thiamethoxam, are being re-evaluated by Health Canada's Pest Management Regulatory Agency (PMRA). An important aspect of the re-evaluation is the potential for effects in non-target organisms, including aquatic organisms. Leaching into surface waters is one of the major concerns surrounding extensive use of neonicotinoids, especially in close proximity to water bodies. The PMRA has classified IMI as 'persistent' with a 'high' leaching potential. Globally, neonicotinoids have been detected in a variety of water bodies, typically at concentrations in the low μg/L range. While IMI has been included in some monitoring exercises, there are currently very few published data for the presence of CLO and THM in Canadian water bodies. The majority of neonicotinoid toxicity studies have been conducted with IMI due to its longer presence on the market and high prevalence of use. Aquatic insects are particularly vulnerable to neonicotinoids and chronic toxicity has been observed at concentrations of IMI below 1 μg/L. Acute toxicity has been reported at concentrations below 20 μg/L for the most sensitive species, including Hyalella azteca, ostracods, and Chironomus riparius. Fish, algae, amphibians, and molluscs are relatively insensitive to IMI. However, the biological effects of THM and CLO have not been as well explored. The Canadian interim water quality guideline for IMI is 0.23 μg/L, but there is currently insufficient use, fate, and toxicological information available to establish guidelines for CLO and THM. Based on concentrations of neonicotinoids reported in surface waters in Canada and globally, there is potential for aquatic invertebrates to be negatively impacted by neonicotinoids. Therefore, it is necessary to address knowledge gaps to inform decisions around guidelines and registration status for neonicotinoid insecticides in Canada to protect our aquatic ecosystems.

Increasing neonicotinoid use and the declining butterfly fauna of lowland California

The butterfly fauna of lowland Northern California has exhibited a marked decline in recent years that previous studies have attributed in part to altered climatic conditions and changes in land use. Here, we ask if a shift in insecticide use towards neonicotinoids is associated with butterfly declines at four sites in the region that have been monitored for four decades. A negative association between butterfly populations and increasing neonicotinoid application is detectable while controlling for land use and other factors, and appears to be more severe for smaller-bodied species. These results suggest that neonicotinoids could influence non-target insect populations occurring in proximity to application locations, and highlights the need for mechanistic work to complement long-term observational data.

Neonicotinoid insecticide travels through a soil food chain, disrupting biological control of non-target pests and decreasing soya bean yield

In this study, published in Journal of Applied Ecology, researchers investigated in laboratory and field studies the influence of the neonicotinoid thiamethoxam, applied as a coating to soya bean seeds, on interactions among soya beans, nontarget molluscan herbivores and their insect predators. In the laboratory, the pest slug Deroceras reticulatum was unaffected by thiamethoxam, but transmitted the toxin to predaceous beetles (Chlaenius tricolor), impairing or killing >60%. In the field, thiamethoxam-based seed treatments depressed activity–density of arthropod predators, thereby relaxing predation of slugs and reducing soya bean densities by 19% and yield by 5%. 5. Neonicotinoid residue analyses revealed that insecticide concentrations declined through the food chain, but levels in field-collected slugs (up to 500 ng g-1 ) were still high enough to harm insect predators. These findings reveal a previously unconsidered ecological pathway through which neonicotinoid use can unintentionally reduce biological control and crop yield. Trophic transfer of neonicotinoids challenges the notion that seed-applied toxins precisely target herbivorous pests and highlights the need to consider predatory arthropods and soil communities in neonicotinoid risk assessment and stewardship.

Effects of neonicotinoids and fipronil on non-target invertebrates.

For terrestrial and aquatic invertebrate species, the known effects of neonicotinoid pesticides and fipronil are described ranging from organismal toxicology and behavioural effects to population-level effects. For earthworms, freshwater and marine species, the relation of findings to regulatory risk assessment is described. Neonicotinoid insecticides exhibit very high toxicity to a wide range of invertebrates, particularly insects, and field-realistic exposure is likely to result in both lethal and a broad range of important sublethal impacts. There is a major knowledge gap regarding impacts on the grand majority of invertebrates, many of which perform essential roles enabling healthy ecosystem functioning. The data on the few non-target species on which field tests have been performed are limited by major flaws in the outdated test protocols. Despite large knowledge gaps and uncertainties, enough knowledge exists to conclude that existing levels of pollution with neonicotinoids and fipronil resulting from presently authorized uses frequently exceed the lowest observed adverse effect concentrations and are thus likely to have large-scale and wide ranging negative biological and ecological impacts on a wide range of non-target invertebrates in terrestrial, aquatic, marine and benthic habitats.

Non-target effects of commonly used plant protection products in roses on the predatory mite Euseius gallicus Kreiter & Tixier (Acari: Phytoseidae)

Euseius gallicus Kreiter & Tixier (Acari: Phytoseidae) is a predatory mite recently available for use against various pests in roses. Study tested in greenhouse trials the impact on the numbers of eggs and motiles of E. gallicus of the most commonly used plant protection products in roses in northern Europe: the acaricides acequinocyl and etoxazole, the insecticides azadirachtin-A, acetamiprid, flonicamid, imidacloprid, indoxacarb, thiacloprid and thiamethoxam and the fungicides boscalid and kresoxim-methyl, cyprodinil + fludioxonil, dodemorph and fluopyram + tebuconazole.The neonicotinoids thiacloprid, thiamethoxam, acetamiprid and imidacloprid had a negative impact on the number of eggs and number of motiles of E. gallicus and were classified as slightly to moderately toxic. Azadirachtin-A, acetamiprid, flonicamid, boscalid and kresoxim-methyl, cyprodinil + fludioxonil, dodemorph and fluopyram + tebuconazole were harmless for E. gallicus. Special attention should be paid to the impact of neonicotinoids and of acequinocyl and etoxazole, and to the application frequency with flonicamid on E. gallicus. 

Insecticide Toxicity to Adelphocoris lineolatus (Hemiptera: Miridae) and Its Nymphal Parasitoid Peristenus spretus (Hymenoptera: Braconidae)

In China, Adelphocoris lineolatus (Goeze) (Hemiptera: Miridae) is an important pest of alfalfa, cotton, and other crops, while Peristenus spretus (Chen & van Achterberg) (Hymenoptera: Braconidae) is the dominant nymphal parasitoid of this mirid bug. In this study, published in the Journal of Economic Entomology, the toxicity of 17 common insecticides to A. lineolatus was evaluated, and the susceptibility of P. spretus to the insecticides with high toxicity to A. lineolatus was tested under laboratory conditions. Of the 17 insecticides tested, 12 (beta cypermethrin, deltamethrin, carbosulfan, acetamiprid, emamectin benzoate, imidacloprid, phoxim, chlorpyrifos, acephate, profenophos, hexaflumuron, and abamectin) had a highly toxic effect on second-instar nymphs of A. lineolatus, with LC50 values ranging from 0.58 to 14.85 mg a.i. (active ingredient) liter-1. Adults of P. spretus were most sensitive to chlorpyrifos, with LC50 values of 0.03 mg a.i. liter-1, followed by phoxim, acetamiprid, profenophos, carbosulfan, acephate, deltamethrin, emamectin benzoate, imidacloprid, beta-cypermethrin, and abamectin, with LC50 values ranging from 0.06 to 3.09, whereas hexaflumuron exhibited the least toxicity to the parasitoid, with LC50 values >500 mg a.i. liter-1. A risk quotient analysis indicated that beta-cypermethrin, emamectin benzoate, abamectin, and hexaflumuron when applied against A. lineolatus were the least toxic to P. spretus.

Combination effects of pyrethroids and neonicotinoids on development and survival of Chironomus riparius

Standard ecotoxicological risk assessments are conducted on individual substances, however monitoring of streams in agricultural areas has shown that pesticides are rarely present alone. Study investigates the potential risks to non-target aquatic organisms exposed to a brief but intense mixture of the neonicotinoid pesticides imidacloprid and thiacloprid and the pyrethroid pesticides deltamethrin and esfenvalerate, compared to single substance exposure. All four of these pesticides have been detected in surface waters at concentrations higher than benchmark values and both classes of pesticides are known to exert adverse effects on non-target aquatic organisms under single substance exposure scenarios. First instar midge larvae of the non-target aquatic organism, Chironomus riparius, were exposed to combinations of these four pesticides at 50% of their LC50 (96 h) values in a 1h pulse. They were then reared to adulthood in uncontaminated conditions and assessed for survival, development time and fecundity. Results show that the risk of disruption to survival and development of non-target aquatic organisms under this scenario is not negligible on account of the significant increases in mortality of C. riparius found in the majority of the pesticide exposures and the delays in development after pyrethroid exposure. While none of the deleterious effects appear to be amplified by combination of the pesticides, there is some evidence for antagonism. 

Declines in insectivorous birds are associated with high neonicotinoid concentrations

In this study, published in Nature, investigated the hypothesis that the most widely used neonicotinoid insecticide, imidacloprid, has a negative impact on insectivorous bird populations. Here researchers show that, in the Netherlands, local population trends were significantly more negative in areas with higher surface-water concentrations of imidacloprid. At imidacloprid concentrations of more than 20 nanograms per litre, bird populations tended to decline by 3.5 per cent on average annually. Additional analyses revealed that this spatial pattern of decline appeared only after the introduction of imidacloprid to the Netherlands, in the mid-1990s. Researchers further show that the recent negative relationship remains after correcting for spatial differences in land-use changes that are known to affect bird populations in farmland. The results suggest that the impact of neonicotinoids on the natural environment is even more substantial than has recently been reported and is reminiscent of the effects of persistent insecticides in the past. Future legislation should take into account the potential cascading effects of neonicotinoids on ecosystems.


