Novel indices reveal that pollinator exposure to pesticides varies across biological compartments and crop surroundings.

Declines in insect pollinators have been linked to a range of causative factors such as disease, loss of habitats, the quality and availability of food, and exposure to pesticides. Here, we analysed an extensive dataset generated from pesticide screening of foraging insects, pollen-nectar stores/beebread, pollen and ingested nectar across three species of bees collected at 128 European sites set in two types of crop. In this paper, we aimed to (i) derive a new index to summarise key aspects of complex pesticide exposure data and (ii) understand the links between pesticide exposures depicted by the different matrices, bee species and apple orchards versus oilseed rape crops. We found that summary indices were highly correlated with the number of pesticides detected in the related matrix but not with which pesticides were present. Matrices collected from apple orchards generally contained a higher number of pesticides (7.6 pesticides per site) than matrices from sites collected from oilseed rape crops (3.5 pesticides), with fungicides being highly represented in apple crops. A greater number of pesticides were found in pollen-nectar stores/beebread and pollen matrices compared with nectar and bee body matrices. Our results show that for a complete assessment of pollinator pesticide exposure, it is necessary to consider several different exposure routes and multiple species of bees across different agricultural systems.

Distribution of infectious and parasitic agents among three sentinel bee species across European agricultural landscapes.

Infectious and parasitic agents (IPAs) and their associated diseases are major environmental stressors that jeopardize bee health, both alone and in interaction with other stressors. Their impact on pollinator communities can be assessed by studying multiple sentinel bee species. Here, we analysed the field exposure of three sentinel managed bee species (Apis mellifera, Bombus terrestris and Osmia bicornis) to 11 IPAs (six RNA viruses, two bacteria, three microsporidia). The sentinel bees were deployed at 128 sites in eight European countries adjacent to either oilseed rape fields or apple orchards during crop bloom. Adult bees of each species were sampled before their placement and after crop bloom. The IPAs were detected and quantified using a harmonised, high-throughput and semi-automatized qPCR workflow. We describe differences among bee species in IPA profiles (richness, diversity, detection frequencies, loads and their change upon field exposure, and exposure risk), with no clear patterns related to the country or focal crop. Our results suggest that the most frequent IPAs in adult bees are more appropriate for assessing the bees' IPA exposure risk. We also report positive correlations of IPA loads supporting the potential IPA transmission among sentinels, suggesting careful consideration should be taken when introducing managed pollinators in ecologically sensitive environments.

Pesticide use negatively affects bumble bees across European landscapes.

Sustainable agriculture requires balancing crop yields with the effects of pesticides on non-target organisms, such as bees and other crop pollinators. Field studies demonstrated that agricultural use of neonicotinoid insecticides can negatively affect wild bee species1,2, leading to restrictions on these compounds3. However, besides neonicotinoids, field-based evidence of the effects of landscape pesticide exposure on wild bees is lacking. Bees encounter many pesticides in agricultural landscapes4-9 and the effects of this landscape exposure on colony growth and development of any bee species remains unknown. Here we show that the many pesticides found in bumble bee-collected pollen are associated with reduced colony performance during crop bloom, especially in simplified landscapes with intensive agricultural practices. Our results from 316 Bombus terrestris colonies at 106 agricultural sites across eight European countries confirm that the regulatory system fails to sufficiently prevent pesticide-related impacts on non-target organisms, even for a eusocial pollinator species in which colony size may buffer against such impacts10,11. These findings support the need for postapproval monitoring of both pesticide exposure and effects to confirm that the regulatory process is sufficiently protective in limiting the collateral environmental damage of agricultural pesticide use.

Emerging threats and opportunities to managed bee species in European agricultural systems: a horizon scan.

Managed bee species provide essential pollination services that contribute to food security worldwide. However, managed bees face a diverse array of threats and anticipating these, and potential opportunities to reduce risks, is essential for the sustainable management of pollination services. We conducted a horizon scanning exercise with 20 experts from across Europe to identify emerging threats and opportunities for managed bees in European agricultural systems. An initial 63 issues were identified, and this was shortlisted to 21 issues through the horizon scanning process. These ranged from local landscape-level management to geopolitical issues on a continental and global scale across seven broad themes-Pesticides & pollutants, Technology, Management practices, Predators & parasites, Environmental stressors, Crop modification, and Political & trade influences. While we conducted this horizon scan within a European context, the opportunities and threats identified will likely be relevant to other regions. A renewed research and policy focus, especially on the highest-ranking issues, is required to maximise the value of these opportunities and mitigate threats to maintain sustainable and healthy managed bee pollinators within agricultural systems.

