The endosymbiont Wolbachia is common among insects and known for the reproductive manipulations it exerts on hosts as well as inhibition of virus replication in their hosts. Recently, we showed that Wolbachia uses host microRNAs to manipulate host gene expression for its efficient maintenance in the dengue mosquito vector, Aedes aegypti . Cytosine methylation is mediated by a group of proteins called DNA (cytosine-5) methyltransferases, which are structurally and functionally conserved from prokaryotes to eukaryotes. The biological functions of cytosine methylation include host defense, genome stability, gene regulation, developmental promotion of organs, and lifespan regulation. Ae. aegypti has only one DNA methyltransferase gene ( AaDnmt2 ) belonging to the cytosine methyltransferase family 2, which is the most deeply conserved and widely distributed gene among metazoans. Here, we show that in mosquitoes the introduced endosymbiont, Wolbachia , significantly suppresses expression of AaDnmt2 , but dengue virus induces expression of AaDnmt2 . Interestingly, we found that aae-miR-2940 microRNA, which is exclusively expressed in Wolbachia -infected mosquitoes, down-regulates the expression of AaDnmt2 . Reversely, overexpression of AaDnmt2 in mosquito cells led to inhibition of Wolbachia replication, but significantly promoted replication of dengue virus, suggesting a causal link between this Wolbachia manipulation and the blocking of dengue replication in Wolbachia -infected mosquitoes. In addition, our findings provide an explanation for hypomethylation of the genome in Wolbachia -infected Ae. aegypti.

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BackgroundBest recognized for its role in manipulating host reproduction, the parasitic gram-negative Wolbachia pipientis is known to colonize a wide range of invertebrates. The endosymbiotic bacterium has recently been shown to cause a life-shortening effect as well as inhibiting replication of arboviruses in Aades aegypti; although the molecular mechanisms behind these effects are largely unknown. MicroRNAs (miRNAs) have been determined to have a wide range of roles in regulating gene expression in eukaryotes. A recent study showed that several A. aegypti mosquito miRNAs are differentially expressed when infected with Wolbachia. Methodology/Principal FindingsBased on the prior knowledge that one of these miRNAs, aae-miR-12, is differentially expressed in mosquitoes infected with Wolbachia, we aimed to determine any significance of this mediation. We also set out to characterize the target genes of this miRNA in the A. aegpyti genome. Bioinformatic approaches predicted a list of potential target genes and subsequent functional analyses confirmed that two of these, DNA replication licensing (MCM6) and monocarboxylate transporter (MCT1), are under the regulative control of aae-miR-12. We also demonstrated that aae-miR-12 is critical in the persistence of Wolbachia in the host cell.Conclusions/SignificanceOur study has identified two target genes of aae-miR-12, a differentially expressed mosquito miRNA in Wolbachia-infected cells, and determined that the miRNA affects Wolbachia density in the host cells.

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