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Recently, the widespread distribution of pesticides detected in the hive has raised serious concerns about pesticide exposure on honey bee (Apis mellifera L.) health. A larval rearing method was adapted to assess the chronic oral toxicity to honey bee larvae of the four most common pesticides detected in pollen and wax--fluvalinate, coumaphos, chlorothalonil, and chloropyrifos--tested alone and in all combinations. All pesticides at hive-residue levels triggered a significant increase in larval mortality compared to untreated larvae by over two fold, with a strong increase after 3 days of exposure. Among these four pesticides, honey bee larvae were most sensitive to chlorothalonil compared to adults. Synergistic toxicity was observed in the binary mixture of chlorothalonil with fluvalinate at the concentrations of 34 mg/L and 3 mg/L, respectively; whereas, when diluted by 10 fold, the interaction switched to antagonism. Chlorothalonil at 34 mg/L was also found to synergize the miticide coumaphos at 8 mg/L. The addition of coumaphos significantly reduced the toxicity of the fluvalinate and chlorothalonil mixture, the only significant non-additive effect in all tested ternary mixtures. We also tested the common 'inert' ingredient N-methyl-2-pyrrolidone at seven concentrations, and documented its high toxicity to larval bees. We have shown that chronic dietary exposure to a fungicide, pesticide mixtures, and a formulation solvent have the potential to impact honey bee populations, and warrants further investigation. We suggest that pesticide mixtures in pollen be evaluated by adding their toxicities together, until complete data on interactions can be accumulated.

As a managed pollinator, the honey bee Apis mellifera is critical to the American agricultural enterprise. Recent colony losses have thus raised concerns; possible explanations for bee decline include nutritional deficiencies and exposures to pesticides and pathogens. We determined that constituents found in honey, including p- coumaric acid, pinocembrin, and pinobanksin 5-methyl ether, specifically induce detoxification genes. These inducers are primarily found not in nectar but in pollen in the case of p- coumaric acid (a monomer of sporopollenin, the principal constituent of pollen cell walls) and propolis, a resinous material gathered and processed by bees to line wax cells. RNA-seq analysis (massively parallel RNA sequencing) revealed that p- coumaric acid specifically up-regulates all classes of detoxification genes as well as select antimicrobial peptide genes. This up-regulation has functional significance in that that adding p- coumaric acid to a diet of sucrose increases midgut metabolism of coumaphos, a widely used in-hive acaricide, by ∼60%. As a major component of pollen grains, p- coumaric acid is ubiquitous in the natural diet of honey bees and may function as a nutraceutical regulating immune and detoxification processes. The widespread apicultural use of honey substitutes, including high-fructose corn syrup, may thus compromise the ability of honey bees to cope with pesticides and pathogens and contribute to colony losses.

Honey bee (Apis mellifera) colonies invest a substantial amount of colony resources in the production of drones during the reproductive season to enable mating with virgin queens from nearby colonies. Recent studies have shown significant differences in the production of sperm cells that are viable (i.e., sperm viability) and can fertilize an ovule among sexually mature drones that are exposed to different environmental conditions during development or as adults. In particular, sperm viability may be negatively affected during drone development from exposure to pesticides in contaminated beeswax. To assess whether sperm viability is negatively affected during drone development from exposure to beeswax contaminated with in-hive pesticides, we compared the viability of sperm collected from drones reared in pesticide-free beeswax with that of drones reared in beeswax contaminated with field-relevant concentrations of the pesticides most commonly found in wax from commercial beekeeping operations in the United States. These pesticides include the miticides fluvalinate, coumaphos and amitraz, and the agro-chemicals chlorothalonil and chlorpyrifos. Sperm from drones collected at 10 and 18 days post emergence were classified as viable or non-viable to calculate sperm viability. For all pesticide treatment groups, drones that were reared in pesticide-laden beeswax had lower sperm viability compared to those reared in pesticide-free beeswax. This difference was especially pronounced among drones reared in miticide-laden wax. Our results reinforce the notion that pesticide contamination of beeswax negatively affects the reproductive quality of drones, which can affect the queens they mate with, ultimately compromising colony health.

