Strong amitraz resistance in Varroa destructor is associated with a sustained GST-dominant detoxification profile

Varroa destructor is a major ectoparasite of honey bees, and the development of amitraz resistance threatens the long-term efficacy of one of the most widely used acaricides in apiculture. This study assessed amitraz susceptibility in Varroa populations from three Polish apiaries and examined whether differences in susceptibility were associated with population-specific detoxification enzyme responses. Amitraz toxicity was evaluated using vial bioassays and probit dose-response modelling, while glutathione S-transferase (GST) and nonspecific esterase activities were measured in surviving mites. The studied populations differed markedly in amitraz susceptibility. Population A1 showed an LC₅₀ of 0.0043 ppm, whereas A2 had an LC₅₀ of 0.0627 ppm and a conservative RR₅₀ lower bound of > 12.54, indicating strong amitraz resistance. In the reference population, mortality was 100% at all tested amitraz concentrations; therefore, the LC₅₀ was reported as < 0.005 ppm. Multivariate analyses revealed distinct population-specific detoxification profiles. A1 exhibited a pronounced esterase-dominant response at the lowest amitraz concentration, whereas the strongly resistant A2 population showed sustained GST-dominant activity across increasing amitraz concentrations at which mites survived. Further analysis demonstrated that enzymatic responses depended on both population origin and amitraz exposure. These findings suggest that amitraz resistance may be associated not with a generalized increase in detoxification capacity, but with the activation and persistence of specific enzymatic pathways, particularly GST-mediated detoxification. Because live honey bees are traded internationally, the occurrence of strongly resistant Varroa populations in exporting countries may have consequences beyond local beekeeping and highlights the need for coordinated resistance surveillance within international bee-trade networks.