Fig. 9: WAKE modulates male-male courtship behaviour by affecting the perception of cVA by ecdysone signalling in Or67d neurons.

High-magnification confocal stack images of a single DA1 glomerulus with putative synapses labelled using brp::GFP in the adult male brain (Or67d-Gal4>UAS-brp::GFP). The neuropil was immunostained using an anti-DLG antibody (magenta). There were significant reductions in the number of brp::GFP puncta signals (green) compared to those in adult flies simultaneously specific expressing EcRi (a) and in wake³²⁰⁹⁹ mutant flies (b) when compared with the corresponding controls, respectively. Moreover, brp::GFP puncta signals were also significantly decreased in flies with WAKE deficiency in IPCs (ilp2-LexA>LexAop-wake^mir) (c) when compared with the corresponding control, n = 10, 11, 19, 18, 11, 10, 7, 15 and 11 (from a1 to c2); data quantified in d; n = 10, 11, 19 and 18 (from left to right) for test group no. 1–4. ^****p < 0.0001, two-tailed unpaired t-test. n = 11 and 17 for test group no. 5 and 6. ^****p <  0.0001, two-tailed Mann–Whitney U-test; showing the distribution of data points for moderate and severe levels within the bar chart of test group no. 6. n = 15 and 11 for test group no. 7 and 8. ^*p < 0.05, two-tailed unpaired t-test. e–g Representative images focusing on the DA1 glomerulus showing the Ca²⁺ response signals for Or67d-Gal4 using UAS-GCaMP6 in the adult male DA1 before (pre) and after simultaneous stimulation with 10% or 100% cVA. e Summary of Ca²⁺ response data from controls (Or67d/UAS-GCaMP6) compared with those from wake³²⁰⁹⁹ flies (f) with downregulated ecdysone signalling in Or67d neurons due to EcR dsRNA expression and those from flies with WAKE-deficient in IPCs (g). Responsiveness to cVA significantly increases when compared with that in flies with EcR knock-down, wake³²⁰⁹⁹ mutants, and flies with WAKE-deficient IPCs; quantified in (h); n = 9, 11, 9, 11 and 14, 8, 14, 8 (from left to right) for test group no. 1–4 and 9–12. ^*p < 0.05 and ^**p < 0.01, two-tailed unpaired t-test. n = 8, 9, 8, and 9 for test group no. 5–8. ^*p < 0.05 and ^**p < 0.01, two-tailed Mann–Whitney U-test. Scatterplots include ± SEM. Source data are provided as a Source Data file. Scale bar, 20 μm.