Significance: Honey bees pollinate the majority of crops, but their survival is under threat. The bee parasitic mite Varroa destructor is a major cause of honey bee decline. The successful control of Varroa mites depends on a small number of in-hive acaricides. Species-specific activation of the chemical coumaphos is a powerful “pro-drug” approach to attain this selective toxicity and protect bees. We identified a coumaphos-resistant Varroa population that escapes toxicity by down-regulating the activating enzyme, a P450 monooxygenase. Decreased activation is a rare but evolutionarily powerful solution to achieve resistance. It demonstrates that the current proinsecticide approach is under threat, as it can be circumvented, and highlights the need to restart the pipeline to develop selective pest control agents.

Insecticide resistance in mosquitoes, largely mediated by cytochrome P450 monooxygenases (CYPs), compromises the efficacy of vector control tools. In this study, chemically-wise selected natural extracts and compounds were screened for their CYP inhibition potential. Among 37 tested plant extracts and fractions, a decarboxylated acidic fraction of industrial hemp ( Cannabis sativa Linnaeus var. ‘Futura 75’) emerged as a promising hit, and phytochemical profiling identified cannabidiol (CBD) as its major component (IC₅₀ = 18.37 μM for CYP9K1). CBD was used as a scaffold to generate semisynthetic analogues; of which a piperazinyl analogue outperformed the natural scaffold demonstrating significantly greater potency (IC₅₀ = 2.50 μM for CYP9K1). Docking studies using homology-derived CYP9K1 models also supported a stronger binding affinity of the piperazinyl analogue relative to CBD. Toxicity assays using pyrethroid-resistant Anopheles gambiae Giles adults confirmed that neither CBD nor the piperazinyl analogue had intrinsic toxicity, yet the semisynthetic analogue significantly enhanced deltamethrin efficacy, showing a threefold synergistic effect. The safety profile of the cannabis compounds for non-target organisms was evaluated through human cell line cytotoxicity tests and bee toxicity assays, suggesting low non-target organism toxicity. Our study describes the identification of a plant-derived synergist lead with strong potential as an insecticide additive to combat metabolic resistance in malaria-transmitting mosquitoes.

Neonicotinoid toxicity to honeybees varies among active ingredients, with the insects being tolerant to thiacloprid but sensitive to imidacloprid. The selective toxicity has been hypothesized to be due to differences in metabolic detoxification by cytochrome P450s. In vitro experiments revealed that CYP9Q3 efficiently metabolises thiacloprid but not imidacloprid, consistent with the toxicity data. Honeybee tolerance to tau-fluvalinate, a pyrethroid acaricide used against Varroa destructor, has also been linked to P450-mediated detoxification. To address the role of CYP9Q3 in vivo in Apis mellifera, we used an RNA interference approach based on double-stranded RNA delivery through the median ocellus of adults to transiently suppress expression of CYP9Q3 in the head. Following confirmation of CYP9Q3 suppression by qPCR, fixed-dose topical bioassays were conducted with the neonicotinoids imidacloprid and thiacloprid and the pyrethroid tau-fluvalinate. Suppression of CYP9Q3 was associated with increased mortality following exposure to thiacloprid and tau-fluvalinate, whereas no increase was observed with imidacloprid. These findings provide in vivo functional evidence supporting the role of CYP9Q3 in honeybee tolerance to thiacloprid and tau-fluvalinate. The RNAi approach offers a valuable tool for investigating the functions of cytochrome P450 enzymes and other genes expressed in the honey bee head that are involved in insecticide susceptibility.