Ecdysone metabolism in Pieris brassicae during the feeding last larval instar

Ecdysone metabolism in Pieris brassicae during the feeding last larval stage was investigated by using ³H-labeled ecdysteroid injections followed by high-performance liquid chromatographic (HPLC

1 Abbreviations: 3DE = 3-dehydroecdysone; 3D20E = 3-dehydro-20-hydroxyecdysone; 2026E = 20,26-dihydroxyecdysone; E = ecdysone; Eoic = ecdysonoic acid; 2026E′ = 3-epi-20,26-dihydroxyecdysone; E′ = 3-epiecdysone; E′oic = 3-epiecdysonoic acid; E′8P = 3-epiecdysone 3-phosphate; 20E′ = 3-epi-20-hydroxyecdysone; 20E′3P = 3-epi-20-hydroxyecdysone 3-phosphate; FT = Fourier transform; HPLC = high-performance liquid chromatography; 20E = 20-hydroxyecdysone; 20Eoic = 20-hydroxyecdysonoic acid; NMR = nuclear magnetic resonance; NP-HPLC = normal phase HPLC; RP-HPLC = reverse phase HPLC; TFA = trifluoroacetic acid; Tris = tris(hydroxymethyl)-aminomethane.

) analysis of metabolites. Metabolites were generally identified by comigration with available references in different HPLC systems. Analysis of compounds for which no reference was available required a large-scale preparation and purification for their identification by ¹H nuclear magnetic resonance spectrometry. The metabolic reactions affect the ecdysone molecule at C-3, C-20, and C-26, leading to molecules which are modified at one, two, or three of these positions. At C-20, hydroxylation leads to 20-hydroxyecdysteroids. At C-26, hydroxylation leads to 26-hydroxyecdysteroids which can be further converted into 26-oic derivatives (ecdysonoic acids) by oxidation. At C-3, there are several possibilities: there may be oxidation into 3-dehydroecdysteroids, or epimerization possibly followed by phosphate conjugation. Thus, injected 20-hydroxyecdysone was converted principally into 20-hydroxyecdysonoic acid, 3-dehydro-20-hydroxyecdysone, and 3-epi-20-hydroxyecdysone 3-phosphate. Labelled ecdysone mainly gave the same metabolites doubled by a homologous series lacking the 20-hydroxyl group.     

Ectopic overexpression of Drosophila lamin C is stage-specific lethal

To gain insight into the function of the developmentally regulated A-type lamins we transformed Drosophila melanogaster with a construct containing the hsp70 promoter followed by the Drosophila lamin C (an analog of vertebrate A-type lamins) cDNA. Lamin C was expressed ectopically after heat shock of embryos and localized to the nucleus. No phenotypic change was observed after lamin C expression in embryos that normally do not contain lamin C. However, ectopic expression of lamin C during most larval (but not pupal) stages stalled growth, inhibited ecdysteroid signaling (in particular during the larval-prepupal transition), resulted in development of melanotic tumors, and finally caused death. During pupation in control animals, when massive apoptosis of larval tissues takes place, lamin C is proteolyzed into a fragment with a size similar to that predicted by caspase cleavage. The ectopically expressed lamin C is identically cleaved, resulting in a large increase of the steady-state level of the lamin C fragment. A null mutation of the dcp-1 gene, one of the two known Drosophila caspase genes, also results in development of melanotic tumors and larval death, suggesting that the ectopically expressed lamin C inhibits apoptosis through competitive inhibition of caspase activity.     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

Notes on gamete production in Lanice conchilega (Annelida,Polychaeta, Terebellidae)

Summary

Male and female gametogenesis in Lanice conchilega are described and illustrated as seen by light microscopy. There is no evidence for size-selectivity in the uptake of male gametes by the hypertrophied reproductive nephromixia, since undissociated motile sperm platelets as well as spermatozoa appear to be taken up by the genital funnels, and such funnels show no sexual dimorphism. Maturation of the coelomic oocytes is marked by a change in shape and the appearance of a surface reticulum of ridges.     

