Evidence to support natural hybridization between Anopheles sinensis and Anopheles kleini (Diptera: Culicidae): possibly a significant mechanism for gene introgression in sympatric populations

Background: Malaria caused by Plasmodium vivax is still a public health problem in the Republic of Korea (ROK), particularly regarding the recent re-emergence of this malarial species near the demilitarized zone in northwestern Paju City, Gyeonggi-do Province. Currently, at least 4 species (An. kleini, An. pullus, An. belenrae and An. lesteri) of the Hyrcanus Group are reported as possible natural vectors of vivax malaria in the ROK, and An. sinensis, which is the most dominant species, has long been incriminated as an important natural vector of this P. vivax. However, An. sinensis was ranked recently as a low potential vector. According to the discovery of natural hybrids between An. sinensis (a low potential vector for P. vivax) and An. kleini (a high potential vector for P. vivax) in Paju City, intensive investigation of this phenomenon is warranted under laboratory conditions. Methods: Mosquitoes were collected during 2010-2012 from Paju City, ROK. Hybridization experiments used iso-female line colonies of these anophelines together with DNA analysis of ribosomal DNA [second internal transcribed spacer (ITS2)] and mitochondrial DNA [cytochrome c oxidase subunit I (COI)] of the parental colonies, F-1-hybrids and repeated backcross progenies were performed intensively by using a PCR-based assay and pyrosequencing technology. Results: The results from hybridization experiments and molecular investigations revealed that the mitochondrial COI gene was introgressed from An. sinensis into An. kleini. The An. sinensis progenies obtained from consecutive repeated backcrosses in both directions, i.e., F-2-(11) progeny [(An. sinensis x An. kleini) x An. sinensis] and F-3-(5) progeny [(An. kleini x An. sinensis) x An. kleini] provided good supportive evidence. Conclusions: This study revealed introgression of the mitochondrial COI gene between An. sinensis and An. kleini through consecutive repeated backcrosses under laboratory conditions. This new body of knowledge will be emphasized in reliable promising strategies in order to replace the population of An. kleini as a high potential vector for P. vivax, with that of a low potential vector, An. sinensis, through the mechanism of gene introgression in nature.