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PubMed2004

Ecdysteroids during early embryonic development in silkworm Bombyx mori: metabolism and functions.

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various molecular species of ecdysteroids in free and conjugated forms. In B. mori eggs, 20-hydroxyecdysone (20E) is a physiologically active molecule. In nondiapause eggs, 20E is produced by the conversion of maternal conjugated ecdysteroids (ecdysteroid-phosphates) and by de novo biosynthesis. In contrast, in diapause eggs, neither of these metabolic processes occurs. In de novo biosynthesis of 20E in B. mori eggs, hydroxylation at the C-20 position of ecdysone, which is catalyzed by ecdysone 20-hydroxylase, is a rate-limiting step. Furthermore, we found that a novel enzyme, called ecdysteroid-phosphate phosphatase (EPPase), specifically catalyzes the conversion of ecdysteroid-phosphates to free ecdysteroids. The developmental changes in the expression pattern of EPPase mRNA correspond closely to changes in the enzyme activity and in the amounts of free ecdysteroids in eggs. EPPase is localized in the cytosol of yolk cells, and the bulk of maternal ecdysteroid-phosphates is bound to vitellin and stored in yolk granules. The vitellin-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase. Therefore, to examine how ecdysteroid-phosphates are hydrolyzed by EPPase during embryonic development further investigations were focused on yolk granules. Recent data indicate that acidification in yolk granules, induced by vacuolar H(+)-ATPase, triggers the dissociation of ecdysteroid-phosphates from the vitellin-ecdysteroid-phosphates complex and the dissociated ecdysteroid-phosphates are released from yolk granules to the cytosol. To explain the process of the increase in the level of 20E during embryonic development in B. mori eggs, a possible model is proposed.

Animals↗
PubMed2007

Ecdysteroid levels in Daphnia magna during a molt cycle: determination by radioimmunoassay (RIA) and liquid chromatography-mass spectrometry (LC-MS).

Despite their abundance and their enormous significance for various ecological processes, endocrine systems of microcrustaceans have been poorly investigated. Here, we used a highly sensitive radioimmunoassay (RIA) to determine free and conjugated ecdysteroid levels in whole-body extracts of adult Daphnia magna during a complete molt cycle. Ecdysteroid levels were predominated by free ecdysteroids. Starting from basal levels in the postmolt stage the concentration of free ecdysteroids increased sharply in the early premolt stage, followed by a sharp decline back to basal levels just prior to ecdysis. Polar and apolar ecdysteroid conjugates were found in rather low amounts with little variation during the molt cycle. Only small amounts of ecdysteroids were found in newly deposited eggs of D. magna, which suggest a sparing investment of maternal ecdysteroids into the eggs for early embryogenesis. As in whole-body extracts, free ecdysteroids were the predominant ecdysteroids found in eggs of D. magna, together with small amounts of polar and intermediary amounts of apolar conjugates. Hence, the pathways leading to polar and apolar ecdysteroid conjugates appear to be of minor importance in D. magna. Analyses of the immunodetectable peak in free ecdysteroids by liquid chromatography-mass spectrometry (LC-MS) revealed that molting is induced most probably by an increased level of 20-hydroxyecdysone. Microcrustaceans of the genus Daphnia are key components in freshwater food webs. Examination of the functional role of ecdysteroids in controlling developmental processes might help to understand the performance of the herbivorous grazer in its environment, in particular with regard to the adverse effects of environmental xenobiotics acting as endocrine disrupters.

Animals↗
PubMed1997

Endogenous ecdysteroid levels and rates of ecdysone acylation by intact ovaries in vitro in relation to ovarian development in adult female 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 reflects 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 as 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 suggested to affect ecdysteroid metabolism/mode of action in other systems were also tested for their effects on the acyltransferase activity of ovaries in vitro.

Acylation↗
PubMed2004

Cross communication between signaling pathways: juvenoid hormones modulate ecdysteroid activity in a crustacean.

