Characterization and partial purification of the Drosophila Kc cell ecdysteroid receptor

The molting hormones of insects, the ecdysteroids, are steroids whose action is mediated by an intracellular receptor. The Kc cell line of Drosophila melanogaster possesses ecdysteroid receptors and exhibits characteristic, receptor-dependent morphological and biochemical responses to the application of ecdysteroids. This paper describes the interaction of muristerone A (2 beta, 3 beta, 5 beta, 11 alpha, 14 alpha(20R,22R)- heptahydroxycholest-7-en-6-one), a phytoecdysteroid, with the Kc cell ecdysteroid receptor. Muristerone A-receptor complexes are not as sensitive to dissociation in high salt buffers as other ecdysteroid-receptor complexes we have examined. This has enabled us to use [3H]muristerone A to follow the Kc cell ecdysteroid receptor during heparin-agarose, DNA-cellulose, and hydroxylapatite chromatography, as well as gel filtration and ion exchange high pressure liquid chromatography. The Drosophila Kc cell ecdysteroid receptor has a Stokes radius of 4.6 nm, a frictional coefficient of 1.4, and a molecular weight of 120,000. A procedure is presented that results in a 750-fold enrichment of the receptor.     

Magnetic DNA affinity purification of ecdysteroid receptor.

A new method for rapid purification to near homogeneity of the ecdysteroid receptor (EcdR) from Drosophila melanogaster nuclear extract is presented. In the first step of the purification procedure the EcdR molecules were radiolabelled with [3H]ponasterone A and the [3H]ponasterone A-EcdR complexes were chromatographed under very mild conditions on Fractogel EMD TMAE(s) ion-exchanger. A 23-fold purified receptor was obtained which can be stored in liquid N2 without loss of activity. The second step involved the use of a magnetic DNA affinity technique where the double stranded hsp 27 oligonucleotide containing EcdR binding sequence was biotin 5'-end labelled and bound to monodisperse superparamagnetic particles coated with streptavidin (Dynabeads M-280 Streptavidin) giving magnetic DNA affinity beads. The chromatographed EcdR-ponasterone A complexes were bound to the magnetic DNA affinity beads and by magnetic separation, wash and elution, a 29,000-fold enriched EcdR preparation was obtained within 1.5 h. This procedure can be applied for other EcdR sources with minor modifications.     

Pyridoxal phosphate inhibits the DNA-binding activity of the ecdysteroid receptor

The effect of pyridoxal 5'-phosphate on the binding of the ecdysteroid receptor from a nuclear extract of Drosophila melanogaster to DNA-cellulose was studied. The binding of hormone-receptor complexes to DNA-cellulose was completely blocked after a 30-min incubation with 3 mM pyridoxal 5'-phosphate at 0-4 degree C. The effect was specific for pyridoxal 5'-phosphate since related compounds (pyridoxal, pyridoxamine 5'-phosphate and pyridoxamine) were not effective or gave only 17% inhibition (pyridoxal). Under standard conditions, none of the compounds tested exerted a significant effect on the stability of [3H](20R,22R)-2 beta,3 beta, 14 alpha,20,22-pentahydroxy-5 beta-cholest-7-en-6-one ([3H]ponasterone A)-receptor complexes. The loss of DNA-binding activity caused by pyridoxal 5'-phosphate is accompanied by changes in the molecular properties of [3H]ponasterone-A-receptor complexes. A shift of [3H]ponasterone-A binding was observed from the 8.0-8.5 S to the 4.5-5.0 S region, when [3H]ponasterone-A-receptor complexes were exposed to pyridoxal 5'-phosphate during sucrose-gradient centrifugation. The inhibition of DNA-cellulose binding by pyridoxal 5'-phosphate can be reversed. Probably, pyridoxal 5'-phosphate forms a Schiff base with a critical lysine group of the ecdysteroid receptor, presumably at its DNA-binding site. The hormone-receptor complexes obtained after removal of pyridoxal 5'-phosphate had the same affinity for DNA-cellulose as 'native' complexes. DNA-cellulose-bound [3H]ponasterone-A complexes were efficiently eluted from DNA-cellulose with pyridoxal 5'-phosphate in 0.1 M KCl resulting in a 104-fold purification of the ecdysteroid receptor. The results reflect possible structural similarities between ecdysteroid and vertebrate steroid receptors.     

