Fig. 4. AstC-mTh neurons gate mating-induced vitellogenesis in response to SP.

a Left, negative images of TRIC labeling in AstC-mTh neurons of virgin (left) and mated (right) females carrying AstC-mTh-Gal4 and TRIC transgenes, indicating intracellular Ca²⁺ transients. Scale bars, 10 μm. Right, relative TRIC (GFP/RFP) intensities from TRIC-expressing AstC-mTh neurons of virgin females and females mated with males of the indicated genotypes. One-way ANOVA followed by Tukey’s test for multiple comparisons among genotypes; **p < 0.01; *p < 0.05; ns (non-significance) p > 0.05. Error bars indicate s.e.m. For adjusted p values, see Supplementary Table 1. b–d Above, experimental protocol. Below, number of follicles of the indicated stage and oviposited eggs (OVI) from females of the indicated genotypes 24 h after mating. For the thermal activation experiments (b, d), females were incubated at 30 °C for 24 h after mating. One-way ANOVA followed by Tukey’s test for multiple comparisons among genotypes; ***p < 0.001; **p < 0.01; *p < 0.05; ns (non-significance) and no labeling, p > 0.05. Error bars indicate s.e.m. For a detailed list of genotypes, see Supplementary Table 2. e A model that explains how SP induces post-mating vitellogenesis via a neuronal pathway composed of SPSN and SAG neurons. SPSN and SAG neurons are electrically active in mature virgin females. Active SAG neurons indirectly stimulate AstC-mTh neurons (arrow with dotted line) to secrete AstC into circulation. Circulating AstC then inhibits JH biosynthesis in the CA and sets up a vitellogenesis blockade. Upon mating, SP silences SPSN and SAG neurons in sequence. This inhibition relieves the AstC-induced vitellogenesis blockade. At the same time, SP also elevates 20E via SPSN. This 20E stimulates the endocrine Inka cells to secrete ETH, which induces JH biosynthesis in the CA. Thus, the neuronal SP pathway stimulates post-mating vitellogenesis in mated females by elevating circulating allatotropin (i.e., ETH) and reducing circulating allatostatin (i.e., AstC).