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.     

The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria

Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g).     

Ecdysteroids in the developing eggs of the desert locust, Schistocerca gregaria

Ecdysteroid levels in the developing eggs of Schistocerca gregaria were determined, at daily intervals, using gas chromatography and electron capture detection of ecdysteroid derivatives. Ecdysone and 20-hydroxyecdysone were present both as the free ecdysteroid and as polar conjugates. Total ecdysteroids reached a maximum of 40 ng/egg with ecdysone contributing the greater part.     

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

Maturing eggs of the desert locust, Schistocerca gregaria, contain a variety of ecdysteroid (insect moulting hormone) conjugates and metabolites, four of which have been previously isolated from polar extracts and identified as ecdysonoic acid, 20-hydroxyecdysonoic acid, 3-acetylecdysone 2-phosphate and ecdysone 2-phosphate. In the present study we have isolated eight additional ecdysteroids from similar late-stage eggs by high-performance liquid chromatography. The 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone, all of which were first identified as ecdysteroid components of newly-laid eggs of S. gregaria, were identified by co-chromatography with authentic compounds and by physicochemical techniques. The remaining compounds were identified as 3-acetyl-20-hydroxyecdysone 2-phosphate, 3-epi-2-deoxyecdysone 3-phosphate, 3-acetylecdysone 22-phosphate and 2-acetylecdysone 22-phosphate by fast atom bombardment mass spectrometry, p.m.r. spectroscopy and analysis of the steroid moieties after enzymic hydrolysis. The latter two compounds, after isolation, are susceptible to nonenzymic acetyl migration and deacetylation to give mixtures of ecdysone 22-phosphate and its 2- and 3-acetate derivatives. The possible role and significance of these ecdysteroid conjugates with respect to the control of hormone titres in insect eggs is discussed.     

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.     

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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 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.     

Metabolism of 26-[14C]hydroxyecdysone 26-phosphate in the tobacco hornworm,Manduca sexta L., to a new ecdysteroid conjugate: 26-[14C]hydroxyecdysone 22-glucoside

Following injection into female Manduca sexta pupae, [¹⁴C]cholesterol is converted to a radiolabeled C₂₁ nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly-laid eggs consist mainly of labeled 26-hydroxyecdysone 26-phosphate. During embryogenesis, as the level of 26-hydroxyecdysone 26-phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0- to 1-hour-old larvae was identified as 26-hydroxyecdysone 22-glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26-hydroxyecdysone 26-phosphate in 0- to 1-hour-old larvae indicates that the 26-hydroxyecdysone 22-glucoside is derived from 26-hydroxyecdysone 26-phosphate. 3-Epi-26-hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3-epi-20,26-dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0- to 1-hour-old larvae contained the highest levels of 3α-ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β-epimers. The ecdysteroid conjugate profiles of ovaries and 0- to 1-hour-old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugated ecdysteroids and nonecdysteroid conjugates (C₂₁-steroid conjugates).     

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.     

Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly,Sarcophaga peregrina

Metabolites of radioactive ecdysone or 20-hydroxyecdysone in larvae and pharate pupae of Sarcophaga peregrina were separated and identified by using thin-layer chromatography, high-performance liquid chromatography, and chemical methods. At the larval stage ecdysone was metabolized to biologically less active ecdysteroids predominantly through 20-hydroxyecydsone, at the pharate pupal stage, to other ecdysteroids which were tentatively identified as 26-hydroxyecdysone, 3-epi-26-hydroxyecdysone, and 3-epi-20,26-dihydroxyecdysone. Ecdysteroid acids were found in the polar metabolites during pharate pupal-pupal transformation, but scarcely detected in the larval metabolites. These acids were presumed to be ecdysonoic acid, 20-hydroxyecdysonoic acid, and their epimers. The conjugates of ecdysteroid that released the free ecdysteroids by enzymatic hydrolysis were produced more in larvae than in pupae, whereas the very polar ecdysteroids that were not affected by the enzyme were found more in pupae. Therefore, there are different metabolic pathways of ecdysone between these two successive developmental stages, and the alteration of the metabolic pathway may serve as one of the important factors in a regulatory mechanism of molting hormone activity which is responsible for normal development of this insect.     

