White pupa: a Ceratitis capitata mutant lacking catecholamines for tanning the puparium

The white pupa mutant of the Mediterranean fruit fly, Ceratitis capitata, fails to tan the puparium, but develops normal larval and adult cuticular structures. We found that the puparium of this mutant underwent minor stiffening at the beginning of pupariation, but subsequently did not increase further in stiffness. By the end of poparium formation, it was fivefold less resistant to compression than the wild type strain, Scanning electron microscopy of cross-sections of puparial exuviae revealed a dense sclerotized cuticle in the wild type, whereas the white pupa cuticle was quite distinct, with the inner two-thirds consisting of unsclerotized lamellae and the outer third being a dense, nonlaminar, amorphous layer. Puparial catecholamine levels were also very low in the white pupa when compared with the wild type strain, in which N-β-alanyldopamine (NBAD) predominated. However, in mutant hemolymph, NBAD, N-acetyldopamine (NADA), and dopamine were about 10 times more concentrated than in the normal phenotype. By injecting 1-¹⁴C-β-alanine as a tracer, we confirmed that N-β-alanyldopamine incorporation into the puparium was much lower in the white pupa than in the wild type strain. However, insoluble cuticle phenoloxidase activity was similar in the two strains. Tanning occurred in vitro when white pupa puparial cuticle, free of epidermis, was incubated with either NBAD or NADA, and melanization occurred when the cuticle was incubated with dopamine, demonstrating that tanning enzymes, but not substrates, were present in white pupa puparial cuticle. Solid state ³C nuclear magnetic resonance spectroscopy revealed that more chitin as well as less protein, catechols and β-alanine were present in the white pupa cuticle relative to the wild type. We conclude that the white pupa mutant is defective in the mechanism that provides hemolymph catecholamines to the puparial cuticle; this defect prevents normal sclerotization and pigmentation.