Identification and ecdysteroid antagonist activity of three oligostilbenes from the seeds of Carex pendula (Cyperaceae)

Methanolic extracts of seeds of several (Carex species were found to antagonise the action of 20-hydroxyecdysone in the Drosophila melanogaster microplate-based B(II) cell bioassay. Bioassay-guided HPLC analysis of seeds of Carex pendula (drooping sedge) provided one previously unknown tetrastilbene (cis-miyabenol A) and two known oligostilbenes (kobophenol B and cis-miyabenol C) as the biologically active compounds (EC50 values were 31, 37 and 19 microM, respectively, vs. 5 x 10(-8) M 20-hydroxyecdysone). The structures and relative stereochemistries of these compounds were deduced by comprehensive ID- and 2D-NMR experiments. These compounds are isolated from Carex pendula for the first time. In vitro experiments with dipteran and lepidopteran ecdysteroid receptor proteins demonstrate that the oligostilbenes are able to compete with radiolabelled ecdysteroid ([3H]ponasterone A) for occupancy of the ligand binding site. IC50/Ki values are similar to the EC50 values obtained in the B(II) bioassay.     

Biological activities of a specific ecdysteroid dimer and of selected monomeric structural analogues in the B(II) bioassay.

The biological activities of selected specific ecdysteroids obtained by photochemical or chemical transformation are compared in the B(II) bioassay, in which the potency reflects the affinity of binding to the ligand-binding site of the Drosophila melanogaster ecdysteroid receptor. The compounds tested represent 14-deoxy, 14-dehydroxy, 14-hydroperoxy and 14-epi derivatives of 20-hydroxyecdysone and were selected on the basis of their close structural relationship to elucidate the contribution of the 14-hydroxy group and the stereochemical configuration at C-14 to ecdysteroid agonist activity. The structure-activity relationship shows that a 14-hydroxy group is not required for activity. However, the alpha-configuration of -H, -OH or -OOH at C-14, which determines the C/D rings trans-annelation, is very significant for activity; it is as important for activity as the well studied A/B rings cis-annelation. Compounds containing a double bond involving C-14 showed low activity with the exception of the specific, and so far unique, ecdysteroid dimer 7,7'-bis-[14-deoxy-8(14)-ene-20-hydroxyecdysone], which was obtained as the main product of the photochemical transformation of 20-hydroxyecdysone. The relatively high biological activity of this dimeric compound is discussed.     

Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis

cDNA for ultraspiracle (USP) from the lepidopteran rice stem borer Chilo suppressalis was cloned using PCR techniques. The deduced amino acid sequence of C. suppressalis USP (CsUSP) was very similar to those of other lepidopteran USPs, especially to the Manduca sexta USP-2 isoform. Northern hybridization analysis detected a 6.5-kb message in the epidermis, fat body, and midgut of wandering larvae. CsUSP mRNA expression in the epidermis varied little during the last larval instar. Gel mobility shift assays showed that in vitro translated C. suppressalis ecdysone receptor (CsEcR) and CsUSP proteins bound to the Pal1 or Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. In a ligand-receptor binding assay, [(3)H]ponasterone A ([(3)H]PoA) did not bind to individual CsEcR or CsUSP protein, but bound strongly to the CsEcR/CsUSP complex. [(3)H]PoA binding to CsEcR/CsUSP complex was competed by 20-hydroxyecdysone and a non-steroidal ecdysteroid agonist, RH-5992, but not by cholesterol, indicating that compounds with molting hormone activity against C. suppressalis can bind specifically to the CsEcR/CsUSP complex.     

Characterization of ecdysteroid receptors in cytosol and naive nuclear preparations of Drosophila Kc cells

Significant ecdysteroid binding activity can be demonstrated in nuclear extracts obtained from hormonally naive Drosophila Kc cells. The kinetic and physical characteristics of this nuclear binding are presented and compared with those exhibited by a high speed cytosol preparation of Kc cells. Examination of the effect of in vivo ecdysteroid exposure on the number of nuclear binding sites revealed that the quantity of nuclear receptors was not detectably altered. In addition, an effective synthesis of the biologically active ecdysteroid radioligand, [3H]ponasterone A, is described.     

Phytoecdysteroids from the juice of Serratula coronata L. (Asteraceae).

