Developmental expression of three genes for larval cuticular proteins of the tobacco hornworm, Manduca sexta

Three cDNA clones coding for the 12.8, 13.3, and 14.6 kDa larval cuticular proteins of the tobacco hornworm, Manduca sexta, were isolated and characterized. Hybridization to abdominal epidermal RNA from different stages showed that the genes for the 12.8 and 13.3 kDa proteins were expressed only during larval life. By contrast, the gene for the 14.6 kDa protein was expressed throughout the segment during the feeding, growing larval stages, then only in the flexible intersegmental regions during the deposition of endocuticle in the pharate pupa and adult. Quantitative RNA dot blot hybridizations showed that the RNA for each protein disappeared during the larval molt when the ecdysteroid titer was high, then reappeared during the preecdysial deposition of endocuticle. All disappeared when the epidermis became pupally committed at the onset of wandering. Exposure of the fourth instar epidermis to 20-hydroxyecdysone (20HE) in vitro under conditions that lead to the formation of a new larval cuticle by 48 hr caused the disappearance of these RNAs by 18 hr. Exposure of Day 2 fifth instar epidermis to 20HE in vitro caused a depression of these RNAs which in the case of the RNAs coding for the 12.8 and 13.3 kDa proteins was partially prevented by simultaneous exposure to methoprene, a juvenile hormone (JH) mimic. By contrast, the RNA for the 14.6 kDa protein was suppressed by exposure to methoprene alone. Thus, each of these larval cuticular genes is turned off by high ecdysteroid; the presence or absence of JH determines whether or not this suppression is permanent in some or all cells.