Expression analyses of neuropeptide GPCR genes in the PG. (A) Developmental expression profile of three neuropeptide GPCRs in the PG from third instar to the first day of the pupal stage. Transcript levels of the GPCR genes are presented as relative expression levels, which are normalized for each gene. Vertical bars represent SE n = 3 batches. HCS, head capsule slippage. (B and C) Effect of 20E on the expression of PTSPR/SPR (B) and BNGR-B4 (C) in the PG in vitro. Lower rectangles in each panel indicate the period of 20E treatment (gray). Vertical bars represent SE n = 3 batches. (D) Current working model on the temporal regulation of the PG activity by various neuropeptides. BMS and BRFa act on BMSR during feeding stage to suppress ecdysteroidogenesis. When enough PTTH is released from the brain, an event probably coupled with the downregulation of BMSR signaling, ecdysteroidogenesis is upregulated to generate an ecdysteroid surge in hemolymph. After the decline of ecdysteroid titer, PTSP and probably BNGR-B4 ligand further suppress ecdysteroidogenesis. TG1, prothoracic ganglion; PTTHR, PTTH receptor. Molecular identity of BNGR-B4 ligand remains unknown.