Amounts Of Ecdysteroids Research Articles

Comparative investigation of the egg ecdysteroids of ticks using radioimmunoassay and metabolic studies

Freshly laid eggs of ticks contain variable amounts of ecdysteroids, either under free form (as in Amblyomma hebraeum and A. variegatum) or the form of apolar conjugates (as in Ornithodoros moubata or Boophilus microplus) or a mixture of both free and conjugated forms ( Rhipicephalus appendiculatus, Hyalomma dromedarii). In a same species, there is also a high variability in the quality and quantity of ecdysteroids incorporated. We would like to discuss the meaning of this accumulation of these compounds in the eggs.

Prothoracic glands and ecdysteroid titre in Galleria mellonella larvae

Changes in the haemolymph ecdysteroid titre during the last larval instar of Galleria are parallelled by changes in the rate of ecdysteroid production by the prothoracic glands in vitro. Extirpation of more than 90% of the prothoracic glands has no significant effect on the ecdysteroid titre in the haemolymph. It was found that the cells remaining after extirpation hypertrophy and produce in vitro comparable amounts of ecdysteroids as the whole glands from intact insects. The production is several times higher than in the corresponding non-hypertrophied cells. The results confirm that prothoracic glands are an indispensable source of ecdysteroids for the induction of pupation in Lepidoptera.

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

Abstract[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.

Diflubenzuron‐Induced alterations during in vitro development of Tenebrio molitor pupal integument

AbstractThe effects of diflubenzuron (DFB) in Tenebrio molitor pupae were first investigated on cuticle secretion induced by 20‐hydroxyecdysone in vitro. The sternal integuments were treated by DFB either 3 days before culture or during culture. DFB, when applied before culture, did not prevent the molting hormone from inducing a new cuticle deposition by integument explants in vitro. However, this cuticle showed several architectural alterations and a thickness reduction. When applied during the culture in the presence of 20‐hydroxyecdysone, DFB at high dose (≥ 20 μg/ml) was able to inhibit cuticle secretion, but lower doses (⩽ 10 μg/ml) resulted in epicuticle deposition. These observations confirm in vivo studies showing antagonistic effects of DFB and ecdysteroids at the level of epidermal cells.In another series of experiments, the DFB effects were analyzed without addition of exogenous molting hormone in vitro. Because it had been observed in previous studies that pupal epidermal explants of Tenebrio secrete low but significant amounts of ecdysteroids in the culture medium, this in vitro secretion was measured by radioimmunoassay after DFB treatment. It was observed that DFB, when applied either before or during culture, significantly reduced the hormonal secretion in vitro. This reduction, observed at the level of epidermal cells, could be homologous with the diminution of the endogenous ecdysteroid peak previously described after in vivo DFB treatment in Tenebrio pupae.

Long‐term cholesterol labeling as a convenient means for measuring ecdysteroid production and catabolism in vivo: Application to the last larval instar of Pieris brassicae

AbstractIn vivo biosynthesis of ecdysteroids during the last larval instar of Pieris brassicae was investigated by administering [3H] cholesterol followed byhigh‐performance liquid chromatography analysis of the resulting [3H] ecdysteroids. The demonstration that the specific activity of the ecdysteroids synthesized at a given time is always identical with that of cholesterol indicates that the cholesterol pool is uniformly labeled, and this allows us to easily calculate the amounts of ecdysteroids produced by animals. The total amount of ecdysone produced throughout the last larval instar was measured as 1.17 nmol/insect. This quantity is more than three‐fold the maximal level of molting hormones (ecdysone +20‐hydroxyecdysone) reached during the instar (0.37 nmol/animal) because a high catabolic activity occurs at the beginning of the hormone production period. Larvae thus differ from pupae, where catabolism is minimal when ecdysone synthesis takes place, resulting in a more “economical” system.

Involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta, orthoptera)

Following their biosynthesis in the follicle cells of vitellogenic ovaries, large amounts of ecdysteroids pass into the oocytes where they accumulate and persist during ovulation and egg-laying. The present paper shows that free ecdysone is unevenly distributed in the oocytes exhibiting the highest concentrations in the region of the posterior pole where the final sequences of nuclear maturation, including germinal vesicle breakdown (GVBD), occur. A correlative study indicates that the concentrations of free ecdysone in this region are particularly high (10 to 20 μ M) during two periods of meiotic reinitiation observed in the oocytes: reinitiation I, leading from prophase I to metaphase I with GVBD; and reinitiation II, from metaphase I to the end of meiosis. In vitro incubations of oocytes in meiotic arrest (prophase I) in the presence of exogenous ecdysone demonstrate that complete reinitiation (including GVBD) can be triggered in a dose-dependent manner by this hormone.

