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International audience; Endocrine-disrupting chemicals encompass a variety of chemicals that may interfere with the endocrine system and produce negative effects on organisms. Among them, bisphenol A is considered a major pollutant in numerous countries. The harmful effects of BPA on environmental and human health are intensely studied. However, the effects of BPA on terrestrial insects are still poorly investigated, despite that several plants can accumulate BPA in their tissues, leading to potential contamination of herbivorous insects. Here, we used the leafworm Spodoptera littoralis, a polyphagous species, to study BPA effects on post-embryonic development. We studied the effects of BPA ingestion at environmental doses (e.g., 0.01, 0.1, and 1 mu g/g of BPA) and high doses (e.g., 25 mu g/g) on larval weight and stage duration, pupal length and sex ratio. BPA effects were investigated in more detail during the last larval instar, a crucial period for preparing pupation and metamorphosis, which are under endocrine control. We monitored the haemolymph concentration of ecdysteroids, hormones controlling moult and metamorphosis, as well as the expression levels of several nuclear receptors involved in the ecdysteroid signalling pathway. Our integrative study showed that, upon exposure doses, BPA can induce various effects on the viability, developmental time, growth and sex ratio. These effects were correlated with a delay of the ecdysteroid peak during the last larval instar and a modification of expression of EcR, USP, E75AB, E75D and Br-c. We provide new evidence about the events that occur after BPA exposure in insect contaminated by food ingestion.

In many animals, male copulation is dependent on the detection and processing of female-produced sex pheromones, which is generally followed by a sexual refractory post-ejaculatory interval (PEI). In the male moth, Agrotis ipsilon, this PEI is characterized by a transient post-mating inhibition of behavioral and central nervous responses to sex pheromone, which prevents males from re-mating until they have refilled their reproductive tracts for a potential new ejaculate. However, the timing and possible factors inducing this rapid olfactory switch-off are still unknown. Here, we determined the initial time delay and duration of the PEI. Moreover, we tested the hypothesis that the brain, the testis and/or the sex accessory glands (SAGs) could produce a factor inducing the PEI. Lastly, we investigated the possible involvement of ecdysteroids, hormones essential for development and reproduction in insects, in this olfactory plasticity. Using brain and SAG cross-injections in virgin and newly-mated males, surgical treatments, wind tunnel behavioral experiments and EIA quantifications of ecdysteroids, we show that the PEI starts very shortly after the onset of copulation, and that SAGs contain a factor, which is produced/accumulated after copulation to induce the PEI. Moreover, SAGs were found to be the main source of ecdysteroids, whose concentration decreased after mating, whereas it increased in the haemolymph. 20-Hydroxyecdysone (20E) was identified as the major ecdysteroid in SAGs of A. ipsilon males. Finally, 20E injections did not reduce the behavioral pheromone response of virgin males. Altogether our data indicate that 20E is probably not involved in the PEI.

We investigated here the ecdysteroid titers and the expression of six genes coding for known enzymes of the ecdysteroid biosynthesis in the testes of last instar larvae of the pest cotton leafworm, Spodoptera littoralis. We showed that the timing of the ecdysteroid profile was the same in testes and in hemolymph, with a small peak at day 2 and a large one at day 4 after ecdysis. Ecdysone and 20-hydroxyecdysone (20E) were detected in both tissues. 20E was the major ecdysteroid in testes and in hemolymph from day 4. Interestingly, the gene expression of the steroidogenetic enzymes, Neverland, and the five cytochrome P450 enzymes encoded by the Halloween genes was confirmed in the testes, and varied during the instar. However, from the data obtained so far, we cannot conclude that the measured ecdysteroids in the testes result from the activity of the genes under study. Indeed, it is suggested that the ecdysone produced centrally in the prothoracic glands, could have been transformed into 20E in the testes, where Sl-shade is well expressed.

