Study On Population Genetic Characteristics Of Aedes Aegypti In China-Laos And China-Myanmar Border Areas Of Yunnan Province

Aedes aegypti is an important invasive mosquito that originated in Africa and is mainly distributed in tropical and subtropical regions.Ae.aegypti invaded into Southeast Asia in the 19 th century.Dengue fever is an acute infectious disease transmitted by Ae.aegypti.It is endemic in more than 100 countries in tropical and subtropical regions of the world.In the past,it was believed that Ae.aegypti was distributed only in the areas south of 22°N latitude in China.However,following the discovery of Ae.aegypti at Jiegao Port in Ruili City,Yunnan Province in 2002,Ae.aegypti was successively caught in other areas of Yunnan Province.Judging from the epidemic situation of dengue fever in recent years,a high density of Ae.aegypti was detected at the epidemic sites,and the distribution areas of Ae.aegypti were consistent with the local dengue fever epidemic areas.Due to the lack of specific drugs and vaccines for the treatment of dengue fever,mosquito vector control is the most effective way to prevent and control dengue fever outbreaks.Therefore,this study reveals the distribution characteristics of the two Aedes species by investigating the breeding containers in China-Laos and China-Myanmar border areas of Yunnan Province.Using microsatellite and mitochondrial DNA markers to study the population genetic characteristics of Ae.aegypti which were collected in border areas of Yunnan Province in 2019 and 2020,in order to clarify the population diversity,the situation of population expansion and invasion sources of Ae.aegypti.Through bioassay of drug resistance to natural populations of Ae.aegypti,and the analyze of the kdr gene,our work aims to master the current drug resistance of Ae.aegypti populations to permethrin,lambda-cyhalothrin and the characteristics of kdr gene mutations.The analysis results are summarized as follows:The investigation of the Ae.albopictus and Ae.aegypti breeding sites found that two kinds of Aedes(Ae.aegypti and Ae.albopictus)in Xishuangbanna Prefecture(Mengla County,Mohan Port,Jinghong City,Menghai County,Daluo Port),Lincang City(Nansan Town,Mengding Town)and Dehong Prefecture(Ruili City).A total of233 breeding sites were discovered,of which buckets accounted for the highest proportion of 45.92%.Genetic characteristics of Ae.aegypti population in border areas of Yunnan Province based on microsatellite markers: When analyzing the populations of 2019 and2020,the average polymorphism information content(PIC)of the 9 pairs of primers selected in this study were 0.546 and 0.545,respectively,showing high polymorphism.67 and 72 alleles were obtained,the number of alleles at each site was 4-14,the frequency of null allele at each site was within an acceptable range,it showed that 9pairs of primers meet the research needs.The Shannon index of the populations in 2019 was between 0.578 and 1.805.The average observed heterozygosity was 0.538,and the expected average heterozygosity was 0.517.The observed heterozygosity was higher than the expected heterozygosity,indicating that the populations were supplemented by foreign individuals.Except the Qingshuihe Port population QSH-2 deviated from Hardy-Weinberg equilibrium,all other populations maintained Hardy-Weinberg equilibrium.The Shannon index of the populations in 2020 was between 0.646 and 1.150.The average observed heterozygosity was 0.514,and the average expected heterozygosity was 0.519.The observed heterozygosity is less than the expected heterozygosity,indicating that the populations had inbreeding.All populations were in Hardy-Weinberg equilibrium.Based on the analysis of clustering methods,it was concluded that the populations of Ae.aegypti in border areas of Yunnan Province in 2019 and 2020 were all divided into two clusters,the first cluster was Xishuangbanna Prefecture populations,and the second clusters was Lincang City,Ruili City populations.The results of the Ae.aegypti population fixation index(FST)in 2019 and 2020 showed that the FST value between populations in the same area was significantly smaller than the FST value between populations in different areas.The test results of the correlation between geographic distance and genetic distance clearly showed that there was a moderately significant correlation between population geographic distance and genetic distance in 2019 and 2020(2019: P<0.05 R=0.6933,P<0.05 R=0.6506).In2019,a total of 6 natural populations produced a bottleneck effect.including the JH-1,JH-4,JH-5 populations in Jinghong City,Xishuangbanna Prefecture;the RL-3population in Ruili City,Dehong