Effects Of Ecdysone Research Articles

Isolation and functional identification of secretin family G-protein coupled receptor from Y-organ of the mud crab, Scylla olivacea

Recently, genes in the superfamily of GPCR are gaining more interest in crustaceans as more evidence shows that they are involved in molting. This study identified four forms of the secretin family of G-protein coupled receptor (GPCR) from the Y-organ of mud crab, Scylla olivacea (ScoGPCR). A full-length sequence of ScoGPCR-B2 was isolated and identified as a lipoprotein receptor while three forms of GPCR in Methuselah-like (Mthl) or B3 subfamilies were reported as ScoGPCR-B3a, -B3b, and -B3c. These four forms exhibit common features of the 7-trans membrane (7TM) domain and distinct aspects in the extracellular region (ECR) at the N-terminus. At the ECR, disulfide bridges are predicted to generate structural stability in all four forms while the putative ScoGPCR-B3 proteins retain conserved Tyr, Trp, Pro, and Phe residues, possibly to form the aromatic-proline interactions and function as key residues for receptor recognition. Expression levels of ScoGPCR-B2 and -B3 in eyestalk, thoracic ganglion, and hindgut between intermolt and premolt stages are similar. Only ScoGPCR-B2 and ScoGPCR-B3a in Y-organ (YO) seem to be premolt-specific responses. An upregulation of ScoGPCR-B2 in YO at the premolt stage is correlated with the demand for cholesterol used in ecdysteroid synthesis, resulting in increased ecdysteroid titers. The effects of ecdysone on YO were pursued by in vitro incubation and revealed that ScoGPCR-B3a and -B3b expressions were induced in a different time frame: early in ScoGPCR-B3b and late in ScoGPCR-B3a. The early response of ScoGPCR-B3b was followed through immunohistology and showed that the newly synthesized protein was located primarily in the cytosol.

From Mosquito Ovaries to Ecdysone; from Ecdysone to Wolbachia: One Woman's Career in Insect Biology.

Simple SummaryMy career in science, and specifically in insect biology, benefitted from post-war efforts to improve science education in the 1950s and 1960s and increased opportunities for women students and faculty; teachers, mentors, associates, colleagues and friends of all sorts helped along the way. Here, I recount the highlights of my research, focusing on studies involving the insect steroid hormone ecdysone, which regulates molting in insect larvae, and reproduction in adults. Ecdysone was the unknown “ovarian factor” involved in egg production in Aedes aegypti mosquitoes. In addition, ecdysone affects the process of cell division in cultured mosquito cells, causing a cell cycle arrest that enhances growth of an obligate symbiotic bacterium, Wolbachia pipientis. Since the mid-1920s, Wolbachia was known to infect ovaries of Culex pipiens mosquitoes. After Wolbachia sequences were discovered in DNA extracted from Drosophila, this obligate intracellular alpha proteobacterium was found to be widespread among insect species, and its effects on reproduction have important applications for control of insect pests. Most recently, effects of ecdysone on Wolbachia replication in mosquito cell lines led to insights that enhance production of infectious bacteria at levels suitable for microinjection into eggs and eventual genetic manipulation.In anautogenous mosquitoes, synchronous development of terminal ovarian follicles after a blood meal provides an important model for studies on insect reproduction. Removal and implantation of ovaries, in vitro culture of dissected tissues and immunological assays for vitellogenin synthesis by the fat body showed that the Aedes aegypti (L.) (Diptera, Culicidae) mosquito ovary produces a factor essential for egg production. The discovery that the ovarian factor was the insect steroid hormone, ecdysone, provided a model for co-option of the larval hormones as reproductive hormones in adult insects. In later work on cultured mosquito cells, ecdysone was shown to arrest the cell cycle, resulting in an accumulation of diploid cells in G1, prior to initiation of DNA synthesis. Some mosquito species, such as Culex pipiens L. (Diptera, Culicidae), harbor the obligate intracellular bacterium, Wolbachia pipientis Hertig (Rickettsiales, Anaplasmataceae), in their reproductive tissues. When maintained in mosquito cell lines, Wolbachia abundance increases in ecdysone-arrested cells. This observation facilitated the recovery of high levels of Wolbachia from cultured cells for microinjection and genetic manipulation. In female Culex pipiens, it will be of interest to explore how hormonal cues that support initiation and progression of the vitellogenic cycle influence Wolbachia replication and transmission to subsequent generations via infected eggs.

