| Dengue fever is an important vector-borne infectious disease caused by the dengue virus.The global incidence of DF has risen sharply in recent years.Southeast Asia is one of the worst affected areas among more than 100 endemic countries.Few cases of imput DF were reported in the historical records of Yunnan province.But since local infection started in 2008,many cases caused by input cases in Yunnan province happened especially since 2013.Aedes aegypti is an important medical insect which is an important vector of the DF.It originated in Africa and is mainly distributed in tropical and subtropical regions.The flight diffusion distance is limited but the adult mosquitoes,pupae,larvae and eggs can achieve passive diffusion distance due to human activities such as plane,ship or automobile transportation.The invasion to Southeast Asia was achieved by shipping in the late 19 th century.Before 2000,the distribution of Ae.aegypti in our country is confined to 22 degree north latitude including southern Taiwan,Hainan,Guangdong,parts of Guangxi and some individual islands.No record was found in Yunnan province but globalization and global warming provides a more convenient condition for the spread of medical insect vector.Since Ae.aegypti was first found in Yunnan in February 2002,the distribution range and population is expanding.Now it's found in Ruili,Mangshi,Mengla,Menghai,Jinghong,Yingjiang,Longchuan,Lushui and Gengma.The invasion and spread of the situation is serious and Ae.aegypti has become an important invasive mosquito specie in Yunnan province.Ae.aegypti and DF is found frequently in the bordering nations of Yunnan province such as Vietnam,Myanmar,Laos and other countries.The invasion has increased the risk of DF outbreak and epidemic in Yunnan.However,most research on the Ae.aegypti of Yunnan province is limited to distribution level,there's a lack of in-depth study on the population genetic differentiation and characteristics,resistance level and mechanism.Due to the reasons listed above,this research use Ae.aegypti in Yunnan province as the research object and carry out the study from two aspects through the field acquisition of natural population in different areas.First is the extraction of genomic DNA,using microsatellite DNA markers and mitochondrial DNA gene CO I?ND4?ND5 for population genetics research,to understand genetic diversity and structure characteristics of Yunnan province,as well as to discuss the intrusion events and the population structure.Second,after the laboratory extend breeding of Ae.Aegypti,we aim to master in the resistance level to commonly used insecticides and drug resistance analysis in Yunnan province through mosquito resistance biological determination,metabolic enzymes and target gene detection,to provide a scientific basis for chemical pesticides prevention and treatment of Ae.aegypti.The results of this study are as follows:1.Microsatellite research resultsThe results showed that the nine microsatellite loci obtained from literature were easy to be amplified and has good stability,the polymorphism was high(PIC = 0.392 ~ 0.886).This indicates that the selected nine microsatellite loci can fully meet the needs of this study and the sample size of each population can meet the requirement of the study.A total of 114 alleles were obtained from 9 microsatellite loci from 28 natural populations of Ae.aegypti,and the average number of alleles was 12.67.From the point of view of the analysis,the average allele per locus was between 3.46 and 7.14 for each.The gene richness of each group was between 3.34 ~ 5.72,and the abundance of Ae.aegypti was similar in Ruili and Jinghong.The average allele of Ae.aegypti was also higher than other border areas in 2016.The population of Ae.aegypti in Yunnan Province has a high genetic diversity,the observed heterozygosis of the 9 microsatellite loci was between 0.108 and 0.939 with an average of 0.579.The expected heterozygosis was at 0.367 to 0.760 while the average expected heterozygosis was 0.592.The average heterozygosis of each population was between 0.401 and 0.689,and the heterozygosis of the eight populations collected in other border areas in 2016 was higher than He while the 20 populations collected in Ruili and Jinghong were lower.Genotypic linkage disequilibrium analysis showed that there were 166 significant linkage sites(P<0.05)in the 1008 loci between the 9 loci of 28 Ae.aegypti natural populations in Yunnan,while other sites were expressed