Experimental males were reared on SY10 food for 2–3 days shortly after eclosion. We aspirated individual flies into the two-choice food arenas (diameter = 31 mm, either a choice diet or a fixed diet, N = 12 for each group) and let them adapt to the environment for 1 day. Then, 6 hr video recording (10:00–16:00, at two frames per second with a resolution at 528 × 320) was taken and analyzed with DTrack, a video analysis package developed in the Pletcher Laboratory. Using the same speed threshold published before (Corrales-Carvajal et al., 2016), we classified the movements of flies into resting or sleeping (speed ≤ 0.2 mm/s), micromovement (0.2 mm/s < speed ≤2 mm/s), and walking (speed > 2 mm/s). For positional preference assay, we aspirated individual male flies into the two-choice food arenas (N = 24 for each genotype) after the designated exposure to the choice diet for 7 days. Three hour video recordings were taken (at two frames per second with a resolution at 528 × 320) and analyzed with DTrack. The fraction of time flies spent on each macronutrient was computed and used for statistical analyses.

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