Ecdysone metabolism and distribution during the pupal-adult development of Manduca sexta

The metabolism and distribution of endogenous ecdysone and injected ³H]ecdysone were studied during the pupal-adult development of Manduca sexta. Well-characterized antisera were used to detect and quantify endogenous metabolites by radioimmunoassay (RIA) following their separation by ion-suppressed reverse phase, and normal phase, high performance liquid chromatography. Identical chromatographic procedures were employed to determine the metabolic fate of the ³H]ecdysone in the haemolymph pool. These studies revealed the sequential appearance in the haemolymph and gut of progressively oxidized metabolites of ecdysone—hydroxylation at C-20 was followed by hydroxylation at C-26. The data are suggestive of both the induction of the steroid hydroxylases (oxidases) by substrate or other effector substances and the possible coordination of developmental events by ecdysteroids other than 20-hydroxyecdysone.In the haemolymph, two highly-polar conjugates of ecdysone were observed together with conjugates of the other free ecdysteroids, especially those hydroxylated at C-26. In contrast, relatively little 20-hydroxycdysone conjugate was detected in the insect. As adult development proceeded, both endogenous and radiolabelled ecdysteroids were increasingly localized in the gut, so that just prior to eclosion most ecdysteroids were present in the meconium of the high gut (rectal pouch). The peak titres and the kinetics of appearance of ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were similar for both haemolymph and gut (and for males and females), but considerably higher levels of C-26 oxidized (acid) metabolites of ecdysone and 20-hydroxyecdysone were localized in the gut. Although levels of highly-polar ecdysteroid conjugates found in the haemolymph and gut were similar, considerable amounts of three less polar ecdysone conjugates, of 3-α-epimers of ecdysone and 20-hydroxyecdysone, and of a substance tentatively identified as 2-deoxyecdysone were found only in the gut. Whether ionized, conjugated, or free, the gut ecdysteroids did not appear to equilibrate with the haemolymph compartment.Differences were observed in the metabolism kinetics of exogenously administered radiolabelled ecdysone when compared to the endogenous ecdysteroids; and some RIA positive gut metabolites did not become significantly radiolabelled. This suggests that injection of ecdysone may not simulate the endogenous secretion of ecdysone or its subsequent metabolism and distribution completely accurately.