Sperm maturation screening and the effect of ecdysone on sperm development of silkworm Bombyx mori

There are two sperm morphs of silkworm, the nucleated spermatozoa (eupyrene) and anucleated spermatozoa (apyrene). Eupyrene sperm cannot complete fertilization successfully without the apyrene sperm. Here a modified rapid and efficient method for sperm identification was developed, after 10 s of fixation in paraformaldehyde and 30 s of 4′6-diamidino-2-phenylindole (DAPI) or propidium Iodide (PI) staining, the sperm bundles can be detected easily using a fluorescence microscope. Sperm maturation process of silkworm from the fifth instar larvae to the adult was described with the above method, the precise time of earliest elongate apyrene bundles was detected on day 2 of pre-pupation, with a ratio of 5% in total sperm bundles, after which the percentage of apyrene sperm bundles increased rapidly and attained a relatively stable ratio of 75% at the end of pupation and nearly 80% after eclosion. Delayed mating leads to apyrene sperm accumulation and damaged fertilization. Previous study showed that ecdysone can increase the frequency of apyrene sperm bundles in vitro. Here 20-hydroxyecdysone (20E) was injected into hemolymph of the 2-d-old fifth instar larvae, the worms entered into mounting period after three days injection, but no apyrene sperm bundles were induced unless day 2 of pre-pupation. Interestingly, maturation of eupyrene sperm bundles were accelerated, and the ratio of eupyrene sperm bundles increased and exhibited a dose-dependent effect after 20E injection, which indicated that the development of eupyrene sperm can be accelerated by ecdysone before pupation of silkworm in vivo. These results will provide new clues for lepidopteran pest control.

The steroid hormone ecdysone regulates growth rate in response to oxygen availability

In almost all animals, physiologically low oxygen (hypoxia) during development slows growth and reduces adult body size. The developmental mechanisms that determine growth under hypoxic conditions are, however, poorly understood. Here we show that the growth and body size response to moderate hypoxia (10% O2) in Drosophila melanogaster is systemically regulated via the steroid hormone ecdysone. Hypoxia increases level of circulating ecdysone and inhibition of ecdysone synthesis ameliorates the negative effect of low oxygen on growth. We also show that the effect of ecdysone on growth under hypoxia is through suppression of the insulin/IGF-signaling pathway, via increased expression of the insulin-binding protein Imp-L2. These data indicate that growth suppression in hypoxic Drosophila larvae is accomplished by a systemic endocrine mechanism that overlaps with the mechanism that slows growth at low nutrition. This suggests the existence of growth-regulatory mechanisms that respond to general environmental perturbation rather than individual environmental factors.

Hyperecdysonism in the Formosan Subterranean Termite and Eastern Subterranean Termite (Isoptera: Rhinotermitidae).

Effects of ecdysone, 20-hydroxyecdysone (20E), and an ecdysone agonist, halofenozide, were tested against the Formosan subterranean termite, Coptotermes formosanus Shiraki, and the eastern subterranean termite, Reticulitermes flavipes (Kollar), in a 12-d no choice assay. Approximately 22-26% of R. flavipes and C. formosanus exhibited symptoms of hyperecdysonism, that is, "jackknife" position, when exposed to ecdysone and 20E at 1,000 ppm, respectively. High mortalities were recorded for both termite species in ecdysone and 20E at 100 and 1,000 ppm, but only at 10,000 ppm for halofenizide. Termites are known to move back to the central nest before the onset of ecdysis, and those that ingested lethal doses of chitin synthesis inhibitors (CSIs) die near the royal pairs, which partially accounts for the success of CSI baits to eliminate subterranean termite colonies. Because ecdysteroids and their agonists induce molting in termites, incorporation of these compounds into baits could potentially achieve the same colony elimination. This study showed that lethal time (12 d) of ecdysteroids and ecdysone agonist is shorter than that of a CSI (45 d); hence, the baiting time should be reduced by more than a month when they are incorporated in termite baits.