Seven phytoecdysteroids have been isolated from Serratula coronata L. One of them is a new phytoecdysteroid, 3-epi-20-hydroxyecdysone. Two further ecdysteroids, 20-hydroxyecdysone 22-acetate and taxisterone, are isolated from this species for the first time in addition to the typical S. coronata ecdysteroids, 20-hydroxyecdysone, ecdysone, ajugasterone C and polypodine B. The juice squeezed from aerial parts of fresh plants of S. coronata was extracted with ethyl acetate. The ecdysteroids were isolated by a combination of chromatographic techniques (mainly HPLC) and identified by 1D and 2D (1)H and (13)C NMR experiments and mass-spectrometry. The biological activities of 3-epi-20-hydroxyecdysone (EC(50)=1.6 x 10(-7) M), taxisterone (EC(50)=9.5 x 10(-8) M) and ajugasterone C (EC(50)=6.2 x 10(-8) M) have been determined in the Drosophila melanogaster B(II) bioassay for ecdysteroid agonist activity.     

Endogenous ecdysteroid levels and rates of ecdysone acylation by intact ovaries in vitro in relation to ovarian development in adult female house crickets,Acheta domesticus

Ecdysteroid titres have been determined in adult female house crickets (Acheta domesticus) in relation to reproductive maturation. Ecdysteroid levels in newly emerged adult females are low except in the gut and carcass, which probably reflect the remnants of the preecdysial ecdysteroid peak. Ecdysteroid levels in all compartments increase markedly once ovarian weight surpasses 10 mg. Apolar ecdysteroid conjugates (ecdysone 22-fatty acyl esters) predominate in ovarian tissue throughout ovarian maturation, but low levels of free ecdysteroid and polar conjugated ecdysteroids are also present. During this period, two peaks of ecdysteroids (mainly free and apolar conjugated ecdysteroids) are observed in the haemolymph, gut, and carcass compartments. The peaks in the haemolymph occur when the ovarian mass reaches 30 and 100 mg. The gut and carcass may be acting as sinks or sites of metabolism for the hormone released from the ovaries. The rate of ecdysone acylation by ovaries was found to be developmentally regulated, increasing from low levels in the immature ovaries of newly emerged females as the ovaries increase in size. A semiquantitative assay has been developed to identify compounds which inhibit the conversion of [³H] ecdysone into 22-fatty acyl [³H] ecdysone by ovaries in vitro. A number of ecdysteroids possessing a free hydroxyl group at C-22 as well as the side-chain stereochemistry of ecdysone effectively inhibit this conversion, probably by acting as competitive substrates. In the cases of 20-hydroxyecdysone and ponasterone A, it was clearly demonstrated that these compounds are converted to a mixture of C-22 fatty acyl esters. Several other compounds which have been sugested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro. Arch. Insect Biochem. Physiol. 35:279-299, 1997.© 1997 Wiley-Liss, Inc.     

Distribution of phytoecdysteroids in the Caryophyllaceae

Certain genera within the Caryophyllaceae (especially Silene and Lychnis) have received a significant amount of attention with regard to the isolation and identification of ecdysteroids. However, the taxonomy of this family is difficult. Hence, the occurrence of phytoecdysteroids in members of the Caryophyllaceae is presented, and combined with new data on ecdysteroid agonist (phytoecdysteroid) and antagonist activities, in order to survey the distribution of phytoecdysteroid-containing species within this large family, and to assess the utility of phytoecdysteroids as chemotaxonomic markers. The new data presented (representing ca. 110 species) have been obtained by the application of sensitive biological/biochemical methods for the detection of ecdysteroid agonists and antagonists, using Drosophila melanogaster B(II) bioassay and ecdysteroid-specific immunoassays. In the antagonist version of the B(II) bioassay, only weak ecdysteroid antagonist activities were detected in a few of the extracts. From both new and previously available data, it was found that phytoecdysteroids were present predominantly in the Genera Lychnis, Petrocoptis, Sagina and Silene. Comparison of ecdysteroid occurrence with a molecular phylogeny for the tribe Sileneae [Taxon 44 (1995) 525] revealed close association of ecdysteroid occurrence with certain groups of this tribe. In 14 species of Silene examined, there is a reasonable, but not absolute, relationship between the presence of ecdysteroids in the seeds and in other plant parts. Where ecdysteroids are present in the plant, highest concentrations are generally present in the roots.     

Expression and down-regulation of cytochrome P450 genes of the CYP4 family by ecdysteroid agonists in Spodoptera littoralis and Drosophila melanogaster

The function of CYP4 genes in insects is poorly understood. Some CYP genes are up-regulated by ecdysteroids and a number of Cyp4 genes in Drosophila melanogaster have been shown by microarray to be down-regulated when the ecdysteroid titre is high, suggesting hormonal regulation. Here, we report the utilization of certain cloned CYP4 cDNAs/fragments to probe their developmental/tissue expression in the Lepidopteran, Spodoptera littoralis, including the effects of ecdysteroid receptor agonists (bis-acyl hydrazines). CYP4L8 is expressed essentially throughout the final larval instar of S. littoralis and, together with CYP4M12, is down-regulated by agonist. Furthermore, expression of these genes occurs in midgut, but is undetectable in brain, fat body, and integument. Similarly, in D. melanogaster, Cyp4ac1, Cyp4ac3, Cyp4ad1 and Cyp4d1 gene expression is drastically down-regulated by ecdysteroid agonist. The significance of the results is discussed in relation to the plausible functions of the CYP4 genes in Lepidoptera and mechanisms of down-regulation.     

