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.     

Allelochemical stimulation of ecdysone 20-monooxygenase in fall armyworm larvae

The influence of dietary allelochemical on ecdysone 20-monooxygenase activity was studied in the fall armyworm, Spodoptera frugiperda (J.E. Smith). Feeding the indoles (indole-3-carbinol, indole-3-acetonitrile), flavonoids (flavone, β-naphthoflavone), monoterpenes (menthol, menthone, peppermint oil), and a coumarin (xanthotoxin) to the larvae stimulated midgut microsomal ecdysone 20-monooxygenase activity from 28 to 200% as compared with the controls. β-Naphthoflavone was the most potent inducer among those tested. Phenobarbital, a well-known cytochrome P450 inducer, also caused a 2-fold increase in the microsomal ecdysone 20-monooxygenase activity. Ecdysone 20-monooxygenase activity was 2.7-fold higher in the microsomal fraction than in the mitochondrial fraction isolated from larval midguts. Microsomal ecdysone 20-monooxygenase activity was highest in the fat body, followed by the midgut and Malpighian tubules. Tissue localization and enzyme inducibility were different between ecdysone 20-monooxygenase and xenobiotic-metabolizing cytochrome P450 monooxygenases, including aldrin epoxidase, biphenyl hydroxylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, p-chloro-N-methylaniline N-demethylase, and phorate sulfoxidase in fall armyworm larvae. © 1995 Wiley-Liss, Inc.     

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.     

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.     

Endocrine background of how 20-hydroxyecdysone agonist, RH 5849, influences diurnal pattern of pupation in shape Spodoptera littoralis

Under laboratory conditions (L16:D8, 24°C, r.h. 80%), applying of an ecdysone agonist, RH 5849, (1,2-dibenzoyl-1-tert-butylhydrazine) onto final instar larvae of Spodoptera littoralis (Boisd.), Noctuidae, Lepidoptera, delayed or advanced the time of pupation depending on the time of treatment. This response was dose-dependent. In addition, non-lethal doses of RH 5849, ranging from 0.05 to 10 µg/larva were used. Following treatment of S. littoralis larvae with 5 µg RH 5849 the endogenous ecdysteroid level was increased or decreased depending on the time of application. Based on the results presented, we suggest that potential users of non-steroidal ecdysone agonists should consider the proper circadian timing of application in the field.     

Effects of the compounds 2-methoxynaphthoquinone, 2-propoxynaphthoquinone, and 2-isopropoxynaphthoquinone on ecdysone 20-monooxygenase activity

The effects of the natural compound 2-methoxy-1,4-naphthoquinone, isolated from the leaves of Impatiens glandulifera and the synthetic compounds 2-propoxy-1,4-naphthoquinone and 2-isopropoxy-1,4-naphthoquinone on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the three compounds. All three compounds were found to inhibit in a dose-dependent fashion the E-20-M activity in the three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 3 x 10(-5) to 7 x 10(-4) M.     

Ecdysteroid Stimulated Protein Synthesis in the Male Accessory Reproductive Glands of Spodoptera litura

Summary

20-hydroxyecdysone stimulates protein synthesis in the male accessory reproductive glands of pharate adults of Spodoptera litura both in vivo and in vitro. Juvenile hormone-I has no synergistic effect when given in conjunction with ecdysone in vivo but it inhibits ecdysone-stimulated protein synthesis in vitro.     

Temporal control of urate oxidase activity during development of the third-instar larva of Drosophila: The role of 20-hydroxyecdysone

The tissue-specific enzyme urate oxidase is confined exclusively to the Malpighian tubules of Drosophila melanogaster and expressed only in the third-instar larva and the adult. Shortly before pupariation urate oxidase activity declines precipitously and is not detectable 24 hours later. That 20-hydroxyecdysone is the factor that triggers the disappearance of urate oxidase activity in late third-instar larvae is demonstrated using the temperature sensitive mutant ecd¹ which at the nonpermissive temperature of 29°C fails to accumulate a sufficient concentration of 20-hydroxyecdysone necessary for puparium formation and thus remains a third-instar larva for 1 to 2 weeks before death. Both the life cycle and the temporal profile of urate oxidase activity in ecd¹ larvae at 19°C is identical to that of the wild type. However, at 29°C ecd¹ third-instar larvae retain high urate oxidase activity. A precipitous decline in urate oxidase activity is observed when ecd¹ larvae at 29°C are fed 20-hydroxyecdysone. These data implicate 20-hydroxyecdysone in the process that controls the rapid decline of urate oxidase activity at the time of puparium formation. In whole homogenates of Malpighian tubules, the urate oxidase polypeptide was identified in SDS-polyacrylamide gels by its Rf with respect to homogeneously pure Drosophila urate oxidase and also by immunoprecipitation with rabbit anti-Drosophila urate oxidase IgG. Throughout development the amount of the urate oxidase polypeptide is correlated with the magnitude of urate oxidase activity.     