Specific detection and quantification of three microsporidia infecting bees, Nosema apis, Nosema ceranae, and Nosema bombi, using probe-based real-time PCR.

Among stressors affecting bee health, Nosema microsporidia are prevalent intracellular parasites. Nosema apis and Nosema ceranae have been described in honey bees (Apis spp.), while Nosema bombi has been described in bumble bees (Bombus spp.). Although available molecular methods serve as a complement to microscopic diagnosis of nosemosis, they do not enable accurate quantification of these three Nosema species. We developed three quantitative real-time PCRs (qPCRs) starting from in silico design of specific primers, probes, and recombinant plasmids, to target the RNA polymerase II subunit B1 (RPB1) gene in the three species. The complete methods, including bee grinding, DNA purification, and qPCR, were validated in honey bee (Apis mellifera) homogenate. Specificity was assessed in silico and in vitro with several types of bee samples. The limit of detection was estimated at 4 log10 copies/honey bee. A small, systematic method bias was corrected for accurate quantification up to 10 log10 copies/honey bee. Method accuracy was also verified in bumble bee (Bombus terrestris) and mason bee (Osmia bicornis) homogenates in the range of 5 to 10 log10 copies/bee. These validated qPCR methods open perspectives in nosemosis diagnosis and in the study of the parasite's eco-dynamics in managed and wild bees.

Lethal, sublethal, and combined effects of pesticides on bees: A meta-analysis and new risk assessment tools.

Multiple stressors threaten bee health, a major one being pesticides. Bees are simultaneously exposed to multiple pesticides that can cause both lethal and sublethal effects. Risk assessment and most research on bee health, however, focus on lethal individual effects. Here, we performed a systematic literature review and meta-analysis that summarizes and re-interprets the available qualitative and quantitative information on the lethal, sublethal, and combined toxicity of a comprehensive range of pesticides on bees. We provide results (1970-2019) for multiple bee species (Bombus, Osmia, Megachile, Melipona, Partamona, Scaptotrigona), although most works focused on Apis mellifera L. (78 %). Our harmonised results document the lethal toxicity of pesticides in bees (n = 377 pesticides) and the types of sublethal testing methods and related effects that cause a sublethal effect (n = 375 sublethal experiments). We identified the most common combinations of pesticides and mode of actions tested, and summarize the experimental methods, magnitude of the interactions, and robustness of available data (n = 361 experiments). We provide open access searchable, comprehensive, and integrated list of pesticides and their levels causing lethal, sublethal, and combined effects. We report major data gaps related to pesticide's sublethal (71 %) and combined (e.g., ~99 %) toxicity. We identified pesticides and mode of actions of greatest concern in terms of sublethal (chlorothalonil, pymetrozine, glyphosate; neonicotinoids) and combined (tau-fluvalinate combinations; acetylcholinesterase inhibitors and neonicotinoids) effects. Although certain pesticides have faced regulatory restrictions in specific countries (chlorothalonil, pymetrozine, neonicotinoids), most are still widely used worldwide (e.g., glyphosate). This work aims at facilitating the implementation of more comprehensive and harmonised research and risk assessments, considering sublethal and combined effects. To ensure safeguarding pollinators and the environment, we advocate for a more refined and holistic assessment that do not only focus on lethality but uses harmonised methods to test sublethal and relevant combinations.

Drivers of colony losses.

Over the past decade, in some regions of the world, honey bee (Apis mellifera L.) colonies have experienced rates of colony loss that are difficult for beekeepers to sustain. The reasons for losses are complex and interacting, with major drivers including Varroaand related viruses, pesticides, nutrition and beekeeper practices. In these endeavors it has also become apparent that defining a dead colony, and singling out the effects of specific drivers of loss, is not so straightforward. Using the class of neonicotinoid pesticides as an example we explain why quantifying risk factor impact at the colony level is at times elusive and in some cases unpractical. In this review, we discuss the caveats of defining and quantifying dead colonies. We also summarize the current leading drivers of colony losses, their interactions and the most recent research on their effects on colony mortality.

The exposure of honey bees (Apis mellifera; Hymenoptera: Apidae) to pesticides: Room for improvement in research.

