Olfactory response (trap assay)

Olfactory preference was determined based on the methods of a previous study with modifications²³. An olfactory test apparatus (Fig. 2) was fabricated from a 500 ml plastic container (Fig. 2a). The ceiling of the screw cap of the apparatus had an opening (8 mm diameter, Fig. 2b) for the rapid transfer of flies from the insect suction tube and 50 tiny holes for ventilation (Fig. 2c) that were too small for the flies to pass through. Two glass vials (Fig. 2d) were placed in the apparatus filled with 10 ml odour solutions (20% apple cider vinegar (Jun-Ringo-Su; Mizkan, Japan), 10% broth (bonito soup stock) (Fuumi-Dashi; Yamasa, Japan), or water) (Fig. 2e). The concentrations of solutions were determined based on the literature²² and preliminary experiments in which they are sufficient for attraction of flies. A hole (2 mm in diameter) was drilled in the lid of the vial cap to provide a path for the flies, and a tube made of a pipette tip was attached to the hole to prevent the flies from escaping (Fig. 2f). In the preliminary observation, 91.25 percent of trapped flies were remained in the trap vials within 24 h (tested with 240 flies divided in 12 vials). Most of flies shows the first choice with olfaction in this assay.

Flies starved for 12 h (including 8 h of periodic rotations in night-time/daytime rotation treatments) were released into the olfactory test apparatus using an insect suction tube. Fifty flies were transferred from the rearing tubes to a centrifuge tube and then to a test apparatus. If there were any dead flies immediately after release, the number was recorded. The apparatus was placed under the same temperature and humidity conditions as the rearing tubes. Flies were allowed to choose two odour source vials for 16 h. After 16 h, the number of flies in each odour source vial and flies not in either vial were recorded. In all experiments, 10 replicates were conducted. The order of each experiment and the positions of the odour source vials (left and right) in each test apparatus were randomly determined using random numbers generated by a computer program. Flies were subjected to one of the experiments only once.

The treatments of flies and odour sources used in each experiment were as follows.

Experiment 1 Preference for Apple cider vinegar (ACV) or broth: Flies with different rotation schedules (no rotation, night-time rotation, daytime rotation); 20% ACV vs 10% broth.

Experiment 2 Response rate to each odour source: Flies with different rotation schedules (no rotation, night-time rotation, daytime rotation); 20% ACV or 10% broth vs water.

Experiment 3 Effect of the rearing environment on the preference: Flies from different media (ACV medium or broth medium) with different rotation schedules (no rotation or night-time rotation); 20% ACV vs 10% broth.