[Population genetic diversity of Culex tritaeniorhynchus in Jining City of Shandong Province based on the mitochondrial cytochrome C oxidase I gene]

Objective: To understand the genetic basis of the adaptation of Culex tritaeniorhynchus to different environmental ecology in Jining City, Shandong Province, so as to provide insights into understanding of the population structure or isolation pattern of Cx. tritaeniorhynchus in the city.

Methods: Seven sampling sites were selected from urban, suburban and rural areas of Jining City, Shandong Province from June to August 2023, and mosquitoes were collected using mosquito-trapping lamps. All collected adult mosquitoes were identified morphologically. Genomic DNA was extracted from a single female Cx. tritaeniorhynchus mosquito, and the mitochondrial cytochrome C oxidase I (COI) gene was amplified using a PCR assay, sequenced and subjected to molecular identification. The number of haplotypes, haplotype diversity (Hd), nucleotide diversity (Pi), and average number of nucleotide differences (K) of Cx. tritaeniorhynchus DNA sequences were estimated among different sampling sites using the software DnaSP 6, and a neutrality test was performed. The fixation index (F_ST), and gene flow (number of migrants, Nm) of Cx. tritaeniorhynchus populations were calculated using the software Arlequin 3.5.2, and subjected to analysis of molecular variance (AMOVA). In addition, a haplotype network diagrams and a phylogenetic tree of Cx. tritaeniorhynchus populations were created using the software PopART and MEGA 11, respectively.

Results: A total of 420 sequences were successfully amplified from the COI gene of Cx. tritaeniorhynchus samples collected from 7 sampling sites in Jining City, and a gene fragment sequence with a length of 603 bp was obtained, with 55 variable sites and 46 haplotypes and without insertion or deletion mutations. Of the 46 haplotypes, H01 was the dominant shared haplotype, and the haplotype frequency increased gradually from urban areas (34.00%) to rural areas (47.00%). The mean Hd, Pi and K values of Cx. tritaeniorhynchus COI genes were 0.814, 0.024 and 14.129, 0.489, 0.016 and 7.941 and 0.641, 0.016 and 10.393 in suburban, urban, and rural areas, respectively, with the highest population diversity of Cx. tritaeniorhynchus in suburban areas and the lowest in urban areas. Paired F_ST analysis among different types of sampling sites showed that the mean F_ST value was 0.029 between urban and suburban areas, indicating more frequent inter-population communication. AMOVA revealed that the percentage of intra-population variation (95.74%) was higher than that of inter-population variation (4.26%). Neutrality tests showed deviation from neutrality in Cx. tritaeniorhynchus populations collected from Nanyang Township (Tajima's D = 2.793, Fu's Fs = 6.429, both P values < 0.05). In addition, the mismatch distribution curves of Cx. tritaeniorhynchus COI gene appeared bimodal or multimodal patterns in Jining City, indicating a relatively stable overall population size.

Conclusions: The mitochondrial COI gene may be used as a molecular marker to investigate the population genetic diversity of Cx. tritaeniorhynchus. The population genetic diversity of Cx. tritaeniorhynchus is higher in the suburban areas of Jining City than in rural and urban areas, and there are frequent genetic exchanges between Cx. tritaeniorhynchus populations from urban and suburban areas.