Ecdysteroid receptors in the neuroendocrine-endocrine axis of a moth

With a combination of thaw-mount autoradiography using a tritiated 20-hydroxyecdysone agonist, ponasterone A, and immunocytochemistry with a monoclonal antibody to 29 K-prothoracicotropic hormone, high affinity binding sites for ecdysteroids were identified in the tissues of the neuroendocrine-endocrine axis inManduca sexta larvae. At specific times during larval-pupal development in fifth stadium larvae, nuclear ecdysteroid binding sites were present in the cerebral prothoracicotropes, the corpora allata and prothoracic glands, the main axis for the regulation and production of ecdysteroids. A stage-specific appearance of ecdysteroid receptors also occurred in cells of fat body, midgut and Malpighian tubules, tissues which convert ecdysone into 20-hydroxyecdysone. Our data identify new target tissues for ecdysteroids and suggest that ecdysteroids could affect their own production at the genomic level via long and short feedback loops.     

Ecdysteroid receptors located in the central nervous system of an insect

Summary Using thaw-mount autoradiography for steroid hormones, we obtained direct evidence for a nuclear localization of ecdysteroid binding sites in target organs of blowfly (Calliphora vicina) larvae. The binding sites revealed properties of ecdysteroid receptors. Endocrine cells of the ring gland were found to be target tissues of ecydysteroids. This observation provides morphological evidence for a network of complex interendocrine regulation. In the central nervous system receptorcontaining neurons were identified which include many, if not all, neurosecretory cells of the brain. A map of ecdysteroid sensitive cells of the larval brain is presented.     

Role of low ecdysteroid levels in the early last larval instar ofBombyx mori

The relationship between juvenile hormone and ecdysteroid titers during the early stage of the last larval instar of the silkworm,Bombyx mori, was examined in this study. When larvae were fed 20-hydroxyecdysone-supplemented mulberry leaves throughout the last larval instar, 100% underwent supernumerary larval molting instead of metamorphosis. The application of juvenile hormone mimic during the early last instar did not induce supernumerary larval molting, but did delay metamorphosis. Temporal and quantitative ecdysteroid titer measurements revealed that in normal larvae the titers maintained very low levels (3–12 ng/ml) during the early stage of the last instar; however, in 20-hydroxyecdysone-treated larvae, levels ranging from 24 to 45 ng/ml were detected, and a major peak (246 ng/ml) was observed on day 6. These results show that very low ecdysteroid titer levels during the early stage of the last larval instar may play an important role in initiating decreases in juvenile hormone titers as well as in directing metamorphosis.     

Variations des taux d'ecdystéroïdes au cours du développement deBombyx mori; rapport entre ces variations et les phases de croissance et de morphogenèse

Summary During the development ofBombyx mori (monovoltin race) ecdysteroid levels were determined in oocytes, eggs, and haemolymph of larvae, and in the haemolymph of pupae. In haemolymph, the only RIA reactive materials are ecdysone and ecdysterone. In oocytes and eggs, other ecdysteroids are also detected. During larval instars, the ecdysteroid levels are low whereas they are very high during morphogenetic periods. During embryonic diapause, the ecdysone titer decreases during the cessation of morphogenesis.     

In vitro biosynthesis of juvenile hormone III by the corpora allata ofCalliphora vomitoria and its role in ovarian maturation and sexual receptivity

Summary JH III is the only JH detected by GLC-MS in medium from in vitro incubations of corpora allata of adult females ofCalliphora vomitoria. When corpora allata were removed from females at various times during the reproductive cycle and the JH III produced by the glands in vitro measured by a JH III radioimmunoassay, an increase in the level of synthesis was found to occur before previtellogenesis (0–24 h). A second increase appeared at the onset of vitellogenesis (72–83 h) and continued until the end of vitellogenesis (96 h) and the occurrence of chorionation (120 h). Since sexual receptivity develops with vitellogenesis, the significantly higher levels of JH III biosynthesis in vitro at this time supports a possible role for JH in the acquisitive of receptivity.     

Reversible juvenile hormone inhibition of ecdysteroid and juvenile hormone synthesis by the ring gland ofDrosophila melanogaster

Juvenile hormone bisepoxide (JHB₃) and juvenile hormone III (JH III) both inhibited the in vitro production of ecdysteroids by ring glands and brain-ring gland complexes from third instar post-feeding larvae ofDrosophila melanogaster in a reversible manner, although JHB₃ had greater efficacy. The JH III and JHB₃ precursor, methyl farnesoate, did not affect ecdysteroid production. The in vitro synthesis of total detectable JH (JHB₃+JH III+methyl farnesoate) by the corpus allatum portion of the isolated ring gland was also inhibited reversibly in the presence of exogenous JHB₃ and JH III, but not by methyl farnesoate. These data indicating negative feedback are in agreement with the accepted dogma of endocrine gland regulation.     

Ecdysteroid conjugates in the ovaries of the silkworm,Bombyx mori: 3-phosphates of 2,22-dideoxy-20-hydroxyecdysone and of bombycosterol

Summary Two novel ecdysteroid conjugates, 2,22-dideoxy-20-hydroxyecdysone 3-phosphate (1) and bombycosterol 3-phosphate (2), as well as four known ecdysteroid 22-phosphate esters, have been isolated and characterized from the ovaries of the silkworm,Bombyx mori.     

