| Mosquitoes are important vectors of numerous infectious diseases of humans,including malaria,dengue fever,yellow fever and West Nile fever.The chemical control of mosquitoes,via the use of insecticides,is the primary means of managing the spread of these diseases,as it is simple,rapid and economical.However,the widespread and improper use of insecticides has resulted in the evolution of resistance,which has become a major obstacle for mosquito-borne diseases management.Researchers firstly used “functional cloning strategy” to study insecticide resistance,primarily through searching the target sites of insecticides or the detoxification enzymes.According to these known function proteins or enzymes,the corresponding encoding genes were cloned.The “functional cloning strategy” has great advantages in the study of monogenic inherited characters.Subsequently,the studies investigating the mechanisms of insecticide resistance through suppression subtractive hybridization(SSH)or serial analysis of gene expression(SAGE)have identified other resistance-relatedgenes.However,insecticide resistance is actually a complex phenotype of polygenic inheritance.Indeed,none of the currently known genes can entirely explain the molecular basis for insecticide resistance.Hence,identifying novel genes associated with insecticide resistance and elucidating the manner in which they regulate this process is critical for effective control of mosquitoes.With the development of high-throughput transcriptome sequencing,it is possible to screen insecticide resistance-related gene from the overall level.In this study,we used Illumina-Solexa transcriptome sequencing(RNA-seq)to obtain a mass of transcriptional information about Culex pipiens pallens in the laboratory deltamethrin-susceptible(Lab-DS)and laboratory deltamethrin-resistant(Lab-DR)strains.We compared the gene expression between the two premixed cDNA libraries.A total of 4,961,197,620 base pairs and 55,124,418 reads were sequenced,mapped to the Culex pipiens quinquefasciatus genome and assembled into 17,679 known genes.We recorded 1,826 significantly differentially expressed genes(DEGs).Among them,1,078 genes were up-regulated and 748 genes were down-regulated in the Lab-DR strain.The criteria of significant difference expression was |log2Ratio| ? 1 and False Discovery Rate(FDR)? 0.001.These DEGs contained cytochrome P450 s,cuticle proteins,UDP-glucuronosyltransferases,lipases,esterases,serine proteases,peptidases,heat shock proteins,ATP-binding cassette transporters and others.Subsequently,these DEGs were for Gene Ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis.The most representative GO and KEGG categories were “metabolic process” and “metabolic pathway”.We refined 8,315 and 8,511 known gene sequences of Culex pipiens quinquefasciatus and predicted 4,957 and 5,048 novel transcript units(NTUs)in the Lab-DS and the Lab-DR strains.To validate the results of gene expression difference analysis,we selected 22 DEGs to quantify their relative expression levels in the Lab-DS and the Lab-DR strains by real time fluorescent quantitative polymerase chain reaction(qRT-PCR).The trend of the gene differential expression analysis by RNA-seq and qRT-PCR was consistent,again increasing the credibility of RNA-seq.On the other hand,we randomly selected 13 NTUs for reverse transcription PCR(RT-PCR)validation.The results confirmed the actual transcriptional expression of these NTUs.We chose 22 DEGs as pyrethroid resistance-related candidate genes according to their reads per kilobase per million mapped reads(RPKM)and fold changes.Some of the 22 DEGs belonged to gene families known to be associated with pyrethroid resistance,while others were novel potential resistance-related genes.We used qRT-PCR to test the relative expression levels of the 22 DEGs between the Lab-DS and the Lab-DR strains in adult female mosquito stage.That we chose the adult female mosquito stage was because the mosquito-borne diseases are mainly transmitted by adult females.Four of the 22 genes(chymotrypsin-2,dimethylaniline monooxygenase,carboxylesterase,venom allergen 5)were significantly overexpressed in the Lab-DR strain compared with the Lab-DS strain(P < 0.05).Combing with the detection results of field populations,we found that only the venom allergen 5 was significantly overexpressed in the resistant strains than the susceptible strains of the laboratory and field populations(P < 0.05).Thus,we selected venom allergen 5 as our candidate gene for the subsequent verification.Full-length cDNAs of the venom allergen 5 was cloned from Culex pipiens pallens by RT-PCR and RACE(Rapid Amplification of cDNA End)techniques,which contained 912 base pairs(bp).The ORF(open reading frame)region had 765 bp and encoded a protein with 254 amino acids(GenBank accession number: KF723295.1).The deduced amino acid sequence shares 100%,70.8%,43.85% and 32.95% identity with the ortholog in Culex pipiens quinquefasciatus,Aedes aegypti,Anopheles gambiae and Drosophila melanogaster.The venom allergen 5 protein contains a SCP(Sperm Coating Protein)-like extracellular protein domain,and belongs to the SCP superfamily.To further investigate the role of venom allergen 5 in deltamethrin resistance,we verified its functionality both in vitro and in vivo.We transiently transfected exogenous pIB/V5-His-venom allergen 5 plasmid DNA into the mosquito cells,and measured the cell viability after different concentrations of deltamethrin treatment using CCK-8 kit.The overexpression of venom allergen 5 significantly decreased the susceptibility of mosquito cells to different concentrations of deltamethrin treatment(P < 0.05).On the other hand,we microinjected designed siRNA40 into the thorax of adult female mosquitoes,and detected the viability of adult female mosquitoes by WHO susceptibility tube bioassay with 0.05% deltamethrin-impregnated drug membranes.The knockdown of venom allergen 5 by RNAi significantly increased the susceptibility of mosquitoes to deltamethrin treatment(P < 0.05).These results indicated that venom allergen 5 may be a novel deltamethrin-resistance gene,which merits further research.We quantified the gene expression level of venom allergen 5 in four strains with different levels of deltamethrin resistance.Their LC50 were 0.03,0.85,3.7 and 7.0 mg/liter,respectively.The relative expression levels of venom allergen 5 were 3.3-,4.7-,and 29.7-fold higher in the three resistant strains than the susceptible strain(P < 0.05).The results of two field populations(Shanghe,Gudao)indicated that the gene expression levels of the venom allergen 5 were 60.58-and 7.34-fold higher in the resistant strains than the susceptible strains(P < 0.05).These results indicated that venom allergen 5 was expected to become a potential marker to monitor and predict the pyrethroid resistance level of field mosquito populations.Finally,we tested the expression levels of venom allergen 5 at different developmental stages(eggs,1st–4th instar larvae,pupae,adult females and males)in the Lab-DS and the Lab-DR strains.The venom allergen 5 was transcribed at all developmental stages.The transcriptional levels were 2.24-,3.3-and 12.3-fold higher in the Lab-DR strain than in the Lab-DS strain at the egg,adult female and male stages(P < 0.05).In this study,we used Illumina-Solexa sequencing to identify 1,826 genes that were expressed differently in the Lab-DS and the Lab-DR strains as a critical theoretical basis for further molecular studies investigating mechanisms of pyrethroid resistance.In addition,we demonstrated that venom allergen 5 participated in the pyrethroid resistance in mosquitoes and was expected to become a target gene in field resistance detection. |
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