Molecular determinants of differential ligand sensitivities of insect ecdysteroid receptors

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormone-independent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.     

Phytoecdysteroids in seeds of Lloydia serotina (Liliaceae)

Seeds of a number of species in the Liliaceae (sensu Brummitt, 1992) were examined for the presence of ecdysteroid agonist and antagonist activities. No species were antagonistic to 20-hydroxyecdysone action on the ecdysteroid-responsive Drosophila melanogaster B_II cell line and only one extract, that of Lloydia serotina, was agonistic. This activity is attributable to the presence of phytoecdysteroids as detected by ecdysteroid-specific radioimmunoassay and the agonist version of the B_II bioassay. HPLC in conjunction with radioimmunoassay and bioassay have been used to determine the ecdysteroid profile. The major ecdysteroids present are identified as 20-hydroxyecdysone and polypodine B (5β,20-dihydroxyecdysone).     

Profile of the ecdysteroid hormone and its receptor in the salivary gland of the adult female tick, Amblyomma hebraeum

We examined the hemolymph ecdysteroid titer (by radioimmunoassay) and profile of the ecdysteroid receptor (EcR/USP; by [3H]ponasterone A binding, gel mobility shift assay, Western blot) in the salivary gland of the ixodid tick, Amblyomma hebraeum Koch (Acari: Ixodidae) throughout the tick feeding period and first 6 days post-engorgement. Throughout the slow phase of feeding, the hemolymph ecdysteroid titer was approximately 18 pg/microliter. The titer peaked at approximately 52 pg/microliter during the rapid phase of feeding, falling back to approximately 22 pg/microliter on the day of engorgement. Ecdysteroid titer rose again to approximately 750 pg/microliter by day 6 post-engorgement. EcR was undetectable by any of the three assays in unfed ticks. Following the onset of feeding, there appeared both specific ponasterone A binding and two major EcR bands detected by Western blot analysis. Both measurements were sustained throughout the feeding period, but declined after detachment when the salivary glands were degenerating. After ticks reached about 100 mg (by which time most females are mated), a discrete DNA-binding band was shown by gel mobility shift assay using Drosophila hsp27 EcRE as a probe. Moreover, the band intensified when hemolymph ecdysteroid titer reached its peak during the rapid phase of feeding; it declined along with decreasing EcR/USP levels, and with specific ligand binding activity following engorgement. This study suggests a role for the small hemolymph ecdysteroid peak during the rapid phase of feeding in initiating salivary gland degeneration.     

Lack of interference of common phytoecdysteroids with production of nitric oxide by immune-activated mammalian macrophages

Effects of selected common phytoecdysteroids on immunobiological responses triggered by lipopolysaccharide and interferon-gamma (IFN-gamma) were tested under in vitro conditions using murine resident peritoneal macrophages. Namely, production of nitric oxide was investigated. The series of test agents encompassed ecdysteroids occurring often as major components of the ecdysteroid fraction in numerous plant extracts: 20-hydroxyecdysone (20E), polypodine B, ajugasterone C, ponasterone A and inokosterone. Their structural variability concerns only variation in the number and position of hydroxyls. Two additional side-chain modified ecdysteroids: makisterone A (with a methyl substituent at position 24) and carthamosterone (with a cyclic side-chain lactone), and three ecdysteroid analogs: poststerone, rubrosterone and dihydrorubrosterone (devoid of side chains) were included into the test series. All test compounds, except of ponasterone A, represent natural substances isolated from the medicinal plant Leuzea carthamoides and are supposed to be significant for the often reported pharmacological activities of preparations derived from this species. However, the tested ecdysteroids did not interfere with the immunobiological activity of the immunocompetent cells. Our results thus differ from the so far reported information.     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfish Procambarus clarkii.

The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of crayfish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.     

