Regulation of cytochrome P-450 dependent steroid hydroxylase activity in Manduca sexta: Effects of the juvenoids hydroprene and methoprene on ecdysone 20-monooxygenase activity

Ecdysone 20-monooxygenase (E.C. 1.14.99.22) is the insect cytochrome P-450 dependent steroid hydroxylase responsible for the conversion of the molting hormone ecdysone to its more active metabolite 20-hydroxyecdysone. Previous studies of this enzyme in the midgut of the tobacco hornworm, Manduca sexta, have revealed that this steroid hydroxylase exhibits an approximate 50-fold increase in activity on day 5 of the last larval stadium of gate II animals. This increase in midgut enzyme activity occurs temporally coincident with the onset of wandering stage development and reprogramming of the larval genome to pupal syntheses, is predicated on mRNA and protein synthesis, and can be elicited in competent head- or thorax-ligated prewandering larvae by injections of ecdysone, 20-hydroxyecdysone, or the non-steroidal ecdysone agonist RH 5849. Since ecdysteroids cannot reprogram larval tissues in the presence of juvenile hormones (JH) or juvenile hormone analogs (JHA), experiments were initiated in the present study to determine the effects of the JHA methoprene and hydroprene on midgut monooxygenase activity in the tobacco hornworm. Gate II last instar larvae of Manduca sexta were topically applied with increasing amounts of methoprene or hydroprene on days 3 or 4 of the last larval stadium; in addition, some larvae were head- or thorax-ligated on day 3 and injected with RH 5849. Midgut ecdysone 20-monooxygenase activity was quantified by radioassay in control and experimental larvae on days 3 and 5. Data from these experiments revealed that the JHA delayed the onset of wandering stage development in whole larvae in a dose-dependent fashion. These JHA also prevented, in a dose-dependent fashion, the 50-fold increase in midgut ecdysone 20-monooxygenase activity in whole animals and head -or thorax-ligated larvae, although the JHA were less effective when applied on day 4 versus day 3. The data suggest that the JHA actions on ecdysteroidogenesis may be occurring directly at the level of the midgut tissues and involve a significant interference with events occurring posttranscriptionally. The JHA effects are consistent with the view that the 50-fold increase in midgut steroid hydroxylase activity is a component of reprogramming and that this midgut enzyme system may be an excellent model for examining the interendocrine regulation of ecdysteroidogenesis by JH.