Haemolymph ecdysteroids level following the injection of ecdysone or ecdysterone; its relation with tegument and midgut response inAeshna cyanea (Insecta,Odonata)

Summary The haemolymph ecdysteroid level after injection of ecdysone or ecdysterone inAeshna cyanea larvae has been determined by a radioimmunoassay method. The rate of excretion appears to be dependent on both the ecdysteroid injected and the time of injection. In case of ecdysone injection, the secretion of the epidermis cuticle and the differentiation of the imaginal midgut epithelium occur when the ecdysteroid level remains low for many days.     

Insect hormones in vertebrates: Anabolic effects of 20-hydroxyecdysone in Japanese quail

Ecdysteroids are hormones controlling cell proliferation, growth and the developmental cycles of insects and other invertebrates¹. They are occasionally present in various unrelated plants for no apparent reason; no phytohormonal function has yet been identified. In certain cases, ecdysteroids are accumulated to high levels in leaves, roots or seeds. Some ecdysteroid-containing plants have been known as medicinal plants for centuries. One of them,Leuzea carthamoides Iljin (Asteraceae), growing in Central Asia, contains 0.4% ecdysteroid in dry roots and 2% in seeds. A pharmacological preparation from this plant, Ecdisten, is already available as a commercial preparation for its anabolic, tonic and other effects, for medical use (review²). It remained problematic, however, whether ecdysteroids were truly responsible for these effects, becauseLeuzea contains a number of other biologically active compounds in addition to ecdysteroids. We extracted and purified ecdysteroids from the seeds ofLeuzea. With 6 g of 96% 20-hydroxyecdysone (20E), we made a large-scale feeding assay with Japanese quail to find out whether ecdysteroid alone could duplicate the anabolic effects of the seeds. We found that the 96% ecdysteroid increased the mass of the developing quails in a dose-dependent manner, with the rate of increase proportional to the ecdysteroid content in the seeds; there was a 115% increase in living mass with 100 mg kg^–1 of pure 20E compared with 109.5% increase with 100–180 mg kg^–1 20E equivalents in the seeds. We conclude that the plethora of growth-promoting, vitamin-like effects induced in vertebrates byLeuzea is mediated by ecdysteroids.     

The association of phytoecdysteroids with flowering in fat hen,Chenopodium album,and other members of the Chenopodiaceae

Very high concentrations of ecdysteroid are associated with flowering inChenopodium album. Highest concentrations are found in anthers, but significant levels are also found in the stamens, carpels and sepals. In contrast, pollen contains only low levels. The ecdysteroid profile is the same in anthers as in whole-plant extracts, with 20-hydroxyecdysone and polypodine B predominating. The results for flowers ofC. album are compared with the patterns determined for other chenopods (C. capitatum, C. polyspermum, C. anthelminticum, C. giganteum, C. quinoa andC. foliosum). The significance of these findings for plant-insect interactions and the relationship to the mode of plant pollination are discussed.     

Ecdysteroid titre and caste determination in the ant,Pheidole pallidula (Nyl.) (Hymenoptera: Formicidae)

Summary In the antPheidole pallidula, ecdysteroid level is higher in worker-biased eggs than queen-biased eggs. Moreover queens laying worker-biased eggs exhibit a higher ecdysteroid level than queens laying queen-biased eggs.     

In vitro secretion of ecdysteroids by Y-organs of the crayfish,Procambarus clarkii

It was demonstrated that excised Y-organs of the crayfish,Procambarus clarkii, synthesize in vitro 3-dehydroecdysone (3-DHE) as the major product, together with small amounts of ecdysone. Both were identified by immunological and spectroscopic methods. The increase of ecdysteroidogenesis in the Y-organs was accompanied by an increase of the major free ecdysteroid, 20-hydroxyecdysone, in the hemolymph. This suggests a physiological role of 3-DHE, the details of which are still to be elucidated.     

Stimulation of polydnavirus replication by 20-hydroxyecdysone

During oviposition the endoparasitic waspCampoletis sonorensis, introduces a polydnavirus into parasitized insects where viral gene expression is required for endoparasite survival. The polydnavirus is integrated into wasp chromosomal DNA and replicates only in the ovary. Ecdysteroids regulate the developmental expression of many insect genes and may regulate polydnavirus replication. Direct verification of viral replication was performed by dot blot hybridization and by amplifying DNA sequences containing the viral integration site; this junction fragment cannot be amplified from integrated virus. Thoracic ligations and in vitro ecdysteroid treatments of wasp ovaries support the hypothesis that polydnavirus DNA replication is regulated by ecdysteroid during parasite development.     

Identification of 2-deoxyecdysone from the mite,Tyrophagus putrescentiae

2-Deoxyecdysone was purified from the miteTyrophagus putrescentiae and monitored by a radioimmunoassay. The compound was identified by GC-MS and LC-MS. This is the first report of an endogenous ecdysteroid from a mite.     

