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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.

Cytochrome P450 monooxygenases (P450) in the honey bee, Apis mellifera, detoxify phytochemicals in honey and pollen. The flavonol quercetin is found ubiquitously and abundantly in pollen and frequently at lower concentrations in honey. Worker jelly consumed during the first 3 d of larval development typically contains flavonols at very low levels, however. RNA-Seq analysis of gene expression in neonates reared for three days on diets with and without quercetin revealed that, in addition to up-regulating multiple detoxifying P450 genes, quercetin is a negative transcriptional regulator of mitochondrion-related nuclear genes and genes encoding subunits of complexes I, III, IV, and V in the oxidative phosphorylation pathway. Thus, a consequence of inefficient metabolism of this phytochemical may be compromised energy production. Several P450s metabolize quercetin in adult workers. Docking in silico of 121 pesticide contaminants of American hives into the active pocket of CYP9Q1, a broadly substrate-specific P450 with high quercetin-metabolizing activity, identified six triazole fungicides, all fungal P450 inhibitors, that dock in the catalytic site. In adults fed combinations of quercetin and the triazole myclobutanil, the expression of five of six mitochondrion-related nuclear genes was down-regulated. Midgut metabolism assays verified that adult bees consuming quercetin with myclobutanil metabolized less quercetin and produced less thoracic ATP, the energy source for flight muscles. Although fungicides lack acute toxicity, they may influence bee health by interfering with quercetin detoxification, thereby compromising mitochondrial regeneration and ATP production. Thus, agricultural use of triazole fungicides may put bees at risk of being unable to extract sufficient energy from their natural food.

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.

Endoparasitism by gall-forming insects dramatically alters the plant phenotype by altering growth patterns and modifying plant organs in ways that appear to directly benefit the gall former. Because these morphological and physiological changes are linked to the presence of the insect, the induced phenotype is said to function as an extension of the parasite, albeit by unknown mechanisms. Here we report the gall-forming aphid-like parasite phylloxera, Daktulosphaira vitifoliae, induces stomata on the adaxial surface of grape leaves where stomata typically do not occur. We characterized the function of the phylloxera-induced stomata by tracing transport of assimilated carbon. Because induction of stomata suggests a significant manipulation of primary metabolism, we also characterized the gall transcriptome to infer the level of global reconfiguration of primary metabolism and the subsequent changes in downstream secondary metabolism. Phylloxera feeding induced stomata formation in proximity to the insect and promoted the assimilation and importation of carbon into the gall. Gene expression related to water, nutrient, and mineral transport; glycolysis; and fermentation increased in leaf-gall tissues. This shift from an autotrophic to a heterotrophic profile occurred concurrently with decreased gene expression for nonmevalonate and terpenoid synthesis and increased gene expression in shikimate and phenylpropanoid biosynthesis, secondary metabolite systems that alter defense status in grapes. These functional insect-induced stomata thus comprise part of an extended phenotype, whereby D. vitifoliae globally reprograms grape leaf development to alter patterns of primary metabolism, nutrient mobilization, and defense investment in favor of the galling habit.

Over evolutionary time, insect herbivores have adapted to the presence of natural toxins and more recently to synthetic insecticides in or on the plants they consume. Biochemical analyses and molecular modeling of the cytochrome P450 monooxygenases (P450s) that metabolize these compounds have provided insight into the many variations affecting their catalytic activity. Phylogenetically distinct P450s may metabolize similar substrates, and phylogenetically similar P450s may metabolize different substrates; as well, some P450s process broad arrays of both phytochemicals and synthetic insecticides, while closely related P450s are restricted to a narrow range of phytochemicals. Mapped on the predicted three-dimensional structures of insect P450s developed from available mammalian P450 crystal structures, differences in multiple regions of the insect proteins reveal the evolutionary processes occurring as P450 genes have duplicated and diverged. Analyses of site-directed mutants in select lepidopteran and dipteran P450s demonstrate that slight changes in the catalytic site, the putative product release channel, and the proximal surface (interacting with electron transfer partners such as cytochrome P450 reductase and cytochrome b5) yield pronounced activity differences. Additionally, changes in the catalytic site and in the linker region preceding the proline-hinge influence P450 folding. With predicted structures available for many mammalian P450s involved in metabolism of xenobiotics, it is possible to record allelic variation relative to catalytically important regions in the overall P450 structure and to predict functionally critical differences. Together with information on the relative levels of allelic variant transcripts, comprehensive characterization of the mechanisms that modulate metabolism of natural and synthetic xenobiotics in insects can yield insights into plant-insect coevolution and into novel approaches for chemical pest management.

