Background Aedes albopictus is the primary vector of dengue fever in China. This mosquito species has a wide distribution range in China and can be found in the tropical climate zones of southern provinces through to temperate climate zones of northern provinces. Insecticides are an important control method, especially during outbreaks of dengue fever, but increasing insecticide resistance raises the risk of failure to control vector-borne diseases. Knockdown resistance (kdr) caused by point mutations in the voltage-gated sodium channel (VGSC) gene is a key mechanism that confers resistance to pyrethroids. In this study we explored the characteristics and possible evolutionary trend of kdr mutation in Ae. albopictus based on analysis of the kdr mutations in field populations of mosquitoes in China. Methods A total of 1549 adult Ae. albopictus were collected from 18 sites in China from 2017 to 2019 and 50 individuals from three sites in the 1990s. A fragment of approximately 350 bp from part of the S6 segment in the VGSC gene domain III was amplified and sequenced. Using TCS software version 1.21A, we constructed haplotypes of the VGSC gene network and calculated outgroup probability of the haplotypes. Data of annual average temperatures (AAT) of the collection sites were acquired from the national database. The correlation between AAT of the collection site and the kdr mutation rate was analyzed by Pearson correlation using SPSS software version 21.0. Results The overall frequency of mutant allele F1534 was 45.6%. Nine mutant alleles were detected at codon 1534 in 15 field populations, namely TCC/TCG (S) (38.9%), TTG/CTG/CTC/TTA (L) (3.7%), TGC (C) (2.9%), CGC (R) (0.3%) and TGG (W) (0.1%). Only one mutant allele, ACC (T), was found at codon 1532, with a frequency of 6.4% in ten field populations. Moreover, multiple mutations at alleles I1532 and F1534 in a sample appeared in five populations. The 1534 mutation rate was significantly positively related to AAT (Pearson correlation: r(18) = 0.624, P = 0.0056), while the 1532 mutation rate was significantly negatively related to AAT (Pearson correlation: r(18) =  − 0.645, P = 0.0038). Thirteen haplotypes were inferred, in which six mutant haplotypes were formed by one step, and one additional mutation formed the other six haplotypes. In the samples from the 1990s, no mutant allele was detected at codon 1532 of the VGSC gene. However, F1534S/TCC was found in HNHK94 with an unexpected frequency of 100%. Conclusions Kdr mutations are widespread in the field populations of Ae. albopictus in China. Two novel mutant alleles, F1534W/TGG and F1534R/CGC, were detected in this study. The 1534 kdr mutation appeared in the population of Ae. albopictus no later than the 1990s. The F1534 mutation rate was positively correlated with AAT, while the I1532 mutation rate was negatively correlated with AAT. These results indicate that iInsecticide usage should be carefully managed to slow down the spread of highly resistant Ae. albopictus populations, especially in the areas with higher AAT. Graphical abstract

Background Dengue fever is an infectious disease that is imported into Shanghai, China and requires prevention and control measures. Controlling the vector Aedes albopictus through insecticide use is a key approach to dengue control. However, the rapid evolution of insecticide resistance in Ae. albopictus has raised concerns about the failure of dengue control efforts. Knockdown resistance (kdr) caused by point mutations in the voltage-gated sodium channel (VGSC) gene is a primary mechanism of pyrethroid resistance. In this study, we investigated the kdr mutations of Ae. albopictus in Shanghai and evaluated the trend in its evolution. Methodology/principal findings We collected 17 populations of Ae. albopictus from 15 districts in Shanghai in 2020, extracted genomic DNA from individual mosquitoes, and amplified Domain II, III, and IV in VGSC using PCR. Following sequencing, we obtained 658 VGSC sequences. We detected the nonsynonymous mutations V1016G, I1532T, and F1534S/C/I, among which V1016G and F1534C/I were reported in Shanghai for the first time and F1534I was a novel mutant allele in Ae. albopictus. The overall mutation frequency was 84.65%, with individual mutation frequencies ranging from 46.81% to 100%, excluding the Fengxian District population, which had a frequency of 0%. The V1016G and I1532T mutation types accounted for 7.14% and 3.42%, respectively. The mutant allele at codon 1534 accounted for 63.98% of all mutations, including TCC/S (62.77%), TGC/C (1.06%), and ATC/I (0.15%). We identified and classified five intron types in Domain III by length, including A (83 bp, 12.07%), B (68 bp, 87.30%), C (80 bp, 0.16%), D (72 bp, 0.16%), and E (70 bp, 0.31%). Individuals with intron B had a significant mutation tendency at codon 1534 relative to intron A (chi-square test, p < 0.0001). We found no correlation between mutation frequency and the amount of pyrethroid used (Pearson correlation, p = 0.4755). Conclusions/significance In recent years, kdr mutations in the Ae. albopictus population in Shanghai have rapidly evolved, as evidenced by an increase in mutation types and significantly increased mutation frequency. The F1534I/ATC mutant allele was found to be a novel mutation, F1534C/TGC was reported for the first time in Shanghai, and intron B in Domain III was significantly associated with mutation frequency at codon 1534. Continuous monitoring of resistance changes and strict regulation of insecticide use are required.

