Dr 36nl incubator

All flies were reared at 25°C in 12 hr light: 12 hr dark (LD) conditions. Male flies (3–5 days old) were loaded into 65mm × 5mm tubes containing 5% sucrose 2% agar food medium. Locomotor activity was recorded using the Drosophila Activity Monitoring System (DAMS, Trikinetics) in 1 minute intervals. Temperature, light and humidity were controlled using a DR-36NL incubator (Percival Scientific). To suppress synaptic output of DN1a split Gal4 (R23E05 AD ∩ R92H07 DBD) and TPN-II split Gal4 (R60H12 AD ∩ VT032805 DBD), males were crossed to virgin UAS-TNT females, and male progeny were assayed.

All flies were reared at 25°C in 12 hr light: 12 hr dark (LD) conditions). Male flies (2–5 days old) were loaded into 65mm × 5mm tubes containing 5% sucrose 2% agar food medium. Locomotor activity was recorded using the Drosophila Activity Monitoring System (DAMS, Trikinetics) in 1-minute intervals. Temperature, light, and humidity were controlled using a DR-36NL incubator (Percival Scientific). To suppress synaptic output of AC Gal4 (TRPA1^SH Gal4) and LPN split-Gal4 (R26A08 AD ∩ R65D05 DBD), males were crossed to virgin UAS-TNT females, and male progeny were assayed. Sleep was defined as ≥5 consecutive minutes of inactivity. Activity and sleep analysis was performed using MatLab 2020b software (Mathworks). Sleep and activity are displayed in 30-minute bins represented as mean ± SEM. For 25°C-30°C behavior comparisons (Fig. 3A, D, F, H), flies were entrained for 3 days to 12:12 LD at 25°C, then gradually transferred to 30°C during the 3^rd night (12 hr +0.4°C/hr linear heating ramp, ZT12-0) for one day. 25°C activity and sleep data are from the 3^rd day. For heat step experiments (Fig. 3 A–C), flies were entrained for 3 days to 12:12 LD at 25°C. On the 4^th day, the temperature was raised to 30°C either from ZT0-3 or ZT6-12 (+0.5°C/min). Quantifications were made between the 30°C day and the preceding 25°C day.