In this study, honey bees ( Apis mellifera L.) were exposed to LD 05 and LD 50 doses of five commonly used acaricides for controlling the parasitic mite, Varroa destructor . LD 50 values at 48 h post-treatment showed that tau-fluvalinate was the most toxic, followed by amitraz, coumaphos, thymol, and formic acid. However, the hazard ratios, which estimate the hive risk level based on a ratio of a standard dose of acaricide per hive to the LD 50 of the acaricide, revealed that tau-fluvalinate was the most hazardous followed by formic acid, coumaphos, amitraz, and thymol. The expression of the honey bee acetylcholinesterase gene increased after treatment with the LD 05 and LD 50 acaricide doses and could distinguish three patterns in the timing and level of increased expression between acaricides: one for amitraz, one for tau-fluvalinate and formic acid, and one for coumaphos and thymol. Conversely, changes in cytochrome P450 gene expression could also be detected in response to all five acaricides, but there were no significant differences between them. Changes in vitellogenin gene expression could only detect the effects of tau-fluvalinate, amitraz, or coumaphos treatment, which were not significantly different from each other. Among the acaricides tested, coumaphos, amitraz, and thymol appear to be the safest acaricides based on their hazard ratios, and a good marker to detect differences between the effects of sub-lethal doses of acaricides is monitoring changes in acetylcholinesterase gene expression.

Although Apis mellifera, the western honey bee, has long encountered pesticides when foraging in agricultural fields, for two decades it has encountered pesticides in-hive in the form of acaricides to control Varroa destructor, a devastating parasitic mite. The pyrethroid tau-fluvalinate and the organophosphate coumaphos have been used for Varroa control, with little knowledge of honey bee detoxification mechanisms. Cytochrome P450-mediated detoxification contributes to pyrethroid tolerance in many insects, but specific P450s responsible for pesticide detoxification in honey bees (indeed, in any hymenopteran pollinator) have not been defined. We expressed and assayed CYP3 clan midgut P450s and demonstrated that CYP9Q1, CYP9Q2, and CYP9Q3 metabolize tau-fluvalinate to a form suitable for further cleavage by the carboxylesterases that also contribute to tau-fluvalinate tolerance. These in vitro assays indicated that all of the three CYP9Q enzymes also detoxify coumaphos. Molecular models demonstrate that coumaphos and tau-fluvalinate fit into the same catalytic pocket, providing a possible explanation for the synergism observed between these two compounds. Induction of CYP9Q2 and CYP9Q3 transcripts by honey extracts suggested that diet-derived phytochemicals may be natural substrates and heterologous expression of CYP9Q3 confirmed activity against quercetin, a flavonoid ubiquitous in honey. Up-regulation by honey constituents suggests that diet may influence the ability of honey bees to detoxify pesticides. Quantitative RT-PCR assays demonstrated that tau-fluvalinate enhances CYP9Q3 transcripts, whereas the pyrethroid bifenthrin enhances CYP9Q1 and CYP9Q2 transcripts and represses CYP9Q3 transcripts. The independent regulation of these P450s can be useful for monitoring and differentiating between pesticide exposures in-hive and in agricultural fields.

The parasitic mite Varroa destructor and the associated viruses it transmits are responsible for most instances of honey bee colony losses in the United States. As such, beekeepers utilize miticides to control Varroa populations. Widespread resistance has developed to the miticides fluvalinate and coumaphos. However, Varroa has largely maintained susceptibility to amitraz despite a long and extensive use history. Anecdotal reports of reduced amitraz effectiveness have been a widely discussed contemporary issue among commercial beekeepers. Amitraz resistance was measured by in vitro bioassays with technical amitraz as well as Apivar® efficacy tests. Amitraz resistance was evaluated in commercial beekeeping operations in Louisiana, New York, and South Dakota with a long history of amitraz use. This research shows that amitraz remains an effective Varroa control product in many operations. However, apiaries across operations displayed a wide range of amitraz resistance from no resistance to high resistance that resulted in Varroa control failure. The resistance ratios from in vitro amitraz bioassays were correlated with reduced Apivar® efficacy, demonstrating bona fide cases of Varroa control failures due to amitraz resistance. Therefore, amitraz resistance monitoring protocols need to be developed. A resistance monitoring network should be established to ensure the sustainability of miticide use for Varroa control.

For a half-century, the commercial wild silkworm, Antheraea pernyi, has been protected by coumaphos, which is an internal organophosphorus insecticide used to kill the potential parasitic fly larvae inside. Knowledge about the detoxification genes of A. pernyi as well as the detoxification mechanism for this species remains severely limited. In this study, we identified 281 detoxification genes (32 GSTs, 48 ABCs, 104 CYPs, and 97 COEs) in the genome of this insect, which are unevenly distributed over 46 chromosomes. When compared to the domesticated silkworm, Bombyx mori, a lepidopteran model species, A. pernyi has a similar number of ABCs, but a greater number of GSTs, CYPs, and COEs. By transcriptome-based expression analysis, we found that coumaphos at a safe concentration level significantly changed the pathways related to ATPase complex function and the transporter complex in A. pernyi. KEGG functional enrichment analysis indicated that protein processing in the endoplasmic reticulum was the most affected pathway after coumaphos treatment. Finally, we identified four significantly up-regulated detoxification genes (ABCB1, ABCB3, ABCG11, and ae43) and one significantly down-regulated detoxification gene (CYP6AE9) in response to coumaphos treatment, suggesting that these five genes may contribute to detoxification of coumaphos in A. pernyi. Our study provides the first set of detoxification genes for wild silkworms from Saturniidae and highlights the importance of detoxification gene repertoire in insect pesticide tolerance.

