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.     

Effects of the pyrimidine-containing cytochrome P-450 inhibitor,fenarimol, on the formation of 20-OH ecdysone in flies

Summary The effect of fenarimol, a pyrimidine-containing cytochrome P-450 inhibitor, was tested in vitro on brain-ring gland complexes ofCalliphora vicina (Dipt., Calliphoridae), and on microsomes prepared from the fat body of 0-h wandering stage larvae ofNeobellieria bullata (Dipt., Sacrophagidae). Fenarimol had no influence on the formation of ecdysone, but it was an effective inhibitor of cytochrome P-450-dependent ecdysone 20-monooxygenase.     

Activité protéosynthétique des glandes prothoraciques et titre d'ecdysone chez des larves permanentes deLocusta migratoria obtenues par irradiation sélective du tissu hématopoïétique

Summary The prothoracic glands of permanent fifth instar larvae ofLocusta migratoria (moulting blocked by selective X-ray treatment of the hemocytopoietic tissue) are in a state of high activity as evidenced by the rate of incorporation of a labelled amino-acid into the proteins synthesized in these glands. Such permanent larvae apparently synthesize no ecdysone nor ecdysterone.     

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.     

Effects of the chitin synthetase inhibitor plumbagin and its 2-demethyl derivative juglone on insect ecdysone 20-monooxygenase activity

The chitin synthetase inhibitor plumbagin and its 2-demethyl derivative juglone were found to inhibit in a dose-response fashion the cytochrome P-450 dependent ecdysone 20-monooxygenase activity associated with adult female Aedes aegypti, wandering stage larvae of Drosophila melanogaster, and fat body and midgut from last instar larvae of Manduca sexta. The concentration of these naphthoquinones required to elicit a 50% inhibition of the steroid hydroxylase activity in all the insects was approximately 1 x 10(-4) M.     

Effects of the adenylate cyclase activator forskolin and its inactive derivative 1,9-dideoxyforskolin on insect cytochrome P-450 dependent steroid hydroxylase activity.

The adenylate cyclase activator forskolin and its pharmacologically inactive derivative 1,9-dideoxyforskolin were found to inhibit in a dose-dependent fashion the ecdysone 20-monooxygenase activity associated with wandering stage larvae of Drosophila melanogaster and fat body and midgut from last instar larvae of the tobacco hornworm, Manduca sexta. The concentrations of these labdane diterpenes required to elicit a 50% inhibition of the cytochrome P-450 dependent steroid hydroxylase activity in the insect tissues ranged from approximately 5 x 10(-6) to 5 x 10(-4) M.     

The insect gut: A new source of ecdysiotropic peptides

Summary Proctodaea of European corn borer (Ostrinia nubilalis) and gypsy moth (Lymantria dispar) last instars (larvae) contain prothoracicotropic factors that stimulate the prothoracic glands (PGs) of the gypsy moth to produce both ecdysone and 3-dehydroecdysone (precursors to the insect molting hormone) in a dose-dependent manner. In a separate in vivo assay, injections of proctodaeal extracts into gypsy moth larvae that were head-ligated before the release of the molt-stimulating brain hormone, PTTH, resulted in a pupal molt.     

Changes in patterns of ecdysteroid secretion by the ring gland ofDrosophila in relation to the sterol composition of the diet

Summary Brain-ring glands fromDrosophila larvae reared on a defined diet containing campesterol (24-methyl-cholesterol) as the major sterol, secreted-in addition to ecdysone-a compound identified previously as a 24-methyl analogue, 20-deoxy-makisterone A. Using ergosterol or cholesterol as the sterol component of the diet, only ecdysone was detectable in cultures of brain-ring glands.     

Juvenilizing effect of ecdysone mimic RH 5849 inGalleria mellonella larvae

The response of the final instar larvae ofG. mellonella to topical application of the non-steroidal ecdysone mimic, RH 5849, was age-related as well as dose-dependent. In young final instar larvae, moderate doses of RH 5849 induced perfect supernumerary larval moults, but doses equal to and higher than 8.5 μg per larva caused premature formation of larval cuticle and were lethal. Application of RH 5849 significantly increased allatotropic activity of the brain, and also activated synthesis of juvenile hormone (JH) by the corpora cardiaca/corpora allata complex. Simultaneous application of RH 5849 and FMev, a potent inhibitor of JH synthesis, to young final instar larvae lowered the incidence of perfect supernumerary larval moults. We conclude that the effect of RH 5849 on the developmental programme inG. mellonella is mediated by the corpora allata.     

Endocrine significance of critical periods during insect development: Analysis of ligation experiments withPieris brassicae last instar larvae

Summary Ecdysone haemolymph levels have been analyzed in neck- or thorax-ligatedPieris larvae in order to explain the physiological significance of critical periods. It appeared that head critical period corresponds to an incomplete activation of prothoracic glands, while the thoracic critical period is related to the secretion of a minimal amount of ecdysone necessary for moulting. DuringPieris' last larval instar, there is no evidence for any noticeable synthesis of ecdysone in isolated abdomens.Acknowledgment. This work was partially supported by CNRS, ATP Mécanisme d'action des Hormones.     