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.     

The role of central aminergic neurons in the action of 20-hydroxyecdysone on neurosecretory cells of Rhodnius prolixus

The enhancement of electrical activity of the neurosecretory cells in the brain and corpus cardiacum of Rhodnius prolixus induced by 20-hydroxyecdysone has been used as a means of examining the role of aminergic neurons in this reflex. The response of the brain and corpus cardiacum from mated ovariectomized females to 20-hydroxyecdysone was blocked by phentolamine and phenoxybenzamine (α-aminergic receptor antagonists) but not by propranolol (a β-aminergic receptor antagonist). Preparations taken from ‘reserpinzed’ females failed to respond to 20-hydroxyecdysone. Dopamine at 10^?7 M was capable of mimicking 20-hydroxyecdysone in activating the neurosecretory system from mated ovariectomised females as well as from ‘reserpinized’ mated ovariectomised females. The response to dopamine was blocked by phentolamine. The neurosecretory system from virgin ovariectomized females failed to respond to 10^?7 or 10^?6 M dopamine, but was activated by 10^?5 M dopamine.It is concluded that the action of 20-hydroxyecdysone onto the neurosecretory cells is indirect and involves aminergic interneurons. The results also suggest that the mating stimuli may function by enhancing the response of neurons to amines.     

Regulation of cytochrome P-450 dependent steroid hydroxylase activity in Manduca sexta: effects of the ecdysone agonist RH 5849 on ecdysone 20-monooxygenase activity

The non-steroidal ecdysone agonist RH 5849 (1,2-dibenzoyl-1-tert-butylhydrazine) was found to inhibit in a dose-response and apparently competitive fashion the cytochrome P-450 dependent ecdysone 20-monooxygenase activity in the midgut of wandering stage last instar larvae of the tobacco hornworn, Manduca sexta. More effectively on a per molar basis than the naturally occurring molting hormones ecdysone and 20-hydroxyecdysone, RH 5849 was also found to elicit the dramatic 50-fold increase in midgut steroid hydroxylase activity (which normally occurs with the onset of the wandering stage) when injected into competent head or thoracic ligated pre-wandering last instar larvae. These data support and extend the potential usefulness of RH 5849 as a pharmacological probe for further investigating the actions of ecdysteroids and their role(s) in the regulation of ecdysteroid monooxygenases.     

On the intracellular localization of the ecdysone 20-monooxygenase in Musca domestica. L

The cytochrome P-450-dependent 20-monooxygenation of ecdysone is catalyzed both by mitochondria and microsomes isolated from Musca domestica (L.) larvae; however, about 50% of the activity is associated with mitochondria, and 37% is associated with microsomes. Pretreatment of larvae with ecdysone results in an increase in Vmax and a decrease in Km values in mitochondria but not in microsomes. Phenobarbital, a known cytochrome P-450 inducer, increases the cytochrome P-450 levels in microsomes without affecting the 20-monooxygenase activity, but both the cytochrome P-450 levels and monooxygenase activity are depressed in mitochondria from phenobarbital-pretreated larvae. The ecdysone 20-monooxygenase activity is equally distributed between mitochondria and microsomes in adult insects. Pretreatment of the insects with ecdysone does not significantly modify the 20-monooxygenase activity of either mitochondrial or microsomal fractions, but the cytochrome P-450 levels are reduced in mitochondria. Phenobarbital also depresses the mitochondrial cytochrome P-450 levels while markedly increasing the microsomal cytochrome P-450 levels. However, no significant changes in ecdysone 20-monooxygenase activity are produced by phenobarbital pretreatment. The effects of ecdysone on adult cytochrome P-450 are mostly evidenced in mitochondria isolated from females, whereas in males the changes are not statistically significant. It is concluded that the mitochondrial ecdysone 20-monooxygenase is under regulatory control by ecdysone in the larval stage, which suggests that only the mitochondrial activity has a physiological role during insect development in M. domestica. In adults, both the mitochondrial and microsomal ecdysone 20-monooxygenase activities are not responsive to ecdysone, which, coupled to their high Km values, indicates that the reaction may not be of physiological importance in adult insects and that the mitochondrial cytochrome P-450 species being depressed by ecdysone in females are possibly not involved in ecdysone metabolism.     

