Genetic structure of Anopheles sinensis populations in Shandong Province based on genetic analysis

As an eastern coastal province with frequent international exchanges, Shandong Province in China occasionally reports imported malaria cases. Anopheles sinensis (An. sinensis) serves as the primary malaria vector in Shandong, making research into its population genetic structure crucial for preventing malaria transmission. In this study, we combined mitochondrial cytochrome c oxidase subunit I ( COI ) sequencing with voltage-gated sodium channel ( VGSC ) gene analysis to investigate the genetic diversity, population differentiation, and resistance mutation profiles of An. sinensis populations across nine geographical locations in Shandong Province. COI analysis of 183 samples identified 121 haplotypes; AMOVA attributed most variation to within-population differences (97.6%; Fst = 0.024). Neutrality tests and mismatch distributions suggest a recent expansion in Linyi population. Maximum-likelihood coalescent analysis of asymmetric migration rates further demonstrated that gene flow among the regional populations is strongly directional: migration from the northwestern plains into the southern plains and into the eastern hilly areas was essentially unidirectional. At VGSC codon 1014, two predominant mutant types, L1014F and F1014/C1014, were detected. Bayesian analyses resolved three clades, with resistance-associated mutations restricted to Clades 1 and 2, and DAPC based on kdr genotype retrieved three principal components and two discriminant axes explaining 98.9% of among-population variance, consistent with the three clades. Using COI and VGSC markers, this study revealed that An. Sinensis populations in Shandong Province exhibit high mitochondrial haplotype diversity, weak to moderate levels of population differentiation, and evidence of recent population expansion. Coalescent estimates of asymmetric migration indicated that these weakly differentiated populations are connected by high levels of gene flow with a directional bias, in which gene flow is predominantly exported from the northwestern plains towards the southern plains and the eastern hilly areas. The VGSC fragment showed significant spatial heterogeneity in the mutation frequency at codon 1014; the L1014F mutation was relatively common across most of Shandong Province but was not detected in the Jining population in this study. This research provides baseline data for future population genomics studies and insecticide resistance monitoring in Shandong Province.