Losses of honey bees have been repeatedly reported from many places worldwide. The widespread use of synthetic pesticides has led to concerns regarding their environmental fate and their effects on pollinators. Based on a standardised review, we report the use of a wide variety of honey bee matrices and sampling methods in the scientific papers studying pesticide exposure. Matrices such as beeswax and beebread were very little analysed despite their capacities for long-term pesticide storage. Moreover, bioavailability and transfer between in-hive matrices were poorly understood and explored. Many pesticides were studied but interactions between molecules or with other stressors were lacking. Sampling methods, targeted matrices and units of measure should have been, to some extent, standardised between publications to ease comparison and cross checking. Data on honey bee exposure to pesticides would have also benefit from the use of commercial formulations in experiments instead of active ingredients, with a special assessment of co-formulants (quantitative exposure and effects). Finally, the air matrix within the colony must be explored in order to complete current knowledge on honey bee pesticide exposure.

A pan-European epidemiological study reveals honey bee colony survival depends on beekeeper education and disease control.

Reports of honey bee population decline has spurred many national efforts to understand the extent of the problem and to identify causative or associated factors. However, our collective understanding of the factors has been hampered by a lack of joined up trans-national effort. Moreover, the impacts of beekeeper knowledge and beekeeping management practices have often been overlooked, despite honey bees being a managed pollinator. Here, we established a standardised active monitoring network for 5 798 apiaries over two consecutive years to quantify honey bee colony mortality across 17 European countries. Our data demonstrate that overwinter losses ranged between 2% and 32%, and that high summer losses were likely to follow high winter losses. Multivariate Poisson regression models revealed that hobbyist beekeepers with small apiaries and little experience in beekeeping had double the winter mortality rate when compared to professional beekeepers. Furthermore, honey bees kept by professional beekeepers never showed signs of disease, unlike apiaries from hobbyist beekeepers that had symptoms of bacterial infection and heavy Varroa infestation. Our data highlight beekeeper background and apicultural practices as major drivers of honey bee colony losses. The benefits of conducting trans-national monitoring schemes and improving beekeeper training are discussed.

A horizon scan of future threats and opportunities for pollinators and pollination.

Background. Pollinators, which provide the agriculturally and ecologically essential service of pollination, are under threat at a global scale. Habitat loss and homogenisation, pesticides, parasites and pathogens, invasive species, and climate change have been identified as past and current threats to pollinators. Actions to mitigate these threats, e.g., agri-environment schemes and pesticide-use moratoriums, exist, but have largely been applied post-hoc. However, future sustainability of pollinators and the service they provide requires anticipation of potential threats and opportunities before they occur, enabling timely implementation of policy and practice to prevent, rather than mitigate, further pollinator declines. Methods.Using a horizon scanning approach we identified issues that are likely to impact pollinators, either positively or negatively, over the coming three decades. Results.Our analysis highlights six high priority, and nine secondary issues. High priorities are: (1) corporate control of global agriculture, (2) novel systemic pesticides, (3) novel RNA viruses, (4) the development of new managed pollinators, (5) more frequent heatwaves and drought under climate change, and (6) the potential positive impact of reduced chemical use on pollinators in non-agricultural settings. Discussion. While current pollinator management approaches are largely driven by mitigating past impacts, we present opportunities for pre-emptive practice, legislation, and policy to sustainably manage pollinators for future generations.

Development and validation of a real-time two-step RT-qPCR TaqMan(®) assay for quantitation of Sacbrood virus (SBV) and its application to a field survey of symptomatic honey bee colonies.

Sacbrood virus (SBV) is the causal agent of a disease of honey bee larvae, resulting in failure to pupate and causing death. The typical clinical symptom of SBV is an accumulation of SBV-rich fluid in swollen sub-cuticular pouches, forming the characteristic fluid-filled sac that gives its name to the disease. Outbreaks of the disease have been reported in different countries, affecting the development of the brood and causing losses in honey bee colonies. Today, few data are available on the SBV viral load in the case of overt disease in larvae, or for the behavioural changes of SBV-infected adult bees. A two-step real-time RT-PCR assay, based on TaqMan(®) technology using a fluorescent probe (FAM-TAMRA) was therefore developed to quantify Sacbrood virus in larvae, pupae and adult bees from symptomatic apiaries. This assay was first validated according to the recent XP-U47-600 standard issued by the French Standards Institute, where the reliability and the repeatability of the results and the performance of the assay were confirmed. The performance of the qPCR assay was validated over the 6 log range of the standard curve (i.e. from 10(2) to 10(8) copies per well) with a measurement uncertainty evaluated at 0.11log10. The detection and quantitation limits were established respectively at 50 copies and 100 copies of SBV genome, for a template volume of 5µl of cDNA. The RT-qPCR assay was applied during a French SBV outbreak in 2012 where larvae with typical SBV signs were collected, along with individuals without clinical signs. The SBV quantitation revealed that, in symptomatic larvae, the virus load was significantly higher than in samples without clinical signs. Combining quantitation with clinical data, a threshold of SBV viral load related to an overt disease was proposed (10(10) SBV genome copies per individual).

