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.

Drivers and pressures behind insect decline in Central and Western Europe based on long-term monitoring data.

Insect declines have been discussed intensively among experts, policymakers, and the public. Albeit, decreasing trends have been reported for a long time for various regions in Europe and North America, but the controversial discussion over the role of specific drivers and pressures still remains. A reason for these uncertainties lies within the complex networks of inter-dependent biotic and abiotic factors as well as anthropogenic activities that influence habitats, communities, populations, and individual organisms. Many recent publications aim to identify both the extent of the observed declines and potential drivers. With this literature analysis, we provide an overview of the drivers and pressures and their inter-relationships, which were concluded in the scientific literature, using some of the best-studied insect groups as examples. We conducted a detailed literature evaluation of publications on Carabidae (Coleoptera) and Lepidoptera trends with data for at least 6 years in countries of Central and Western Europe, with a focus on agricultural landscapes. From the 82 publications identified as relevant, we extracted all reported trends and classified the respective factors described according to the DPSIR model. Further, we analysed the level of scientific verification (presumed vs correlated vs examined) within these papers for these cited stressors. The extracted trends for both species groups underline the reported overall declining trend. Whether negative or positive trends were reported in the papers, our semi-quantitative analysis shows that changes in insect populations are primarily anthropogenically driven by agriculture, climate change, nature conservation activities, urbanisation, and other anthropogenic activities. Most of the identified pressures were found to act on habitat level, only a fraction attributed to direct effects to the insects. While our analysis gives an overview of existing research concerning abundance and biodiversity trends of carabids and lepidopterans, it also shows gaps in scientific data in this area, in particular in monitoring the pressures along with the monitoring of abundance trends. The scientific basis for assessing biodiversity changes in the landscape is essential to help all stakeholders involved to shape, e.g. agriculture and other human activities, in a more sustainable way, balancing human needs such as food production with conservation of nature.

Acute toxicity of pesticide mixtures to honey bees is generally additive, and well predicted by Concentration Addition.

Understanding the frequency of non-additive effects of pesticides (synergism and antagonism) is important in the context of risk assessment. The goal of this study was to investigate the prevalence of non-additive effects of pesticides to honey bees (Apis mellifera). We investigated a large set of mixtures including insecticides and fungicides of different chemical modes of action and classes. The mixtures included represent a relevant sample of pesticides that are currently used globally. We investigated whether the experimental toxicity of the mixtures could be predicted based on the Concentration Addition (CA) model for acute contact and oral adult bee toxicity tests. We measured the degree of deviation from the additivity predictions of the experimental toxicity based on the well-known Mixture Deviation Ratio (MDR). Further, we investigated the appropriate MDR thresholds that should be used for the identification of non-additive effects based on acceptable rates for false positive (alpha) and true positive (beta) findings. We found that a deviation factor of MDR = 5 is a sound reference for labeling potential non-additive effects in acute adult bee experimental designs when assuming a typical Coefficient of Variation (CV%) = 100 in the determination of the LD50 of a pesticide (a factor of 2× deviation in the LD 50 resulting from inter-experimental variability). We found that only 2.4 % and 9 % of the mixtures evaluated had an MDR > 5 and MDR < 0.2, respectively. The frequency and magnitude of deviation from additivity found for bees in this study are consistent with those of other terrestrial and aquatic taxa. Our findings suggest that additivity is a good baseline for predicting the toxicity of pesticide mixtures to bees, and that the rare cases of synergy of pesticide mixtures to bees are not random but have a mechanistic basis.

A toxicogenomics approach reveals characteristics supporting the honey bee (Apis mellifera L.) safety profile of the butenolide insecticide flupyradifurone.

Flupyradifurone, a novel butenolide insecticide, selectively targets insect nicotinic acetylcholine receptors (nAChRs), comparable to structurally different insecticidal chemotypes such as neonicotinoids and sulfoximines. However, flupyradifurone was shown in acute toxicity tests to be several orders of magnitude less toxic to western honey bee (Apis mellifera L.) than many other insecticides targeting insect nAChRs. The underlying reasons for this difference in toxicity remains unknown and were investigated here. Pharmacokinetic studies after contact application of [14C]flupyradifurone to honey bees revealed slow uptake, with internalized compound degraded into a few metabolites that are all practically non-toxic to honey bees in both oral and contact bioassays. Furthermore, receptor binding studies revealed a lack of high-affinity binding of these metabolites to honey bee nAChRs. Screening of a library of 27 heterologously expressed honey bee cytochrome P450 enzymes (P450s) identified three P450s involved in the detoxification of flupyradifurone: CYP6AQ1, CYP9Q2 and CYP9Q3. Transgenic Drosophila lines ectopically expressing CYP9Q2 and CYP9Q3 were significantly less susceptible to flupyradifurone when compared to control flies, confirming the importance of these P450s for flupyradifurone metabolism in honey bees. Biochemical assays using the fluorescent probe substrate 7-benzyloxymethoxy-4-(trifluoromethyl)-coumarin (BOMFC) indicated a weak, non-competitive inhibition of BOMFC metabolism by flupyradifurone. In contrast, the azole fungicides prochloraz and propiconazole were strong nanomolar inhibitors of these flupyradifurone metabolizing P450s, explaining their highly synergistic effects in combination with flupyradifurone as demonstrated in acute laboratory contact toxicity tests of adult bees. Interestingly, the azole fungicide prothioconazole is only slightly synergistic in combination with flupyradifurone - an observation supported by molecular P450 inhibition assays. Such molecular assays have value in the prediction of potential risks posed to bees by flupyradifurone mixture partners under applied conditions. Quantitative PCR confirmed the expression of the identified P450 genes in all honey bee life-stages, with highest expression levels observed in late larvae and adults, suggesting honey bees have the capacity to metabolize flupyradifurone across all life-stages. These findings provide a biochemical explanation for the low intrinsic toxicity of flupyradifurone to honey bees and offer a new, more holistic approach to support bee pollinator risk assessment by molecular means.

