Fig. 1. Protein deprivation induces protein-seeking behavior in mated female flies via octopamine signaling.

a Schematic illustration of the DAMS-based locomotion assay. Briefly, virgin female flies were collected shortly after eclosion and raised in the presence of sucrose and yeast extract (S + YE) for 3–5 days. Afterwards, female flies were either mated with males for 1 day or kept as virgins. These mated or virgin females were then transferred individually to polycarbonate tubes (5 mm (D) × 65 mm (L)) and assayed in DAMS. b Midline-crossing activity in 30-min bins of wild-type Canton-S-mated female flies assayed in the presence of 5% sucrose alone (“S”, light orange) or 5% sucrose plus 2% yeast extract (“S + YE”, dark orange) (n = 49–53). Yellow bars represent light-on period of 12 h in this and all other figures. c Average daily midline-crossing activity of flies assayed in b (n = 49–53). d Average daily midline-crossing activity of flies assayed in b from Day 1 to Day 4 (n = 49–53). e–g Average daily midline-crossing activity of flies assayed on different protein-rich food types (n = 38–41, 58–69, 24–28, respectively). h, i Midline-crossing activity in 30-min bins of wild-type Canton-S male flies (h) and virgin female flies (i) assayed in the presence of 5% sucrose alone (“S”, light orange) or 5% sucrose plus 2% yeast extract (“S + YE”, dark orange) (n = 44–53). j Schematic illustration of the video recording-based locomotion assay. Briefly, individual flies were introduced into a behavioral chamber in the presence of a small food patch located in the center, and their positions and behaviors were recorded and analyzed by a custom computer program. k Spatial distribution of protein-deprived Canton-S flies assayed in the presence of sucrose (left) or sucrose plus yeast extract (right) (the heat maps showed the average duration for flies to stay in each pixel; n = 13 (left) and 17 (right); for SEM see Supplementary Fig. S5). Color temperature represents average time spent on each pixel for the duration of the assay (11 h). l Total walking distance of protein-deprived flies assayed in i (n = 13 and 17). m On-food and off-food walking speed of protein-deprived flies assayed in i (n = 13 and 17). n A summary of the behavioral analysis. Briefly, protein deprivation enhances flies’ protein-seeking behavior, by increasing their tendency to approach protein-rich food, to reduce their movement during their visits to protein-rich food, and to reduce their willingness to leave protein-rich food. o–v Average daily midline-crossing activity of flies assayed in S and S + YE food (o, n = 27–42; p, n = 26–32; q, n = 28–39; r, n = 25–33; s, n = 44–55; t, n = 22–32; u, n = 21–39; v, n = 32–41). Mianserin (MI) (1 mg/mL) was mixed in food in t. w A working model. Protein deprivation induces protein-seeking behavior in mated female flies via octopamine signaling. Female flies’ mated experience is crucial for this behavioral response via SP-SPR signaling. All error bars represent SEM. Student’s t test and one-way ANOVA were applied to statistical analysis. NS, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For the raw activity data from Fig. 1, see Supplementary Data S2. Materials and Methods were described in Supplementary Data S1