A review of the direct and indirect effects of neonicotinoids and fipronil on vertebrate wildlife.
Concerns over the role of pesticides affecting vertebrate wildlife populations have recently focussed on systemic products which exert broad-spectrum toxicity. Given that the neonicotinoids have become the fastest-growing class of insecticides globally, we review here 150 studies of their direct (toxic) and indirect (e.g. food chain) effects on vertebrate wildlife--mammals, birds, fish, amphibians and reptiles. We focus on two neonicotinoids, imidacloprid and clothianidin, and a third insecticide, fipronil, which also acts in the same systemic manner. Imidacloprid and fipronil were found to be toxic to many birds and most fish, respectively. All three insecticides exert sub-lethal effects, ranging from genotoxic and cytotoxic effects, and impaired immune function, to reduced growth and reproductive success, often at concentrations well below those associated with mortality. Use of imidacloprid and clothianidin as seed treatments on some crops poses risks to small birds, and ingestion of even a few treated seeds could cause mortality or reproductive impairment to sensitive bird species. In contrast, environmental concentrations of imidacloprid and clothianidin appear to be at levels below those which will cause mortality to freshwater vertebrates, although sub-lethal effects may occur. Some recorded environmental concentrations of fipronil, however, may be sufficiently high to harm fish. Indirect effects are rarely considered in risk assessment processes and there is a paucity of data, despite the potential to exert population-level effects. Our research revealed two field case studies of indirect effects. In one, reductions in invertebrate prey from both imidacloprid and fipronil uses led to impaired growth in a fish species, and in another, reductions in populations in two lizard species were linked to effects of fipronil on termite prey. Evidence presented here suggests that the systemic insecticides, neonicotinoids and fipronil, are capable of exerting direct and indirect effects on terrestrial and aquatic vertebrate wildlife, thus warranting further review of their environmental safety.

Risks of large-scale use of systemic insecticides to ecosystem functioning and services

In this 2015 review, published in Environmental Science and Research, researchers look over the state of knowledge regarding the potential impacts of these insecticides on ecosystem functioning and services provided by terrestrial and aquatic ecosystems including soil and freshwater functions, fisheries, biological pest control, and pollination services. While empirical studies examining the specific impacts of neonicotinoids and fipronil to ecosystem services have focused largely on the negative impacts to beneficial insect species (honeybees) and the impact on pollination service of food crops, these researchers document broader evidence of the effects on ecosystem functions regulating soil and water quality, pest control, pollination, ecosystem resilience, and community diversity. In particular, microbes, invertebrates, and fish play critical roles as decomposers, pollinators, consumers, and predators, which collectively maintain healthy communities and ecosystem integrity. Several examples in this review demonstrate evidence of the negative impacts of systemic insecticides on decomposition, nutrient cycling, soil respiration, and invertebrate populations valued by humans. Invertebrates, particularly earthworms that are important for soil processes, wild and domestic insect pollinators which are important for plant and crop production, and several freshwater taxa which are involved in aquatic nutrient cycling, were all found to be highly susceptible to lethal and sublethal effects of neonicotinoids and/or fipronil at environmentally relevant concentrations. By contrast, most microbes and fish do not appear to be as sensitive under normal exposure scenarios, though the effects on fish may be important in certain realms such as combined fish-rice farming systems and through food chain effects. The researchers highlight the economic and cultural concerns around agriculture and aquaculture production and the role these insecticides may have in threatening food security. Overall, they recommend improved sustainable agricultural practices that restrict systemic insecticide use to maintain and support several ecosystem services that humans fundamentally depend on.

Neonicotinoid insecticides inhibit cholinergic neurotransmission in a molluscan (Lymnaea stagnalis) nervous system

Neonicotinoids are highly potent and selective systemic insecticides, but their widespread use also has a growing impact on non-target animals and contaminates the environment, including surface waters. Study tested the neonicotinoid insecticides commercially available in Hungary (acetamiprid, Mospilan; imidacloprid, Kohinor; thiamethoxam, Actara; clothianidin, Apacs; thiacloprid, Calypso) on cholinergic synapses that exist between the VD4 and RPeD1 neurons in the central nervous system of the pond snail Lymnaea stagnalis. In the concentration range used (0.01-1 mg/ml), neither chemical acted as an acetylcholine (ACh) agonist; instead, both displayed antagonist activity, inhibiting the cholinergic excitatory components of the VD4-RPeD1 connection. Thiacloprid (0.01 mg/ml) blocked almost 90% of excitatory postsynaptic potentials (EPSPs), while the less effective thiamethoxam (0.1 mg/ml) reduced the synaptic responses by about 15%. The ACh-evoked membrane responses of the RPeD1 neuron were similarly inhibited by the neonicotinoids, confirming that the same ACh receptor (AChR) target was involved. Authors conclude that neonicotinoids act on nicotinergic acetylcholine receptors (nAChRs) in the snail CNS. This has been established previously in the insect CNS; however, this data indicate differences in the background mechanism or the nAChR binding site in the snail. Study provide the first results concerning neonicotinoid-related toxic effects on the neuronal connections in the molluscan nervous system.

Sex allocation theory reveals a hidden cost of neonicotinoid exposure in a parasitoid wasp

This study demonstrates for the first time to our knowledge, that neonicotinoids disrupt the crucial reproductive behaviour of facultative sex allocation at sub-lethal, field-relevant doses in the parasitoid wasp Nasonia vitripennis.Female N. vitripennis allocate the sex of their offspring in line with Local Mate Competition (LMC) theory. Females exposed to neonicotinoids are less able to allocate sex optimally and that this failure imposes a significant fitness cost. This work highlights that understanding the ecological consequences of neonicotinoid deployment requires not just measures of mortality or even fecundity reduction among non-target species, but also measures that capture broader fitness costs, in this case offspring sex allocation. Our work also highlights new avenues for exploring how females obtain information when allocating sex under LMC.

Neonicotinoid contamination of global surface waters and associated risk to aquatic invertebrates: A review

In this 2015 review, published in Environmental International, scientists synthesized the current state of knowledge on the reported concentrations of neonicotinoids in surface waters from 29 studies in 9 countries world-wide in tandem with published data on their acute and chronic toxicity to 49 species of aquatic insects and crustaceans spanning 12 invertebrate orders. Strong evidence exists that water-borne neonicotinoid exposures are frequent, long-term and at levels which commonly exceed several existing water quality guidelines. Imidacloprid is by far the most widely studied neonicotinoid (66% of the 214 toxicity tests reviewed) with differences in sensitivity among aquatic invertebrate species ranging several orders of magnitude; other neonicotinoids display analogous modes of action and similar toxicities, although comparative data are limited. Overall, neonicotinoids can exert adverse effects on survival, growth, emergence, mobility, and behavior of many sensitive aquatic invertebrate taxa at concentrations at or below 1μg/L under acute exposure and 0.1μg/L for chronic exposure. Using probabilistic approaches (species sensitivity distributions), researchers recommend here that ecological thresholds for neonicotinoid water concentrations need to be below 0.2μg/L (short-term acute) or 0.035μg/L (long-term chronic) to avoid lasting effects on aquatic invertebrate communities. The application of safety factors may still be warranted considering potential issues of slow recovery, additive or synergistic effects and multiple stressors that can occur in the field. Our analysis revealed that 81% (22/27) and 74% (14/19) of global surface water studies reporting maximum and average individual neonicotinoid concentrations respectively, exceeded these thresholds of 0.2 and 0.035μg/L. Therefore, it appears that environmentally relevant concentrations of neonicotinoids in surface waters worldwide are well within the range where both short- and long-term impacts on aquatic invertebrate species are possible over broad spatial scales.

Neonicotinoid concentrations in arable soils after seed treatment applications in preceding years

Concentrations of the neonicotinoid insecticides clothianidin, thiamethoxam and imidacloprid were determined in arable soils from a variety of locations in England. In soil samples taken from the central area of fields, concentrations of clothianidin ranged from 0.02 to 13.6 µg kg(-1). Thiamethoxam concentrations were between <0.02 and 1.50 µg kg(-1), and imidacloprid concentrations between <0.09 and 10.7 µg kg(-1) . Concentrations of clothianidin and thiamethoxam were lower in soil samples taken from the edges of fields than from the centres of fields, but this difference was less pronounced for imidacloprid.This work gives a clear indication of the levels of neonicotinoids in arable soils after typical use of these compounds as seed dressings in the United Kingdom. There was evidence that imidacloprid was more persistent in the soils studied than clothianidin and thiamethoxam. As clothianidin and thiamethoxam have largely superseded imidacloprid in the United Kingdom, neonicotinoid levels were lower than suggested by predictions based on imidacloprid alone.

Pyrethroid pesticide effects on behavioral responses of aquatic isopods to danger cues

The 2014 study, published in Environmental Science and Pollution Research, sought to evaluate the behavioral responses of non-target organisms in order to determine whether phototactic responses of isopods to danger cues are altered as a function of exposure to the pyrethroid pesticides λ-cyhalothrin and bifenthrin. Experiments conducted on Gnorimosphaeroma oregonensisidentified sublethal behavioral responses to pyrethroids, λ-cyhalothrin and bifenthrin. Experimental setup tested isopod phototactic responses across six treatments: control, pyrethroid, hemolymph, predator, hemolymph + pyrethroid, and predator + pyrethroid. Isopods exhibited no preference for phototactic responses in the control and pyrethroid treatments. When exposed to danger cues (hemolymph or predator), isopods exhibited significant negative phototaxis, as expected. When exposure to danger cues was combined with pyrethroids, isopods again exhibited no preference for phototactic response. Experiments indicate that pyrethroids diminish isopod’s negatively phototactic response to danger cues.