We investigated the distinct physical controls causing entombment and asphyxiation, the key mechanisms influencing salmonid egg-to-emergence (EtE) survival. Entombment occurs when sediment blocks the interstitial pathways (macropores) that larvae use to emerge from the streambed, while asphyxiation is related to low oxygen flux, which is a function of interstitial flow velocity. EtE survival has been related to substrate composition and flow velocity. However, in streambed sediments these variables are correlated, and few studies have examined the sensitivity of EtE survival to changes in velocity and oxygen flux at fixed substrate composition. EtE survival has not yet been directly related to the size and density of macropores. We incubated brook trout ( Salvelinus fontinalis ) embryos in artificial redds with different sediment compositions and hydraulic gradients to examine independently the effects of substrate composition, macropore geometry, and flow velocity on EtE survival, emergence timing, and fry condition. In situ measurements of macropore size were obtained using a computed tomography scanner. Despite high oxygen concentrations, we observed that entombment or blockage effects caused high embryo mortality in fines-rich substrates with few large macropores, and triggered early emergence of rare survivors. These outcomes could not be mitigated by increased flow velocity and oxygen flux to the egg pocket.

In the present work, juvenile development and physiological state of mygalomorph Brachypelma albopilosum were investigated by means of individual rearing under controlled conditions. Males required 4-5 years for development from first juvenile instar to adulthood, passing through 8 to 12 juvenile molts. Females developed to adults in 5-6 years with a variable juvenile molt number from 9 to 13. The development and growth of males and females took place in a similar way until the last juvenile molt leading to subadults. Ecdysteroids, total lipid, cholesterol, and protein concentrations increased along with the different development instars in both males and females. After the last juvenile molt, spiders presented morphological and biochemical sex differences. Subadult and adulthood males were smaller in size and weight than females; hemolymph levels of ecdysteroids, total lipids, cholesterol, and glucose were higher in males. These physiological and biochemical differences can be correlated to the different sexual development between males and females.

Variations in ecdysteroid titers play crucial roles in arthropods by initiating and regulating molting and metamorphosis. The recent identification of genes coding for cytochrome P450 enzymes involved in Drosophila ecdysteroidogenesis provides new molecular tools to investigate the regulation of insect hormone production. In the present study, we used an enzyme immunoassay to show that the molting hormone titer is strictly correlated with the steroidogenic capacity of the ring gland. A temporal correlation between dynamics of ecdysone production and expression of genes encoding steroidogenic enzymes was observed during the third instar, suggesting that the timing of hormone production depends on transcriptional regulation of the biosynthetic enzymes. Using clonal analysis, levels of two steroidogenic enzymes, Phantom (PHM) and Disembodied (DIB), were shown to be very reduced in ftz transcription factor 1 (ftz-f1) mutant ring gland cells whereas there was no effect of the without children (woc) mutation, suggesting that FTZ-F1 regulates phm and dib expression. Since βFTZ-F1 is the homolog of the vertebrate steroidogenic factor 1 (SF1), which plays a key role in the differentiation of vertebrate steroidogenic organs through transcriptional regulation of steroidogenic enzymes, this study emphasizes the strong parallels between insects and vertebrates with respect to the regulatory mechanisms of steroidogenesis.

International audience; In most animals, steroid hormones are crucial regulators of physiology and developmental life transitions. Steroid synthesis depends on extrinsic parameters and autoregulatory processes to fine-tune the dynamics of hormone production. In Drosophila, transient increases of the steroid prohormone ecdysone, produced at each larval stage, are necessary to trigger moulting and metamorphosis. Binding of the active ecdysone (20-hydroxyecdysone) to its receptor (EcR) is followed by the sequential expression of the nuclear receptors E75, DHR3 and beta Ftz-f1, representing a model for steroid hormone signalling. Here, we have combined genetic and imaging approaches to investigate the precise role of this signalling cascade within theprothoracic gland (PG), where ecdysone synthesis takes place. We show that these receptors operate through an apparent unconventional hierarchy in the PG to control ecdysone biosynthesis. At metamorphosis onset, DHR3 emerges as the downstream component that represses steroidogenic enzymes and requires an early effect of EcR for this repression. To avoid premature repression of steroidogenesis, E75 counteracts DHR3 activity, whereas EcR and beta Ftz-f1 act early in development through a forward process to moderate DHR3 levels. Our findings suggest that within the steroidogenic tissue, a given 20-hydroxyecdysone peak induces autoregulatory processes to sharpen ecdysone production and to confer competence for ecdysteroid biosynthesis at the next developmental phase, providing novel insights into steroid hormone kinetics.

