Abstract
Reverse genetics in the mosquito Anopheles gambiae by RNAi mediated gene silencing has led in recent years to an advanced understanding of the mosquito immune response against infections with bacteria and malaria parasites. We developed RNAi screens in An. gambiae hemocyte-like cells using a library of double-stranded RNAs targeting 109 genes expressed highly or specifically in mosquito hemocytes to identify novel regulators of the hemocyte immune response. Assays included phagocytosis of bacterial bioparticles, expression of the antimicrobial peptide CEC1, and basal and induced expression of the mosquito complement factor LRIM1. A cell viability screen was also carried out to assess dsRNA cytotoxicity and to identify genes involved in cell growth and survival. Our results identify 22 novel immune regulators, including proteins putatively involved in phagosome assembly and maturation (Ca2+ channel, v-ATPase and cyclin-dependent protein kinase), pattern recognition (fibrinogen-domain lectins and Nimrod), immune modulation (peptidase and serine protease homolog), immune signaling (Eiger and LPS-induced factor), cell adhesion and communication (Laminin B1 and Ninjurin) and immune homeostasis (Lipophorin receptor). The development of robust functional cell-based assays paves the way for genome-wide functional screens to study the mosquito immune response to infections with human pathogens.
Highlights
Anopheles gambiae is a major vector of Plasmodium falciparum malaria in sub-Saharan Africa and a secondary vector of other parasitic and viral diseases [1]
The mosquito immune system relies on innate humoral and cellular reactions to fight infections, including those by malaria parasites that must pass through mosquitoes before they can infect humans
We use a technique to silence in mosquito cultured cells genes that are highly and/or expressed in mosquito hemocytes, the equivalent of human white blood cells, as a means to investigate their function in reactions of the mosquito immune system
Summary
Anopheles gambiae is a major vector of Plasmodium falciparum malaria in sub-Saharan Africa and a secondary vector of other parasitic and viral diseases [1]. Numerous mosquito agonist and antagonist effectors of Plasmodium and bacteria have been identified, principally by RNAi-mediated reverse genetic tests using dsRNA (double-stranded RNA) injections into adult mosquitoes [5] These factors operate in complex molecular networks that involve pathogen recognition by secreted or membrane bound receptors, activation of immune signaling pathways, and synthesis or activation of effectors that contribute to lysis, melanization or phagocytosis of the invading pathogens [2,6,7]. Many of these factors are produced by hemocytes and function in the hemolymph [8,9,10]. We have generated a dsRNA library targeting 109 genes or predominantly expressed in circulating hemocytes and optimized cell-based RNAi screens to investigate the role
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