Profiles of free and conjugated ecdysteroids and ecdysteroid acids during pupal-adult development of Manduca sexta.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 12:63-74 (1989) Profiles of Free and Conjugated Ecdysteroids and Ecdysteroid Acids During Pupal-Adult Development of Manduca sexta R u b e n Lozano, Malcolm J. Thompson, James A. Svoboda, a n d William R. L u s b y Insect and Nematode Hormone Laboratoy, Agricultural Research Service, USDA, Beltsville, Ma yland The levels of individual free and conjugated ecdysteroids and ecdysteroid acids, labeled from ['4Clcholesterol, in five different age groups of male Manduca sexta during pupal-adult development were determined by HPLC. Eight free ecdysteroids, eight ecdysteroid phosphates, and two ecdysteroid acids were identified. Newly ecdysed pupae contained predominantly 3-epiecdysteroids in each of the free, conjugated, and acidic ecdysteroid fractions. The titer of each ecdysteroid fraction rose sharply by day4, and this was particularly noteworthy with respect to free ecdysone and 3-epi-20-hydroxyecdysonoic acid. This stage demonstrated high degrees of ecdysone biosynthesis, oxidative catabolism, and phosphorylation. As development proceeded to day 16, total ecdysteroid titer remained constant; a decreasing free ecdysteroid titer was accompanieid by increasing titers of both conjugates and acids resulting from the metabolic processes of hydroxylation, oxidation, epimerization, and phosphorylation. The predominant metabolites throughout development were 3-epi-20-hydroxyecdysonoicacid and the phosphate conjugates of 3-epi-20hydroxyecdysone and 3-epi-20,26-dihydroxyecdysone. The ultimate inactivation of the ecdysteroids of M. sexta during pupal-adult development is possibly mediated by two pairs of metabolically-linked processes, one leading to a 3-epiecdysteroid acid, and the other to 3-epiecdysteroid phosphates. Key words: ['4C]cholesterol, HPLC, ecdysteroidtiters, ecdysteroid metabolism, epimerization, hydroxylation, oxidation, epiecdysteroid acids, epiecdysteroid phosphates INTRODUCTION Physiological events that occur during pupal-adult metamorphosis of insects can be correlated with the presence of ecdysteroid hormones whose titer and Acknowledgments: We gratefully thank Lynda J. Liska and Kenneth R. Wilzer, Jr., for their technical support, Sherry A. Cohen for her secretarial assistance, and Martin L. Weinrich for his preparation of the manuscript. We also wish to thank John M. Roman of Program Resources, IRC., NCI-FCRS, Frederick, MD, for the FAB-mass spectra. Received April 21,1989; accepted August 3,1989. Address reprint requests to Dr. JamesA. Svoboda, USDA, ARS, PSI, Insect Hormone Laboratory, Bldg. 467, BARC-East, Beltsville, MD 20705. 0 1989 Alan R. Liss, Inc. 64 Lozano et al. activity during this closed stage are regulated by biosynthesis, enzymatic degradation, and compartmentalization of the hormonally active compounds [l-31. The pupal-adult development of Munducu sextu is characterizedby quantitative, qualitative, and temporally specific changes in the ecdysteroid titer . Six of the free ecdysteroids occurring in M. sextu during this period were originally isolated and identified by this laboratory using mass spectrometryand NMR spectroscopy . More recently, a detailed study of the metabolism kinetics and distribution of ecdysteroids during M. sextu pupal-adult development has been reported by Warren and Gilbert . They defined a hemolymph ecdysteroid profile resulting from the synthesis and secretion of ecdysone by the prothoracic glands and the sequential oxidative metabolism of ecdysone by peripheral tissues. As metamorphosis progressed, ecdysteroids were increasinly localized in the gut. We have been performing in vivo studies of ecdysteroid metabolism in M. sextu using alternate methodology, namely, long-term ['4C]cholesterol labeling