RNAi targeting ABCB1-like efflux transporters improves miticide efficacy in resistant Varroa mites

The ectoparasitic mite Varroa destructor is the gravest threat to managed honeybees, and its control relies on a limited number of chemical miticides. Among these, amitraz is widely used because of its strong efficacy against mites and relatively low toxicity to bees. However, increasing resistance to amitraz in Varroa populations threatens its long-term effectiveness. While mutations in the mite's β2 octopamine receptor are strongly associated with amitraz resistance, additional mechanisms influencing toxicant uptake and efflux are believed to also contribute. ATP-binding cassette (ABC) transporters, including ABCB1/P-glycoproteins, are well-established mediators of xenobiotic efflux and pesticide tolerance across arthropods, making them promising targets for silencing via RNA interference (RNAi) to combat miticide resistance.

We cloned a full-length Varroa ABCB1-like transporter (VdABCB1) and synthesized dsRNAs targeting its coding sequence. Adult mites were treated with dsRNA prior to amitraz exposure in laboratory bioassays. Mite survival was analyzed longitudinally, and resistance-associated β2 octopamine receptor genotypes were determined.  Varroa transcriptomic responses to dsRNA were assessed by RNA sequencing. Honeybee safety was evaluated in cage assays following chronic oral dsRNA exposure, including conservative tests co-administered with a known ABC transporter substrate.

Mites exposed to ABCB1 dsRNA showed greater amitraz-induced mortality than those treated with non-specific dsRNA. This effect was observed across multiple trials and amitraz concentrations. Transcriptomic analyses of mites revealed a significant knockdown of ABCB1-like transcripts following RNAi treatment. Chronic dietary exposure to ABCB1 dsRNA did not impact honeybee survival. Toxicity assays with ABCB1 dsRNA on its own or together with acetamiprid (ABC transporter substrate) as a high-risk interaction control showed minimal adverse effects on bees.

RNAi suppression of Varroa ABCB1-like transporters increased amitraz mortality in resistant mites, identifying transporter-mediated efflux as a modifiable component of amitraz resistance. These findings demonstrate that targeted disruption of detoxification pathways can enhance miticide efficacy while minimizing off-target effects in bees. RNAi-based synergists therefore represent a selective resistance management strategy that could extend the effective lifespan of amitraz and other miticides relied on by the beekeeping industry.