A Developmental Analysis of Ecdysteroids During the Metamorphosis ofDrosophila Melanogaster

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Abstract
A combination of ion suppression reverse phase high pressure liquid chromatography (RP-HPLC) and differential radioimmunoassay (RIA) was used to identify and quantify ecdysteroids from Drosophila melanogaster during the development of the white puparium to the adult. The whole body ecdysteroid titer revealed a large peak approximately 30 hours after pupariation. RP-HPLC-RIA analysis of the brain-ring gland complex secretion in vitro revealed the presence of: ecdysone, 20-deoxymakisterone A, and a yet uncharacterized low polarity ecdysteroid (LP-1). The metabolism of ecdysone and 20-deoxymakisterone A was followed by analysis of the ecdysteroids during development. The white puparium (0 hour) yielded a four-fold greater quantity of free 20-hydroxyecdysone and makisterone A than ecdysone and 20-deoxymakisterone A, respectively, indicating high 20-monooxygenase activity during this stage. Another metabolite of ecdysone, 20,26-dihydroxyecdysone, was present at a quantity intermediate between that of ecdysone and 20-hydroxyecdysone. Highly polar ecdysteroids (conjugates) treated with an esterase-phosphatase mixture yielded the free ecdysteroids, ecdysone, 20-deoxymakisterone A, 20-hydroxyecdysone, and makisterone A, suggesting the presence of novel ecdysteroid conjugates of 20-deoxymakisterone A and makisterone A. At 18 hours post-pupariation the levels of all free ecdysteroids were reduced when compared to the 0 hour white puparium with the highly polar ecdysteroids predominating. By 30 hour after pupariation the level of free ecdysteroids had increased, indicating net ecdysteroid synthesis. Both ecdysone and 20-deoxymakisterone A were in greater concentrations than 20-hydroxyecdysone and makisterone A, indicating a reduced level of 20-monooxygenase activity at this stage. At 48 hours post-pupariation Drosophila contained decreasing amounts of ecdysteroids except for 20,26-dihydroxy- ecdysone and low polar products. 20, 26-Dihydroxyecdysone was the only known ecdysteroid to remain at a high level during the rest of adult development.