of ecdysteroids. The collection and isolation of minute quantities of radiolabeled steroids from various ages of M. sextu ovaries, eggs, and larvae have allowed us to trace the metabolic fate of maternally acquired ecdysteroids throughout embryogenesis . Our studies have now extended to the longterm labeling of ecdysteroids during the pupal-adult development of male M. sextu. Of particular significance, two ecdysteroid acids, 20-hydroxyecdysonoic acid and its 3a-epimer, were isolated and identified from insects 8 days following pupation as metabolites of ['4C]cholesterol administered to M. sextu as fifth-instar larvae [8,9]. Ecdysteroid acids and 3-epiecdysteroids are generally considered as inactivation products of the active molting hormone, 20-hydroxyecdysone [lo], resulting from C-26 oxidation and C-3 epimerization. These metabolic processes, as well as the formation and hydrolysis of conjugates, are thus critical factors in the regulation of ecdysteroid hormone titer during insect development. This report follaws the metabolic fate of radiolabeled free, acidic, and conjugated ecdysteroids from ['4C]cholesterol during pupal-adult development of M. sextu, with particular emphasis on the kinetics of the formation of ecdysteroid acids and C-26 oxidized 3-epiecdysteroids. MATERIALS AND METHODS As described previously , each newly molted fifth-instar larva, reared on an artificial diet, was injected with one microcurie of purified commercial [4-'4C]cholesterol (Amersham Corporation,' Arlington Heights, IL; specific activity 53.7 mCi/mmol). Male pupae, staged from ecdysis (day 0), were collected at various ages (0, 4, 8, 12, and 16 days) for analysis in groups of 16-65 in number. Adult eclosion of control insects occurred approximately on day 18. Whole animals were extracted with CHC13/methanol,methanol, and 70% aqueous methanol . Ecdysteroids were fractionated according to class (free, acid, or conjugate) and were then analyzed by HPLC.* Separation and purification procedures using Florisil, XAD-16, XAD-2, DEAE-Sephadex A-50, and C18 Sep-Pak column chromatography have been detailed elsewhere [8,9]. 'Mention of a company name or proprietary product does not necessarily constitute endorsement by the U.S. Department of Agriculture. *Abbreviations used: E = ecdysone; 20E = 20-hydroxyecdysone; 20EA = 20-hydroxyecdysonoic acid; 26E = 26-hydroxyecdysone; 2026E = 20,26-dihydroxyecdysone; EP, 26EP, etc. = ecdysone phosphate, 26-hydroxyecdysone phosphate, etc.; E', E'A, E'P, etc. = 3-epiecdysone, 3-epiecdysonoicacid, 3-epiecdysone phosphate, etc; X-P = unknown ecdysteroid phosphates. Ecdysteroid Profiles in Manduca 65 HPLC/Radioassay Free ecdysteroids were analyzed by reverse-phase HPLC on an ODs-Hypersil x 25 cm, 5 pm particles; Shandon, Sewickley, PA) eluted with 38%aqueous methanol. Ecdysteroid acids and conjugates were analyzed by ion-suppression reverse-phase HPLC, also on an ODs-Hypersil c18 column (that provided better resolution than c8 columns used previously) and were eluted with 28% methanol in 0.03 M aqueous NaH2P04solution (pH 5). Effluent absorbance was detected at 254 nm. Analyses were performed at 33°C and a flow rate of 1.0 ml/min. For radioassay, fractions (0.5 ml) were collected throughout the chromatogram and mixed with scintillation fluid, and their radioactivity was measured in a liquid scintillation counter (Beckman LS 5801). Radiolabeled metabolites were identified by comparison with ecdysteroid standards and with ecdysteroid metabolites from M. sexta that had been characterized previously by NMR and mass spectral analyses . Ecdysteroid content was quantitated by HPLC. Concentrations of free ecdysteroids and ecdysteroid acids were determined by comparison to standards of each metabolite. Concentrations of ecdysteroid phosphates were determined by comparison to 26-hydroxyecdysone 26-phosphate. c 