Introduction
Varroa destructor is currently the most serious parasitic threat to honey bee health. The intensive use of synthetic acaricides has led to the emergence of resistant mite populations, creating an urgent need for alternative control strategies. This study evaluates the acaricidal activity of essential oils (EOs) from Origanum vulgare subsp. viridulum, Thymus capitatus, Thymus longicaulis, and Salvia rosmarinus against V. destructor through in vitro testing.

Methods
Each EO was dissolved in HPLC-grade acetone (2 mg/ml) and applied at a volume of 50 µl inside Eppendorf tubes. The open tubes were placed in an incubator at 35 °C and rotated multiple times to facilitate the EO impregnation of the tube walls while allowing for acetone evaporation. Subsequently, five Varroa mites were introduced into each tube, which was then resealed and returned to the incubator. Control groups included a negative control (acetone only) and a positive control (Amitraz diluted in acetone). Mortality was assessed manually after 1 hr in the incubator by probing individual mites with an entomological pin. Mites showing no movement were classified as dead, while those unable to walk but still capable of moving at least one leg were classified as inactive.

Results and conclusions

The control groups confirmed assay reliability, with 100% survival in the negative control and 100% mortality in the Amitraz-treated group. Among the tested EOs, O. vulgare subsp. viridulum and T. longicaulis exhibited the highest acaricidal activity, each killing 94% of mites. T. capitatus showed slightly lower but still significant efficacy (92%). In contrast, S. rosmarinus demonstrated a more moderate effect, killing 32% of mites. These findings suggest that oregano and thyme essential oils have strong acaricidal properties and could be promising candidates for Varroa control in beekeeping.