Ecdysone Concentration Research Articles

LC/MS/MS identification of 20-hydroxyecdysone in a scorpion ( Liocheles australasiae) and its binding affinity to in vitro-translated molting hormone receptors

Recent advances in mass spectrometry (MS) technology have facilitated the detection and quantification of minor components in organisms and the environment. In this study, we successfully identified 20-hydroxyecdysone (20E) in first instar nymphs (7 days after hatching) of the scorpion Liocheles australasiae, using tandem mass spectrometry combined with high-performance liquid chromatography (LC/MS/MS). This substance was not found in adults after the fifth stage. Other possible molting hormone candidates such as makisterone A (MaA) and ponasterone A (PoA), both of which are reported to be the molting hormones of a few arthropod species, were not detected in this scorpion. The ligand–receptor binding of 20E and its analogs was quantitatively evaluated against the in vitro-translated molting hormone receptor, the heterodimer of ecdysone receptor (EcR) and the retinoid X receptor (RXR) of L. australasiae (LaEcR/LaRXR). The concentrations of ecdysone (E), MaA, 20E, and PoA that are required to inhibit 50% of [ 3H]PoA binding to the LaEcR/LaRXR complex were determined to be 1.9, 0.69, 0.05, and 0.017 μM, respectively. The activity profiles of these 4 ecdysteroids are consistent with those obtained for the molting hormone receptors of several insects. The binding of a non-steroidal E agonist, tebufenozide, to EcR was not observed even at high concentrations, indicating that the structure of the ligand-binding pocket of LaEcR is not favorable for interaction with tebufenozide.

UV Protectants for the Biopesticide Based on Photostabilization of Ecdysone

The ultraviolet protectant (UV protectant) properties of different natural and synthetic compounds were investigated for a biopesticide based on ecdysone. This study examined the photostabilization of ecdysone when exposed to ultraviolet light in the presence of some ultraviolet protectants. Ecdysone solutions with and without UV protectants in methanol were applied onto the surface of glass slides. At particular intervals, the remaining concentration of ecdysone was analyzed by HPLC. Using first-order kinetic equation, the dissipation half-life values (DT50) for the degradation of ecdysone under ultraviolet radiation were obtained. The larvicidal activity was evaluated against the larvae of Martianus dermestoides Chevrolat. It indicated that the addition of congo red, yeast, starch and arabia gum provided moderate degree of photostabilization of ecdysone and that addition of lignin provided the best photostabilization of ecdysone, among these UV protectants studied. Toxicity of the ecdysone with UV protectants was higher to the larvae of M. dermestoides Chevrolat compared to the ecdysone alone as indicated by the lower EC50 value. The dissipation half-life values of ecdysone after irradiation under ultraviolet light and the larvicidal activity suggested that the addition of lignin (in 1:l mol ratio) can provide better photostabilization of ecdysone molecule.

A microarray analysis of genes involved in relating egg production to nutritional intake in Drosophila melanogaster

Egg chambers of Drosophila are reabsorbed under conditions of nutritional shortage by inducing apoptosis at stages 8 and 9, midway through oogenesis. Nutritional shortage leads to an increase in ecdysone concentration in flies. Apoptosis at stage 8/9 is also induced by 20-hydroxyecdysone injection into the females maintained with adequate nutrition. The expression pattern in the ovary of some ecdysone response genes, E75A, BR-C, is different according to the nutritional environment and the overexpression of these genes induces apoptosis. Apoptosis is suppressed by Juvenile hormone analog treatment of females under nutritional shortage. We predict nutritional and stress response genes control hormone levels and the increase in ecdysone concentration in the flies following starvation induces the ovarian apoptosis. We therefore used a microarray approach to identify the genes involved in receiving the nutritional signal from the environment and translating it in the ovary, thus initiating and executing apoptosis.