Metabolism of ecdysone and 20-hydroxyecdysone in the camel tick, Hyalomma dromedarii (Acari: Ixodidae).

Camel ticks, Hyalomma dromedarii , utilized inoculated 14C cholesterol to synthesize apolar ecdysteroids, unknown polar ecdysteroids, and fractions coeluting with ecdysone and 20-hydroxyecdysone (20-OH ecdysone). Following inoculation into feeding virgin females, most 3H ecdysone was metabolized to nonimmunoreactive apolar compounds; lesser amounts were metabolized to 20-OH ecdysone, 20,26-dihydroxyecdysone, and possibly other unidentified ecdysteroids. Identification of these compounds was supported by coelution with authentic reference standards, coelution of radioactivity, radioimmunoassay, nuclear magnetic resonance, and infrared spectrometry of the purified fractions. Assay of the apolar ecdysteroids in extracts from ticks inoculated with 14C acetate demonstrated conjugation with carbon-containing compounds. Hydrolysis of the highly apolar ecdysteroids released 20-OH ecdysone, ecdysone, and 20,26-dihydroxyecdysone. Methylation of the hydrolysis products revealed long-chain fatty acids, especially palmitic, stearic, oleic, and linoleic acids, all confirmed by gas chromatography/mass spectrometry. The possible sites of metabolism of ecdysteroids in these ticks was investigated. Most of the ecdysteroid metabolites produced were found in the midgut (60.4%) and in the combined epidermis and fat-body tissues (31.1%). Small amounts of ecdysteroids were found in the previtellogenic ovaries of these virgin female ticks (1.5%). Ecdysone and 20-OH ecdysone were found in the hemolymph, but no apolar ecdysteroids were found in the circulatory fluids.

Ecdysteroid levels during the larval development of the spider crab Hyas araneus

Ecdysteroid levels were determined by radioimmunoassay during the larval development of the spider crab Hyas araneus L. In each of the three larval instars (zoea I, zoea II, megalopa) a minimum in ecdysteroid levels was found during early postmoult and a maximum during premoult. The amounts of ecdysteroids increased during development and reached maxima of 93, 137, and 165 pg ecdysone equivalents per larva in zoea I, zoea II, and megalopa, respectively. Ecdysteroid concentrations per unit of body dry weight or carbon increased during each moulting cycle, but the average values showed decreasing tendency from the first to the last instar. The main ecdysteroid in all larval instars and during the moulting cycle was 20-OH-ecdysone, and practically no ecdysone could be detected by HPLC-RIA.

Ecdysteroid titers and Y-organ activity during late anecdysis and proecdysis in the fiddler crab, Uca pugilator

The titer of ecdysone in hemolymph and the ratio of ecdysone to other radioimmunoassay(RIA)-active hemolymph ecdysteroids were compared to in vitro secretion of ecdysone in Y-organs removed from eyestalkless fiddler crabs at various times following eyestalk ablation. Using high-pressure liquid chromatography (HPLC) and RIA it was established that ecdysone, 20-hydroxyecdysone, and RIA-active metabolites are present in the hemolymph at the end of anecdysis and throughout proecdysis. There was little correlation between in vitro secretory activity and total ecdysone in circulation. Ratios of ecdysone and 20-hydroxyecdysone changed during proecdysis as did the percentage of total RIA activity attributable to both. Positive correlations were observed between in vitro Y-organ secretion rates and amounts of ecdysteroids extracted from hemolymph and added to incubation media.

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.

Ecdysteroids in Developing ovarian follicles of Hyalophora cecropia

AbstractDeveloping 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.

Occurrence of Ecdysteroids in Specific Body Organs of the Camel Tick, Hyalomma Dromedarii, and the American Dog Tick, Dermacentor Variabilis (Acari: Ixodidae), With Notes on their Synthesis from Cholesterol1

Evidence suggesting significant accumulations of ecdysteroids in the vicinity of the synganglion and associated nerve plexi of virgin feeding Dermacentor variabilis and Hyalomma dromedarii is described. Excision of the nerve plexi surrounding the synganglion resulted in a drastic drop in ecdysteroid titer. The presence of 20-hydroxyecdysone in the synganglia/nerve plexi of these ticks was demonstrated by high-performance liquid chromatography. Significant quantities, from 1.36–1.93mg/tick, were also found in the hemolymph. Other studies indicated biosynthesis of ecdysteroids, especially 20-hydroxyecdysone, from cholesterol. Large accumulations of 14C-labeled material were found in the central nervous system of H. dromedarii adults, especially in the cortical zone and, to a lesser extent, in the vicinity of the lateral nerve plexus, following inoculation of 14C cholesterol into nymphs. Whether these findings reflect synthesis of ecdysteroids in and/or near the synganglion or merely storage of steroids and sterols in these structures is unknown. Radioimmunoassay revealed ecdysteroid activity in all other tissues and organs examined, but in considerably smaller amounts than in the synganglion/nerve plexi and hemolymph. Relatively large amounts of ecdysteroid were found in the foveal glands, which may indicate hormonal regulation of these structures.

