Vented 75 cm2 tissue culture flasks

Mixed stage and mixed sex D. gallinae mites were collected from commercial egglaying facilities and stored in vented 75 cm 2 tissue culture flasks (Corning Inc, Corning, NY, USA) at room temperature for seven days, after which they were stored at 4°C for 3 weeks without access to food. For oral delivery of dsRNA and siRNA to D. gallinae mites, approx. 100 mites were housed in an in vitro feeding device constructed as described previously (29) . Each replicate feeding device contained 200 µl of freshlycollected heparinised goose blood (20 units/ml) with dsRNA at concentrations described in each experiment. For each dsRNA feeding experiment, biological replicates consisted of independent group of mites in replicate feeding devices (n = 3 -6 depending on experiment). Feeding devices were placed in a Sanyo MLR-351H relative humidity incubator for 3h at 39°C; 21h at 25°C both at 85% relative humidity.
After 24 h, fed adult female mites were recovered from each replicate feeding divice and transferred to separate labelled 1.5ml tubes, which were held at 25°C in dark conditions for the duration of the experiment. Mites from each replicate group were flash-frozen in liquid nitrogen at time-points indicated in each experiment and stored at -70°C for later RNA extraction.