Identification of the molting hormone of the sweet potato ( Bemisia tabaci) and greenhouse ( Trialeurodes vaporariorum) whitefly

In order to identify the whitefly molting hormone, whole body extracts of mature 4th instar and newly formed pharate adult Bemisia tabaci (Biotype B) and Trialeurodes vaporariorum were prepared and subjected to reverse phase high performance liquid chromatography (RPHPLC). Ecdysteroid content of fractions was determined by enzymeimmunoassay (EIA). The only detectable ecdysteroids that were present in significant amounts in whitefly extracts were ecdysone and 20-hydroxyecdysone. The concentrations of 20-hydroxyecdysone in B. tabaci and T. vaporariorum extracts, respectively, were 40 and 15 times greater than the concentrations of ecdysone. The identity of the two ecdysteroids was confirmed by normal phase high performance liquid chromatography (NPHPLC). When ecdysteroid content of RPHPLC fractions was assayed by radioimmunoassay (RIA), small amounts of polar ecdysteroids were also detected indicating that these ecdysteroids have a very low affinity for the antiserum used in the EIA. Ecdysteroid at 10.4 mM administered by feeding stimulated 2nd instar whitefly nymphs to molt. Based on our results, it appears that 20-hydroxyecdysone is the whitefly molting hormone.

Effect of ecdysone application on the growth and biochemical changes in Chlorella vulgaris cells

This study was conducted on the influence of ecdysone upon the growth and the content of phosphorus, nucleic acids, protein, chlorophylls and sugar in the cell of alga Chlorella vulgaris Beijerinck ( Chlorophyceae). Ecdysone had the stimulatory effect on growth and metabolism of algae between the third and ninth day of cultivation after treatment in the range from 10 –15 to 10 –7 M. The highest growth of the analysed parameters was observed at the concentration of 10 –9 M. C. vulgaris cells treated with ecdysone demonstrate the inhibition of protein secretion. The protein secretion was intensively inhibited at the concentration of 10 –7 M, after the ninth day of cultivation. It was proportional to the concentration of ecdysone in the culture.

Ecdysone-regulation of synthesis and processing of fat body protein 1, the larval serum protein receptor of Drosophila melanogaster.

At the end of the third larval instar of Drosophila melanogaster, larval serum proteins 1 and 2 (LSP-1 and -2) are taken up by cells of the fat body. Here, we show that the product of the ecdysteroid-inducible gene Fbp-1 (Fat Body Protein 1) is the receptor that binds LSP-1. Transcription and translation of Fbp-1 is stage-specifically restricted to the end of the third larval instar, starting around 99 h after egg laying. Expression of Fbp-1 is induced by a low level of 20-hydroxy-ecdysone (>/= 10-7 m). After translation, the FBP-1 protein is thought to be proteolytically cleaved in three subsequent steps. The final cleavage step is delayed by 6 h and relies on a higher concentration of ecdysone (>/= 10-5 m). Therefore, 20-hydroxy-ecdysone regulates Fbp-1 expression and function at two different levels. To the best of our knowledge, this study is the first to date to demonstrate two distinct functions for different concentrations of a steroid hormone on a single biochemical process.

Regeneration ofSarcophagaImaginal Discsin Vitro:Implication of 20-Hydroxyecdysone

When the 3/4 sectors of leg imaginal discs ofSarcophagawere culturedin vitroin the presence of 2.5 × 10−8M20-hydroxyecdysone, wound healing and restoration of their morphology occurred. This concentration of ecdysone was critical for wound healing and was 40 times lower than that necessary for inducing differentiation of imaginal discsin vitro.Lost positional values revealed by expression of thewinglessgene were found to show partial recovery under these conditions. These results suggest that a low titer of ecdysone is essential for the regeneration of imaginal discs.

RELATED CHANGES IN HEMOLYMPH ACID-BASE STATUS, ELECTROLYTES, AND ECDYSONE IN INTERMOLT CRAYFISH (PROCAMBARUS CLARKII) AT 23°C DURING EXTRACELLULAR ACIDOSIS INDUCED BY EXPOSURE TO AIR, HYPEROXIA, OR ACID

ABSTRACT The relationships between hemolymph acidic-basic equivalents (pH, PCO2, and [HCO3, + C032-]), electrolytes (Na, K, Ca, Mg, and Cl), and ecdysone concentration were investigated in the crayfish Procambarus clarkii at 23°C during extracellular acidosis resulting from: (1) 24-h aerial exposure, (2) 96-h hyperoxic exposure (PO2 > 550 mm Hg), or (3) 96-h acid exposure (pH 4.0). A control series failed to reveal any significant effects of repetitive hemolymph sampling. In air, crayfish developed an initial (3-h) respiratory acidosis that was completely compensated within 24 h by metabolic base [HCO3 + CO32 ] accumulation. Circulating Ca and Cl both decreased at 24 h, while K increased. In this case, there was evidence that acid-base balance was corrected by ion exchange with the intracellular compartment. Hyperoxia was characterized by an initial (3-h) respiratory acidosis followed by a metabolic acidosis. The combined acidosis remained uncompensated, and circulating ecdysone decreased after 24 h. Acid exposure produced a purely metabolic acidosis that was partially corrected by a respiratory alkalosis between 48 and 72 h and was accompanied by an increase in circulating ecdysone. Ca and K decreased. Collectively, the experiments established a relationship between extracellular ecdysone and pH. Meanwhile, Ca remained relatively constant in all 3 treatments.