Impacts of a neonicotinoid, neonicotinoid–pyrethroid premix, and anthranilic diamide insecticide on four species of turf-inhabiting beneficial insects

In this 2014 study, published in Ecotoxicology, researchers compared the impact of a neonicotinoid (clothianidin), a premix (clothianidin + bifenthrin), and an anthranilic diamide (chlorantraniliprole), the main insecticide classes used for multiple targeting, on four species of beneficial insects: Harpalus pennsylvanicus, an omnivorous ground beetle, Tiphia vernalis, an ectoparasitoid of scarab grubs, Copidosoma bakeri, a polyembryonic endoparasitoid of black cutworms, and Bombus impatiens, a native bumble bee. Ground beetles that ingested food treated with clothianidin or the premix suffered high mortality, as did C. bakeri wasps exposed to dry residues of those insecticides. Exposure to those insecticides on potted turf cores reduced parasitism by T. vernalis. Bumble bee colonies confined to forage on white clover (Trifolium repens L.) in weedy turf that had been treated with clothianidin or the premix had reduced numbers of workers, honey pots, and immature bees. Premix residues incapacitated H. pennsylvanicus and C. bakeri slightly faster than clothianidin alone, but otherwise we detected no synergistic or additive effects. Chlorantraniliprole had no apparent adverse effects on any of the beneficial species. Implications for controlling turf pests with least disruption of non-target invertebrates are discussed.

Lethal and behavioral effects of pesticides on the insect predator Macrolophus pygmaeus

The 2014 study published in Chemosphere examines the lethal and sublethal effects of six insecticides and a fungicide on the beneficial insect Macrolophus pygmaeus. They found “Thiacloprid and metaflumizone caused 100% and 80% mortality, respectively, and were classified as harmful. Indoxacarb and spinosad resulted in close to 30% mortality to the predator, and were classified as slightly harmful, while the fungicide copper hydroxide caused 58% mortality and was rated as moderately harmful. Chlorantraniliprole and thiacloprid were selected for further sublethal testing by exposing M. pygmaeus to two routes of pesticide intake: pesticide residues and feeding on sprayed food. Thiacloprid led to an increase in resting and preening time of the predator, and a decrease in plant feeding. Chlorantraniliprole resulted in a decrease in plant feeding, but no other behaviors were affected.”

Impacts of orchard pesticides on Galendromus occidentalis: Lethal and sublethal effects

This 2014 study published in Crop Protection tests the lethal and sublethal effects of fifteen pesticides on the principal mite predator in Washington apple orchards.Their studies provide support for the impact of sublethal effects of “reduced-risk pesticides” on beneficial species. “At the 1_ dose, only spinetoram and lambda-cyhalothrin caused >75% acute mortality of females. Carbaryl, azinphos methyl, spinosad, spirotetramat, cyantraniliprole, and sulfur had relatively little effect on mortality, but moderate to high effects on fecundity. Egg viability was most affected by carbaryl, spinosad, novaluron, spirotetramat, and sulfur. Lambda-cyhalothrin, spinosad, and sulfur were the most toxic compounds to larvae. Materials such as sulfur and spinetoram had widely divergent toxicity to adults versus larvae. The cumulative impact of these effects was best integrated by the numbers of live larvae of the F1 generation. Using this measurement, spirotetramat, sulfur, spinetoram, acetamiprid, lambda-cyhalothrin, carbaryl and novaluron caused the greatest percentage reduction compared to the check, yet only spinetoram and lambda-cyhalothrin would have been identified as harmful in acute bioassays.”

The impact of insecticides applied in apple orchards on the predatory mite Kampimodromus aberrans (Acari: Phytoseiidae)

This 2013 study, published in Experimental and Applied Acarology, examines the side effects of insecticides on a predatory mite in apple orchards, finding that they did not in fact diminish their populations as intended. “Spider mite (Panonychus ulmi) populations reached higher densities on plots treated with etofenprox and tau-fluvalinate than in the other treatments. Single or multiple applications of neonicotinoids caused no detrimental effects on predatory mites. In the laboratory, spinosad and tau-fluvalinate caused 100 % mortality. Etofenprox caused a significant mortality and reduced fecundity. The remaining insecticides did not affect female survival except for imidacloprid. Thiamethoxam, clothianidin, thiacloprid, chlorpyrifos, lufenuron and methoxyfenozide were associated with a significant reduction in fecundity. No effect on fecundity was found for indoxacarb or acetamiprid. Escape rate of K. aberrans in laboratory was relatively high for etofenprox and spinosad, and to a lesser extent thiacloprid. The use of etofenprox, tau-fluvalinate and spinosad was detrimental for K. aberrans and the first two insecticides induced spider mite population increases. The remaining insecticides caused no negative effects on predatory mites in field trials.”

Imidacloprid affects the functional response of predator Podisus nigrispinus (Dallas) (Heteroptera: Pentatomidae) to strains ofSpodoptera frugiperda (J.E. Smith) on Bt cotto

This 2013 study examines the impact of imidacloprid on an important biological control agent for many crops in South and Central America. The species, Podisus nigrispinus feeds on several pests including Spodoptera frugiperda.Researchers found that although the pest S. frugiperda became more aggressive, the beneficial insect P. nigrispinus showed significantly lower predation rates when exposed to imidacloprid.

Effect of imidacloprid on the biochemical contents of kidneys in male Swiss albino mice

This 2013 study published in The Bioscan examines mice that were orally administered varied doses of imidacloprid to determine their effects.” Kidney is the target organ for a wide variety of toxic agents as it acts as a blood filter during the excretory process. As kidneys receive high blood flow, the insecticides might be delivered to these organs in relatively high amounts through systemic (blood) circulation.” The study found that “The present findings on the toxicity of imidacloprid in mice suggest significant decrease in the level of protein, DNA and RNA in kidneys. Though six groups of the experimental mice were treated with varied concentrations of imidacloprid, the decrease of protein, DNA and RNA was not dose dependent in any of the 3 test organs.”

Detection and analysis of neonicotinoids in river waters – Development of a passive sampler for three commonly used insecticides

This 2013 study published in Chemosphere examined a new method to detect and analyze five of the most common neonicotinoid compounds in rivers around Sydney, Australia, where 93% of samples contained two or more neonicotinoids. “As a consequence of their high water solubility and persistence in soil they pose a risk of water contamination, particularly after storm events that produce runoff pulses and by leaching to the groundwater.”

Trace level determination of pyrethroid and neonicotinoid insecticides in beebread using acetonitrile-based extraction followed by analysis with ultra-high-performance liquid chromatography–tandem mass spectrometry

This 2013 study is an analysis of 32 samples of beebread from various regions in France to discover contamination levels of pyrethroids and neonicotinoids. The study detected 14 target pesticides and metabolites at sublethal levels, while 7 target substances were detected in beebread samples collected from hives. The study found that “The most frequently detected pesticides belonged to the neonicotinoid family and were generally present at low concentrations, but in some cases exceeded 170ng/g (acetamiprid and thiacloprid). Some pyrethroids were also detected (lambda-cyhalothrine and bifenthrine), but at very low levels.”

Pesticides reduce regional biodiversity of stream invertebrates

This 2013 study, published in Proceedings of the National Academy of Sciences "analyzed the effects of pesticides on the regional taxa richness of stream invertebrates in Europe (Germany and France) and Australia (southern Victoria). Pesticides caused statistically significant effects on both the species and family richness in both regions, with losses in taxa up to 42% of the recorded taxonomic pools. Furthermore, the effects in Europe were detected at concentrations that current legislation considers environmentally protective. Thus, the current ecological risk assessment of pesticides falls short of protecting biodiversity, and new approaches linking ecology and ecotoxicology are needed."

An overview of the environmental risks posed by neonicotinoid insecticides

This 2013 study, published in the Journal of Applied Ecology, determines that neonicotinoid pesticides have broad ranging negative impacts not only on beneficial pollinators, but on overall biodiversity and ecosystem health. "They are water soluble and prone to leaching into waterways. Being systemic, they are found in nectar and pollen of treated crops. Reported levels in soils, waterways, field margin plants and floral resources overlap substantially with concentrations that are sufficient to control pests in crops, and commonly exceed the LC50 (the concentration which kills 50% of individuals) for beneficial organisms. Concentrations in nectar and pollen in crops are sufficient to impact substantially on colony reproduction in bumblebees. Although vertebrates are less susceptible than arthropods, consumption of small numbers of dressed seeds offers a route to direct mortality in birds and mammals... Major knowledge gaps remain, but current use of neonicotinoids is likely to be impacting on a broad range of non-target taxa including pollinators and soil and aquatic invertebrates and hence threatens a range of ecosystem services."

Wildlife ecotoxicology of pesticides: can we track effects to the population level and beyond?

This 2013 study published in Science, examines the role of pesticides, including bee-killing pesticides, on wildlife. "One of the major challenges in wildlife ecotoxicology... is to trace the effects and side effects of chemicals, from their cellular targets through levels of increasing complexity to communities of species and the function of ecosystems. Here we provide an integrated view of the existing knowledge regarding pesticides of the past and present. This includes synthetic chemicals and biological compounds applied in agriculture..."