Demographics of the European apicultural industry.

Over the last few years, many European and North American countries have reported a high rate of disorders (mortality, dwindling and disappearance) affecting honeybee colonies (Apis mellifera). Although beekeeping has become an increasingly professional activity in recent years, the beekeeping industry remains poorly documented in Europe. The European Union Reference Laboratory for Honeybee Health sent a detailed questionnaire to each Member State, in addition to Kosovo and Norway, to determine the demographics and state of their beekeeping industries. Based on data supplied by the National Reference Laboratory for honeybee diseases in each European country, a European database was created to describe the beekeeping industry including the number and types of beekeepers, operation size, industry production, and health (notifiable diseases, mortalities). The total number of beekeepers in Europe was estimated at 620,000. European honey production was evaluated at around 220,000 tons in 2010. The price of honey varied from 1.5 to 40 €/kg depending on the country and on the distribution network. The estimated colony winter mortality varied from 7 to 28% depending on the country and the origin of the data (institutional survey or beekeeping associations). This survey documents the high heterogeneity of the apicultural industry within the European Union. The high proportion of non-professional beekeepers and the small mean number of colonies per beekeeper were the only common characteristics at European level. The tremendous variation in European apicultural industries has implication for any comprehensive epidemiological or economic analysis of the industry. This variability needs to be taken into account for such analysis as well as for future policy development. The industry would be served if beekeeping registration was uniformly implemented across member states. Better information on the package bee and queen production would help in understanding the ability of the industry to replace lost honey bee stocks.

Improving molecular discrimination of Nosema apis and Nosema ceranae.

Nosema apis and Nosema ceranae are the causative agents of nosemosis, a contagious honeybee disease that weakens bee colonies. The species are discriminated through several PCR-based methods including a multiplex PCR recommended by the World Organization for Animal Health (OIE). In this study, the OIE protocol was compared to two other PCR protocols using different PCR kits with the same primer pairs as described in OIE. The results showed that the three PCR protocols have similar sensitivity but only the kit dedicated to multiplex PCR could detect small quantities of one Nosema species when greater quantities of the other were also present. However, singleplex PCR methods are currently the most sensitive methods for discerning each species. These results have important implications for epidemiology and the understanding of the disease.

An assessment of honeybee colony matrices, Apis mellifera (Hymenoptera: Apidae) to monitor pesticide presence in continental France.

The frequency of occurrence and relative concentration of 44 pesticides in apicultural (Apis mellifera) matrices collected from five French locations (24 apiaries) were assessed from 2002 to 2005. The number and nature of the pesticides investigated varied with the matrices examined-living honeybees, pollen loads, honey, and beeswax. Pollen loads and beeswax had the highest frequency of pesticide occurrence among the apiary matrices examined in the present study, whereas honey samples had the lowest. The imidacloprid group and the fipronil group were detected in sufficient amounts in all matrices to allow statistical comparisons. Some seasonal variation was shown when residues were identified in pollen loads. Given the results (highest frequency of presence) and practical aspects (easy to collect; matrix with no turnover, unlike with bees that are naturally renewed), pollen loads were the best matrix for assessing the presence of pesticide residues in the environment in our given conditions.

Influence of pesticide residues on honey bee (Hymenoptera: Apidae) colony health in France.

A 3-yr field survey was carried out in France, from 2002 to 2005, to study honey bee (Apis mellifera L.) colony health in relation to pesticide residues found in the colonies. This study was motivated by recent massive losses of honey bee colonies, and our objective was to examine the possible relationship between low levels of pesticide residues in apicultural matrices (honey, pollen collected by honey bees, beeswax) and colony health as measured by colony mortality and adult and brood population abundance. When all apicultural matrices were pooled together, the number of pesticide residue detected per sampling period (four sampling periods per year) and per apiary ranged from 0 to 9, with the most frequent being two (29.6%). No pesticide residues were detected during 12.7% of the sampling periods. Residues of imidacloprid and 6- chloronicotinic acid were the most frequently detected in pollen loads, honey, and honey bee matrices. Several pairs of active ingredients were present concurrently within honey bees and in pollen loads but not in beeswax and honey samples. No statistical relationship was found between colony mortality and pesticide residues. When pesticide residues from all matrices were pooled together, a mixed model analysis did not show a significant relationship between the presence of pesticide residues and the abundance of brood and adults, and no statistical relationship was found between colony mortality and pesticide residues. Thus, although certain pesticide residues were detected in apicultural matrices and occasionally with another pesticide residual, more work is needed to determine the role these residues play in affecting colony health.

