Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata

Fifth (last) instar nymphs of th e tick Ornithodoros moubata convert ingested 20-hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to th e more polar conjugates API. Only small quantities of free hormone were transferred to th e hemolymph and the carcass within t h e first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [³H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of API conjugates. API obtained in second instar nymphs fed with [3H]ecdysone ([³H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of APT remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., API mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle. Apolar products (mainly AP2) that accumulated in eggs of females injected with [³H]E or [³H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to API, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs. We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.     

Metabolism of ecdysteroids in the female tickAmblyomma hebraeum (Ixodoidea,Ixodidae): accumulation of free ecdysone and 20-hydroxyecdysone in the eggs

Summary [³H]-20-hydroxyecdysone ([³H]-20E) injected intoAmblyomma hebraeum females 7 days before the beginning of oviposition,viz. at the beginning of vitellogenesis, was converted to 3 polar peaks of unknown nature called 1, 2 and 3, and to apolar conjugates AP1, AP2 and AP3. AP2 have the same retention times as the esters of 20E with long chain fatty acids described inOrnithodoros moubata (Diehl et al. 1985). However, principally unmetabolized 20E was incorporated into the ovaries, and 16% of the injected labelling was recovered in the eggs, 3/4 being free 20E. When 20E was injected during oviposition, it was not converted to the polar products but only to the apolar products. At this time, 76% of the total radiolabel injected accumulated in the egg-batch, principally in the form of free unmetabolized 20E.After injection of [³H]-ecdysone (³H]-E), the three polar metabolites 1, 2 and 3, probably 20-deoxy homologues of 1, 2 and 3 described above were always produced irrespective of the time of injection. In addition, E was metabolized to 20E and to the apolar conjugates AP1, AP2, and AP3. E, 20E and peak 2 were incorporated into the ovary within the first day after injection. These 3 compounds were found in freshly laid eggs in variable proportions, the quantity of E decreasing with time while 20E and peak 2 increased. At the end of oviposition, ca. 60% of the injected radiolabel had been incorporated into the eggs. Apolar products and polar metabolites accumulating in the body were apparently not used as a source of free hormone for the eggs.Our results with tritiated ecdysteroids confirm our data concerning endogenous ecdysteroids of the eggs ofA. hebraeum (Connat et al. 1985). This species, in contrast to 2 other female ticks,Ornithodoros moubata andBoophilus microplus, incorporates free E and 20E instead of ecdysteroid conjugates into its eggs. The role of these free ecdysteroids remains to be elucidated.Abbreviations ES ecdysteroids - E ecdysone - 20E 20-hydroxyecdysone - HPLC high-performance liquid chromatography - RP-18 reyerse phase with C18 aliphatic chains grafted on the silica - RIA radio-immunoassay Part of this work was realized for the PhD thesis of E.M. Dotson     

Metabolism of [3H]-ecdysone in embryos and larvae of the tick Ornithodoros moubata

The fate of [³H]-ecdysone ([³H]-E) was investigated in hanging drop cultures of embryos and larvae of the tick Ornithodoros moubata using HPLC. The hormone was metabolized more slowly during described periods of increasing endogenous ecdysteroid (ES) titers than during periods of low titers except for young embryos. Three different classes of metabolites were produced: (1) apolar products (AP) corresponding to C-22 fatty acid ester conjugates of E and, in some cases, of 20-hydroxyecdysone (20E), (2) unidentified polar products (PP) more polar than E, one peak of which had the same retention times as 20,26-dihydroxyecdysone, and finally, (3) 20E verified by comigration of cold standards on RP-18 and silica columns. Hydroxylation of E to 20E first became evident in cultures of 2 day old embryos and was present in all cultures of older animals. Highest production of free 20E occurred during increasing endogenous ES titers in embryos and during the ES peak in larvae. Conjugation of E to AP occurred in all stages investigated, but was more pronounced during periods of low endogenous ES titers, and may correspond to a detoxification mechanism. In contrast, PP were produced during high 20E production in embryos and during periods of high and decreasing endogenous titers in larvae. © 1993 Wiley-Liss, Inc.     