Summary We have characterised the major circulating ecdysteroid in the South African spiny lobster, Jasus lalandii, as 20-hydroxyecdysone (20E) by means of chromatographic separation and an enzyme immunoassay (EIA). The titre of ecdysteroids in the haemolymph, as quantified by EIA, varied greatly during the moult cycle: it was highest in premoult and lowest in postmoult stages. A significant decrease in ecdysteroid levels was measured shortly before ecdysis. Since the major circulating ecdysteroid in J. lalandii is apparently 20E, which arises from peripheral 20-hydroxylation of ecdysone, we concluded that the Y-organs of this spiny lobster are synthesising ecdysone or/and 3-dehydroecdysone. In order to establish an in vitro assay with which to characterise moult-inhibiting hormone (MIH) activity in the sinus gland, we have incubated Y-organs from J. lalandii in vitro in the presence of crude extracts of homospecific sinus glands and measured the secreted ecdysteroid titres by EIA. Our experiments show th...

Ecdysteroid biosynthesis was analyzed in vitro using dissociated Y-organ cells from the shore crab Carcinus maenas . 3-Dehydroecdysone (3DE) was detected as a minor secretory product, in addition to the formerly identified end-products 25-deoxyecdysone and ecdysone (E). In conversion studies, 3DE was formed from tritiated 5 β -ketodiol (2,22,25-trideoxyecdysone), 2,22deoxyecdysone and 2-deoxyecdysone but not from E. Further experiments were performed in order to understand the interconversions between 3-oxo and 3 β -OH compounds in the crab Y-organ. The enzyme involved in 3 β -dehydrogenation was not ecdysone oxidase, a soluble enzyme found in peripheral tissues of many arthropods but it presented strong similarities with 3 β -hydroxysteroiddehydrogenase enzymes from vertebrates: it was membrane-bound and NAD + -dependent. Moreover, a NADH-dependent 3 β -reduction of several 3-oxo-ecdysteroids was obtained using the same microsomal fraction (100 000× g pellet) of Y-organs, indicating that the reaction might be reversible. As this activity was specific of molting glands, we hypothesize that there is at least one 3 β -hydroxysteroid dehydrogenase enzyme involved in the biosynthetic pathway of ecdysteroids.

Limiting Your Options Many species are dependent on specific resources that may limit their range of hosts to that of a few or even a single species. The restriction of the fly Drosophila pachea to senita cactus is a classic case of an ecological specialization. Lang et al. (p. 1658 ) reveal how multiple amino acid substitutions in the neverland gene have rendered D. pachea dependent upon the lathosterol of the senita cactus. Thus, relatively few genetic changes can play a large role in determining a species' ecological niche.

Summary Ecdysteroids secreted in vitro by Y-organs of the white shrimp Penaeus vannamei were quantified at different stages of the molt cycle by enzyme immunoassay (EIA). The production was maximal during the first hour of incubation, and the highest values were obtained with Y-organs from animals at molt stages D1′″. and D2-D3. Ecdysteroid production increased in parallel with the rise in hemolymph ecdysteroid levels, which peaked at the late premolt stage D1′″. A preliminary characterization showed that 20-hydroxyecdysone was the major circulating ecdysteroid. Ecdysteroids present in the incubation median of Y-organs were measured after HPLC separation. Three major immunoreactive compounds comigrated with standard ecdysteroids, 20-hydroxyecdysone, ecdysone and 3-dehydroecdysone; a fourth, less polar immunoreactive product was also detected. 3-Dehydroecdysone appeared to be the predominant secretory product, and the other compounds could arise from its metabolism by contaminating epidermis cells in the Y...

