Fig. 6: WAKE modulates male-male courtship behaviour through IIS.

a Relative Ilp2 immunofluorescence signals in IPCs with WAKE downregulation after 24 h of starvation (a1–a3) (n = 9 for each), and after feeding for 30 min (a4-6) (n = 9 for each); quantification in b; n = 9 for test group no. 1–3, p = 0.0002, one-way ANOVA using F-test, post hoc Tukey’s multiple comparisons test, ^*p < 0.05, and ^***p < 0.005, n = 9 for test group no. 4–6, p > 0.05 (n.s.), one-way ANOVA using F-test, F(2, 24) = 0.07273, p = 0.9301. c The concentration of Ilp2HF in haemolymph with WAKE-deficient in IPCs. n = 5 for each, ^*p < 0.05, two-tailed unpaired t-test. d Insulin signalling pathways indicating the targets of inhibitors used in further experiments. e In wake³²⁰⁹⁹ flies or those with WAKE-deficient in IPCs, simultaneous treatment with different inhibitors. n = 18, 18, 18, 17, 18 (from left to right) for test group no. 1–5, n = 19, 18, 18, 17 for test group no. 10–13. p > 0.05 (n.s.), ^***p < 0.005, two-tailed unpaired t-test. n = 18, 18, 19, 18 for test group no. 6–9. ^*p < 0.05 and ^**p < 0.01, two-tailed Mann–Whitney U-test. f In WAKE-deficient background simultaneous expression of InR^DN, PI3K^DN, or chico dsRNA, respectively. n = 18 for test group no. 1, 2, 7, 8, ^****p < 0.0001, two-tailed unpaired t-test. n = 18 for test group no. 3–6, n = 25, 17 for test group no. 9, 10, ^****p < 0.0001, two-tailed Mann–Whitney U-test. n = 27, 22, 27, 24 for test group no. 11–14, p > 0.05 (n.s.), two-tailed unpaired t-test. g Downregulation of Rdl in IPCs, simultaneous treatment with Wortmannin or those with heterozygous ilp2, 3, 5 mutants. n = 25, 18, 18, 18, 18, and 18 for test group no. 1–4, 7 and 8. ^*p < 0.05, ^**p < 0.01 and ^****p < 0.0001, two-tailed Mann–Whitney U-test. n = 18, 16 for test group 5 and 6, ^****p < 0.0001, two-tailed unpaired t-test. Scatterplots include ± SEM. Source data are provided as a Source Data file.