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.

Chronic toxicity and physiological changes induced in the honey bee by the exposure to fipronil and Bacillus thuringiensis spores alone or combined.

In the agricultural environment, honey bees may be exposed to combinations of pesticides. Until now, the effects of these combinations on honey bee health have been poorly investigated. In this study, we assessed the impacts of biological and chemical insecticides, combining low dietary concentrations of Bacillus thuringiensis (Bt) spores (100 and 1000µg/L) with the chemical insecticide fipronil (1µg/L). In order to assess the possible effects of Cry toxins, the Bt kurstaki strain (Btk) was compared with a Bt strain devoid of toxin-encoding plasmids (Bt Cry(-)). The oral exposure to fipronil and Bt spores from both strains for 10 days did not elicit significant effects on the feeding behavior and survival after 25 days. Local and systemic physiological effects were investigated by measuring the activities of enzymes involved in the intermediary and detoxication metabolisms at two sampling dates (day 10 and day 20). Attention was focused on head and midgut glutathione-S-transferase (GST), midgut alkaline phosphatase (ALP), abdomen glyceraldehyde-3-phosphate dehydrogenase (GAPD) and glucose-6-phosphate dehydrogenase (G6PD). We found that Bt Cry(-) and Btk spores induced physiological modifications by differentially modulating enzyme activities. Fipronil influenced the enzyme activities differently at days 10 and 20 and, when combined with Bt spores, elicited modulations of some spore-induced physiological responses. These results show that an apparent absence of toxicity may hide physiological disruptions that could be potentially damaging for the bees, especially in the case of combined exposures to other environmental stressors.

Non-monotonic dose-response relationships and endocrine disruptors: a qualitative method of assessment.

Experimental studies investigating the effects of endocrine disruptors frequently identify potential unconventional dose-response relationships called non-monotonic dose-response (NMDR) relationships. Standardized approaches for investigating NMDR relationships in a risk assessment context are missing. The aim of this work was to develop criteria for assessing the strength of NMDR relationships. A literature search was conducted to identify published studies that report NMDR relationships with endocrine disruptors. Fifty-one experimental studies that investigated various effects associated with endocrine disruption elicited by many substances were selected. Scoring criteria were applied by adaptation of an approach previously used for identification of hormesis-type dose-response relationships. Out of the 148 NMDR relationships analyzed, 82 were categorized with this method as having a "moderate" to "high" level of plausibility for various effects. Numerous modes of action described in the literature can explain such phenomena. NMDR can arise from numerous molecular mechanisms such as opposing effects induced by multiple receptors differing by their affinity, receptor desensitization, negative feedback with increasing dose, or dose-dependent metabolism modulation. A stepwise decision tree was developed as a tool to standardize the analysis of NMDR relationships observed in the literature with the final aim to use these results in a Risk Assessment purpose. This decision tree was finally applied to studies focused on the effects of bisphenol A.

Sensitive analytical methods for 22 relevant insecticides of 3 chemical families in honey by GC-MS/MS and LC-MS/MS.

Several methods for analyzing pesticides in honey have been developed. However, they do not always reach the sufficiently low limits of quantification (LOQ) needed to quantify pesticides toxic to honey bees at low doses. To properly evaluate the toxicity of pesticides, LOQ have to reach at least 1 ng/g. In this context, we developed extraction and analytical methods for the simultaneous detection of 22 relevant insecticides belonging to three chemical families (neonicotinoids, pyrethroids, and pyrazoles) in honey. The insecticides were extracted with the QuEChERS method that consists in an extraction and a purification with mixtures of salts adapted to the matrix and the substances to be extracted. Analyses were performed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) for the pyrazoles and the pyrethroids and by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) for the neonicotinoids and ethiprole. Calibration curves were built from various honey types fortified at different concentrations. Linear responses were obtained between 0.2 and 5 ng/g. Limits of detection (LOD) ranged between 0.07 and 0.2 ng/g, and LOQ ranged between 0.2 and 0.5 ng/g. The mean extraction yields ranged between 63 % and 139 % with RSD <25 %. A complete validation of the methods also examined recovery rates and specificity. These methods were applied to 90 honey samples collected during a 2009-2010 field study in two apiaries placed in different anthropic contexts.

Differential proteomic analysis of midguts from Nosema ceranae-infected honeybees reveals manipulation of key host functions.

