| ObjectiveTo understand the distribution of Anopheles species in Henan Province,and to explore whether DNA barcoding with different genetic markers is different in the identification of Anopheles species.The genetic and evolutionary relationships among different geographical populations of An.sinensis were studied to understand the population structure.To grasp the level and the molecular characteristics of resistance of An.sinensis to four insecticides in Henan Province,the insecticide resistance of An.sinensis was investigated in four sites,and the target resistance genes of kdr and Ace-1 were detected.The population genetic evolution relationship between resistant and sensitive mosquitoes of An.Sinensis were analysed based on COI gene,deepening the understanding of the difference of resistance status and the occurrence and law of gene mutation in the process of population evolution,so as to provide scientific basis for vector monitoring and disease prevention and controling.Methods1.Collecting An.mosquito samples from different areas in Henan Province from2019 to 2021,extracting DNA and identifying species of Anopheles mosquitoes based on species-specific ITS2 gene.Then the sequence of different mosquito species was analysed using DNA barcoding technology based on two-gene(ITS2 and COI)markers.Calculating genetic distances and building phylogenetic trees,then comparing the species identification results.2.An.sinensis was selected,and genetic evolutionary relationships between various groups were analyzed by calculating the genetic diversity index,conducting neutral tests and AMOVA analysis with Mega,Dna SP,Win Arl,Pop Art and other bioanalytic software.3.The adult mosquito filter paper contact tube method recommended by WHO was used to determinate the resistance of four sites in Henan Province from July to September 2021,then the target resistance genes of kdr and Ace-1 were amplified to detect mutation frequency.Chi-square tests were conducted for the difference of mosquitoes mortality rates in different regions and gene mutation rates in different populations.The COI genes of deltamethrin sensitive mosquitoes and resistant mosquitoes were amplified,and then the genetic diversity was analyzed by using various biological analysis software.Results1.A total of 150 Anopheles mosquitoes were identified based on ITS2 specific fragment length,including 108 An.sinensis,13 An.pullus,23 An.belenrae and 6 An.anthropophagus.The intra-species variation of ITS2 in the four Anopheles species was much smaller than the inter-specific variation,which could accurately identify mosquito species.The DNA barcoding based on COI gene could accurately identify An.sinensis,An.anthropophagus and An.pullus,but couldn’t identify An.sinensis and An.belenrae because of the distances of intra-and inter-specific between them were closely.2.The haploid diversity(Hd)and nucleotide diversity(Pi)of 108 An.sinensis from 15 geographical populations were 0.993 and 0.01089,respectively,with no significant differences in neutral test(P>0.1).The genetic differentiation index(Fst)among the 15 populations was 0.02434(P<0.05).Most of the Fst among the 15populations were not significant,but the Fst of Kaifeng and Jiaozuo populations was0.27982(P<0.05),showing obvious genetic differentiation.AMOVA analysis showed that among the total genetic variation of An.sinensis,the proportion of intra-population variation was as high as 97.57%,while the proportion of inter-population variation was only 2.43%,suggesting that the variation was mainly from within the population.The interspecific and intraspecific genetic distances of 15 An.sinensis populations ranged from 0.00668 to 0.01487,and from 0.00556 to 0.01477,respectively.The analysis of the correlation between genetic distance and geographical distance between populations showed that there was no correlation relationship and the haploid network diagram did not form the aggregation cluster of geographical units.3.The 24h mortality rates of An.sinensis to deltamethrin,beta-cyfluthrin and propoxur at four sites in Henan province were 3.28%-85.85%,which had reached the confirmed resistance(R).The 24h mortality rates to malathion ranged from 64.23%to99.21%,and the resistance levels ranged from susceptible(S)to resistance(R).There were statistically significant differences in 60-min knockdown rates and 24h mortality rates of different insecticides in the four sites(X~2=32.635～143.433,P<0.05).4.Three mutations of kdr gene were detected:1014F,1014C and 1014W.Only one mutation was found in Ace-1 gene:119S.The differences in mutation rates among regions were statistically significant(x~22=311.369,P<0.01;x~22=149.42,P<0.01).In Tanghe,Nanyang,there were statistically significant differences in both mutation frequency of kdr gene and Ace-1 gene between resistant and sensitive mosquitoes in beta-cyfluthrin(c2=8.393,P<0.01)and propoxur(x~22=12.863,P<0.01),respectively.5.The inter-specific genetic distance between deltamethrin-resistant and sensitive populations was 0.009,but the intra-species genetic distance was 0.009 and 0.01,respectively.The results of Fst and neutral test were not significantly negative(P>0.1),and the haploid network diagram failed to form two branches.Conclusion1.DNA barcoding based on two-gene markers of ITS2 and COI can be used to identify mosquito species accurately.Four species of Anopheles were identified:An.sinensis,An.pullus,An.anthropophagus and An.belenrae.Significantly,An.belenrae were first found in Henan Province.2.The population of An.sinensis in Henan province showed a coexisting distribution structure of gene flow and isolation.3.Three mutations of kdr gene were detected:1014F,1014C and 1014W.1014W was first detected in Henan Province,and 1014F was the main mutant gene.There were differences in insecticide resistance mechanisms in different regions,and the development of resistance may be the result of a combination of multiple factors. |
