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.     

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.     

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.     

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.     

Identification of mucondialdehyde as a novel stress metabolite

In a survey of antifungal stress compounds induced by cupric chloride we found that leaves ofChenopodium album exuded a highly fungitoxic metabolite mucondialdehyde (trans-2,trans-4-hexadienedial), which was associated with 13-oxo-9,11-tridecadienoic acids (cis-9,trans-11 andtrans-9,trans-11 isomers) presumably resulting from -scission of 13-hydroperoxy-octadecadi(tri)enoic acid. The biogenesis and role as a general defensive agent in plants are briefly discussed.     

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.     

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.     

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     

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.     

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.     

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.     

Titers of ecdysone, 20-hydroxyecdysone and juvenile hormone III throughout the life cycle of a hemimetabolous insect,the ovoviviparous cockroachNauphoeta cinerea

Summary Titers of ecdysone, 20-hydroxyecdysone and juvenile hormone III were measured in whole body extracts or hemolymph of embryos, first, penultimate and last stadium nymphs, and adult females ofNaupoheta cinerea. We used a gas-chromatography/mass spectrometry method for quantifying juvenile hormone and a radio-immunoassay for ecdysteroid determination. Juvenile hormone III is particularly abundant in the embryonic stage (up to 960 ng/g), at a low level in first and penultimate stadium nymphs (2–10 ng/ml) and almost absent in the last nymphal stadium; in the adult female the juvenile hormone titer rises to 180 ng/ml in hemolymph during rapid oocyte growth. The titers of ecdysone and 20-hydroxyecdysone undergo similar fluctuations in the embryonic and nymphal stages, being highest at the time of cuticle formation in the embryo and a few days before the nymphal and adult molts (around 100–200 ng/ml for exdysone and 2–4 g/ml for 20-hydroxyecdysone).Acknowledgments. We thank Mrs A. Tschan for rearing the cockroaches, Mr M. Kaltenrieder for drawing the graphs, Mr G.C. Jamieson and Mrs C. Reuter for GC/MS analyses. We are also grateful to the Swiss National Science Foundation (grant no. 3.291-0.82 to B. Lanzrein) and the United States National Science Foundation (grant no. PCM 82-08665 to D.A. Schooley) for their financial support.     

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.     

The biotransformation of tritiated 3-dehydroecdysone by crayfish,Procambarus clarkii

The injection of 3-dehydroecdysone (3dhE, 5 /g), the major ecdysteroid secreted by the Y-organ of crayfishProcambarus clarkii, resulted in apolysis within about 5 days. The hormonal response at the molecular level was investigated by injection of the radio-labeled compound; within 3 h of injection of [³H]3dhE, most radio-isotope was found in the extracted epidermal tissues and identified as ecdysone, 20-hydroxyecdysone (20E), and their 3-hydroxy epimers. The biotransformation was undoubtedly performed in the peripheral area of the Y-organ. Cleavage of the polar conjugates, using an enzyme fromHelix pomatia, gave all of the above ecdysteroids including 3dhE. It was also found that the biosynthetic site of 3dhE was different from that of ecdysone at the subcellular level of the Y-organ.     

Secretion of chitinase byAphanocladium album,a hyperparasite of wheat rust

Summary Aphanocladium album, a hyperparasite of wheat rust,Puccinia graminis vartritici, secretes chitinase when grown on a chitin-containing medium. This enzyme was purified and shown to attack the mycelium of the parasitized rust fungus.Supported, in part, by a grant from the Naef-Roth-Foundation.     

A new,unexpected marine source of a molting hormone. Isolation of ecdysterone in large amounts from the zoanthidGerardia savaglia

Summary Ecdysterone was found in large amounts in the Mediterranean zoanthidGerardia savaglia both immediately after its collection and after it had been kept for 15 months in an aquarium. This is the first time that an ecdysteroid has been found in a marine animal which does not belong to the phylum Arthropoda.Acknowledgments. We are grateful to Dr E. Volpi, Trento, for collecting, and keeping alive, the zoanthid, and to Prof. L. Rossi, Torino, both for the identification and for useful comments, and to Prof. L. Colombo, Padova, for stimulating discussions. This work was funded by the Provincia Autonoma di Trento, Assessorato Agricoltura e Foreste, within our program for biological control of phytopathogenic fungi, by C.N.R., Roma, and by M.P.I., through the Centro Interuniversitario di Biologia Marina in Livorno.Laboratorio di Chimica.Stazione Sperimentale Agraria.To whom reprint requests should be addressed.     

Dissociated cell suspensions ofCarcinus maenas Y-organs as a tool to study ecdysteroid production and its regulation

In vitro ecdysteroid production by dissociated Y-organ cells of the shore crabCarcinus maenas was characterized during short-term incubations. Under optimized conditions (M199 adjusted to crab osmolality and with the addition of 10% foetal calf serum), ecdysteroid production by dispersed cells increased linearly during 4-hour incubations, with little intra-assay variation. 25-deoxyecdysone was mainly produced. PurifiedCarcinus molt inhbiting hormone (CamMIH) produced a dose-dependent inhibition of ecdysteroid production by dispersed cells. The cells were about 50 times more sensitive than whole glands to MIH. Other structurally-related peptides were tested.     

Photodecomposition of orellanine and orellinine,the fungal toxins ofCortinarius orellanus Fries andCortinarius speciossimus

Summary The photosensitivity of orellanine, the main toxin ofCortinarius orellanus Fries mushrooms, and its transformation to orelline via orellinine is discussed. All three substances were found in methanolic extracts ofCortinarius orellanus andCortinarius speciossimus mushrooms. The problem of homogeneity of orellanine is also discussed.Acknowledgment. The authors wish to express their gratitude to Prof. Dr W. Steglich from the Institut für Organische Chemie und Biochemie der Universität Bonn (Federal Republic of Germany) for having provided them with a sample of dried material ofC. speciossisimus.     

Notable diversity in hemoglobin expression patterns among species of the deep-sea clam, <Emphasis Type="Italic">Calyptogena</Emphasis>

The deep-sea clams Calyptogena nautilei and C. tsubasa, which live in the cold-seep area at a depth of 3570 m in the Nankai Trough, Japan, have abundant hemoglobins (Hbs) in erythrocytes, similar to other Calyptogena species. We determined the cDNA-derived amino acid sequences of Hbs from two Calyptogena species. C. tsubasa was found to contain two dimeric Hbs, Hb I consisting of 145 amino acid residues and Hb II with 137 residues, similar to known Hbs from C. soyoae and C. kaikoi. Sequence identity was over 90% among the orthologous chains of Calyptogena Hbs. On the other hand, surprisingly, C. nautilei contained two monomeric Hbs, Hb III containing 141 residues and Hb IV with 134 residues. In addition, Hbs III and IV showed only 33–42% sequence identity with Hbs I and II from other Calyptogena species. The distal (E7) histidine, one of the functionally important residues of the heme protein, is replaced by glutamine in all Hb chains of Calyptogena species. A phylogenetic analysis indicated that C. nautilei Hb III is closer to Hb I from other Calyptogena species. We suppose that a Hb gene was duplicated at least three times in an immediate ancestor of Calyptogena and, presumably depending on physiological conditions different Hb sets are being expressed: dimeric Hbs I and II in C. soyoae, C. kaikoi and C. tsubasa, and monomeric Hbs III and IV in C. nautilei. Received 13 May 2003; received after revision 5 June 2003; accepted 12 June 2003     

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.