Effects of 20-Hydroxyecdysone on the Reversible Mitochondrial Transhydrogenase in the Tobacco Hornworm,Manduca sexta

The reversible, mitochondrial membrane-associated transhydrogenase from the midgut of Manduca sexta (L.) (Lepidoptera: Sphingidae) catalyzes hydride-ion transfer between NADP(H) and NAD(H). The effects of ecdysone and 20-hydroxyecdysone were evaluated and compared to both the NADH-NADP+ and NADPH-NAD+ transhydrogenations. In the direction of NADPH-formation, the developmentally significant transhydrogenations occur as non-energy- or energy-linked reactions. The energy-linked activity couples with either electron transport-dependent NADH or succinate utilization, or ATP hydrolysis by Mg++ -dependent ATPase. Upon the addition of ecdysone alone, all energy-linked reactions in the direction of NADPH formation exhibited a notable increase in activity level over the control reaction. The addition of 20-hydroxyecdysone yielded no significant increase in the activity of any of the transhydrogenations. Synergistic addition of both ecdysone and 20-hydroxyecdysone resulted in no significant effect on transhydrogenase activity. The results of this study make evident a relationship between the presence of ecdysone and 20-hydroxyecdysone on the overall activity of M. sexta midgut mitochondrial transhydrogenations. The potential mediation of the energy-linked mitochondrial transhydrogenations involved with NADPH synthesis through the developmental relationship of ecdysone and 20-hydroxyecdysone is considered.

Induction of somatic growth in juvenile crayfish Cherax quadricarinatus (Decapoda, Parastacidae), by ecdysone and insulin growth factor

The effect of ecdysone and IGF-1 on the somatic growth of juvenile crayfish Cherax quadricarinatus, was examined both in vitro and in vivo. After 24h of incubating explants of abdominal muscle, protein synthesis was augmented by exposure to 0.15μM of ecdysone, 0.02μM of IGF-1 or the combination of both hormones. The in vivo experiments were done by feeding the juveniles a pellet food supplemented with ecdysone, IGF-1 or a combination of both hormones, at doses equivalents to those that had an effect in the in vitro experiment. Animals fed IGF-1 or IGF-1 and ecdysone enriched pellets showed a higher weight gain than controls, while no differences were detected in the case of juveniles fed pellets enriched with ecdysone alone. In close correspondence with these results, and also with the in vitro results, a significant increment in protein content was found in muscle of crayfish treated with IGF-1, alone or in combination with ecdysone. On the other hand, significant lower lipid content was found in the hepatopancreas of both groups treated with IGF-1. Our results confirm the in vivo anabolic effects of IGF-1 on crustaceans, in terms of enhancing growth and protein synthesis, therefore identifying this hormone as a potential tool for crayfish culture.