as independent inheritance(P> 0.05).The Hardy Weinberg equilibrium test showed that only the Jinghong(GLR)and Menghai(M1H)populations in the 28 populations had a Hallowan equilibrium deviation.TPM and SMM were used to detect the genetic bottleneck effect.The results showed that there were 10 natural populations with bottleneck effect.Among them,six out of 10 populations collected in Ruili area(MNL,JDL,JGH,JCG,GMG,and CXT)had a bottleneck effect,accounting for 60% of the population of Ruili region.Four out of the eight natural populations collected in other border areas in 2016 had a bottleneck effect,accounting for 50%.But the 10 populations collected in Jinghong did not have the effect.The results of inbred lines show that the inbred lines of seven populations of the eight populations collected in other border areas in 2016 are negative,indicating that the inbreeding rates of these seven populations are low and the inbreeding coefficients of the other populations are positive,which means that these populations have different degrees of inbred.PING analysis showed that only 17 pairs of fixed indices(FST)did not show significant genetic differentiation among the groups,and the remaining 361 pairs of fixed indices(FST)showed significant genetic differentiation.With the use of the STRUCTURE software to analyze the clustering of each individual,combined with the data of the group within the group,the results showed that when K =3,the population of 28 Ae.aegypti in different regions of Yunnan Province was better divided into three categories.10 populations collected in Ruili,10 populations collected in Jinghong and 8 species collected in other border areas in 2016 each were better divided into one category.The results were the same when based on Nei's standard genetic distance application UPGMA construction system developmental tree and PCoA map analysis.Molecular variance analysis(AMOVA)showed significant differentiation among different groups,accounting for 14.51%.The genetic differentiation among different populations in the same area accounted for 6.01% of the total population.The genetic differentiation among individuals was the most significant,accounting for 77.76%.Correlation analysis showed a positive correlation between geographical distance and genetic differentiation(r = 0.4948,P<0.0001).Due to the comprehensive analysis listed above,we found that the 2002 Ruili invasion and the 2013 Jinghong invasion should be a different invasion,and there has still been a continuous multi-point invasion in other border areas in 2006.2.Mt DNA research resultsIn this study,the gene diversity indices of mitochondrial genes CO I,ND4 and ND5 of 20 different Ae.aegypti populations in Ruili and Jinghong of Yunnan Province were analyzed.The results showed that the content of CO I gene A + T was 66.9%,with 381 polymorphic loci,45.36% of the total number of polymorphic loci(including 66 ethnic assimilation sites and 315 simple information loci),14 species with haplotype,total population haplotype diversity.The index(Hd)was 0.5583 and the nucleotide diversity index(?)was 0.02265.The content of A + T of ND4 gene was 70.0% and 334 polymorphic loci were obtained,accounting for 55.76% of the total number of bases(including 126 self-seeded sites and 191 simple information loci).A total of 18(Hd)was 0.47156,and the average nucleotide diversity(?)was 0.01860.The content of A + T in the ND5 gene was 74.6%,and 369 polymorphic loci were obtained,accounting for 60.89% of the total number of bases(including 109 seedling sites and 254 simple information loci),and 5 haplotypes.The total population haplotype diversity index(Hd)was 0.5098 and the average nucleotide diversity was 0.07223.Because of the haplotypes of ND5 gene fragments were very few(only 5 species),this study only used CO I and ND4 to study the genetic differentiation and genetic structure of 20 Ae.aegypti populations in Ruili and Jinghong areas.Genetic diversity based on CO I gene showed that GGH population of Jinghong was not significantly different from MNL,BFC,HDH and BNS in Ruili,but there were significant genetic differentiation comparing to the five populations GXS,JBQ,YSC,DMY,GSZ,NKH,etc.in Jinghong area.The results of genetic differentiation based on ND4 showed that there was no obvious genetic differentiation between the HDH population of Ruili and the three populations of LJY,JBQ and YSC in Jinghong.More interestingly,the other 9 populations in Ruili area had obvious Genetic