In the search of alternatives for controlling Aethina tumida Murray, we recently proposed the BAA trap which uses boric acid and an attractant which mimics the process of fermentation caused by Kodamaea ohmeri in the hive. This yeast is excreted in the feces of A. tumida causing the fermentation of pollen and honey of infested hives and releasing compounds that function as aggregation pheromones to A. tumida. Since the boron is the toxic element in boric acid, the aim of this article is to assess the amount of boron residues in honey and beeswax from hives treated with the BAA trap. For this aim, the amount of bioaccumulated boron in products of untreated hives was first determined and then compared with the amount of boron of products from hives treated with the BAA trap in two distinct climatic and soil conditions. The study was conducted in the cities of Padilla, Tamaulipas, and Valladolid, Yucatan (Mexico) from August 2014 to March 2015. The quantity of boron in honey was significantly less in Yucatan than in Tamaulipas; this agrees with the boron deficiency among Luvisol and Leptosol soils found in Yucatan compared to the Vertisol soil found in Tamaulipas. In fact, the honey from Yucatan has lower boron levels than those reported in the literature. The BAA treatment was applied for four months, results show that the BAA trap does not have any residual effect in either honey or wax; i.e., there is no significant difference in boron content before and after treatment. On the other hand, the organophosphate pesticide coumaphos was found in 100% of wax samples and in 64% of honey samples collected from Yucatan. The concentration of coumaphos in honey ranges from 0.005 to 0.040 mg/kg, which are below Maximum Residue Limit (MRL) allowed in the European Union (0.1 mg/kg) but 7.14% of samples exceeded the MRL allowed in Canada (0.02 mg/kg).

To study the acaricide resistance status and possible mechanisms of action in conferring resistance to commonly used acaricides (deltamethrin and coumaphos), Hyalomma anatolicum ticks were collected from 6 dairy farms of Hisar and Charkhi Dadri districts of Haryana. By using standard larval packet test, H. anatolicum tick larvae of Charkhi Dadri isolates were found to be susceptible (100% mortality) to both the acaricides. Level-I resistance against coumaphos was recorded from four isolates, whereas, level-II was observed in only one isolate, collected from Hisar. One isolates (Kaimri) from Hisar also showed level-I resistance against deltamethrin. Biochemically, the ticks having higher values of resistance factor (RF) against coumaphos were found to possess increased enzymatic activity of α-esterase, β-esterase, glutathione-S-transferase (GST) and mono-oxygenase enzymes, whereas, the monoamine oxidase did not show any constant trend. However, the RF showed a statistical significant correlation with GST only. Native PAGE analysis of H. anatolicum ticks revealed the presence of nine types of esterases (EST-1 h to EST-9 h) by using napthyl acetate as substrate. In the inhibitory assay, esterases were found to be inhibited by PMSF, indicating the presence of serine residue at catalytic triad. The partial cds of carboxylesterase and domain II of sodium channel genes were sequenced to determine any proposed mutations in resistant isolates of H. anatolicum ticks, however, no mutations were observed in either gene, indicating that increased expression of detoxification enzymes as a possible mechanism for resistance development, in the current study.

Pesticides that target cholinergic neurotransmission are highly effective, but their use has been implicated in insect pollinator population decline. Honeybees are exposed to two widely used classes of cholinergic pesticide: neonicotinoids (nicotinic receptor agonists) and organophosphate miticides (acetylcholinesterase inhibitors). Although sublethal levels of neonicotinoids are known to disrupt honeybee learning and behaviour, the neurophysiological basis of these effects has not been shown. Here, using recordings from mushroom body Kenyon cells in acutely isolated honeybee brain, we show that the neonicotinoids imidacloprid and clothianidin, and the organophosphate miticide coumaphos oxon, cause a depolarization-block of neuronal firing and inhibit nicotinic responses. These effects are observed at concentrations that are encountered by foraging honeybees and within the hive, and are additive with combined application. Our findings demonstrate a neuronal mechanism that may account for the cognitive impairments caused by neonicotinoids, and predict that exposure to multiple pesticides that target cholinergic signalling will cause enhanced toxicity to pollinators. Exposure to pesticides can disrupt foraging and navigation behaviour in bees. Palmer et al . use electrophysiology to show that two neonicotinoids and an organophosphate miticide cause neuronal dysfunction in the honeybee brain at environmentally relevant concentrations.