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In agricultural ecosystems, bees are exposed to combinations of pesticides that may have been applied at different times. For example, bees visiting a flowering crop may be chronically exposed to low concentrations of systemic insecticides applied before bloom and then to a pulse of fungicide, considered safe for bees, applied during bloom. In this study, we simulate this scenario under laboratory conditions with females of the solitary bee, Osmia bicornis L. We studied the effects of chronic exposure to the neonicotinoid insecticide, Confidor® (imidacloprid) at a realistic concentration, and of a pulse (1 day) exposure of the fungicide Folicur® SE (tebuconazole) at field application rate. Syrup consumption, survival, and four biomarkers: acetylcholinesterase (AChE), carboxylesterase (CaE), glutathione S-transferase (GST), and alkaline phosphatase (ALP) were evaluated at two different time points. An integrated biological response (IBRv2) index was elaborated with the biomarker results. The fungicide pulse had no impact on survival but temporarily reduced syrup consumption and increased the IBRv2 index, indicating potential molecular alterations. The neonicotinoid significantly reduced syrup consumption, survival, and the neurological activity of the enzymes. The co-exposure neonicotinoid-fungicide did not increase toxicity at the tested concentrations. AChE proved to be an efficient biomarker for the detection of early effects for both the insecticide and the fungicide. Our results highlight the importance of assessing individual and sub-individual endpoints to better understand pesticide effects on bees.

The recent EU ban of the three most widely used neonicotinoids (imidacloprid, thiamethoxam and clothianidin) to all outdoors applications has stimulated the introduction of new insecticides into the market. Sulfoxaflor is a new systemic insecticide that, like neonicotinoids, acts as a modulator of nicotinic acetylcholine receptors. In agro-environments, bees can be exposed to this compound via contaminated pollen and nectar for long periods of time. Therefore, it is important to assess the potential effects of chronic exposure to sulfoxaflor, alone and in combination with fungicides, on pollinators. In this study, we tested the effects of chronic exposure to two field concentrations of sulfoxaflor (20 and 100 ppb) alone and in combination with four concentrations of the fungicide fluxapyroxad (7500, 15,000, 30,000 and 60,000 ppb) on syrup consumption and longevity in females of the solitary bee Osmia bicornis L. Exposure to 20 ppb of sulfoxaflor, alone and in combination with the fungicide, stimulated syrup consumption, but did not affect longevity. In contrast, syrup consumption decreased in bees exposed to 100 ppb, all of which died after 2–6 days of exposure. We found no evidence of synergism between the two compounds at any of the two sulfoxaflor concentrations tested. Comparison of our findings with the literature, confirms that O. bicornis is more sensitive to sulfoxaflor than honey bees. Our results highlight the need to include different bee species in risk assessment schemes., This study was supported by a grant from the University of Bologna to FS (Grant RFO2019_SGOLASTRA), the Spanish Ministry of Science, Innovation and Universities (Grant R+D RTI2018-098399-B-I00) and the postdoctoral fellowship from the French Association POLLINIS (www.pollinis.org) to CA.

The Thematic Group “Plant Health” (TG-PH) of DISTAL, established as part of the “Departments of Excellence” project, has been active since October 2020. To date, 13 research groups with over 40 people including teaching staff, researchers, technicians, postdocs and doctoral students have joined the TG-PH. The common goal of the TG’s activities is to protect and enhance the role of “Plant Health” with a plurality of approaches and specific activities which include: (i) the development of strategies to prevent, monitor and control plant pests; (ii) new agronomic practices and plant nutrition approaches to reduce the impact on ecosystems; (iii) the selection and characterization of new varieties more resilient to biotic/abiotic stresses; (iv) the implementation of sustainable agricultural and food policies. Most of these activities are consistent with initiatives to effectively address major global policy issues such as reducing the use of pesticides and fertilizers, increasing biodiversity in order to ensure healthy, affordable and sustainable food supply to society compliant with food quality and safety. The high interdisciplinarity of the TG-PH is ensured by the wide range of scientific sectors to which the research groups involved belong. The common commitment is to pursue the sustainability objectives of the European Green Deal by bridging the gap between research and practice, accelerating the process of transferring research results and following up with networking. Moreover, TG-PH members share the common belief that only a stronger synergy between this knowledge and expertise coupled with a strong public-private partnership (PPP) will allow for the translation of the results of basic research into concrete solutions useful for the various stakeholders and society at large.

Bees and other pollinators play vital roles in biodiversity and food security, and they are a source of income and inspiration. Yet, bees’ biodiversity and abundance are decreasing every day. Their decline, driven by pesticides (1–3), poses serious threats to the environment, ecosystems, and human health. The European pesticide authorization framework states that a pesticide can only be put on the market if it has no harmful effects on human health or animal health and no unacceptable effects on the environment (4). EU ministries met in June to decide what constitutes an “acceptable” effect of a pesticide on bees (5). Despite scientific evidence indicating that the current level of protection is barely adequate, they tentatively agreed to provide even less protection moving forward (5). We call on EU governments to apply the maximum level of protection of pollinators from pesticides (6).

