Fig. 3: TOR and TrpA1-mediated nutrient and ROS sensing regulate gut Tk release in mated female flies.

a, Tk peptide-staining intensity in midgut regions of Tk> controls and Tk>slif-RNAi animals fed for 18 h with yeast (two-tailed Mann–Whitney U-tests). b, Representative in situ hybridisation images localizing Tk, pros and slif transcripts in R5 EECs. Scale bar, 50 µm. Z projection of confocal substacks. Similar localisation patterns were observed in five independent gut samples from different animals. c,d, Expression of Tk in midguts (c) and anti-Tk staining in R5 (d) of controls and animals with reduced TOR signalling (Tor-RNAi or Tsc1 and Tsc2 overexpression) in Tk⁺ EECs fed for 15 h with yeast (c, one-way ANOVA with Dunnett’s test; d, Kruskal–Wallis with Dunn’s test). e, Representative images of Tk immunostaining and TrpA1::GFP (anti-GFP) coexpression in R5. Scale bars, 5 µm. Similar coexpression patterns were observed in five independent gut samples. In R5, 140 of 143 Tk⁺ EECs across three midguts coexpressed detectable TrpA1::GFP. f, Calcium activity (GFP/tdTomato ratio) and anti-Tk staining in Tk⁺ EECs throughout the midgut on a normal adult-optimized diet (ND) and medium supplemented with 5% H₂O₂ for 18 h, in controls and in animals with RNAi against the TrpA1 transcript in Tk⁺ EECs (two-tailed Mann–Whitney U-tests). g, Anti-Tk staining intensity in R5 in animals fed for 15 h on a yeast diet, with and without TrpA1 knockdown in Tk⁺ EECs (two-tailed Mann–Whitney U-test). h, One-hour dye-feeding assay for sugar consumption for controls and slif knockdown in Tk⁺ EECs, following 15 h of yeast feeding (Kruskal–Wallis with Dunn’s test). GD, long-hairpin RNAi P-element collection from Vienna Drosophila Resource Center. i, Sugar consumption over a 90-min dye-feeding assay of controls and animals with reduced TOR signalling Tk⁺ EECs, following 15 h of yeast feeding (Kruskal–Wallis, Dunn’s test). Sample sizes (n) and P values are indicated in each plot. In a,d,f,g, n represents EECs. In c, n represents midgut samples, and, in h,i, n represents animals per group. Plots in a,d,f,g show median and 95% confidence interval, and plots in c show mean ± s.e.m. Box plots show the median, the bounds of the box (25th and 75th percentiles) and whiskers representing minimum and maximum values. NS, P > 0.05.

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