Induction of autotomy in the American bird grasshopperSchistocerca americana (Drury) by the ecdysone agonist RH-5849 and investigation of its mode of action

The non-steroidal ecdysone agonist RH-5849 (1,2-dibenzoyl-l-tert-butylhydrazine) was found to be an effective neurotoxicant on injection into the American bird grasshopper,Schistocerca americana (Drury). Treated grasshoppers became immediately hyperactive, followed by loss of coordination, paralysis and eventually death. We also discovered that this compound induced bilateral autotomy of the metathoracic legs during the early stages of intoxication. However, no evidence of ecdysonergic or morphogenetic activities was observed. Synergism studies with neurotoxins of known mode of action suggested that RH-5849 has a mechanism of action similar to that of 4-amino pyridine, which blocks potassium channels.     

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.     

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.     

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.     

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.     

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.     

Endogenous inhibitor of ecdysone synthesis in crabs

Summary 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-hydroxyl-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.Acknowledgments. We thank Prof. M. Oka (Nagasaki University) and Dr J. Cappuzo (Oceanographic Inst.) for information on Y-organ and molting stages, Dr J. Termini and Mr J. Cesarelli (Columbia University) for organ excision of blue crabs and experiments in the early stage of this study, the Japan Sea Farming Association for aqua culture of crabs, Prof. Y. Umebachi (Kanazawa University) for a gift of 3-OH-l-Kyn, and Mr J. Rudloe (Gulf Specimen Co.) for collection and shipping of some selected crabs. This study was partially supported by NIH grant AI 10 187 (to K. N., at Columbia University).     

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.     

Effect of xanthurenic acid on P-450-dependent biotransformation by molting glands in vitro

Incubation of molting glands from the crayfishProcambarus clarkii (Y-organ) and the silkwormBombyx mori (prothoracic gland) with 23,24-[²H₄]-2-deoxyecdysone resulted in the production of deutero-ecdysone; this biotransformation was inhibited in the presence of xanthurenic acid. When the experiments were performed under an¹⁸O₂ atmosphere, the¹⁸O atom was introduced into ecdysone, as confirmed by mass spectrometry. We therefore suggest that xanthurenic acid inhibits P-450-dependent hydroxylation of 2-deoxyecdysone. However, deutero-2-deoxyecdysone was not converted to 3-dehydroecdysone when using Y-organs in vitro, although it is a major product. We therefore conclude that the biosynthetic pathway of ecdysteroids inP. clarkii branches at an early step.     

RH-5849, a nonsteroidal ecdysone agonist,does not mimic makisterone-A inDysdercus koenigii

The response of final instar nymphs ofDysdercus koenigii to topical application of the non-steroidal ecdysone agonist, RH-5849, was dose dependent. The candidate compound produced mortality even at moderate doses, but precocious adult development was not observed. Similar results were obtained after oral administration or injection. Conversely, injections of makisterone-A (the principal moulting hormone ofDysdercus) into 5th instar nymphs resulted in precocious adult development within 4 days. We conclude that RH-5849 does not mimic makisterone-A, as is the case with ecdysone, and that toxicity is mediated instead through non-endocrine targets in this insect species.