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Many serine proteinase inhibitors of the serpin superfamily have evolved in vertebrates and invertebrates to regulate serine proteinase cascades that mediate the host defense responses. We have isolated an immuneresponsive serpin from the tobacco hornworm, Manduca sexta. This inhibitor, M. sexta serpin-3, contains a reactive site loop strikingly similar to the proteolytic activation site in prophenoloxidase (pro-PO). Molecular cloning and sequence comparison indicate that serpin-3 is orthologous to Drosophila melanogaster serpin 27A, a regulator of melanization. M. sexta serpin-3 is constitutively present in hemolymph at a low concentration of 5-12 g/ml and increases to 30 -75 g/ml after a microbial challenge. Recombinant serpin-3 efficiently blocks pro-PO activation in the hemolymph, and it forms SDS-stable acyl-enzyme complexes with purified pro-PO-activating proteinases (PAPs) from M. sexta. PAP-serpin-3 complexes were isolated by immunoaffinity chromatography from hemolymph activated by treatment with Micrococcus luteus. Kinetic analysis of PAP-serpin-3 association strongly suggests that serpin-3 is a physiological regulator of the pro-PO activation reaction.

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Newly synthesized insect cuticle is soft and pale but becomes stronger (sclerotized) and often darker (pigmented) over several hours or days. The first step in the sclerotization and pigmentation pathways is the hydroxylation of tyrosine to produce 3,4-dihydroxyphenylalanine (DOPA). Tyrosine hydroxylase (TH) is known to catalyze this reaction during pigmentation, but a role for TH in sclerotization has not been documented. The goal of this study was to determine whether TH is required for cuticle sclerotization in the red flour beetle, Tribolium castaneum. We used quantitative RT-PCR to verify that TH expression occurs at the time of cuticle tanning and immunohistochemistry to confirm that TH is expressed in the epithelial cells underlying sclerotized cuticle. In addition, we found that a reduction in TH function (mediated by RNA interference) resulted in a decrease in cuticle pigmentation and a decrease in the hardness of both pigmented and colorless cuticle. These results demonstrate a requirement for TH in sclerotization as well as brown pigmentation of insect cuticle.

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