ABSTRACT
Anopheles dirus is refractory to a rodent malaria parasite, Plasmodium yoelii, and melanized oocysts are manifested in infected mosquitoes. Prophenoloxidase (PPO) is a zymogen whose active form mediates melanotic encapsulation of invading pathogens in mosquitoes. In this study, we cloned cDNA fragments of four An. dirus PPOs, that are orthologs of Anopheles gambiae PPO2, PPO4, PPO5 and PPO6. AdPPO4 expression in hemocytes was induced in response to P. yoelii infection. RNA interference using double stranded RNA of AdPPO4 led to depletion of its mRNA and other PPO transcripts. This depletion increased P. yoelii infection prevalence and oocyst intensity, and abolished the melanization of oocysts as well. Therefore, An. dirus PPOs may play a role in the refractoriness to P. yoelii.
Subject(s)
Anopheles/enzymology , Anopheles/parasitology , Catechol Oxidase/metabolism , Enzyme Precursors/metabolism , Plasmodium yoelii/growth & development , Amino Acid Sequence , Animals , Anopheles/classification , Anopheles/genetics , Catechol Oxidase/chemistry , Catechol Oxidase/genetics , Cloning, Molecular , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Female , Insect Vectors/classification , Insect Vectors/enzymology , Insect Vectors/genetics , Insect Vectors/parasitology , Melanins/metabolism , Mice , Phylogeny , Plasmodium yoelii/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Transcription, GeneticABSTRACT
Although knowledge of the mosquito immune response has recently improved, less is known about the impact of antimalarial drugs on mosquito immunity. In the present study, we found that nitroquine, an effective antimalaria drug, could also induce melanotic encapsulation of Plasmodium by Anopheles stephensi. The melanization rate of the nitroquine treated group was 60.8%. To explore the effect of nitroquine on mosquito immunity, we determined the increase in activity of phenoloxidases (PO) enzyme, the main component of melanotic encapsulation, with nitroquine treatment. Moreover, we cloned prophenoloxidase (PPO) gene, which is accepted as the inactive phenoloxidase form and observed inducible expression of this gene with nitroquine treatment by real-time PCR. Our data implied that up-regulation of PPO gene and PO activity might be correlated with nitroquine. Nevertheless, nitroquine had no effect on the transcription of PPO gene or the activity of PO enzyme in the mosquito fed on a normal blood meal. In our study, we also observed the degenerative effect of 0.1% nitroquine on Plasmodium in the mosquito. This suggests that the degeneration of Plasmodium induced by nitroquine might result in the exposure of pattern-recognition ligands which can active the immune reaction, up-regulate PPO gene expression and PO activity, and induce the melanization.
Subject(s)
Anopheles/parasitology , Antimalarials/pharmacology , Catechol Oxidase/metabolism , Enzyme Precursors/metabolism , Monophenol Monooxygenase/metabolism , Plasmodium yoelii/immunology , Quinazolines/pharmacology , Amino Acid Sequence , Animals , Anopheles/enzymology , Anopheles/immunology , Catechol Oxidase/chemistry , Catechol Oxidase/genetics , Cloning, Molecular , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Female , Hemolymph/enzymology , Melanins/metabolism , Mice , Molecular Sequence Data , Plasmodium yoelii/drug effects , Plasmodium yoelii/physiology , Polymerase Chain Reaction , Random Allocation , Up-RegulationABSTRACT
On the basis of the research on interaction between plasmodium and mosquito vector, the mechanism of innate immune defense responses in anopheline mosquitoes against plasmodium infection has been studied. The innate immune defense may be applied to confine and kill malaria parasites under migration and development, to an effective control strategy on malaria vectors.