Identification and functional characterization of CYP3002B2, a cytochrome P450 associated with amitraz and flumethrin resistance in the major bee parasite Varroa destructor

Beekeeping worldwide is increasingly threatened by the parasitic mite Varroa destructor, whose management relies heavily on synthetic acaricides such as amitraz and flumethrin. However, the growing incidence of acaricide resistance in V. destructor presents a significant global challenge to apiculture. In this study, we investigated the mechanisms underlying resistance to these compounds in a V. destructor population exhibiting reduced susceptibility to both amitraz and flumethrin. Specifically, bioassays revealed that the resistant population (IL-R) displayed 35.0 % mortality in response to amitraz and 39.5 % mortality to flumethrin, in contrast to >90 % mortality observed in the susceptible IL-L and ATH-S populations. The resistance phenotype was not strongly associated with any of the known target site mutations; the putative amitraz resistance mutation F290L in the Octβ2R gene, and the pyrethroid resistant mutation L925V in the vgsc gene, were found at low frequencies (8.6 % and 13.6 % respectively). Transcriptomic analysis, comparing gene expression levels between the resistant population and two susceptible populations, revealed that resistance is associated with the overexpression of several cuticle genes and the cytochrome P450 gene CYP3002B2. CYP3002B2 was functionally expressed in E. coli, exhibiting catalytic activity against multiple model substrates and effectively metabolizing both amitraz and flumethrin. The predominant product of amitraz metabolism is likely an inactive, hydroxylated form of the insecticide, rather than any of the known activated/toxic metabolites of amitraz. These findings are crucial for evidence-based V. destructor management, as CYP3002B2 is the first detoxification enzyme shown to metabolize two major acaricides from different modes of action classes.