Many invasive pathogens effectively bypass the insect defenses to ensure the completion of their life cycle. Among those, an invasive microsporidian species, Nosema ceranae, can cause nosemosis in honeybees. N. ceranae was first described in the Asian honeybee Apis cerana and is suspected to be involved in Western honeybee (Apis mellifera) declines worldwide. The midgut of honeybees is the first barrier against N. ceranae attacks. To bring proteomics data on honeybee/N. ceranae crosstalk and more precisely to decipher the worker honeybee midgut response after an oral inoculation of N. ceranae (10days post-infection), we used 2D-DIGE (2-Dimensional Differential In-Gel Electrophoresis) combined with mass spectrometry. Forty-five protein spots produced by the infected worker honeybee group were shown to be differentially expressed when compared to the uninfected group; 14 were subsequently identified by mass spectrometry. N. ceranae mainly caused a modulation of proteins involved in three key host biological functions: (i) energy production, (ii) innate immunity (reactive oxygen stress) and (iii) protein regulation. The modulation of these host biological functions suggests that N. ceranae creates a zone of "metabolic habitat modification" in the honeybee midgut favoring its development by enhancing availability of nutrients and reducing the worker honeybee defense.

Potential Health Benefits of a Diet Rich in Organic Fruit and Vegetables versus a Diet Based on Conventional Produce: A Systematic Review.

CONTEXT: Over the past decade, the production and consumption of organic food (OF) have received increasing interest. Scientific studies have shown better quality of organic fruit and vegetables (FV) in terms of nutrients and pesticide contents, but it appears difficult to conclude if there are potentially greater health benefits of these products compared with conventional food (CF). OBJECTIVE: To determine whether the current scientific literature demonstrates that a diet rich in organic FV is healthier than 1 based on conventional produce. METHODS: A systematic search was conducted using the PubMed and Web of Science databases for articles published between January 2003 and December 2022. Articles were analyzed uniformly by 2 reviewer, using a specific template summary sheet, and scored from 1 to 5. The level of evidence and the quality of studies in humans were assessed using the Jadad score and the French National Authority for Health method. RESULTS: A total of 12 human studies were included. Studies often reported contradictory or even opposite results, with methodological limitations. Only 6 of the 12 studies found significant associations between OF and the health outcomes evaluated. CONCLUSION: The current data do not enable a firm conclusion about a greater health benefit for a diet rich in FV based on products grown organically compared with conventional farming. There is a paucity of available data and considerable heterogeneity in study designs (participants, exposures, durations, health outcomes, and residual confounding factors). Well-designed interventional studies are required.

Development of biomarkers of exposure to xenobiotics in the honey bee Apis mellifera: application to the systemic insecticide thiamethoxam.

This study describes the development of acetylcholinesterase (AChE), carboxylesterases (CaE1, CaE2, CaE3), glutathion-S-transferase (GST), alkaline phosphatase (ALP) and catalase (CAT) as enzyme biomarkers of exposure to xenobiotics such as thiamethoxam in the honey bee Apis mellifera. Extraction efficiency, stability under freezing and biological variability were studied. The extraction procedure achieved good recovery rates in one extraction step and ranged from 65 percent (AChE) to 97.3 percent (GST). Most of the enzymes were stable at -20°C, except ALP that displayed a slight but progressive decrease in its activity. Modifications of enzyme activities were considered after exposure to thiamethoxam at the lethal dose 50 percent (LD(50), 51.16 ng bee(-1)) and two sublethal doses, LD(50)/10 (5.12 ng bee(-1)) and LD(50)/20 (2.56 ng bee(-1)). The biomarker responses revealed that, even at the lowest dose used, exposure to thiamethoxam elicited sublethal effects and modified the activity of CaEs, GST, CAT and ALP. Different patterns of biomarker responses were observed: no response for AChE, an increase for GST and CAT, and differential effects for CaEs isoforms with a decrease in CaE1 and CaE3 and an increase in CaE2. ALP and CaE3 displayed contrasting variations but only at 2.56 ng bee(-1). We consider that this profile of biomarker variation could represent a useful fingerprint to characterise exposure to thiamethoxam in the honey bee A. mellifera. This battery of honey bee biomarkers might be a promising option to biomonitor the health of aerial and terrestrial ecosystems and to generate valuable information on the modes of action of pesticides.

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.

Interactions between Nosema microspores and a neonicotinoid weaken honeybees (Apis mellifera).

