Synthesis and secretion of ecdysteroids by the prothoracic glands of Manduca sexta

Abstract

[ 3H] or [ 14C]cholesterol was administered to Manduca sexta larvae by injection and the prothoracic glands of the injected animals were then incubated in vitro. Labelled steroids in the glands and medium were analyzed by reverse-phase and normal phase high-performance liquid chromatography, and differential radioimmunoassay, and the data revealed the conversion of the labelled cholesterol to 7-dehydrocholesterol and then to ecdysone, 26-hydroxyecdysone and a less polar ecdysteroid with a modified A-ring. Similar, but more efficient incorporation of radiolabeled cholesterol into 7-dehydrocholesterol and ecdysteroids was attained by including the [ 3H]cholesterol in the incubation medium when prothoracic glands were incubated in vitro. Analysis by gas-liquid chromatography of the content of endogenous cholesterol and 7-dehydrocholesterol in the prothoracic glands during the 5th larval instar revealed changes in temporal accord with the production of ecdysteroids by these glands, the ratio of the two steroids being consistent with data obtained from the radiotracer studies. By incubating prothoracic glands with [ 3H]7-dehydrocholesterol, it was demonstrated that 7-dehydrocholesterol is not reduced back to cholesterol and that the former is an obligatory intermediate in ecdysteroid biosynthesis. The principal component of the A-ring modified ecdysteroid peak is 3-dehydroecdysone, a putative secretory product of the prothoracic glands and a possible immediate precursor of ecdysone.