Comparative studies on ecdysone metabolism between mature larvae and pharate pupae in the fleshfly,Sarcophaga peregrina

Metabolites of radioactive ecdysone or 20-hydroxyecdysone in larvae and pharate pupae of Sarcophaga peregrina were separated and identified by using thin-layer chromatography, high-performance liquid chromatography, and chemical methods. At the larval stage ecdysone was metabolized to biologically less active ecdysteroids predominantly through 20-hydroxyecydsone, at the pharate pupal stage, to other ecdysteroids which were tentatively identified as 26-hydroxyecdysone, 3-epi-26-hydroxyecdysone, and 3-epi-20,26-dihydroxyecdysone. Ecdysteroid acids were found in the polar metabolites during pharate pupal-pupal transformation, but scarcely detected in the larval metabolites. These acids were presumed to be ecdysonoic acid, 20-hydroxyecdysonoic acid, and their epimers. The conjugates of ecdysteroid that released the free ecdysteroids by enzymatic hydrolysis were produced more in larvae than in pupae, whereas the very polar ecdysteroids that were not affected by the enzyme were found more in pupae. Therefore, there are different metabolic pathways of ecdysone between these two successive developmental stages, and the alteration of the metabolic pathway may serve as one of the important factors in a regulatory mechanism of molting hormone activity which is responsible for normal development of this insect.     

Ecdysteroid levels throughout the life cycle of the desert locust, Schistocerca gregaria

Moulting hormone levels for all stages of the life cycle of the desert locust, Schistocerca gregaria, have been determined using gas chromatography with electron capture detection of the trimethylsilylated hormones. During larval development, the major hormone detected is 20-hydroxyecdysone with smaller quantities of ecdysone present. In mature adult females the major ecdysteroid observed is a polar conjugate of ecdysone, with smaller quantities of conjugated 20-hydroxyecdysone also present. During embryonic development the pattern changes from a high proportion of conjugated ecdysone in the early stages to give more free hormone and a higher proportion of 20-hydroxyecdysone in later stages. The highest titre of 20-hydroxyecdysone found in this insect is during the 5th larval instar. Maximal levels of ecdysteroid per insect are found in mature females just before oviposition, while the highest level of ecdysteroid per g of tissue is found in the eggs.     

棉铃虫蛹期血淋巴的蜕皮甾类

目前为止仅在少数几种昆虫中研究过蛹期的蜕皮激素。关于蜕皮甾类的性质分析,结果也颇不一致。本文采用放射免疫分析、薄层层析、高压液相色谱及质谱对棉铃虫Heliothis armigera蛹血淋巴内的蜕皮激素进行了研究。结果如下:1.物理-化学方法证明蛹血淋巴内存在二种蜕皮甾类:蜕皮酮和20-羟基蜕皮酮。2.蛹期蜕皮甾类滴度呈一宽峰,高峰出现在化蛹后的第5天(3435ng/ml)。3.在高峰时,蜕皮酮与20-羟基蜕皮酮的比例为1:3.57,说明20-羟基蜕皮酮是主要的蜕皮甾类。4.比较雌雄两性蛹的蜕皮甾类滴度,未见明显差异。研究表明在棉铃虫中影响成虫发育的主要激素是20-羟基蜕皮酮而不是蜕皮酮。     

Ecdysteroids during pupal development of female Xyleborus ferrugineus

Ecdysteroid titers were estimated on the whole body homogenates of Xyleborus ferrugineus (Fabr.) female pupae during development by radioimmunoassay. A distinct peak of ecdysteroids was observed at 36-hr pupal development (743 pg/mg body wt). Titer declined to 299 pg/mg by the pharate adult stage and to 193 pg/mg body wt just before adult emergence. Qualitative studies by HPLC revealed a ratio of 3:1 ecdysone to 20-hydroxyecdysone in the initial pupal stage. Pharate adults had mainly 20-hydroxyecdysone. The observed single peak in ecdysteroid titer agrees with findings in other studied coleopteran species.

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Zusammenfassung Der Ecdysteroidtiter weiblicher Puppen von Xyleborus ferrugineus (Fabr.) wurde geschätzt, indem ganze Tiere homogenisiert und radioimmunologisch untersucht wurden. Ein ausgeprägtes Maximum an Ecdysteroiden wurde bei 36 Stunden Puppenent-wicklung beobachtet (743 pg/mg Körpergewicht). Der Titer nahm ab auf 299 pg/mg im Pharatstadium und auf 193 pg/mg unmittelbar vor Schlüpfen der Adulten. Qualitative Studien mit HPLC ergaben in frischen Puppen ein Verhältnis von 3:1 Ecdyson zu 20-Hydrooxyecdyson. Pharatstadien enthielten vor allem 20-Hydrooxyecdyson. Das beobachtete einzige Maximum im Titer stimmt überein mit den Resultaten bei andern untersuchten Coleopteren.