Global pollinators, like honeybees, are declining in abundance and diversity, which can adversely affect natural ecosystems and agriculture. Therefore, we tested the current hypotheses describing honeybee losses as a multifactorial syndrome, by investigating integrative effects of an infectious organism and an insecticide on honeybee health. We demonstrated that the interaction between the microsporidia Nosema and a neonicotinoid (imidacloprid) significantly weakened honeybees. In the short term, the combination of both agents caused the highest individual mortality rates and energetic stress. By quantifying the strength of immunity at both the individual and social levels, we showed that neither the haemocyte number nor the phenoloxidase activity of individuals was affected by the different treatments. However, the activity of glucose oxidase, enabling bees to sterilize colony and brood food, was significantly decreased only by the combination of both factors compared with control, Nosema or imidacloprid groups, suggesting a synergistic interaction and in the long term a higher susceptibility of the colony to pathogens. This provides the first evidences that interaction between an infectious organism and a chemical can also threaten pollinators, interactions that are widely used to eliminate insect pests in integrative pest management.

Nosema spp. infection alters pheromone production in honey bees (Apis mellifera).

Pheromones in social insects play a key role in the regulation of group homoeostasis. It is well-established that parasites can modify hormone signaling of their host, but less is known about the effect of parasites on pheromone signaling in insect societies. We, thus, tested in honey bees (Apis mellifera) the effect of the widespread parasite Nosema spp. on the production of ethyl oleate (EO), the only identified primer pheromone in honey bee workers. Since environmental stressors like pesticides also can weaken honey bees, we also analyzed the effect of imidacloprid, a neonicotinoid widely used in agriculture, on EO production. We show that, contrary to imidacloprid, Nosema spp. significantly altered EO production. In addition, the level of Nosema infection was correlated positively with the level of EO production. Since EO is involved in the regulation of division of labor among workers, our result suggests that the changes in EO signaling induced by parasitism have the potential to disturb the colony homoeostasis.

Phenylpyrazole insecticides induce cytotoxicity by altering mechanisms involved in cellular energy supply in the human epithelial cell model Caco-2.

Phenylpyrazoles are relatively new insecticides designed to manage problematic insect resistance and public health hazards encountered with older pesticide families. In vitro cytotoxicity induced by the phenylpyrazole insecticides, Ethiprol and Fipronil, and Fipronil metabolites, sulfone and sulfide, was studied in Caco-2 cells. This cellular model was chosen because it made possible to mimic the primary site of oral exposure to xenobiotics, the intestinal epithelium. Assessment of the barrier function of Caco-2 epithelium was assessed by TEER measurement and showed a major loss of barrier integrity after exposure to Fipronil and its metabolites, but not to Ethiprol. The disruption of the epithelial barrier was attributed to severe ATP depletion independent of cell viability, as revealed by LDH release. The origin of energetic metabolism failure was investigated and revealed a transient enhancement of tetrazolium salt reduction and an increase in lactate production by Caco-2 cells, suggesting an increase in glucose metabolism by pesticides. Cellular symptoms observed in these experiments lead us to hypothesize that phenylpyrazole insecticides interacted with mitochondria.

Fireproofing of domestic upholstered furniture: Migration of flame retardants and potential risks.

Flame retardants (FRs) are widely incorporated in polyurethane foams to decrease their fire reaction. Currently, the risks associated with the use of FRs in domestic upholstered furniture (UF) are evaluated according to FRs volatility and potency to be emitted into the atmosphere. However, exposure via contact and dermal penetration, mediated by sweat, has not been considered so far. Our study provides an identification of the latest-generation of FRs most commonly used in UF, and an evaluation of their potency to migrate into artificial sweat. First of all, an extensive literature search, along with surveys with professionals, led to the identification of twenty-two FRs and synergists commonly used in France and Europe. Then, migration into artificial sweat of various FRs embedded into synthetic or commercially available polymer matrix was studied and evidenced. These results were analysed in the light of their potential effects on human health and the environment. Based on the migration's data, it is not possible to clearly rule out potential effects of FRs on human and environment health. Therefore, the authors consider that the use of FRs in domestic upholstery does not seem to be justified due to potential risks and a lack of clear benefits.

Is acetylcholinesterase a pertinent biomarker to detect exposure of pyrethroids? A study case with deltamethrin.