In many countries, the Western honey bee is used as surrogate in pesticide risk assessments for bees. However, uncertainty remains in the estimation of pesticide risk to non-Apis bees because their potential routes of exposure to pesticides, life histories, and ecologies differ from honey bees. We applied the vulnerability concept in pesticide risk assessment to 10 bee species including the honey bee, two bumble bee species, and seven solitary bee species with different nesting strategies. The trait-based vulnerability considers the evaluation of a species both at the level of the organism (exposure and effect) and the population (recovery) that goes beyond the sensitivity of individuals to a toxicant assessed in standard laboratory toxicity studies by including effects on populations in the field. Based on expert judgement, each trait was classified by its relationship to the vulnerability to pesticide exposure, effects (intrinsic sensitivity), and population recovery. The results suggested that the non-Apis bees included in our approach are potentially more vulnerable to pesticides than the honey bee due to traits governing exposure and population recovery potential. Our analysis highlights many uncertainties related to the interaction between bee ecology and the potential exposures and population-level effects of pesticides, emphasizing the need for more research to identify suitable surrogate species for higher-tier bee risk assessments. This article is protected by copyright. All rights reserved.

CONTEXT There is growing evidence that agroecology can reconcile the environmental, economic, and social pillars of agricultural sustainability. However, teaching and learning agroecology is challenging, especially since most agricultural graduate programs in Europe are not adapted to teach the diversity of its related practices. OBJECTIVE To improve agroecology learning, we built the online simulation game SEGAE. This article illustrates the game's relevance for learning agroecology. METHODS The game is based on a modeling framework that gamifies the implementation of agroecological practices in an integrated crop-livestock farm and assesses their impacts on sustainability. To do so, SEGAE is based on an output-oriented approach that represents impacts of practices on various indicators. These impacts are included in a matrix, which is associated with a dynamic graphical interface accessible to players. Two examples of game sessions were developed to illustrate the game's potential. RESULTS AND CONCLUSIONS In the first example, players can gain knowledge about agroecological practices by implementing practices that improve soil quality and assessing their impacts on sustainability. Results of this example place the farm's improved overall sustainability into perspective with its reduced food production potential. In the second example, players can improve their skills in transition management and acquire a systems approach by converting the farm to organic farming within five years. Results of this example prompt discussion of the steps needed to obtain organic certification and the coherence between crop and animal production needed to foster sustainability. SIGNIFICANCE SEGAE was designed to strengthen European training in agroecology, and active contributions from users would help to improve this tool, extend it to new farming systems and forge connections within the community of teachers working on agroecology.

The sulfoximine insecticide sulfoxaflor is regarded as a potential substitute for neonicotinoids that were recently banned in the EU due to their side effects on bees. Like neonicotinoids, sulfoxaflor acts as a competitive modulator of nicotinic acetylcholine receptors. In agricultural environments, bees are commonly exposed to combinations of pesticides, and neonicotinoids are known to interact synergistically with fungicides. The objective of our study is to assess the acute oral toxicity of sulfoxaflor alone and in combination with a single dose of fluxapyroxad, a succinate dehydrogenase inhibitor (SDHI) fungicide, in three bee species:Apis mellifera,Bombus terrestrisandOsmia bicornis. Because synergism may be dose-dependent, we tested a range of sulfoxaflor doses. Synergistic effects were assessed using three different approaches: Bliss criterion of drugs independence, ratio test comparing LD50s and model deviation ratio.Osmia bicorniswas the most sensitive species to sulfoxaflor and bothO. bicornisandA. melliferashowed significant synergism between the insecticide and the fungicide. For the most part, these synergistic effects were weak and only occurred at early assessment times and intermediate sulfoxaflor doses. The potential ecological relevance of these effects should be confirmed in field and/or cage studies. Overall, our laboratory results demonstrate that sulfoxaflor is somewhat less toxic than the recently banned neonicotinoids imidacloprid, thiamethoxam and clothianidin, but much more toxic than other neonicotinoids (acetamiprid, thiacloprid) still in use in the EU at the time this study was conducted.

Agroecology represents a pertinent option to improve the sustainability of agriculture. To promote its application, agroecological concepts should be taught to students and professionals in the agricultural sector. However, most agricultural courses are not adapted to teach these concepts due to little interactivity or interdisciplinarity, and a lack of a systems approach to farm management. Serious games help to fill these gaps by simulating complex models in which players can learn by doing. We thus developed a serious computer game, called SEGAE (SErious Game for AgroEcology learning), which represents a mixed crop&ndash, livestock farm and assesses impacts of farming practices on indicators related to environmental, economic, and social sustainability. Its pedagogical interest was evaluated through two types of surveys given to university students who played the game during a one-week workshop: A knowledge survey on agroecology, and a feedback survey based on flow theory. Results showed that students increased their knowledge of agroecology significantly, particularly those who had had little knowledge of crop production. More than 86% of the students enjoyed the game, appreciating its interaction and feedback. Thus, SEGAE is an interesting tool to help students acquire knowledge of agroecology in a fun way by facilitating interdisciplinary and collaborative learning.

