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Abstract：
BACKGROUND: Culex pipiens pallens and Culex pipiens quinquefasciatus are the dominant species of Culex mosquitoes in China and important disease vectors. Long-term use of insecticides can cause mutations in the voltage-gated sodium channel (vgsc) gene of mosquitoes, but little is known about the current status and evolutionary origins of vgsc gene in different geographic populations. Therefore, this study aimed to determine the current status of vgsc genes in Cx. p. pallens and Cx. p. quinquefasciatus in China and to investigate the evolutionary inheritance of neighboring downstream introns of the vgsc gene to determine the impact of insecticides on long-term evolution.
METHODS: Sampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China. Genomic DNA was extracted from 1308 mosquitoes, the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction, and the sequence was used to evaluate allele frequency and knockdown resistance (kdr) frequency. MEGA 11 was used to construct neighbor-joining (NJ) tree. PopART was used to build a TCS network.
RESULTS: There were 6 alleles and 6 genotypes at the L1014 locus, which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F, TTC/F, TCT/S and TCA/S. The geographic populations with a kdr frequency less than 20.00% were mainly concentrated in the regions north of 38° N, and the geographic populations with a kdr frequency greater than 80.00% were concentrated in the regions south of 30° N. kdr frequency increased with decreasing latitude. And within the same latitude, the frequency of kdr in large cities is relatively high. Mutations were correlated with the number of introns. The mutant allele TCA/S has only one intron, the mutant allele TTT/F has three introns, and the wild-type allele TTA/L has 17 introns.
CONCLUSIONS: Cx. p. pallens and Cx. p. quinquefasciatus have developed resistance to insecticides in most regions of China. The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene. Mutations may originate from multiple mutation events rather than from a single origin, and populations lacking mutations may be genetically isolated.
METHODS: Sampling was conducted from July to September 2021 in representative habitats of 22 provincial-level administrative divisions in China. Genomic DNA was extracted from 1308 mosquitoes, the IIS6 fragment of the vgsc gene on the nerve cell membrane was amplified using polymerase chain reaction, and the sequence was used to evaluate allele frequency and knockdown resistance (kdr) frequency. MEGA 11 was used to construct neighbor-joining (NJ) tree. PopART was used to build a TCS network.
RESULTS: There were 6 alleles and 6 genotypes at the L1014 locus, which included the wild-type alleles TTA/L and CTA/L and the mutant alleles TTT/F, TTC/F, TCT/S and TCA/S. The geographic populations with a kdr frequency less than 20.00% were mainly concentrated in the regions north of 38° N, and the geographic populations with a kdr frequency greater than 80.00% were concentrated in the regions south of 30° N. kdr frequency increased with decreasing latitude. And within the same latitude, the frequency of kdr in large cities is relatively high. Mutations were correlated with the number of introns. The mutant allele TCA/S has only one intron, the mutant allele TTT/F has three introns, and the wild-type allele TTA/L has 17 introns.
CONCLUSIONS: Cx. p. pallens and Cx. p. quinquefasciatus have developed resistance to insecticides in most regions of China. The neighboring downstream introns of the vgsc gene gradually decreased to one intron with the mutation of the vgsc gene. Mutations may originate from multiple mutation events rather than from a single origin, and populations lacking mutations may be genetically isolated.
摘要：
背景：淡色库蚊和淡色库蚊是中国库蚊的优势种和重要的病媒。长期使用杀虫剂可引起蚊子电压门控钠通道(vgsc)基因突变,但是对不同地理种群中vgsc基因的现状和进化起源知之甚少。因此,本研究旨在确定Cx中vgsc基因的现状。p、苍白和CX。p.quinquefasciatus在中国的研究，并研究vgsc基因邻近下游内含子的进化遗传，以确定杀虫剂对长期进化的影响。
方法：于2021年7月至9月在中国22个省级行政区划的代表性栖息地进行采样。从1308只蚊子中提取基因组DNA，使用聚合酶链反应扩增神经细胞膜上vgsc基因的IIS6片段，并使用该序列评估等位基因频率和敲低抗性（kdr）频率。MEGA11用于构建邻居连接(NJ)树。PopART用于构建TCS网络。
结果：在L1014位点有6个等位基因和6个基因型，其中包括野生型等位基因TTA/L和CTA/L和突变等位基因TTT/F，TTC/F,TCT/S和TCA/Skdr频率小于20.00%的地理种群主要集中在北纬38°以北地区,kdr频率大于80.00%的地理种群集中在30°N以南地区。kdr频率随纬度的降低而增加。在同一纬度内，大城市kdr的频率相对较高。突变与内含子的数量相关。突变等位基因TCA/S只有一个内含子,突变等位基因TTT/F有三个内含子，野生型等位基因TTA/L有17个内含子。
结论：Cx。p、苍白和CX。p。中国大部分地区都对杀虫剂产生了抗药性。vgsc基因的邻近下游内含子逐渐减少到一个内含子，并带有vgsc基因突变。突变可能源于多个突变事件，而不是单一来源。缺乏突变的群体可能是基因分离的。
方法：于2021年7月至9月在中国22个省级行政区划的代表性栖息地进行采样。从1308只蚊子中提取基因组DNA，使用聚合酶链反应扩增神经细胞膜上vgsc基因的IIS6片段，并使用该序列评估等位基因频率和敲低抗性（kdr）频率。MEGA11用于构建邻居连接(NJ)树。PopART用于构建TCS网络。
结果：在L1014位点有6个等位基因和6个基因型，其中包括野生型等位基因TTA/L和CTA/L和突变等位基因TTT/F，TTC/F,TCT/S和TCA/Skdr频率小于20.00%的地理种群主要集中在北纬38°以北地区,kdr频率大于80.00%的地理种群集中在30°N以南地区。kdr频率随纬度的降低而增加。在同一纬度内，大城市kdr的频率相对较高。突变与内含子的数量相关。突变等位基因TCA/S只有一个内含子,突变等位基因TTT/F有三个内含子，野生型等位基因TTA/L有17个内含子。
结论：Cx。p、苍白和CX。p。中国大部分地区都对杀虫剂产生了抗药性。vgsc基因的邻近下游内含子逐渐减少到一个内含子，并带有vgsc基因突变。突变可能源于多个突变事件，而不是单一来源。缺乏突变的群体可能是基因分离的。
