Fig. 8: WAKE modulates male-male courtship behaviour by ecdysone signalling in Or67d neurons.

a1 Activation of IIS enhancement of JH biosynthesis in the CA significantly decreased the relative expression of the ecdysone responsive-genes Br-C and E75, n = 4 for each group. Moreover, relative expression of Br-C and E75 also significantly decreased with WAKE deficiency in IPCs, n = 5 and 6 for each group. a2 Levels of 20E in wake³²⁰⁹⁹ flies were significantly lower than those in the revertant control (n.d. means not detected). b EcR knockdown in fru-P1 neurons blocked ecdysone-dependent signalling, effectively prompting male-male courtship behaviour. Downregulation of WAKE in IPCs and simultaneous expression of EcRA in fru-P1 neurons significantly suppressed male-male courtship behaviour. n = 18 for test group no. 1–3 and 8–10. p <  0.0001, Kruskal–Wallis test. ^**p < 0.01 and ^****p < 0.0001, post hoc Dunn’s multiple comparisons test. n = 15 for test group no. 4–7. ^***p < 0.001, two-tailed unpaired t-test. c Directly or conditional knockdown of EcRs in Or67d neurons blocked ecdysone-dependent signalling, effectively prompting male-male courtship behaviour. n = 18 for test group no. 1–3. p < 0.0001, one-way ANOVA. ^****p < 0.0001, post hoc Tukey’s multiple comparisons test. n = 18 for test group no. 4–6. p < 0.0001, Kruskal–Wallis test. ^****p < 0.0001, post hoc Dunn’s multiple comparisons test. Downregulation of WAKE in IPCs and simultaneous expression of EcRA in Or67d neurons significantly suppressed male-male courtship behaviour. n = 18 for test group no. 7 and 8. ^**P <  0.01, two-tailed unpaired t-test. d There were no significant effects on male–female courtship behaviour in wake³²⁰⁹⁹ males treated with 20E or the males expressing EcRA in Or67d neurons. n = 19 and 18 for test group no. 1 and 2. p > 0.05 (n.s.), two-tailed Mann–Whitney U-test. n = 17, 18, 18 for test group no. 3–5. p > 0.05 (n.s.), Kruskal–Wallis test. Scatterplots include ± SEM. Source data are provided as a Source Data file.