Methyl farnesoate is a juvenoid hormone that regulates a variety of processes in crustaceans including male sex determination among daphnids (Branchiopoda, Cladocera). The synthetic juvenoids pyriproxyfen and fenoxycarb mimic the action of methyl farnesoate in daphnids. In the present study we tested the hypothesis that juvenoids also can regulate ecdysteroid activity in a crustacean (Daphnia magna). Methyl farnesoate, pyriproxyfen, and fenoxycarb all disrupted ecdysteroid-regulated aspects of embryo development in daphnids. Exposure of ecdysteroid-responsive cells to 20-hydroxyecdysone reduced cell proliferation and increased mRNA levels of the ecdysone receptor and its partner protein ultraspiracle. Co-treatment of cells with the juvenoid pyriproxyfen attenuated all of these ecdysteroid mediated responses. While juvenoids functioned as anti-ecdysteroids in both intact embryos and in cultured cells, 20-hydroxyecdysone showed no evidence of acting as an anti-juvenoid. The combined effects of pyroproxyfen with the ecdysteroid synthesis inhibitor fenarimol and the ecdysteroid receptor antagonist testosterone were evaluated in an effort to discern whether the action of the juvenoids were additive with those of know anti-ecdysteroids. The anti-ecdysteroid effects of pyriproxyfen were non-additive with those of either anti-ecdysteroid. Rather, joint effects conformed to a model of synergy. These results demonstrated that juvenoids elicit anti-ecdysteroidal activity in a crustacean through a unique mechanism of action. A model involving receptor partner deprivation is proposed that explains the synergistic interactions observed.

Animals↗
PubMed2003

Practical uses for ecdysteroids in mammals including humans: an update.

Ecdysteroids are widely used as inducers for gene-switch systems based on insect ecdysteroid receptors and genes of interest placed under the control of ecdysteroid-response elements. We review here these systems, which are currently mainly used in vitro with cultured cells in order to analyse the role of a wide array of genes, but which are expected to represent the basis for future gene therapy strategies. Such developments raise several questions, which are addressed in detail. First, the metabolic fate of ecdysteroids in mammals, including humans, is only poorly known, and the rapid catabolism of ecdysteroids may impede their use as in vivo inducers. A second set of questions arose in fact much earlier with the pioneering "heterophylic" studies of Burdette in the early sixties on the pharmacological effects of ecdysteroids on mammals. These and subsequent studies showed a wide range of effects, most of them being beneficial for the organism (e.g. hypoglycaemic, hypocholesterolaemic, anabolic). These effects are reviewed and critically analysed, and some hypotheses are proposed to explain the putative mechanisms involved. All of these pharmacological effects have led to the development of a wide array of ecdysteroid-containing preparations, which are primarily used for their anabolic and/or "adaptogenic" properties on humans (or horses or dogs). In the same way, increasing numbers of patents have been deposited concerning various beneficial effects of ecdysteroids in many medical or cosmetic domains, which make ecdysteroids very attractive candidates for several practical uses. It may be questioned whether all these pharmacological actions are compatible with the development of ecdysteroid-inducible gene switches for gene therapy, and also if ecdysteroids should be classified among doping substances.

Animals↗
PubMed1999

Ecdysteroids during ovarian development and embryogenesis in solitary and gregarious schistocerca gregaria

Maternal ecdysteroids identified in the vitellogenic oocytes of Schistocerca gregaria included more than 80% polar conjugates, up to 5% free ecdysteroids, and up to 15% non-hydrolyzable polar metabolites. The representations of ecdysone (E), 20-hydroxyecdysone (20E), and 2-deoxyecdysone (2dE) in the conjugates was about 16:3:1, and in the free ecdysteroids about 3:1:1. The quantity of ecdysteroids in the ovaries before egg-laying reached 2.3 ng 20E equiv. per mg tissue in the solitary, and 8.9 ng/mg in the gregarious females. Newly laid eggs contained 14 ng and 89 ng, respectively, of 20E equiv. per egg. Nearly all egg ecdysteroids were in form of conjugates and their content declined during the first half of embryonic development. The amount of ecdysteroids sharply increased to over 70 ng 20E equiv./egg in the solitary, and to nearly 400 ng/egg in the gregarious phase. In the second half of embryonic development, the representation of conjugates in total ecdysteroids was reduced to 45-55%, whereas that of free E + 20E rose to 30-40%. Free 2dE remained low but, in the gregarious embryos, free 26E increased to 10% of all ecdysteroids. The conjugates of solitary embryos contained nearly exclusively E and 20E (in ratio 2:1), whereas those of the gregarious embryos included E, 20E, 2dE, and 26E (in ratio 12:7:4:1). Towards the end of embryonic development, the amounts of conjugates and of free ecdysteroids decreased, while that of polar metabolites rose. A sharp drop in ecdysteroid content was associated with hatching but the more than five times higher ecdysteroid level in the gregarious than in the solitary phase was maintained in the newly hatched larvae. Arch. Copyright 1999 Wiley-Liss, Inc.

