| Mosquitoes,the best known diseases vector,transmit diseases including malaria,dengue,yellow fever,West Nile fever,etc.Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and West Nile virus.Currently,there are no commercially available vaccines against malaria,West Nile fever and filariasis;andthe emergence of resistance to treatment drugs was demonstrated by many reports.So vector control is considered important for the control of diseases,the core strategies of which mainly rely on insecticides.Pyrethroid is a kind of insecticide that is widely used for mosquito control.Unfortunately,many countries rely much heavily on the use of insecticides in controlling mosquito vectors.As a result,the resistance becomes a major obstacle for mosquito control.The resistance appears to be inherited quantitatively.The genetic basis of insecticide resistance remains poorly understood.In this study,a 380 cM genetic map of resistance to deltamethrin(DM)was conducted in F2 intercross segregation populations using amplified fragment length polymorphism markers(AFLP)in Cx.pipiens pallens.The final map covered 53 markers.Lengths of three LGs were 125.5,173.6 and 82.1 cM,and the numbers of markers in three LGs were 18,31 and 4,respectively.The average spacing size in the distance between two markers was 7 cMand the largest distance between two markers was 44 cM in LG 3.We developed the first AFLP-based genetic map in Cx.pipiens pallens.QTL analysis was performed using CIM algorithm,based on the genetic map of resistance to DM.Seven QTLs were identified.The LOD score peak of each of these QTLs exceeded 3.0 and were from 3.58 to 9.57.The DR-4 had the highest LOD score(9.57),while the DR-3 had the lowest score(3.58).The identified QTLs accounted for 95%of the phenotypic variance.DR-6 was the major QTL and accounted for approximately 61%of the variance.Most of the identified QTLs(5)were located in linkage group 2.The major finding of this study is that resistance to DM is under the control of one major QTL,but several QTLs play more minor roles in Cx.pipien pallens,which supported that resistance to insecticides operates through a polygenetic inheritance.12 AFLP markers in the map were cloned and the genomic locations of these marker sequences were determined by applying the genome sequence of the closely related Cx.pipiens quinquefasciatus.All were located on different supercontigs in regions of high sequence identity(>90%).The marker L1B1.151 in supercontig 3.67 was mapped to a location on chromosome 2,and the unique correspondences of LG2 and Chromosome 2 was confirmed.Since 10 AFLP markers(L3A9.119,L1B1.151,L3A8.177,L2A5.138,L1B2.90,L1A16.95,L1A16.146,L1A42.114,L4B1.175 and L3A8.139)were mapped on the same linkage group,we could anticipate that all of these supercontigs(3.289,3.67,3.388,3.98,3.21,3.560,3.45,3.20,3.492,3.453)could be mapped to chromosome 2.This will form the basis of future to fill the gaps in the current genome assembly using a combination of linkage mapping and physical mapping.We searched for potential candidate genes in the flank regions of these 12 markers in the genome sequence,and excluded conserved hypothetical proteins.By combination the molecular functions and biological processes of genes with a keyword search(insecticide resistance),we selected potential candidate genes,involving P450 families,serine protease families,ribosomal protein families,transcription factor families.Protease M1 zinc metalloprotease was found around the genomic sequence surrounding the marker L3A8.177.The range of its expression level between the susceptible and resistant strains was 25-fold in Cx.pipiens pallens.Then the relationship between DM resistance and the candidate gene was identified using RNAi technology in vivo.Knockdown of the gene decreased the sensitivity of mosquitoes to DM in the susceptible strain.The results suggested that Protease Ml zinc metalloprotease was a novel deltamethrin-resistance-associated gene.We cloned the ORF regions of protease Ml zinc metalloprotease in both susceptible and resistant strains.The ORF region has 924 bp and encodes for a protein with 308 amino acids(GenBank:KX275212).The protease M1 zinc metalloprotease of Cx.pipiens pallens shared the highest homology with Cx.pipiens quinquefasciatus(94%identity).An ORF sequence analysis carried out in two strains showed that only point mutation was found at the 806th base,and the mutation(806T>C)was synonymous.The genes CYP6CP1 and protease M1 zinc metalloprotease were in the flank region of L3A8.177 and had significantly different expression levels between susceptible and resistant strains.The expressions profiles of CYP6CP1 and protease M1 zinc metalloprotease were similar in various structures of mosquiotoes.The expression level of CYP6CP1 was up-regulated when protease Ml zinc metalloprotease was knocked down in susceptible strains,while the level of protease M1 zinc metalloprotease was not changed significantly when CYP6CP1 was knocked down in resistant ones.So we speculated that protease M1 zinc metalloprotease may be involved in DM resistance by affecting the transcriptional level of CYP6CP1.The expression levels of protease M1 zinc metalloprotease in five strains with different levels of DM resistance were identified.With the increase of resistance levels,transcriptional expressions of the gene gradually decreased.We used the correlation analysis and found a significant negative correlation between relative quantification of protease Ml zinc metalloproteaseand the LC50(r=-0.97,P<0.05).It was worth mentioning that protease Ml zinc metalloprotease was significantly up-regulated at different stages.Moreover,the gene was up-regulated in the susceptible strain compared to the corresponding resistant strainin 4 field populations(2.01-fold in SH,2.43-fold in GD,1.20-fold in HM,1.60-fold in JN,respectively).The gene may be used as a potential molecular marker to monitor the pyrethroid resistancein fieldpopulations.In the study,we conducted the QTL mapping conferring DM resistance and identified a novel DM-resistance-associated gene,which was responsible for the critical theoretical basis for further genetic studies investigating mechanisms of insecticide resistance. |
PDF Full Text Request