1 8 column (4.6 mm Ecdysteroid Conjugate Analysis Each component of the ecdysteroid conjugate fraction was isolated by HPLC collection, purified on a SEP-PAK, and analyzed by negative-ion fast atom bombardment mass spectrometry. An aliquot of each conjugate was hydrolyzed with an enzyme mixture ; after purification, each deconjugated ecdysteroid was identified by TLC  and HPLC. RESULTS The results presented are from single experiments with samples of five different groups of male M . sexta taken at 0, 4, 8, 12, and 16 days following pupation. Free Ecdysteroid Analysis A typical reverse-phase HPLC/radioassay of a free ecdysteroid fraction, obtained from insects on day 4 following pupation, is shown in Figure 1.Seven radiolabeled free ecdysteroids were identified in this sample; E was present in the greatest quantity. From all age groups examined, a total of eight radiolabeled free ecdysteroids were identified: E, 20E, 26E, 2026E, and their four corresponding 3a-epimers. The profile of individual free ecdysteroid titers through day 16 of M. sexta pupal-adult development is shown in Figure 2A. The profile of total free ecdysteroid titer is shown in Figure 3. The levels of E’ present in all age groups were very low (20-40 ng/g) and are not shown in Figure 2A. Also not included (for graphic simplicity) is 26E, which was detected only at day 4 at 0.20 pg/g. In 0 day pupae, 85% of the total free ecdysteroids were the 3-epiecdysteroids, 20E‘, 26E’, and 2026E’, in relatively small amounts of 0.31, 0.29, and 0.38 pg/g of pupae, respectively. By day 4, there was a 2.5-fold increase in free ecdysteroid titer, as expressed by the rising concentrations of E (1.86 pg/g), 66 Lozano et al. 1.005 AU Fig. 1. Reverse-phase HPLC/radioassay of free ecdysteroids from male M. sexta 4 days postputation. UV absorbance was detected at 254 nm. Shaded areas indicated radioactivity. Component eluting at 20 min was not identified. 20E (0.33pg/g), 26E (0.20 pg/g), and 2026E (0.23 pg/g). The levels of 20E’, 26E’, and 2026E’ decreased, and free 3-epiecdysteroid content formed only 13%of the total. At 8 days, the titer of total free ecdysteroids remained the same quantitatively, but it exhibited dramatic qualitative changes. Decreasing levels of E and 26E were accompanied by much increased levels of 20E (1.07 pg/g) and 2026E (1.21 pg/g). At 12 days following pupation, a decline in free ecdysteroid titer occurred, particularly that of 20E, whereas 2026E (1.08 pg/g) became predominant and 3-epiecdysteroidcontent increased. By 16 days, the free ecdysteroid titer decreased again, especially the level of 2026E. However, the titer of 3-epiecdysteroids continued to become more predominant, forming 66% of the total free ecdysteroids at this stage. 2026E’, which occurred in all age groups, was the most abundant free ecdysteroid in both 0 and 16 day samples. Ecdysteroid Conjugate Analysis A typical ion-suppression reverse-phase HPLC/radioassayof an ecdysteroid conjugate fraction, obtained from insects 4 days post-pupation, is shown in Figure 4A. Nine radiolabeled compounds were detected in this sample and were representative of all of the conjugates found in the other age groups. Analysis by FAB mass spectrometry of the first conjugate, eluting at 10 min, produced diagnostic peaks at m/z 79 (PO,)- and 97 (H2P04)-,indicative of a phosphate ester. The spectrum also showed major peaks in the high mass region at m/z 575 (M-H)- and 559 (M-OH)-, signifying a phosphate conjugate of a dihydroxyecdysone. Enzymatic hydrolysis of the conjugate released 2026E as the ecdysteroid moiety. Therefore, the initial conjugate was identified as 2026EP. Similarly, mass spectral analyses of the other isolated conjugates established that they were all ecdysteroid phosphates of which the position(s) of the phosphate moiety is presently not known. The identities of the ecdysteroid moieties are shown for each conjugate in Figure 4A and were corroborated by determination of the mass spectra of the intact conjugates. Ecdysteroid Profiles in Manduca 0 4 8 12 16 12 16 67 a9 0.8 a 20E’P ai 2026E’P X-P 26EP . 