Mosquito innate immunity: involvement of β 1,3-glucan recognition protein in melanotic encapsulation immune responses in Armigeres subalbatus

Beta 1,3-glucan recognition proteins (GRP) have specific affinity for β 1,3-glucan, a component on the surface of fungi and bacteria. By interacting with β 1,3-glucan, GRP initiates activation of prophenoloxidase, a key enzyme in the signaling pathway leading to melanotic encapsulation in invertebrates. In this study, we characterize a novel hemocyte-specific GRP from the mosquito, Armigeres subalbatus (AsGRP). The 1.57 kb cDNA clone encodes a 499 deduced amino acid sequence, which contains a region that displays significant similarity to the glucanase-like regions of other GRPs and Gram-negative bacteria binding proteins found in other organisms. AsGRP is constitutively expressed in the hemolymph of adult female mosquitoes, and is upregulated following challenge with Escherichia coli, Micrococcus luteus, and the filarial worm Dirofilaria immitis. AsGRP specifically recognizes curdlan (insoluble β 1,3-glucan), but not mannose or N-acetyl-d-glucosamine. AsGRP binds a low percentage of E. coli, most M. luteus and D. immitis microfilariae. AsGRP double-stranded RNA interference strongly inhibits melanotic encapsulation of D. immitis in Ar. subalbatus. These results suggest that AsGRP has the capacity to bind to a variety of pathogens, functions as a pattern recognition receptor, and is required for effective melanotic encapsulation immune responses in Ar. subalbatus.