Honey bee mortality and colony losses have been reported worldwide. Although this phenomenon is caused by a combination of factors, agrochemicals have received special attention due to their potential effects on bees. In agricultural and urban environments bees are exposed to several compounds that may interact in unexpected ways, but information on the extent of pesticide exposure remains unclear. Several monitoring studies have been conducted to evaluate the field-realistic exposure of bees to pesticides after their release on the market. However, their outputs are difficult to compare and harmonize due to differences in the analytical methodologies and the sampling protocols (e.g. number of screened compounds and analysed samples, and detection limits (LODs)). Here, we hypothesize that the analytical methodologies used in the monitoring studies may strongly affect the pesticide occurrences in pollen underestimating the real pesticide exposure. By mean of a systematic literature review, we have collected relevant information on pesticide contaminations in the honey bee-collected pollen. Our findings showed that the pesticide occurrences were associated with the analytical methodologies and the real pesticide exposure has likely been underestimated in some monitoring studies. For four highly toxic compounds, the LOD used in these monitoring studies exceeded the doses that cause toxic effects on honey bees. We recommend that, especially for the highly toxic compounds, the LODs used in the monitoring studies should be low enough to exclude lethal or sublethal effects on bees and avoid “false negative” samples.

Neonicotinoid insecticides have been signaled as an important driver of widespread declines in bee diversity and abundance. Neonicotinoids were registered in the 1990s and by 2010 accounted for one third of the global insecticide market. Following a moratorium in 2013, their use on open-field crops was completely banned in the EU in 2018. Pesticide regulation should be based on solid and updated scientific evidence, whereby products showing unacceptable effects on the environment are not approved. Clearly, pesticide regulation failed to detect the ecological threats posed by neonicotinoids. We argue that at the time neonicotinoids were authorized, risk assessment (RA) protocols were inadequate to detect some of the risks associated with neonicotinoid properties, including high efficacy, long persistence, high systemicity, high mobility, and application versatility. We advocate for the adoption of a more holistic RA approach that should account for: a) temporal and spatial dimensions of pesticide exposure; b) co-exposure to multiple compounds; c) differences among bee species with different life histories in levels of exposure and sensitivity; and d) sublethal effects (mostly ignored in current RA procedures). We also argue that regulatory studies conducted to support pesticide registration should be publicly available, and that pesticide regulation should not be discontinued once a product has been authorized. We should use the knowledge acquired through the neonicotinoid experience as an opportunity to profoundly revise bee RA schemes. These efforts should be initiated promptly; the neonicotinoid story has also taught us that the regulatory system is reluctant to react.

Besides the wind, insects are the main pollinating agents and many of them are hymenopterans. However, among the 20,000 species of bees (Superfamily Apoidea), only a very restricted group has been domesticated for commercial crop pollination, including social and solitary species. The honey bee Apis mellifera L. has been the first domesticated pollinator species, anyway, many cultivated crops benefit largely from the activity of other pollinators, especially when honey bees cannot provide a sufficient pollination service. This occurrence is particularly frequent in crops blooming in early spring or in crops with flowers not enough attractive to honey bees, i.e. because of low nectar and pollen production or for particular flower shapes. In addition to these ecological aspects, which are the result of a long history of coevolution between insects and flowers, modern agriculture and the well-known difficulties that honey bees are suffering of, create new concern that a general decline in the native pollinator populations will have unavoidable consequences on crop production due to insufficient pollination service. The most striking contradiction of the “industrial” extensive monocultures is that on one side they put in forth the need of enormous number of pollinators to satisfy the vast number of flowers contemporarily ready to be pollinated; on the other side, they cancel from the agroecosystem uncultivated meadows, edges and forests, which serve as refuge and conservation areas. The immediate repercussion on pollinating insects is the lack of continuity in blooms: on large areas, flowers are extremely abundant during the short blooming period of the main crops and nearly almost absent in the remaining part of their reproductive season (what is called “green desert”). In addition, very often a dramatic reduction of adequate nesting sites and materials may strongly limit the reproductive success, while, up today, the knowledge about the acute and chronic effects of pesticides on native bees is still insufficient.