Journal Article↗
PubMed2000

Ecdysteroid titer, ovary status, and dominance in adult worker and queen bumble bees (Bombus terrestris).

The role of ecdysteroids in the regulation of dominance and reproduction in social Hymenoptera is little explored. In the current study we compared ecdysteroid titers in hemolymph of individual queen and worker bumble bees (Bombus terrestris) that differ in their behavior, reproductive status and social environment. Egg-laying queens that head colonies and have ovaries exhibiting all stages of follicle development, had a higher ecdysteroid titer than virgin queens whose ovaries contained only follicles at initial stages. In workers, the relationship between ecdysteroid titers and follicle development appears to be more complex and to be influenced by the bee's social status and social environment. Shortly after emergence, young workers had only follicles at the initial stages of oogenesis and they exhibited a low ecdysteroid titer. No significant correlation was detected between ovary status and ecdysteroid titer in workers, with some workers showing activated ovaries but low ecdysteroid titers. However, at six days of age, a trend towards higher ecdysteroid titer was observed for workers in queenless groups, a condition characterized by rapid follicle development relative to queenright conditions. In these queenless groups, high social status was associated with high ecdysteroid titers. By contrast, in queenright workers ecdysteroid titers were low, even for bees with presumably high social status that had activated ovaries and were observed performing oviposition behavior. This study suggests that ecdysteroids are involved in regulation of reproduction in B. terrestris.

Journal Article↗
PubMed1990

Ecdysteroid fluctuations during embryogenesis in the giant freshwater prawn, Macrobrachium rosenbergii.

Ecdysteroid levels during the embryogenesis of the giant freshwater prawn, Macrobrachium rosenbergii, were determined by radioimmunoassay and high-performance liquid chromatography. Ecdysteroids consisting of significant amounts of 20-hydroxyecdysone and high-polarity products (HPP) and lesser amounts of ecdysone and low-polarity products (LPP) were detected in mature ovaries and newly laid eggs. All ecdysteroid groups decreased gradually during the nauplius phase. With the formation of the compound eye and the appearance of the carapace and other body-like structures, marking morphogenesis to the zoeal stage, embryos showed the beginning of a continuous and dramatic increase in ecdysteroid concentrations sustained until larval hatchout. Ecdysteroid levels at hatchout were above 20-fold greater than ecdysteroid levels in newly laid eggs. More specifically, HPP and 20-hydroxyecdysone increased concomitantly, with a decrease in 20-hydroxyecdysone only at the end of the embryogenic period, while ecdysone and LPP levels remained low or undetectable. It may be postulated that the presence of ecdysteroids in ovaries and eggs represents a reserve of maternal ecdysteroids which are necessary at the commencement of embryonic development; with the differentiation of embryonic tissue capable of ecdysteroid synthesis, ecdysteroids increase rapidly to play a role in later embryonic development.

Animals↗
PubMed2005

Reproductive division of labor, dominance, and ecdysteroid levels in hemolymph and ovary of the bumble bee Bombus terrestris.

To determine whether ecdysteroids are associated with reproductive division of labor in Bombus terrestris, we measured their levels in hemolymph and ovaries of queens and workers. Queens heading colonies had large active ovaries with high ecdysteroid content, whereas virgin gynes and mated queens before and after diapause had undeveloped ovaries with low ecdysteroid content. The hemolymph ecdysteroid titer was rather variable, but in a pooled analysis of mated queens before and after diapause versus colony-heading queens, ecdysteroid titers were higher in the latter group. In workers, agonistic behavior, ovarian activity, ovarian ecdysteroid content, and hemolymph ecdysteroid titers were positively correlated, and were lowest when a queen was present. In queenless workers, ecdysteroid levels were elevated in dominant workers, and were also influenced by the presence of brood and by group demography; hormone levels were higher in bees kept in larger groups. These findings are consistent with the premise that in B. terrestris the ovary is the primary site of ecdysteroid synthesis, and they show that ecdysteroids levels vary with the social environment.

Animals↗
PubMed1991

Ecdysteroid synthesis and molting by the tobacco hornworm, Manduca sexta, in the absence of prothoracic glands.