Reproductive Effects of Two Neonicotinoid Insecticides on Mouse Sperm Function and Early Embryonic Development In Vitro

This 2013 study examined the role of two neonicotinoids, Acetamiprid and imidacloprid on reproduction " by using an integrated testing strategy for reproductive toxicology, which covered sperm quality, sperm penetration into oocytes and preimplantation embryonic development. Direct chemical exposure (500 mM or 5 mM) on spermatozoa during capacitation was performed, and in vitro fertilization (IVF) process, zygotes and 2-cell embryos were respectively incubated with chemical-supplemented medium until blastocyst formation to evaluate the reproductive toxicity of these chemicals and monitor the stages mainly affected. Generally, treatment of 500 mM or 5 mM chemicals for 30 min did not change sperm motility and DNA integrity significantly but the fertilization ability in in vitro fertilization (IVF) process, indicating that IVF process could detect and distinguish subtle effect of spermatozoa exposed to different chemicals. Culture experiment in the presence of chemicals in medium showed that fertilization process and zygotes are adversely affected by direct exposure of chemicals (P,0.05), in an order of nicotine.IMI.ACE, whereas developmental progression of 2-cell stage embryos was similar to controls (P.0.05). These findings unveiled the hazardous effects of neonicotinoid pesticides exposure on mammalian sperm fertilization ability as well as embryonic development, raising the concerns that neonicotinoid pesticides may pose reproductive risks on human reproductive health, especially in professional populations.

Immunotoxic effects of imidacloprid following 28 days of oral exposure in BALB/c mice

This 2013 study, published in Environmental Toxicology and Pharmacology, evaluated "immunotoxic effects of imidacloprid in female BALB/c mice. Imidacloprid was administered orally daily at 10, 5, or 2.5 mg/kg over 28 days. Specific parameters of humoral and cellular immune response including hemagglutinating antibody (HA) titer to sheep red blood cells (SRBC; T-dependent antigen), delayed type hypersensitivity (DTH) response to SRBC, and T-lymphocyte proliferation in response to phytohemagglutinin (PHA) were evaluated. The results showed that imidacloprid at high dose, specifically suppressed cell-mediated immune response as was evident from decreased DTH response and decreased stimulation index of T-lymphocytes to PHA. At this dose, there were also prominent histopathological alterations in spleen and liver. Histopathological analysis of footpad sections of mice revealed dose-related suppression of DTH response. Imidacloprid at low dose of 2.5 mg/kg/day did not produce any significant alterations in cellular and humoral immune response and it seemed to be an appropriate dose for assessment of ‘no observable adverse effects level’ for immunotoxicity in BALB/c mice. The results also indicated that imidacloprid has immunosuppressive effects at doses >5 mg/kg, which could potentially be attributed to direct cytotoxic effects of IMD against T cells (particularly TH cells) and that long-term exposure could be detrimental to the immune system."

Assessment of imidacloprid toxicity on reproductive organ system of adult male rats

Study investigated "the toxicity of low doses of imidacloprid (IMI) on the reproductive organ systems of adult male rats. The treatment groups received 0.5 (IMI-0.5), 2 (IMI-2) or 8 mg IMI/kg body weight by oral gavage (IMI-8) for three months. The deterioration in sperm motility in IMI-8 group and epidydimal sperm concentration in IMI-2 and IMI-8 groups and abnormality in sperm morphology in IMI-8 were significant. The levels of testosterone (T) and GSH decreased significantly in group IMI-8 compared to the control group. Upon treatment with IMI, apoptotic index increased significantly only in germ cells of the seminiferous tubules of IMI-8 group when compared to control. Fragmentation was striking in the seminal DNA from the IMI-8 group, but it was much less obvious in the IMI-2 one. IMI exposure resulted in elevation of all fatty acids analyzed, but the increases were significant only in stearic, oleic, linoleic and arachidonic acids. The ratios of 20:4/20:3 and 20:4/18:2 were decreased and 16:1n-9/16:0 ratio was increased. In conclusion, the present animal experiments revealed that the treatment with IMI at NOAEL dose-levels caused deterioration in sperm parameters, decreased T level, increased apoptosis of germ cells, seminal DNA fragmentation, the depletion of antioxidants and change in disturbance of fatty acid composition. All these changes indicate the suppression of testicular function."

Chronic exposure to imidacloprid induces inflammation and oxidative stress in the liver & central nervous system of rats

This 2012 study, published in Pesticide Chemistry and Physiology, examined "oxidant and inflammatory responses to chronic exposure of imidacloprid... in rats. Wistar rats were randomly allocated into two groups as control and imidacloprid-exposed group (n = 10 rat/each group).1 mg/kg/BW/day imidacloprid was administrated orally by gavage for 30 days. After exposure, rats were euthanized and liver and brain samples were surgically removed for analyses. Imidacloprid application caused a significant increase in nitric oxide production in brain (p < 0.05) and liver (p < 0.001). The quantitative analyses of mRNA confirmed the finding that imidacloprid induced the mRNA transcriptions of the three isoforms of nitric oxide synthases (iNOS, eNOS, nNOS) in brain and two isoforms (iNOS, eNOS) in the liver. Exposure to imidacloprid caused significant lipid peroxidation in plasma, brain (p < 0.001) and liver (p < 0.003). While the superoxide-generating enzyme xanthine oxidase activity was elevated in both tissues (p < 0.001), myeloperoxidase activity was increased only in the liver (p < 0.001). Antioxidant enzyme activities showed various alterations following exposure, but a significantly depleted antioxidant glutathione level was detected in brain (p < 0.008). Evidence of chronic inflammation by imidacloprid was observed as induction of pro-inflammatory cytokines such as TNF-a, IL-1b, IL-6, IL- 12 and IFN-c in the liver and brain. In conclusion, chronic imidacloprid exposure causes oxidative stress and inflammation by altering antioxidant systems and inducing pro-inflammatory cytokine production in the liver and central nervous system of non-target organisms.

The molecular basis of simple relationships between exposure concentration and toxic effects with time

This 2013 review, published in Toxicology, " re-introduces an old approach that takes into account the biochemical mode of action and their resulting biological effects over time of exposure. Empirical evidence demonstrates that the Druckrey–Küpfmüller toxicity model, which was validated for chemical carcinogens in the early 1960s, is also applicable to a wide range of toxic compounds in ecotoxicology. According to this model, the character of a poison is primarily determined by the reversibility of critical receptor binding. Chemicals showing irreversible or slowly reversible binding to specific receptors will produce cumulative effects with time of exposure, and whenever the effects are also irreversible (e.g. death) they are reinforced over time; these chemicals have time-cumulative toxicity. Compounds having non-specific receptor binding, or involving slowly reversible binding to some receptors that do not contribute to toxicity, may also be time-dependent; however, their effects depend primarily on the exposure concentration, with time playing a minor role. Consequently, the mechanism of toxic action has important implications for risk assessment. Traditional risk approaches cannot predict the impacts of toxicants with time-cumulative toxicity in the environment. New assessment procedures are needed to evaluate the risk that the latter chemicals pose on humans and the environment."

Water polluted with imidacloprid linked to low numbers of aquatic insects

This 2013 study found 70% fewer invertebrate species in water polluted with imidacloprid compared to clean water in the Netherlands. Mayflies, midges and molluscs, were all severely impacted with potential harm to their predators like birds. Researchers compiled and analyzed information from 700 sites between 1998 and 2009 to determine the impacts of water quality on wildlife. For highly polluted waters that exceeded the Dutch pollution limits, only 17 species were found on average in comparison to 52 species in clean water.

Effects of aldicarb and neonicotinoid seed treatments on twospotted spider mite on cotton 

This 2013 study, published in the Journal of Economic Entomology, indicates that neonicotinoid seed treatments increase infestation of pest species in cotton crops. "Twelve field experiments and one laboratory experiment were conducted to determine the effects of furrow applied aldicarb and seed treatments of thiamethoxam, imidacloprid, Avicta (thiamethoxam + abamectin), Aeris (imidacloprid + thiodicarb), and acephate on twospotted spider mite, Tetranychus urticae Koch, on cotton, Gossypium hirsutum L. For the field experiments, data were pooled across all experiments for analysis.eris, thiamethoxam, and imidacloprid treatments resulted in twospotted spider mite densities greater than those in the untreated check, aldicarb, and acephate treatments. However, cotton treated with Avicta (thiamethoxam + abameetin) had 34% fewer mites than other neonicotinoid seed treatments when infestations occurred near cotyledon stage. Untreated check and aldicarb treatments had the lowest mite densities. Only aldicarb reduced mite densities below that in the untreated check. In a laboratory trial, the fecundity of twospotted spider mite was measured. While neonicotinoid seed treatments increased mite densities in the field, they did not increase fecundity in the laboratory experiment."

Lethal and sublethal effects of imidacloprid and buprofezin on the sweetpotato whitefly parasitoid

This 2013 study, published in Crop Protection, demonstrated that the longevity and fecundity of the parasitoid, E.mundus, were reduced significantly by the two insecticides, though the sex ratio of E.mundus offspring was not affected. Population parameters of the parasitoid such as R0rm and T were also significantly reduced by the insecticides. Our results indicated that, in addition to lethal effects, sublethal effects should also be considered when these insecticides are applied.