Solitary bees are frequently exposed to pesticides, which are considered as one of the main stress factors that may lead to population declines. A strong immune defence is vital for the fitness of bees. However, the immune system can be weakened by environmental factors that may render bees more vulnerable to parasites and pathogens. Here we demonstrate for the first time that field-realistic concentrations of the commonly used neonicotinoid insecticide thiacloprid can severely affect the immunocompetence of Osmia bicornis. In detail, males exposed to thiacloprid solutions of 200 and 555 µg/kg showed a reduction in hemocyte density. Moreover, functional aspects of the immune defence - the antimicrobial activity of the hemolymph - were impaired in males. In females, however, only a concentration of 555 µg/kg elicited similar immunosuppressive effects. Although males are smaller than females, they consumed more food solution. This leads to a 2.77 times higher exposure in males, probably explaining the different concentration thresholds observed between the sexes. In contrast to honeybees, dietary exposure to thiacloprid did not affect melanisation or wound healing in O. bicornis. Our results demonstrate that neonicotinoid insecticides can negatively affect the immunocompetence of O. bicornis, possibly leading to an impaired disease resistance capacity.

Current pesticide risk assessment for bees relies on a single (social) species, the western honey bee, Apis mellifera L. (Hymenoptera: Apidae). However, most of the >20,000 bee species worldwide are solitary. Differences in life history traits between solitary bees (SB) and honey bees (HB) are likely to determine differences in routes and levels of pesticide exposure. The objectives of this review are to: 1) compare SB and HB life history traits relevant for risk assessment; 2) summarize current knowledge about levels of pesticide exposure for SB and HB; 3) identify knowledge gaps and research needs; 4) evaluate whether current HB risk assessment schemes cover routes and levels of exposure of SB; and 5) identify potential SB model species for risk assessment. Most SB exposure routes seem well covered by current HB risk assessment schemes. Exceptions to this are exposure routes related to nesting substrates and nesting materials used by SB. Exposure via soil is of particular concern because most SB species nest underground. Six SB species (Hymenoptera: Megachilidae - Osmia bicornis L., O. cornifrons Radoszkowski, O. cornuta Latreille, O. lignaria Say, Megachile rotundata F., and Halictidae - Nomia melanderi Cockerell) are commercially available and could be used in risk assessment. Of these, only N. melanderi nests underground, and the rest are cavity-nesters. However, the three Osmia species collect soil to build their nests. Life history traits of cavity-nesting species make them particularly suitable for semifield and, to a lesser extent, field tests. Future studies should address basic biology, rearing methods and levels of exposure of ground-nesting SB species.

Several anthropogenic contaminants, including pesticides and heavy metals, can affect honey bee health. The effects of mixtures of heavy metals and pesticides are rarely studied in bees, even though bees are likely to be exposed to these contaminants in both agricultural and urban environments. In this study, the lethal toxicity of Cr alone and in combination with the neonicotinoid insecticide clothianidin and the ergosterol-biosynthesis-inhibiting fungicide propiconazole was assessed in Apis mellifera adults. The LD 50 and lowest benchmark dose of Cr as Cr(NO 3 ) 3 , revealed a low acute oral toxicity on honey bee foragers (2049 and 379 mg L −1 , respectively) and the Cr retention ( i.e. bee ability to retain the heavy metal in the body) was generally low compared to other metals. A modified method based on the binomial proportion test was developed to analyse synergistic and antagonistic interactions between the three tested contaminants. The combination of an ecologically-relevant field concentration of chromium with clothianidin and propiconazole did not increase bee mortality. On the contrary, the presence of Cr in mixture with propiconazole elicited a slight antagonistic effect.

Pesticide use is one of the main causes of pollinator declines in agricultural ecosystems. Traditionally, most laboratory studies on bee ecotoxicology test acute exposure to single compounds. However, under field conditions, bees are often chronically exposed to a variety of chemicals, with potential synergistic effects. We studied the effects of field-realistic concentrations of three pesticides measured in pollen and nectar of commercial melon fields on the solitary bee Osmia bicornis L. We orally exposed females of this species throughout their life span to 8 treatments combining two neonicotinoid insecticides (acetamiprid, imidacloprid) and a triazole fungicide (myclobutanil) via pollen and sugar syrup. We measured pollen and syrup consumption, longevity, ovary maturation and thermogenesis. Although bees consumed larger amounts of syrup than pollen, pesticide intake via syrup and pollen were similar. At the tested concentrations, no synergistic effects emerged, and we found no effects on longevity and ovary maturation. However, all treatments containing imidacloprid resulted in suppressed syrup consumption and drastic decreases in thoracic temperature and bee activity. Our results have important implications for pesticide regulation. If we had measured only lethal effects we would have wrongly concluded that the pesticide combinations containing imidacloprid were safe to O. bicornis. The incorporation of tests specifically intended to detect sublethal effects in bee risk assessment schemes should be an urgent priority. In this way, the effects of pesticide exposure on the dynamics of bee populations in agroecosystems will be better assessed.