0.6 c a - D a 03 0.4 0.3 02 0. I 0 2.e 2.4 2.2 . - 2 0, ~ a c c 1.8 1.0 1.4 ID L c 1.2 0 V P 0 2a ,” 0.8 L 0.8 0.4 V w 0.2 0 0 4 8 Age of pupae (days after ecdysis) Fig. 2. Profiles of titers of (A) free ecdysteroids, (B)ecdysteroid conjugates, and (C) ecdysteroid acids during pupal-adult development of M. sextu males. Titers were quantitated by HPLC/ radioassay analyses. Not shown in 2A are minor quantities of 26E and E’; not shown in 2B is a minor amount of E‘P (see text). 68 Lozano et al. A CD \ CD a Y e C al C 0 0 ‘0 0 L. 5 e u) > U 0 w 0 4 8 12 10 Age of pupae ( d a y s after ecdysis) Fig. 3. Profiles of titers of total free ecdysteroids, total conjugates, total acids, and total ecdysteroids (sum of free, conjugates, and acids). Total concentration (&g) is expressed as the sum of the individual ecdysteroid concentrations (Fig. 2 . ) The two 2026E’Pconjugates, eluting at 12 and 13 min, occurred in an approximate 2:l ratio in every age group. They could possibly be an isomeric pair, namely, the 25R and 25s isomers, rather than being a pair of 2026E’ conjugates with phosphate groups at different positions. In their previous study, Warren and Gilbert  observed the probable C-25 isomers of free 2026E occurring in a constant 2:l ratio throughout M. sextu pupal development. Under our HPLC conditions, we could not resolve the C-25 isomers of 2026E, 2026E’, or 2026EP in our samples, if both isomers were indeed present at all. The radiolabeled peak at 17 min (X-P, Fig. 4A) occurred in significant amounts only on days 8, 12, and 16 (0.10-0.18 pg/g). The mass spectrum of X-P revealed the presence of a phosphate group. However, hydrolysis of X-P revealed a mixture of unknown free ecdysteroids. The 26EP conjugate at 24.5 min had the same retention time as authentic 26-hydroxyecdysone 26-phosphate derived Ecdysteroid Profiles in Manduca 69 2 0 2 6 E’ P A 2 ’P I 26EP 2 0 2 6E’P 1 , 0 0 5 AU EP E‘P $6 ....... 2.:s<. : I 4 2 8 I 36 I 40 I I I 44 46 52 100020E’A - B 800- E P 0 >- 600- I t ? !- 0 .01 AU 400- 9 n 4 a B 2oo- TIME (min) Fig. 4. Ion-suppression reverse-phase HPLChadioassays of (A) ecdysteroid conjugates from male M. sexta 4 days post-pupation and (B) ecdysteroid acids from males 16 days post-pupation. UV absorbance was detected at 254 nm. Shaded areas indicate radioactivity. from M. sextu eggs [ll]. The 26EP conjugates at 24.5 and 26.5 min might be the 25R- and 25s-isomeric pair. In summary, we identified phosphate conjugates of E, E‘, 20E’, 26E, 2026E, and 2026E’. The profile of conjugated ecdysteroids throughout pupal-adult development is shown in Figures 2B (individual ecdysteroid conjugates) and 3 (total conjugates). For graphic simplicity, E’P is not included in Figure 2B; it was only detected in 4 day insects at 40 ng/g. A relatively small amount of total conjugates was present in 0 day pupae and consisted mainly of 20E’P (0.13 pg/g) and 2026E’P (0.10 pg/g). The titer increased fivefold by 4 days, with large amounts of 20E’P (0.39 Kglg), 26EP (0.40 @g), and 2026E’P (0.44 pg/g) and 70 Lozano et al. smaller amounts of EP and E’P. By 8 days, the titer fell, with decreases in the levels of EP, E’P, and 26EP. From day 8 through day 16, the concentration of 26EP continued to decline, but the total titer gradually rose, with increases in 2026EP and 2026E’P and significant amounts of the unknown mixture, X-P. 20E‘P and 2026E’Pwere the major conjugates in each age group. Ecdysteroid Acid Analysis A typical ion-suppression reverse-phase HPLChadioassay of an ecdysteroid acid fraction, obtained from a 16 day sample, is shown in Figure 4B. 20EA and 20E’A, characterized by NMR and mass spectral analyses , were the only ecdysteroid acids detected in substantial quantities. They were found in every age group and accounted for 95-100% of the total radioactivity in each ecdysteroid acid fraction. The profile of ecdysteroid acid titer is shown in Figures 2C (individual ecdysteroid acids) and 3 (total acids). 