Fig. 1
Protocol developed for studying the transmission of DWV between Varroa destructor (Vd) females and honey bees (HB) in in vitro conditions. During step 1, white-eyed pupae were inoculated with 5 × 102 copies of DWV variant A or B (DWV-A or DWV-B), or injected with control phosphate buffer (PB). After 4 days, a total of 62 mites sampled from the infested colony were transferred to the treated brown-eyed pupae (22 mites on 22 PB-injected pupae, 21 mites on 21 DWV-A-inoculated pupae and 19 mites on 19 DWV-B-inoculated pupae). Nine additional mites were also sampled to check their initial DWV status, and 21 bee pupae were left without mites (7 per DWV condition). The mites and pupae were kept in an incubator (34.5 °C, 70% relative humidity) for 4 additional days until the bee reached the imaginal stage. A sample of 22 mites and bees was frozen for later molecular analysis (8 in the PB control, 8 in the DWV-A treatment and 6 in the DWV-B condition) along with the 21 Varroa-free bees. The remaining 40 mites (14 in the PB control and 13 in each of the two test conditions) were used in a second step and transferred to untreated fifth instar larvae from the healthy colony in gelatin capsules. The capsules were then kept in an incubator until emergence. These bees (when alive) and mites were then sampled and stored at − 80 °C until molecular analyses were performed. The analyses later revealed a pre-existing DWV-B infection interfering with our treatments. DWV, Deformed wing virus; EB, emerging bees