| Objective:To understand the resistance targets genes mutations of Anopheles sinensis in different areas of Guizhou Province,to analyze the genetic diversity,genetic differentiation,population expansion and phylogenetic relationship of Anopheles sinensis population,so as to provide basic data and scientific guidance for the study of resistance mechanism and the control of Anopheles sinensis in Guizhou Province.Methods:Adult mosquitoes were collected around rice fields in Guizhou Province by lamp trapping method.Female Anopheles sinensis were selected and tested the insecticide resistance using WHO standard test kits.Then genomic DNA was extracted to amplify mtDNA-COI gene for molecular identification of mosquito species.The kdr and ace-1 genes were amplified by PCR from DNA samples identified as Anopheles sinensis and sequenced in two directions.The results were spliced and compared by DNA Star software.The mutations of the related sites were detected and the gene frequency and genotype frequency were calculated.DNA SP 5.0 software was used to analyze the polymorphism of mtDNA-COI gene,calculate the size of gene flow of population differentiation coefficient,carry out neutral test and mismatch analysis.MEGA 7.1 software was used to calculate the genetic distances among different populations,and analyze the correlation between them and geographical distances,and phylogenetic evolutionary tree was constructed based on population genetic distances.Results:The knockdown rate after exposure to insecticide for 1 hour was 34.31%~86.21%and the mortality rate was 37.93%~89.74%of Anopheles sinensis in Guizhou Province.By morphological and molecular identification,551samples of Anopheles sinensis were obtained from 12 populations and 551sequences of kdr,ace-1 and mtDNA-COI genes were analysised.Five alleles were detected at 1014 locus in kdr gene,which were 1014L(70.50%),1014F(24.44%),1014C(0.63%),1014S(2.17%)and 1014W(2.26%)respectively.The frequencies ranged from 2.00%to 57.69%.A total of 18 genotypes were found,and the frequencies ranged from 0.18%to 56.48%.Two alleles were detected at 119 locus in ace-1 gene,119G(49.64%)and 119S(50.36%)respectively.The frequency of mutant genes ranged from 6.00%to 77.00%.Five genotypes were found with genotype frequencies ranging from 0.18%to 38.47%.A total of 329 haplotypes were detected in 551 mtDNA-COI gene sequences,accounting for 59.71%of the total sample number.The haplotypes accounted for more than 70%of the total sample number.The haplotype diversity index was more than 0.99,the nucleotide diversity was more than 0.015,the Fst of 12 populations ranged from 0.0008 to0.4207,and the Nm ranged from 0.0861 to 75.239.The neutrality test statistics were negative,and the distribution curve of the dislocation points in population mismatch analysis showed a single peak.The genetic distance ranged from 0.0071to 0.0529 within the population,from 0.0079 to 0.0522 among the populations,and the correlation coefficient between genetic distance and geographical distance was R~2=0.1658,and there was a positive correlation.The phylogenetic evolutionary trees of DY and ZJ populations were relatively independent compared with other populations.Conclusion:Resistance to pyrethroid insecticides was observed in Anopheles sinensis of Guizhou Province.Four gene mutations were detected at1014 locus in kdr gene with a low frequency of mutation and 1014F as the dominant mutation gene;G119S mutation at 119 locus in ace-1 gene was more frequent and widely distributed,and there was a certain risk of insecticide resistance;genetic diversity was higher,genetic differentiation between most populations was smaller,and gene exchange was smaller.It is relatively abundant.It has experienced population expansion in history,and there is a certain geographical isolation,showing a pattern of co-existence of communication and isolation. |
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