Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.     

Role of ecdysone 20-monooxygenase in regulation of 20-hydroxyecdysone levels by juvenile hormone and biogenic amines in Drosophila

The effects of increased levels of dopamine (feeding flies with dopamine precursor, l-dihydroxyphenylalanine) and octopamine (feeding flies with octopamine) on ecdysone 20-monooxygenase activity in young (2 days old) wild type females (the strain wt) of Drosophila virilis have been studied. l-dihydroxyphenylalanine and octopamine feeding increases ecdysone 20-monooxygenase activity by a factor of 1.6 and 1.7, respectively. Ecdysone 20-monooxygenase activity in the young (1 day old) octopamineless females of the strain Tβh ^nM18 , in females of the strain P845 (precursor of Tβh ^nM18 strain) and in wild type females (Canton S) of Drosophila melanogaster have been measured. The absence of octopamine leads to a considerable decrease in the enzyme activity. We have also studied the effects of juvenile hormone application on ecdysone 20-monooxygenase activity in 2-day-old wt females of D. virilis and demonstrated that an increase in juvenile hormone titre leads to an increase in the enzyme activity. We discuss the supposition that ecdysone 20-monooxygenase occupies a key position in the regulation of 20-hydroxyecdysone titre under the conditions that lead to changes in juvenile hormone titre and biogenic amine levels.     

Plasticity of the ecdysone receptor DNA binding domain

Ecdysteroids coordinate molting and metamorphosis in insects via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and the ultraspiracle (Usp) protein. Here we show how the DNA-recognition alpha-helix and the T box region of the EcR DNA-binding domain (EcRDBD) contribute to the specific interaction with the natural response element and to the stabilization of the EcRDBD molecule. The data indicate a remarkable mutational tolerance with respect to the DNA-binding function of the EcRDBD. This is particularly manifested in the heterocomplexes formed between the EcRDBD mutants and the wild-type Usp DNA-binding domain (UspDBD). Circular dichroism (CD) spectra and protein unfolding experiments indicate that, in contrast to the UspDBD, the EcRDBD is characterized by a lower alpha-helix content and a lower stability. As such, the EcRDBD appears to be an intrinsically unstructured protein-like molecule with a high degree of intramolecular plasticity. Because recently published crystal structures indicate that the ligand binding domain of the EcR is also characterized by the extreme adaptability, we suggest that plasticity of the EcR domains may be a key factor that allows a single EcR molecule to mediate diverse biological effects.     

Identification of 20-hydroxyecdysone and ecdysone from the pupa of the gypsy moth,Lymantria dispar

Normal and reverse-phase high-performance liquid chromatography in conjunction with radioimmunoassay and mass spectrometry were used to identify the free and conjugated ecdysteroids (after enzymatic hydrolysis) from day-4 pupae of the gypsy moth, Lymantria dispar L. Seven ecdysteroids were searched for, but only 20-hydroxyecdysone (964 ng/g fresh weight) and ecdysone (367 ng/g fresh weight) were detected. Analysis of conjugated ecdysteriods after liberation by hydrolysis also indicated the presence of 20-hydroxyecdysone (21.6 ng/g fresh weight) and ecdysone (2.4 ng/g fresh weight). Neither 26-hydroxyecdysone nor the 3α-epimers of 20-hydroxyecdysone or ecdysone were detected.     

An ecdysone receptor from the pentatomomorphan, Nezara viridula, shows similar affinities for moulting hormones makisterone A and 20-hydroxyecdysone