Ecdysone 20-hydroxylation inManduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20-monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The Km5 (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10−5 and 3.67 × 10−7 M, respectively. The Vmax was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher Vmax, at physiological ecdysone concentrations (10−7 − 10−8 M) it is only one-eighth to one-tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20-hydroxylation in M. sexta midgut. © 1996 Wiley-Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Occurrence and hormonal action of ecdysone on gametogenesis and energy utilization in the leech Nephelopsis obscura (Erpobdellidae)

AbstractThe present study provides novel information on the presence of compounds with ecdysone‐like activity and its involvement in the control of gonadal development in a leech. The presence of ecdysone was demonstrated in leech (Nephelopsis obscura) extract by means of high‐performance liquid chromatography (HPLC) in combination with radioimmunoassy. Leech extract was found to bind specifically to an anti‐ecdysone antiserum in a similar manner as standard ecdysone. Further HPLC purification demonstrated the presence of an immunoreactive fraction coeluting exactly with the standard ecdysone, strongly suggesting the presence of ecdysone in N. obscura. Addition of ecdysone to the ambient medium not only advanced leech gametogenesis but also influenced energy utilization. Both low (60 μg·L−1) and high (600 m̈g·L−1 concentrations of ecdysone increased testisac number, spermatogonia, 4th stage spermatogonia, ovisac size, and 2nd stage oogonia within 14 days compared to 35 days in the controls. Ecdysone also caused significant decreases in glycogen, total lipid, and triacylglycerols correlated with the energetic demands of gametogenesis. The findings provide support for the hypothesis that endogenous ecdysone is involved in the regulation of reproduction in N. obscura. © 1995 Wiley‐Liss, Inc.

A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone

Two successive pulses of ecdysone signal the ends of larval and prepupal development in Drosophila, inducing early and late puffs in the salivary gland polytene chromosomes. Early puff induction in prepupae is dependent on a preceding period of protein synthesis and low ecdysone concentration. We demonstrate here that the competence acquired during this interval can be provided by βFTZ-F1, a nuclear hormone receptor superfamily member derived from the 75CD midprepupal puff. We show that βFTZ-F1 represses its own transcription and is repressed by ecdysone, explaining its bried expression in mid-prepupae. We further show that ectopic βFTZ-F1 expression leads to enhanced levels of ecdysone-induced BR-C, E74, and E75 early gene transcription and premature induction of the stage-specific 93F early puff and E93 transcription. These findings indicate that βFTZ-F1 plays a central role in the prepupal genetic response to ecdysone and provide a molecular mechanism for stage-specific responses to steroid hormones.

Temporal coordination of regulatory gene expression by the steroid hormone ecdysone.

In Drosophila, pulses of the steroid hormone ecdysone function as temporal signals that trigger the major postembryonic developmental transitions. The best characterized of these pulses activates a series of puffs in the polytene chromosomes as it triggers metamorphosis. A small set of early puffs is induced as a primary response to the hormone. These puffs encode regulatory proteins that both repress their own expression and activate a large set of late secondary response genes. We have used Northern blot analysis of RNA isolated from staged animals and cultured organs to study the transcription of three primary response regulatory genes, E75, BR-C and EcR. Remarkably, their patterns of transcription in late larvae can be defined in terms of two responses to different ecdysone concentrations. The class I transcripts (E74B and EcR) are induced in mid-third instar larvae in response to the low, but increasing, titer of ecdysone. As the hormone concentration peaks in late third instar larvae, these transcripts are repressed and the class II RNAs (E74A, E75A and E75B) are induced. The BR-C RNAs appear to have both class I and class II characteristics. These data demonstrate that the relatively simple profile of a hormone pulse contains critical temporal information that is transduced into waves of primary response regulatory gene activity.