Occurrence of 28- and 27-Carbon ecdysteroids and sterols in developing worker pupae of the leaf-cutting ant acromyrmex octospinosus (reich) (hymenoptera,formicidae: Attini)

The major ecdysteroids in large worker pupae of the leaf-cutting ant Acromyrmex octospinosus were characterized at the peak ecdysteroid concentration by using high-performance liquid chromatography, enzyme immunoassay, and mass spectrometry. In decreasing amounts, they were determined to be makisterone A, an unidentified C₂₈ ecdysteroid bearing a molecular weight of 494, 20-hydroxyecdysone (ratio of 1 to 6 as compared to makisterone A), and putative but negligible ecdysone. The presence of both C₂₈ and C₂₇ ecdysteroids is discussed in relation to the content of 4-desmethylsterols determined by gas chromatography and mass spectrometry to be ergosta-5,7,24 (28)-trien-3β-ol, ergosterol, ergosta-5,7-dien-3β-ol and ergosta-7,24(28)-dien-3β-ol for the main sterols, and with a small amount of cholesterol.     

Protein kinase and protein phosphatase expression in the central nervous system of G93A mSOD over-expressing mice

The expressions of 78 protein kinases, 24 protein phosphatases and 31 phosphoproteins were investigated by Kinetworks trade mark analysis in brain and spinal cord tissue of transgenic mice over-expressing G93A mutant superoxide dismutase (mSOD), a murine model of amyotrophic lateral sclerosis (ALS). In the brains of affected mSOD mice, we observed increased expression of cAMP-dependent protein kinase (PKA, 111% increase compared with control), and protein phosphatase 2B Aalpha-catalytic subunit (calcineurin, 109% increase), and reductions in the levels of PAK3 (76% decrease) and protein phosphatase 2C Cbeta-subunit (32% decrease). Increased Ser259 phosphorylation of Raf1 (126% increase) in mSOD mice correlated with higher expression of p73 Raf1 (147% increase). There was also increased p73 Raf1 (69% increase) and Ser259 phosphorylation (45% increase) in the spinal cords of mSOD mice. While adducin underwent enhanced phosphorylation (alphaS724, 90% increase; gammaS662, 290% increase) in mSOD brain, its phosphorylation was lower in the mSOD spinal cord (alphaS724, 53% decrease; gammaS662, 46% decrease). In spinal cords of affected mSOD mice, we also observed elevated expression of casein kinase 1delta (CK1delta, 157% increase), JAK2 (84% increase), PKA (183% increase), protein kinase C (PKC) delta (123% increase), p124 PKC micro (142% increase), and RhoA kinase (221% increase), and enhanced phosphorylation of extracellular regulated kinases 1 (ERK1, T202/Y204, 90% increase), and 2 (ERK2, T185/Y187, 73% increase), p38 MAP kinase (T180/Y182, 1570% increase), and PKBalpha (T308, 154% increase; S473, 61% increase). There was also reduced phosphorylation of RB (S780, 45% decrease; S807/S811, 65% decrease), Src (Y418, 63% decrease) and p40 SAPK/JNKbeta (T183/Y185, 43% decrease). Variability in the expression of kinases, phosphatases and phosphorylation of their substrates was observed even in mutant animals having a similar phenotype. The expression and phosphorylation differences between mSOD and control mice were dissimilar to those between ALS patients and controls. This finding indicates that the activation of protein kinases and phosphoproteins is different with neuron loss in the mSOD mouse compared with that seen in patients with the sporadic form of ALS.     