20EA (0.23 pg/g) and 20E‘A (0.73 pg/g) were both present in 0 day pupae. Total acid titer increased as development progressed. Each acid concentration, especially that of 20E’A, had increased by day 4.The level of 20EA tripled from day 4 to 8, appeared to peak on day 12 (1.77 pglg), and then declined on day 16. The level of 20E’A exhibited little significant variation from days 4 to 12, but then it rose sharply by day 16 (2.57 pg/g). DISCUSSION Our long-term [14C]cholesterollabeling of ecdysteroids has resulted in the identification of eight free ecdysteroids, two ecdysteroid acids, and eight ecdysteroid phosphates in M. sextu during larval-pupal-adult development. Significant among these compounds are several C-26-oxidized and C-3-epimerized ecdysteroids whose metabolism kinetics during insect metamorphosis have not been examined by HPLC/RIA because of limitations imposed by the relatively low-binding affinities of the antisera for these ecdysteroids . Our results show an approximatelyconstant titer (6-7 pg/g)of total ecdysteroids after day 8 (Fig. 3). However, various RIA studies [6,12-141 have shown a declining ecdysteriod titer in the later stages of pupal development, because the metabolic processes of 26-oxidation and 3-epimerization predominate in the later stages, and the resulting 26-oxidized 3-epiecdysteroids are not detected by RIA. Our methods and results thus provide an advantage in that the declining titer can be accounted for by the isolation and identification of ecdysteroid metabolites such as 26E’, 2026E’, 2026E’P, and 20E‘A. We also have identified 20E’P as a major metabolite during M. sextu pupal development. Although this 3-epiecdysteriod is not oxidized at C-26, it had not been detected previously by H-2 antisera , suggesting that the phosphate group of 20E’P is located in the side chain, possibly at C-22. However, because of the sensitivity of their HPLC/RIA methodology and the specificity of their compartmental analyses of hemolymph and gut, Warren and Gilbert  have been able to identify certain ecdysteroid metabolites in M. sexta that we could not identify in this study. These include 2-deoxy-E, ecdysonoic acid, 20E-conjugates, and three E-conjugates of different polarity. Our failure to detect these compounds is due partly to their occurrence in Ecdysteroid Profiles in Manduca 71 rather low concentrations in whole insects. Also, the relative apolarity of 2-deoxy-E and the E-conjugates may have caused their retention on the HPLC columns under our elution conditions. Nevertheless, certain observations on the titers of the major ecdysteroid metabolites can be made from our results. In newly pupated M. sextu, 3-epiecdysteroidspredominated in the free, conjugate, and acid fractions. Freshly ecdysed pupae of another lepidopteran, Pieris brussicue, have also been shown to contain mainly 3-epiecdysteroids (20E’ and 20E’A) . The formation of 3-epiecdysteroidsappears to be characteristic before molting or hatching in these two insects. Newly emerged first-instar larvae of M. sexta also contained large amounts of 3-epiecdysteroids (26E’, 2026E’, 20E’A) . Between days 0 and 4 of pupal-adult development, the titer of each of the free, conjugate, and acid fractions rose sharply because of a large synthesis of E. The 20-monooxygenase and 26-hydroxylation/oxidation enzyme systems were presumably active at this stage, particularly increasing the level of 20E’A. Conjugation was very prominent as a variety of ecdysteroid phosphates were synthesized, especially 20E’P, 26EC and 2026E’P. The major