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The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3 beta-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.

It is generally accepted that the prothoracic glands of insects produce ecdysone, which is converted by a 20-monooxygenase in peripheral tissues to the major molting hormone, 20-hydroxyecdysone. Incubation in vitro of the prothoracic glands of larval or pupal Manduca sexta in the presence of a hemolymph protein fraction (HPF) increased the ecdysteroid content of the medium almost 8-fold. A comparable increase was noted when HPF was added to medium preconditioned with prothoracic glands but from which the glands had been removed. We used a differential RIA to show that a major product of the prothoracic glands in vitro cross-reacts with antiserum (20-hydroxyecdysone-2-succinylthyroglobulin amide; H-2) that retains affinity to ecdysterolds having a modified A ring. However, this product did not bind to antiserum (ecdysone-22-succinylthyroglobulin amide; H-22) that has affity mainly for ecdysteroids modified at the side chain. We employed radiolabeled precursor studies with prothoracic glands in vitro and a combination of analytical techniques (NMR, CD, MS) to demonstrate that the major ecdysteroid released from the glands is a mixture of 2-dehydroecdysone and 3-dehydroecdysone (1:2), which is rapidly reduced to ecdysone in the presence of HPF. We postulate that the active component of HPF is 3,(2fi)-forming-3(2)-ketoecdysteroid reductase. These results may explain several anomalous observations pertaining to the molting of insect fragments in the absence of prothoracic glands and suggest a complex system for the control of insect molting and metamorphosis.It has been more than four decades since it was demonstrated that the prothoracic glands play a critical role in insect molting (1) and 33 years since ecdysone, the presumed molting hormone, was crystallized from extracts of 500 kg of Bombyx mori pupae (2). Thirteen years ago the relationship between ecdysone and the prothoracic glands was apparently clarified when it was reported that ecdysone was the principal product of the prothoracic glands of Manduca sexta (3) and B. mori larvae (4). In the intervening years, a central dogma of insect endocrinology has emerged-i.e., a neuropeptide from the insect brain, prothoracicotropic hormone (P1TH), stimulates the prothoracic glands by way of a cyclic nucleotide-protein kinase cascade leading to the synthesis and release of ecdysone (5, 6). Ecdysone is then converted to the principal insect molting hormone, 20-hydroxyecdysone, by an ecdysone 20-monooxygenase in tissues peripheral to the prothoracic glands, and a critical titer of 20-hydroxyecdysone leads to the initiation of the molting process. We report here the existence of a regulatory system that appears to play a critica role in establishing the ecdysteroid titer so crucial for normal insect molting, development, and metamorphosis. MATERIALS AND METHODSAnimals. M. sexta larvae were reared on an artificial diet at 25-26TC and high humidity (60%) under long-day conditions (light/dark, 16:8) as described (7). Antheraea polyphemus pupae were obtained commerci...

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