Drosophila Genetic Manipulation and ETC Inhibition

Wild-type flies (white Dahomey, wDAH), RNA interference (RNAi) and GAL4 driver lines were collected and cultured as in [41 (link)]. Briefly, flies were maintained on standard media (1% Drosophila agar type II (Dutscher Scientific, #789,150), 1.5% sucrose (Sigma, #S27480), 3% glucose (Sigma, #16,325), 3.5% dried yeast (Dutscher Scientific, #789,093), 1.5% maise (TRS), 1% wheat (MP Biomedicals, #0,290,328,805), 1% soy (Santa Cruz Biotechnology, #Sc-215897A), 3% treacle (Bidvest, #90028S), 0.5% propionic acid (VWR, #8.00605.2500), 0.1% Nipagin (Sigma, #H5501)), collected using CO₂ anaesthesia within 24 h of eclosion and maintained at a density of 20 flies per vial at 25 °C. Female flies 2–5 days old, unless otherwise stated, were used for all experiments. UAS-levy-RNAi (CG17280, 101,523) and UAS-ND-75-RNAi (CG2286, 100,733) were obtained from the Vienna Drosophila Resource Centre (VDRC), while daughterless-GAL4 (daGAL4) was acquired from the Bloomington Drosophila Stock Centre (BDSC). ETC inhibitors, rotenone (Santa Cruz Biotechnology, #Sc-203242) and cyanide (Sigma, #60,178), were dissolved in ethanol and water and added to the fly food at a final concentration of 600 µM and 18 mM, respectively.