Changes of acid phosphatase content and activity in the fat body and the hemolymph of the flesh fly Neobellieria (sarcophaga) bullata during metamorphosis

Changes in the specific and total activity of the lysosomal marker enzyme acid phosphatase (Acph) and in the amount of enzyme protein were examined in the fat body and the hemolymph from the last larval molt to the larval-pupal apolysis. The specific activity showed minor changes during the last larval period. In contrast, the total activity of the enzyme was low during the feeding period and higher during the wandering stage and strikingly increased at the time of puparium formation. We purified a protein having para-nitrophenyl phosphate phosphatase (Acph) activity and raised antisera against it. The amount of Acph protein in the fat body and hemolymph was examined using an ELISA. The specific Acph content showed little variation, but the total amount of the enzyme protein showed a stepwise increase in both organs during last larval stage and was markedly elevated in the pupal stage in the fat body. In contrast, a considerable decrease in the amount of Acph protein was observed in the hemolymph during this period. These data were in agreement with immunohistochemical observations showing an accumulation of the enzyme protein in fat body cells during the prepupal stage with a concomitant disappearance of the enzyme from the hemolymph. Inhibition of ecdysteroid secretion by water stress prevented the changes both in total enzyme activity and in the amount of Acph protein. However, Acph protein content and enzyme activity could be restored when the water stress was followed by a 20-hydroxyecdysone (20-HE) treatment. Taken together, our data show that Acph is secreted by fat body cells into the hemolymph during the larval stage, where it is stored in an inactive form. Increase in the 20-HE titer at the end of last larval stage reverses this process, and the enzyme is taken up by the fat body cells, where it becomes activated and appears in auto- and heterophagic vacuoles. Arch. Insect Biochem. Physiol. 34:369–390, 1997. © 1997 Wiley-Liss, Inc.     

CD109 antigen-like gene is induced by ecdysone signaling and involved in the cellular immunity of Helicoverpa armigera

ABSTRACT

Cellular immunity is evolutionarily conserved in invertebrates and vertebrates. In insects, cellular immune response is provided by the hemocytes, and its molecular mechanisms are currently not fully understood. Here, we identified a CD109 antigen-like gene (HaCD109) from Helicoverpa armigera which is highly expressed in the hemocytes of larvae. Stimulation by Escherichia coli and chromatography beads significantly upregulated HaCD109 expression. In vivo HaCD109 silencing significantly increased bacterial load in larval hemolymphs and reduced the hemocyte spread. 20-Hydroxyecdysone (20E) can induce HaCD109 expression through its receptors, EcR and USP. In vivo HaCD109 silencing nearly abolished 20E-induced bacterial clearance and hemocyte spread. These results suggested that HaCD109 plays an important role in cellular immunity, and the 20E-induced cellular immune response in H. armigera requires HaCD109 involvement. Our study contributes to the understanding of regulatory mechanisms for innate immune response and provides new insights into the interaction between innate immunity and steroid hormone signaling.     

In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say)

Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1-50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50 x treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.     

Differential regulation of the Cdk5-dependent phosphorylation sites of inhibitor-1 and DARPP-32 by depolarization

While cyclin-dependent kinase 5 (Cdk5) is of growing importance to neuronal signaling, its regulation remains relatively unexplored. Examination of the mechanism by which NMDA modulates the phosphorylation of protein phosphatase inhibitor-1 at Ser6 and Ser67 and dopamine- and cAMP-regulated phosphoprotein M _r 32 000 at Thr75 revealed that generalized depolarization, rather than specific activation of NMDA receptors, was sufficient to induce decreases in these Cdk5 sites. Although no evidence for the involvement of the Cdk5 cofactors p35 or p39, or for L- and T-type voltage-gated Ca²⁺ channels, was found, evaluation of the role of phosphatases and extracellular cations revealed differential regulation of the three sites. NMDA-induced decreases in the phosphorylation of Thr75 of dopamine- and cAMP-regulated phosphoprotein M _r 32 000 required protein phosphatase 1/2A activity and extracellular Ca²⁺. In contrast, the effects on Ser6 and Ser67 of inhibitor-1 were not cation specific; either Na⁺ or Ca²⁺ sufficed. Furthermore, while the decrease in phosphorylation of Ser6 was partially dependent on protein phosphatase 2B, that of Ser67 was independent of the major protein serine/threonine phosphatases, likely indicating the presence of a pathway by which NMDA inhibits Cdk5 activity. Thus, in the striatum the regulation of phosphorylation of Cdk5-dependent sites by NMDA occurs through multiple distinct pathways.     

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.