Ovarian ecdysteroids and their secretion in late-pharate adults of Galleria mellonella

Approximately two-thirds of the total amount of ecdysteroids in late—pharate adults of the wax moth, Galleria mellonella, were found in the ovaries and one-third in the ovariectomized body. Chemical analysis of these ecdysteroids by thin-layer and high-pressure liquid chromatography, coupled with an ecdysteroid radioimmunoassay, revealed the presence of 2-deoxyecdysone, ecdysone and 20-hydroxyecdysone, as well as high and low polarity unknowns. The predominant identifiable ecdysteroid in both the ovaries and ovariectomized body was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone, respectively. Incubation of late-pharate adult ovaries in culture medium revealed that they synthesize and secrete ecdysteroids in vitro. The in vitro distribution of ecdysteroids between ovaries and incubation medium was similar to that observed between ovaries and ovariectomized bodies in situ and the predominant identifiable moiety both retained and released by the ovaries in vitro was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone. Collectively, these results support the idea that the ecdysteroids synthesized by the ovaries of late-pharate adult Galleria are both stored and secreted and that the quantity of a specifically secreted ecdysteroid is precisely controlled. This apparent regulation of the distribution of ovarian ecdysteroids raises the possibility that the stored and secreted forms have distinct functions in the reproductive physiology of this insect.     

昆虫蜕皮激素生物合成及其神经肽调控

蜕皮激素是对节肢动物体内类固醇激素的统称,昆虫的蜕皮激素主要由内分泌器官前胸腺合成,具有诱发幼虫周期性蜕皮以及最终变态蜕皮的生理功能。近期的研究工作阐明了前胸腺中原先被称为"黑箱"的一系列酶促反应步骤,此外促前胸腺激素受体的成功鉴定使人们对PTTH信号转导通路调控前胸腺蜕皮激素合成有了更深入的理解。     

Azadirachtin affects growth and endocrine events in larvae of the tobacco hornworm, Manduca sexta

Injection of azadirachtin in freshly emerged last-instar larvae of Manduca sexta elicited different reactions according to the dose administered. At low doses, pupation occurred in most of the cases, but the resulting pupae were defective for the most part. Individuals treated with higher doses usually did not fully complete development, moulting to supernumerary larvae or dying as larvae (sometimes at the wandering stage) after varying periods of survival. The haemolymph ecdysteroid titre of individuals treated with 2 μg azadirachtin/g bodyweight showed characteristic changes which are presumed to cause the disorders in the last stages that normally lead to pupation. Injection of moulting hormone in azadirachtin-treated individuals at certain times during the penultimate stage elicited no reduction of the azadirachtin-induced effects. It is shown that azadirachtin is able to inhibit development even when individuals performed a complete moult after the treatment.     

Ecdysteroids in Developing ovarian follicles of Hyalophora cecropia

Developing ovarian follicles of the silkmoth Hyalophora cecropia accumulate large amounts of ecdysteroids during oogenesis. As measured by an ecdysteroid radioimmunoassay (RIA), this accumulation begins near the end of vitellogenesis, just prior to nurse cell collapse, and continues through the beginning of chorion formation. Analysis of ovarian ecdysteroids by a combination of high-performance liquid chromatography and RIA demonstrates that the major proportion of these are present in a highly polar form, most likely as conjugates; ecdysone and 20-hydroxyecdysone were present as well, in much lower proportions. Light microscopic autoradiographs of photoactivated follicles after in vivo incubation with [³H]ecdysone indicate that within the oocyte ecdysteroids are associated with the yolk sphere membranes.     

ECDYSTEROIDS IN THE LARVA OF HAEMAPHYSALIS LONGICORNIS NEUMANN (ACARI: IXODIDAE)*

Abstract The ecdysteroids (ECs) concentration and their components in the larvae of Haemaphysalis longicornis were determined by radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). ECs ware found in feeding and engorged larvae and absent in unfed larvae. During feeding and the first 3 days after engorgement, ECs were in low level (less than 14. 35pg EE /larva). On the 5th day, they began to increase sharply (29. 87pg EE /larva) and reached their peak (56. 04pg EE /larva) on the 7th day. Then they declined to low value again (19. 38pg EE /larva). HPLC analysis revealed that the ECs components of the RIA peak could be 20-hydroxyecdysone (20E) and ecdysone (E) on the ratio of 20E: E = 3. 07: 1, which meant 20E was probably the main effective hormone.     