Probable occurrence of ecdysteroid fatty acid esters in different classes of arthropods

We investigated the fate of injected [³H]ecdysone or [³H]20-hydroxyecdysone in various species of ticks, spiders, scorpions, myriapods, crustaceans and insects. Most of these arthropods were able to convert the ecdysteroids to esterase-labile metabolites with a very apolar behaviour in reverse-phase HPLC. Some of them have retention times similar to the apolar conjugates AP2 of the tick, Ornithodoros moubata, which have been identified recently as ecdysteroids esterified as C₂₂ with palmitic, stearic, oleic or linoleic acid [Diehl et al. (1985a) Int. J. invert. Reprod. Devl.8, 1–13]. Others are less apolar and could correspond to the AP1 from O. moubata. The possible function of these metabolites remains to be established. They could represent inactivation products and/or a hormone storage-form for embryos.     

Ecdysone metabolism in adult crickets,Gryllus bimaculatus

The fate of injected [³H]ecdysone has been investigated in female and male adults of the Mediterranean field cricket, Gryllus binaculatus (de Geer). The metabolism is similar in both sexes and at various stages of adult life. Several classes of apolar metabolites (A1–A5) represent the major compounds. The amount of polar conjugates is low in all tissues, as are the concentrations of 20-hydroxyecdysone. Ovaries are the only organs capable of storing considerable amounts of ecdysteroids. The amount of radiolabelled ecdysteroid activity (mostly [³H]ecdysone) excreted during the first 24 hr after injection is high.The chemical identity of the apolar metabolites is not yet known. A2, which is the major apolar compound, has recently been identified as a complex of ecdysone conjugates with abundant long-chain fatty acids (Hoffman et al., 1985 Life Sci.37, 185–192). Incubations with tissue homogenates in vitro have shown that several organs are capable of converting ecdysone into apolar compounds. Apolar ecdysteroid acyl esters represent a newly identified class of ecdysone conjugates from insects. Their role in regulation of free ecdysteroid titres during the reproductive period in female crickets is discussed.     

Ecdysteroid titre and metabolism to novel apolar derivatives in adult female Boophilus microplus (Ixodidae)

The free ecdysteroid titre determined by radioimmunoassay in adult female Boophilus microplus showed a peak just prior to full engorgement and detachment of the ticks and decreased subsequently to a very low value. In contrast, the titre of polar ecdysteroid conjugates was very low. Ecdysone was the major ecdysteroid at peak titre and was accompanied by much lower levels of 20-hydroxyecdysone. In newly detached ticks, injected [³H]ecdysone was metabolized primarily (80%) into much less polar compounds, which could be resolved into at least three groups by reversed-phase h.p.l.c. These [³H] “apolar” metabolites were transferred to the newly laid eggs, where they accounted for the vast preponderance of ecdysteroids, the level of free hormone being low. Hydrolysis of the three groups of compounds with an esterase preparation from porcine liver yielding [³H]ecdysone, together with the release of [³H] ecdysteroid and fatty acids upon alkaline saponification of the compounds, suggests that they are of a fatty acyl ester nature. The chemical transformation of these “esters” into the corresponding acetonide derivatives indicates that the 2- and 3-hydroxyls of ecdysone remain unsubstituted in these compounds. Several tick tissues, including Malpighian tubules, ovaries, gut, and fat body, metabolized [³H]ecdysone in vitro forming the “apolar esters” as major products. The maternal ecdysteroid “esters” may function as storage forms of hormone (presumably hormonally inactive), which could be hydrolysed enzymically during embryogenesis releasing free ecdysteroids. Such enzymic hydrolysis of [³H]ecdysone “esters” by homogenates from developing eggs of B. microplus has been demonstrated.     

