Falcon biodishxl

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.

Behavioral experiments were prepared and performed under red light conditions during the day. At least 20 min prior to the start of behavioral experiments larvae were kept in darkness. Additionally, for experiments in which neurotransmission was blocked by inducing high temperature larvae were kept for at least 10 min in a water bath at 32 °C. Larvae were taken out of a food vial with a spoon and cleaned with tap water. Larvae were kept in a water drop at room temperature or at 32 °C for 5–10 min prior to the experiment. Thirty larvae were placed circular in the middle of a 24.5 × 24.5 cm petri dish (BD Falcon BioDishXL, BD Biosciences) filled with 2% agarose (Agarose Standard, Roth). To increase the contrast a black aluminum plate was placed underneath the agarose. The agarose plate was either at room temperature or at 32 °C.

All experiments were performed on 2% agarose plates. 2% agarose (Agarose Standard, Roth) was filled into 24.5 × 24.5 cm plates (BD Falcon BioDishXL, BD Biosciences). At the bottom of these plates we placed a black aluminum plate, in order to increase the contrast. The agarose had to cool down to room temperature before experiments were performed.
At least 20 min before the experiment started food vials (containing larvae) were stored in darkness. With a spoon larvae were transferred into a petri dish. With tap water the larvae were cleaned from the food. With a fine paintbrush 30 larvae were collected and kept in a water drop for up to 10 min. The 30 larvae were transferred into the middle of the agarose plate. All experiments were prepared under red illumination. We performed 10 trials per genotype and assay. The only exception were the outdoor experiments, in which we were comparing light avoidance in the morning (3 trials) and in the afternoon (4 trails).