International audience; This article presents data designed by European researchers who performed a literature review and interpreted the results to determine impact factors of many agroecological practices on a wide variety of sustainability indicators. The impact factors are represented in a matrix that connects practices to indicators. The indicators are related to environmental, economic and social sustainability of a typical European integrated crop-livestock farm. The data are included in the serious game SEGAE to learn agroecology, as described in “SEGAE: a serious game to learn agroecology” [1]. The data can be modified to adapt the game to other agricultural systems. Finally, the data can be re-used in research projects as a basis to assess impacts of agroecological practices.

Simple Summary Methods to rear Osmia bees to pollinate fruit trees have been developed in various parts of the world. These bees are excellent pollinators but evidence that their populations can be sustained in orchards and their use results in increased fruit production is scarce. We released an Osmia cornuta population at one end of an almond orchard. Then, we surveyed the pollinators visiting the almond flowers and measured fruit set in trees located at increasing distances from the nesting stations. We found that fruit production was higher in the trees that received more Osmia visits. Importantly, this result was obtained against a strong background of honeybees, which were 10 times more abundant than Osmia. The Osmia population obtained at the end of the flowering period was 1.28 larger than the population initially released. Our study demonstrates that Osmia populations can be sustained in orchard environments and that even a small population of a highly effective pollinator may have a significant impact on fruit set. Our results are encouraging for the use of Osmia populations and for the implementation of measures to promote wild pollinators in agricultural environments. Abstract Osmia spp. are excellent orchard pollinators but evidence that their populations can be sustained in orchard environments and their use results in increased fruit production is scarce. We released an Osmia cornuta population in an almond orchard and measured its population dynamics, as well as visitation rates and fruit set at increasing distances from the nesting stations. Honeybees were 10 times more abundant than O. cornuta. However, the best models relating fruit set and bee visitation included only O. cornuta visitation, which explained 41% and 40% of the initial and final fruit set. Distance from the nesting stations explained 27.7% and 22.1% of the variability in initial and final fruit set. Of the 198 females released, 99 (54.4%) established and produced an average of 9.15 cells. Female population growth was 1.28. By comparing our results with those of previous O. cornuta studies we identify two important populational bottlenecks (female establishment and male-biased progeny sex ratios). Our study demonstrates that even a small population of a highly effective pollinator may have a significant impact on fruit set. Our results are encouraging for the use of Osmia managed populations and for the implementation of measures to promote wild pollinators in agricultural environments.

BACKGROUND Neonicotinoid insecticides have been identified as an important factor contributing to bee diversity declines. Nonetheless, uncertainties remain about their impact under field conditions. Most studies have been conducted on Apis mellifera and tested single compounds. However, in agricultural environments, bees are often exposed to multiple pesticides. We explore the synergistic mortality between a neonicotinoid (clothianidin) and an ergosterol-biosynthesis-inhibiting fungicide (propiconazole) in three bee species (A. mellifera, Bombus terrestris, Osmia bicornis) following oral exposure in the laboratory. RESULTS We developed a new approach based on the binomial proportion test to analyse synergistic interactions. We estimated uptake of clothianidin per foraging bout in honey bees foraging on seed-coated rapeseed fields. We found significant synergistic mortality in all three bee species exposed to non-lethal doses of propiconazole and their respective LD10 of clothianidin. Significant synergism was only found at the first assessment times in A. mellifera (4 and 24 h) and B. terrestris (4 h), but persisted throughout the experiment (96 h) in O. bicornis. O. bicornis was also the most sensitive species to clothianidin. CONCLUSION Our results underscore the importance to test pesticide combinations likely to occur in agricultural environments, and to include several bee species in environmental risk assessment schemes. © 2016 Society of Chemical Industry

Pollinators in agroecosystems are often exposed to pesticide mixtures. Even at low concentrations, the effects of these mixtures on bee populations are difficult to predict due to potential synergistic interactions. In this paper, we orally exposed newly emerged females of the solitary bee Osmia bicornis to environmentally realistic levels of clothianidin (neonicotinoid insecticide) and propiconazole (fungicide), singly and in combination. The amount of feeding solution consumed was highest in bees exposed to the neonicotinoid, and lowest in bees exposed to the pesticide mixture. Ovary maturation and longevity of bees of the neonicotinoid and the fungicide treatments did not differ from those of control bees. By contrast, bees exposed to the pesticide mixture showed slow ovary maturation and decreased longevity. We found a synergistic interaction between the neonicotinoid and the fungicide on survival probability. We also found an interaction between treatment and emergence time (an indicator of physiological condition) on longevity. Longevity was negatively correlated to physiological condition only in the fungicide and the mixture treatments. Delayed ovary maturation and premature death imply a shortened nesting period (highly correlated to fecundity in Osmia ). Our findings provide a mechanism to explain the observed dynamics of solitary bee populations exposed to multiple chemical residues in agricultural environments.