Ecdysteroids in females and eggs of the Ixodid tickAmblyomma hebraeum

Summary A mated Amblyomma hebraeum female will engorge on a host for about 8 days before detaching and beginning the maturation of its single egg batch which is laid during a period of about 30 days. The feeding period is characterized by an important synthesis of endocuticular material occurring before the rapid feeding phase. This latter phase, correlated with an enormous weight uptake, shows an increase of ecdysteroid levels measured in the whole animal by RIA. However, the hemolymphatic levels of ecdysteroids remain very low (12 pg 20-hydroxyecdysone equivalent (20-OH-E eq.) per μ1. Within 4 days after detachment, the salivary glands degenerate. Ecdysteroid levels in the whole animal continue to increase, reaching high values (about 500 ng 20-OH-E eq./tick) at the moment of oviposition which begins 10–14 days after dropping. During the same period, hemolymphatic ecdysteroid levels increase, rising to a peak (600 pg 20-OH-E eq./μ1) 1 day prior to the beginning of oviposition. Then, the levels decrease and stabilize around 250 pg 20-OH-E eq./μl during egg-laying. Freshly laid eggs contain large amounts of ecdysteroids (2744 pg 20-OH-E eq./mg). 20-Hydroxyecdysone and ecdysone have been found to be the major free ecdysteroids in hemolymph, ovaries and eggs (verified by the HPLC-RIA technique and GC-MF of silylated HPLC fractions). Helix juice (or esterase) labile ecdysteroid conjugates do not seem to be present to any noticeable extent in hemolymph, ovaries and eggs.

Ovarian ecdysteroids and their secretion in late-pharate adults of Galleria mellonella

Approximately two-thirds of the total amount of ecdysteroids in late—pharate adults of the wax moth, Galleria mellonella, were found in the ovaries and one-third in the ovariectomized body. Chemical analysis of these ecdysteroids by thin-layer and high-pressure liquid chromatography, coupled with an ecdysteroid radioimmunoassay, revealed the presence of 2-deoxyecdysone, ecdysone and 20-hydroxyecdysone, as well as high and low polarity unknowns. The predominant identifiable ecdysteroid in both the ovaries and ovariectomized body was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone, respectively. Incubation of late-pharate adult ovaries in culture medium revealed that they synthesize and secrete ecdysteroids in vitro. The in vitro distribution of ecdysteroids between ovaries and incubation medium was similar to that observed between ovaries and ovariectomized bodies in situ and the predominant identifiable moiety both retained and released by the ovaries in vitro was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone. Collectively, these results support the idea that the ecdysteroids synthesized by the ovaries of late-pharate adult Galleria are both stored and secreted and that the quantity of a specifically secreted ecdysteroid is precisely controlled. This apparent regulation of the distribution of ovarian ecdysteroids raises the possibility that the stored and secreted forms have distinct functions in the reproductive physiology of this insect.

Distribution and metabolism of ecdysteroids in the adult yellow mealworm beetle, Tenebrio molitor

By a combination of different analytical methods, ecdysone and highly polar metabolites (HPP) were shown to be the predominant ecdysteroids in adult Tenebrio molitor. A distinct difference in the amount of ecdysteroid between males and females was observed. Tritium-labelled ecdysone was rapidly metabolized. Several compounds could be separated by HPTLC. Most of the [ 3H]material recovered from the insect was found in the HPP fraction. Since most of the injected [ 3H]material was excreted in the faeces as ecdysone (approx 65%), and HPP (approx 30%), a turnover of ecdysone into other ecdysteroids and vice versa seems possible. A new two-dimensional TLC method consisting of an initial run in a narrow strip of reversed phase media and another run in a conventional silica gel layer demonstrated that the main components of the tritiated materials were ecdysone and HPP, and that 20-hydroxyecdysone and 22,25-dideoxyecdysone were virtually absent. In males and females two HPP classes could be differentiated HPP I(′I) and HPP II(′II) and in females a third class was also present, HPP ′III. HPP I(′I) was the only class that was hydrolysable by several enzyme preparations (approx 30% of HPP), and was presumed to consist of glucuronides, β-glucosides and sulphate conjugates and was composed of ecdysone and two compounds with a higher polarity (C A and C B).

