Action of Different Ecdysteroids on the Regulation of mRNAs for the Ecdysone Receptor, MHR3, Dopa Decarboxylase, and a Larval Cuticle Protein in the Larval Epidermis of the Tobacco Hornworm, Manduca sexta

Abstract

To determine which ecdysteroids may be biologically active in the larval epidermis of the tobacco hornworm, Manduca sexta,we studied the action of several known ecdysteroids and metabolites on the expression of the genes encoding the ecdysone receptor (EcR), Manducahormone receptor 3 (MHR3), dopa decarboxylase (DDC), and a larval cuticle protein (LCP-14). Both Day 2 fourth- and Day 2 fifth-instar larval epidermis contained significant 3β-reductase activity which metabolized 3-dehydroecdysone (3DE) and 3-dehydro-20-hydroxyecdysone (3D20E) to ecdysone (E) and 20-hydroxyecdysone (20E), respectively, but had only very low amounts of ecdysone oxidase activity (E to 3DE) and no detectable ecdysone 20-monooxygenase activity (E to 20E). When the expression of the various genes was studied in the epidermis in vitro,20E and 3D20E had similar effects, whereas E, 3DE, 26-hydroxyecdysone and 20,26-dihydroxyecdysone were ineffective. Exposure of Day 2 fifth-instar epidermis to 500 ng/ml of either 20E or 3D20E for 24 hr caused a rapid, biphasic increase in EcR-B1 mRNA. By contrast, EcR-A mRNA showed a less rapid initial increase followed by a slow steady rise and was less responsive to 3D20E. Ecdysone in a 1:1 mixture with 20E effectively halved the concentration of 20E needed to induce EcR-B1 mRNA but showed no synergism in the induction of EcR-A mRNA. The induction of MHR3 mRNA and of DDC mRNA in Day 2 fourth-instar epidermis as well as the suppression of DDC and LCP-14 gene expression by 3D20E was indistinguishable from that of 20E. Therefore, for Manducalarval epidermis, only 20E and 3D20E are biologically active ecdysteroids. Since the 3D20E can be converted to 20E by the epidermis, its effects are likely mediated by 20E.