1. Diapause is a dynamic process of low metabolic activity that allows insects to survive periods of harsh conditions. Notwithstanding the lowered metabolism, and because diapausing insects have no access to food, diapause has an energetic cost that may affect post-diapause performance. 2. Previous studies on the solitary bee Osmia lignaria have shown that prolonged pre-wintering periods (the time during which individuals already in diapause remain at warm temperatures) are associated with elevated lipid consumption, fat body depletion, and body weight loss. The present study investigated whether prolonged pre-wintering also affects reproduction, i.e. whether the costs associated with diapause could have an effect on post-diapause performance in this species. 3. Females were exposed to a range of pre-wintering conditions, and ovary development and individual post-wintering performance were monitored throughout their adult life span. 4. No evidence of an effect of pre-wintering duration on post-diapause reproductive success was found. Expected differences in the timing of establishment were not observed because ovary maturation was, surprisingly, not arrested during pre-wintering. Prolonged pre-wintering duration did not result in decreased life span, probably because emerging females could rapidly replenish their metabolic reserves through feeding. However, there was a very strong effect of the duration of the pre-emergence period on the likelihood of nest establishment. 5. Longevity, the main factor determining fecundity in Osmia, is subjected to high levels of intrinsic variability, even among females of similar size exposed to identical conditions during development and nesting.

International audience; Modern agriculture often involves the use of pesticides to protect crops. These substances are harmful to target organisms (pests and pathogens). Nevertheless, they can also damage non-target animals, such as pollinators and entomophagous arthropods. It is obvious that the undesirable side effects of pesticides on the environment should be reduced to a minimum. Western honey bees (Apis mellifera) are very important organisms from an agricultural perspective and are vulnerable to pesticide-induced impacts. They contribute actively to the pollination of cultivated crops and wild vegetation, making food production possible. Of course, since Apis mellifera occupies the same ecological niche as many other species of pollinators, the loss of honey bees caused by environmental pollutants suggests that other insects may experience a similar outcome. Because pesticides can harm honey bees and other pollinators, it is important to register pesticides that are as selective as possible. In this manuscript, we describe a selection of methods used for studying pesticide toxicity/selectiveness towards Apis mellifera. These methods may be used in risk assessment schemes and in scientific research aimed to explain acute and chronic effects of any target compound on Apis mellifera.

Adult honey bees (Apis mellifera) usually maintain colony brood rearing temperature between 34-35°C by thermoregulation. The brood may, however, also be subjected to suboptimal temperature. Here weinvestigated whether a decrease of brood rearing temperature mayhave effects on larval mortality, adult emergence, longevity, morphology and susceptibility to poisoning by pesticides (dimethoate).Using the in vitrorearing protocol of Aupinel (2005), we were able for the first time to control the brood temperature not only during the pupalstage but also during the larval stage. Honey bee larvae were reared in vitroat 35°C (optimal) and 33°C (suboptimal) from 12 h after hatching for 15 days. Dimethoate was tested by ingestion either on 4-day old larvae or on 7-day old adults. Our results showed that lower rearing temperature had no significant effects on larval mortality and adult emergence, but adult bee mortality was strongly affected. Moreover, adult workers emerging at 33°C were significantly more susceptible to dimethoate. Larval LD50 (48 h) was, however, 28 times higher at33°C than at 35°C. The striking differences between larvae and adults may beexplained by differential larval metabolism at 33°C and resulting slower active ingredient absorption. We conclude that adult honey bees reared at even slightly suboptimal brood temperature may be more susceptible to pesticide poisoning and be characterised by reduced longevity. Thus, low temperature brood rearing could be another stress factor for colonies.

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 and haemolymph 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.

International audience; AbstractPollen diet is essential to nurse bees to develop hypopharyngeal glands (HPGs) and produce royal jelly to feed larvae. The effect of different diet regimes combined with pesticide exposure on the development of HPGs in honeybees was studied. In particular, a high-quality pollen mixture (high pollen diversity and protein content) and a low-quality diet (low pollen diversity and protein content) were combined with the chronic administration of thiamethoxam (10 and 40 μg/L). The impact of these stressors was evaluated by measuring the size of HPG acini and the total protein content of bee heads in 8- and 12-day-old bees. Bees fed with sub-lethal concentrations of thiamethoxam and with the low-quality diet exhibited smaller and irregularly shaped acini. The total protein content in bee head was also affected by pesticide administration, while the pollen quality did not cause significant modifications. In conclusion, these results highlighted the importance of the food quality, considered as nutritional value and pesticide contamination, for the physiological development of honeybees.