Control of a Metamorphic Switch

When a single hormone--such as the steroid hormone ecdysone in insects--can cause a metamorphic reorganization of the organism from a larva to a pupa or from the pupal stage to the adult form, accidental activation of its signaling could be a catastrophe. Varghese and Cohen present findings showing that a microRNA-mediated feedback loop appears to help form a reliable switch mechanism for activating ecdysone signaling. The authors noted that the Drosophila gene encoding the ecdysone receptor contains predicted binding sites for the microRNA miR-14 . In transfected cells, expression of miR-14 inhibited expression of a reporter construct containing the ecdysone receptor promoter sequence. Mutant flies lacking miR-14 showed increased abundance of the ecdysone receptor, and some of the defects in miR-14 mutants were alleviated in flies in which one functional copy of the ecdysone receptor gene was removed. Thus, miR-14 appears to limit expression of the ecdysone receptor. Experiments with explanted larval fat bodies indicated that ecdysone increased the abundance of mRNA encoding the ecdysone receptor, an effect that was lost in cells depleted of the ecdysone receptor by RNAi (the primary transcript from the ecdysone receptor gene was not affected by the RNAi construct, so its abundance could be measured to estimate transcriptional activity of the gene). Furthermore, treatment of explants with ecdysone decreases the abundance of miR-14 , again with a requirement for the ecdysone receptor. The authors interpret these findings in light of the dramatic all-or-none biological effects of ecdysone. The hormone itself participates in a positive feedback loop by promoting transcription of the ecdysone receptor gene. Expression of the ecdysone receptor gene is normally opposed by miR-14 , but expression of miR-14 is also subject to repression by ecdysone signals. Thus, noise in the system--in the form of a transient increase in ecdysone receptor expression--may be buffered by the need to overcome miR-14 -mediated repression of ecdysone signaling. A more sustained activation of ecdysone signaling that lasts long enough for the decay of existing miR-14 is expected to exceed this threshold and release full activation of the self-sustaining ecdysone transcriptional autoregulatory loop. J. Varghese, S. M. Cohen, MicroRNA miR-14 acts to modulate a positive autoregulatory loop controlling steroid hormone signaling in Drosophila . Genes Dev. 21 , 2277-2282 (2007). [Abstract] [Full Text]

Flies Reveal New Nuclear Receptor Signaling Mechanism

Ecdysteroids are hormones with important roles in regulating development and metamorphosis in insects. Like mammalian steroid hormones, they act to regulate transcription through a nuclear receptor, the ecdysone receptor (EcR). The EcR forms heterodimers with Ultraspiracle (USP), which is related to another nuclear receptor family member, the vertebrate retinoid X receptor (RXR). However, the EcR appears not to mediate all effects of ecdysone and related ecdysteroids found in insect hemolymph. Baker et al . therefore examined the function of DHR38, an insect nuclear receptor that heterodimerizes with USP. The authors constructed a reporter system that used the better-characterized RXR as a partner in place of USP and showed that transcriptional activity of the complex was activated in response to various ecdysteroids. Activation of complexes of DHR38 with RXR required ligand-induced activation of both receptors, and the same appeared to be true of complexes with USP. Particularly surprising were experiments showing that DHR38, although responsive to ecdysteroids, appears not to bind the ligands directly. An X-ray crystal structure of the protein confirmed that the ligand-binding pocket and the coactivator binding sites normally conserved in nuclear receptors are unlikely to function in DHR38. The authors speculate that a cofactor acting with the receptors at sites of transcriptional activation might confer sensitivity to ecdysteroids. Because DHR38 is related to a subfamily of mammalian orphan nuclear receptors (the NGFI-B subfamily), the new paradigm for activation revealed by DHR38 in flies may be relevant to signaling by vertebrate nuclear receptors as well. K. D. Baker, L. M. Shewchuk, T. Kozlova, M. Makishima, A. Hassell, B. Wisely, J. A. Caravella, M. H. Lambert, J. L. Reinking, H. Krause, C. S. Thummel, T. M. Willson, D. J. Mangelsdorf, The Drosophila orphan nuclear receptor DHR38 mediates an atypical ecdysteroid signaling pathway. Cell 113 , 731-742 (2003). [Online Journal]

Cloning and functional analysis of pyruvate kinase promoter region from Drosophila melanogaster.

