Regulation of melanization of tobacco hornworm larval cuticle in vitro.

When tobacco hornworm larvae (Manduca sexta) are allatectomized 5-6 hr before head capsule slippage in the molt to the fifth (final) larval instar, the new cuticle melanizes 3 hr before ecdysis. After explantation between 7 and 3 hr before the onset of melanization, the new cuticle was found to melanize in vitro in Grace's medium only if beta-alanine was removed. When explanted at the onset of melanization, the presence of beta-alanine had no effect on melanization. The addition of either dopa or dopamine was found to be necessary for complete melanization of pieces explanted before the onset of melanization with 0.3 mM of either dopa or dopamine being optimal. Both of these compounds were incorporated into the cuticular melanin. In this optimal medium, melanization occurred over about a 9-hr period after a 5- to 6-hr lag period presumably required for adjustment to the medium. Fifty ng/ml 20-hydroxyecdysone was found to inhibit melanization of pieces explanted 7 hr but not 3 hr before melanization. The hormone neither inhibited uptake of dopa into the epidermis nor prevented melanization in the cuticle once the prophenoloxidase in the premelanin granules was activated. Therefore, 20-hydroxyecdysone may inhibit the activation of the phenoloxidase in the pre-melanin granules, or may inhibit the incorporation of dopa into the granules.