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To determine the precursors and the fate of 26-hydroxyecdysone in eggs, the fate of labeled putative ecdysteroid precursors was examined in the tobacco hornworm,Manduca sexta. Following injection of [(14)C]cholesterol, 22,25[(14)C]dideoxyecdysome or [(3)H]ecdysone into female pupae (day 16), only [(14)C]cholesterol was incorporated and metabolized. It was converted to labeled nonecdysteroid and ecdysteroid conjugates, of which the latter in ovaries and 48- to 64-hr-old eggs is mainly 26-hydroxyecdysone 26-phosphate (>85% in ovaries). Quantitation of the ecdysteroid conjugate by reversed-phase high performance liquid chromatography (RP-HPLC) showed that the levels of 26-hydroxyecdysone 26-phosphate were 31 μg/g of ovaries from 4-day-old females and 25 μg/g and 17 μg/g of 48- to 64-hr-old and 72- to 88-hr-old eggs, respectively. The RP-HPLC of the conjugate fraction of 48- to 64-hr-old eggs showed an additional peak of radioactive material eluting about three min before the 26-hydroxyecdysone 26-phosphate. The quantity of this material increased in the 72-to 88-hr-old eggs, though it was not detected in the analyses of the conjugate fraction from ovaries. Additional peaks of radioactive material eluting before the 26-hydroxyecdysone 26-phosphate peak were observed in the chromatogram of the conjugates of 72- to 88-hr-old eggs. These radioactive materials need to be identified to determine the ultimate fate of ecdysteroids in the developing embryos of the tobacco hornworm. No radioactive free ecdysteroids were detected in either egg age group.

The levels of both free and conjugated ecdysteroids, maternally labeled from [14C]cholesterol, of six different age groups of Manduca sexta eggs were quantitatively determined. Eggs 0-1-h old contain about 2.5 and 35 pglg of the 2-and 26-phosphates of 26-hydroxyecdysone, respectively, and 1 pglg of 26-hydroxyecdysone. During embryogenesis the 26hydroxyecdysone 26-phosphate is hydrolyzed to 26-hydroxyecdysone, which reaches a peak titer in I-18-h-old eggs; the level of 26-hydroxyecdysone 2-phosphate remains rather constant. Additionally, other metabolic modifications such as hydroxylation, conjugation, epimerization, and oxidation are occurring; and as the level of the 26-hydroxyecdysone 26-phosphate decreases there is a progression of other ecdysteroids. C-20 hydroxylation first appears in 24-40-h-old eggs and reaches peak activity in 48-64h-old eggs, where 20-hydroxyecdysone and 20, 26-dihydroxyecdysone are both present at peak titer but the latter is the major free ecdysteroid. Ecdysone is observed at measurable levels only in the three age groups of eggs between 1 and 64 hold. C-3 epimerase activity also appears at 24-40 h and continually increases throughout embryogenesis to the point that 3-epi-26hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone are the major free ecdysteroids in 96-h-old eggs. A new ecdysteroid conjugate, 26-hydroxyecdysone 22-glucoside, first appears at 24-40h and becomes the major conjugate in 72-88-h-old eggs; it represents an apparent end-product as its peak titer is reached and maintained throughout the final embryonic stages. 20-Hydroxyecdysonoic acid occurs in 48-64h-old eggs, and along with 3-epi-20-hydroxyecdysonoic and ecdysonoic acids in 72-88-h-old eggs. 20-Hydroxyecdysonoic acid peaks during the latter time interval, and as its titer subsequently falls, there i s a concurrent increase in the level of 3-epi-20-hydroxyecdysonoic which was identified as the second major component of the ecdysteroid conjugate fraction of 0-I-h-old larvae. O u r results indicate that there is little or n o Received October 5,1987; accepted December 28,1987. Address reprint requests to Malcolm J. Thompson INTRODUCTIONThe maternal ecdysteroid conjugates of ovaries and newly laid eggs of the tobacco hornworm, Munducu sextu, have been identified as the 26-and 2-phosphate of 26-hydroxyecdysone of which the 2-phosphate accounts for 3% and 9% of total conjugates in 24-and 96-h-old ovaries, respectively [1,2]. During embryogenesis the major ecdysteroid conjugate is hydrolyzed, and

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