Pyruvate kinase (PK; EC 2.7.1.40) is a key glycolytic enzyme of Drosophila melanogaster. It catalyzes the conversion of phosphoenolpyruvate into pyruvate with the transfer of a phosphate group to ADP to form ATP. The ATP provides energy for cell growth and metabolism, and pyruvate participates in many metabolic reactions. Therefore, PK plays an important role in cell metabolism. Southern blot analysis, PCR, and sequencing were used to determine the content of a Drosophila pyruvate kinase (Pyk) genomic clone, lambdaPK61. The results indicated that the insert of lambdaPK61 comprised 8330 bp upstream of and 7186 bp downstream of the transcription start point of the Pyk gene. The size of the insert was 15,516 bp in total, which contained six genes including Pyk. Deletion mapping was applied to identify the promoter region and cis-acting elements 5' of PyK. Ten serial deletions produced by PCR were inserted upstream of the reporter gene (LacZ) to form recombinant plasmids, which were then transfected into Drosophila S2 cells. The results revealed that the regions -1475 approximately -1033 and -1033 approximately -534 of the 5' end of PyK possessed positive regulatory function for Pyk expression; i.e., increased gene expression. There were redundant putative cis-acting elements, including ecdysone response element (EcRE), E74A, and broad complex zinc finger (BRCZ) binding sites. Both E74A and BRCZ belong to the early genes regulated by ecdysone. This result suggested that Pyk might be regulated by ecdysone, directly or indirectly. However, the results of the developmental profile of Pyk expression by Northern blot analysis suggested that the effects of ecdysone on Pyk were repressive, not inductive. In addition, it was found that in these regions, there were many cis-acting elements related to egg and embryo development. Both -258 approximately -254 and -167 approximately -163 contained a CAAT box, and deletion of these regions decreased reporter gene expression. Therefore, it is suggested that both CAAT boxes are functional and that the promoter of Pyk might be located in the region of -258 approximately +109. No TATA box or downstream promoter element were identified around the transcription start site of Pyk. Additionally, PyK might share a regulatory region with an unknown neighboring gene. It was concluded that Pyk has the characteristics of a housekeeping gene.

Ecdysone-regulated chromosome puffing in the salivary glands of the Mediterranean fruit fly, Ceratitis capitata

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.

The ecdysoid RH5849 induces yolk polypeptide synthesis in male flies

Summary RH5849 is a benzoyl hydrazine analog which has been reported to mimic several effects of the arthropod steroid hormone ecdysone to which it is chemically totally unrelated. In adult Diptera, ecdysone is the hormone that triggers vitellogenin synthesis. We report here that RH5849, upon oral ingestion, is able to induce vitellogenin synthesis in male Drosophila, Neobellieria, Phormia and Lucilia. This contrasts to data in the literature which showed that RH5849 could not mimic the pupariation-inducing effect of ecdysone in last instar fly larvae. RH5849 neither exerts a juvenile hormone mimicking effect nor behaves as an anti-juvenile hormone in both the Colorado potato beetle and Galleria.

Effects of ecdysone and neurotrophins on network formation in embryonic neurones in primary culture

Biology of the CellVolume 86, Issue 2-3 p. 194-194 EFFECTS OF ECDYSONE AND NEUROTROPHINS ON NETWORK FORMATION IN EMBRYONIC NEURONES IN PRIMARY CULTURE Laure DEBURE, Laure DEBURE Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorJanine LE GUEN, Janine LE GUEN Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorColette VAILLANT, Colette VAILLANT Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorVeronique ANGEVIN, Veronique ANGEVIN Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorChristophe MERLE, Christophe MERLE Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorChristophe MARCHAL, Christophe MARCHAL Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorYves PICHON, Yves PICHON Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this author Laure DEBURE, Laure DEBURE Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorJanine LE GUEN, Janine LE GUEN Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorColette VAILLANT, Colette VAILLANT Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorVeronique ANGEVIN, Veronique ANGEVIN Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorChristophe MERLE, Christophe MERLE Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorChristophe MARCHAL, Christophe MARCHAL Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this authorYves PICHON, Yves PICHON Equipe Canaux et Récepteurs Membranaires, URA 256, RENNES F-35042, FRANCESearch for more papers by this author First published: 1996 https://doi.org/10.1016/0248-4900(96)84817-9AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume86, Issue2-31996Pages 194-194 RelatedInformation

