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[This corrects the article DOI: 10.1371/journal.pntd.0008986.].

The Culex vishnui subgroups, particularly Culex tritaeniorhynchus, are considered the primary vectors of the Japanese encephalitis virus (JEV) in Asia. Recent molecular phylogenetic analyses of JEV isolates from Asian countries have shown that JEVs with diverse genetic variants are present in Asia. Furthermore, some JEV strains have been found to have crossed the East China Sea and been introduced into Japan. In this study, the possibility of overseas migration of the JE vector mosquito, Cx. tritaeniorhynchus was examined from the genetic, physical, and meteorological perspectives. Molecular phylogenetic analysis was performed based on both whole coding sequences and on the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) gene of Cx. vishnui subgroups collected from Asian countries. Culex tritaeniorhymchus was classified into two genetically independent taxa by COI sequences: the Japanese type (Ct-J), which inhabits Japan except for the Amami Islands of southern Japan, and the continental type (Ct-C), which inhabits the Asian region except for Japan. It was confirmed that approximately 10% of Cx. tritaeniorhynchus trapped during the summer in western Kyushu were Ct-C, and that they could fly for up to 38 h continuously. The meteorological analysis also confirmed that the atmospheric flow occurring over the continent coincided with the date of Ct-C capture. This is the first report showing the existence of two taxa in Cx. tritaeniorhynchus. Their physical and physiological characteristics suggest the possibility of long-distance migration from overseas regions to Japan across the East China Sea. Future efforts are expected to provide evidence to support the occurrence of long-distance migration of Cx. tritaeniorhynchus with JEV.

Japanese encephalitis virus (JEV) is maintained in an enzootic cycle between swine, water birds, and mosquitoes. JEV has circulated indigenously in Asia, with Culex tritaeniorhynchus as the primary vector. In some areas where the primary vector is scarce or absent, sporadic cases of Japanese encephalitis have been reported, with Aedes japonicus japonicus presumed to have the potential as a secondary vector. As one of the world's most invasive culicid species, Ae. j. japonicus carries a considerable health risk for spreading diseases to wider areas, including Europe and North America. Thus, evaluation of its competency as a JEV vector, particularly in a native population, will be essential in preventing potential disease spread. In this study, the two mosquito species' vector competence in transmitting three JEV genotypes (I, III, and V) was assessed, with Cx. tritaeniorhynchus serving as a point of reference. The mosquitoes were virus-fed and the infection rate (IR), dissemination rate (DR), and transmission rate (TR) evaluated individually by either RT-qPCR or focus forming assay. Results showed striking differences between the two species, with IR of 95% (261/274) and 9% (16/177) in Cx. tritaeniorhynchus and Ae. j. japonicus, respectively. Both mosquitoes were susceptible to all three JEV genotypes with significant differences in IR and mean viral titer. Results confirm the primary vector's competence, but the fact that JEV was able to establish in Ae. j. japonicus is of public health significance, and with 2%-16% transmission rate it has the potential to successfully transmit JEV to the next host. This may explain the human cases and infrequent detection in primary vector-free areas. Importantly, Ae. j. japonicus could be a relevant vector spreading the disease into new areas, indicating the need for security measures in areas where the mosquito is distributed or where it may be introduced.

In insects, the voltage-gated sodium channel (VGSC) is the primary target site of pyrethroid insecticides. Various amino acid substitutions in the VGSC protein, which are selected under insecticide pressure, are known to confer insecticide resistance. In the genome, the VGSC gene consists of more than 30 exons sparsely distributed across a large genomic region, which often exceeds 100 kbp. Due to this complex genomic structure, it is often challenging to genotype full coding nucleotide sequences (CDSs) of VGSC from individual genomic DNA (gDNA). In this study, we designed biotinylated oligonucleotide probes from CDSs of VGSC of Asian tiger mosquito, Aedes albopictus. The probe set effectively concentrated (>80,000-fold) all targeted regions of gene VGSC from pooled barcoded Illumina libraries each constructed from individual A. albopictus gDNAs. The probe set also captured all orthologous VGSC CDSs, except some tiny exons, from the gDNA of other Culicinae mosquitos, A. aegypti and Culex pipiens complex, with comparable efficiency as a result of the high nucleotide-level conservation of VGSC. To improve efficiency of the downstream bioinformatic process, we developed an automated pipeline-MoNaS (Mosquito Na+ channel mutation Search)-which calls amino acid substitutions in the VGSC from NGS reads and compares those to known resistance mutations. The proposed method and our bioinformatic tool should facilitate the discovery of novel amino acid variants conferring insecticide resistance on VGSC and population genetic studies on resistance alleles (with respect to the origin, selection, and migration etc.) in both clinically and agriculturally important insect pests.