International audience | Pollinators have to cope with a wide range of stressful, not necessarily lethal factors limiting their performance and the ecological services they provide. Among these stressors are pesticides, chemicals that are originally designed to target crop-harming organisms but that also disrupt various functions in pollinators, including flight, communication, orientation and memory. Although all these functions are crucial for reproductive individuals when searching for mates or nesting places, it remains poorly understood how pesticides affect reproduction in pollinators. In this study, we investigated how a widely used fungicide, boscalid, affects reproduction in honey bees (Apis mellifera), an eusocial insect in which a single individual, the queen, fulfills the reproductive functions of the whole colony. Boscalid is a succinate dehydrogenase inhibitor (SDHI) fungicide mainly used on rapeseed flowers to target mitochondrial respiration in fungi but it is also suspected to disrupt foraging-linked functions in bees. We found that immature queen exposure to sublethal, field relevant doses of boscalid disrupted reproduction, as indicated by a dramatic increase in queen mortality during and shortly after the nuptial flights period and a decreased number of spermatozoa stored in the spermatheca of surviving queens. However, we did not observe a decreased paternity frequency in exposed queens that successfully established a colony. Queen exposure to boscalid had detrimental consequences on the colonies they later established regarding brood production, Varroa destructor infection and pollen storage but not nectar storage and population size. These perturbations at the colony-level correspond to nutritional stress conditions, and may have resulted from queen reduced energy provisioning to the eggs. Accordingly, we found that exposed queens had decreased gene expression levels of vitellogenin, a protein involved in egg-yolk formation. Overall, our results indicate that boscalid decreases honey bee queen reproductive quality, thus supporting the need to include reproduction in the traits measured during pesticide risk assessment procedures.

Pollinators have to cope with a wide range of stressful, not necessarily lethal factors limiting their performance and the ecological services they provide. Among these stressors are pesticides, chemicals that are originally designed to target crop-harming organisms but that also disrupt various functions in pollinators, including flight, communication, orientation and memory. Although all these functions are crucial for reproductive individuals when searching for mates or nesting places, it remains poorly understood how pesticides affect reproduction in pollinators. In this study, we investigated how a widely used fungicide, boscalid, affects reproduction in honey bees (Apis mellifera), an eusocial insect in which a single individual, the queen, fulfills the reproductive functions of the whole colony. Boscalid is a succinate dehydrogenase inhibitor (SDHI) fungicide mainly used on rapeseed flowers to target mitochondrial respiration in fungi but it is also suspected to disrupt foraging-linked functions in bees. We found that immature queen exposure to sublethal, field relevant doses of boscalid disrupted reproduction, as indicated by a dramatic increase in queen mortality during and shortly after the nuptial flights period and a decreased number of spermatozoa stored in the spermatheca of surviving queens. However, we did not observe a decreased paternity frequency in exposed queens that successfully established a colony. Queen exposure to boscalid had detrimental consequences on the colonies they later established regarding brood production, Varroa destructor infection and pollen storage but not nectar storage and population size. These perturbations at the colony-level correspond to nutritional stress conditions, and may have resulted from queen reduced energy provisioning to the eggs. Accordingly, we found that exposed queens had decreased gene expression levels of vitellogenin, a protein involved in egg-yolk formation. Overall, our results indicate that boscalid decreases honey bee queen reproductive quality, thus supporting the need to include reproduction in the traits measured during pesticide risk assessment procedures.

