Fig. 5. SAG neurons function upstream of AstC-mTh neurons and gate vitellogenesis during reproductive maturation.

a, d Above, experimental protocol. Below, number of stage 8–11 and 12–14 follicles per virgin female of the indicated genotypes at the indicated hours after eclosion (HAE). The letters above the columns indicate significant differences (p < 0.05) for comparisons of time points and genotypes (two-way ANOVA with Bonferroni post-hoc test for multiple comparisons); a–d for stage 8–11 and x-z for stage 12–14. For adjusted p values, see Supplementary Table 1. Error bars in a–e indicate s.e.m. To target SAG neurons, we used SAG1-Gal4, which combines the VT50405-p65AD and VT7068-GAL4DBD transgenes. We also used SAG-LexA (i.e., VT50405-LexA) or SAG-Gal4 (i.e., VT50405-Gal4). Although the CNS expression driven by SAG1-Gal4 is slightly more restricted than that of the VT50405 transgene alone (i.e., SAG-LexA or SAG-Gal4)¹⁶, SAG1-Gal4 or SAG-LexA induced Kir2.1 expression produced similar vitellogenesis phenotypes (compare the magenta circles in a and d). b, c Above, experimental protocol. Below, number of mature eggs per virgin female of the indicated genotypes 3 days after eclosion under 30 °C. The letters above the columns indicate significant differences (p < 0.05) for comparisons of genotypes (one-way ANOVA followed by Tukey’s test for multiple comparisons). e Relative TRIC (GFP) intensities from SAG neurons of TRIC virgin females showing Ca²⁺ activity at the indicated HAE. 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. For a detailed list of genotypes, see Supplementary Table 2. f A model that explains how endocrine and neuronal events stimulate and terminate vitellogenesis during reproductive maturation. During adult molting, the Inka cells secrete ETH to trigger eclosion behaviors. The resulting increase in circulating ETH induces JH biosynthesis and generates a post-eclosion JH surge. This stimulates vitellogenesis shortly after eclosion. As female completes reproductive maturation, circulating ETH levels fall and SAG neurons augment the activation of AstC-mTh neurons, driving them to secrete AstC. This then inhibits JH biosynthesis and terminates vitellogenesis.