Ecdysteroid titers during larval reproduction of the dipteran insectHeteropeza pygmae

Summary Ecdysteroid titers were measured in whole-body extracts of pedogenetically reproducing larvae of the dipteran insectHeteropeza pygmaea and in the dietary fungus. The titers are very low in the first 3 days of larval growth, but increase during the last 2 days. The level of 20-hydroxyecdysone is then significantly higher than that of ecdysone. Measurements of the titers in the fungus gave no conclusive results.Supported by the Swiss National Science Foundation, grants No. 3.218-0.77 to D.F.W. and 3.714-0.80 to B.L. We thank Xiang-Xiong Zhu for help with the RIA and Mrs G. Rhyner for technical assistance. Gifts of ecdysone antiserum from Dr J. D. O'Connor and of 23,24-[³H₂]-ecdysone from Dr J. Koolman are also gratefully acknowledged.     

Effects of the adenylate cyclase activator forskolin and its inactive derivative 1,9-dideoxyforskolin on insect cytochrome P-450 dependent steroid hydroxylase activity

The adenylate cyclase activator forskolin and its pharmacologically inactive derivative 1,9-dideoxyforskolin were found to inhibit in a dose-dependent fashion the ecdysone 20-monooxygenase activity associated with wandering stage larvae ofDrosophila melanogaster and fat body and midgut from last instar larvae of the tobacco hornworm,Manduca sexta. The concentrations of these labdane diterpenes required to elicit a 50% inhibition of the cytochrome P-450 dependent steroid hydroxylase activity in the insect tissues ranged from approximately 5×10^–6 to 5×10^–4 M.     

Oenocyte and prothoracic gland activity inManduca sexta under varying photoperiod and light conditions

Summary Manduca sexta larvae were subjected to diapause-inducing and diapause-preventing photoperiods, using two types of fluorescents (Indorsun and Blacklight-blue). The oenocytes, prothoracic glands (PTG) and ecdysone levels were examined in 3-day-old 5th instar larvae, 2-day-old and 10-day-old pupae. Our results indicate that oenocytes and PTG cells tend to be more active under long photoperiods while oenocytes only are active under short photoperiods in pupae in diapause. UV light has a definite effect on oenocytes while PTG cells seem to be unaffected. Ecdysone and ecdysterone levels vary with PTG and oenocyte activity at the pupal stage. The significance of these findings is discussed.This work was supported by a research associateship to L.M. by the People's Republic of China and a grant from NSERC to B.J.R.P.     

Induction of a special Balbiani ring by position effect in the salivary gland chromosomes ofAcricotopus lucidus

Summary Chromosome mutations were induced by X-ray treatment of males ofAcricotopus lucicus. One of the larvae of the F₁-generation showed a dislocation of a segment of the centromeric region into an euchromatic part of the same chromosome as a consequence of mutation. In the new position, in some salivary gland cells, this segment expressed a distinct Balbiani ring. In other cells this Balbiani ring regressed, probably through the influence of ecdysone.     

Synthesis of ecdyson-14C and ecdysterone-14C from cholesterol-14C in cockroaches (Periplaneta americana) without molting glands

Summary The synthesis of molting hormone of cockroaches of which the prothoracic glands had previously been extirpated was investigated after injection of cholesterol-¹⁴C. It could be proved by means of radio thin-layer chromatography of extracts that ecdysterone and in small amounts ecdysone, are synthesized in Periplaneta americana larvae without prothoracic glands. The results demonstrate that other tissues are also able to perform the molting hormone besides the molting gland itself.Supported by the Sächsische Akademie der Wissenschaften zu Leipzig.     

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.     

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3 beta-reductase mediated conversion to ecdysone in representative insects

The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3 beta-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.     

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3β-reductase mediated conversion to ecdysone in representative insects

Summary The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.     

Endogenous inhibitor of ecdysone synthesis in crabs

Attempts to isolate the molt-inhibiting hormone (MIH) of crustaceans from crab eyestalks (ES) resulted in the characterization of xanthurenic acid as an inhibitor of ecdysone biosynthesis in the cultured Y-organ-complex (YOC) homogenate. It was also found that 3-hydroxy-L-kynurenine present in the ES is transformed into xanthurenic acid in the YOC and body fluid. Its mode of inhibitory action in ecdysone biosynthesis is probably inactivation of cytochrome P-450.     

Occurrence of ecdysone in the blood of the chelicerate arthropod,Limulus polyphemus

Summary A molt promoting substance, assumed to be ecdysone, was discovered inLimulus haemolymph. Preliminary bioassay suggests a titre of 9 ng ecdysone per ml of haemolymph.Supported by the University of Minnesota Graduate School, NSF Grant No. GB 16607 and USPHS Grant No. HO 07336.