| Aedes albopictus,also known as "Asian tiger mosquito",originated from southeast Asia,is widely distributed in tropical,subtropical and temperate regions around the world,and is one of the most common mosquito species in China.It is distributed in Liaoning province in the north,Hainan province in the south and Tibet in the west.Aedes albopictus is an important vector of dengue virus,chikungunya virus,yellow fever virus and zika virus.The application of chemical insecticides to control mosquitoes is one of the important measures for the prevention and control of mosquito-borne diseases.Studies have shown that F1534 S mutation of knockdown resistance(kdr)gene is closely related to pyrethroid insecticide resistance phenotype.In this study,Aedes albopictus was collected from all over China to detect the type and frequency of kdr mutation.Meanwhile,polymorphic microsatellite DNA was used to calculate population genetic differences and structures.To comprehensively analyze and explore the origin of kdr mutation in the population,as well as the rule of diffusion between groups.The results can provide theoretical basis for scientific and effective control of Aedes albopictus.The main results are as follows:1.Total of 1392 Aedes albopictus mosquitoes were collected from 15 sites in China through morphological and molecular identification,and the groups were divided according to the collection areas for follow-up study.2.By comparing the sequence of partial VGSC gene fragment of Aedes albopictus,the results showed that there were non-synonymous mutations at I1532 and F1534 of S6 fragment in coding region 3,and there were synonymous mutations at P1516,F1528,T1539,L1540 and N1541 of 5 sites in total.Among the non-synonymous mutations,the two alleles and frequencies at 1532 were as follows: wild-type ATC/I(92.89%)and mutant ACC/T(7.11%).The 10 alleles and frequencies at 1534 loci were wild type TTC/F(41.99%),mutant type TTG/L(1.24%),CTG/L(0.15%),CTC/L(0.39%),TTA/L(0.89%),TCC/S(40.91%),TCG/S(0.15%),TGC/C(0.97%),CGC/R(0.43%)and TGG/W(0.12%).Among the synonymous mutation loci,there were two types of alleles at 1539 and 1540,namely ACT/T,ACG/T,CTG/L and CTT/L.There was only one type of 1516,1528 and 1541 loci,respectively CCA/P,TTT/F and AAT/N.The mutation frequencies of the 5 sites were 17.13%,12.03%,3.77%,51.51% and 51.80%,respectively.3.Firstly,52 individuals(3.74%)of Aedes albopictusVGSC gene 1532 and 1534 were found to be mutated at the same time,and no individuals were found to be mutated homozygous,only existing in 5 field populations of Yunnan jinghong,Shanghai baoshan,Shanghai yangpu,Yangpu gongqing and Zhejiang Hangzhou.The correlation between simultaneous mutations at both sites and drug resistance needs to be further determined.4.The mutation frequency of kdr F1534 S in Aedes albopictuswas calculated,which was 38.01%.F1534 S mutation was not found in populations of Shaanxi Xian,Guangdong Shantou and Shandong Jining.The results suggested that the resistance level was the highest in Zhejiang Hangzhou population.5.The 11 populations of Aedes albopictus were detected inferred from 8 polymorphic microsatellite DNA loci.The number ofalleles was ranged from 7 to 22,with an average of 11.8750.Allele richness was ranged from 3.5070 to 10.5720.The results suggested that these microsatellite DNA loci were suitable molecular markers for the analysis of population genetic variation.6.The expected heterozygosityand observed heterozygosity were ranged from 0.5085(Hainan Sansha)to 0.6626(Hainan Haikou),0.3179(ShanghaiBaoshan)to 0.5664(Guangdong Shantou),with an average of 0.6154 and 0.3749,respectively.The inbreeding coefficient was ranged from 0.1020(Guangdong Shantou)to 0.5190(ShanghaiBaoshan),with an average of 0.3906.The results suggested that the inbreeding opportunities of the population were more and the heterozygosity was less.7.The Fst value of Aedes albopictus population was 0.1048,and the gene flow was 2.1366.In general,there was little genetic difference between the populations and no significant variation.The genetic difference between Guangdong Shantou population and Hainan Sansha population was the greatest(Fst= 0.2836),which indicated that the genetic differentiation between the two populations was obviously.On the contrary,the Fst value was zero pairwise population between ShanghaiBaoshan withZhejiang Hangzhou.AMOVA analysis showed that genetic variation mainly existed in the population,accounting for 89.53% of the total variation,and inter-population variation accounted for 10.47% of the total variation.Bayesian analysisshowed that all individuals could be divided into three clades,total of mosquitoes in Hainan Sansha population was belonged to clade I,73.33% of samples in Guangdong Shantou populationwas assigned to the clade II.The rest individuals of the other9 populations were to the clade II and III,with frequency was no significant difference.The results suggested that there was no genetic variation among the populations except Hainan Sansha and Guangdong Shantou populations.8.The comprehensive analysis of the relationship between population genetic variation and kdr mutations of Ae.albopictus.There was enough gene flow among large-scale populations of Ae.albopictus in China.The genetic differences among populations were not related to geographical distance.The migration and spread of kdr mutations in different populations were expected to be fast.Combining with weak genetic structure and mutation types of VGSC gene,the diffusion route was from south to north and the northeast,respectively. |
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