differentiation.The results indicate that there may be mutual communication between Ae.aegypti populations in Ruili and Jinghong.Genetic differentiation based on CO I and ND4 genes showed that the genetic differentiation among populations in Jinghong area was significantly lower than that in Ruili area.This result is similar to that of microsatellite studies.Although the mitochondrial CO I and ND4 haplotypes can be differentiated into two branches by the distribution pattern of NBR and NJ phylogenetic tree,the distribution of haplotype populations cannot be carried out very clearly.Tajima'S D and Fu's FS neutral test showed significant negative values.The nucleotide mismatch distribution showed that a single peak structure occurred in the distribution curves.These results indicate that Ae.aegypti experienced obvious population expansion in these areas.3.Resistance biology test resultsThe laboratory bred sensitive strains of Ae.aegypti in Yunnan was taken as a reference to biologically determine the larvae by sensitive baseline method.Among the three insecticides tested,the toxicity of propoxur was the lowest(LC50 = 1.02882mg/L,95% CI: 0.97708~1.08173 mg/L),while dichlorvos was higher(LC50 = 0.0367 mg / L,95% CI: 0.03580 ~ 0.03770 mg / L)and the toxicity of beta-cypermethrin was the highest(LC50 = 0.00102 mg / L,95% CI: 0.00088 ~ 0.00118 mg / L).The results of the biology test showed that among the 12 species of Ae.aegypti larvae collected in 2016 in Xishuangbanna and Dehong,the multiplicity of beta-cypermethrin was between 11.31 and 41.56.Although the resistance level of propoxur and dichlorvos increased slightly,the criteria for the resistant population had not yet been reached.The diagnostic dose of mosquitoes for three insecticides was 983.41 mg / L of propoxur,21.95 mg / L of dichlorvos and 1493.37 mg / L of beta-cypermethrin respectively.The biology of adult mosquitoes of Ae.aegypti was studied by diagnostic dose method.The results were also shown to be resistant only to beta-cypermethrin,and that the antibiotic resistance of the mosquitoes was the same as that of larvae.4.Metabolic detoxification enzyme activity test resultsIn this study,the metabolic detoxification activity of the natural populations of Ae.aegypti was detected by WHO's recommended biochemical assay.The results showed that the ACHE activity was 22.36-31.60 after being suppressed by propoxur while only J-3 and J-5 were slightly higher than 30%.The active mean of EST(1-N)was 111.1717 ± 34.9588 nmole of 1-naphthol / min / mg protein with ?-Naphthyl Acetate as substrate while(1-N)activity of wild populations was between 41.7940 nmole of 1-naphthol / min / mg protein ~ 198.5297 nmole of 1-naphthol / min / mg protein.The activity of EST(2-N)was 135.4011 ± 47.3301 nmole of 2-naphthol / min / mg protein with ?-Naphthyl Acetate as the substrate while the activity of EST(2-N)in wild population was 39.7117 nmole of 2-naphthol / min / mg protein ~ 214.0106 nmole of 2-naphthol /min/mg protein.The MFO activity of the susceptible strain was 3.08 ± 3.77 nmol cytochrome C / mg protein,and the MFO activity of the wild population was between 5.43 nmol cytochrome C / mg protein ~ 20.11 nmol cytochrome C / mg protein.The MFO activity of the wild population was higher than that of the sensitive population compared with the susceptible strain.The GST activity of the susceptible strain was 1.2504 ± 0.3961 nmol of CDNB / min / mg protein,and the GST activity of the wild population was between 0.6178 nmol of CDNB / min / mg protein ~ 3.4939 nmol of CDNB / min / mg protein.The results showed that the activities of MFO only increased in all wild populations,while other metabolic enzymes perform differently in different populations.It suggests that the resistance to Pyrethroids of Ae.aegypti in Yunnan may be related to the increase of MFO enzyme activity.The reason for the difference between the enzyme activity level and the biological assay is to be further studied.5.Target resistance test resultsIn this study,it was found that the amino acid sequence of the 1534 locus of the Amino acid channel of Ae.aegypti in Yunnan was changed from F to C,and the corresponding base TTC mutation was TGC.No other mutations were found.The correlation analysis indicates that the mutation at this point was only positively correlated with the resistance of the cypermethrin to the insecticide,and was not found to be related to the resistance to propoxur and dichlorvos. |
PDF Full Text Request