MiR-13664 Regulates Deltamethrin Resistance In Culex Pipiens Pallens

Vector-borne diseases account for 17% of all communicable diseases.As an important vector,mosquitoes can spread a variety of serious mosquito-borne diseases,such as malaria,dengue fever,zika,encephalitis and so on.Because of lacking effective vaccines and therapeutic drugs,it’s very important to develop effective methods to control mosquito vectors.Chemical control is widely used for mosquito control because it’s efficient,and be convenient to use.With the large-scale and continuous use of insecticides,mosquito vectors develop resistance to insecticides,which has become a major obstacle to mosquito control.In order to effectively manage the resistance of mosquito,we have to deeply understand the development and the regulatory mechanism of resistance.Metabolic resistance is recognized as one of the most important resistance mechanisms.MicroRNAs(miRNAs)can bind to the target sites of message RNA(mRNA)and participate in gene post-transcriptional regulation,which affect various relevant regulatory processes in vivo.Based on the high-throughput miRNA sequencing data of Culex pipiens pallens in our laboratory,we used the miRDeep prediction method to predict novel miRNAs.We selected 9 miRNAs with relatively high scores and used real-time quantitative PCR(qRT-PCR)to detect their relative expression levels in the deltamethrin-sensitive(DS-strain)and deltamethrin resistant(DR-strain)strains of Cx.pipiens pallens.The results showed that the novel miRNA,miR-13664,was highly expressed in the DS-strain.We overexpressed or inhibited the expression ofmiR-13664 using microinjection,and we observed changes in mosquitoes sensitivity to deltamethrin.The results suggested that miR-13664 may be associated with resistance.To verifiy whether the novel miR-13664 is a miRNA,we performed Argonaute interference experiments.Argonaute 1(Ago1)is involved in the miRNA pathway,and Argonaute 3(Ago3)is involved in the piRNA pathway.Using the Ago1 and Ago3 siRNA to knock down the expression of related genes of Cx.pipiens pallens,We found that the expression level of miR-13664 was reduced after Ago1 knockdown compared with the negative control(NC),but miR-13664 expression level was not affected by Ago3 knockdown,indicating that Ago1 is essential for the biosynthesis of the novel miRNA(miR-13664).The software prediction showed that the seed domain of miR-13664 could be paired with the 3’ untranslated region(3’UTR)of CYP302A1 、 CYP314A1 、CYP307B1.Then,we confirmed the expression levels of three genes using qRT-PCR.Then 3’ UTR regions of these three genes were cloned and compare with the miR-13664 seed domain.Only 3’ UTR regions of CYP314A1 was perfectly complementary to the miR-13664 seed domain and was highly expressed in DR-strain,Which suggested that CYP314A1 might be the target gene of miR-13664.We identified the regulatory relationship between miR-13664 and CYP314A1 using the dual luciferase reporter system and in vivo gene microinjection technology.The results show that miR-13664 regulate the expression of CYP314A1.And then we interfered CYP314A1 expression to observe the sensitivity of mosquito to deltamethrin,the results show that down-regulation of CYP314A1 led to increased sensitivity to deltamethrin in mosquito.The present study reported that novel miR-13664 of Cx.pipiens pallens is involved in deltamethrin resistance in mosquito through regulating the expression ofthe target cytochrome P450.This study further reveals the molecular mechanism of pyrethroid resistance in mosquito,which could help to generate new strategies for mosquito vectors management.