When a pair of prothoracic glands (PGs) were removed from Manduca sexta pupae on the day of pupation, the hemolymph ecdysteroid titer remained at a low level. When a portion of the gland pair was extirpated from pupae after the critical period for prothoracicotropic hormone release, the maximum hemolymph ecdysteroid titer was reduced in proportion to the mass of the PGs removed. These findings clearly showed that the PGs in intact pupae are responsible for the elevated ecdysteroid titer required to elicit adult development on schedule. When brains were removed on the day of pupation, the initiation of adult development was delayed for weeks or months. In contrast, pupae whose PGs were removed on the day of pupation initiated development only 7 days late, indicating the existence of an additional source of pupal ecdysteroids. Further, abdomens of male M. sexta that were isolated on the day of pupation initiated adult development spontaneously within 70 days. The implantation of day 0 pupal brains into these isolated abdomens accelerated the initiation of adult development and elicited synchronous adult development. The hemolymph ecdysteroid titer of those isolated abdomens receiving implants of brains increased within 5 days and reached a maximum level of 1.5 micrograms/ml. The analysis of hemolymph ecdysteroids by reverse-phase HPLC revealed that ecdysone was the major moiety and that the ecdysteroid composition was similar to that of normal, intact pupae that had just initiated adult development. These results demonstrate that the PGs are not requisite for adult development. An increased hemolymph ecdysteroid titer was also observed in isolated abdomens from which the testes were removed and in abdomens devoid of their digestive tract. Indeed, in the latter case, the ecdysteroid titer attained much higher levels than those observed for abdomens with intact guts. Despite numerous attempts to identify the tissue(s) in the isolated abdomens responsible for the increase in ecdysteroid titer, its identity remains unknown.

Animals↗
PubMed1991

Ecdysteroid receptors in the central nervous system of Manduca sexta: their changes in distribution and quantity during larval-pupal development.

Ecdysteroids act initially by binding to nuclear and possibly also extranuclear receptors. The presence and expression of these receptors in the insect brain was investigated in the present study as a means of defining these neurons involved in ecdysteroid-regulated processes at different developmental stages. Early in the fifth larval stadium of Manduca sexta, when endogenous ecdysteroid levels are low, receptors for ecdysteroids in cerebral neurons are either absent or present at low levels. Receptors can be reliably detected only on day 0 and are not found again until day 3.5, at the beginning of the commitment peak in the ecdysteroid titer, when they occur in a small stage-specific population of cells. At this time, ecdysteroid receptors are found mainly in nuclei but are also observed at low levels in cytoplasm. By day 4.8, ecdysteroid receptors are exclusively nuclear, and the number of target cells has increased dramatically in several brain regions, including those with known neurosecretory cell groups. This population and organization of ecdysteroid target cells is constant up to day 6, after which time the number of target neurons declines. By day 7.8, only 10% of the number of labelled neurons seen on days 4.8-6.8 remain in peripheral areas. In the pupal brains, ecdysteroid receptors reappear in a new population of neurons. The results indicate changes in the genomic regulation of a varying neuron population by ecdysteroids during fifth stadium development.

Animals↗
PubMed2006

Ecdysteroid hormone titer and its relationship to vitellogenesis in the soft tick, Ornithodoros moubata (Acari: Argasidae).

A blood meal is required for reproduction in most argasid female ticks. The blood meal appears to stimulate an organ in the posterior end to produce a fat body stimulating factor (FSF), which is thought to be an ecdysteroid, to induce vitellogenin (Vg) synthesis. In this study, the relationship of vitellogenesis and ecdysteroids was investigated by measuring Vg and ecdysteroid titers while observing oocyte development and oviposition in mated and virgin females. Oviposition occurred from day 10 after engorgement in mated females and continued up to 40-50 days, whereas egg maturation and oviposition did not occur in virgin females. Vg titers in the hemolymph peaked on day 6 after engorgement and subsequently declined in mated females. Interestingly, Vg synthesis occurred and ovarian development progressed to the development of early vitellogenic oocytes in virgin females but oocyte maturation and oviposition did not occur. Topical application of ecdysteroids induced oviposition in fed virgin females indicating that ecdysteroids may induce oviposition. Concentrations of ecdysteroids for 20 days after engorgement revealed several peaks in mated female whole body extracts, but no peaks in virgin female extracts. In the hemolymph of only mated females, ecdysteroid titers showed two peaks that followed the early peak of ecdysteroids in the whole body on day 4 and 6 after engorgement. In addition, ecdysteroids in the reproductive tissues increased with the development of the ovary in mated females and this increase coincided with the latter peaks of the whole body. These observations indicate that physiological elevation of ecdysteroids accelerate Vg synthesis, and may induce egg maturation and stimulate oviposition in fed mated Ornithodoros moubata females.