Identification of the endogenous apolar ecdysteroid conjugates present in newly-laid eggs of the house cricket (Acheta domesticus) as 22-long-chain fatty acyl esters of ecdysone

Newly-laid eggs of the house cricket Acheta domesticus contain significant amounts of apolar ecdysteroid conjugates, which can be hydrolysed by prolonged incubation with a mixture of Helix pomatia gut hydrolases. The ecdysteroid released on hydrolysis of the apolar conjugates has been purified and identified as ecdysone by co-chromatography on normal-phase and reversed-phase HPLC and by fast-atom bombardment mass spectrometry.Starting with only 22 g newly-laid eggs (containing 16 μg conjugated ecdysone), the ecdysone conjugates have been purified by open column chromatography and four successive HPLC purification steps to give essentially pure apolar conjugates with a yield of 57%. The conjugates are shown to be a mixture of ecdysone 22-fatty acyl esters by co-chromatography with authentic reference compounds and by fast-atom bombardment mass spectrometry. The fatty acyl composition of the conjugates is very similar to that produced by the ovaries of A. domesticus from [³H]ecdysone in vitro (Whiting and Dinan, Biochem. J.252, 95–103, 1988). The major fatty acyl esters are the 22-palmitate (C_16:0), 22-oleate (C_18:1) and 22-linoleate (C_18:2), with smaller amounts of the myristate (C_14:0), stearate (C_18:0) and arachidate (C_20:0) esters.This report constitutes the first identification from an insect source of endogenous ecdysteroid 22-fatty acyl esters, which have previously been identified in ticks and as metabolites of exogenous [³H]ecdysone in several arthropod species.     

Distribution and metabolism of exogenous ecdysteroids in the egyptian cotton leafworm Spodoptera littoralis (lepidoptera: noctuidae)

After ingestion of various amounts of either [³H]ecdysone or [³H]20-hydroxyecdysone (0.8 ng to 10 μg) by sixth instar larvae of the Egyptian cotton leafworm Spodoptera littoralis, apolar metabolites are rapidly detected in the gut and frass. Hydrolysis of the apolar products with Helix hydrolases releases solely [³H]ecdysone or [³H]20-hydroxyecdysone, respectively. This, coupled with the formation of chemical derivatives (acetonide and acetate) which cochromatograph with authentic reference compounds on hptlc and hplc demonstrates that these apolar metabolites consist of ecdysone or 20-hydroxyecdysone esterified at C-22 with common long-chain fatty acids. The major fatty acids have been identified by RP-hplc and their contribution to the mixture determined. In contrast, [³H]ecdysone injected into the haemolymph of S. littoralis is metabolized to yield 20-hydroxyecdysone, ecdysonoic acid, and 20-hydroxyecdysonoic acid. Thus, two different pathways exist for the metabolism of ecdysteroids in this species. In addition to an essentially polar pathway operating on injected and endogenous ecdysteroids, exogenous ecdysteroids entering the gut of S. littoralis are detoxified, yielding apolar ecdysteroid 22-fatty acyl esters which are rapidly excreted. The significance of these results in relation to the effects of ingested ecdysteroids on S. littoralis is discussed. Arch. Insect Biochem. Physiol. 34:329–346, 1997. © 1997 Wiley-Liss, Inc.     

Ecdysteroids in relation to adult development and reproduction in female Rhipicephalus appendiculatus (Acari; Ixodidae)

