The Role Of MiR-2?13?71 Cluster In Resistance To Deltamethrin In Culex Pipiens Pallens

Vector-borne infections account for 17%of the global burden of infectious disease.Mosquitoes,as the best known disease vector,could transmit numerous mosquito-borne diseases,including malaria,dengue fever,West Nile fever.Mosquito control is the most important element in global strategies for mosquito-borne diseases.Chemical control is the core mean of mosquito control due to its high-efficiency,long-lasting,low toxicity and convenience.Prolonged and excessive exposure to insecticides could result in mosquitoes developing resistance,which has become the major obstacle for mosquito control.It is the problem to be solved for researchers to make a scientific and rational management controls of insecticide resistance.To understand the mechanism of insecticide resistance deeply could help to provide a scientific theory of insecticide resistance management.Metabolic resistance is known to be a main mechanism in insecticide resistance.Metabolic enzyme especially cytochrome P450 is associated with insecticide resistance.The recent researches on P450 are focused on transcriptional regulation,however,post-transcriptional regulation largely remains scarce.MicroRNAs(miRNAs)are a key component in post-transcriptional regulation and play significant roles in various biological processes.Emerging evidence reveals that miRNAs in insects play a notable in cellular immune response.The roles of miRNAs in insecticide resistance have received little attention.In our previous study,differentially expressed miRNAs of Culex pipiens pallens were obtained between deltamethrin-susceptible(DS-)strain and deltamethrin-resistant(DR-)strain by high-throughput sequencing.MiR-71,upregulated in DS-strain,participated in deltamethrin resistance through its target gene cytochrome P450 325BG3(CYP325BG3).When maximum inter-miRNA distance(MID)was 10 kb,miR-71 was found to be clustered miR-2 and miR-13,namely miR-2?13?71 cluster.In this study,we aim to the regulation of miR-2?13?71 cluster in insecticide resistance.The expression levels of miR-2?13?71 cluster between DS-strain and DR-strain were investigated by the quantitative real-time PCR(qRT-PCR).The results suggested miR-2 and miR-13 were 2.32-and 1.86-fold higher(P<0.05)in the DS strain compared with the DR strain,in line with miR-71.The effects of knocking down of miR-2 or miR-13 or miR-71 to the susceptibility of female mosquitoes to deltamethrin treatment were tested after injection the miR-2 or miR-13 or miR-71 inhibitor in DS-strain by American CDC bottle bioassay.The results showed that knocking down of miR-2 or miR-13 or miR-71 in DS-strain decreased susceptibility to deltamethrin,the mortality rate of female mosquito was decreased.The susceptibility to deltamethrin was increased in female mosquitoes by injecting miRNA mimics,the mortality rate of female mosquito was increased.The results suggested that miR-2?13?71 cluster was associated with insecticide resistance.Target prediction for miR-2 and miR-13,both miRNA seed region sequences were complementary to the 3'-untranslated(UTR)sequences of CYP9J35.The expression level of CYP9J35 was 3.78 fold higher in the DR strain compared with the DS strain(P<0.05).The dual fluorescent report system was introduced to verify the interaction between miR-2 or miR-13 and its predicted target gene CYP9J35 in vitro.The results indicated that miR-2 and miR-13 mimics decreased luciferase activity by 38.1%and 31.3%(P<0.05)in the CYP9J35 PMIR-UTR group,compared with the NC control group.Compared to the control group,the levels of CYP9J35 in miR-2 or miR-13 mimic injected group decreased by 33%or 40%(P<0.05).However,the expression level of CYP9J35 was 2.0-or 1.8-fold higher in the miR-2 or miR-13 inhibitor group than that in the control group.Taken together,miR-2 and miR-13 could directly regulate CYP9J35 in vitro and in vivo.The expression level of miR-2 and miR-13 decreased by 43%and 49%(P<0.05),when miR-71 was overexpression in DR-strain.However,the expression of miR-2 and miR-13 were 2.01-and 2.49-fold higher(P<0.05)when miR-71 was inhibited in DS-strain(P<0.05)and vice versa.The results showed that miR-2?13?71 cluster may have intrinsic regulation that balance a certain expression level in the miRNA cluster and keep the stability of the miRNA cluster function of insecticide resistance.The expression level of CYP325BG3,target gene of miR-71,was decreased in miR-71 mimic groups,while CYP9J35(target gene of miR-2 and miR-13)was 4.1 fold higher compared with NC(P<0.05).The expression level of CYP9J35 was decreased,while CYP325BG3 was 2.5 fold higher(P<0.05)in miR-2 and miR-13 mimic injected group than that in NC group.The changeable expression of target genes further explained the intrinsic regulation in miR-2?13?71 cluster.MiR-2?13?71 cluster had a network regulation in regulating target genes CYP325BG3 and CYP9J35.Knocking down of CYP9J35 in DR-strain,decrease transcript of CYP9J35 made female mosquitoes more sensitive to deltamethrin.The group injected siCYP9J35 showed higher mortality rate than the other two groups.The results suggested that CYP9J35 was a new insecticide resistance associated P450 gene.The open reading frame(ORF)of CYP9J35 was cloned in DS-strain and DR-strain.The ORF was 1506 bp and encoded for a protein with 501 amino acids(GenBank number:KY780946,2017).With the sequence analysis on CYP9J35 in two strains,the three synonymous mutations were detected simultaneously:933T>A?939G>A and 945T>C,and the mutation frequencies were 70%in DR-strain and 26%in DS-strain.In 1143th base in the exon region of CYP9J35 had two different mutations.1143C>A was synonymous mutation in DS-strain,1143C>A was nonsynonymous mutation in DR-strain which the mutation induced a switch from phenylalanine to leucine in 381th amino acids.Whether this nonsynonymous mutation could change the activity of metabolic enzyme,it was needed to be further investigated.In this study,we first report the role of miR-2?13?71 cluster in pyrethroid resistance in mosquito and identified the regulatory network involving miR-2/13 and miR-71 participating in pyrethroid resistance through regulating CYP9J35 and CYP325BG3.The results provided the evidences with post-transcriptional regulation in metabolic enzyme.The four mutation sites and five mutation types of CYP9J35 ORF between DS-strain and DR-strain gave the clues of evolution of insecticide resistance.