Fig. 3

Generalized experimental workflow. (1) Varroa mites were collected from an apiary located at the Onna village office in Okinawa, Japan, March, 2020. Mites were shaken off the honey bees by sprinkling powdered sugar on the frame. Bees were collected into a tray and shaken to remove mites. The tray containing powdered sugar and Varroa mites was transported back to the laboratory, within 30 minutes, discarding dead mites on arrival. (2) Individual mites were placed in separate 1.5-mL microcentrifuge tubes and (3) were immersed in 500 µL of a preservative solution or snap-frozen and stored at -80 °C until processing. (4) Incubation periods were 5, 10, 15, and 21 days in respective preservation methods. (5) Preservation solution was discarded after the incubation period, and (6) samples were immersed in liquid nitrogen for a minute, then (7) crushed with a sterile pestle that was also immersed in liquid nitrogen. Each sample was split in two tubes, 8a) mite dust on the pestle was washed with ATL buffer into a new tube and used for DNA extraction with a QIAamp DNA extraction kit and 8b) TRIzol was added to the tube containing mite dust and subjected to RNA extraction using TRIzol