In view of the paucity of information on ecdysteroids during tick development, the profiles of the free ecdysteroids, together with the polar and apolar conjugates have been established by radioimmunoassay during development of adult females of the hard tick, Rhipicephalus appendiculatus. The free ecdysteroid titre increased sharply to a peak approximately 3 days post-engorgement, a day preceding beginning of oviposition. This titre decreased to a low level, which was maintained throughout oviposition. Although the titre of polar ecdysteroid conjugates was appreciably less than that of the free ecdysteroids during the peak, the general profile of such conjugates was similar to that of the free ecdysteroids. In the case of the apolar ecdysteroid conjugates, the titre increased simultaneously with production of free ecdysteroids, but was maintained at a relatively high level until the end of oviposition, when it sharply declined. The apolar conjugates were the predominant form of ecdysteroids present during most of oviposition. The free ecdysteroids as well as the polar and apolar conjugates were shown to contain 20-hydroxyecdysone accompanied by smaller amounts of ecdysone by high-performance liquid chromatography-RIA (HPLC-RIA) and gas chromatography/mass spectrometry (selected ion monitoring; GC/MS [SIM]). © 1996 Wiley-Liss, Inc.     

In vitro ecdysteroid conjugation by enzymes of Manduca sexta midgut cytosol

In incubations with 80,000g supernatant of Manduca sexta midgut homogenates, [³H]ecdysone was converted to 3-[³H]epiecdysone and tritiumlabeled highly polar metabolites. C₁₈ SEP-PAK cartridges were found suitable for the separation and purification of the free ecdysteroids and of the highly polar metabolites. Eighty to ninety percent of the metabolites were hydrolyzed by enzyme mixtures (mainly β-glucuronidase, sulphatase, and acid phosphatase) from molluscs, even when β-glucuronidase activity was completely inhibited by D-saccharic acid 1,4-lactone, or various human acid phosphatases (free of sulphatase activity). In each experiment, the hydrolysate contained a much higher proportion of 3-epiecydsone than the free (unconjugated) ecdysteroid fraction. [³H]ecdysone was not metabolized in anaerobic incubations of midgut supernatant that had been filtered through Sephadex G-25. Addition of 5 mM ATP and 5 mM Mg²⁺ restored the conjugate formation in incubations of Sephadex-filtered supernatant. Four ecdysone conjugates and two 3-epiecdysone conjugates were resolved by reversedphase ion-pair high-performance liquid chromatography. It is concluded that the midgut cytosol contains several ATP:ecdysteriod phosphotransferases. This is the first demonstration of the formation of ecdysteroid phosphoconjugates in a cell-free system.     

Metabolism of [3H]-ecdysone in Schistocerca gregaria; formation of ecdysteroid acids together with free and phosphorylated ecdysteroid acetates

The metabolism of [³H]-ecdysone has been investigated at times of low and high endogenous ecdysteroid tit re, in early and late fifth-instar Schistocerca gregaria larvae, respectively. Ecdysone-3-acetate, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were identified as metabolites in both the free form and as polar conjugates. Comparison of the intact polar conjugates of the ecdysteroid acetates on two HPLC systems with the corresponding authentic compounds indicated that they were 3-acetylecdysone-2-phosphate and 3-acetyl-20-hydroxyecdysone-2-phosphate. Other major polar metabolites were identified as ecdysonoic acid and 20-hydroxyecdysonoic acid. Ecdysone metabolism in fifth-instar S. gregaria is apparently an age-dependent process. Early in the instar, excretion of both free and conjugated ecdysteroids, as well as ecdysteroid 26-acids, occurs. At this stage the level of ecdysteroid acetates in the conjugated (phosphate) form is high, in contrast to the free ecdysteroids, where ecdysone predominates. When the endogenous hormone titre is high, the formation of ecdysteroid acetates is less, the major excreted matabolites at that stage being conjugated 20-hydroxyecdysone together with ecdysteroid-26-acids, but little free ecdysteroids. Acetylation of ecdysone occurs primarily in the gastric caecae. Ecdysone-3-acetate (mainly as polar conjugate) is also a major product of ingested ecdysone in early fifth-instar Locusta migratoria.     

Formation of apolar ecdysteroid conjugates by ovaries of the house cricket Acheta domesticus in vitro.