     

Haemolymph ecdysteroid in the housefly,Musca domestica,during oögenesis and its relationship with vitellogenin levels

Ecdysteroid titre in the haemolymph of the housefly, Musca domestica, cycled during oögenesis and peaked at ～50 pg/μl during stages 5, 6 and 7. Levels of 10–20 pg/μl were found in houseflies with pre- and post-vitellogenic ovaries. Removal of the corpus allatum and corpus cardiacum complex resulted in low ecdysteroid levels (10 pg/μl). Ovariectomized flies also had lower ecdysteroid levels than the controls at 2 days (5 pg/μl) after emergence but not at 6 days (22 pg/μl). It is possible that the ecdysteroid peak that occurred during stages 5, 6 and 7 was produced by the ovaries because ovaries secreted and synthesized ecdysteroid in vitro. Endogenous haemolymph ecdysteroid levels had a linear correlation with the amount of vitellogenin that held for hormone concentrations of 5–43 pg/μl. Furthermore, the injection of 20-hydroxyecdysone at doses of 10 ng?1.0 μg/fly increased the amount of vitellogenin from 6 h to 12 h after injection; by 24 h, the vitellogenin returned to control levels. When 20-hydroxyecdysone was injected into ovariectomized flies, it was rapidly degraded and 96% was cleared from the haemolymph within 1 h.     

Free ecdysteroids in adult male crickets, Gryllus bimaculatus

ABSTRACT. Ecdysteroid titres were determined in testes, fat body, muscles, haemolymph, carcass tissue, spermatophores, and faeces of males of the Mediterranean field cricket, Gryllus bimaculatus de Geer, throughout its adult life span. Considerable amounts of free ecdysteroids are concentrated in the testes and the fat body. The ecdysteroid titres were only slightly influenced by environmental temperature. In all tissues except the fat body, ecdysone and 20-hydroxyecdysone were the predominant ecdysteroids present. In faeces, highest ecdysteroid concentrations were found at the time of lowest levels in tissues.     

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

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

Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.     

Haemolymph ecdysteroid levels and cellular events in the intermoult/moult sequence of Calpodes ethlius

The haemolymph ecdysteroid titre of the last larval and pupal stadia of Calpodes ethlius was determined by radioimmunoassay. During the last larval stadium, four significant ecdysteroid peaks are present, two of which have been reported for other Lepidoptera. The first peak occurs 12 hr after ecdysis and correlates temporally with nucleolar activity, RNA synthesis and organelle formation in the fat body and epidermis. It correlates also with fat body DNA synthesis, polyploidy and the initiation of a low rate of lipid synthesis. Another peak, at 78 hr, starts its increase when the prothoracic glands no longer require the influence of the brain to produce ecdysone for pupation, and marks the first critical period. It correlates with the initiation of epidermal DNA synthesis and mitosis, and with the progressive determination of pupal characteristics (change in commitment, reprogramming). This ecdysteroid peak may also be involved in the massive intermoult syntheses in the epidermis (lamellate cuticle, wax) and the fat body (lipid, protein). The largest ecdysteroid peak is seen at 162 hr, 6 hr after the tissues no longer require the prothoracic glands for pupation (second critical period). It correlates temporally with the cessation of massive synthetic activity in both epidermis and fat body and initiates preparation for pupal synthesis in both tissues. At this time the ratio of ecdysone: 20-hydroxyecdysone is ～ 1 : 6.6.In common with other Lepidoptera, a single large ecdysteroid peak occurs during the first half of the pupal stadium. Comparisons between these events and the ecdysteroid titre are made between Calpodes and other insects.     

Effects of a fat body extract on larval midgut cells and growth of lepidoptera

Treatment with fat body extract (FBX) from pupae of the tobacco hornworm, Manduca sexta, caused mortality in larvae of two pest lepidopterans, the gypsy moth, Lymantria dispar, and the cotton leafworm, Spodoptera littoralis. In FBX-treated larvae, the feeding rate was depressed, causing reduced weight gain and then larval death. Their midgut showed formation of multicellular layers of midgut epidermis, indicating stem-cell hyperplasia. Hence, the integument of FBX-treated larvae had a double cuticle, indicating induction of premature molting. But radioimmunoassay measurements confirmed that the amount of ecdysteroids in FBX was too low to be responsible for the molt-inducing effects observed after treatment with FBX. With midgut stem cell cultures in vitro, addition of FBX to the culture medium stimulated cell proliferation and differentiation in a concentration-dependent manner. This effect was compared with those of insect molting hormones, ecdysone and 20-hydroxyecdysone; an ecdysteroid agonist, RH-2485; and a purified protein from FBX (multiplication factor). This article describes the mode of action of FBX and possible interplay between fat body factor(s) and insect hormones in the development and metamorphosis of the insect midgut.     

Haemolymph ecdysteroid titres in diapause- and non-diapause-destined larvae and pupae of Sarcophaga argyrostoma

No differences were observed between the rates of development of larvae and pupae from diapause- and non-diapause-destined lines of Sarcophaga argyrostoma except that those destined for diapause have a longer post-feeding, wandering, larval phase associated with a lower haemolymph ecdysteroid titre, as measured by radioimmunoassay. Following pupariation, both cultures show a high haemolymph titre associated with larval/pupal apolysis. The developing culture displays an ecdysteroid peak at 72 h after pupariation which may be involved with pupal/adult apolysis and the initiation of pharate-adult development. This peak is reduced in the diapause-destined culture. Following the initiation of pharate adult development, there is a very large peak at 85–90 h. Those pupae entering diapause display very low titres as a result of the failure of the brain/prothoracic gland axis to release ecdysone. There are no quantitative or qualitative differences between the titres of specific ecdysteroids in the prepupae of the two lines as determined by reverse-phase high-performance liquid chromatography. A preliminary examination of the levels of free and conjugated ecdysteroids has provided the basis for proposing a mechanism of ecdysone metabolism in this insect.     

