Evaluation of the evolutionary genetics and population structure of Culex pipiens pallens in Shandong province, China based on knockdown resistance (kdr) mutations and the mtDNA-COI gene
Authors: Zang ChuanHui , wang XueJun , Cheng Peng , Liu LiJuan , Guo XiuXia , Wang HaiFang , Lou ZiWei , Lei JingJing , Wang WenQian , Wang YiTing , Gong MaoQing , Liu HongMei 
Published: February 2023

Background
Mosquitoes are important vectors for a range of diseases, contributing to high rates of morbidity and mortality in the human population. Culex pipiens pallens is the most widely prevalent species of Culex mosquito in northern China and a major vector for both West Nile virus and Bancroftian filariasis. Efforts at controlling the mosquito-mediated spread of these diseases largely center around insecticide application, contributing to rising rates of resistance in the mosquito population. Cx. p. pallens have been found to harbor rising rates of knockdown resistance (kdr) to pyrethroid insecticides resulting from a range of haplotypes and mutations associated with the para-sodium channel gene. While these kdr mutations are known to be critical to pyrethroid resistance, their evolutionary origins remain poorly understood. Efforts to clarify the origins of these mutations have the potential to guide further vector control and disease prevention efforts. Accordingly, the present study was designed to study the evolutionary genetics of these kdr mutations and their association with the population structure of Cx. p. pallens found in Shandong province, China.
Methods
Adult Culex females from Shandong province were collected and subjected to morphological identification under a dissection microscope. DNA samples were then collected from these mosquitos, and the Vgsc gene was amplified via PCR and sequenced to assess kdr allele frequencies, intron polymorphisms, and kdr codon evolution. In addition, population genetic diversity and related population characteristics were assessed by amplifying and sequencing the mitochondrial cytochrome C oxidase I gene (COI).
Results
In total, 263 morphologically similar Cx. p. pallens specimens were used for DNA barcoding AND sequencing analyses to assess kdr allele frequencies in nine Culex populations. The kdr codon L1014 in the para-type sodium channel gene exhibited two non-synonymous mutations (L1014F and L1014S) in the analyzed population. Both mutations were present in the eastern hilly area and northwest plain regions of Shandong Province, with the L1014F mutation being the most common in the latter of these regions. However, only the L1014F mutation was detected in the southern mountainous area and Dongying regions of Shandong Province, and the mutation frequency in these areas was low. Analyses of the sequence of the mitochondrial COI gene revealed significant levels of genetic differentiation when comparing Culex populations from Qingdao and other analyzed cities. Spatial kdr mutation patterns are likely attributable to some combination of prolonged insecticide-mediated selection coupled with the genetic isolation of these mosquito populations.
Conclusions
These data suggest that multiple kdr alleles associated with insecticide resistance are present within the Cx. p. pallens populations of Shandong Province, China. The geographical distributions of kdr mutations in this province are likely the result of prolonged and extensive insecticide application in agricultural contexts together with frequent mosquito population migrations. In contrast, the low-frequency kdr mutation rates detected in central Shandong likely stem from the limited selection pressure in this area and the relative genetic isolation of these populations. Overall, the comparison of the genetic distribution patterns of both a neutral reference gene and a functional gene associated with insecticide resistance provides valuable insight into the demographic and selection factors that affect population structure.