The newly laid eggs of the house cricket Acheta domesticus contain apolar ecdysteroid conjugates, which we have hypothesized to be ecdysone long-chain fatty acyl esters [Whiting & Dinan (1988) J. Insect Physiol., in the press]. The ovaries of mature adult female A. domesticus in vitro convert [3H]ecdysone into apolar conjugates identical with those found in newly laid eggs. Comparison of the radioactive metabolites produced on incubation of [3H]ecdysone with various organs of adult female A. domesticus in vitro indicate that the fat-body is the major producer of polar ecdysteroid metabolites at this stage of development, whereas the ovaries are the major site of production of apolar metabolites. Apolar metabolites are also produced to a lesser extent by the crop, gut sections and the fat-body. Hydrolysis of radioactive metabolites produced by the ovaries with Helix enzymes releases only [3H]ecdysone, and thus ecdysone is not metabolized before conjugation by the ovaries. Formation of chemical derivatives (acetonide and acetates) of these 3H-labelled apolar conjugates strongly indicates that the position of conjugation is through the hydroxy group at C-22 of ecdysone. Extensive chromatographic analysis of the 3H-labelled apolar metabolites produced by the ovaries by t.l.c. and h.p.l.c. and comparison with authenticated reference compounds have conclusively demonstrated that the conjugates consist of ecdysone esterified at C-22 to a mixture of common long-chain fatty acids. The major fatty acyl esters have been identified and their percentage contribution to the mixture determined: laurate (0.5%), myristate (2.8%), palmitate (25.8%), stearate (8.4%), arachidate (1.0%), oleate (15.7%), linoleate (38.8%) and linolenate (2.1%). In addition there are three minor unidentified peaks, one of which has been tentatively identified as ecdysone 22-palmitoleate (2.6%). Comparison of this percentage composition with the previously published fatty acid composition of A. domesticus haemolymph [Wang & Patton (1969) J. Insect Physiol. 15, 851-860] reveals remarkable similarities, indicating that the acyl transferase(s) forming the conjugates have a broad specificity with regard to the fatty acyl substrate.     

Meeting announcements

The levels of individual free and conjugated ecdysteroids and ecdysteroid acids, labeled from [¹⁴C]cholesterol, 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 day 4, 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-hydroxyecdysonoic acid and the phosphate conjugates of 3-epi-20-hydroxyecdysone 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.     

Metabolism of maternal ecdysteroid-22-phosphates in developing embryos of the desert locust,Schistocerca gregaria

The ecdysteroid composition of Schistocerca gregaria eggs at different stages of development was determined by analysis of ecdysteroids labelled maternally from [4-¹⁴C]cholesterol. At all stages studied, highly polar ecdysteroid derivatives predominated, but changes in their composition occurred between day 10 of development and hatching (day 17). During this period, polar conjugates of ecdysone-3-acetate and 3-epi-2-deoxyecdysone appeared together with ecdysteroid acids. At day 17, the polar conjugate of [¹⁴C]ecdysone-3-acetate represented 36% of the total conjugated steroids. Separate in vivo studies on the metabolism of [¹⁴C]ecdysteroid conjugates isolated from newly-laid eggs and consisting primarily of the 22-phosphates of ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone showed that ecdysteroid phosphates could be hydrolysed to give primarily free ecdysone during embryogenesis. Developing eggs can metabolize [³H]ecdysone to ecdysonoic acid, 3-acetylecdysone-2-phosphate and to a lesser extent ecdysone-22-phosphate and 20-hydroxyecdysonoic acid. A polar conjugate of 20-hydroxyecdysone-3-acetate, possibly the 2-phosphate derivative, was detected as a minor metabolite of ecdysone. A scheme of the proposed pathways involved in the metabolism of ecdysteroid-22-phosphates in the developing eggs of S. gregaria is presented.     

