Differential effects of nonsteroidal ecdysteroid agonists in Coleoptera and Lepidoptera: Analysis of evagination and receptor binding in imaginal discs

The effects of 20-hydroxyecdysone (20E) and the first nonsteroidal ecdysteroid agonists, RH-5849 and tebufenozide (=RH-5992), were tested on in vitro cultured mesothoracical imaginal wing discs of last-instar larvae of the Colorado potato beetle, Leptinotarsa decemlineata Say, and the greater mar moth, Galleria mellonella L. In general, the response of the nonsteroidal agonists was qualitatively similar to that induced by 20E, although the concentrations required to induce evagination varied widely. The EC(50) of 20E, RH-5849 and tebufenozide, that indicates the compound concentration which elicits evagination in 50% of the isolated imaginal discs, was 60.7, 461 and 757 nM, respectively, in L. decemlineata. In vying discs of G. mellonella the order of biological activity (expressed as EC(50)) resulted in tebufenozide (8.9 nM) > 20E (321 nM) > RH-5849 (865 nM). In a second series of experiments, a competition receptor binding assay was developed with whole imaginal discs, and showed that the I-50 value of 20E, RH-5849 and tebufenozide to compete 50% of the specifically bound amount of H-3-ponasterone A in imaginal discs of L. decemlineata was 425, 740 and 1316 nM, respectively, In wing discs of G. mellonella that respective value was 106, 911 and 22 nM. The current results showed that the binding affinity (I-50) of 20E reached a similar level as the concentration to elicit a biological response (EC(50)) in imaginal wing discs of the two insect species; a similar conclusion could be drawn for the two nonsteroidal ecdysteroid agonists. In this regard, it is suggested that the imaginal disc assay allows us to measure the potency of an ecdysteroid active compound on the one hand and its affinity to bind on the ecdysteroid receptors (EcRs) in competition with H-3-ponasterone A on the other. In addition, the current data suggest that the specific activity and insect selectivity of the nonsteroidal agonists, especially the high toxicity of tebufenozide for Lepidoptera and the nonsusceptibility of Coleoptera, is related to selective binding on the EcRs. Copyright (C) 1996 Elsevier Science Ltd.