The honey bee Apis mellifera, the test species used in the current environmental risk assessment procedure, is generally considered as extremely sensitive to pesticides when compared to other bee species, although a quantitative approach for comparing the difference in sensitivity among bees has not yet been reported. A systematic review of the relevant literature on the topic followed by a meta-analysis has been performed. Both the contact and oral acute LD50 and the chronic LC50 reported in laboratory studies for as many substances as possible have been extracted from the papers in order to compare the sensitivity to pesticides of honey bees and other bee species (Apiformes). The sensitivity ratio R between the endpoint for the species a (A. mellifera) and the species s (bees other than A. mellifera) was calculated for a total of 150 case studies including 19 bee species. A ratio higher than 1 indicated that the species s was more sensitive to pesticides than honey bees. The meta-analysis showed a high variability of sensitivity among bee species (R from 0.001 to 2085.7), however, in approximately 95 % of the cases the sensitivity ratio was below 10. The effect of pesticides in domestic and wild bees is dependent on the intrinsic sensitivity of single bee species as well as their specific life cycle, nesting activity and foraging behaviour. Current data indicates a need for more comparative information between honey bees and non-Apis bees as well as separate pesticide risk assessment procedures for non-Apis bees.

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.

The impact of climate change on insect populations depends on specific life cycle traits and physiological adaptations. The solitary bee Osmia lignaria winters as a pre-emergent adult, and requires a period of cold temperature for winter diapause completion. It is a univoltine species, and diapause induction does not depend on photoperiod. To understand the potential effects of longer summers on O. lignaria populations, we exposed individuals to three treatments simulating early, mid and late winter arrivals, and measured respiration rates, metabolic expenditure, weight loss, fat body depletion, lipid levels and winter mortality. The early-winter treatment disrupted diapause development, but had no apparent negative effects on fitness. In contrast, late-winter bees had a greater energetic expenditure (1.5-fold), weight (1.4-fold) and lipid (2-fold) loss, greater fat body depletion, and a 19% increase in mortality compared to mid-winter bees. We also monitored adult eclosion and arrival of winter temperatures under natural conditions in four years. We found a positive correlation between mean degree-day accumulation during pre-wintering (a measure of asynchrony between adult eclosion and winter arrival) and yearly winter mortality. Individually, bees experiencing greater degree-day accumulations exhibited reduced post-winter longevity. Timing of adult eclosion in O. lignaria is dependent on the duration of the prepupal period, which occurs in mid-summer, is also diapause-mediated, and is longer in populations from southerly latitudes. In a global warming scenario, we expect long summer diapause phenotypes to replace short summer diapause phenotypes, effectively maintaining short pre-wintering periods in spite of delayed winter arrivals.

Most insects from temperate areas enter diapause ahead of winter. Species diapausing in a feeding stage and accumulating metabolic reserves during permissive pre-wintering conditions are expected to enter diapause shortly before the onset of winter. In contrast, species diapausing in a non-feeding stage are expected to lower their metabolism as soon as possible to avoid excessive consumption of metabolic reserves. The solitary bee Osmia lignaria winters as a non-feeding adult within its cocoon, but previous studies show important weight losses and increased winter mortality in populations pre-wintered for extended periods. We measured respiration rates to assess diapause initiation and maintenance during pre-wintering, and tested whether timing of adult eclosion affected fitness by measuring fat body depletion, winter mortality and post-winter longevity. We worked with different cohorts of a population reared under natural conditions, and manipulated pre-wintering duration in a population reared under artificial conditions. In agreement with our expectation, O. lignaria lower their metabolic rates within a few days of adult eclosion, but nonetheless suffer strong weight loss during pre-wintering. Early developing individuals suffer greater weight loss and fat body depletion, and have short post-winter longevity. Although, we found no differences in winter mortality among treatments, our results indicate that increased mortality may occur in years with late winter arrivals. We discuss fundamental ecophysiological differences between adult and prepupal diapause within the Megachilidae, and hypothesize that species wintering as adults will be more negatively affected by a situation of extended summers under a scenario of global warming.

The interplay between insect and plant traits outlines the patterns of pollen transfer and the subsequent plant reproductive fitness. We studied the factors that affect the pollination efficiency of a pollinator community of Dictamnus albus L. by evaluating insect behaviour and morphological characteristics in relation to flowering phenology. In order to extrapolate the pollinator importance of single taxa and of the whole pollinator guild, we calculated an index distinguishing between potential (PPI) and realized (RPI) pollinator importance. Although the pollinator species spectrum appeared rather constant, we found high intra- and inter-annual variability of pollinator frequency and importance within the insect community. Flower visitation rate strictly depended on insect abundance and on the overlap between their flying period and flower blooming. All the pollinators visited flowers from the bottom to the top of the racemes, excluding intra-plant geitonogamous pollination, and most of them showed high pollen fidelity. Only medium large-sized bees could contact the upward bending stiles while feeding on nectar, highlighting a specialisation of the plant towards bigger pollinators. Moreover, we found evidence of functional specialisation, since all pollinators were restricted to a single taxonomic group (order: Hymenoptera; superfamily: Apoidea). Both the PPI and RPI indices indicate Habropoda tarsata as the most important pollinator of D. albus. Following hand cross-pollination experiments we revealed the presence of pollination limitation in 1 of the 3 years of field study. We discuss this result in relation to flowering abundance and to possible mismatches of phenological periods between plants and insects.