Metabolism of ecdysteroid by testes of the tobacco budworm,Heliothis virescens

Testes from late last stage larvae of the tobacco budworm, Heliothis virescens, were incubated with [³H]ecdysone and [³H]cholesterol. [³H]Ecdysone was converted to six other major ecdysteroids, identified by cochromatography in reverse-phase high-pressure liquid chromatography (RPHPLC); four of them were verified by normal-phase HPLC. A highly polar fraction, moderately polar ecdysteroids (20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, and 20-hydroxyecdysone) and low-polarity ecdysteroids, including 2-deoxyecdysone, were detected after incubation with [³H]ecdysone. Compounds that reacted positively to antibodies to progesterone and testosterone were detected in the low-polarity fractions. Testes were incubated in fractions corresponding to each of the major ecdysteroid peaks derived from [³H]ecdysone metabolism. Although most of the radioactive ecdysteroid fractions were further metabolized to high- and low-polarity endpoints, 88% of the [³H]20-hydroxyecdysone peak apparently remained unmetabolized. 20-Hydroxyecdysone may be the primary ecdysteroid product of testes of H. virescens. [³H]Cholesterol was not metabolized to any appreciable extent.     

Endogenous ecdysteroid levels and rates of ecdysone acylation by intact ovaries in vitro in relation to ovarian development in adult female house crickets,Acheta domesticus

Ecdysteroid titres have been determined in adult female house crickets (Acheta domesticus) in relation to reproductive maturation. Ecdysteroid levels in newly emerged adult females are low except in the gut and carcass, which probably reflect the remnants of the preecdysial ecdysteroid peak. Ecdysteroid levels in all compartments increase markedly once ovarian weight surpasses 10 mg. Apolar ecdysteroid conjugates (ecdysone 22-fatty acyl esters) predominate in ovarian tissue throughout ovarian maturation, but low levels of free ecdysteroid and polar conjugated ecdysteroids are also present. During this period, two peaks of ecdysteroids (mainly free and apolar conjugated ecdysteroids) are observed in the haemolymph, gut, and carcass compartments. The peaks in the haemolymph occur when the ovarian mass reaches 30 and 100 mg. The gut and carcass may be acting as sinks or sites of metabolism for the hormone released from the ovaries. The rate of ecdysone acylation by ovaries was found to be developmentally regulated, increasing from low levels in the immature ovaries of newly emerged females as the ovaries increase in size. A semiquantitative assay has been developed to identify compounds which inhibit the conversion of [³H] ecdysone into 22-fatty acyl [³H] ecdysone by ovaries in vitro. A number of ecdysteroids possessing a free hydroxyl group at C-22 as well as the side-chain stereochemistry of ecdysone effectively inhibit this conversion, probably by acting as competitive substrates. In the cases of 20-hydroxyecdysone and ponasterone A, it was clearly demonstrated that these compounds are converted to a mixture of C-22 fatty acyl esters. Several other compounds which have been sugested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro. Arch. Insect Biochem. Physiol. 35:279-299, 1997.© 1997 Wiley-Liss, Inc.     

Metabolism of [3H] Gibberellin A5 in Cell Suspension Cultures of Pharbitis nil

Gibberellin A₅ (GA₅), a native GA of immature seeds of Pharbitis nil, was fed to Pharbitis nil cell suspension cultures as [C-l, ³H] GA₅ (3.1 Ci/mmol), and its metabolism over a 48 hr period was investigated. Radioactivity in free GA metabolites was 13.1%, with 79.9% in GA glucosyl conjugate-like metabolites. Only 7.0% of the radioactivity remained as [³H] GA₅. Tentative identifications were based on comparison with retention times of authentic free GAs and/or glucosyl conjugates after sequential chromatography on Si gel partition column → gradient-eluted C18 HPLC-radiocounting (RC) → isocratic-eluted C18 HPLC-RC, and showed that [³H] GA₅ was converted to [³H] GA₁ (2%), [³H] GA₃ (4%), [³H] GA₆ (2%), [³H] GA₂₂ (1%) and their glucosyl conjugates, and also to [³H] GA₈ glucoside, and [³H] GA₅ glucosyl conjugates. The major conjugate-like substances were [³H] GA₁ and [³H] GA₃ glucosyl esters, at 15% and 34%, respectively, of the total extractable radioactivity.     