The presence of the neonicotinoid imidacloprid in nectar, honey, pollen, beebread and beeswax has been implicated in declines worldwide in the health of the western honey bee Apis mellifera. Certain phytochemicals, including quercetin and p-coumaric acid, are ubiquitous in the honey bee diet and are known to upregulate cytochrome P450 genes encoding enzymes that detoxify insecticides. Thus, the possibility exists that these dietary phytochemicals interact with ingested imidacloprid to ameliorate toxicity by enhancing its detoxification. Quercetin and p-coumaric acid were incorporated in a phytochemical-free artificial diet individually and together along with imidacloprid at a range of field-realistic concentrations. In acute toxicity bioassays, honey bee 24- and 48- hour imidacloprid LC50 values were determined in the presence of the phytochemicals. Additionally, chronic toxicity bioassays were conducted using varying concentrations of imidacloprid in diets with the phytochemicals to test impacts of phytochemicals on longevity. In acute toxicity bioassays, the phytochemicals had no effect on imidacloprid LC50 values. In chronic toxicity longevity bioassays, phytochemicals enhanced honey bee survival at low imidacloprid concentrations (15 and 45 ppb) but had a negative effect at higher concentrations (105 ppb and 135 ppb). p-Coumaric acid alone increased honey bee longevity at concentrations of 15 and 45 ppb imidacloprid (hazard ratio (HR): 0.83 and 0.70, respectively). Quercetin alone and in combination with p-coumaric acid similarly enhanced longevity at 45 ppb imidacloprid (HR:0.81 and HR:0.77, respectively). However, p-coumaric acid in combination with 105 ppb imidacloprid and quercetin in combination with 135 ppb imidacloprid increased honey bee HR by approximately 30% (HR:1.33 and HR:1.30, respectively). The biphasic concentration-dependent response of honey bees to imidacloprid in the presence of two ubiquitous dietary phytochemicals indicates that there are limits to the protective effects of the natural diet of honey bees against neonicotinoids based on their own inherent toxicity.

For the past decade, migratory beekeepers who provide honey bees for pollination services have experienced substantial colony losses on a recurring basis that have been attributed in part to exposure to insecticides, fungicides, or their combinations applied to crops. The phytochemicals p -coumaric acid and quercetin, which occur naturally in a wide variety of bee foods, including beebread and many types of honey, can enhance adult bee longevity and reduce the toxicity of certain pesticides. How variation in concentrations of natural dietary constituents affects interactions with xenobiotics, including synthetic pesticides, encountered in agroecosystems remains an open question. We tested the effects of these two phytochemicals at a range of natural concentrations on impacts of consuming propiconazole and chlorantraniliprole, a triazole fungicide and an insecticide frequently applied as a tank mix to almond trees during bloom in California’s Central Valley. Propiconazole, even at low field concentrations, significantly reduced survival and longevity when consumed by adult bees in a sugar-based diet. The effects of propiconazole in combination with chlorantraniliprole enhanced mortality risk. The detrimental effects of the two pesticides were for the most part reduced when either or both of the phytochemicals were present in the diet. These findings suggest that honey bees may depend on non-nutritive but physiologically active phytochemical components of their natural foods for ameliorating xenobiotic stress, although only over a certain range of concentrations; particularly at the high end of the natural range, certain combinations can incur additive toxicity. Thus, efforts to develop nectar or pollen substitutes with phytochemicals to boost insecticide tolerance or immunity or to evaluate toxicity of pesticides to pollinators should take concentration-dependent effects of phytochemicals into consideration.

Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored. We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. These results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey.

Colony collapse disorder (CCD) is a mysterious disappearance of honey bees that has beset beekeepers in the United States since late 2006. Pathogens and other environmental stresses, including pesticides, have been linked to CCD, but a causal relationship has not yet been demonstrated. Because the gut acts as a primary interface between the honey bee and its environment as a site of entry for pathogens and toxins, we used whole-genome microarrays to compare gene expression between guts of bees from CCD colonies originating on both the east and west coasts of the United States and guts of bees from healthy colonies sampled before the emergence of CCD. Considerable variation in gene expression was associated with the geographical origin of bees, but a consensus list of 65 transcripts was identified as potential markers for CCD status. Overall, elevated expression of pesticide response genes was not observed. Genes involved in immune response showed no clear trend in expression pattern despite the increased prevalence of viruses and other pathogens in CCD colonies. Microarray analysis revealed unusual ribosomal RNA fragments that were conspicuously more abundant in the guts of CCD bees. The presence of these fragments may be a possible consequence of picorna-like viral infection, including deformed wing virus and Israeli acute paralysis virus, and may be related to arrested translation. Ribosomal fragment abundance and presence of multiple viruses may prove to be useful diagnostic markers for colonies afflicted with CCD.