Dong, ShengzhangFu, XiaonanDong, YuemeiSimoes, Maria L.Zhu, JinsongDimopoulos, George2020-09-092020-09-092020-041553-7366e1008453http://hdl.handle.net/10919/99933Malaria, caused by the protozoan parasite Plasmodium and transmitted by Anopheles mosquitoes, represents a major threat to human health. Plasmodium's infection cycle in the Anopheles vector is critical for transmission of the parasite between humans. The midgut-stage bottleneck of infection is largely imposed by the mosquito's innate immune system. microRNAs (miRNAs, small noncoding RNAs that bind to target RNAs to regulate gene expression) are also involved in regulating immunity and the anti-Plasmodium defense in mosquitoes. Here, we characterized the mosquito's miRNA responses to Plasmodium infection using an improved crosslinking and immunoprecipitation (CLIP) method, termed covalent ligation of endogenous Argonaute-bound RNAs (CLEAR)-CLIP. Three candidate miRNAs' influence on P. falciparum infection and midgut microbiota was studied through transgenically expressed miRNA sponges (miR-SPs) in midgut and fat body tissues. MiR-SPs mediated conditional depletion of aga-miR-14 or aga-miR-305, but not aga-miR-8, increased mosquito resistance to both P. falciparum and P. berghei infection, and enhanced the mosquitoes' antibacterial defenses. Transcriptome analysis revealed that depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes in mosquito midguts. The overall fitness cost of conditionally expressed miR-SPs was low, with only one of eight fitness parameters being adversely affected. Taken together, our results demonstrate that targeting mosquito miRNA by conditional expression of miR-SPs may have potential for the development of malaria control through genetically engineered mosquitoes. Author summary Malaria is caused by the Plasmodium parasite that is transmitted by Anopheles mosquitoes. The mosquito's innate immune system plays an important role in controlling parasite infection. We have identified mosquito microRNAs (miRNAs) that are involved in regulating mosquito immunity to parasite infection. Transgenic mosquitoes that deplete the immunity-related miRNAs aga-miR-14 or aga-miR-305 through miRNA sponges, show increased resistance to both human and rodent parasite infection, and enhanced antibacterial defenses. Depletion of aga-miR-14 or aga-miR-305 resulted in an increased expression of multiple immunity-related and anti-Plasmodium genes, and the overall fitness cost of transgenic mosquitoes upon depletion of aga-miR-14 or aga-miR-305 was negligible. We show that targeting mosquito miRNA by transgenic expression of miRNA sponges may have potential for the development of malaria control through genetically engineered mosquitoes.application/pdfenCreative Commons Attribution 4.0 InternationalArticle - Refereedhttps://doi.org/10.1371/journal.ppat.1008453164323301981553-7374