It has been suggested that Pentatomomorpha utilise the C₂₈ ecdysteroid, makisterone A (MakA), as the major moulting hormone rather than the more common C₂₇ hormone, 20-hydroxyecdsyone (20E). The present study is the first to examine this postulate at the level of the ecdysone receptor protein, a heterodimer of nuclear receptors EcR and USP. cDNAs encoding two alternatively spliced isoforms of EcR and a single USP were isolated from a high-quality cDNA library prepared from a representative pentatomomorphan, Nezara viridula (Nv). NvEcR and NvUSP were found to group phylogenetically with heteropteran and other insect EcRs and USP/RXRs, respectively. Sequence comparison and phylogenetic analysis of these proteins found them to be distinct from those belonging to other hemipteran ecdysone receptors characterised to date. Co-expression of the His₆-tagged ligand binding regions (LBRs) of the two NvEcR variants with the FLAG-tagged LBR of NvUSP was achieved in insect cells employing appropriately constructed baculoviruses. The corresponding heterodimers, designated NvE10 and NvE11, were purified by affinity chromatography utilising the His₆ tags on their NvEcR subunits. The heterodimers displayed nanomolar affinity for [³H]ponasterone A (K_d = 6.8-7.5 nM), characteristic of ecdysone receptors. MakA has a similar affinity to 20E for both NvE10 and NvE11, consistent with MakA being a major moulting hormone in N. viridula.     

The role of 20-hydroxyecdysone in housefly sex pheromone biosynthesis

Houseflies ovariectomized within 12 h after emergence do not produce (Z)-9-tricosene nor demonstrate the shift from alkene to alkane synthesis that is typcal of flies with developing ovaries. A single injection of 20-hydroxyecdysone at doses of 0.1 to 10 μg will induce the pattern in ovariectomized insects that is characteristic of flies with ovaries. Furthermore, this pattern persists for 3 days, but by 6 days after hormone injection, the synthesis of (Z)-9-tricosene stops and more alkenes are produced than alkanes. A post-hormone treatment time of 16 h was required before detectable amounts of (Z)-9-tricosene appeared on ovariectomized flies. Multiple injections of 20-hydroxyecdysone at doses of 50 ng into ovariectomized flies induced (Z)-9-tricosene synthesis and a shift in alkene to alkane synthesis. Thus, 20-hydroxyecdysone was able to act as an ovarian substitute in ovariectomized flies by stimulating pheromone synthesis.     

Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria.

The four major ecdysteroid (insect moulting hormone) conjugates present in the newly laid eggs of the desert locust, Schistocera gregaria, have been purified by reversed-phase and anion-exchange high-performance liquid chromatography. The steroid moieties were identified as ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Phosphate analysis of acid-hydrolysed samples showed a steroid:phosphate ratio of approx. 1:1 for all four compounds. The intact conjugates were identified as ecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate and 2-deoxy-20-hydroxyecdysone 22-phosphate by fast atom bombardment mass spectrometry and 1H, 13C and 31P n.m.r. The significance of ecdysteroid phosphates as a source of free hormone during embryogenesis is discussed.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone during pupal—adult metamorphosis of Mamestra configurata wlk

Large peaks of ecdysone (E, 2,875 ng/g live wt) and 20-hydroxyecdysone (20-HE, 2,150 ng/g live wt) occur on days 8 and 12, respectively, of postdiapause pupal-adult metamorphosis at 20°C in the bertha armyworm, Mamestra configurata, and then decline to low levels (< 100 ng/g live wt) prior to eclosion of the moth (50% eclosion at day 31.8). These peaks of E and 20-HE can be suppressed by treating the developing pupae with a physiological dose (2,500 ng/g live wt) of 20-HE. Suppression of E and 20-HE by 20-HE treatment was dose dependent, rapid (within 24 h of treatment) and permanent. The peaks of E and 20-HE were suppressed by 20-HE treatment on days 1, 3, 5, and 7 but the 20-HE peak was not suppressed by treatment on days 9 or 11. It is proposed that the mechanism by which 20-HE suppresses the production of E and thereby its own production forms a negative feedback loop that operates during the first 0.4 units of pupal-adult development in M. configurata. The function of the transitory peaks of E and 20-HE that form this feedback loop is currently unknown. Since most adults from pupae that had their ecdysteroid levels experimentally suppressed by 20-HE treatment were morphologically normal, it seems that the peaks of E and 20-HE have little or no function in controlling morphological development in pupae of M. configurata.     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

Protective role of 20-hydroxyecdysone against lead stress in Chlorella vulgaris cultures

