a, Top panel: Schematic design to achieve RNAi from embryonic to adult stage. Middle panel: Schematic design to achieve RNAi only at pupal stage. At lower temperature, Gal80 inhibits Gal4 activity. At 29°C, Gal80 becomes inactive and cannot suppress Gal4. Bottom panel: Schematic design to achieve RNAi only at adult stage. b, Validating ac-Gal4+tub-Gal80ts system by driving UAS-GFP expression at high temperature (29°C) and low temperature (18°C) in larval midgut and proventriculus. This experiment was only performed once. c, RT-qPCR to measure the mdg4 silencing efficiency for the ac-Gal4+tub-Gal80ts system at 29°C based on the two-tailed t-test. Newly eclosed flies raised at 29°C during pupal stage were used to extract RNA. Data are normalized to rp49 (RpL32) expression; the bars report mean ± standard deviation for two biological replicates. d, RT-qPCR to measure the mdg4 silencing efficiency for the ac-Gal4+tub-Gal80ts system at 25°C based on the two-tailed t-test. Adult flies being shifted to 25°C for 5 days were used to extract RNA. Data are normalized to rp49 (RpL32) expression; the bars report mean ± standard deviation for three biological replicates.