Metabolism of injected ecdysone in female Sarcophaga bullata (Diptera)

1. 1. Radioactive ecdysone injected into mature females of Sarcophaga bullata was rapidly metabolized firstly to ecdysterone and later to highly polar products and to a series of not identified ecdysteroid compounds. 2. 2. The ovaries were the only tissue capable of storing considerable amounts of ecdysteroids and this in the form of conjugates. From the other tissues of the flies, most of the tritiated ecdysteroids were excreted with the faeces. Only in the abdomen without the ovaries, a low amount of ecdysteroids remained present. 3. 3. The highly polar products (HPP) of the abdomen without the ovaries were extracted, examined by HPLC and HPTLC and shown to consist of two fractions: HPP B and HPP C. 4. 4. Enzyme treatment of HPP revealed that apparently fraction B consisted of glucuronide, β-glucoside and sulfate conjugates of ecdysteroids, whereas fraction C could not be hydrolysed with enzymes.

Ecdysteroids are bound to vitellin in newly laid eggs of locusta

The follicle cells of vitellogenic ovaries of Locusts migratoria have been reported to synthesize impressive amounts of ecdysteroids which accumulate inside the oöcytes where they persist during egg-laying; these ecdysteroids are conjugated to more than 95%, and it is believed that their hydrolysis in the egg is the source of the peaks of free ecdysone observed in early embryonic development. The present paper shows that, in the eggs, the ecdysteroid conjugates are bound to a 520000-dalton macromolecule which shares several characteristics with the major yolk protein vitellin and is precipitated by an anti-vitellin antibody. The physiological relevance of the binding of ovarian ecdysteroid conjugates is discussed in respect to the transfer of maternal ecdysteroids to the embryo.

Ecdysteroids During Embryogenesis inLocusta migratoria

Synopsis. Ovaries of Locusta migratoria synthesize large amounts of ecdysteroids at the end of oocyte maturation. The predominant ecdysteroids in mature ovaries are conjugated 2-deoxyecdysone (100 fxM) and conjugated ecdysone (50 fxM) which outnumber the corresponding free compounds by 50-100 fold. These various ecdysteroids persist during ovulation and are recovered from newly-laid eggs. The conjugated maternal ecdysteroids are gradually metabolized as embryonic development proceeds; they have disappeared as such on day 6 after oviposition, that is after blastokinesis and shortly after dorsal closure. Concomitantly to this metabolism *of the maternal conjugated ecdysteroids, other ecdy? steroid conjugates appear in the eggs which have different chromatographic behaviors and some of which are conjugates of ecdysone metabolites formed by the embryo. The data available so far are compatible with the hypothesis that the maternal conjugates are hy? drolysed to free 2-deoxyecdysone and ecdysone by the embryo during early stages of development and subsequently conjugated to inactivation compounds. During the later stages of embryonic development however, a de novo synthesis of ecdysone is probable, the maternal conjugates having been metabolized during the earlier stages.

Ecdysteroids in the adult male blowflyCalliphora vicina

Male adult blowflies contain ecdysteroids. A considerable amount of ecdysteroid is concentrated in the testes and gives rise to a high local concentration. Analysis by high performance liquid chromatography and thin-layer chromatography indicate that ecdysterone is the most prominent ecdysteroid in male flies. The results can explain several hormone-dependant processes.

Ecdysone during ovarian development in Locusta migratoria

Considerable amounts of ecdysteroids are produced during each ovarian cycle in adult females of Locusta when vitellogenesis is almost completed. The hormonal molecules are synthesized at the end of the maturation of the terminal oöcytes during each cycle, at the time when vitellogenesis is almost completed. No synthesis takes place in the absence of ovarian development (allatectomy, ovariectomy), whereas extirpation of the prothoracic glands at the beginning of adult life does not affect ecdysteroid production. More than 95% of the total ecdysteroid content of female adults can be recovered from the ovaries. In vitro studies show that the ovaries produce ecdysteroids and convert labelled cholesterol into ecdysone. Microsurgical experiments indicate that this synthesis takes place in the follicle cells surrounding the oöcyte. The newly synthesized ecdysteroids do not enter massively into the blood, but pass into the oöplasm where they are progressively converted to polar compounds; as a result, at the end of each ovarian cycle, egg-laying corresponds to the disappearance of ecdysteroids from the female insects, the hormonal molecules can easily be recovered from the eggs. A gas chromatographic analysis coupled to mass spectrometry shows that the principal ecdysteroid synthesized by the adult females of Locusta is by far ecdysone. Ecdysterone, the paramount ecdysteroid of the larvae of Locusta, is not present in noticeable amounts in the female adult of this species.