Anteroposterior gradient during nymphal-adult moulting cycle of the tropical bont tick,Amblyomma variegatum (Acarina: Ixodidae)

Summary The structure of the extensible (alloscutum) and inextensible (scutum) integument of the nymph, Amblyomma variegatum was examined during the whole bloodmeal and the nymphal-adult moulting cycle. Integumental events were tentatively correlated with the ecdysteroid levels measured by radioimmunoassay. We observed that all the integumental events were realised along an anteroposterior gradient. During the 5 days corresponding to the bloodmeal, although the hormone concentration was low, a new endocuticle was deposited on both the alloscutum and scutum. Furthermore, mitoses were initiated in the capitulum. On days 1–2 after the meal, ecdysteroid titres began to increase and reached a first peak corresponding to 4.1 ng 20-hydroxyecdysone equivalents/tick on the 4th day after the ticks dropped off their host. At this time the epidermis of the capitulum was detached and the outline of the adult capitulum was already visible. Mitotic activity in the alloscutum was initiated. On day 6 post-drop, the frontal apolysis was achieved and the ecdysteroid titres declined to basal values. A second peak much higher than the first one (maximum value of 33.7 ng/tick) and identified principally as 20-hydroxyecdysone by HPLC/RIA was noted on the 13th day post-drop. During the period of increase in the ecdysteroid levels (days 9–10 post-drop), the mitotic phase ended in the alloscutum and the apolysis began. Epicuticle was deposited after day 12 postdrop. Then, while the titre fell to low values (about 1.6 ng/tick, days 16–20 post-drop), the exocuticle was deposited and the nymphal cuticle was digested. All adult structures were functional 3 days before ecdysis. In young male as in female adults the mean value of the ecdysteroid levels corresponded to about 2.5 ng/tick. Finally, hydrolysis of tick whole extracts with esterase demonstrated a low increase of RIA-positive material, demonstrating the probable presence of natural ecdysteroid fatty-acid conjugates in this species.     

Ecdysteroids in the developing eggs of the desert locust, Schistocerca gregaria

Ecdysteroid levels in the developing eggs of Schistocerca gregaria were determined, at daily intervals, using gas chromatography and electron capture detection of ecdysteroid derivatives. Ecdysone and 20-hydroxyecdysone were present both as the free ecdysteroid and as polar conjugates. Total ecdysteroids reached a maximum of 40 ng/egg with ecdysone contributing the greater part.     

The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria

Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g).     

Ecdysteroid profiles for hemolymph and testes from larvae,pupae, and pharate adults of the European corn borer,Ostrinia nubilalis hubner

Total ecdysteroid levels as well as concentrations of several individual ecdysteroids were determined for hemolymph and testes of fifth instars, pupae, and pharate adults of the European corn borer, Ostrinia nubilalis (Hubner). For total levels, the patterns of fluctuation in hemolymph and testes were similar, but the concentrations in testes were lower than those in hemolymph. In both hemolymph and testes there were two ecdysteroid peaks: the first just prior to the formation of the pharate pupa, the second just prior to the formation of the pharate adult. An examination of ecdysteroid profiles revealed some important differences. Ecdysone was either absent or present at extremely low levels in larval testes, whereas in hemolymph there was a premolt ecdysone peak. In pupal testes, ecdysone was present, but levels of 26-hydroxyecdysone were much lower than those in hemolymph. Thus, in regard to ecdysteroids, testes have the ability to control their own internal milieu.     

Physiological requirements for 20-hydroxyecdysone-induced rectal sac distention in the pupa of the silkworm, Bombyx mori

Successful insect development is achieved via appropriate fluctuation of ecdysteroid levels. When an insect's ecdysteroid level is disrupted, physiological and developmental defects occur. In the pupa of the silkworm, Bombyx mori, the rectal sac is an essential organ that operates as a repository for degraded ecdysteroids, and it can be distended by administration of 20-hydroxyecdysone (20E). Our previous study showed that rectal sac distention appears 4 days after 20E administration. Hemolymph ecdysteroid levels, however, decrease to lower level during this period. Thus, the timing of the rectal sac distention does not match with that of ecdysteroid elevation. Here, we examine how 20E induces rectal sac distention. A ligature experiment and ecdysteroid quantification showed that continuous 20E stimulation induces rectal sac distention. Thorax tissue contributed to the continuous 20E stimulation needed to induce distention. Ecdysteroid released from the thorax tissue may be converted to 20E by ecdysone 20-hydroxylase to produce continuous 20E stimulation. Thus, the ecdysone metabolic pathway plays a critical role in rectal sac distention.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.