Hybridization and gene flow between two malaria vectors, Anopheles sinensis and Anopheles kleini: a potential challenge to malaria control strategies in the Republic of Korea

Vectors of pathogens of medical and veterinary importance can rapidly adapt to anthropogenic selection pressures, i.e., insecticide-based interventions, through genetic variations, de novo mutations, and gene introgressions. Therefore, identifying mechanisms underlying insecticide resistance is essential for effective vector control and pest management. In the Republic of Korea (ROK), malaria is primarily transmitted by mosquitoes belonging to members of the Anopheles Hyrcanus Group. Among these, An. sinensis and An. kleini are at an incipient stage of speciation. Although hybrid individuals between these two species are thought to occur at low frequencies in natural populations, studies examining their genetic differentiation and potential gene introgression are scarce. To address this gap, this study investigated the possibility of introgressive hybridization between these two taxa. Mosquitoes were collected from US military installations/training sites in northern Gyeonggi and Gangwon provinces near the demilitarized zone that separates the ROK and the Democratic People's Republic of Korea (DPRK), where An. sinensis and An. kleini species are sympatric, in addition to allopatric sites (Humphreys US Army Garrison, Pyeongtaek city and a cattle shed near Daegu city where only An. sinensis were collected. A species-specific molecular marker was developed based on the resistance to the dieldrin ( rdl ) gene to distinguish between the two species and their hybrids. Additionally, microsatellite markers were used to assess the genetic structure of the populations. Analysis using the rdl -based marker suggested potential gene flow between An. sinensis and An. kleini , which may correlate with the presence of the rdl 296S mutation in both species. Microsatellite analyses demonstrated that the two species form distinct genetic clusters with marked differentiation, while also displaying signals consistent with hybridization and backcrossing; however, the contribution of shared ancestral variation or incomplete lineage sorting cannot be completely excluded given the targeted marker set. These findings suggest that the genetic boundary between An. sinensis and An. kleini is permeable and complex, likely shaped by incomplete reproductive isolation, ongoing gene flow, or retained ancestral polymorphisms. Continuous surveillance and genome-wide studies are required to disentangle these evolutionary processes and support effective malaria vector control strategies in the ROK.