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.

Fig. 1.

Untargeted GC-MS analysis of tau-fluvalinate metabolism. (A) Chromatograms for controls (black) and for tau-fluvalinate treatments (red). These are compounds extracted from frass of control insects and of tau-fluvalinate–treated insects. Products were BFSTA-derivatized and then analyzed by GC-MS. (B) Mass spectrum of the arrow-marked red peak specific for the tau-fluvalinate treatments. The insert represents the fragment of tau-fluvalinate metabolite (338 Da).

Fig. 2.

Docking of tau-fluvalinate in the CYP9Q2 catalytic site. (A) The docking mode for tau-fluvalinate (elemental colors in stick format) in the predicted CYP9Q2 catalytic site is shown with substrate contacts within 4.5 Å of this substrate. Overlaid with this are the predicted CYP9Q1 and CYP9Q3 catalytic sites with Phe123, Phe305, Phe374, Phe491, and Lys221 conserved in two or three CYP9Q proteins, shown in yellow, and other side chains conserved in all three CYP9Q proteins, shown in green. The heme and its bound oxygen are shown in space-filling format. (B and C) The predicted docking modes for tau-fluvalinate (B) and coumaphos (C) in CYP9Q2 are shown in elemental colors, with amino acid side chains within 4.5 Å of this substrate shown in red for acidic residues, blue for basic residues, green for hydrophobic residues, and cyan for hydrophilic residues.

Fig. 3.

Induction of CYP9Q subfamily genes. (A) Relative expression levels of CYP9Q subfamily transcripts in midguts of workers fed with ethyl acetate/methanol fractions (10× concentrated) of ethyl acetate extract of honey using solvent treatments as controls. (B) Relative expression levels of CYP9Q subfamily transcripts in midguts of workers treated with 15 μg per bee of tau-fluvalinate, 0.1 μg per bee of cypermethrin, and 0.1 μg per bee of bifenthrin with acetone treatments as controls. The data shown are mean ± SEM (three biological replicates).