Identification of breeding habitats and kdr mutations in Anopheles spp. in South Korea

Malaria is still endemic in South Korea. However, limited information is available on the current Anopheles breeding sites and the occurrence of insecticide resistance-associated genetic mutations and their distribution needed to control the malaria vector efficiently. This study explored breeding sites of Anopheline adults in Gimpo-si, near the demilitarized zone (DMZ) in Gyeonggi-do province, South Korea, from 2022 to 2023. Genetic diversity was investigated based on the internal transcribed spacer (ITS2), cytochrome c oxidase subunit I (COI), and knockdown resistance (kdr) genes of Anopheles mosquitoes. A natural environment associated with the seasonal abundance of Anopheles larvae was characterized. Two breeding sites of Anopheles larvae and adults were found at a stream margin or shallow freshwater near the forest in Wolgot-myeon in Gimpo-si without cattle shed within 1 km and in Naega-myeon in Ganghwa-gun with cow shed within 100 m in 2022 and 2023, respectively. Both sites were located between the newly cultivated lands and the forest. Besides, both breeding sites were in the valley at a slight elevation of 60-70 m from ground lands and maintained the shadow all day. Overall, the Wolgot-myeon breeding site showed various Anopheles spp. larvae, including Anopheles sinensis. Naega-myeon, an additional breeding site found in 2023, had Anopheles sineroides larvae, and approximately 59.7% (89/149) of An. sinensis adults inhabited within a 100-m distance. The total collection, including larvae and adults, revealed that An. sinensis, Anopheles pullus, Anopheles kleini, An. sineroides, Anopheles belenrae, and Anopheles lindesayi accounted for 44.2% (118/267), 0.7% (2/267), 0.7% (2/267), 22.1% (59/267), 1.9% (5/267), and 30.3% (81/267), respectively. Furthermore, various kdr mutant genotypes (F/F, C/C, L/F, L/C and F/C) in An. sinensis, and the first kdr allele mutant (L/F1014) in An. belenrae were identified in South Korea. Two breeding sites of Anopheles larvae were studied in Wolgot-myeon and Naega-myeon. Various Anopheles spp. larvae were detected in both habitats, but overall, An. sinensis was the most prevalent adults in both study sites. The occurrence of kdr allele mutant of An. belenrae in South Korea was reported. Rigorous larvae monitoring of Anopheles spp., continuously updating information on Anopheles breeding sites, and understanding the environmental conditions of Anopheles habitats are required to develop an effective malaria control progra