Mild chronic exposure to pesticides alters physiological markers of honey bee health without perturbing the core gut microbiota.

Recent studies highlighted that exposure to glyphosate can affect specific members of the core gut microbiota of honey bee workers. However, in this study, bees were exposed to relatively high glyphosate concentrations. Here, we chronically exposed newly emerged honey bees to imidacloprid, glyphosate and difenoconazole, individually and in a ternary mixture, at an environmental concentration of 0.1 µg/L. We studied the effects of these exposures on the establishment of the gut microbiota, the physiological status, the longevity, and food consumption of the host. The core bacterial species were not affected by the exposure to the three pesticides. Negative effects were observed but they were restricted to few transient non-core bacterial species. However, in the absence of the core microbiota, the pesticides induced physiological disruption by directly altering the detoxification system, the antioxidant defenses, and the metabolism of the host. Our study indicates that even mild exposure to pesticides can directly alter the physiological homeostasis of newly emerged honey bees and particularly if the individuals exhibit a dysbiosis (i.e. mostly lack the core microbiota). This highlights the importance of an early establishment of a healthy gut bacterial community to strengthen the natural defenses of the honey bee against xenobiotic stressors.

Toxicity of the Pesticides Imidacloprid, Difenoconazole and Glyphosate Alone and in Binary and Ternary Mixtures to Winter Honey Bees: Effects on Survival and Antioxidative Defenses.

To explain losses of bees that could occur after the winter season, we studied the effects of the insecticide imidacloprid, the herbicide glyphosate and the fungicide difenoconazole, alone and in binary and ternary mixtures, on winter honey bees orally exposed to food containing these pesticides at concentrations of 0, 0.01, 0.1, 1 and 10 µg/L. Attention was focused on bee survival, food consumption and oxidative stress. The effects on oxidative stress were assessed by determining the activity of enzymes involved in antioxidant defenses (superoxide dismutase, catalase, glutathione-S-transferase, glutathione reductase, glutathione peroxidase and glucose-6-phosphate dehydrogenase) in the head, abdomen and midgut; oxidative damage reflected by both lipid peroxidation and protein carbonylation was also evaluated. In general, no significant effect on food consumption was observed. Pesticide mixtures were more toxic than individual substances, and the highest mortalities were induced at intermediate doses of 0.1 and 1 µg/L. The toxicity was not always linked to the exposure level and the number of substances in the mixtures. Mixtures did not systematically induce synergistic effects, as antagonism, subadditivity and additivity were also observed. The tested pesticides, alone and in mixtures, triggered important, systemic oxidative stress that could largely explain pesticide toxicity to honey bees.

Toxicological status changes the susceptibility of the honey bee Apis mellifera to a single fungicidal spray application.

During all their life stages, bees are exposed to residual concentrations of pesticides, such as insecticides, herbicides, and fungicides, stored in beehive matrices. Fungicides are authorized for use during crop blooms because of their low acute toxicity to honey bees. Thus, a bee that might have been previously exposed to pesticides through contaminated food may be subjected to fungicide spraying when it initiates its first flight outside the hive. In this study, we assessed the effects of acute exposure to the fungicide in bees with different toxicological statuses. Three days after emergence, bees were subjected to chronic exposure to the insecticide imidacloprid and the herbicide glyphosate, either individually or in a binary mixture, at environmental concentrations of 0.01 and 0.1 µg/L in food (0.0083 and 0.083 µg/kg) for 30 days. Seven days after the beginning of chronic exposure to the pesticides (10 days after emergence), the bees were subjected to spraying with the fungicide difenoconazole at the registered field dosage. The results showed a delayed significant decrease in survival when honey bees were treated with the fungicide. Fungicide toxicity increased when honey bees were chronically exposed to glyphosate at the lowest concentration, decreased when they were exposed to imidacloprid, and did not significantly change when they were exposed to the binary mixture regardless of the concentration. Bees exposed to all of these pesticide combinations showed physiological disruptions, revealed by the modulation of several life history traits related mainly to metabolism, even when no effect of the other pesticides on fungicide toxicity was observed. These results show that the toxicity of active substances may be misestimated in the pesticide registration procedure, especially for fungicides.

Physiological effects of the interaction between Nosema ceranae and sequential and overlapping exposure to glyphosate and difenoconazole in the honey bee Apis mellifera.

Pathogens and pollutants, such as pesticides, are potential stressors to all living organisms, including honey bees. Herbicides and fungicides are among the most prevalent pesticides in beehive matrices, and their interaction with Nosema ceranae is not well understood. In this study, the interactions between N. ceranae, the herbicide glyphosate and the fungicide difenoconazole were studied under combined sequential and overlapping exposure to the pesticides at a concentration of 0.1 µg/L in food. In the sequential exposure experiment, newly emerged bees were exposed to the herbicide from day 3 to day 13 after emerging and to the fungicide from day 13 to day 23. In the overlapping exposure experiment, bees were exposed to the herbicide from day 3 to day 13 and to the fungicide from day 7 to day 17. Infection by Nosema in early adult life stages (a few hours post emergence) greatly affected the survival of honey bees and elicited much higher mortality than was induced by pesticides either alone or in combination. Overlapping exposure to both pesticides induced higher mortality than was caused by sequential or individual exposure. Overlapping, but not sequential, exposure to pesticides synergistically increased the adverse effect of N. ceranae on honey bee longevity. The combination of Nosema and pesticides had a strong impact on physiological markers of the nervous system, detoxification, antioxidant defenses and social immunity of honey bees.

Mixtures of an insecticide, a fungicide and a herbicide induce high toxicities and systemic physiological disturbances in winter Apis mellifera honey bees.

Multiple pesticides originating from plant protection treatments and the treatment of pests infecting honey bees are frequently detected in beehive matrices. Therefore, winter honey bees, which have a long life span, could be exposed to these pesticides for longer periods than summer honey bees. In this study, winter honey bees were exposed through food to the insecticide imidacloprid, the fungicide difenoconazole and the herbicide glyphosate, alone or in binary and ternary mixtures, at environmental concentrations (0 (controls), 0.1, 1 and 10 µg/L) for 20 days. The survival of the honey bees was significantly reduced after exposure to these 3 pesticides individually and in combination. Overall, the combinations had a higher impact than the pesticides alone with a maximum mortality of 52.9% after 20 days of exposure to the insecticide-fungicide binary mixture at 1 µg/L. The analyses of the surviving bees showed that these different pesticide combinations had a systemic global impact on the physiological state of the honey bees, as revealed by the modulation of head, midgut and abdomen glutathione-S-transferase, head acetylcholinesterase, abdomen glucose-6-phosphate dehydrogenase and midgut alkaline phosphatase, which are involved in the detoxification of xenobiotics, the nervous system, defenses against oxidative stress, metabolism and immunity, respectively. These results demonstrate the importance of studying the effects of chemical cocktails based on low realistic exposure levels and developing long-term tests to reveal possible lethal and adverse sublethal interactions in honey bees and other insect pollinators.