Animals↗
PubMed1998

Manipulation of fifth-instar host (Manduca sexta) ecdysteroid levels by the parasitoid wasp Cotesia congregata.

Although 5th (last) instar parasitized Manduca sexta larvae undergo developmental arrest and do not wander, they exhibit a small hemolymph ecdysteroid peak (300-400pg/&mgr;l) which begins one day prior to the parasitoid's molt to the 3rd (last) instar and concomitant emergence from the host. Ecdysteroids present in this peak were 20-hydroxyecdysone, 20,26-dihydroxyecdysone and one or more very polar ecdysteroids, as well as small amounts of 26-hydroxyecdysone and ecdysone. In parasitized day-1 and -2 5th instars ligated just behind the 1st abdominal proleg, hemolymph ecdysteroid levels increased in both anterior and posterior portions (100-500pg/&mgr;l), while in unparasitized larvae, hormone levels only increased in the anterior portion (100-350pg/&mgr;l). Thus, the ecdysteroid peak observed in host 5th instars was probably produced, at least in part, by the parasitoids. It may serve to promote Cotesia congregata's molt from the second to the third instar and/or to facilitate parasitoid emergence from the host. In parasitized day-1 and -2 5th instars ligated between the last thoracic and 1st abdominal segments, hemolymph ecdysteroid titers reached much higher levels (500-3500pg/&mgr;l) in the anterior portion (no parasitoids present) than in the posterior portion (150-450pg/&mgr;l). Therefore, it appears that the parasitoid's regulation of hemolymph ecdysteroid titers occurs at two levels. First, parasitization neutralizes the host's ability to maintain its normal hemolymph ecdysteroid levels. Second, in a separate action, the parasitoid manipulates the ecdysteroid-producing machinery so that hemolymph levels are maintained at the 200-400pg/&mgr;l characteristic of day 3-4 hosts. This is the first report of a parasitoid's ability to interfere with the normal inhibitory mechanisms which prevent prothoracic gland production of ecdysteroid at inappropriate periods of insect growth and development.

Journal Article↗
PubMed1993

Excessive abundance and dynamics of unusual ecdysteroids in Pycnogonum litorale ström (Arthropoda, Pantopoda).

In embryos, larvae, and juveniles of Pycnogonum litorale, unusual ecdysteroids have been found at excessively high levels. Concentrations and dynamics of the following eight ecdysteroids were determined by HPLC: 20-hydroxyecdysone, 20-hydroxyecdysone 22-glycolate, (25R) and (25S) isomers of 20,26-dihydroxyecdysone 22-acetate, 22-deoxy-20,26-dihydroxyecdysone, 20-hydroxyecdysone 22-acetate, 22-deoxy-20-hydroxyecdysone, and ecdysone 22-glycolate. The excessively high content of ecdysteroids is found in all developmental stages. Juvenile pycnogonids contain the highest total ecdysteroid amounts ever found in arthropods: 0.1% of their body dry weight. 20-Hydroxyecdysone 22-acetate is the prominent ecdysteroid and represents at all stages of both sexes, 66.2-85.8% of the total ecdysteroids. This compound, as well as all other ecdysteroids without a free 22-OH group, are presumed to be inactive as molting hormones. In contrast to these compounds, 20-hydroxyecdysone is present only in amounts similar to those in other arthropods. Furthermore, it is the only ecdysteroid with its peak at apolysis. It is regarded as the molt-promoting hormone. The origin and function of the other ecdysteroids in different developmental stages are discussed.

Aging↗
PubMed1991

Circadian regulation of synthesis of ecdysteroids by prothoracic glands of the insect Rhodnius prolixus: evidence of a dual oscillator system.

Rhythmic synthesis of moulting hormones (ecdysteroids) by prothoracic glands (PGs) of the insect Rhodnius prolixus during the last larval instar was studied in vitro following explantations every 4-5 hr for up to 96 hr. Ecdysteroid synthesis was measured by radioimmunoassay as the quantity of ecdysteroid produced during 4 hr in vitro. A massive daily rhythm is seen, with synthesis at night being three- to fivefold higher than during the day. This rhythm of ecdysteroid synthesis by PGs free-runs in continuous darkness with a temperature-compensated period length close to 24 hr and is therefore controlled by a circadian system. This is the first report of circadian regulation of synthesis of an invertebrate hormone. The synthesis rhythm also free-runs in continuous light, but with an inverted phase and shorter period length. It is argued that the circadian system controlling synthesis comprises two oscillators which free-run in antiphase, occupy different anatomical locations, and are coupled by a humoral factor, possibly prothoracicotropic hormone. The ecdysteroid synthesis rhythm in PGs appears to drive the previously reported circadian rhythm in the haemolymph ecdysteroid titre. It is concluded that the circadian system controlling synthesis of ecdysteroids constitutes a pacemaker which drives various rhythms in the target cells of ecdysteroids via the rhythm in the haemolymph titre. Ecdysteroids are viewed as "hormonal Zeitgebers," imposing temporal order on development.