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

[Departement_IRSTEA]Ecotechnologies [TR1_IRSTEA]INSPIRE [ADD1_IRSTEA]Équiper l'agriculture | International audience | Pesticide residues (112 compounds) were quantified by GC-MS/MS or LC-MS/MS in 85 bee bread samples and 154 pollen samples obtained from five apiaries each with three or four colonies (genotype Buckfast) in Luxembourg over the period 2011-2013. Thiacloprid, chlorfenvinphos, tebuconazole, and methiocarb were found most frequently in bee bread while thiacloprid, permethrin-cis, and permethrin-trans were detected most frequently in the pollen samples. Three neonicotinoid insecticides (clothianidin, imidacloprid, and thiamethoxam) that were restricted by an EU regulation in 2013 after our sampling campaign was finished were each found in less than 8% of the pollen or bee bread samples. The maximum concentrations of thiacloprid, metazachlor, and methiocarb measured in the pollen collected by a group of honeybee colonies (n=5) without survivors within the 3-year period of observation were 86.20 +/- 10.74ng/g, 2.80 +/- 1.26ng/g, and below the limit of quantification, respectively. The maximum concentrations of the same compounds measured in the pollen collected by a group of honeybee colonies with significantly (P=0.02) more survivors (7 out of 9) than expected, if the survivors had been distributed randomly among the groups of colonies, were 11.98 +/- 2.28ng/g, 0.44 +/- 0.29ng/g, and 8.49 +/- 4.13ng/g, respectively. No honeybee colony that gathered pollen containing more than 23ng/g thiacloprid survived the 3-year project period. There was no statistically significant association between pesticide residues in the bee bread and the survival of the colonies. Actions already taken or planned and potential further actions to protect bees from exposure to pesticides are discussed.

Pollinating bees are stressed by highly variable environmental conditions, malnutrition, parasites and pathogens, but may also by getting in contact with microorganisms or entomopathogenic nematodes that are used to control plant pests and diseases. While foraging for water, food, or nest material social as well as solitary bees have direct contact or even consume the plant protection product with its active substance (e.g., viruses, bacteria, fungi, etc.). Here, we summarize the results of cage, microcolony, observation hive assays, semi-field and field studies using full-size queen-right colonies. By now, some species and subspecies of the Western and Eastern honey bee (Apis mellifera, A. cerana), few species of bumble bees, very few stingless bee species and only a single species of leafcutter bees have been studied as non-target host organisms. Survival and reproduction are the major criteria that have been evaluated. Especially sublethal effects on the bees' physiology, immune response and metabolisms will be targets of future investigations. By studying infectivity and pathogenic mechanisms, individual strains of the microorganism and impact on different bee species are future challenges, especially under field conditions. Overall, it became evident that honey bees, bumble bees and few stingless bee species may not be suitable surrogate species to make general conclusions for biological mechanisms of bee-microorganism interactions of other social bee species. Solitary bees have been studied on leafcutter bees (Megachile rotundata) only, which shows that this huge group of bees (∼20,000 species worldwide) is right at the beginning to get an insight into the interaction of wild pollinators and microbial plant protection organisms.

The Covid-19 outbreak had a critical impact on a massive amount of human activities as well as the global health system. On the other hand, the lockdown and related suspension of working activities reduced pollution emissions. The use of biomonitoring is an efficient and quite recent tool to assess environmental pollution through the analysis of a proper bioindicator, such as bees. This study set out to ascertain the impact of the Covid-19 pandemic lockdown on the environmental occurrence of eleven heavy metals in the Campania region (Italy) by analyzing bees and bee products. A further aim of this study was the assessment of the Honeybee Contamination Index (HCI) in three different areas of the Campania region and its comparison with other Italian areas to depict the current environmental pollutants levels of heavy metals. The results showed that the levels of heavy metals bioaccumulated by bees during the pandemic lockdown (T1) were statistically lower than the sampling times after Covid-19 restrictions and the resumption of some or all activities (T2 and T3). A comparable trend was observed in wax and pollen. However, bee, pollen, and wax showed higher levels of Cd and Hg in T1 than T2 and T3. The analysis of the HCI showed a low contamination level of the sampling sites for Cd and Pb, and an intermediate-high level as regards Ni and Cr. The biomonitoring study highlighted a decrease of heavy metals in the environmental compartments due to the intense pandemic restrictions. Therefore, Apis mellifera and other bee products remain a reliable and alternative tool for environmental pollution assessment.

