Neonicotinoid Exposure and Climbing Ability in Drosophila

Adult D. melanogaster flies were collected within a day following eclosion and placed in normal fly food (less than 30 flies/vial). Flies were transferred daily to new fly food. 2–5 days after eclosion, flies were briefly anesthetised with CO₂, and the sexes were separated and sorted into fly vials containing 1.0% agar food for starvation (10 flies/vial). After 16 h starvation, flies were transferred to vials containing neonicotinoid-containing food without anesthesia and cultured for 6 h (10 flies/vial). Neonicotinoid-containing foods were prepared by mixing 10 μL of diluted neonicotinoids dissolved in DMSO with 990 μL of a solution containing 1% agar and 5% sucrose for each vial. After 6 h cultured in vials containing neonicotinoid-containing food, flies were gently tapped down to the surface of the food, and flies that climbed within 20 s after tapping were recorded by a video camera (GZ-F270-W, JVCKENWOOD, Japan). The maximum climbing heights of the flies within 20 s after tapping were measured using ImageJ1.53v (National Institute of Health, USA). Since the height from the surface of the food to the vial top is 8 cm, the maximum climbing height is 8 cm.