Hemolymph Ecdysone Concentration as a Function of Sexual Maturity in the Male Snow Crab (Chionoecetes opilio)

Ecdysone concentrations in the hemolymph of juvenile and morphometrically mature male snow crab (Chionoecetes opilio) were determined by radioimmunoassay. A logarithmic transformation of the allometric relationship of the dry weight of the chelae versus the carapace width was used to identify morphometric maturity. Results indicate that concentrations of ecdysone found in the hemolymph of juvenile crab are higher than those found in morphometrically mature crab, thus providing a biochemical basis for the observation that the onset of sexual maturity seems to coincide with a terminal molt.

Ecdysone coordinates the timing and amounts of E74A and E74B transcription in Drosophila.

Pulses of the steroid hormone ecdysone function as temporal signals to coordinate the development of both larval and adult tissues in Drosophila. Ecdysone acts by triggering a genetic regulatory hierarchy that can be visualized as puffs in the larval polytene chromosomes. In an effort to understand how the ecdysone signal is transduced to result in sequential gene activation, we are studying the transcriptional control of E74, an early gene that appears to play a regulatory role in the hierarchy. Northern blot analysis of RNA isolated from staged animals or cultured organs was used to characterize the effects of ecdysone on E74 transcription. Ecdysone directly activates both E74A and E74B promoters. E74B mRNA precedes that of E74A, each mRNA appearing with delay times that agree with their primary transcript lengths and our previous transcription elongation rate measurement of approximately 1.1 kb/min. The earlier appearance of E74B transcripts is enhanced by its activation at an approximately 25-fold lower ecdysone concentration than E74A. E74B is further distinguished from E74A by its repression at a significantly higher ecdysone concentration than that required for its induction, close to the concentration required for E74A activation. These regulatory properties lead to an ecdysone-induced switch in E74 expression, with an initial burst of E74B transcription followed by a burst of E74A transcription. We also show that the patterns of ecdysone-induced E74A and E74B transcription vary in four ecdysone target tissues. These studies provide a means to translate the profile of a hormone pulse into different amounts and times of regulatory gene expression that, in turn, could direct different developmental responses in a temporally and spatially regulated manner.

Ecdysteroids in the embryos and sera of the crabs, Cancer magister and C. anthonyi

Ecdysteroids in the embryos and sera of ovigerous brachyuran crabs, Cancer magister and C. anthonyi, were measured and characterized by radioimmunoassay (RIA) and high-performance liquid chromatography (HPLC). C. magister embryos displayed a biphasic pattern of ecdysteroid fluctuation during development; titers decreased until mid embryogenesis and then increased and peaked prior to hatching. HPLC-RIA analysis indicated increasing ecdysone concentrations from mid embryogenesis to hatching. Endogenous biosynthesis of ecdysone by the embryos is suggested. In contrast, ecdysteroid titers in the embryos of C. anthonyi showed a steady decrease from very high initial concentrations. The decrease in titers of ecdysone and 20-hydroxyecdysone is suggestive of utilization of maternally derived ecdysteroids rather than endogenous biosynthesis during the shorter embryogenic period for C. anthonyi. Ecdysteroid concentrations did not differ with respect to location of the embryo within the egg mass. Serum ecdysteroids in C. magister females generally showed a monotonic pattern during brooding. However, for C. anthonyi females, increasing and decreasing titers were observed during the brood and interbrood periods, respectively. These fluctuations suggest mobilization of the ecdysteroids to the ovaries for subsequent storage and utilization during embryogenesis. The evolutionary significance of these differing patterns of ecdysteroid metabolism in these congeners is discussed.

Ecdysteroids in Daphnia magna: their role in moulting and reproduction and their levels upon exposure to cadmium

The role of ecdysteroids in moulting and reproduction of Daphnia magna has been investigated. Further we examined whether previously observed moulting and reproduction disturbances in cadmium-exposed animals could be attributed to changes in ecdysteroid titres. Six concentrations of ecdysone and 20-hydroxyecdysone, ranging from 1 × 10 −8 M to 5 × 10 −6 M, were administered to daphnids exogenously. Treatment with high concentrations of both ecdysteroids resulted in unsuccessful exuviations and a decline in the number of progeny per female. The size of the neonates, however, appeared to be unaffected. 20-Hydroxyecdysone was demonstrated to be more effective than ecdysone. The presence of ecdysteroids in whole-body extracts of adult females was demonstrated using an enzyme-immunoassay. Additionally, ecdysteroid concentrations in daphnids were found to be affected by cadmium exposure at concentrations of 5.0, 10.0 and 20.0 μg/l. We concluded tentatively that changed ecdysteroid concentrations may have caused the moulting impairments in cadmium-exposed daphnids. On the other hand, there was no indication that the endocrine response of D. magna to cadmium exposure was also responsible for the cadmium-induced decline in neonate-size.