The frequent exposure of bees to a wide variety of fungicides, on crops where they forage, can be considered a stressor factor for these pollinators. The organisms are exposed both to the fungicide active ingredients and to the adjuvants of commercial formulations. All these ingredients are brought to the hive by bee foragers through contaminated pollen and nectar, thus exposing also immature individuals during larval phase. This work aimed to compare the effects of larval exposure to the fungicide pyraclostrobin (active ingredient and commercial formulation) and its influence on the cytotoxicity to midguts in adults, which were inoculated with the Nosema ceranae spores in the post-emergence stage. Under laboratory conditions, Apis mellifera larvae received an artificial diet containing fungicide solution from the third to the sixth day of the feeding phase. One-day-old adult workers ingested 100,000 infectious N. ceranae spores mixed in sucrose solution. Effects on midgut were evaluated through cellular biomarkers of stress and cell death. The exposure to the fungicide (active ingredient and commercial formulation) did not affect the larval post-embryonic development and survival of adult bees. However, this exposure induced cytotoxicity in the cells of the midgut, showed by the increase in DNA fragmentation and alteration in the HSP70 immunolabeling pattern. Without the pathogen, the midgut cytotoxic effects and HSP70 immunolabeling of the organisms exposed to the commercial formulation were lower when compared to the exposure to its active ingredient. However, in the presence of the pathogen, the cytotoxic effects of the commercial formulation to the adult bees’ midgut were potentialized. The pathogen N. ceranae increased the damage to the intestinal epithelium of adult bees. Thus, realistic doses of pyraclostrobin present in beebread consumed by larvae can affect the health and induce physiological implications to the midgut functions of the adult bees.

Pesticide exposure is regarded as a contributing factor to the high gross loss rates of managed colonies of Apis mellifera. Pesticides enter the hive through contaminated nectar and pollen carried by returning forager honey bees or placed in the hive by beekeepers when managing hive pests. We used an in vitro rearing method to characterize the effects of seven pesticides on developing brood subjected dietary exposure at worse-case environmental concentrations detected in wax and pollen. The pesticides tested included acaricides (amitraz, coumaphos, fluvalinate), insecticides (chlorpyrifos, imidacloprid), one fungicide (chlorothalonil), and one herbicide (glyphosate). The larvae were exposed chronically for six days of mimicking exposure during the entire larval feeding period, which is the worst possible scenario of larval exposure. Survival, duration of immature development, the weight of newly emerged adult, morphologies of the antenna and the hypopharyngeal gland, and gene expression were recorded. Survival of bees exposed to amitraz, coumaphos, fluvalinate, chlorpyrifos, and chlorothalonil was the most sensitive endpoint despite observed changes in many developmental and physiological parameters across the seven pesticides. Our findings suggest that pesticide exposure during larvae development may affect the survival and health of immature honey bees, thus contributing to overall colony stress or loss. Additionally, pesticide exposure altered gene expression of detoxification enzymes. However, the tested exposure scenario is unlikely to be representative of real-world conditions but emphasizes the importance of proper hive management to minimize pesticide contamination of the hive environment or simulates a future scenario of increased contamination.

In order to study the distribution of pesticide residues in beekeeping matrices, samples of live in-hive worker honey bees (Apis mellifera), fresh stored pollen and beeswax were collected during 2016–2017 from 45 apiaries located in different landscape contexts in Spain. A total of 133 samples were screened for 63 pesticides or their degradation products to estimate the pesticide exposure to honey bee health through the calculation of the hazard quotient (HQ). The influence of the surrounding environment on the content of pesticides in pollen was assessed by comparing the concentrations of pesticide residues found in apiaries from intensive farming landscapes to those found in apiaries located in mountainous, grassland and urban contexts. Beeswax revealed high levels of miticides used in beekeeping such as coumaphos, chlorfenvinphos, fluvalinate and acrinathrin, which were detected in more than 75% of samples. Pollen was predominantly contaminated by miticides but also by insecticides used in agriculture such as chlorpyrifos and acetamiprid, which showed concentrations significantly higher in apiaries located in intensive farming contexts. Pesticides residues were less frequent and at lower concentrations in live honey bees. Beeswax showed the highest average hazard scores (HQ > 5000) to honey bees. Pollen samples contained the largest number of pesticide residues and relevant hazard (HQ > 50) to bees. Acrinathrin was the most important contributor to the hazard quotient scores in wax and pollen samples. The contributions of the pesticides dimethoate and chlorpyrifos to HQ were considered relevant in samples.

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