The metabolism of tritiated 20-hydroxyecdysone was investigated in vitellogenic females of Labidura riparia. It proceeds mainly through conjugation reactions, in particular through acetylation at C-3 and phosphate ester formation at C-2. The acetylphosphate derivative was the major conjugate found in different tissues. This ecdysteroid derivative appears to be present as the major endogenous conjugate in hemolymph of vitellogenic females but only as a minor one in ovaries.

Ecdysteroids are steroid hormones, which coordinate major developmental transitions in insects. Both the rises and falls in circulating levels of active hormones are important for coordinating molting and metamorphosis, making both ecdysteroid biosynthesis and inactivation of physiological relevance. We demonstrate that Drosophila melanogaster Cyp18a1 encodes a cytochrome P450 enzyme (CYP) with 26-hydroxylase activity, a prominent step in ecdysteroid catabolism. A clear ortholog of Cyp18a1 exists in most insects and crustaceans. When Cyp18a1 is transfected in Drosophila S2 cells, extensive conversion of 20-hydroxyecdysone (20E) into 20-hydroxyecdysonoic acid is observed. This is a multi-step process, which involves the formation of 20,26-dihydroxyecdysone as an intermediate. In Drosophila larvae, Cyp18a1 is expressed in many target tissues of 20E. We examined the consequences of Cyp18a1 inactivation on Drosophila development. Null alleles generated by excision of a P element and RNAi knockdown of Cyp18a1 both result in pupal lethality, possibly as a consequence of impaired ecdysteroid degradation. Our data suggest that the inactivation of 20E is essential for proper development and that CYP18A1 is a key enzyme in this process.

6 páginas, 5 figuras -- PAGS nros. 487-492, Grbic, Miodrag et.al., The spider mite Tetranychus urticae is a cosmopolitan agricultural pest with an extensive host plant range and an extreme record of pesticide resistance. Here we present the completely sequenced and annotated spider mite genome, representing the first complete chelicerate genome. At 90 megabases T. urticae has the smallest sequenced arthropod genome. Compared with other arthropods, the spider mite genome shows unique changes in the hormonal environment and organization of the Hox complex, and also reveals evolutionary innovation of silk production. We find strong signatures of polyphagy and detoxification in gene families associated with feeding on different hosts and in new gene families acquired by lateral gene transfer. Deep transcriptome analysis of mites feeding on different plants shows how this pest responds to a changing host environment. The T. urticae genome thus offers new insights into arthropod evolution and plant–herbivore interactions, and provides unique opportunities for developing novel plant protection strategies, M.G. and V.G. acknowledge support from NSERC Strategic Grant STPGP 322206-05, Marie Curie Incoming International Fellowship, OECD Co-operative Research Programme: Biological resource management for Sustainable Agricultural Systems JA00053351, and Ontario Research Fund–Global Leadership in Genomics and Life Sciences GL2-01-035. The genome and transcriptome sequencing projects were funded by the Government of Canada through Genome Canada and the Ontario Genomics Institute (OGI-046), JGI Community Sequencing Program grant 777506 to M.G., a University of Utah SEED grant (to R.M.C.), and National Science Foundation (NSF) grant 0820985 (to R.M.C., Principal Investigator L. Sieburth); work conducted by the US Department of Energy Joint Genome Institute is supported by the Office of Science of the US Department of Energy under contract No. DE-AC02-05CH11231

Summary Neuroendocrine controls of vitellogenesis, gonadotrophic cycles and ecdysteroid levels were examined in Labidura riparia using microsurgical manipulations, hormonal treatments, enzyme immunoassay (EIA) and high performance liquid chromatography (HPLC). In normal females high levels of ecdysteroids occur both in ovaries and hemolymph at the end of each vitellogenic cycle. In allatectomized (CA−) and pars intercerebralis cauterized females (PI−) vitellogenesis is suppressed, and ovarian ecdysteroids are at a low level. Juvenile hormone (JH) treatments restore vitellogenesis and high ecdysteroid levels in both experimental cases. Pars lateralis cauterized females (PL−) display a continuous vitellogenesis; however, ecdysteroid levels are lower in ovaries and higher in hemolymph than in normal females. HPLC/EIA analysis of ovarian ecdysteroids was performed, and the effects of different hormonal treatments are described. Discussing the interactions between JH, ecdysteroids, gonadotrophic activity and n...