Flower strip networks offer promising long term effects on pollinator species richness in intensively cultivated agricultural areas.

BACKGROUND: Intensively cultivated agricultural landscapes often suffer from substantial pollinator losses, which may be leading to decreasing pollination services for crops and wild flowering plants. Conservation measures that are easy to implement and accepted by farmers are needed to halt a further loss of pollinators in large areas under intensive agricultural management. Here we report the results of a replicated long-term study involving networks of mostly perennial flower strips covering 10% of a conventionally managed agricultural landscape in southwestern Germany. RESULTS: We demonstrate the considerable success of these measures for wild bee and butterfly species richness over an observation period of 5 years. Overall species richness of bees and butterflies but also the numbers of specialist bee species clearly increased in the ecological enhancement areas as compared to the control areas without ecological enhancement measures. A three to five-fold increase in species richness was found after more than 2 years of enhancement of the areas with flower strips. Oligolectic bee species increased significantly only after the third year. CONCLUSIONS: In our long-term field experiment we used a large variety of seed mixtures and temporal variation in seeding time, ensured continuity of the flower-strips by using perennial seed mixtures and distributed the measures over c. 10% of the landscape. This led to an increase in pollinator abundance, suggesting that these measures may be instrumental for the successful support of pollinators. These measures may ensure the availability of a network of diverse habitats and foraging resources for pollinators throughout the year, as well as nesting sites for many species. The measures are applied in-field and are suitable for application in areas under intensive agriculture. We propose that flower strip networks should be implemented much more in the upcoming CAP (common agricultural policy) reform in the European Union and promoted more by advisory services for farmers.

A long-term field study on the effects of dietary exposure of clothianidin to varroosis-weakened honey bee colonies.

Clothianidin is a commonly used systemic insecticide in seed treatments. Residues of clothianidin can occur in nectar and pollen as a result of within-plant-translocation. Foraging bees can collect contaminated nectar or pollen. Concerns have been brought forward that exposure to pesticide residues might affect colonies especially if they are weakened by varroosis. However, there are few scientific studies investigating such multiple-stressor scenarios in the context of the entire colony. To close this gapa field trial with 24 colonies was set up. The study design comprised four groups of six colonies each fed with uncontaminated sugar syrup ('C0'), or syrup spiked with 10 µg L-1 clothianidin ('C10'), 50 µg L-1 clothianidin ('C50') or 200 µg L-1 clothianidin ('C200'). C10 represented a residue concentration that may exceptionally occur and therefore a worst-case scenario, the higher dietary concentrations exceed and do not reflect fieldrealistic levels. A substantial load of 8 mites of Varroa destructor per ten gram bees in autumn was adjusted. The colonies were followed up for 328 days. The amount of brood and the strength of each colony were regularly assessed. Colony health, bee mortality, overwintering success, hive weights, and levels of in-hive residues were determined. Varroosis turned out to be the significant key factor for the endpoint colony strength. Clothianidin did not have a statistically significant impact on C0, C10 and C50 colonies. No statistical evidence was found for an interaction between varroosis andexposure to clothianidin.

Investigation of full-scale ozonation at a municipal wastewater treatment plant using a toxicity-based evaluation concept.

Effluents from municipal wastewater treatment plants (WWTPs) are known to be point sources of micropollutants for surface waters. The aim of this study was to examine a reconstructed full-scale ozonation equipped with a pump-injector system for ozone (O3) dosage and a fluidized moving-bed reactor as biological posttreatment at a municipal WWTP utilizing an effect-directed approach. This approach consists of chemical analysis in combination with toxicological tests for the assessment of treatment efficiency of the plant. Chemical analysis showed elimination rates > 80% for pharmaceuticals and industrial chemicals. Analysis of endocrine disruptors was limited due to substance concentrations below the limit of detection (LOD). Estrogenic activity was detected by the Arxula Adeninivorans yeast estrogen screen (A-YES) at low concentrations (pg to ng EEQ/l range). Estrogenic activity was reduced by more than 90% after ozonation. In contrast, androgenic activity (measured in the Adeninivorans yeast androgen screen, A-YAS) was still found after O3 treatment and after biological posttreatment, which is consistent with the data obtained by chemical analysis. Furthermore, no marked genotoxic or cytotoxic effects were observed after ozonation using the alkaline comet and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid (MTT) assays, respectively. Results suggest that the applied specific O3 dose of 0.4 mgO3/mgDOC is a safe operation setup in terms of toxicologically relevant transformation products. In addition, no adverse effects on primary producers, as evidenced by algae growth inhibition tests, were detected. The monitored biofilm growth in the biological posttreatment exhibited a steady state after one month. Based on computational fluid dynamics (CFD) simulations and biomass, one might conclude that O3 did not apparently enter biological posttreatment to a great extent and that hydraulic retention time in the O3 reactor was sufficient. Our data demonstrate the effectiveness of a full-scale O3 treatment in combination with a fluidized moving-bed reactor as biological posttreatment for the reduction of a majority of micropollutants without the release of relevant toxic transformation products as assessed by a chemical and toxicity-based approach.