Ecdysone and 20-hydroxyecdysone supplements to the diet affect larval development in the silkworm, Bombyx mori, differently

Ecdysone and 20-hydroxyecdysone were added to artificial diets of the silkworm, Bombyx mori, to evaluate their effects on larval development and the ecdysteroid titer. Ecdysone at more than 50 p.p.m. induced extra larval ecdyses. In contrast, extra larval ecdyses did not occur at any concentration of 20-hydroxyecdysone. 20-Hydroxyecdysone present at a high concentration from hatching onward caused mortality during the second instar. In larvae reared on an ecdysone-supplemented diet the ecdysteroid titer rapidly increased, then declined sharply in each instar. In larvae reared on 20-hydroxyecdysone-supplemented diet, the ecdysteroid titer increased and remained high throughout the second instar. These results suggest that the effect of ecdysone on the mechanism regulating the ecdysteroid titer is qualitatively different from that of 20-hydroxyecdysone.

Effects of ecdysone, juvenile hormone and haemolymph transfer on cryoprotectant metabolism in diapausing and non-diapausing pupae of Pieris brassicae

Hormone applications and haemolymph transfer were used to study the control of cryoprotectant synthesis in diapause and non-diapause Pieris brassicae. Diapause pupae are 24% heavier than non-diapause pupae 5 days after pupation and have 10 times more sorbitol and 8 times more mannitol in the absence of low temperature exposure. In diapause pupae, sorbitol increases 8-fold from 5 to 55 days at 20°C. Other carbohydrates such as glucose, mannose, glycerol, mannitol and inositol also increase during this time. Injection of ecdysone (500 ng) terminates diapause at all stages investigated but does not affect development or carbohydrate levels in non-diapause pupae. Diapause pupae show significant decreases in glucose, mannose, sorbitol and mannitol after ecdysone injection, whilst glycerol increases. Juvenile hormone application (500 ng) has no effect on the development of diapause or non-diapause pupae but the latter show significant increases in glucose and glycerol whilst sorbitol levels are unchanged. In contrast, diapause pupae show significant increases in sorbitol whilst glycerol decreases. Haemolymph transfer did not influence carbohydrate levels. Implications for the relationship between cryoprotectant synthesis and diapause are discussed and a model is proposed.

26-[125I]iodoponasterone A is a potent ecdysone and a sensitive radioligand for ecdysone receptors.

The effects of ecdysone, the steroid molting hormone of arthropods, are of considerable interest both to insect physiologists and to those studying steroid-regulated gene expression. Yet progress in understanding ecdysone receptors has been inhibited by the lack of a suitable highly radioactive hormone analog with high affinity for the receptor. Here we report that the synthetic ecdysteroid 26-iodoponasterone A is one of the most active ecdysones known, inducing half-maximal morphological transformation in Drosophila Kc167 cells when present at 0.5 nM. 26-[125I]Iodoponasterone A can be prepared at a specific activity of 2175 Ci/mmol (1 Ci = 37 GBq) by reaction of the precursor 26-mesylinokosterone with carrier-free Na125I. The radiolabeled material binds to Kc167 cell ecdysone receptors specifically and with affinity (Kd ca. 3.8 X 10(-10) M). Thus, 26-[125I]iodoponasterone A appears to be a superior radioligand for ecdysone receptors on grounds both of affinity and of specific activity. Its ready availability should greatly facilitate studies of these receptors.

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

AbstractThe 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.

Opposite effects of ecdysone and 20-hydroxyecdysone on in vitro uterus motility of a tsetse fly.