ecdysteroids on day 4 were E and 20E’A, demonstrating that this early stage of pupal-adult development and rapid E biosynthesis was also characterized by a variety of catabolic reactions (oxidation, phosphorylation, epimerization), occurring in conjunction with each other, that may regulate the titer of free ecdysteroids, particularly E and 20E. The high catabolic activity decreased the level of free 3-epiecdysteroids as they were metabolized either to 20E’A or to phosphate conjugates. As noted previously  and verified here, the appearance of maximal levels of 26E on day 4 particularly in the phosphorylated form indicated that considerable 26-hydroxylation activity occurred early in pupal-adult development. In fact, the peak titers of 26E and 26EP on day 4 coincided with the peak titers of E and EP. As further development to days 8 and 12 apparently promoted higher 20-monooxygenase activity, the titers of 26E and 26EP declined in favor of increased levels of 2026E, 2026EP, and 20EA. The appearance of the peak titers of 26E and 26EP on day 4 could indicate a specific physiological role for 26E. Male pupal hemolymph of the European corn borer, Ostriniu nubildis, has been found to exhibit near-coincidental peak titers of E and 26E . We believe that 26E also is a physiologically active hormone during the embryonic development of M. sextu [71. Day 8 was characterized by dramatic qualitative changes in the free ecdysteroid titer presumably due to high oxidative metabolism by the 20-monooxygenase and 26-hydroxylase/oxidasesystems. The total ecdysteroid titer rose slightly; a large increase in the level of 20EA was accompanied by a small decrease in the conjugate titer. Days 4-8 thus were marked by additional synthesis of E; rapid oxidation of E to 20E, 2026E, and 20EA; deconjugation of EP, E‘P, and 26EP, but no changes in 20E’P or 2026E’P; and an inactive 3-epimerase system, indicated by the lack of titer increases for any of the 3-epiecdysteroids. Conjugation may play a dual role during this period. Because of their deconjugation between days 4 and 8, EP and 26EP could serve as storage or intermediate metabolites to 20E and 2026E, whereas the constant or rising levels of 20E’P, 2026EP, and 2026E’P suggest their possible role as inactivation products. 72 Lozano et al. The total ecdysteroid titer remained unchanged from days 8 to 12 to 16; free ecdysteroid titer decreased, while acid and conjugate titers increased. These stages were characterized by more oxidative metabolism, particularly 26-hydroxylation/oxidation,increased 3-epimerization, and phosphorylation. As a result of these processes, 26-oxidized 3-epiecdysteroids, and a 3-epiecdysteroid acid especially, predominated in 16 day pupae: 2026E’ in the free fraction, 2026E’P in the conjugate fraction, and 20E’A in the acid fraction. Any significant ecdysone synthesis apparently ceased by day 8. The present study shows that the ecdysteroid titer in M . sextu, during the ages of pupal-adult development examined here, is characterized mostly by the rapid synthesis of E followed by a series of C-20 and C-26 hydroxylations, C-26 oxidation, C-3 epimerization, and phosphorylation. E, which peaked on day 4, is metabolized, resulting in a progression of peak titers of oxidized, epimerized, and phosphorylated ecdysteroid metabolites that produced the following additional major ecdysteroids: 26E and 26EP peaked on day 4, 20E on day 8, 2026E between days 8 and 12, and 20EA on day 12; and 20E’A, 2026E‘, 20E’P, 2026EP, and 2026E’P all peaked on day 16. Similar oxidative sequences have been observed previously in pupae of M . sextu [5,6], Drosophilu melanoguster , and 0. nubilutis . The metabolic progression of ecdysone toward a 3-epiecdysteroid acid is probably a general occurrence during pupal-adult metamorphosis . As noted previously , the accumulation and resulting peak titers