Research On Knockdown Resistance Gene Mutations And Detection Technology Of Aedes Albopictus In China

Aedes albopictus is one of the most common mosquito species in China.It is very invasive and can spread many kinds of viral infectious diseases such as dengue fever,yellow fever,Zika virus disease and Chikungunya fever.To minimize the risk of human infection with these infectious diseases,effective control of Ae.albopictus is of great importance.In recent years,dengue fever broke out in many provinces and cities in China,and the scope of the outbreak is expanding,which makes the demand for prevention and control increasing.At present,the control of mosquitoes after dengue outbreak depends largely on the use of chemical insecticides.Pyrethroid insecticides are widely used because of their high insecticidal activity and low toxicity to mammals.However,the continuous and large-scale use of insecticides to kill Ae.albopictus is equivalent to giving the population a selection pressure.In order to resist this selection pressure,some specific sites of the target gene of Ae.albopictus will mutate,leading to the occurrence and development of resistance to insecticides.Insecticide resistance is a huge obstacle to mosquito control.Therefore,it is very important to monitor the mutation of the resistance target gene of Ae.albopictus population in the field and to elucidate the mechanism of the resistance of mosquitoesIn this study,39 geographical populations of Ae.albopictus were collected nationwide.Based on the mosquito samples collected in the field,the gene mutation and population genetic parameters of VGSC genes at loci V1016,11532 and F1534,which are closely related to the resistance of pyrethroid insecticides,were studied by using molecular biological methods.Secondly,a more efficient and economic mutation detection method was established for locus F1534.The results are summarized as follows:1.In Beijing,Chongqing,Jiangsu,Hunan and Yunnan provinces,407 individuals of 12 Ae.albopictus field populations were collected to detect the genotypes,alleles and frequencies of loci V1016,11532 and F1534 of VGSC gene,so as to reveal the level of resistance to pyrethroids.The results of sequencing showed that there were three genotypes at locus V1016,i.e.wild homozygous V/V(277/68.06%),wild/mutant heterozygous V/G(102/25.06%)and mutant homozygous G/G(28/6.88%).Three genotypes were also found at locus 11532,namely I/I(281/69.04%),I/T(98/24.08%)and T/T(28/6.8%).Compared with loci V1016 and 11532,locus F1534 is more diverse and complex.Six genotypes were detected,including wild-type F/F(199/48.89%),wild/mutant F/S(66/16.22%)and F/C(20/4.91%).There were two types of mutant homozygotes,namely S/S(49/12.04%)and C/C(37/9.09%),and there was another mutant heterozygote,namely S/C(36/8.85%)of heterozygotes of serine and cysteine.The results of molecular analysis indicated that all the above populations were resistant to pyrethroid insecticides.The results of population genetic studies of the above 12 populations showed that all the measurable populations except HZYZ population were in line with Hardy Weinberg equilibrium.There was no linkage disequilibrium in all loci.For locus V1016,the populations of BJCP,BJTZ,BJDX,CQNC,CQQJ,HNYZ,HNZJJ and YNDL were inbreeding,while the populations of BJFT,CQTN,CQSPB and JSNJ were far inbreeding.At 11532 locus,there were inbreeding in BJDX,BJFT and YNDL,and there were distant inbreeding in BJCP,BJTZ and JSNJ.However,at F1534 locus,except CQTN and JSNJ,there were inbreeding in other geographical regions.The population differentiation between HNYZ,HNZJJ and the four administrative districts in Beijing were more significant.YNDL was significantly different from the other populations except the four administrative districts in Beijing.The calculation results of Nm value showed that gene exchange were frequent among different geographical populations.2.A total of 817 samples were collected from 27 Ae.albopictus populations with mutation data of VGSC gene at loci 11532 and F1534 in Hangzhou and Ningbo,Zhejiang province and other areas.The genotype,allele and frequency of VGSC gene at locus V1016 were detected,so as to re veal the level of resistance to pyrethroids.In general,there were 3 genotypes at locus V1016,i.e.wild homozygotes V/V(577/70.62%),wild/mutant heterozygotes V/G(181/22.15%)and mutant homozygotes G/G(59/7.22%).Except for the populations HKXX,HKYX and ZJNB,V1016 mutations were found in the remaining 24 populations.QZSQ has the highest mutation rate.3.165 samples including 8 genotypes at the F1534 locus in the VGSC gene were used to establish the AS-PCR method.DNA sequencing in this study was taken as the gold standard,and used to determine the accuracy of AS-PCR.The designed AS-PCR technique showed high specificity for distinguishing the mutations at the F1534 locus,in addition to the genotype L/L(TTG/L),the accuracy for F/F,F/S,F/C,F/L(CTC/L),S/S,C/C and S/C were 100%,95.35%,100%,100%,100%,100%and 100%,respectively.