We have synthesized a tritiated form of 5β-cholest-7-ene-3,6-dione (5β-diketone) of high specific activity (9.2 TBq/mmol) in ten steps from deoxychenocholic acid, which possesses the desired A/B cis ring junction (5β-H configuration). We have conceived a synthetic pathway which permits labelling by tritiation at the final step and allowing for the introduction of the tritium label into both the side chain and the nucleus. We have examined the ability of insect endocrine glands (prothoracic glands) of Pieris Brassicae to use this molecule as a precursor of 3-dehydroecdysone and of ecdysone biosynthesis. No conversion can be detected. In the myriapod Lithobius forficatus only reduction of the 3-ketone can be observed. It seems that the 5β-diketone is not an intermediate in the ecdysone biosynthetic pathway.

Ecdysteroids secreted in vitro by the Y-organs of Orconectes limosus were measured by radioimmunoassay after HPLC-separation of culture media. Ecdysone and 3-dehydroecdysone were found as secretory products. Metabolic studies with [3H]ecdysone and [3H]3-dehydroecdysone showed that Y-organs are not able to convert 3DE into E or vice versa. The putative precursors 5β-ketodiol and 5-β-diketol (“3-oxoketodiol”) were both converted by Y-organs. [3H]5-β-ketodiol was converted into E. [3H]5β-diketol was converted into 3DE and E. Ketodiol was also observed as a conversion product of 5β-diketol. Our data suggest, that in Orconectes 5β-diketol is a good candidate for an intermediate in the ecdysteroid biosynthetic pathway and that there is a branching of the pathway leading to 3DE as well as to E, due to the activity of a 3-oxoecdysteroid-3β-reductase specific for less hydroxylated compounds.

Ecdysteroids exert many pharmacological effects in mammals (including humans), most of which appear beneficial, but their mechanism of action is far from understood. Whether they act directly and/or after the formation of metabolites is still an open question. The need to investigate this question has gained extra impetus because of the recent development of ecdysteroid-based gene-therapy systems for mammals. In order to investigate the metabolic fate of ecdysteroids in mice, [1α,2α- 3 H]20-hydroxyecdysone was prepared and injected intraperitoneally to mice. Their excretory products (urine + faeces) were collected and the different tritiated metabolites were isolated and identified. The pattern of ecdysteroid metabolites is very complex, but no conjugates were found, in contrast to the classical fate of the (less polar) endogenous vertebrate steroid hormones. Primary reactions involve dehydroxylation at C-14 and side-chain cleavage between C-20 and C-22, thereby yielding 14-deoxy-20-hydroxyecdysone, poststerone and 14-deoxypoststerone. These metabolites then undergo several reactions of reduction involving, in particular, the 6-keto-group. A novel major metabolite has been identified as 2β,3β,6α,22 R ,25-pentahydroxy-5β-cholest-8(14)-ene. The formation of this and the other major metabolites is discussed in relation to the various effects of ecdysteroids already demonstrated on vertebrates.