Background Mosquito control is needed to prevent dengue fever, which is mainly spread by Aedes albopictus in China. Application of insecticides is one of the main mosquito control methods; however, this approach can fail due to the knockdown resistance (kdr) gene mutation that causes decreased sensitivity to insecticides in Ae. albopictus. The kdr mutation patterns among different regions in China differ significantly. However, the underlying mechanism and factors that influence kdr mutation remain unclear. To explore the potential influence of genetic background on the development of insecticide resistance in Ae. albopictus, we analyzed the genetic structure of Ae. albopictus populations in China and its correlation with major kdr mutations. Methods We collected Ae. albopictus from 17 sites in 11 provinces (municipalities) across China from 2016 to 2021 and extracted the genomic DNA from individual adult mosquitoes. We selected eight microsatellite loci for genotyping, and based on microsatellite scores, we estimated intraspecific genetic diversity, population structure, and effective population size. The association between the intrapopulation genetic variation and F1534 mutation rate was evaluated by the Pearson correlation coefficient. Results Based on variation analysis of the microsatellite loci of 453 mosquitoes representing 17 populations throughout China, more than 90% of the variation occurred within individuals, whereas only about 9% of the variation occurred among populations, indicating that field populations of Ae. albopictus are highly polymorphic. The northern populations tended to belong to gene pool I (BJFT 60.4%, SXXA 58.4%, SDJN 56.1%, SXYC 46.8%), the eastern populations tended to belong to pool III (SH 49.5%, JZHZ 48.1%), and the southern populations tended to belong to three different gene pools. Moreover, we observed that the greater the fixation index (FST), the lower the wild-type frequency of F1534 of VSGC. Conclusions The degree of genetic differentiation among Ae. albopictus populations in China was low. These populations were divided into three gene pools, in which the northern and eastern pools are relatively homogeneous, while the southern gene pool is heterogeneous. The possible correlation between its genetic variations and kdr mutations is also noteworthy. Graphical Abstract

Background: Aedes albopictus is the main vector of dengue fever in China, distributed from north to south in China. Insecticides are an important method to control the mosquitoes, especially in the outbreak of dengue fever, but insecticide resistance raises the risk of failure to control vector-borne diseases. Knockdown resistance (kdr) caused by point mutations in the VGSC gene is a key mechanism that confers resistance to pyrethroids. To explore the characteristics and possible evolution trend of kdr mutation in Ae. albopictus, we analyzed the kdr mutations of field populations in China in this study.Methods: A total of 1 549 Ae. albopictus were collected from 18 sites in China from 2017 to 2019, as well as 50 individuals from three sites in the 1990s. A fragment of approximately 350 bp from part of S6 segment in the VGSC gene domain III was amplified and sequenced. The haplotypes of VGSC gene were recorded and the parsimony network was constructed using TCS 1.21. The data of annual average temperatures (AAT) of collection sites was acquired from national database. The correlation between AAT of the collection site and the kdr mutation rate was analyzed by Pearson Correlation using SPSS 21.0. Results: The overall frequency of mutant allele F1534 is 45.62%. Nine mutant alleles were detected at codon 1534 in fifteen field populations, namely TCC/TCG (S) (38.86%), TTG/CTG/CTC/TTA (L) (3.71%), TGC (C) (2.68%), CGC (R) (0.27%) and TGG (W) (0.10%). Only one mutant allele ACC (T) was found at codon 1532 with frequency of 6.39% in ten field populations. Moreover, multiple mutations at I1532 and F1534 in a sample appeared in five populations. The 1534 mutation rate was significantly positive related to AAT (Coefficient=0.624, p=0.0056), while the 1532 mutation rate was significantly negative related to AAT (Coefficient=-0.645, p=0.0038). Thirteen haplotypes were inferred, in which six mutant haplotypes were formed by one step, and the other six haplotypes were formed by one more mutations. In the samples from 1990s, no mutant allele was detected at codon 1532 of VGSC gene. However, F1534S/TCC was found in HNHK94 with an unexpected frequency of 100%.ConclusionsKdr mutations are widespread in the field populations of Ae. albopictus in China. Two novel mutant alleles F1534W/TGG and F1534R/CGC were the detected. The 1534 kdr mutation appeared in the population of Ae. albopictus no later than 1990s. F1534 mutation rate is positive correlated to AAT, while I1532 mutation rate is negative correlated to AAT. Insecticide using should be carefully managed to slow down the spread of high-resistance Ae. albopictus populations.

Background: Aedes albopictus is considered to be the main vector of dengue fever and Chikungunya fever in China. Mosquito control is an important component of the integrated programs to prevent dengue fever transmission. In this study, to provide technical support for future genetic control of mosquitoes, the genetic structure of Ae. albopictus population in China was analyzed and the correlation between genetic variations within Ae. albopictus population and kdr mutation was explored. Methods: Seventeen collection sites from 11 provinces (municipalities) of Ae. albopictus were collected from 2016 to 2021 in China, including most regions in the distribution area. Genomic DNA was extracted from individual adult mosquito using the DNAzol reagent. Eight microsatellite loci were finally selected for genotyping. Population genetic structure and demographic inference were analyzed using the software. Pearson correlation coefficient R was used to examine the association between the intra-population genetic variation and kdr mutation. Results: The considered eight microsatellite loci, scored in 453 mosquitoes across the 17 localities from North to South of China. The variation analysis showed that most variation occurred within individuals, whereas only about 9% of the total variation was detected among populations. Three gene pools were inferred by Bayesian analysis, in which the overall trend was that the northern populations belong to gene pool I (red), the eastern populations belong to III (blue), and the southern populations belong to three different gene pools. We also observed that the greater the genetic difference (FST), the lower the wild-type frequency of F1534 of VSGC gene in the same population. Conclusions: The degree of genetic divergence of Ae. albopictus in China was very low among the populations. These populations can be divided into three genetic gene pools. The gene pool of the northern and eastern Chinese populations was relatively simple, and southern populations were heterogeneous. The greater the genetic difference within the population, the larger the mutation rate of knockdown resistance, indicating that frequent migration among Ae. albopictus populations in China maybe a major factor in the increasing insecticide resistance.