Exogenous makisterone A accelerates early embryonic development in the milkweed bugOncopeltus fasciatus

Summary Reproducing females ofOncopeltus fasciatus which were treated with exogenous makisterone A and 20-hydroxyecdysone laid eggs with considerably elevated ecdysteroid contents. The early embryonic development was markedly accelerated when the mother was treated with makisterone A, whereas 20-hydroxyecdysone had no influence.Supported by grants of the Deutsche Forschungsgemeinschaft (Do 163/7) to A. D.The technical assistance of Ms Heike Bender is acknowledged. We thank Dr K.-D. Spindler for providing us with antiserum for the RIA.     

Reversion of virus N-1 resistant mutant of blue-green algaNostoc muscorum

Summary The reversion of N-1 virus resistant strain of the algaNostoc muscorum was studied by inoculating parent virus in the resistant culture at various incubations. A fraction of virus resistant cells reverted to wild sensitive type with the reversion rate of 3.99×10^–6/cell/generation.We gratefully acknowledge Dr H.K. Pande and Dr S. Patnaik, Crops and Soils Division.     

Histidine-induced injury to cultured liver cells,effects of histidine derivatives and of iron chelators

The amino acid histidine is an excellent buffer and is therefore included in several organ preservation solutions used in transplantation medicine. However, when used at concentrations as in these solutions, histidine has a marked injurious potential. Therefore, we here assessed the mechanism of histidine-induced cell injury and searched for ways to use the buffering power of histidine but avoid histidine toxicity. When cultured hepatocytes were incubated in HTK solution or in modified Krebs-Henseleit buffer containing 198 mM L-histidine at 37°C, most cells lost viability within 3 h (LDH release 86 ± 7% and 89 ± 5%, respectively). This injury was accompanied by marked lipid peroxidation, and was strongly inhibited by hypoxia, by the antioxidants trolox, butylated hydroxytoluene and N-acetylcysteine and by the membrane-permeable iron chelators 2,2′-dipyridyl, 1,10-phenanthroline, LK 614, LK 616 and deferoxamine. Thus, histidine-induced cell injury appears to be mediated by an iron-dependent formation of reactive oxygen species. D-Histidine, imidazol and L-histidine methyl ester also elicited marked injury, while the N-substituted derivatives Nα-acetyl-L-histidine and tert-butyl-oxycarbonylhistidine and histidine-containing dipeptides showed almost no toxicity. Histidine toxicity, its iron dependence and the superiority of Nα-acetyl-L-histidine were also evident during/after cold (4°C) incubations. Therefore, we suggest the addition of iron chelators to histidine-containing solutions, and/or replacing histidine with Nα-acetyl-L-histidine in organ preservation solutions. Received 23 October 2006; accepted 21 November 2006     

On the distribution of phytoecdysteroids in plants

The occurrence and levels of phytoecdysteroids in the seeds and other parts of plants grown from the seeds of 180 randomly selected plant species were assessed and compared. Ecdysteroids are frequently detectable in leaves and flowers, but less so in stems, roots and seeds. The seeds of 290 species were assessed for the presence of hydrolysable ecdysteroid conjugates. Low levels of conjugates were detected in a significant number of species, large amounts being present only when levels of free ecdysteroids were high. Individual plants of Arabidopsis thaliana were assessed for the presence of phytoecdysteroids. While plants of this species are generally ecdysteroid negative, individual plants in the population accumulate low levels of ecdysteroids. Extracts of seeds of 50 "ecdysteroid-negative" species were concentrated and partially purified to determine if they possess ecdysteroids at levels below the usual detection levels. Ecdysteroids were detectable by radioimmunoassay in almost all of these concentrated samples. Thus, all lines of evidence point to the conclusion that all species of plants have the capacity to produce at least low levels of phytoecdysteroids. This has important implications for the protection of crop species through enhancing ecdysteroid levels by breeding/genetic modification strategies. Received 2 April 2001; received after revision 4 May 2001; accepted 22 May 2001     

Ecdysteroids in vitro promote differentiation in the accessory glands of male mealworm beetles

Summary Physiological peak doses of 20-hydroxyecdysone were added to organ cultures of young pupal accessory glands of maleTenebrio molitor. During subsequent culture in vitro or in vivo, the glands accumulated adult-specific antigens. Control organ cultures showed no such antigen accumulation. In this system, ecdysteroid controls not only cell cycles but also differentiation.21 October 1986This research was supported in part by a National Institutes of Health Research Service Award F32 AM7515 (KAG) and NIH grants GM26140 and AI15662 (GMH). We thank Toshinobu Yaginuma for help in developing the transplantation technique.     

Juvenile hormones inThermobia domestica females: Identification and quantification during biological cycles and after precocene application

Summary JH I and JH III immunoreactive substances were detected in the hemolymph of imaginal females of the primitive insectThermobia domestica. Periodic changes in the levels of these hormones were investigated in correlation with molting and reproductive cycles in inseminated, virgin and precocene-treated females. The presumed influence of JH III on the various phases of vitellogenesis is discussed, also taking into account the periodic changes of the hemolymphatic ecdysteroid levels.