International audience; Ecdysteroids are polyhyclroxylated steroids that function as molting hormones in insects. 20-Hydroxyecdysone (a 27C-ecdysteroid) is classically considered as the major steroid hormone of Drosophila melanogaster, but this insect also contains 28C-ecdysteroids. This arises from both the use of several dietary sterols as precursors for the synthesis of its steroid hormones, and its inability to dealkylate the 28C-phytosterols to produce cholesterol. The nature of Drosophila ecdysteroids has been re-investigated using both high-performance liquid chromatography coupled to enzyme immunoassay and a particularly sensitive nano-liquid chromatography-mass spectrometry methodology, while taking advantage of recently available ecdysteroid standards isolated from plants. in vitro incubations of the larval steroidogenic organ, the ring-gland, reveals the synthesis of ecdysone, 20-deoxy-makisterone A and a third less polar compound identified as the 24-epimer of the latter, while wandering larvae contain the three corresponding 20-hydroxylated ecdysteroids. This pattern results from the simultaneous use of higher plant sterols (from maize) and fungal sterols (from yeast). The physiological relevance of all these ecdysteroids, which display different affinities to the ecdysteroid receptors, is still a matter of debate.

Resistance of a laboratory selected DDT strain of Drosophila melanogaster (RalDDT R ) has been found to be monofactorial and correlated to an increased level of activity of the cytochrome P450-dependent mixed function oxidase (MFO). Both strains metabolize DDT and deltamethrin via MFO activity. However, the resistant strain does it more rapidly. The amount of DDT metabolites, including kelthane, bis-4-chlorophenyl acid, bis-4-chlorophenyl-ethanol, and 1,1-bis ( p -chlorophenyl)2,2-dichloroethane, is approximately 9-fold greater with RalDDT R microsomes than with the wild-type strain Raleigh (Ral). Production of deltamethrin metabolites is 2.7-fold higher within the resistant strain. As compared to insecticides, lauric acid and the two steroids used as substrates in this study present many more sites for MFO metabolic action. Lauric acid is hydroxylated on positions 11 and 12 by both strains, but the amount of metabolites formed is 10-fold higher with RalDDT R microsomes. The 2,22-dideoxyecdysone is converted to two polar metabolites when incubated with RalDDT R microsomal preparations. These unidentified metabolites are neither 2-deoxyecdysone nor ecdysone. Also reported for the first time is the metabolization of testosterone by insect microsomes, which gives 13 oxiderivatives formed at different rates, depending on the strains.

Using the developmental mutant ecd1, the biological activity of 20-hydroxyecdysone (20E) and 20E metabolites 3-dehydro-20-hydroxyecdysone (3D20E), 3-epi-20-hydroxyecdysone (3D20E), 3-epi-20-hydroxyecdysone-3-phosphate (20E′3P), 20,26-dihydroxyecdysone (20,26E), and 20-hydroxyecdysonoic acid (20Eoic) was tested for their ability to induce the transcription of the steroid-inducible gene P1 in the Drosophila larval fat body. 3D20E was the most efficient ecdysteroid in the initiation of P1 gene transcription therefore the formation of 3D20E and the 3-epimer could not be regarded as an inactivation pathway in Drosophila larvae. Formation of 20,26E and 20Eoic may be an inactivation pathway in this biological model.

Protophormia terraenovae is a forensically important fly whose development time is studied by forensic entomologists to establish the time elapsed since death (post-mortem interval, PMI). Quantity and nature of ecdysteroid hormones present in P. terraenovae pupae were analysed in order to determine if they could be correlated to the age of pupae found on corpses and thereby could give information on the PMI. Ecdysteroid levels were quantified during the pupal-adult development of synchronised animals using enzyme immunoassay (EIA), a sensitive method allowing acurate quantification in one pupa. Two types of pupae were compared: "fresh" pupae, kept frozen until analysis and "experimentally dried" pupae, which were left for several weeks at ambient temperature. A peak of ecdysteroids was detected between 36 and 96 h after pupariation in fresh animals. It was not observed in "experimentally dried" pupae. High-pressure liquid chromatography (HPLC) analyses combined with EIA showed that 20-hydroxyecdysone (20E) was the major free ecdysteroid at various pupal ages. Enzymatic hydrolysis experiments revealed the presence of apolar conjugates at all ages tested. However, neither qualitative nor quantitative difference was detected between early and late pupae. This study gives precise information on the nature and quantity of ecdysteroids in the course of pupal development of a calliphorid fly. The limits of using ecdysteroid measurement as a tool in forensic entomology are discussed.