A four-year investigation comparing organic and conventional horticultural vegetable rotation systems was carried out in Central Italy using soil arthropods as bioindicators. The indexes of biodiversity showed, in general, higher values on organic in comparison with conventional management, but the differences were strongly affected by the crops. An index specifically designed to select the Carabid species, which benefit from organic versus conventional, was calculated in order to understand faunistic data on this group. Our data demonstrated that the spray intensity adversely affects the Carabid species richness, as proved by the lowest diversity values recorded in the conventional plots. The experiment, planned by following a “system approach research,” was suitable to understand the ecological sustainability in organic farming in comparison with conventional. The better conservation of arthropod fauna in the organic seems to be coherent also with the enhancement of organic matter in this system. For this reason, a holistic effect of the organic system can provide a reasonable interpretation of the better conservation of the living component and, in particular, of Carabid beetle biodiversity, during the four-year period.

In this paper we assessed, under laboratory conditions, the toxicity of an active substance on solitary bee larvae of Osmia cornuta (Hymenoptera: Megachilidae). A field-realistic dose of the systemic insecticide spirotetramat was applied to the mass provisions. The insecticide’s effects on several life-cycle parameters were studied in males and females. Our results showed a significantly shorter post-emergence longevity in bees exposed to spirotetramat during the larval stage, compared to the control. The observed reduction in longevity was 18 and 15%, respectively, in males and females. Mortality rate and other biological traits (larval and spinning duration, emergence time, food/body conversion rate) did not show significant differences between the two treatments. The method described in this study can be used to test the effects of toxic substances (i.e. agrochemicals) on bees. Moreover, it can constitute a basis for the development of a standardised protocol in the first tier of the Environmental Risk Assessment for solitary bees.

Osmia (Osmia) bees are strictly univoltine and winter as diapausing adults. In these species, the timing of adult eclosion with the onset of wintering conditions is critical, because adults exposed to long pre-wintering periods show increased lipid loss and winter mortality. Populations from warm areas fly in February-March and are exposed to longer growth seasons than populations from colder areas, which fly in April-May. Given their inability to produce an extra generation, early-flying populations should develop more slowly than late-flying populations and thus avoid the negative consequences of long pre-wintering periods. In this study we compare the development under natural and laboratory conditions of phenologically-distinct populations in two Osmia species. Early-flying populations took ∼2 months longer to develop than late-flying populations. Differences between populations in larval and pupal period duration were very small, whereas the prepupal period was much longer in early-flying populations. In contrast to the larval and pupal stages, the prepupal stage showed a non-linear response to temperature, was strongly affected by thermoperiod, and exhibited minimum respiration rates. Coupled with other lines of evidence, these results suggest that the prepupal period in Osmia corresponds to a summer diapause, and its duration may be under local selection to synchronize adult eclosion with the onset of winter temperatures. We discuss the implications of our results relative to current expectations of global warming.

Osmia lignaria is a solitary bee that over-winters as a fully eclosed, cocooned, unfed adult. Our objective is to understand the effect of wintering temperature on diapause maintenance and termination in this species. We measure respiration rates and weight loss in individuals exposed to various wintering temperatures (0, 4, 7, 22 degrees C, outdoors) and durations (28, 84, 140, 196, 252 days). We use time to emerge and respiration response (respiration rate measured at 22 degrees C) as indicators of diapause intensity. Adults spontaneously lower their respiration rates to approximately 0.1 ml/gh within 1 month after adult eclosion, indicating obligatory diapause. Non-wintered individuals maintain low respiration rates, but lose weight rapidly and die by mid-winter. In wintered adults, two phases can be distinguished. First, respiration response undergoes a rapid increase and then reaches a plateau. This phase is similar in bees wintered at 0, 4 and 7 degrees C. In the second phase, respiration response undergoes an exponential increase, which is more pronounced at the warmer temperatures. Composite exponential functions provide a good fit to the observed respiration patterns. Adults whose respiration response has reached 0.45 ml/gh emerge promptly when exposed to 20 degrees C, indicating diapause completion. Individuals wintered for short periods do not reach such respiration levels. When exposed to 20 degrees C these individuals lower their metabolic rate, and their emergence time is extended. The relationship between respiration rates and emergence time follows a negative exponential function. We propose two alternative models of diapause termination to interpret these results.

Megachile disjunctiformis Cockerell 1911 (Hymenoptera Megachilidae) is recorded for the first time in Europe. After previous observations in 2011 and 2016 near Bologna, Italy, three specimens (one female and two males) were captured in three different locations of the Bologna area in 2017 and identified. The species is known from China, Japan, Taiwan, Hong Kong and Korea. In this paper a description of the specimens and comparison with other similar species are provided. Furthermore visited plants are indicated. The finding localities are relatively close to two important Bologna trading areas, suggesting that these may be the original sites of introduction in Italy. Little is known about this species and its biology in Italy will be subject of future investigations.