Ecdysteroid receptors in a tumorous blood cell line of Drosophila melanogaster

The tumorous Drosophila melanogaster blood cell line BII has been studied for evidence for the presence of ecdysteroid receptors. The [³H]ponasterone A (pon A)* used in this study has been extensively purified, and the location of the tritium in the molecule has been partially determined. BII cells do not metabolise ecdysteroids. Intact cells demonstrate a considerable specific uptake of [³H]pon A which is saturable, apparently showing two specific components: a very high affinity component (K_D = 0.3 nM) and a high affinity component (K_D = 2 nM). The specific binding of [³H]pon A to whole cells is compatible with unlabelled ecdysteroids, but not with mammalian steroid hormones. The association rate constant (k_a) for [³H]pon A was determined to be 3 × 10⁷M^?1min^?1 at 21 °C, while the dissociation rate constant (k_d) for the specifically bound [³H]pon A was found to be 4.4 × 10^?3/min. Together, the kinetic rate constants yield a value of 0.15 nM for the K_D. The receptors have been partially characterised in a cell-free extract prepared by sonification of the cells. The optimum pH for extraction and hormone binding is 8.2. Scatchard plots of binding data indicate that the cell-free extract also contains two high affinity specific binding components (k_D = 0.1 nM and K_D = 1 nM). The hgih affinity binders are macromolecular, as shown by chromatography on Sephadex G-25, and are susceptible to protease digestion, heat, and treatment with N-ethylmaleimide. Sucrose density centrifugation of the labelled receptor shows one peak at approximately 6S. The stability of the receptor preparation has been studied and conditions have been empirically determined (10% w/v sucrose, 25 mM dithioerthreitol, and 10 mM citrate), whereby the binding capacity of the unlabelled receptor is stable for at least 8 weeks if frozen at ?20°C.     

Presence of membrane ecdysone receptor in the anterior silk gland of the silkworm Bombyx mori.

Nongenomic action of an insect steroid hormone, 20-hydroxyecdysone (20E), has been implicated in several 20E-dependent events including the programmed cell death of Bombyx anterior silk glands (ASGs), but no information is available for the mode of the action. We provide evidence for a putative membrane receptor located in the plasma membrane of the ASGs. Membrane fractions prepared from the ASGs exhibit high binding activity to [3H]ponasterone A (PonA). The membrane fractions did not contain conventional ecdysone receptor as revealed by Western blot analysis using antibody raised against Bombyx ecdysone receptor A (EcR-A). The binding activity was not solubilized with 1 m NaCl or 0.05% (w/v) MEGA-8, indicating that the binding sites were localized in the membrane. Differential solubilization and temperature-induced phase separation in Triton X-114 showed that the binding sites might be integrated membrane proteins. These results indicated that the binding sites are located in plasma membrane proteins, which we putatively referred to as membrane ecdysone receptor (mEcR). The mEcR exhibited saturable binding for [3H]PonA (Kd = 17.3 nm, Bmax = 0.82 pmol.mg(-1) protein). Association and dissociation kinetics revealed that [3H]PonA associated with and dissociated from mEcR within minutes. The combined results support the existence of a plasmalemmal ecdysteroid receptor, which may act in concert with the conventional EcR in various 20E-dependent developmental events.     

Ecdysteroid agonist and antagonist activities in species of the Solanaceae

Previously, it has been shown that certain withanolides from Iochroma gesnerioides (Solanaceae) possess ecdysteroid antagonistic activity. Phytoecdysteroids (agonists) are widely distributed in the plant world, but solanaceous species have not been extensively examined for their presence. We have now surveyed 128 species of solanaceous plants for the presence of ecdysteroid agonist and antagonist activities using the Drosophila melanogaster B(II) cell line bioassay. Only weak antagonistic activity was associated with a few of the methanolic extracts, including those from species known to contain high levels of withanolides. Therefore, the major withanolides are inactive per se, but they may be activated after ingestion by invertebrate predators. Several extracts possessed ecdysteroid agonist activity as a consequence of the presence of phytoecdysteroids. Phytoecdysteroid-accumulating species are at least as common in the Solanaceae as they are in plants in general. Preliminary characterization of the identities of the phytoecdysteroids present in the most active extracts has been performed by hplc separations on normal- and reversed-phase systems in conjunction with ecdysteroid-specific radioimmunoassay and bioassay. Each of the phytoecdysteroid-accumulating species examined (Browallia speciosa, Nierembergia hippomanica var violacea, N. solanacea and Solanum nigrum) contain a cocktail of ecdysteroids, of which 20-hydroxyecdysone and polypodine B (5beta,20-dihydroxyecdysone) are major components.     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfishProcambarus clarkii

Summary The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of cryafish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.