Eco-toxicity of Neonicotinoid: a case study on the impact of imidacloprid using Drawida willsi earthworm as bioindicator

Many pesticides are used in the world and in India to boost the crop productivity, among which imidacloprid; a neonicotinoid is widely used now a day due to its low toxicity. These pesticides no doubt have protected the crops from the dangerous pest but in turn are becoming dangerous for soil health. Therefore an experiment was conducted to find out the eco-toxicity of imidacloprid. For this, earthworm (Drawida willsi, Michaelsen) and soil was collected from such agricultural field where there had no record of input of agrochemicals. Different concentrations of imidacloprid were prepared in dilution with acetone and sprayed on the soil surface. Five replicates for each concentration of the pesticides were prepared. Then ten numbers of juvenile, immature and adult earthworms were added separately into all the replicates of different concentrated samples. All the samples were kept in the laboratory under close vigil for 96 hours. Number of earthworm death with respect to doses and replicates were recorded and the 96h LC50 values for juvenile, immature and adult earthworm were calculated by Finney's Probit Method (Finney, 1971). It was found that the 96 h LC50 values with their 95% confidence limit of juvenile, immature and adult earthworm were 4.43, 7.96 and 12.45 mg a.i. imidacloprid/kg dry soil respectively. Although the recommended dose of imidacloprid was lower than the 96 h LC50 values of D. willsi for imidacloprid, but still it could affect the soil biota by altering its vital rates and metabolism due to bioaccumulation of the agrochemicals.

Studies on the electrolytes and microelements in Wistar rat following multiple exposures to acetamiprid

This 2012 study, published in Toxicology and Industrial Health, performed a "subacute toxicity study of acetamiprid... in 72 female Wistar rats randomly divided into four groups (18 each). Acetamiprid was administered orally at the dose rate of 0, 25, 100 and 200 mg/kg of body weight to rats of groups I, II, III and IV, respectively. Group I served as control. Calcium, phosphorous, sodium, potassium, chloride, zinc, copper, iron and cobalt concentrations in plasma were significantly (p  0.05) increased in acetamiprid administered groups. However, no alteration was observed in plasma manganese concentration in acetamiprid-treated rats. The repeated oral toxicity study on acetamiprid in present investigation suggested that it has toxic potential and it is a high-risk insecticide."

Effects of binary mixtures on the life traits of Daphnia magna

This 2011 study, published in Ecotoxicology and Environmental Safety, assesses "the joint effect of chemical mixtures to the life—history traits of Daphnia magna Straus. For that a binary mixture of two neonicotinoid insecticides, imidacloprid and thiacloprid, and another one of imidacloprid with nickel chloride were tested. Theoretical models have been developed and applied in studies with chemical mixtures, predicting toxicity based on their modes of action: concentration addition (CA) and independent joint action (IA) models. Still there are cases where deviations are observed (e.g. synergistic or antagonistic behaviors, dose ratio or level dependency). In this study, the effects of the individual compounds and their mixtures were studied in a chronic test where reproduction, survival and body length were evaluated in D. magna. Regarding single compound effects, it was observed that the most toxic was nickel chloride followed by thiacloprid and imidacloprid. For the mixture exposure of imidacloprid and thiacloprid, a synergistic pattern was observed in the sublethal doses used for the number of neonates produced, while for the body length the best fit was shown with the CA model. In the mixture exposure of imidacloprid and nickel, no deviation from the IA was observed for the neonate production data; for the body length parameter, a synergistic pattern was observed in low doses of the chemicals while an antagonistic pattern was observed."

Pesticide exposure and inducible antipredator responses in the zooplankton grazer, Daphnia magna Straus

This 2010 study, published in Chemosphere, studies "the effects of the pesticide imidacloprid on the responses of Daphnia magnato a combination of predator-release kairomones from trout and alarm cues from conspecifics, simulating different levels of perceived predation risk. The joint effects of simultaneous exposure to both types of stressors were assessed both by traditional analysis of variance and by employing conceptual models for the evaluation of contaminant mixture exposures. Results demonstrated that pesticide exposure can significantly increase the costs of inducible antipredator defences and impair life-history responses of daphnids to fish predation pressure. Since trait-mediated effects are well-known to play a key role in population dynamics, the combined direct and indirect effects of sub-lethal concentrations of pesticides could induce maladaptive responses in zooplankton populations in the field, reducing their long-term viability."

The significance of the Druckrey-Küpfmüller equation for risk assessment - The toxicity of neonicotinoid insecticides to arthropods is reinforced by exposure time 

This 2010 study, published in Toxicology, examines The Druckrey-Küpfmüller equation which "explains why toxicity may occur after prolonged exposure to very low toxicant levels. Recently, similar dose-response characteristics have been established for the toxicity of the neonicotinoid insecticides imidacloprid and thiacloprid to arthropods. This observation is highly relevant for environmental risk assessment. Traditional approaches that consider toxic effects at fixed exposure times are unable to allow extrapolation from measured endpoints to effects that may occur at other times of exposure. Time-to-effect approaches that provide information on the doses and exposure times needed to produce toxic effects on tested organisms are required for prediction of toxic effects for any combination of concentration and time in the environment." See: Daily News Blog.

Assessing the effects of the neonicotinoid insecticide imidacloprid in the cholinergic synapses of the stellate cells of the mouse cochlear nucleus using whole-cell patch-clamp recording

This 2010 study, published in Neurotoxicology, sought to "determine to what extent imidacloprid (IMI) affects the nAChRs of the stellate cells of mouse cochlear nucleus (CN), using whole-cell patch-clamp recording. Puff application of 1 mM IMI had no significant effect on the membrane properties of the neurons tested, while a concentration of 10 mM caused a significant depolarizing shift in themembrane potential and resulted in increases in the fluctuation of the membrane potential and in the frequency of miniature postsynaptic potentials (mpps) within less than a minute of exposure. IMI at concentrations 50 mMcaused a significant depolarizing shift in themembrane potential, accompanied by amarked increase in the frequency of action potential. IMI decreased the membrane input resistance and the membrane time constants. Bath application of 50 mM d-tubocurarine (d-TC) reversibly blocked the depolarizing shift of the resting membrane potential and the spontaneous firing induced by IMI application in current clamp and blocked the inward currents through nicotinic receptors induced by IMI application in voltage clamp. Similarly, 100 nMa-bungarotoxin (a-BgTx) blocked the spontaneous firing induced by IMI (n = 3). The amplitude of the 100 mM IMI-induced inward current at 60 mV holding potential was 115.0  16.2 pA (n = 7). IMI at a concentration of 10 mM produced 11.3  3.4 pA inward current (n = 4). We conclude that exposure to IMI at concentrations 10 mM for <1 min can change the membrane properties of neurons that have nAChRs and, as a consequence, their function.

Acute oxidant and inflammatory effects of imidacloprid on the mammalian central nervous system and liver in rats

In this 2010 study, published in Pesticide Toxicology and Physiology, researchered examined "potential acute neuro and liver toxic effects of imidacloprid... in rats as a model of mammalian using antioxidant–oxidant and inflammatory system. 10 lM imidacloprid was administrated intravenously and 2 h post-administration, the rats were sacrificed, liver and brains were surgically removed. Exposure to imidacloprid led to significant increases in nitric oxide concentrations in brain, liver and plasma samples. The quantitative mRNA transcriptional analyses demonstrated that imidacloprid-elevated production of NO levels due to the induction of iNOS in liver, but neither nNOS nor iNOS were induced in brain. The oxidant-generating enzymes xanthine oxidase and myeloperoxidase activities in both tissues were elevated and significant lipid peroxidation in liver and plasma was observed. The antioxidant catalase, superoxide dismutase and glutathione peroxidase activities were differently responded to imidacloprid administration. Significant intracellular glutathione depletion was also measured in both tissues. Imidacloprid treatment upregulated inflammatory cytokines TNF-a, IL-6 and IL-1b mRNA transcriptions by 2.5- to 5.2-fold increases in both brain and liver. Conversely, anti-inflammatory mediator IL-10 mRNA was down-regulated in both organs. These results suggest that imidacloprid cause oxidative stress and inflammation in central nervous system and liver in non-target organisms in rats."

The impact of neonicotinoid insecticides on bumblebees, Honey bees and other non-target invertebrates
This 2009 report, published by BugLife,, reviews existing approvals research and independent research on the effects of neonicotinoid pesticides on Honey bees, bumblebees and other non-target invertebrates, and investigates the current approvals mechanism and its standards.

Parasites and Viruses
bee flower

Management of Arthropod Pathogen Vectors in North America: Minimizing Adverse Effects on Pollinators.
Tick and mosquito management is important to public health protection. At the same time, growing concerns about declines of pollinator species raise the question of whether vector control practices might affect pollinator populations. We report the results of a task force of the North American Pollinator Protection Campaign (NAPPC) that examined potential effects of vector management practices on pollinators, and how these programs could be adjusted to minimize negative effects on pollinating species. The main types of vector control practices that might affect pollinators are landscape manipulation, biocontrol, and pesticide applications. Some current practices already minimize effects of vector control on pollinators (e.g., short-lived pesticides and application-targeting technologies). Nontarget effects can be further diminished by taking pollinator protection into account in the planning stages of vector management programs. Effects of vector control on pollinator species often depend on specific local conditions (e.g., proximity of locations with abundant vectors to concentrations of floral resources), so planning is most effective when it includes collaborations of local vector management professionals with local experts on pollinators. Interventions can then be designed to avoid pollinators (e.g., targeting applications to avoid blooming times and pollinator nesting habitats), while still optimizing public health protection. Research on efficient targeting of interventions, and on effects on pollinators of emerging technologies, will help mitigate potential deleterious effects on pollinators in future management programs. In particular, models that can predict effects of integrated pest management on vector-borne pathogen transmission, along with effects on pollinator populations, would be useful for collaborative decision-making.

