Cellular and Molecular Life Sciences

, Volume 68, Issue 11, pp 1929–1939 | Cite as

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

  • Chunju An
  • Aidan Budd
  • Michael R. Kanost
  • Kristin Michel
Research Article

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.

Keywords

Anopheles gambiae Innate immunity Serpin Serine proteinase Malaria 

Abbreviations

dKD

Double knockdown

IEARpNA

Acetyl-Ile-Glu-Ala-Arg-p-nitroanilide

KD

Knockdown

KS

Kolmogorov–Smirnov

LLA

Late-life acting

MS

Mass spectrometry

PPO

Prophenoloxidase

Notes

Acknowledgments

We thank Dr. M. Gorman for purified M. sexta PPO and T. Graves, K. Kjos, R. Woolsey, and G. Hammon for mosquito rearing. Thanks go to Drs. J. Tomich and Y. Hiromasa at the KSU Proteomics Core Laboratory for protein digestion and mass spectrometry. Real-time PCR analyses were performed at the COBRE Core I, KSU. This work was supported by NIH grants 3P20RR017708-07S1 and P20RR017686 subawards to K.M. and GM41247 to M.K. This is contribution 10-368-J from the Kansas Agricultural Experiment Station.

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Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • Chunju An
    • 1
  • Aidan Budd
    • 3
  • Michael R. Kanost
    • 4
  • Kristin Michel
    • 2
  1. 1.Division of BiologyKansas State UniversityManhattanUSA
  2. 2.Division of BiologyKansas State UniversityManhattanUSA
  3. 3.European Molecular Biology LaboratoryHeidelbergGermany
  4. 4.Department of BiochemistryKansas State UniversityManhattanUSA

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