A cytochrome P450 enzyme cDNA (CYP4C15) has been previously cloned from a cDNA library of crayfish steroidogenic glands (Y-organs). The conceptual translation of the CYP4C15 cDNA sequence was analyzed for regions of putative high antigenicity and a mixture of two synthetic peptides was chosen for the production of a specific polyclonal antibody. Western blot analysis on Y-organ subcellular fractions indicated an endoplasmic reticulum location of CYP4C15, in agreement with the structural feature of the predicted protein, i.e. the presence of a hydrophobic N-terminal segment. The protein is only expressed in Y-organs, thus showing a similar distribution to the corresponding mRNA. From this tissue specific expression, it has been postulated that CYP4C15 would play a role in ecdysteroid biosynthesis rather than detoxification and the variations of its expression during a molt cycle were carefully examined. CYP4C15 is not detectable in intermolt animals, expression levels are maximal during early premolt and decrease during late premolt. The results are discussed in relation to the variations of hemolymphatic ecdysteroid titers and steroidogenic capacities of the Y-organs during the molt cycle.

Although the involvement of 3-oxo-delta 4 compounds as intermediates in arthropod ecdysteroid biosynthesis has been postulated for a long time, it has not yet been directly demonstrated. In the present study, 3-oxo-delta 4-steroids have been synthesized and incubated in vitro with dissociated moulting gland cells from the crab Carcinus maenas. The tritiated compounds were converted into 3-dehydroecdysone, ecdysone and/or 25-deoxyecdysone, i.e. final ecdysteroids. This means that the 3-oxo-delta 4 compounds had undergone a 5 beta-reduction, to give the 5 beta-conformation of ecdysteroids. Our results suggest that the 3-oxo-delta 4-steroid 4,7-cholestadien-14 alpha-ol-3,6-dione may be an intermediate in the biosynthetic pathway. The 5 beta-reduction reaction involves a cytosolic enzyme which requires NADPH as electron donor and seems specific for 3-oxo-delta 4 substrates. This reaction was the most active in crab Y-organs, as compared with other tissues. The characteristics of the 5 beta-reductase (subcellular localization, substrate and cofactor requirements) appear similar to those of the vertebrate 3-oxo-delta 4-steroid 5 beta-reductase involved in steroid hormone catabolism and bile acid biosynthesis.

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

Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) have complementary advantages in the study of radioactive metabolites of ecdysteroids. While lacking the high resolution of HPLC, radio-TLC gives a rapid, quantitative picture of the total range of metabolites present in a single elution. This is illustrated by recent studies on the metabolism of dietary [3H]ecdysone by Heliothis species. In-line radioactivity monitoring of HPLC has advantages of speed, resolution and reduced manipulation over conventional fraction counting.

Several 3-dehydro- (or 3-oxo-) ecdysteroids have been prepared by enzymatic and/or chemical means. Methods for their purification using various high-performance liquid chromatography systems are described. Proton and carbon nuclear magnetic resonance analyses show that 3-dehydroecdysteroids when dissolved in water or methanol (but not in chloroform) present a temperature-dependent equilibrium between two forms. The possible structure of these two forms is discussed.

HPLC allowed separation of twelve major labeled compounds after injection of 3H-ecdysone into Pieris pharate pupae. These compounds were identified as six pairs of metabolites (3 alpha and 3 beta epimers), comprising ecdysone, 20-hydroxyecdysone, 26-hydroxyecdysone, 20,26-dihydroxy-ecdysone and the polar metabolites P and 20-hydroxy-P. These last two products could not be enzymatically split by any hydrolase tested and are weak acids arising respectively from 26-hydroxyecdysone and 20,26-dihydroxyecdysone. They might be 26-oic compounds. Epimerization appears as a fundamental inactivation process in Pieris and could well be a general characteristic of closed systems (eggs and pupae). No significant amounts of hydrolyzable conjugates were detected in our biological system (pharate pupae and pupae).