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.

Recherches sur les ecdysteroïdes présents dans les cocons de la sangsueHirudo medicinalisau cours de l'embryogenèse / Investigations on the ecdysteroids in the cocoons of the leechHirudo medicinalisduring embryogenesis

Summary In earlier studies, we demonstrated that leeches contain ecdysone and 20-hy- droxyecdysone. The titre of these molecules was found to fluctuate during the moult/intermoult cycle which is suggestive of a role of ecdysteroids in the control of cuticulogenesis in Hirudinea similar to that observed in Arthropods. We have now extended our investigations to embryonic development in the leech Hirudo medicinalis. During this period of development, which lasts some 30 days, 5–25 embryos grow inside a large cocoon at the expense of a proteolipidic gel (‘albumen’) synthesized by clitellian glandular cells of the parent leech. We have found that the albumen already contains ecdysteroids before the onset of embryogenesis (‘parental ecdysteroids’). At this time, and during the early stages of embryogenesis, several as yet unidentified low polar ecdysteroids predominate in the albumen; the titre of these molecules shows a dramatic decrease in the albumen at mid-embryogenesis which is concomitant with a marked rise in the concentration of free ecdysone and 20-hydroxyecdysone. In the embryos, this stage coincides with a remarkable transformation of the first structures (‘the cryptolarval metamorphosis’). At all stages investigated the albumen contains significant amounts of Helix hydrolysable conjugates. At least in early stages, the hydrolysis of these conjugates yields free ecdysone and a low polarity ecdysteroid. We suggest that in Hirudo the ecdysteroids synthesized before egg-laying by the adult leech play a role in the control of embryonic development and possibly also in the as yet not understood cycle(s) of embryonic cuticulogenesis.

Ecdysteroids in the Camel Tick, Hyalomma Dromedarii (Acari: Ixodidae), and Comparison with Sex Pheromone Activity1

The ecdysteroid 20-hydroxyecdysone (20-OH ecdysone) and 2 other radioimmunoassay (RIA) positive ecdysteroids were found in unfed and feeding adult Hyalomma dromedarii ticks. Increases in total ecdysteroids per tick occurred during female feeding, especially following mating and repletion. Ecdysteroid content in unfed and feeding males remained approximately the same (estimated by RIA) or declined (estimated by HPLC). Changes in concentration of 20-OH ecdysone resembled the changes in total ecdysteroid content in most cases. High-performance liquid chromatography (HPLC) gave substantially higher estimates of total ecdysteroid content in the various tick life stages or physiological states than did RIA. Increases in ecdysteroid concentrations during the first few days following female eclosion may indicate a regulatory role for stimulation of sex pheromone production. No apparent relationship was observed between ecdysteroid concentrations and sex pheromone secretion during feeding. The greatest increases in all ecdysteroids, including 20-OH ecdysone, occurred following mating and during repletion.

Ecdysteroids in the American dog tick, Dermacentor variabilis (Acari: Ixodidae), during different periods of tick development.

Ecdysteroids occur in all life stages of the American dog tick, Dermacentor variabilis . 20-hydroxyecdysone (20-OH ecdysone) and 2 other radioimmunoassay (RIA) positive fractions were found in engorged nymphs and adult ticks. 20-OH ecdysone and at least 1 RIA-positive fraction were found in the embryos and engorged larvae. No 20-OH ecdysone was found in the unfed larvae or unfed nymphs. Increases in total ecdysteroid content per tick occurred during ecdysial periods, adult feeding, and especially during female repletion and oviposition. In contrast, the total concentration declined gradually in fasting adults. In most cases, changes in the concentration of 20-OH ecdysone resembled the changes in total ecdysteroid content. With 1 exception, high-performance liquid chromatography (HPLC) gave substantially higher estimates of total ecdysteroid content in the various tick life stages or physiological states than did RIA. No relationship between the changes in the concentrations of ecdysteroids and sex pheromone (2,6-dichlorophenol) were observed. However, the foveal (pheromone) glands appeared to respond to exogenous 20-OH ecdysone; ultrastructural changes characteristic of actively secreting glands were found in unfed females following exposure to this hormone.