Species Identification of the Major Japanese Encephalitis Vectors within the Culex vishnui Subgroup (Diptera: Culicidae) in Thailand Using Geometric Morphometrics and DNA Barcoding

Simple Summary Culex pseudovishnui, Cx. tritaeniorhynchus, and Cx. vishnui are members of the Cx. vishnui subgroup and are vectors of Japanese encephalitis (JE) in Thailand. However, their extremely similar morphologies make identification difficult. Thus, geometric morphometrics (GM) and DNA barcoding were applied to identify Cx. pseudovishnui, Cx. tritaeniorhynchus, and Cx. vishnui in Thailand. Our results of cross-validation reclassification demonstrated that GM based on wing shape was relatively high potential for distinguishing three Culex species. Meanwhile, DNA barcoding was highly effective at identifying all three species based on the DNA barcode gap. Japanese encephalitis (JE) is a viral infection of the brain caused by the Japanese encephalitis virus, which spreads globally, particularly in 24 countries of Southeast Asia and the Western Pacific region. In Thailand, the primary vectors of JE are Cx. pseudovishnui, Cx. tritaeniorhynchus, and Cx. vishnui of the Cx. vishnui subgroup. The morphologies of three mosquito species are extremely similar, making identification challenging. Thus, geometric morphometrics (GM) and DNA barcoding were applied for species identification. The results of cross-validation reclassification revealed that the GM technique based on wing shape analysis had relatively high potential for distinguishing Cx. pseudovishnui, Cx. tritaeniorhynchus, and Cx. vishnui (total performance = 88.34% of correctly assigned individuals). While the DNA barcoding yielded excellent results in identifying these Culex species based on the DNA barcode gap (average intraspecific genetic distance = 0.78% +/- 0.39% and average interspecific genetic distance = 6.14% +/- 0.79%). However, in the absence of the required facilities for DNA barcoding, GM techniques can be employed in conjunction with morphological methods to enhance the reliability of species identification. Based on the results of this study, our approach can help guide efforts to identify members of the Cx. vishnui subgroup, which will be useful for the effective vector control of JE in Thailand.