Performance of honey bee colonies under a long-lasting dietary exposure to sublethal concentrations of the neonicotinoid insecticide thiacloprid.

BACKGROUND: Substantial honey bee colony losses have occurred periodically in the last decades. The drivers for these losses are not fully understood. The influence of pests and pathogens are beyond dispute, but in addition, chronic exposure to sublethal concentrations of pesticides has been suggested to affect the performance of honey bee colonies. This study aims to elucidate the potential effects of a chronic exposure to sublethal concentrations (one realistic worst-case concentration) of the neonicotinoid thiacloprid to honey bee colonies in a three year replicated colony feeding study. RESULTS: Thiacloprid did not significantly affect the colony strength. No differences between treatment and control were observed for the mortality of bees, the infestation with the parasitic mite Varroa destructor and the infection levels of viruses. No colony losses occurred during the overwintering seasons. Furthermore, thiacloprid did not influence the constitutive expression of the immunity-related hymenoptaecin gene. However, upregulation of hymenoptaecin expression as a response to bacterial challenge was less pronounced in exposed bees than in control bees. CONCLUSION: Under field conditions, bee colonies are not adversely affected by a long-lasting exposure to sublethal concentrations of thiacloprid. No indications were found that field-realistic and higher doses exerted a biologically significant effect on colony performance. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Active zone proteins are dynamically associated with synaptic ribbons in rat pinealocytes.

Synaptic ribbons (SRs) are prominent organelles that are abundant in the ribbon synapses of sensory neurons where they represent a specialization of the cytomatrix at the active zone (CAZ). SRs occur not only in neurons, but also in neuroendocrine pinealocytes where their function is still obscure. In this study, we report that pinealocyte SRs are associated with CAZ proteins such as Bassoon, Piccolo, CtBP1, Munc13-1, and the motorprotein KIF3A and, therefore, consist of a protein complex that resembles the ribbon complex of retinal and other sensory ribbon synapses. The pinealocyte ribbon complex is biochemically dynamic. Its protein composition changes in favor of Bassoon, Piccolo, and Munc13-1 at night and in favor of KIF3A during the day, whereas CtBP1 is equally present during the night and day. The diurnal dynamics of the ribbon complex persist under constant darkness and decrease after stimulus deprivation of the pineal gland by constant light. Our findings indicate that neuroendocrine pinealocytes possess a protein complex that resembles the CAZ of ribbon synapses in sensory organs and whose dynamics are under circadian regulation.

The relevance of sublethal effects in honey bee testing for pesticide risk assessment.

The option of an evaluation and assessment of possible sublethal effects of pesticides on bees has been a subject of discussion by scientists and regulatory authorities. Effects considered included learning behaviour and orientation capacity. This discussion was enhanced by the French bee issue and allegations against systemic insecticides that were linked to the hypothesis that sublethal intoxication might even have led to reported colony losses. This paper considers whether and, if so, how sublethal effects should be incorporated into risk assessment, by addressing a number of questions: What is meant by a sublethal effect? Which sublethal effects should be measured, when and how? How are sublethal effects to be included in risk assessments? 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.

Influence of photoperiodic history on clock genes and the circadian pacemaker in the rat retina.

The influence of seasonal lighting conditions on expression of clock genes and the circadian pacemaker was investigated in the rat retina. For this purpose, the 24-h profiles of nine clock genes (bmal1, clock, per1, per2, per3, dec1, dec2, cry1 and cry 2) and the arylalkylamine N-acetyltransferase gene as an indicator of the circadian pacemaker output were compared between light-dark periods of 8 : 16 and 16 : 8 h. The photoperiod influenced the daily patterns of the amount of transcript for per1, per3, dec2 and arylalkylamine N-acetyltransferase. This indicates that photoperiodic information modulates clock gene expression in addition to the circadian pacemaker of the retina. Under constant darkness, photoperiod-dependent changes in the daily profile of the level of transcript persisted for the arylalkylamine N-acetyltransferase gene but not for any of the clock genes. Hence, quantitative expression of each clock gene is influenced by the photoperiod only under the acute light-dark cycle, whereas the pacemaker is capable of storing photoperiodic information from past cycles.