of E and 2026E suggests that they, in addition to 20E, may be active hormones during each of their respective intervals of peak titer. The subsequent metabolism of 2026E to the presumably less active 20EA and 2026E’ is in support of a postulated role for 2026E. 20EA and 20E’A are logical inactivation end-products of this metabolic sequence because of their anionic nature that facilitates their excretion via the meconium of the hindgut. On day 16, 20E’A was found in the largest concentration of any ecdysteroid during development. As the titer of 20EA increased throughout and appeared to peak on day 12, more 20EA was probably transported into the gut, where 3-epimerase activity is extremely high , promoting the conversion of 20EA to 20E’A (as well as the conversion of 2026E to 2026E‘ and thereon to 20E’A). These results suggest that activity of the 3-epimerase was significant only at the beginning and end of pupal-adult development. Conversely, minimal activity occurred during the interval from days 4 to 8, coincident with the stage of peak titers of 20E and 2026E. This perhaps reflects the critical role played by this gut enzyme in M . sextu during those late stages of development, i.e., late fifth-instar, pre-adult-eclosion, and late embryogenesis , when ecdysteroids are prepared for excretion by becoming more concentrated in the gut. Conjugation of ecdysteroids occurred via phosphorylation. Phosphate esters of E and 26E were mentioned above as possible storage products that occur during a period of high E biosynthesis (day 4), thus providing for the release of free E and 26E to be metabolized by the 20-monooxygenase when high titers of 20E and 2026E are required to fill their postulated roles (at day 8). However, two other conjugates, 20E ‘P and 2026E‘C predominated during development. Their titers, and that of 2026EE either increased or remained fairly constant throughout, suggesting that conjugation, linked with 3-epimerization, served to inac- Ecdysteroid Profiles in Manduca 73 tivate the free ecdysteroids, 20E and 2026E. The primary role of conjugation in M . sextu as an inactivation process during pupal-adult development is in contrast to its primary role as a storage mechanism in embryonic development during which 26EP was synthesized in adult ovaries and then hydrolyzed during embryogenesis to release the presumably active hormone, free 26E . In M . sextu pupal-adult development, conjugation and epimerization enzymes played relatively minor roles during the time (days 8 to 12) of peak titer of the active hormone 20E and possibly active 2026E, hormones whose titers were more critically regulated by inactivation via the oxidative sequence leading to 20EA. Conversely, epimerization and conjugation attained more significant inactivation roles during intervals of low ecdysone biosynthesis and presumably low 20-monooxygenase activity (days 0, 12, 16). Perhaps such intervals initiate the induction of the epimerase and phosphorylase systems, or else they merely reflect the increased transport of metabolized ecdysteroids into the gut, a site where the two enzymes may be highly active . Inactivation of the ecdysteroid molting hormones in M. sextu near the end of pupal-adult development is therefore characterized by two pairs of metabolically linked processes: l) oxidation and epimerization leading to a 3-epiecdysteroid acid (20E'A); and 2) epimerization and phosphorylation leading to 3-epiecdysteroid phosphates (20E'P and 2026E'P). LITERATURE CITED 1. Gilbert LI, Bollenbacher WE, Goodman W, Smith SL, Agui N, Granger N, Sedlak BJ: Hormones controlling insect metamorphosis. Recent Prog Horn Res 36,401 (1980). 2. Smith SL: Regulation of ecdysteroid titer: Synthesis. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press, Oxford, vol 7, pp 295-341 (1985). 3. 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