Nitric oxide is an essential component of the hemocyte-mediated mosquito immune response against bacteria.

Nitric oxide is a signaling and immune effector molecule synthesized by the enzyme nitric oxide synthase. In mosquitoes, nitric oxide functions as a parasite antagonist in the midgut but little is known about its function in the hemocoel. Here, we characterized the temporal and spatial expression of the Anopheles gambiae nitric oxide synthase gene and explored the role nitric oxide plays in the antibacterial response in the mosquito hemocoel. Quantitative PCR and Western blot analyses showed that nitric oxide synthase is expressed in hemocytes and fat body, and is upregulated in response to systemic infection with Escherichia coli and Micrococcus luteus. Diaphorase staining and immunofluorescence showed that nitric oxide synthase is abundant in the granulocyte subpopulation of hemocytes, and both the staining intensity and the percentage of cells that stain for nitric oxide synthase significantly increase after a bacterial challenge. When nitric oxide production was inhibited, the mosquito's ability to kill E. coli was significantly reduced. Accordingly, inhibiting nitric oxide production increased the mortality rate of mosquitoes with systemic E. coli infections. Taken altogether, these data show that nitric oxide is a crucial player in the antibacterial immune response in the mosquito hemocoel.