Ecdysteroid titres and location in developing eggs of Schistocerca gregaria

Ecdysteroid levels in the separated embryo and yolk fractions of Schistocerca gregaria eggs have been determined at each of the developmental stages. The major hormones present both in the free and conjugated state are ecdysone, 20-hydroxyecdysone and 2-deoxyecdysone. At the beginning of embryonic development the ecdysteroids occur only in the yolk whereas, after blastokinesis, they are found in the embryo. The levels of conjugates fall during embryonic development, whereas a decrease of free hormone titres in early embryogenesis is followed by a marked increase in late embryos (stage 26 and 28). The possible role of ecdysteroids in relation to the morphogenetic processes of egg development and the site of origin of the free ecdysteroid peaks are discussed.     

Ecdysteroid levels throughout the life cycle of the desert locust, Schistocerca gregaria

Moulting hormone levels for all stages of the life cycle of the desert locust, Schistocerca gregaria, have been determined using gas chromatography with electron capture detection of the trimethylsilylated hormones. During larval development, the major hormone detected is 20-hydroxyecdysone with smaller quantities of ecdysone present. In mature adult females the major ecdysteroid observed is a polar conjugate of ecdysone, with smaller quantities of conjugated 20-hydroxyecdysone also present. During embryonic development the pattern changes from a high proportion of conjugated ecdysone in the early stages to give more free hormone and a higher proportion of 20-hydroxyecdysone in later stages. The highest titre of 20-hydroxyecdysone found in this insect is during the 5th larval instar. Maximal levels of ecdysteroid per insect are found in mature females just before oviposition, while the highest level of ecdysteroid per g of tissue is found in the eggs.     

Ecdysone metabolism and distribution during the pupal-adult development of Manduca sexta

The metabolism and distribution of endogenous ecdysone and injected [³H]ecdysone were studied during the pupal-adult development of Manduca sexta. Well-characterized antisera were used to detect and quantify endogenous metabolites by radioimmunoassay (RIA) following their separation by ion-suppressed reverse phase, and normal phase, high performance liquid chromatography. Identical chromatographic procedures were employed to determine the metabolic fate of the [³H]ecdysone in the haemolymph pool. These studies revealed the sequential appearance in the haemolymph and gut of progressively oxidized metabolites of ecdysone—hydroxylation at C-20 was followed by hydroxylation at C-26. The data are suggestive of both the induction of the steroid hydroxylases (oxidases) by substrate or other effector substances and the possible coordination of developmental events by ecdysteroids other than 20-hydroxyecdysone.In the haemolymph, two highly-polar conjugates of ecdysone were observed together with conjugates of the other free ecdysteroids, especially those hydroxylated at C-26. In contrast, relatively little 20-hydroxycdysone conjugate was detected in the insect. As adult development proceeded, both endogenous and radiolabelled ecdysteroids were increasingly localized in the gut, so that just prior to eclosion most ecdysteroids were present in the meconium of the high gut (rectal pouch). The peak titres and the kinetics of appearance of ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxyecdysone were similar for both haemolymph and gut (and for males and females), but considerably higher levels of C-26 oxidized (acid) metabolites of ecdysone and 20-hydroxyecdysone were localized in the gut. Although levels of highly-polar ecdysteroid conjugates found in the haemolymph and gut were similar, considerable amounts of three less polar ecdysone conjugates, of 3-α-epimers of ecdysone and 20-hydroxyecdysone, and of a substance tentatively identified as 2-deoxyecdysone were found only in the gut. Whether ionized, conjugated, or free, the gut ecdysteroids did not appear to equilibrate with the haemolymph compartment.Differences were observed in the metabolism kinetics of exogenously administered radiolabelled ecdysone when compared to the endogenous ecdysteroids; and some RIA positive gut metabolites did not become significantly radiolabelled. This suggests that injection of ecdysone may not simulate the endogenous secretion of ecdysone or its subsequent metabolism and distribution completely accurately.     

