Article

Characterization of a regulatory unit that controls melanization and affects longevity of mosquitoes

Division of Biology, Kansas State University, 271 Chalmers Hall, Manhattan, KS 66506, USA.
Cellular and Molecular Life Sciences CMLS (Impact Factor: 5.86). 10/2010; 68(11):1929-39. DOI: 10.1007/s00018-010-0543-z
Source: PubMed

ABSTRACT Melanization is an innate immune response in arthropods that encapsulates and kills invading pathogens. One of its rate-limiting steps is the activation of prophenoloxidase (PPO), which is controlled by an extracellular proteinase cascade and serpin inhibitors. The molecular composition of this system is largely unknown in mosquitoes with the exception of serpin-2 (SRPN2), which was previously identified as a key negative regulator of melanization. Using reverse genetic and biochemical techniques, we identified the Anopheles gambiae clip-serine proteinase CLIPB9 as a PPO-activating proteinase, which is inhibited by SRPN2. Double knockdown of SRPN2 and CLIPB9 reversed the pleiotrophic phenotype induced by SRPN2 silencing. This study identifies the first inhibitory serpin-serine proteinase pair in mosquitoes and defines a regulatory unit of melanization. Additionally, the interaction of CLIPB9 and SRPN2 affects the life span of adult female mosquitoes and therefore constitutes a well-defined potential molecular target for novel late-life acting insecticides.

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    • "For membrane feeding assays, hyper-reticulocytosis was induced in mice by injection of 6 mg/mL of phenylhydrazine 3 days prior to infection with 10 7 parasitized red blood cells (pRBCs). Three days after infection, exflagellation of male gametocytes was checked as described previously (Blagborough and Sinden, 2009). Blood containing infectious P. berghei gametocytes was then collected by cardiac puncture, split into two equal aliquots and mixed with pooled serum from mice immunized with either AgSRPN2 or GFP (each n = 8) at a dilution of 1:5 serum:blood and fed to A. stephensi mosquitoes through plastic membrane feeders. "
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    • "AgCLIPB9, the first PAP to be identified in mosquitoes, is inhibited by AgSRPN2. Double knockdown of AgSRPN2 and AgCLIPB9 partially reversed the phenotypes induced by AgSRPN2 silencing (An et al., 2010). One interpretation of the partial reversion of the AgSRPN2 depletion phenotype is that AgSRPN2 has additional PAP proteinase targets, which are currently being identified. "
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