Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae

The absence of juvenile hormone at the time of head cap slippage during the last-larval moult of the tabacco hornworm, Manduca sexta, causes deposition of premelanin granules into the outer regions of the newly forming endocuticle beginning 13 h later. These granules were found to contain an inactive phenoloxidase which becomes activated about 9 h later, 4 h before body melanization begins. The onset of melanization was not accelerated by melanization and reddish colouration hormone from Bombyx heads, extracts of pharate-adult corpora cardiaca or pharate-larval ventral nerve cords (sources of eclosion hormone), or extracts of pharate-larval suboesophageal ganglia or corpora cardiaca-corpora allata complexes. Instead the fall of the ecdysteroid titre to below 250 ng/ml 20-hydroxyecdysone equivalents appeared to be the cue that allowed melanization about 4.5 h later. Up to, but not after, this time both melanization and ecdysis could be delayed by exogenous 20-hydroxyecdysone in a dose-dependent fashion above 0.1 μg per larva. In vitro studies published elsewhere indicate that 20-hydroxyecdysone prevents the activation of the premelanin granules. Thus the granules can be deposited at the proper time in the newly forming endocuticle but their melanization is regulated by the declining ecdysteroid titre and it thus synchronized with other events occurring just before ecdysis.