长角血蜱幼虫中的蜕皮甾类(英文)

本文用放射免疫分析和高效液相色谱法对长角血蜱幼虫中的蜕皮甾类进行了测定。　蜕皮甾类存在于吸血和饱血幼虫,并且随生理时期而变化。吸血期和饱血后前３天,激素含量低（小于 １４． ３５ｐｇ ＥＥ／只）;饱血后５天迅速上升（２９．８７ｐｇ ＥＥ／只）;饱血后７天达到高峰（５６． ０４ｐｇ ＥＥ／只）;高峰后又下降到低水平（１９．３８ ｐｇＥＥ／只）。高峰期的蜕皮甾类主要为两种成分,２０Ｅ和Ｅ（２０Ｅ∶Ｅ＝３．０７∶１）。２０Ｅ可能是幼虫发育中起主要作用的激素。     

Apolar conjugates of ecdysteroids are not used as a storage form of molting hormone in the argasid tick Ornithodoros moubata

Fifth (last) instar nymphs of the tick Ornithodoros moubata convert ingested 20-hydroxyecdysone (20E) to apolar conjugates AP2, which are then converted to the more polar conjugates AP1. Only small quantities of free hormone were transferred to the hemolymph and the carcass within the first 2 days after the blood meal. The proportion of radiolabel in these two compartments was highest at the time of the endogenous ecdysteroid peak; however, no traces of free [³H]20E were detected. The conversion probably occurs principally in the intestinal cells. Eleven days after ingestion, 84% of the radiolabel is located in the digestive tract, mainly in the form of AP1 conjugates. AP1 obtained in second instar nymphs fed with [³H]ecdysone ([³H]E) remain stable throughout the following nymphal instars. The ecdysteroid moiety of AP1 remained unchanged. The hydrolysis, although not complete, always yielded a peak comigrating with the reference E but never 20E or any other clearly distinct peaks that may have corresponded to metabolites of 20E. Less label per individual was present in adults, but its nature remained the same, viz., AP1 mainly located in the digestive tract. In females, 2.5% of the label was transferred to the progeny during the first ovipositional cycle. Apolar products (mainly AP2) that accumulated in eggs of females injected with [³H]E or [³H]20E during vitellogenesis remained unchanged during the whole embryonic development. During the molting cycle of larvae, there was only a slight conversion of AP2 to AP1, but esterase hydrolysis of these products released the same percentages of E and 20E as in the freshly laid eggs. We conclude that in this tick species apolar conjugates of ecdysteroids are inactivation metabolites that are not reutilized during the development of the animal. These metabolites are mainly retained in the tick, probably because of its peculiar blocked midgut. Several studies have shown that in other arthropod species (ticks, spiders, and insects), these apolar metabolites are excreted in the feces.     

A re-investigation on the ecdysteroids during embryogenesis in the desert locust, Schistocerca gregaria

Comparison of gas chromatography and high-pressure liquid chromatography methods for determination of ecdysteroids in insect eggs revealed that published values for Schistocerca gregaria were very low. The error has been traced to incomplete hydrolysis of conjugates. Revised values for the levels of ecdysone, 20-hydroxy-ecdysone and 2-deoxy-ecdysone are given here. The same general pattern of falling ecdysteroid titre during early stages of embryo development followed by an increase and a final decrease is observed. Experiments have been made on the effect of phosphate, ionic strength and a specific β-glucuronidase competitive inhibitor on the hydrolysis of the polar ecdysteroid conjugates, in an attempt to provide more evidence on the chemical nature of these compounds.     

滞育和非滞育棉铃虫血淋巴类固醇蜕皮素含量变化的比较

采用放射免疫分析法对不同时期的注定滞育和非滞育棉铃虫的血淋巴中的类固醇蜕皮素的含量进行了测定,发现在预蛹期间,注定滞育的棉铃虫的类固醇蜕皮素含量高于非滞育的棉铃虫,化蛹后,注定滞育的棉铃虫的类固醇蜕皮素含量则迅速降到极低的水平,明显低于非滞育棉铃虫。用20-羟基蜕皮素处理不同时期的滞育蛹,均能打破滞育。由此可见,类固醇蜕皮素含量的降低或缺乏乃是导致棉铃虫滞育的关键因子之一。     

Prothoracic gland involution related to moulting hormone levels during the metamorphosis of Tenebrio molitor L.

Prothoracic gland (PG) of Tenebrio shows ultrastructural changes which can be correlated with ecdysteroid levels (measured by radioimmunoassay) during larval-pupal development. However, the gland cells begin to degenerate before pupal-adult ecdysis: the PG involution is completed before the moulting hormone peak which triggers pupal-adult development. These facts strongly suggest that another endocrine organ produces moulting hormone needed for adult development.     

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.

     

An indirect mechanism by which a protein from the male gonad hastens salivary gland degeneration in the female ixodid tick, Amblyomma hebraeum.

