Dam system

The locomotion activity was measured by the DAM system (TriKinetics Inc, Waltham, MA), which allows a measure of fly locomotion capabilities based on their negative geotactic response, as the number of climbing performances in 30 min; 10–15 ageing-synchronized male flies (2–3 days, 7 days, or 15 days of age) were gathered and placed in each monitor for each genotype for each experiment. Briefly, the DAM system (TriKinetics Inc) records activity from individual flies maintained in sealed tubes placed in activity monitors. An infrared beam directed through the midpoint of each tube measures an ‘activity event’ each time a fly crosses the beam. The number of climbing events was scored for 30 min, tapping flies to the bottom every 40 s. Events detected over the course of each consecutive sampling interval are summed and recorded over the course of 30 min for each fly.

For the flying assay in Fig. 4c, twenty adult male flies at 14 days old were placed in individual vials (93 mm height × 350 mm² area) at noon and left at rest for 20 min. Flies were dropped down to the bottom by gently tapping, and flight events during 30 s were recorded. Locomotor behavior in Supplementary Fig. 4b was recorded in polycarbonate tubes containing light-shielded fly food with 100 µM retinal using the DAM system (Trikinetics)^{42 (link)}. The behavior of single male flies preconditioned at 25 °C under a 12-hr light:dark cycle condition for 14 days was individually recorded for 1 additional day. Light irradiation was performed during the light period. For a climbing assay in Fig. 4e, vials (25 mm diameter × 180 mm height) containing 20–25 flies were tapped gently on the table and left standing for 18 s. The number of flies that climbed at least 60 mm was recorded^{43 (link)}.

Mosquitoes were reared at 28 °C, 80% relative humidity under a 12:12 h light:dark regime, and measurements of flight activity were performed using a Drosophila Activity Monitoring (DAM) System (TriKinetics, LAM25) and DMASystem3 software (TriKinetics) using large tubes designed for mosquitoes (TriKinetics, PGT 25 × 125 mm Pyrex Glass). Individual 4–7-day-old, non-blood-fed virgin ♀ and non-mated ♂ mosquitoes were introduced into the monitoring tubes, which contained 10% sucrose (Sigma, Cat. No. S0389) at both ends of the tube as the food source. The DAM System was positioned vertically during the assays. Flight activity was measured over a period of 24 h by automatically calculating the number of times that mosquitoes passed through the infrared beam in the center of the tubes. The walls of the monitoring tubes were coated with Sigmacote (Sigma, Cat. No. SL2) to inhibit mosquitoes from walking upward. For preparing the wingless mosquitoes, the animals were anesthetized on ice, and the wings were removed using Vannas Scissor (World Precision Instruments, Cat. No. 14003). The wingless mosquitoes were allowed to recover for 12 h before recording. Mosquitoes were manually checked after flight activity recording to ensure survival. Data acquisition was performed using the DAM System (TriKinetics) (Fig. 2D, Supplementary Video 5, and Supplementary Table 6).