76 The Fate of Radiolabeled Steroids in Ovaries and Eggs of the Tobacco Hornworm, Manduca sexta Malcolm J. Thompson', James A. Svoboda, Mark F. Feldlaufer and Ruben Lozano Insect Physiology Laboratory, Agricultural Research Service, USDA, BeltsviHe, Maryland To determine the precursors and the fate of 26-hydroxyecdysone in eggs, the fate of labeled putative ecdysteroid precursors was examined in the tobacco horn- worm, Manduca sexta. Following injection of [~4C]cholesterol, 22,25114C]dideoxyecdysone or [3H]ec- dysone into female pupae (day 16}, only [~4C]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 2Ghydroxyecdysone 26-phosphate (>85% in ovaries}. Quantitation of the ecdysteroid conjugate by reversed-phase high performance liquid chromatography {RP-HPLC) showed that the levels of 2~hydroxyecdysone 2Gphosphate were 31 pg/g of ovaries from 4-day-old females and 25 ~g/g and 17 pglg 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 ad- ditional 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 2Ghydroxyecdy- sone 26-phosphate peak were observed in the chromato- gram 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 em- bryos of the tobacco hornworm. No radioactive free ecdy- steroids were detected in either egg age group. Lipids 21, 76-81 (1986}. The ovaries and newly laid eggs of various insect species contain comparatively large quantities of ecdysteroid con- jugates and only minute amounts of free ecdysteroids {1-3}. In ovaries and newly laid eggs (0- to 1-hr-old} of the tobacco hornworm, Manduca sexta, conjugates account for more than 95% of the total ecdysteroids present {4). These conjugates, unlike conjugates in eggs of other in- sect species, are comprised primarily of 26-hydroxyecdy- sone (21 ~g/g of eggs} and ecdysone {0.73 ~g/g). 26-Hydroxyecdysone 26-phosphate {Fig. 1} has been iden- tified as the major conjugate in 48- to 64-hr-old eggs of M. sexta (5}. In batch-collected, 1- to 18-hr-old eggs stored at --20 C until analyzed, more than 63% of the ec- *To whom correspondence should be addressed at the Insect PhysiologyLaboratory,Building467, BARC-East,Beltsville,MD 20705. Nomenclature--Ecdysone: 2~, 3~, 14a,22R,25-pentahydroxy-513- cholest-7-en-6-one; 20-hydroxyecdysone: 2/3,3/3,14a,20R,22R,25-hexa- hydroxy-5~-cholest-7-en-6-one; 26-hydroxyecdysone: 2~,3/3,14a,22R, 25,26-hexahydroxy-5/~-cholest-7-en-6-one; 20,26-dihydroxyecdysone: 2~,3/~, 14a,20R,22R,25,26-heptahydroxy-5~-cholest-7-en-6-one; 3-epi-20,26-dihydroxyecdysone: 2/3,3a,14a,20R,22R,25,26-heptahy- droxy-5~-cholest-7-en-6-one; 3-epi-26-hydroxyecdysone: 2~,3a,14a, 22R,25,26-hdexahydroxy-5~-cholest-7~n-6-one; 22,25-dideoxyecdy- sone: 2~,3~,14a-trihydroxy-5~-cholest-7-en-6-one, and 26-hydroxy- ecdysone 26-phosphate: 2~,3~,14a,22R,25,26-hexahydroxy-5~- cholest-7-en-6-one 26-phosphate. dysteroids are in the free form with 26-hydroxyecdysone accounting for 97% and ecdysone accounting for 3% (4). Interestingly, 26-hydroxyecdysone first isolated from 48- to 64-hr-old tobacco hornworm eggs {6) is devoid of molting hormone activity in the house fly bioassay (7} and has been reported to occur at such a high level only in eggs of the tobacco hornworm. Although low levels of other ecdysteroids such as 20-hydroxyecdysone, 20,26-dihydroxyecdysone, 3-epi-26-hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone{Fig. 1} as well as six other unidentified ecdysteroids have been isolated from kg quantities of tobacco hornworm eggs of different age groups 17},nothing is known of the precursors or the fate of 26-hydroxyecdysone in these eggs. We now have ex- amined putative ecdysteroid precursors in Manduca sexto~ [4-~4C]Cholesterol, 22,25-[4-~4C]dideoxyecdysone or [23,23-3H]ecdysone was injected into female tobacco horn- worm pupae on day 16 after pupation. Steroid analyses of ovaries from 4-day-old adult females (ca. five days after injection} showed that only the [14C]cholesterol was incor- porated and appreciably metabolized. In this paper we will discuss the metabolism of ['4C]cholesterol in M. sexta and its incorporation into ecdysteroid and nonecdysteroid conjugates in ovaries and eggs. We also will discuss re- cent developments concerning the fate and/or state of labeled ecdysteroid conjugates of different age groups of tobacco hornworm embryonated eggs. MATERIALS AND METHODS Instrumentation. RP-HPLC analyses were performed with a Spectra-physics SP8700 solvent delivery system equipped with an SP8500 dynamic mixer. Absorbance of the effluent was monitored at 254 nm with a Waters model 441 absorbance detector and automatically re- corded by a Shimadzu model C-R1B. Samples were counted in a Packard TriCarb 460CD Liquid Scintillation System or Beckman LS 5801. Samples were analyzed by high performance thin layer chromatography {HP-TLC) (precoated plates for Nano TLC, Silica Gel 60F 254, E. Merck, Darmstadt, West Germany}. Steroids. [23,24-3H]Ecdysone was a gift of Z~mconCorp. {Palo Alto, California). After dilution with unlabeled ec- dysone, it was purified to >95% radiopurity by RP-HPLC on a radial compression C18 column {Waters Assoc., Midland, Massachusetts}. The [3H]ecdysone was eluted isocratically with a mixture of methanol (Burdick and Jackson, American Scientific Products, Columbia, Maryland} and glass-distilled water {38:62, v/v} at 2 ml/ min. Fifty ~g of ecdysone, 2.49 • 105 dpm, were injected per pupa. 22,25-[4-14C]Dideoxyecdysone was prepared from [4-14C]cholesterol according to procedures used for the synthesis of unlabeled compound {8}, and 9.5 • 104 dpm (50 ~g} were injected into each pupa. [4-14C]Cholesterol was purchased from Amersham Corp. {Arlingte'. Heights, Illinois}. After purification by column chrom:mgraphy the radiochemical purity was >99%. One ~Ci was injected into each pupa. LIPIDS, Vol, 21, No. 1 (1986)