Hormonal regulation of sequential larval cuticular gene expression

As the tobacco hornworm ( Manduca sexta ) larva feeds and grows, the abdominal epidermis synthesizes larval endocuticular proteins. During a molt this synthesis temporarily ceases while the cells deposit a new epicuticle; then these proteins reappear in a sequential manner, some before ecdysis, others after ecdysis. At the time of metamorphosis, the epidermis becomes pupally committed and ceases synthesizing these endocuticular proteins. Northern and dot blot hybridization analysis using cDNA clones for three different low‐molecular‐weight larval cuticular proteins showed that RNA transcription of all three ceased by the time of head cap slippage during the molt, then begins again just before ecdysis, one showing a different time course from the other two. All responded to the commitment peak of ecdysteroid in the absence of juvenile hormone (JH) by a permanent cessation of transcription. During the final day of feeding (day 3) of the 5th instar the cuticular lamellae become 5–10 times thinner coincidentally with a change in cuticular protein synthesis involving a cessation of synthesis of many of the endocuticular proteins and the appearance of at least three new proteins — 14.5, 15, and 27 kd (kilodaltons). Incubation of day 15th instar epidermis with 10–100 ng/ml 20‐hydroxyecdysone (20HE) induced the synthesis of the 27‐kd protein and the transcription of the mRNAs for the 14.5‐ and 15‐kd proteins. Incubation in the absence of hormones or with 20HE in the presence of JH prevented the appearance of these new proteins and the deposition of thin lamellae. The mRNAs for three endocuticular proteins whose synthesis ceases on day 3 showed differing responses to 20HE and JH. Thus, the sequential program of larval endocuticular gene expression is controlled primarily by hormonal titers in both the molt and the intermolt periods.