Animals↗
PubMed1987

Analysis of ecdysteroids in different developmental stages of Hymenolepis diminuta.

Prepatent and patent adult Hymenolepis diminuta from the intestines of rats, H. diminuta eggs recovered from the faeces of rats harbouring patent infections, and infective cysticercoids from the beetle intermediate host were analysed for free and conjugated ecdysteroids. Adult worms and eggs contained both free ecdysteroids and hydrolysable polar conjugated ecdysteroids, with comparatively large amounts of immunoreactive material also being detected following hydrolysis of the possible apolar conjugated ecdysteroid fraction. Free ecdysteroids were not detected in the cysticercoid sample. The concentration of free ecdysteroids in H. diminuta eggs was higher than that detected in the tissues of the adult worms. Ecdysone and 20-hydroxyecdysone were the major identified compounds of the free ecdysteroid fraction, whereas in the hydrolysed polar conjugated ecdysteroid fraction these two compounds were accompanied by 20,26-dihydroxyecdysone. The free ecdysteroid fraction also contained comparatively large amounts of unidentified immunoreactive material.

Animals↗
PubMed1987

Identity and tissue localization of free and conjugated ecdysteroids in adults of Dirofilaria immitis and Ascaris suum.

Adult males and females of the dog heartworm, Dirofilaria immitis, and of the swine parasite, Ascaris suum, were extracted, the free and polar conjugated ecdysteroid fractions separated and the latter hydrolysed enzymically. The ecdysteroids released by hydrolysis of the conjugates and the free hormones were analysed by radioimmunoassay, high-performance liquid chromatography on reversed phase and adsorption columns monitoring fractions by radioimmunoassay, and by gas-liquid chromatography/mass spectrometry (selected ion monitoring). In both species, males and females contained free and polar conjugated ecdysteroids, with evidence for the presence primarily of ecdysone and 20-hydroxyecdysone together with smaller amounts of 20,26-dihydroxyecdysone. Males and females of both species were then dissected into body fluid, reproductive system, gut and remaining body wall compartments, the ecdysteroids extracted, fractionated and analysed by radioimmunoassay and high-performance liquid chromatography monitoring fractions by radioimmunoassay. The results for both sexes in the two species were similar and indicated that ecdysteroids were not detectable in body fluids and that free ecdysteroids occurred in the reproductive system and the body wall, whereas polar conjugated ecdysteroids were detected in the reproductive system and the gut; a minor portion of the free ecdysteroids in A. suum was also apparently present in the gut. Further localization of the ecdysteroids in the body wall of A. suum females suggested that negligible immunoreactivity was associated with the circumpharyngeal nerve ring. The possible significance of the results is discussed.

Animals↗
PubMed1996

A new nonsteroidal chemical class of ligand for the ecdysteroid receptor 3, 5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide shows apparent insect molting hormone activities at molecular and cellular levels.

Insect molting hormones, ecdysteroids, found in natural products so far have steroidal structures. However, nonsteroidal ecdysteroid agonists show promise as a new type of insecticide and may be useful as probes for the biochemical study of ecdysteroids. 3, 5-Di-tert-butyl-4-hydroxy-N-isobutyl-benzamide (DTBHIB), discovered by the combination of the automated ecdysteroid receptor binding assay and the ecdysteroid responsive gene expression assay, showed both ecdysteroid receptor binding activity and induction activities of ecdysteroid responsive genes. Furthermore, the inhibition of the Kc cell proliferation and the induction of the cell process protrusion were observed in the presence of DTBHIB in a dose response manner. A similar compound of DTBHIB, however, could neither bind the receptor nor induce the responsive gene. These results provide strong evidence for the ecdysteroid agonist activity of DTBHIB, which has a novel nonsteroidal chemical structure, and might be a valuable lead chemical for higher potential ecdysteroid agonists.

Animals↗