Prefecture;the QSH-2 population in Qingshuihe Port of Lincang City,and one Burmese population.Among the 17 populations collected in2020,the JH-11 population in Jinghong City,Xishuangbanna Prefecture and the DL-2population at Daluo Port had a bottleneck effect.Genetic characteristics of Ae.aegypti populations in border areas of Yunnan Province based on mitochondrial DNA molecular markers: A total of 10 haplotypes were found in the COⅠ fragment of the populations in 2019.The haplotype diversity index(Hd)was 0.626,and the nucleotide diversity index(π)was 0.01135.A total of 20 haplotypes were found in the COⅠ fragments of the Ae.aegypti populations collected in2020.The haplotype diversity index(Hd)was 0.592,and the nucleotide diversity index(π)was 0.01034.A total of 17 haplotypes were found in the ND4 fragment of the populations in 2019,the haplotype diversity index(Hd)was 0.706,and the nucleotide diversity index(π)was 0.01704.The 2020 populations of ND4 fragments obtain a total of 24 haplotypes,the haplotype diversity index(Hd)was 0.736,and the nucleotide diversity index(π)was 0.01642.The haplotype network diagram was drawn through the Network software,and the results showed that there were two dominant haplotypes of the COⅠ fragment and the ND4 fragment of the populations in 2019 and 2020.One haplotype was mainly distributed in Xishuangbanna Prefecture,Yunnan Province,and the other haplotype was mainly found in populations of Lincang City and Ruili City.Based on the COⅠ and ND4 gene analysis,the collected populations in 2019 confirmed that there were JH-3 and JH-5 populations in Jinghong City,Xishuangbanna Prefecture,MD-3 population in Mengding Town,Lincang City,QSH-1 and QSH-2 at Qingshui River Port and RL-2 population in Ruili City experienced bottleneck effect and then expanded.Analyzing the COⅠ and ND4 genes of the populations collected in2020,we found that the JH-12 population in Jinghong City,Xishuangbanna,the RL-7population in Ruili City,the MD-6 population in Mengding Town of Lincang City experienced bottleneck effects.The nucleotide mismatch distribution showed that a single peak structure occurred in the distribution curves.These results indicated that Ae.aegypti experienced obvious population expansion in these areas.The results of Ae.aegypti kdr gene mutation analysis: Three loci mutations were found,which were S989 P,V1016G and F1534 C.The analysis of allele frequencies at S989 locus in different populations showed that the mutation rate of Ae.aegypti collected in Jinghong City was the highest in two years,while Ruili City had the lowest mutation rate among all the areas we had surveyed.The allele frequency of F1534 locus was different from that of S989 locus.The mutation frequency of Jinghong city was the highest,while the mutation rate of Lincang populations was the lowest in two years.Except for 1 individual in the Myanmar population who is heterozygous,the other populations collected in 2019 are all homozygous mutations at the locus 1016.Among the 15 populations collected in 2020,except for one individual in DL-4 population of Daluo Port in Xishuangbanna Prefecture and one in MD-9 population of Mengding Town,Lincang City were wild-type homozygous,all the others were homozygous mutations.15 natural populations were resistant to both permethrin and lambda-cyhalothrin.A significant positive correlation between the frequency of the F1534 C point mutations and the bioassay mortality permethrin and lambda-cyhalothrin was observed.However,no relationship between 989 loci and population mortality is found.Conclusion:(1)The Ae.aegypti invaded into Lincang City and Ruili City,Yunnan Province,China via multiple routes from Myanmar.The populations in Xishuangbanna Prefecture did not expand from Lincang City or Ruili City.It may come from the Lancang-Mekong freight transport or invaded with the movement of people.(2)Ae.aegypti in China-Laos and China-Myanmar border areas of Yunnan Province can be divided into two clustered: Xishuangbanna Prefecture population as one cluster,Lincang City and Ruili City as the other cluster.(3)The population diversity of Ae.aegypti changed little in different years,but human activities had an impact on the genetic exchange of Ae.aegypti.The population diversity of Ae.aegypti collected in 2019 and 2020 was relatively high,and there was still great potential for the Ae.aegypti to expand.(4)The Study on the kdr gene of Ae.aegypti populations in the China-Laos and China-Myanmar border areas in Yunnan Province find three mutation sites: S989 P,V1016G and F1534 C.Ae.aegypti in border areas of Yunnan Province had developed resistance to permethrin and lambda-cyhalothrin.