Gut microbiota composition is associated with environmental landscape in honey bees.
There is growing recognition that the gut microbial community regulates a wide variety of important functions in its animal hosts, including host health. However, the complex interactions between gut microbes and environment are still unclear. Honey bees are ecologically and economically important pollinators that host a core gut microbial community that is thought to be constant across populations. Here, we examined whether the composition of the gut microbial community of honey bees is affected by the environmental landscape the bees are exposed to. We placed honey bee colonies reared under identical conditions in two main landscape types for 6 weeks: either oilseed rape farmland or agricultural farmland distant to fields of flowering oilseed rape. The gut bacterial communities of adult bees from the colonies were then characterized and compared based on amplicon sequencing of the 16S rRNA gene. While previous studies have delineated a characteristic core set of bacteria inhabiting the honey bee gut, our results suggest that the broad environment that bees are exposed to has some influence on the relative abundance of some members of that microbial community. This includes known dominant taxa thought to have functions in nutrition and health. Our results provide evidence for an influence of landscape exposure on honey bee microbial community and highlight the potential effect of exposure to different environmental parameters, such as forage type and neonicotinoid pesticides, on key honey bee gut bacteria. This work emphasizes the complexity of the relationship between the host, its gut bacteria, and the environment and identifies target microbial taxa for functional analyses.

Persistence of subclinical deformed wing virus infections in honeybees following Varroa mite removal and a bee population turnover.
Deformed wing virus (DWV) is a lethal virus of honeybees (Apis mellifera) implicated in elevated colony mortality rates worldwide and facilitated through vector transmission by the ectoparasitic mite Varroa destructor. Clinical, symptomatic DWV infections are almost exclusively associated with high virus titres during pupal development, usually acquired through feeding by Varroa mites when reproducing on bee pupae. Control of the mite population, generally through acaricide treatment, is essential for breaking the DWV epidemic and minimizing colony losses. In this study, we evaluated the effectiveness of remedial mite control on clearing DWV from a colony. DWV titres in adult bees and pupae were monitored at 2 week intervals through summer and autumn in acaricide-treated and untreated colonies. The DWV titres in Apistan treated colonies was reduced 1000-fold relative to untreated colonies, which coincided with both the removal of mites and also a turnover of the bee population in the colony. This adult bee population turnover is probably more critical than previously realized for effective clearing of DWV infections. After this initial reduction, subclinical DWV titres persisted and even increased again gradually during autumn, demonstrating that alternative non-Varroa transmission routes can maintain the DWV titres at significant subclinical levels even after mite removal. The implications of these results for practical recommendations to mitigate deleterious subclinical DWV infections and improving honeybee health management are discussed.

Effects of Oral Exposure to Fungicides on Honey Bee Nutrition and Virus Levels

Sublethal exposure to fungicides can affect honey bees (Apis mellifera L.) in ways that resemble malnutrition. These include reduced brood rearing, queen loss, and increased pathogen levels. We examined the effects of oral exposure to the fungicides boscalid and pyraclostrobin on factors affecting colony nutrition and immune function including pollen consumption, protein digestion, hemolymph protein titers, and changes in virus levels. Because the fungicides are respiratory inhibitors, we also measured ATP concentrations in flight muscle. The effects were evaluated in 3- and 7-d-old worker bees at high fungicide concentrations in cage studies, and at field-relevant concentrations in colony studies. Though fungicide levels differed greatly between the cage and colony studies, similar effects were observed. Hemolymph protein concentrations were comparable between bees feeding on pollen with and without added fungicides. However, in both cage and colony studies, bees consumed less pollen containing fungicides and digested less of the protein. Bees fed fungicide-treated pollen also had lower ATP concentrations and higher virus titers. The combination of effects we detected could produce symptoms that are similar to those from poor nutrition and weaken colonies making them more vulnerable to loss from additional stressors such as parasites and pathogens.

Effects of Nosema ceranae and thiametoxam in Apis mellifera: A comparative study in Africanized and Carniolan honey bees

Multiple stressors, such as chemicals and pathogens, are likely to be detrimental for the health and lifespan of Apis mellifera, a bee species frequently exposed to both factors in the field and inside hives. The main objective of the present study was to evaluate comparatively the health of Carniolan and Africanized honey bees (AHB) co-exposed to thiamethoxam and Nosema ceranae (N. ceranae) spores. Newly-emerged worker honey bees were exposed solely with different sublethal doses of thiamethoxam (2% and 0.2% of LD50 for AHB), which could be consumed by bees under field conditions. Toxicity tests for the Carniolan bees were performed, and the LD50 of thiamethoxam for Carniolan honey bees was 7.86 ng bee-1. Thiamethoxam exposure had no negative impact on Nosema development in experimental conditions, but it clearly inhibited cell death in the midgut of thiamethoxam and Nosema-exposed bees, as demonstrated by immunohistochemical data. Indeed, thiamethoxam exposure only had a minor synergistic toxic effect on midgut tissue when applied as a low dose simultaneously with N. ceranae to AHB and Carniolan honey bees, in comparison with the effect caused by both stressors separately. Our data provides insights into the effects of the neonicotenoid thiamethoxam on the AHB and Carniolan honey bee life span, as well as the effects of simultaneous application of thiamethoxam and N. ceranae spores to honey bees.

Effects, but no interactions, of ubiquitous pesticide and parasite stressors on honey bee (Apis mellifera) lifespan and behaviour in a colony environment

Interactions between pesticides and parasites are believed to be responsible for increased mortality of honey bee (Apis mellifera) colonies in the northern hemisphere. Previous efforts have employed experimental approaches using small groups under laboratory conditions to investigate influence of these stressors on honey bee physiology and behaviour, although both the colony level and field conditions play a key role for eusocial honey bees. Here, authors challenged honey bee workers under in vivo colony conditions with sublethal doses of the neonicotinoid thiacloprid, the miticide tau-fluvalinate and the endoparasite Nosema ceranae, to investigate potential effects on longevity and behaviour using observation hives. In contrast to previous laboratory studies, these results do not suggest interactions among stressors, but rather lone effects of pesticides and the parasite on mortality and behaviour, respectively. These effects appear to be weak due to different outcomes at the two study sites, thereby suggesting that the role of thiacloprid, tau-fluvalinate and N. ceranae and interactions among them may have been overemphasized. In the future, investigations into the effects of honey bee stressors should prioritize the use of colonies maintained under a variety of environmental conditions in order to obtain more biologically relevant data.

Interactive effects of pesticide exposure and pathogen infection on bee health - a critical analysis

Pesticide exposure and pathogen infection are recognised as potential stressors impacting upon bee populations and recently there has been a surge in research on pesticide-disease interactions to reflect environmentally realistic scenarios better. Authors critically analyse the findings on pesticide-disease interactions, including effects on the survival, pathogen loads and immunity of bees, and assess the suitability of various endpoints to inform our mechanistic understanding of these interactions. Study shows that pesticide exposure and pathogen infection have not yet been found to interact to affect worker survival under field-realistic scenarios. Colony-level implications of pesticide effects on Nosema infections, viral loads and honey bee immunity remain unclear as these effects have been observed in a laboratory setting only using a small range of pesticide exposures, generally exceeding those likely to occur in the natural environment, and assessing a highly selected series of immune-related endpoints. Future research priorities include the need for a better understanding of pesticide effects on the antimicrobial peptide (AMP) component of an individual's immune response and on social defence behaviours. Interactions between pesticide exposure and bacterial and fungal infections have yet to be addressed. The paucity of studies in non-Apis bee species is a further major knowledge gap.

Bees under stress: sublethal doses of a neonicotinoid pesticide and pathogens interact to elevate honey bee mortality across the life cycle

Microbial pathogens are thought to have a profound impact on insect populations. Honey bees are suffering from elevated colony losses in the northern hemisphere possibly because of a variety of emergent microbial pathogens, with which pesticides may interact to exacerbate their impacts. To reveal such potential interactions, authors administered at sublethal and field realistic doses one neonicotinoid pesticide (thiacloprid) and two common microbial pathogens, the invasive microsporidian Nosema ceranae and black queen cell virus (BQCV), individually to larval and adult honey bees in the laboratory. Through fully crossed experiments in which treatments were administered singly or in combination, study found an additive interaction between BQCV and thiacloprid on host larval survival likely because the pesticide significantly elevated viral loads. In adult bees, two synergistic interactions increased individual mortality: between N. ceranae and BQCV, and between N. ceranae and thiacloprid. The combination of two pathogens had a more profound effect on elevating adult mortality than N. ceranae plus thiacloprid. Common microbial pathogens appear to be major threats to honey bees, while sublethal doses of pesticide may enhance their deleterious effects on honey bee larvae and adults. It remains an open question as to whether these interactions can affect colony survival.

Bee declines driven by combined stress from parasites, pesticides, and lack of flowers

In this review, published in Science, the authors recognize that bees are subject to numerous pressures in the modern world. The abundance and diversity of flowers has declined, bees are chronically exposed to cocktails of agrochemicals, and they are simultaneously exposed to novel parasites accidentally spread by humans. Climate change is likely to exacerbate these problems in the future. Stressors do not act in isolation; for example pesticide exposure can impair both detoxification mechanisms and immune responses, rendering bees more susceptible to parasites. It seems certain that chronic exposure to multiple, interacting stressors is driving honey bee colony losses and declines of wild pollinators, but such interactions are not addressed by current regulatory procedures and studying these interactions experimentally poses a major challenge. In the meantime, taking steps to reduce stress on bees would seem prudent; incorporating flower-rich habitat into farmland, reducing pesticide use through adopting more sustainable farming methods, and enforcing effective quarantine measures on bee movements are all practical measures that should be adopted. Effective monitoring of wild pollinator populations is urgently needed to inform management strategies into the future.