Ecdysone metabolism in the host-parasitoid-system Trichoplusia ni/Chelonus sp

In unparasitized 4th and 5th-instar larvae of Trichoplusia ni and in 4th-instar larvae parasitized by Chelonus sp. 20-hydroxyecdysone, 20,26-dihydroxyec-dysone, and 20-hydroxyecdysonoic acid were the predominant metabolites formed 2 h after injection of [³H]ecdysone. Other unidentified metabolites were seen, but none seemed to be specific for either parasitized or unparasitized larvae. The major difference between parasitized and unparasitized larvae was seen with respect to the quantity of apolar (unidentified) and polar metabolites (20-hydroxyecdysonoic acid and unidentified ones), which were produced to a greater extent in parasitized larvae. Ecdysone was rapidly converted into 20-hydroxyecdysone and the other polar metabolites in all stages investigated, and the parasitoid seemed not to affect the conversion of ecdysone into 20-hydroxyecdysone. When analyzing the fate of [³H]ecdysone in host and parasite separately, at a stage when the parasite drinks hemolymph of its host, we observed that 10–20% of the radioactivity was recovered from the parasitoid. Analysis of the parasitoid's ecdysteroids revealed that ecdysone and 20-hydroxyecdysone represented only a small proportion of the recovered labeled ecdysteroids, the majority being apolar and polar metabolites. Our data suggest that the parasitoid takes up ecdysteroids from its host, converts them, and to some extent releases apolar metabolites into the host.     

Gibberellins and Gravitropism in Maize Shoots : Endogenous Gibberellin-Like Substances and Movement and Metabolism of [3H]Gibberellin A20

[³H]Gibberellin A₂₀ (GA₂₀) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [³H]GA₂₀ to [³H]GA₁⁻ and [³H]GA₂₉-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [³H]GA₁, [³H]GA₂₉, and [³H]GA₈. The tentative identification of these putative [³H]GA glucosyl conjugates was further supported by the release of the free [³H]GA moiety after cleavage with cellulase. Within 12 hours of the [³H]GA₂₀ feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [³H]GA₂₀, especially [³H]GA₁, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the `Tanginbozu' dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA₂₀-like substance and lower halves containing mainly GA₁-like and GA₁₉-like substances. Gibberellins A₁ (10 nanograms per gram) and A₂₀ (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.     

Metabolism of tritiated gibberellin a(20) in maize

After the application of 2.36 Curies per millimole [2,3-³H]gibberellin A₂₀ (GA₂₀) to 21-day-old maize (Zea mays L., hybrid CM7 × CM49) plants, etiolated maize seedlings, or maturing maize cobs, a number of ³H-metabolites were observed. The principal acidic (pH 3.0), ethyl acetate-soluble metabolite was identified as [³H]GA₁ on the basis of co-chromatography with standard [³H]GA₁ on SiO₂ partition, high resolution isocratic elution reverse phase C₁₈ high performance liquid chromatography and gas-liquid chromatography radiocounting. Two other acidic metabolites were identified similarly as [³H]GA₈ and C/D ring-rearranged [³H]GA₂₀, although gas-liquid chromatography radiocounting was not performed on these metabolites. Numerous acidic, butanol-soluble (e.g. ethyl acetate-insoluble) metabolites were observed with retention times on C₁₈ high performance liquid chromatography radiocounting similar to those of authentic glucosyl conjugates of GA₁ and GA₈, or with retention times where conjugates of GA₂₀ would be expected to elute. Conversion to [³H]GA₁ was greatest (23% of methanol extractable radioactivity) in 21-day-old maize plants. In etiolated maize seedlings, the C/D ring-rearranged [³H]GA₂₀-like metabolite was the major acidic product, while conversion to [³H]GA₁ was low.