Negative geotaxis climbing assay

Locomotor activity of the flies was tested using the geotaxis assay described elsewhere (Gargano et al., 2005; Piazza et al., 2009). Flies were collected on the day of eclosion under brief CO₂ anesthesia (1-2 min), sorted in groups of ten (five males and five females) and allowed to recover at least 18 h at 25°C prior to the assay. Next day, flies were transferred into vials marked by lines forming four equally spaced quadrants. Each vial was gently tapped to bring flies to the bottom of the vial and to initiate the negative geotaxis response. Flies were allowed to climb up for 4 s before a photograph was taken to record the position of each fly in the vial. Each fly was assigned a rating based on the quadrant it reached; flies that did not climb and stayed at the bottom were scored as zero. The weighted average of three consecutive trials was calculated for each vial, representing the climbing index for that vial.