Treatment of cultured C. vulgaris cells with 10(-6)-10(-4) M lead decreased their growth and chemical composition during the first 48 h of cultivation. However, at concentrations above 10(-4) M, lead is cytotoxic to Chlorella vulgaris cells, resulting in cellular fragmentation and lysis. In contrast, at concentrations below 10(-6) M lead had no influence on the growth and metabolism of C. vulgaris cells. 20-Hydroxyecdysone (20E) (10(-10)-10(-8) M) increased growth and chemical composition of C. vulgaris cells over a concentration range. Levels per cell of chlorophylls, protein, sugars are all increased by 20E treatment, when compared to non-treated control cells. However, the cultures treated with 20E and lead show a lower stimulation than the cultures treated with 20E alone. The effects of 20E mixed with lead on the growth and the level of cellular lead, chlorophyll, sugar and protein in C. vulgaris are also reported. The decreased growth and composition of C. vulgaris cells treated with lead was restored by the 20E. Application of 20E to C. vulgaris cultures reduced the impact of lead stress on growth, prevented chlorophyll, sugar and protein loss and increased phytochelatins synthesis. Furthermore, 20E did not restore toxic effect of lead on C. vulgaris cells. The combined treatment with lead and 20E appeared to have a stimulatory effect on the above parameters during the 48 h of cultivation, as compared to the control. 20E reduced the toxicity of lead and the growth recovered to the level of cells treated with 20E alone. Concentration-dependent stimulation was observed with increasing concentration of 20E and decreasing concentration of lead.     

The influence of forskolin on the metabolism of ecdysone and 20-hydroxyecdysone in isolated fat body of the blowfly, Calliphora vicina

Rates of ecdysone and 20-hydroxyecdysone metabolism were measured by radio-assay in an in vitro system containing fat body isolated from blowfly larvae. The addition of forskolin which is known to stimulate artificially the intracellular adenylate cyclase system led to decreased rates of conversion of ecdysone into 20-hydroxyecdysone (= hormone activation) and of 20-hydroxyecdysone further to other metabolites (= hormone inactivation). The effect of forskolin was dose-dependent and reversible. Extracts prepared from larval brains were also tested. Some of them had the same effect as forskolin. It is concluded that the reactions leading from ecdysone to 20-hydroxyecdysone and further to hormonally inactive ecdysteroids are modulated by the intracellular level of cyclic nucleotides. We propose that a neurohormone is involved in the control of the reactions of the ecdysone metabolism. The observed new principle may contribute to the control of the titer of moulting hormone.     

Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle

The Ecdysone receptor (EcR) is distributed between cytoplasm and nucleus in CHO cells. Nuclear localization is increased by the ligand Muristerone A. The most important heterodimerization partner Ultraspiracle (Usp) is localized predominantly in the nucleus. We used the diethylentriamine nitric oxide adduct DETA/NO, which releases NO and destroys the zinc-finger structure of nuclear receptors, to investigate whether nuclear EcR and Usp interact with DNA. If expressed separately, Usp and EcR in the absence of hormone do not interact with DNA. The hormone-induced increase in nuclear EcR is due to enhanced DNA binding. In the presence of Usp, EcR is shifted nearly quantitatively into the nucleus. Only a fraction (approximately 30%) of the heterodimer is sensitive to DETA/NO. Interaction of the heterodimer with DNA is mediated mainly by the C-domain of EcR. Deletion of the DNA-binding domain of Usp only slightly reduces nuclear localization of EcR/Usp, although the nuclear localization signal of Usp is not present anymore. The results indicate that EcR and Usp can enter the nucleus independently, but cotransport of both receptors mediated by dimerization via the ligand binding domains is possible even in the absence of hormone.     

Cultured mosquito cells Aedes albopictus C6/36 (Dip., Culicidae) responsive to 20-hydroxyecdysone and non-steroidal ecdysone agonist

Abstract: A cell line derived from an important mosquito, Aedes albopictus Skuse, was responsive to treatments with the insect moulting hormone 20-hydroxyecdysone (20E) and two new insecticides, RH-5849 and RH-5992, by cell clumping and inhibition of cell proliferation. The Aedes C6/36 cells showed typical numerous long extensions, and fluorescent BODIPY^®-phalloidin staining demonstrated that they resulted from F-actin polymerization. In addition, the ultrastructure of treated cells showed a high degree of vacuolarization that is in accord with protein synthesis. SDS-PAGE demonstrated that three proteins were induced that may belong to the hsp70 family. In conclusion, an insect cell line that responds to 20E may be useful for screening and identifying hormone analogues and novel insecticides.     

