Agarose standard

Experiments were performed during the animals’ subjective day. Larvae were in darkness at least 20 min before an experiment. Larvae were collected in water droplets for up to 10 min before an experiment. The testing plate was a 24.5 × 24.5 cm petri dish containing 2% agarose (Agarose Standard, Roth).

All experiments were set, performed and analyzed as detailed previously^{64 (link)}. Adult flies were allowed to lay eggs for 24 hours under 12 h light-dark (LD) conditions. 2-day-old larvae entrained to LD conditions were placed into constant darkness (DD) for 2 days. Behavior experiments were performed using 3^rd instar foraging larvae (4-day-old). We took as premise that after solely two days in free-running state, the internal clock faithfully reflects the circadian times the animals were entrained to. Therefore, experimental time-points are indicated by circadian time (CT), respectively as CT 0, CT 6, CT 12 and CT 18. For each CT, the experiment was designed to start 1 hour before and to end 1 hour after the given time point (i.e. CT 5 - CT 7 for CT 6 experiments). For each time-point, experiments were repeated ten times. Each experiment included thirty larvae collected from the food and washed twice with tap water at room temperature. A behavioral plate was prepared by using a 24.5 × 24.5 cm petri dish (BD Falcon BioDishXL, BD Biosciences) with an aluminum plate on the bottom to create contrast, covered homogenously with 2,5% agarose (Agarose Standard, Roth). After the agarose cooled down to room temperature, larvae were placed in the middle of the plate for behavioral recording. All experiments were performed under red-light illumination.

DNA fragments were separated by electrophoresis at 110 V for 30–50 min on a TAE-buffered 1% (wt/vol) agarose gel (Agarose Standard; Carl Roth) with 1:10,000 SYBR Safe stain (Thermo Fisher Scientific). Before loading, samples were mixed with Gel Loading Dye (Purple, 6X; NEB). Plus DNA Ladder (1 kb; NEB) was loaded in parallel for size determination.