Honeybee Colony Disorder in Crop Areas: The Role of Pesticides and Viruses

The present study, published in PLOS ONE in 2014, aims to determine the role of both pesticide exposure and virus load on the appraisal of unexplained honeybee colony disorders in field conditions. From July 2011 to May 2012, 330 colonies were monitored. Three acaricides, 5 insecticides and 13 fungicides were detected in the analyzed samples. A significant correlation was found between the presence of fungicide residues and honeybee colony disorders. A significant positive link could also be established between the observation of disorder and the abundance of crop surface around the beehive. According to the results, the researchers state that the role of fungicides as a potential stressor for honeybee colonies should be further studied, either by their direct and/or indirect impacts on bees and bee colonies.

Impact of chronic exposure to a pyrethroid pesticide on bumblebees and interactions with a trypanosome parasite

Researchers focus on the impacts of chronic exposure to the commonly used pyrethroid pesticide lambda (λ)-cyhalothrin on the bumblebee Bombus terrestris at both the individual and colony level. Furthermore, they investigated the interactions of pesticide exposure with a highly prevalent trypanosome parasite Crithidia bombi. Pesticide-treated colonies produced workers with a significantly lower body mass. However, out of the twelve variables of colony development measured, this was the only metric that was significantly affected by pesticide treatment and there was no subsequent significant impact on the reproductive output of colonies. Lambda-cyhalothrin had no significant impact on the susceptibility of workers to C. bombi, or intensity of parasitic infection. Pesticide exposure did not cause differential survival in workers or males, even when workers were additionally challenged with C. bombi. Chronic exposure to λ-cyhalothrin has a significant impact on worker size, a key aspect of bumblebee colony function, particularly under conditions of limited food resources. This could indicate that under times of resource limitation, colonies exposed to this pesticide in the field may fail. However, the lack of other impacts found in this study indicates that further field trials are needed to elucidate this.

Transcriptome Analyses of the Honeybee Response to Nosema ceranae and Insecticides

In this 2014 study, published in PLOS ONE, researchers investigated the molecular response of honeybees exposed to N. ceranae, to insecticides (fipronil or imidacloprid), and to a combination of both stressors. Midgut transcriptional changes induced by these stressors were measured in two independent experiments. Although N. ceranae-insecticide combinations induced a significant increase in honeybee mortality, researchers observed that they did not lead to a synergistic effect. According to gene expression profiles, chronic exposure to insecticides had no significant impact on detoxifying genes but repressed the expression of immunity-related genes. Honeybees treated with N. ceranae, alone or in combination with an insecticide, showed a strong alteration of midgut immunity together with modifications affecting cuticle coatings and trehalose metabolism. An increasing impact of treatments on gene expression profiles with time was identified suggesting an absence of stress recovery which could be linked to the higher mortality rates observed.

Evaluation of the Distribution and Impacts of Parasites, Pathogens, and Pesticides on Honey Bee (Apis mellifera) Populations in East Africa

In this 2014 study, published in PLOS ONE, researchers initiated a nationwide survey encompassing 24 locations across Kenya in 2010 to evaluate the numbers and sizes of honey bee colonies, assess the presence of parasites (Varroa mites and Nosemamicrosporidia) and viruses, identify and quantify pesticide contaminants in hives, and assay for levels of hygienic behavior. The results suggest that Varroa, the three viruses, and Nosema have been relatively recently introduced into Kenya, but these factors do not yet appear to be impacting Kenyan bee populations. Thus chemical control for Varroa and Nosema are not necessary for Kenyan bees at this time. This study provides baseline data for future analyses of the possible mechanisms underlying resistance to and the long-term impacts of these factors on African bee populations.

Thiacloprid–Nosema ceranae interactions in honey bees: Host survivorship but not parasite reproduction is dependent on pesticide dose

In this 2014 study, researchers demonstrated that a synergistic effect on mortality by the low toxic, commonly used neonicotinoid thiacloprid and the nearly ubiquitous gut parasite Nosema ceranae is dependent on the pesticide dose. Furthermore, thiacloprid had a negative influence on N. ceranae reproduction. These results highlight that interactions among honey bee health stressors can be dynamic and should be studied across a broader range of combinations.

A Causal Analysis of Observed Declines in Managed Honey Bees (Apis mellifera)

This 2013 study, published in Human and Ecological Risk Assessment: An International Journal, summarized the results of a workshop that was convened during which bee experts were introduced to a formal causal analysis approach to compare 39 candidate causes against specified criteria to evaluate their relationship to the reduced overwinter survivability observed since 2006 of commercial bees used in the California almond industry. Candidate causes were categorized as probable, possible, or unlikely; several candidate causes were categorized as indeterminate due to lack of information. Due to time limitations, a full causal analysis was not completed at the workshop. In this article, examples are provided to illustrate the process and provide preliminary findings, using three candidate causes. Varroa mites plus viruses were judged to be a “probable cause” of the reduced survival, while nutrient deficiency was judged to be a “possible cause.” Neonicotinoid pesticides were judged to be “unlikely” as the sole cause of this reduced survival, although they could possibly be a contributing factor.

Crop Pollination Exposes Honey Bees to Pesticides Which Alters Their Susceptibility to the Gut Pathogen Nosema ceranae

This 2013 study published in the journal PLoS ONE importantly evaluated field relevant combinations and loads of pesticides and their effect on honey bee health. “We collected pollen from bee hives in seven major crops to determine 1) what types of pesticides bees are exposed to when rented for pollination of various crops and 2) how field-relevant pesticide blends affect bees’ susceptibility to the gut parasite Nosema ceranae.” The study found that there were 35 different pesticides in the sampled pollen, and found high fungicide loads. “While fungicides are typically seen as fairly safe for honey bees, we found an increased probability of Nosema infection in bees that consumed pollen with a higher fungicide load. Our results highlight a need for research on sub-lethal effects of fungicides and other chemicals that bees placed in an agricultural setting are exposed to.”

Towards a systems approach for understanding honeybee decline: a stocktaking and synthesis of existing models

This 2013 study published in Journal of Applied Ecology reviewed "existing honeybee models that are relevant to examining the effects of different stressors on colony growth and survival. Most of these models describe honeybee colony dynamics, foraging behaviour or honeybee – varroa mite – virus interactions. We found that many, but not all, processes within honeybee colonies, epidemiology and foraging are well understood and described in the models, but there is no model that couples in-hive dynamics and pathology with foraging dynamics in realistic landscapes."

Neonicotinoid clothianidin adversely affects insect immunity and promotes replication of a viral pathogen in honey bees

Large-scale losses of honey bee colonies represent a poorly understood problem of global importance. Both biotic and abiotic factors are involved in this phenomenon that is often associated with high loads of parasites and pathogens. A stronger impact of pathogens in honey bees exposed to neonicotinoid insecticides has been reported, but the causal link between insecticide exposure and the possible immune alteration of honey bees remains elusive. Here, authors demonstrate that the neonicotinoid insecticide clothianidin negatively modulates NF-κB immune signaling in insects and adversely affects honey bee antiviral defenses controlled by this transcription factor. Authors identified in insects a negative modulator of NF-κB activation, which is a leucine-rich repeat protein. Exposure to clothianidin, by enhancing the transcription of the gene encoding this inhibitor, reduces immune defenses and promotes the replication of the deformed wing virus in honey bees bearing covert infections. This honey bee immunosuppression is similarly induced by a different neonicotinoid, imidacloprid, but not by the organophosphate chlorpyriphos, which does not affect NF-κB signaling. The occurrence at sublethal doses of this insecticide-induced viral proliferation suggests that the studied neonicotinoids might have a negative effect at the field level. These experiments uncover a further level of regulation of the immune response in insects and set the stage for studies on neural modulation of immunity in animals. Furthermore, this study has implications for the conservation of bees, as it will contribute to the definition of more appropriate guidelines for testing chronic or sublethal effects of pesticides used in agriculture.

Pesticide exposure in honey bees results in increased levels of the gut pathogen Nosema

Study exposed honey bee colonies during three brood generations to sub-lethal doses of a widely used pesticide, imidacloprid, and then subsequently challenged newly emerged bees with the gut parasite, Nosema spp. The pesticide dosages used were below levels demonstrated to cause effects on longevity or foraging in adult honey bees. Nosema infections increased significantly in the bees from pesticide-treated hives when compared to bees from control hives demonstrating an indirect effect of pesticides on pathogen growth in honey bees. Interactions between pesticides and pathogens could be a major contributor to increased mortality of honey bee colonies, including colony collapse disorder, and other pollinator declines worldwide.

A New Threat to Honey Bees, the Parasitic Phorid Fly Apocephalus borealis

Study provides the first documentation that the phorid fly Apocephalus borealis, previously known to parasitize bumble bees, also infects and eventually kills honey bees and may pose an emerging threat to North American apiculture. Parasitized honey bees show hive abandonment behavior, leaving their hives at night and dying shortly thereafter. On average, seven days later up to 13 phorid larvae emerge from each dead bee and pupate away from the bee. Using DNA barcoding, study confirmed that phorids that emerged from honey bees and bumble bees were the same species. Phorid parasitism may affect hive viability since 77% of sites sampled in the San Francisco Bay Area were infected by the fly and microarray analyses detected phorids in commercial hives in South Dakota and California's Central Valley. Study concludes that understanding details of phorid infection may shed light on similar hive abandonment behaviors seen in CCD.