Ecdysteroids in Carausius eggs during embryonic development

Peaks of ecdysteroids were observed during the different phases of embryonic development of intact Carausius eggs or eggs precociously deprived of their exochorion and cultivated under paraffin oil. Several groups of ecdysteroids were separated and analyzed by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) combined with radioimmunoassay. Ecdysteroids were similar in the two categories of eggs, including high-polarity products (essentially conjugates hydrolyzable by Helix pomatia digestive juice, or alkaline phosphatase), possible ecdysonoic acids (unhydrolyzable polar substances), free hormones, and nonpolar ecdysteroids. Four ecdysteroids were identified by co-elution during HPLC with reference compounds of 20,26-dihydroxyecdysone, 20-hydroxyecdysone, ecdysone, and 2-deoxy-20-hydroxyecdysone. Concentrations of these substances (free and conjugated forms) were studied during the different stages of embryonic development: 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone were the major free ecdysteroids. They showed parallel variations with large peaks at stages VI₈ and VII₆ whereas ecdysone titers were consistently low. Injected labelled ecdysone was converted efficiently into 20-hydroxyecdysone, and both compounds underwent 26-hydroxylation and/or conjugation to polar or apolar metabolites.     

Profile of free and conjugated ecdysteroids and ecdysteroid acids during embryonic development of Manduca sexta (L.) following maternal incorporation of [14C]cholesterol

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 μ/g of the 2- and 26-phosphates of 26-hydroxyecdysone, respectively, and 1 μg/g of 26-hydroxyecdysone. During embryogenesis of 26-hydroxyedcdysone 26-phosphate is hydrolyzed to 26-hydroxyecdysone, which reaches a peak titer in 1–18-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 48–64-h-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 h-old. C-3 epimerase activity also appears at 24–40 h and continually increases throughout embryogenesis to the point that 3-epi-26-hydroxyecdysone 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 72–80-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 48–64-h-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 is 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 0–1-h-old larvae. Our results indicate that there is little or no biosynthesis of ecdysteroids during embryogenesis; that the materal ecdysteroid conjugate 26-hydroxyecdysone 26-phosphate serves as source for 26-hydroxyecdysone and the numerous metabolites; that 26-hydroxyecdysone and 20,26-dihydroxyecdysone may be the active hormones during embryonic development; and that glucosylation, epimerization, and formation of acids cosntitute inactivation processes. A scheme of the proposed pathways involved in the metabolism of 26–hydroxyecdysone 26-phosphate in the developing eggs of m. sexta is presented.     

Ecdysteroids in Developing ovarian follicles of Hyalophora cecropia

Developing ovarian follicles of the silkmoth Hyalophora cecropia accumulate large amounts of ecdysteroids during oogenesis. As measured by an ecdysteroid radioimmunoassay (RIA), this accumulation begins near the end of vitellogenesis, just prior to nurse cell collapse, and continues through the beginning of chorion formation. Analysis of ovarian ecdysteroids by a combination of high-performance liquid chromatography and RIA demonstrates that the major proportion of these are present in a highly polar form, most likely as conjugates; ecdysone and 20-hydroxyecdysone were present as well, in much lower proportions. Light microscopic autoradiographs of photoactivated follicles after in vivo incubation with [³H]ecdysone indicate that within the oocyte ecdysteroids are associated with the yolk sphere membranes.     