An improved purification procedure is described for high-performance liquid chromatography (HLPC) analysis of ecdysteroids. It can be used for both qu antitative analyses of ecdysone and 20-hydroxlyecdysone, and for various metabolic studies. The procedure comprises two steps only: chloroform-water partition and adsorption/purification of the water phase solutes on a SEP-PAK® C 18 cartridge. The 60% methanol eluate can be used after evaporation for direct HPLC quantitative analysis of ecdysteroids on a C 18 -bonded column. The detection limit is about 10 ng of hormones for biological extracts. Makisterone A added before extraction is used as internal standard. Application of this procedure to metabolic studies requires all the metabolites to be retained on the SEP-PAD cartridge, as is the case for the insect system investigated, i.e ., Pieris brassicae (Lepidoptera) pupae. Examples of separations are given and the limits of the procedure are discussed.

The metabolism of ecdysone and 20-hydroxyecdysone has been investigated in various animal species, including representatives of arthropods, molluscs, annelids and mammals. Some of the major metabolites have been isolated and characterized by mass spectrometry and two-dimensional proton NMR. It appears that metabolic pathways differ strongly between species and that each class has evolved specific reactions.

Insects convert ecdysone and 20-hydroxyecdysone into their corresponding 26-oic derivatives, named ecdysonoic acid and 20-hydroxyecdysonoic acid respectively. The conversion takes piace in several tissues and can either be the only pathway for converting ecdysone into highly polar ecdysteroids, or coexist with various conjugating mechanisms. 20-Hydroxyecdysonoic acid was isolated from Pieris brassicae pupae as its methyl ester derivative. Its chemical structure was identified by Cl/D mass spectrometry and compared with a synthetic compound (20-hydroxy-25-deoxyecdysonoic acid) chemically prepared by oxidation of inokosterone (20,26-dihydroxy-25-deoxyecdysone). Natural ecdysonoic acids appear to exist as a mixture of 25R and 25S isomers. The significance of this pathway is discussed in comparison with similar reactions occuring in the metabolism of steroid hormones in vertebrates.

Various biochemical parameters including DNA, RNA, proteins, uric acid, and pteridines have been examined during the pupal-adult transformation of Pieris brassicae. Special consideration was given to two tissues: the wings and abdominal fat body. They showed characteristic patterns of development with respect to macromolecular syntheses that could be related to histological data and tentatively correlated with ecdysone levels during the same period. All these data were collected in order to know the in situ behaviour of some organs before the in vitro study of ecdysone action.

The formation and fate of 3-dehydroecdysteroids were studied in vitro in tissue extracts from Pieris brassicae (Lepidoptera). Three cytosolic enzymes are involved: an ecdysone oxidase converts 3 beta-hydroxyecdysteroids into 3-dehydroecdysteroids, and two different reductases, requiring NADPH as cofactor, transform 3-dehydroecdysteroids into the 3 alpha-hydroxy- or the 3 beta-hydroxy forms, respectively. 3 alpha-Hydroxyecdysteroid formation is only detected in the gut; all tissues contain the two other activities. It appears that conversion of 3 beta- to 3 alpha-hydroxyecdysteroids proceeds through 3-dehydro compounds. The biological significance of these reactions is discussed.

Ecdysone haemolymph levels and in vivo development of imaginal wing discs have been studied during the last larval instar of Pieris brassicae. During this period, β-ecdysone variations show two successive peaks, the first one related to the induction of wandering stage, and the second (main) one to pupal cuticle synthesis. The observed situation is very similar to that of Manduca sexta. Imaginal wing disc growth is composed of several genetically programmed steps that need the presence of ecdysone, but do not appear very closely linked to circulating hormone levels. It seems that ecdysone haemolymph peaks should be considered as periods where ecdysone levels are above a threshold value.