NOS overexpression during larval feeding inhibits ecdysone biosynthesis. (A) NOS activity in the PG reduces ecdysteroid production. The presence of ecdysteroids is reduced in larvae with NOS overexpression in the PG (phm > NOS) compared to control (phm > LacZ) larvae. Ecdysone levels were measured by ELISA assay for independent isolation triplicates. (B) NOS expression in the PG reduces ecdysone signaling. Transcription of E74B is reduced in larvae with NOS overexpression in the PG (phm > NOS) compared to control (phm > LacZ) larvae. Transcription levels measured by qRT-PCR in triplicate, normalized to control expression levels at 116 hr AED. (C) NOS activity in the PG reduces Halloween gene transcription. Relative expression of spok and dib in control (phm > LacZ) larvae and larvae with NOS overexpression in the PG (phm > NOS) are depicted. Transcription levels were measured by qRT-PCR in triplicate, normalized to control transcription levels. (D) Expression of NOS early during the larval feeding period (76 and 80 hr AED) substantially delays larval development, while NOS expression late during the wandering period (96 and 104 hr AED) does not delay development. NOS was systemically expressed by heat shock (hs > NOS) once at 76, 80, 96, or 104 hr AED, and time to pupariation was measured. (E) Expression of NOS early during the larval feeding period restricts imaginal disc growth, while NOS expression late during the wandering period does not inhibit growth. NOS was systemically expressed by heat shock (hs > NOS) at either 76 or 104 hr AED, and eye imaginal disc size was measured at 116 hr AED. All phm > LacZ- and phm > NOS-expressing larvae were raised at 21°. Statistical analysis: A and E, mean of triplicates ± SD calculated by two-tailed Student’s t-test. (B and C) Mean of triplicates ± SEM, calculated by paired one-tailed t-test. (D) Mean of triplicates ± SEM. *P < 0.05, **P < 0.01, ****P < 0.001.