Possible role for covalent modification in the reversible activation of ecdysone 20-monooxygenase activity

Evidence is presented for the reversible activation-inactivation of the microsomal ecdysone 20-monooxygenase from fat body of the cotton leafworm, Spodoptera littoralis, in a manner commensurate with reversible changes in its phosphorylation state. The activity of the monooxygenase was higher following preincubation with fluoride (an inhibitor of phosphoprotein phosphatases) than in its absence. Preincubation with alkaline phosphatase or with cAMP-dependent protein kinase resulted in appreciable diminution or enhancement, respectively, in monooxygenase activity. Activation of ecdysone 20-monooxygenase activity could also be effected by incubation with a cytosolic fraction in the presence of cAMP, ATP, and fluoride; this activation was prevented by a cAMP-dependent protein kinase inhibitor. Similarly, inactivation of the monooxygenase was achieved by preincubation with cytosol, the effect being enhanced by Ca²⁺-calmodulin or by Mg²⁺ ions. The combined results provide indirect evidence that the microsomal ecdysone 20-monooxygenase exists in an active phosphorylated form and an inactive dephosphorylated form, interconvertible by a cAMP-dependent protein kinase and a phosphoprotein phosphatase.     

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     

Response of cultured Aedes aegypti fat bodies to 20-hydroxyecdysone

Fat bodies from non-blood-fed Aedes aegypti, stimulated in vitro by 10⁻⁴ M and 10⁻⁶ M of 20-hydroxyecdysone, were found to synthesize and release vitellogenin into the culture medium. Vitellogenin-specific monoclonal antibodies were utilized in an enzyme-linked immunosorbent assay procedure for quantification of vitellogenin in small aliquots of medium taken periodically from the culture. A minimal exposure of 5 h to 20-hydroxyecdysone was shown to be needed before the fat bodies would respond. Time-course of vitellogenin production in vitro was found to be identical to that observed in vivo. Vitellogenin-titre profiles were also investigated in cultured fat bodies from blood-fed A. aegypti. In all cases, response patterns were not affected by the presence or absence of 20-hydroxyecdysone after the fat bodies had been stimulated by blood meal to produce vitellogenin. We suggest here that initiation and control of vitellogenin synthesis is a programmed response to 20-hydroxyecdysone.     

Highly flexible ligand binding pocket of ecdysone receptor: a single amino acid change leads to discrimination between two groups of nonsteroidal ecdysone agonists

The insect steroid hormone 20-hydroxyecdysone works through a ligand-activated nuclear receptor, the ecdysone receptor (EcR), which plays critical roles in insect development and reproduction. The EcR has been exploited to develop insecticides to control pests and gene switches for gene regulation. Recently reported crystal structures of the EcR protein show different but partially overlapping binding cavities for ecdysteroid (ECD) and diacylhydrazine (DAH) ligands, providing an explanation for the differential activity of DAH ligands in insects. 1-Aroyl-4-(arylamino)-1,2,3,4-tetrahydroquinoline (THQ) ligands were recently discovered as ecdysone agonists. Mutagenesis of the EcR (from Choristoneura fumiferana, CfEcR) ligand binding domain followed by screening in a reporter assay led to the identification of CfEcR mutants, which responded well to THQ ligands but poorly to both ECD and DAH ligands. These mutants were further improved by introducing a second mutation, A110P, which was previously reported to cause ECD insensitivity. Testing of these V128F/A110P and V128Y/A110P mutants in a C57BL/6 mouse model coactivator interaction assay and in insect cells showed that this mutant EcR is activated by THQ ligands but not by ECD or DAH ligands. The CfEcR and its V128F/A110P mutant were used to demonstrate simultaneous regulation of two reporter genes using THQ and DAH ligands.