Integrative Morphological and Molecular Classification of Culex vishnui Larvae from Diverse Ecological Habitats of Uttar Pradesh

Mosquito-borne infections remain critical global health challenges in tropical regions. Culex vishnui is recognized as a principal vector of Japanese Encephalitis (JE) across Asia. The present study integrates morphological and molecular tools to classify C. vishnui larvae from varied ecological habitats in Uttar Pradesh, India. Larvae were collected from rice fields, ponds, marshes, and drains during post-monsoon months. Environmental parameters such as temperature, pH, and conductivity were recorded at each site. Morphological identification followed standard keys by Bram and Harbach, while COI gene sequencing confirmed species identity. Results revealed highest larval density in rice fields and minimal density in drains. A clear inverse correlation was observed between electrical conductivity and larval abundance. Molecular analysis indicated 99.2–99.8 % COI similarity with C. vishnui sequences in GenBank. Comparative evaluation with literature from 2018– 2025 supported these findings and confirmed ecological consistency across Asian landscapes. The integrative methodology established morphological–molecular coherence, improving accuracy in vector taxonomy and providing valuable insight for Japanese Encephalitis control programs.

Culex vishnui; Morphological Identification; COI Barcoding; Habitat Ecology; Japanese Encephalitis; Uttar Pradesh

1. Amerasinghe, F., Indrajith, N., & Ariyasena, T. (1995). Physico-chemical characteristics of mosquito breeding habitats in Sri Lanka. Ceylon Journal of Science, 24(1), 13–29. [Google Scholar] [Crossref]

2. Bashar, K., Rahman, M. S., Nodi, I. J., & Howlader, A. J. (2016). Species composition and habitat characterization of mosquito larvae in semi-urban areas of Bangladesh. Pathogens and Global Health, 110(2), 48–61. https://doi.org/10.1080/20477724.2016.1151068 [Google Scholar] [Crossref]

3. Bram, R. A. (1967). Contributions to the mosquito fauna of Southeast Asia II: The genus Culex in Thailand (Diptera: Culicidae). Contributions of the American Entomological Institute, 2(1), 1–296. [Google Scholar] [Crossref]

4. Bursalı, F., Öncü, C., Mutluay, N., Yücel, M., & Kütük, A. (2024). Population genetics and molecular variation of Culex tritaeniorhynchus across Türkiye. Pathogens, 13(5), 400–410. https://doi.org/10.3390/pathogens13050400 [Google Scholar] [Crossref]

5. Chung, H.H., Lee, C.F., Wang, H.C., Tsai, Y.C., Chen, C.L., & Cheng, H.Y. (2024). Molecular identification and ecological assessment of the Culex vishnui subgroup in Taiwan using COI markers. Insects, 15(2), 120– 130. https://doi.org/10.3390/insects15020120 [Google Scholar] [Crossref]

6. Cywinska, A., Hunter, F. F., & Hebert, P. D. N. (2006). Identifying Canadian mosquito species through DNA barcodes. Medical and Veterinary Entomology, 20(4), 413–424. https://doi.org/10.1111/j.13652915.2006.00653.x [Google Scholar] [Crossref]

7. Folmer, O., Black, M., Hoeh, W., Lutz, R., & Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3(5), 294–299. [Google Scholar] [Crossref]

8. Green, M. R., & Sambrook, J. (2017). Isolation of high-molecular-weight DNA using phenol–chloroform extraction. Cold Spring Harbor Protocols, 2017(6), 357–359. https://doi.org/10.1101/pdb.prot093450 [Google Scholar] [Crossref]

9. Harbach, R. E. (2007). The Culicidae (Diptera): Taxonomy, classification, and phylogeny. Zootaxa, 1668, 591–638. https://doi.org/10.11646/zootaxa.1668.1.28 [Google Scholar] [Crossref]

10. Hebert, P. D. N., Cywinska, A., Ball, S. L., & DeWaard, J. R. (2003). Biological identifications through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences, 270(1512), 313–321. https://doi.org/10.1098/rspb.2002.2218 [Google Scholar] [Crossref]

11. Jeon, J., Park, J., Kim, H., & Seo, Y. (2024). Two distinct clades of Culex tritaeniorhynchus with Wolbachia infection identified by mitochondrial COI gene sequences. Microorganisms, 12(3), 589–598. https://doi.org/10.3390/microorganisms12030589 [Google Scholar] [Crossref]

12. Kanojia, P. C. (2007). Ecological study on mosquito vectors of Japanese Encephalitis virus in Bellary District, Karnataka. Indian Journal of Medical Research, 126(2), 152–157. [Google Scholar] [Crossref]

13. Karthika, P., Rajavel, A. R., Natarajan, R., & Jambulingam, P. (2018). COI-based DNA barcoding and phylogenetic analysis of five Japanese Encephalitis vectors in India. Acta Tropica, 185, 225–232. https://doi.org/10.1016/j.actatropica.2018.05.017 [Google Scholar] [Crossref]

14. Laskar, M. A., Rahman, M. H., Saikia, P. K., & Gogoi, P. (2025). Epidemiological overview of Japanese [Google Scholar] [Crossref]

15. Encephalitis vectors in North-East India: Challenges and management perspectives. IJID Regions, 8(1), 77–85. https://doi.org/10.1016/j.ijregi.2025.01.005 [Google Scholar] [Crossref]

16. Lessard, B. D., Abu Hassan, A., Jeffrey, J., & Sofian-Azirun, M. (2021). DNA barcoding of Culex mosquitoes from Malaysia reveals new distributional records and genetic variation. Insects, 12(4), 345–353. https://doi.org/10.3390/insects12040345 [Google Scholar] [Crossref]

17. Rattanarithikul, R., Harrison, B. A., Harbach, R. E., Panthusiri, P., Coleman, R. E., & Richardson, J. H. (2023). Illustrated keys to the mosquitoes of Thailand: Morphological updates and field guide revision. Southeast Asian Journal of Tropical Medicine and Public Health, 54(2), 150–178. [Google Scholar] [Crossref]

18. Yin, Q., Wang, X., Chen, Z., Zhang, H., & Liu, J. (2025). Spatiotemporal modeling of Japanese Encephalitis virus transmission dynamics: Linking hosts, vectors, and environment. Viruses, 17(1), 35–46. https://doi.org/10.3390/v17010035 [Google Scholar] [Crossref]

19. Zhang, Y., Liu, H., Li, X., Zhou, G., Chen, R., & Wang, S. (2024). Genetic population structure and divergence of Culex tritaeniorhynchus across East Asia inferred from COI gene sequences. BMC Genomics, 25(3), 330–341. https://doi.org/10.1186/s12864-024-09933-3 [Google Scholar] [Crossref]

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