In order to examine the possible effects of ecdysteroids on parturition, we studied in vitro the influence of ecdysone and 20-hydroxyecdysone on the motility of isolated uterus from virgin and pregnant female tsetse fly (Glossina fuscipes). Ecdysone initiates phasic uterine contractions or enhances the frequency of preexisting contractile activity. In contrast, uterine contractions are decreased or abolished by 20-hydroxyecdysone. Pharmacological data indicate that tsetse fly uterus exhibits myogenic and nerve-evoked contractions. Ecdysteroids mainly act on nervous structures that control muscle contractions. Our results provide evidence for a specific action of ecdysteroids on a nerve-muscle target involved in female reproduction.

Effects of ecdysone on the metamorphosis and ecdysis prevention induced by precocene II in Rhodnius prolixus.

Abstract The effects of precocene II (10 to 300 μg/ml of blood), given orally, on the development of third- and fourth-instar larvae of Rhodnius prolixus were studied. Precocene acted as a feeding deterrent, reducing the amount of blood eaten, and caused a high mortality at high doses. Molting was delayed in a dose-dependent manner and in several cases ecdysis was completely prevented. Large numbers of insects underwent precocious metamorphosis. A significant correlation between the prolongation of the molting cycle and morphogenetic adultoid characteristics was observed. Ecdysone given orally (5 μg/ml of blood) counteracted the ecdysis prevention and also reduced the duration of the molting cycle which is prolonged by precocene II. However, ecdysone did not preclude the adultiform production. Precocious adults with slight imaginal characters were able to feed and initiate the next ecdysis. Adultoids with distinct imaginal characteristics were short-lived and did not feed despite repeated opportunities.

Ecdysone-inducible functions of larval fat bodies in Drosophila.

Late in the third instar larval stage of Drosophila melanogaster, the titer of the steroid hormone ecdysone increases sharply. This increase is blocked in the temperature-sensitive mutant ecd(1) after a temperature shift from 20 degrees C to 29 degrees C. The mutant was used to prepare three samples of late third instar larvae with different titers of ecdysone; the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysone-supplemented food, and also high in a third sample that was kept at 20 degrees C, providing a control for normal development. The effect of the high titer of ecdysone on proteins of the larval fat bodies was examined by comparing two-dimensional gel electrophoresis patterns of total proteins in stained gels. There were proteins at five positions in the gels for the high-ecdysone samples that were not detected at the corresponding positions in the gel for the low-ecdysone sample. The effect of ecdysone on these proteins was further studied by injecting [(35)S]methionine into the larvae at both early and late third instar stages, in order to label proteins synthesized before and after the increase in ecdysone titer. The results indicate that ecdysone induces two major responses in the fat bodies; certain proteins that were synthesized earlier in the fat bodies and secreted into the hemolymph are incorporated back into the fat bodies, and other proteins are newly synthesized. Attempts to induce prematurely the synthesis of the new proteins by exposing early third instar larvae to exogenous ecdysone were unsuccessful, suggesting that development must proceed further before the fat bodies can respond to ecdysone. By in vitro translation of RNA isolated from fat bodies of low-and high-ecdysone samples of larvae, it was shown that ecdysone greatly increases the amount of translatable messenger RNA for one of the newly synthesized proteins. A clone of DNA complementary to the induced messenger RNA has been isolated from a population of lambda bacteriophage carrying segments of the Drosophila genome. Using the cloned DNA to measure amounts of complementary poly(A)-RNA in the fat bodies by DNA.RNA hybridization, we detected about 50 times more complementary poly(A)-RNA in the high-ecdysone sample of larvae than in the low-ecdysone sample. This finding provides direct evidence that ecdysone induces an increase in the amount of the messenger RNA. The ecdysone-induced appearance of a major messenger RNA in late third instar larval fat bodies represents a developmental response to ecdysone that appears to be gene-specific, tissue-specific, and stage-specific, and it has exceptionally favorable features for further molecular studies of the control of gene expression by a steroid hormone.