Iridovirus and Microsporidian Linked to Honey Bee Colony Decline 

In 2010 Colony Collapse Disorder (CCD), again devastated honey bee colonies in the USA, indicating that the problem is neither diminishing nor has it been resolved. Many CCD investigations, using sensitive genome-based methods, have found small RNA bee viruses and the microsporidia, Nosema apis and N. ceranae in healthy and collapsing colonies alike with no single pathogen firmly linked to honey bee losses. These findings implicate co-infection by invertebrate iridescent virus (IIV) (Iridoviridae) and Nosema with honey bee colony decline, giving credence to older research pointing to IIV, interacting with Nosema and mites, as probable cause of bee losses in the USA, Europe, and Asia. See: Daily News Blog

Nosema ceranae, a newly identified pathogen of Apis mellifera in the U.S. and Asia 

Nosemosis (Nosema disease) is one of the most serious and prevalent adult honey bee diseases worldwide. For years, Nosema apis was thought to be the only microsporidia infecting domestic bee colonies. However, recently it was discovered that N. ceranae could jump from Asian honey bees (Apis cerana) to European honey bees (Apis mellifera) that are widely used for crop pollination. The data presented in the studies demonstrated that N. ceranae infection is widespread in the U.S., China and Australia and that infection with N. ceranae was more common than infection with N. apis in European honey bees. The finding about the prevalence of N. ceranae in the U.S. and Asian bee populations in conjunction with findings in other parts of the world invites further research of the evolutionary history of N. ceranae infection in European honey bees.

Winter losses of honeybee colonies (Apis mellifera): The role of infestations with Aethina tumida and Varroa destructor

Multiple infections and infestations of honeybee colonies with pathogens and parasites are inevitable due to the ubiquitous ectoparasitic mite Varroa destructor and might be one of the mechanisms underlying winter losses. Here the authors investigated the role of adult small hive beetles, Aethina tumida, alone and in combination with V. destructor for winter losses and infections with the microsporidian endoparasite Nosema ceranae. High losses occurred in all groups highly infested with V. destructor, supporting the central role of this mite in colony losses. Data suggest that A. tumida alone is unlikely to contribute to losses of overwintering honeybee colonies.

Changes in Gene Expression Relating to Colony Collapse Disorder in honey bees, Apis mellifera 

Colony collapse disorder (CCD) is a mysterious disappearance of honey bees that has beset beekeepers in the United States since late in 2006. Pathogens and other environmental stresses, including pesticides, have been linked to CCD, but a causal relationship has not yet been demonstrated. Considerable variation in gene expression was associated with the geographical origin of bees, but a consensus list of 65 transcripts was identified as potential genetic markers for CCD status. Reduced expression of two genes associated with detoxification and a mixed response from genes involved in immune function were observed. Unusual ribosomal RNA fragments were also conspicuously more abundant in CCD bee guts. The presence of these fragments may be a possible consequence of picornavirus infection. Ribosomal fragment abundance and viral presence may prove useful as diagnostic markers for colonies afflicted with CCD.

Honeybee Sacbrood virus infects adult small hive beetles, Aethina tumida (Coleoptera: Nitidulidae)

The Small Hive Beetle (SHB) is a recently discovered pest that invades honey bee colonies and causes damage to comb, stored honey and pollen. A laboratory experiment was conducted to investigate whether SHB could harbor honey bee virus(es) via feeding on virus infected brood and thereby serving as a vector of viruses in honey bee colonies. Study demonstrated for the first time that SHB could become infected with honey bee Sacbrood virus (SBV) via the food-borne transmission route. This study should raise awareness among scientists, beekeepers, and regulatory personnel to the threats of SHB not only for its directly negative impact on bee health but also for its ability to transmit viral diseases in bee colonies.

Entombed pollen: A new condition in honey bee colonies associated with increased risk of colony mortality 

Here authors describe a new phenomenon, entombed pollen, which is highly associated with increased colony mortality. Entombed pollen appears as sunken, wax-covered cells amidst "normal", uncapped cells of stored pollen, and the pollen contained within these cells is brick red in color. The increased incidence of entombed pollen in reused wax comb suggests that there is a transmittable factor common to the phenomenon and colony mortality. In addition, there were elevated pesticide levels, notably of the fungicide chlorothalonil, in entombed pollen. Additional studies are needed to determine if there is a causal relationship between entombed pollen, chemical residues, and colony mortality.

Differential gene expression of the honey bee Apis mellifera associated with Varroa destructor infection

The parasitic mite, Varroa destructor, is the most serious pest of the western honey bee, Apis mellifera, and has caused the death of millions of colonies worldwide. Authors investigated whether Varroa infestation induces changes in Apis mellifera gene expression, and whether there are genotypic differences in the bee’s tolerance, as first steps toward unraveling mechanisms of host response and differences in susceptibility to Varroa parasitism. Results suggest that differences in behavior, rather than in the immune system, underlie Varroa tolerance in honey bees. They provide a first step toward better understanding molecular pathways involved in this particular host-parasite relationship.

A metagenomic survey of microbes in honey bee colony collapse disorder 

In Colony Collapse Disorder (CCD), honey bee colonies inexplicably lose all of their workers. CCD has resulted in a loss of 50-90% of colonies in beekeeping operations across the United States. The observation that irradiated combs from affected colonies can be repopulated with naïve bees suggests an infectious basis for CCD. One organism, Israeli acute paralysis virus (IAPV) of bees, was strongly correlated with CCD. The prevalence of IAPV sequences in CCD operations, as well as the temporal and geographic overlap of CCD and importation of IAPV infected bees from Australia, indicates that IAPV is a significant marker for CCD.

Honeybee colony collapse due to Nosema ceranae in professional apiaries 

Honeybee colony collapse is a sanitary and ecological worldwide problem. To date there has not been a consensus about its origins. This report describes the clinical features of two professional bee-keepers affecting by this syndrome. Anamnesis, clinical examination and analyses support that the depopulation in both cases was due to the infection by Nosema ceranae (Microsporidia), an emerging pathogen of Apis mellifera. No other significant pathogens or pesticides (neonicotinoids) were detected and the bees had not been foraging in corn or sunflower crops. The treatment with fumagillin avoided the loss of surviving weak colonies. This is the first case report of honeybee colony collapse due to N. ceranae in professional apiaries in field conditions reported worldwide.

Recent Honey Bee Colony Declines 

In 2006, commercial migratory beekeepers along the East Coast of the United States began reporting sharp declines in their honey bee colonies. Current reports indicate that beekeepers in 35 states have been affected. Recent surveys indicate that about one-half of surveyed beekeepers have experienced "abnormal" or "severe" colony losses. To date, the potential causes of CCD, as reported by the scientists who are researching this phenomenon, include but may not be limited to the following: parasites, mites, and disease loads in the bees and brood; emergence of new or newly more virulent pathogens; poor nutrition among adult bees; lack of genetic diversity and lineage of bees; level of stress in adult bees (e.g., transportation and confinement of bees, or other environmental or biological stressors); chemical residue/contamination in the wax, food stores, and/or bees; and a combination of these and/or other factors.

IAPV, a bee-affecting virus associated with Colony Collapse Disorder can be silenced by dsRNA ingestion

Colony Collapse Disorder (CCD) has been associated with Israeli acute paralysis virus (IAPV). CCD poses a serious threat to apiculture and agriculture as a whole, due to the consequent inability to provide the necessary amount of bees for pollination of critical crops. Here we report on RNAi-silencing of IAPV infection by feeding bees with double-stranded RNA, as an efficient and feasible way of controlling this viral disease. The association of CCD with IAPV is discussed, as well as the potential of controlling CCD. 

Behavioral attributes and parental care of Varroa mites parasitizing honeybee brood 

Varroa jacobsoni, an ectoparasite of the Asian honeybee Apis cerana, has been introduced world-wide, and is currently decimating colonies of the European honeybee Apis mellifera. Study describes here how a single fertilized female, infesting a brood cell, can produce two to four adult fertilized females within the limited time span of bee development (270 h in worker and 330 h in drone cells), despite the disturbance caused by cocoon spinning and subsequent morphological changes of the bee.

Nutritional stress due to habitat loss may explain recent honeybee colony collapses 

In spite of the tremendous public interest in the recent large honeybee losses attributed to colony collapse disorder, there is still no definitive explanation for the phenomenon. With the hypothesis that nutritional stress due to habitat loss has played an important role in honeybee colony collapse, study analyzes the land use data in United States to show that the colony loss suffered by each state is significantly predicted by the extent of its open land relative to its developed land area. Study also discusses how increasing loss of foraging resources could be synergistically acting with emerging diseases to stress honeybee populations and the importance therefore for preserving natural areas that act as important pollinator habitats.

Effects of neonicotinoid pesticide pollution of Dutch surface water on non-target species abundance 

MSc Thesis by Teresa C. van Dijk, 2010. Sustainable Development Track Land use, Environment and Biodiversity (SD: LEB), Utrecht University

Special Interest

Global Bee Colony Disorders and other Threats to Insect Pollinators United Nations Environment Programme (UNEP)

Scientists working for the United Nations (UN) reveal in a report published March 10, 2011 that the collapse of honeybee colonies is now a global phenomenon that could have devastating consequences. Declines in managed bee colonies, seen increasingly in Europe and the US in the past decade, are now being observed in China, Japan and Egypt according to the report.