The possibility to use acetylcholinesterase as biomarker of exposure to deltamethrin insecticide in the honeybee, Apis mellifera were considered. Joined actions of deltamethrin and pirimicarb (carbamate), alone or in association (dual treatment), were investigated on AChE activity in surviving and dead honeybees in order to test its reliability as biomarker. All treatments induced a reduction in tissue AChE activity in dead bees. In surviving bees, deltamethrin treatment induced an important increase of AChE activity that is not abolished by pirimicarb treatment. The analysis of AChE forms revealed an increase in the soluble form in surviving and dead bees and an increase of the membrane form in surviving bees. No direct effect of deltamethrin on soluble and membrane AChE was observed in vitro. The important increase in AChE activity in response to deltamethrin, not altered by pirimicarb treatment, suggests that AChE activity could represent a robust biomarker specific to deltamethrin exposure in living bees.

Impairment of learning and memory performances induced by BPA: Evidences from the literature of a MoA mediated through an ED.

Many rodent studies and a few non-human primate data report impairments of spatial and non-spatial memory induced by exposure to bisphenol A (BPA), which are associated with neural modifications, particularly in processes involved in synaptic plasticity. BPA-induced alterations involve disruption of the estrogenic pathway as established by reversal of BPA-induced effects with estrogenic receptor antagonist or by interference of BPA with administered estradiol in ovariectomized animals. Sex differences in hormonal impregnation during critical periods of development and their influence on maturation of learning and memory processes may explain the sexual dimorphism observed in BPA-induced effects in some studies. Altogether, these data highly support the plausibility that alteration of learning and memory and synaptic plasticity by BPA is essentially mediated by disturbance of the estrogenic pathways. As memory function in humans involves similar signaling pathways, this mode of action of BPA has the potential to alter human cognitive abilities.

Efficiency of an air curtain as an anti-insect barrier: the honey bee as a model insect.

BACKGROUND: Vector-borne diseases are of high concern for human, animal and plant health. In humans, such diseases are often transmitted by flying insects. Flying insects stop their flight when their kinetic energy cannot compensate for the wind speed. Here, the efficiency of an air curtain in preventing insects from entering a building was studied using the honey bee as a model. RESULTS: Bees were trained to visit a food source placed in a building. The air curtain was tested with strongly motivated bees, when the visiting activity was very high. Airflow velocity was modulated by setting an air curtain device at different voltages. At the nominal voltage, the anti-insect efficiency was 99.9 ± 0.2% compared with both the number of bees at a given time in the absence of the air curtain and the number of bees before the activation of the air curtain. The efficiency decreased as the airflow velocity decreased. CONCLUSION: The results show that an air curtain operating at an airflow velocity of 7.5 m s-1 may prevent a strong flyer with high kinetic energy, such as the honey bee, from entering a building. Thus, air curtains offer an alternative approach for combating vector-borne diseases. © 2018 Society of Chemical Industry.

Regulatory identification of BPA as an endocrine disruptor: Context and methodology.

BPA is one of the most investigated substances for its endocrine disruptor (ED) properties and it is at the same time in the center of many ED-related controversies. The analysis on how BPA fits to the regulatory identification as an ED is a challenge in terms of methodology. It is also a great opportunity to test the regulatory framework with a uniquely data-rich substance and learn valuable lessons for future cases. From this extensive database, it was considered important to engage in a detailed analysis so as to provide specific and strong evidences of ED while reflecting accurately the complexity of the response as well the multiplicity of adverse effects. An appropriate delineation of the scope of the analysis was therefore critical. Four effects namely, alterations of estrous cyclicity, mammary gland development, brain development and memory function, and metabolism, were considered to provide solid evidence of ED-mediated effects of BPA.

Nosema ceranae, Fipronil and their combination compromise honey bee reproduction via changes in male physiology.

The honey bee is threatened by biological agents and pesticides that can act in combination to induce synergistic effects on its physiology and lifespan. The synergistic effects of a parasite/pesticide combination have been demonstrated on workers and queens, but no studies have been performed on drones despite their essential contribution to colony sustainability by providing semen diversity and quality. The effects of the Nosema ceranae/fipronil combination on the life traits and physiology of mature drones were examined following exposure under semi-field conditions. The results showed that the microsporidia alone induced moderate and localized effects in the midgut, whereas fipronil alone induced moderate and generalized effects. The parasite/insecticide combination drastically affected both physiology and survival, exhibiting an important and significant generalized action that could jeopardize mating success. In terms of fertility, semen was strongly impacted regardless of stressor, suggesting that drone reproductive functions are very sensitive to stress factors. These findings suggest that drone health and fertility impairment might contribute to poorly mated queens, leading to the storage of poor quality semen and poor spermathecae diversity. Thus, the queens failures observed in recent years might result from the continuous exposure of drones to multiple environmental stressors.