CYP9Q-mediated detoxification of acaricides in the honey bee (Apis mellifera)

Abstract

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.Apidae | miticide | transcriptional regulation

Honey constituents up-regulate detoxification and immunity genes in the western honey bee Apis mellifera

“…Remarkably, among these 31 genes (Table 1), 12 xenobioticmetabolizing genes, including seven genes encoding phase I enzymes, four genes encoding phase II enzymes (three of which comprise one-quarter of all uridine-diphosphate-glucosyl transferases in the honey bee genome) (20), and two genes encoding phase III enzymes, were up-regulated by p-coumaric acid. In addition to CYP9Q3, known to metabolize pesticides (16), four CYP6AS enzymes known to metabolize honey flavonoids (10) and CYP6BD1 were induced 1.9-to 3.11-fold. Also up-regulated by almost twofold was the gene encoding abaecin (Dataset S1), an antimicrobial peptide mediating immunity against bacteria (21).…”

“…To use an ecologically reasonable concentration and at the same time maximize the likelihood of observing a functional response, we selected a concentration of 1 mg/g, approximately double the concentrations reported in certain pollen (0.41 mg/g) (22) and beebread (0.367 mg/g) (23). Midguts were removed and assayed for rates of substrate disappearance of coumaphos, an acaricide known to be metabolized by CYP9Q3 (16), which is included among the genes up-regulated by p-coumaric acid in the RNA-Seq analysis. Activities of control and p-coumaric acid-induced midguts toward coumaphos were 2.57 and 4.10 nmol/min per midgut, respectively.…”

“…Honey bees metabolize phytochemicals found in honey and pollen as well as acaricides used in-hive for management of Varroa destructor, an ectoparasitic mite of honey bees, via a number of CYP6 and CYP9 family members. Quercetin, a flavonoid constituent of honey and pollen, is metabolized by three enzymes in the CYP6AS subfamily and two enzymes in the CYP9Q subfamily (15,16), whereas the acaricides coumaphos and τ-fluvalinate are detoxified by three enzymes in the CYP9Q subfamily (16). Regulation of these detoxification genes in A. mellifera differs in some respects from P450 regulation in other insects (17) in that CYP6AS3, which metabolizes quercetin, is not inducible by its substrate or by phenobarbital, a classic experimental inducer of insect P450 transcription.…”

Resistance of developing honeybee larvae during chronic exposure to dietary nicotine

“…However, an expansion is observed in the CYP6AS subfamily (Mao et al 2009) and a similar expansion in the CYP6 family in lepidopteran genomes is related to the xenobiotic detoxification ability of these insects (Li et al, 2003). The dominance and redundancy of the CYP6AS family in the honeybee genome may in part be responsible for the ability of bees to detoxify both natural (Mao et al, 2009) and synthetic (Mao et al, 2011;Suchail et al, 2003) xenobiotics present in their diet. An alternative -but not mutually exclusive -explanation for the resistance to pesticides suggested by our results could be the higher genetic variability of African honeybee populations.…”

“…The latter may be due to reduced nutrient assimilation (Bentz and Barbosa, 1990); alternatively, detoxification may be energetically costly, using resources that could otherwise be diverted to growth and development. Tolerance of dietary toxins is most likely due to the presence of metabolic detoxification mechanisms mediated in part by P450 enzymes Mao et al, 2011).…”