Locomotion analysis

Adult geotaxis was measured in adult male flies aged 2–3 days as previously described.⁵⁹ Eight cohorts of ten adult males (80 total flies) per genotype were separated after eclosion and allowed to recover from CO₂ for 24 h on standard fly medium. After 24 h, each cohort was moved to a chamber made from two clear plastic vials taped together, with a line drawn around the lower vial 8 cm from the bottom. Each cohort was allowed to acclimate in the chamber for 5 min before assays began. Negative geotaxis was measured as percent of the cohort that crossed the 8 cm line within 10 s after being tapped to the bottom of the chamber. Each cohort was subjected to ten rounds of negative geotaxis assay, with a 1-min rest period between each. The percent of flies that crossed the 8 cm line after each round was averaged to produce a pass rate per cohort. Larval crawling was assayed in third-instar larvae of both sexes, as previously described.^60,61 Larvae were briefly washed in room temperature water before placing onto the center of a 5 cm Petri dish containing 2% agarose, with five animals from a single genotype placed together (n = 10 larvae per genotype). The Petri dish was placed over a grid and velocity was measured as average distance (in mm) traveled during the first 30 s following placement.