Pesticide residues in beeswax samples collected from honey bee colonies (Apis mellifera L.) in France.

In 2002 a field survey was initiated in French apiaries in order to monitor the health of honey bee colonies (Apis mellifera L.). Studied apiaries were evenly distributed across five sites located in continental France. Beeswax samples were collected once a year over 2 years from a total of 125 honey bee colonies. Multiresidue analyses were performed on these samples in order to identify residues of 16 insecticides and acaricides and two fungicides. Residues of 14 of the searched-for compounds were found in samples. Tau-fluvalinate, coumaphos and endosulfan residues were the most frequently occurring residues (61.9, 52.2 and 23.4% of samples respectively). Coumaphos was found in the highest average quantities (792.6 microg kg(-1)). Residues of cypermethrin, lindane and deltamethrin were found in 21.9, 4.3 and 2.4% of samples respectively. Statistical tests showed no difference between years of sampling, with the exception of the frequency of pyrethroid residues. Beeswax contamination was the result of both in-hive acaricide treatments and, to a much lesser extent, environmental pollution.

A survey of pesticide residues in pollen loads collected by honey bees in France.

In 2002, a field survey was initiated on French apiaries to monitor weakness of honey bee, Apis mellifera L., colonies. Apiaries were evenly distributed in five sites located on continental France. Five colonies were randomly selected in each apiary, leading to a total of 125 studied honey bee colonies. For 3 yr (starting in autumn 2002), colonies were visited four times per year: after winter, before summer, during summer, and before winter. Pollen loads from traps were collected at each visit. Multiresidue analyses were performed in pollen to search residues of 36 different molecules. Specific analyses were conducted to search fipronil and metabolites and also imidacloprid and metabolites. Residues of 19 searched compounds were found in samples. Contamination by pesticides ranged from 50 to 0%. Coumaphos and tau-fluvalinate residues were the most concentrated of all residues (mean concentrations were 925.0 and 487.2 microg/kg, respectively). Fipronil and metabolite contents were superior to the limit of detection in 16 samples. Residues of fipronil were found in 10 samples. Nine samples contained the sulfone compound, and three samples contained the desulfinyl compound. Residues of imidacloprid and 6-chloronicotinic acid were found in 69% of samples. Imidacloprid contents were quantified in 11 samples with values ranging from 1.1 to 5.7 microg/kg. 6-Chloronicotinic acid content was superior to the limit of quantification in 28 samples with values ranging from 0.6 to 9.3 microg/kg. Statistical tests showed no difference between places of sampling with the exception of fipronil. Possible origins of these contaminations, concentration and toxicity of pesticides, and the possible consequences for bees are discussed.

Experimental study on the toxicity of imidacloprid given in syrup to honey bee (Apis mellifera) colonies.

Two groups of eight honey bee colonies were fed with two different concentrations of imidacloprid in saccharose syrup during summer (each colony was given 1 litre of saccharose syrup containing 0.5 microg litre(-1) or 5 microg litre(-1) of imidacloprid on 13 occasions). Their development and survival were followed in parallel with control hives (unfed or fed with saccharose syrup) until the end of the following winter. The parameters followed were: adult bee activity (number of bee entering the hive and pollen carrying activity), adult bee population level, capped brood area, frequency of parasitic and other diseases, mortality, number of frames with brood after wintering and a global score of colonies after wintering. The only parameters linked to feeding with imidacloprid-supplemented saccharose syrup when compared with feeding with non-supplemented syrup were: a statistically non-significant higher activity index of adult bees, a significantly higher frequency of pollen carrying during the feeding period and a larger number of capped brood cells. When imidacloprid was no longer applied, activity and pollen carrying were re-established at a similar level for all groups. Repeated feeding with syrup supplemented with imidacloprid did not provoke any immediate or any delayed mortality before, during or following the next winter, whereas such severe effects are described by several French bee keepers as a consequence of imidacloprid use for seed dressing in neighbouring cultures. In any case, during the whole study, mortality was very low in all groups, with no difference between imidacloprid-fed and control colonies. Further research should now address several hypotheses: the troubles described by bee keepers have causes other than imidacloprid; if such troubles are really due to this insecticide, they may only be observed either when bees consume contaminated pollen, when no other sources of food are available, in the presence of synergic factors (that still need to be identified), with some particular races of bees or when colonies are not strong and healthy.