Ecdysteroids during oögenesis in the ovoviviparous cockroach Nauphoeta cinerea

By using thin-layer chromatography and high-pressure liquid chromatography combined with radioimmunoassay as well as gas chromatography-mass spectrometry we have identified and quantified ecdysteroids in ovaries and haemolymph of adult female Nauphoeta cinerea. Our analyses demonstrate the presence of ecdysone and 20-hydroxyecdysone, the latter being clearly predominant in all stages investigated. Titre determinations of free ecdysteroids in ovaries show that the 20-hydroxyecdysone concentration is highest (approximately 400 ng/g) at the beginning of chorion formation, suggesting an involvement in this process. Towards ovulation, the titre of free ecdysteroid drops and is low in the newly ovulated egg case. Measurement of immunoreactive highly polar products demonstrates that their concentration remains on a low level throughout the oöcyte maturation period; hydrolysis experiments with Helix pomatia enzymes reveal that, compared to the free ecdysteroids in the ovary, only small quantities of ecdysteroids are present as Helix hydrolysable conjugates. If one compares the quantities of free ecdysteroids in the ovary with those in the haemolymph it becomes apparent that the concentration in the haemolymph is about 10 times lower than that in the ovary.In vitro incubation of follicle cells from oöcytes at stages around chorion formation reveals that these cells are able to produce ecdysone and 20-hydroxyecdysone, and incubation with [³H]-ecdysone demonstrates that ecdysone is efficiently converted to 20-hydroxyecdysone in a stage-dependent manner. These observations strongly suggest that the follicle cells are the site of ecdysteroid biosynthesis and of C-20-ecdysone hydroxylation.A comparison of these findings with observations made of other insects such as locusts and mosquitoes demonstrates significant differences in quality, composition, titre fluctuation and distribution of ecdysteroids in adult females from different species and suggests that these ecdysteroids might fulfil multiple and various biological functions.     

Metabolism of [14C]cholesterol in Manduca sexta pupae: Isolation and identification of sterol sulfates,free ecdysteroids,and ecdysteroid acids

[¹⁴C]Cholesterol was injected into fifth-instar larvae of Manduca sexta, and the metabolites were isolated and identified from 8-day-old male and female pupae. A major portion of the metabolized cholesterol was esterified either with a sulfate group or with fatty acids. The predominant ecdysteroid metabolites were 20-hydroxyecdysone, 20,26-dihydroxyecdysone, 20-hydroxyecdysonoic acid, and 3-epi-20-hydroxyecdysonoic acid. Smaller amounts of ecdysteroids were identified as conjugates of 26-hydroxyecdysone, 3-epi-20-hydroxyecdysone, 20,26-dihydroxyecdysone, and its 3α-epimer. The metabolic profiles were similar for both male and female pupae. The two ecdysteroid acids were identified by nuclear magnetic resonance spectroscopy and chemical ionization mass spectrometry and by mass spectral analyses of their methyl esters. Detection of 3-epi-20-hydroxyecdysonoic acid as a major metabolite is significant, as its occurrence has been scarcely reported. 3-Epiecdysteroid acid formation is discussed as a possible ecdysteroid-inactivating pathway that may be operating specifically in lepidopterous insects or in particular developmental stages such as eggs or pupae.     

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.     

Haemolymph ecdysteroids in the solitary and gregarious larvae of Schistocerca gregaria

In the penultimate and last instar larvae of Schistocerca gregaria, 20-hydroxyecdysone (20E) makes up 74–84% of detected ecdysteroids in the females, and 63–74% in the males. Remaining ecdysteroids include ecdysone, a compound with HPLC and TLC retention times of makisterone A, and highly polar metabolites. Except for the last instar females, the contents of ecdysone and the unknown compound are higher in the solitary phase, while that of polar metabolites is higher in the gregarious phase. The phases also differ in that the molt-inducing ecdysteroid peaks last longer in the gregarious than in the solitary larvae. Peak concentrations reach 3.0–4.0 μg 20E equiv./ml in penultimate female instar, 2.5–3.0 μg/ml in penultimate male instar, and 1.5–2.0 μg/ml in the last larval instar of both sexes. © 1996 Wiley-Liss, Inc.