DNA barcoding of five Japanese encephalitis mosquito vectors (Culex fuscocephala, Culex gelidus, Culex tritaeniorhynchus, Culex pseudovishnui and Culex vishnui)

Culex mosquitoes can act as vectors of several important diseases, including Japanese encephalitis, West Nile virus, St. Louis encephalitis and equine encephalitis. Besides the neurological sequelae caused in humans, Japanese encephalitis can lead to abortion in sows and encephalitis in horses. Effective vector control and early diagnosis, along with continuous serosurveillance in animals, are crucial to fight this arboviral disease. However, the success of vector control operations is linked with the fast and reliable identification of targeted species, and knowledge about their biology and ecology. Since the DNA barcoding of Culex vectors of Japanese encephalitis is scarcely explored, here we evaluated the efficacy of this tool to identify and analyze the variations among five overlooked Culex vectors of Japanese encephalitis, Culex fuscocephala, Culex gelidus, Culex tritaeniorhynchus, Culex pseudovishnui and Culex vishnui, relying to the analysis of mitochondrial COl gene. Variations in their base pair range were elucidated by the entropy H-x plot. The differences among individual conspecifics and on base pair range across the same were studied. The C (501-750 bp) region showed a moderate variation among all the selected species. C. tritaeniorhynchus exhibited the highest variation in all the ranges. The observed genetic divergence was partially non-discriminatory. i.e., the overall intra- and inter nucleotide divergence was 0.0920 (0.92%) and 0.125 (1.25%), respectively. However, 10X rule fits accurately intraspecies divergence < 3% for the five selected Culex species. The analysis of individual scatter plots showed threshold values (10X) of 0.008 (0.08%), 0.005 (0.05%), 0.123 (1.23%), 0.033 (0.33%) and 0.019 (0.19%) for C. fuscocephala, C. gelidus, C. tritaeniorhynchus, C. pseudovishnui and C. vishnui, respectively. The C. tritaeniorhynchus haplotypes KU497604, KU497603, AB690847 and AB690854 exhibited the highest divergence range, i.e., from 0.465 -0.546. Comparatively, the intra-divergence among the other haplotypes of C. tritaeniorhynchus ranged from 0-0.056. The maximum parsimony tree was formed by distinctive conspecific clusters with appreciable branch values illustrating their close congruence and extensive genetic deviations. Overall, this study adds valuable knowledge to the molecular biology and systematics of five overlooked mosquito species acting as major vectors of Japanese encephalitis in Asian countries.