In the adult female tick, Amblyomma hebraeum Koch (Acari: Ixodidae), salivary gland degeneration is triggered by an ecdysteroid, provided the female is above a critical weight (approximately 300-400 mg). In mated females, salivary gland degeneration is virtually complete within 4 days of detachment from the host. In virgin females, salivary gland degeneration is delayed by 4 days. This delay can be reversed by the injection of a male reproductive tract homogenate directly into the hemocoel. In this study, we consider a possible mechanism of action for this "male factor." Once mated, male factor likely gains access to its target tissue(s) as a humoral factor. Male factor, however, appears not to act by sensitizing the salivary glands to the action of ecdysteroids. Instead, it appears to act by accelerating the appearance of ecdysteroids in the hemolymph.     

Juvenile hormone regulates the titer of a hemolymph factor that enhances ecdysone synthesis by insect (Manduca sexta) prothoracic glands in vitro

The hemolymph of last instar Manduca sexta larvae contains a protein factor that enhances ecdysone synthesis by prothoracic glands in vitro. The titer of the factor fluctuates during development in a pattern that suggests that it is regulated by juvenile hormone (JH). In untreated control larvae, the titer drops from 2.17 U ml^?1 on day 1 to 0.27 U ml^?1 on day 3. When larvae were treated with (7S)-hydroprene (a JH analog), the titer remained elevated (2.09 U ml^?1 on day 3). JH I, however, was ineffective in preventing the precommitment drop in the titer of the factor. After pupal commitment, the titer of the factor increases in untreated larvae from 0.84 U ml^?1 on day 5 to 1.62 U ml^?1 on day 7. This increase was blocked when the sources of JH (the corpora allata) were removed on day 5 by head ligation. When head-ligated day 5 larvae were treated with either (7S)-hydroprene or JH I, the titer of the factor was driven to a level (1.88 U ml^?1 and 2.05 U ml^?1, respectively) that was not significantly different from that found in untreated day 7 larvae (1.62 U ml^?1). The combined results indicate the titer of the hemolymph factor is regulated by JH.     

Ecdysone metabolism in the host-parasitoid-system Trichoplusia ni/Chelonus sp

In unparasitized 4th and 5th-instar larvae of Trichoplusia ni and in 4th-instar larvae parasitized by Chelonus sp. 20-hydroxyecdysone, 20,26-dihydroxyec-dysone, and 20-hydroxyecdysonoic acid were the predominant metabolites formed 2 h after injection of [³H]ecdysone. Other unidentified metabolites were seen, but none seemed to be specific for either parasitized or unparasitized larvae. The major difference between parasitized and unparasitized larvae was seen with respect to the quantity of apolar (unidentified) and polar metabolites (20-hydroxyecdysonoic acid and unidentified ones), which were produced to a greater extent in parasitized larvae. Ecdysone was rapidly converted into 20-hydroxyecdysone and the other polar metabolites in all stages investigated, and the parasitoid seemed not to affect the conversion of ecdysone into 20-hydroxyecdysone. When analyzing the fate of [³H]ecdysone in host and parasite separately, at a stage when the parasite drinks hemolymph of its host, we observed that 10–20% of the radioactivity was recovered from the parasitoid. Analysis of the parasitoid's ecdysteroids revealed that ecdysone and 20-hydroxyecdysone represented only a small proportion of the recovered labeled ecdysteroids, the majority being apolar and polar metabolites. Our data suggest that the parasitoid takes up ecdysteroids from its host, converts them, and to some extent releases apolar metabolites into the host.     

THE ROLE OF ECDYSTEROIDS IN THE REPRODUCTIVE DIAPAUSE OF DERMACENTOR NIVEUS NEUMANN

Abstract  The ecdysteroid levels in hemolymph, ovary, synganglion and whole body of diapausing female Dermacentor niveus were detected by HPLC, and compared with the results of nondiapausing female. It is revealed that the ecdysteroid levels in hemolymph and ovary of diapausing female are similar basically to that of nondiapausing female in the first few days after engorgement. From the 10th day after engorgement, the ecdysteroid levels of diapausing female decreased and even became distinctly lower than that of nondiapausing female. The paucity of ecdysteroids in these individuals would influence the normal development of oocytes. In order to explore the effect of ecdyateroids on the diapausing female, we injected 20-hydroxyecdysone with different dosages at different time into the ticks, and found that after just complete engorgement the injection with large dosages (10000 and 1375 ng/tick) caused death of the ticks. From 10th to 20th day after engorgement the ecdysteroid levels of diapausing female are lower than that of nondiapausing one before oviposition, the injection with certain dosages 50, 70 and 100 ng/tick> of 20E can accelerate vitellogenesis and terminate reproductive diapause, but the amount of eggs produced by them is less than that produced by nondiapausing female. The termination of diapause in female of ixcdid tick by exogenous ecdysteroids is reported for the first time.