A total of three mutations were detected in this study: one was a nonsynonymous mutation, in which leucine L1014 was mutated to phenylalanine L1014F (TTA-TTT) or serine L1014S (TTA-TCA). The second was a base double mutation of L1014F/L1014S, in which leucine L1014 is mutated to both phenylalanine and serine F1014S. The third was a base synonymous mutation of L1014L (TTA-CTA). Consistent with the results of existing studies [22,23,24,25], the L1014F mutation is not conserved. The existence of multiple mutation types at locus 1014 suggested that the mutations in Cx. p. pallens/Cx. p. quinquefasciatus in China tend to be diversified, and the genotype is polymorphic. Increased mutational orientation of locus 1014 under insecticide selective pressure and enhanced adaptation to the environment. The present study revealed significant geographic variation in the type, number and frequency of mutations. The allelic and genotypic composition of Cx. p. pallens/Cx. p. quinquefasciatus varies with latitude, from single to diverse, before shifting to single. The genotypes are more monotypic north of 38° N latitude. The genotypes in the 30–38° N region are diverse. Populations south of 30° N latitude are homogeneous in terms of allelic and genotypic composition. Populations north of 38° N had a high percentage of L/L, while populations south of 30° N had more mutant genotypes, with no wild pureblood genotypes detected in YNJH and HNSY and less than 5% of wild types in HBWH and ZJNB. Populations south of 30° N latitude had a high mutation frequency and a small percentage of sensitive genotypes. The adaptability of the mutant phenotypes of Cx. p. pallens/Cx. p. quinquefasciatus is increasing under the selective pressure of insecticides. Mutant genes can be stably inherited in populations, suggesting that genes may be conserved in the wild.

In this study, the synonymous mutation CTA/L was detected only in the Liaocheng population in Shandong, China. Most of the mutations at locus 1014 of Aedes albopictus from the Anhui and Hunan Zhuzhou populations were CTA/L. It is possible that CTA/L is nonmutated in Aedes albopictus because locus 1014 has a CTA/L background. In contrast, the wild-type Cx. p. pallens/Cx. p. quinquefasciatus is TTA/L, and although CTA and TTA both encode leucine, CTA may be a mutation in Cx. p. pallens/Cx. p. quinquefasciatus. In the Zibo population, the CTA and TTC allele genotypes were not detected, in contrast to the results of Wei's study [24]. This difference may be due to the following reasons. The studies used different sampling habitats and sampling times. Convenient transportation, frequent transportation of goods, and well-developed tourism increase the chances of mosquitoes' passive movement, which increases the possibility of gene exchange between populations and changes the frequency of allele genotypes of the populations. And the populations had a low content of mutant alleles.

Resistance has developed in Cx. p. pallens/Cx. p. quinquefasciatus in most areas. The laboratory strain of Cx. p. pallens (BJ strain) had a resistance frequency of 15.96% and a mortality rate of 50.00% under exposure to a diagnostic dose of deltamethrin of 0.025%. The BJ strain was resistant to deltamethrin [26]. Combining the BJ bioassay results and the frequency, it can be seen that field populations with a frequency < 20.00% have developed resistance to pyrethroid insecticides. The greater the frequency is, the lower the sensitivity to pyrethroid insecticides. This indicates that the light-colored Culex mosquitoes in most parts of the country have become resistant to the insecticide. Even populations with low frequencies may have developed resistance. When the frequency of vgsc alleles in Culex pipiens is low, cytochrome P450-mediated metabolic resistance dominates. With continued selection pressure, as the number of screening generations increases, the expression of P450 genes tends to stabilize, genetic differences between individuals in the population decrease, the actual heritability of drug resistance in the population increases, and vgsc mutations dominate. A combination of biological and molecular methods should be used to determine the level of resistance in populations, especially those with low frequencies, in a comprehensive manner.

Wild population resistance is regional in nature. The frequency increases as the latitude decreases. This study revealed that the mutation in Cx. p. pallens/Cx. p. quinquefasciatus in China exhibits significant spatial heterogeneity. The number and frequency of mutations showed significant geographic differences. Geographic populations with frequencies < 20.00% were mainly concentrated north of 38° N latitude. However, geographic populations south of 30° N latitude have frequencies > 80.00%. The populations south of 30° N latitude, such as the HNSY, YNJH, CQSPB and HBWH populations, had frequencies > 90.00%. No wild-type allele genotypes were detected in the HNSY population, with a mutation frequency of 100.00%. This could be because temperatures are higher at lower latitudes, and temperature is one of the most important factors affecting the growth cycle of mosquitoes and the level of breeding. Tire-causing Cx. p. quinquefasciatus were caught throughout the year in Hainan and Guangdong. In Jilin and Liaoning, the peak occurred in August, and after October, there were basically no catches of Cx. p. pallens [27]. Mosquitoes overwinter differently, affecting the number of mosquitoes in the second year, which in turn affects the genetics of resistance. The more mosquitoes that overwinter, the more mosquitoes there will be in the following year. The greater the percentage of resistant mosquitoes that survive overwintering is, the greater the probability of resistance inheritance. Shandong's Cx. p. pallens overwinter at high temperatures and high humidity and in light-sheltered kilns, and the larvae cannot overwinter [28]. Cx. p. quinquefasciatus in Jiangsu overwinters in underground garages, stairwells on the ground floor of buildings, and private houses on the outskirts of the city in different forms [29]. On the other hand, Yunnan Province has a high incidence of dengue fever [30], and Chongqing had a dengue outbreak in 2019 [31]. Therefore, the frequency, dose and duration of insecticide use are greater in these areas than in others. Insecticide residue levels are increased at mosquito breeding sites. The duration of mosquito exposure to insecticides increases, leading to increased mosquito resistance. The higher kdr frequency also converges across urban areas. The frequency of pesticide use is related not only to natural factors such as latitude and temperature but also to social factors such as economic, demographic. BJCP, SHSJ, and HBWH are large cities in terms of population, economy, and transportation. These cities are densely populated and serve as important transportation hubs with high logistics and people flow. Developed regions have a long history of insecticide use, and mosquitoes are exposed to insecticides for long periods of time, increasing cumulative toxicity. The frequency of insecticide use is greater in large cities within the same latitude. Mosquitoes are more likely to develop insecticide resistance.

This study only included mosquitoes collected in 2021 and lacked previous data. In recent years, due to its relationship with dengue fever, research on Aedes albopictus has been increasing, and research on Cx. p. pallens has correspondingly decreased. The main focus of this study was to investigate the haplotypes of adjacent introns downstream of locus 1014 using the appropriate sequences. The detected vgsc genes may not necessarily contain the complete introns required for research, making it impossible to obtain additional historical sequence data for Cx. p. pallens. Another approach is to compare functional genes (such as vgsc genes) with neutral markers (such as mitochondrial DNA), which may more accurately demonstrate the combined impact of demographic and selection factors on population structure.