Molecular Identification of Onchocerciasis Vectors (Diptera: Simuliidae) from the Central Himalayan Landscape of India: A DNA Barcode Approach.

Background: Black flies (Diptera: Simuliidae) are a notorious group of blood-sucking insects acting as vectors of various diseases in humans and other animals, most notable being Onchocerciasis. Due to its medical and veterinary significance, accurate and quick species identification is of utmost importance in the field of black fly research. DNA barcoding is one such taxonomic tool, aiding in quick and efficient species identification using molecular methods. Despite sporadic reports of ocular and cutaneous Onchocerciasis, especially from North-East India, Indian Simuliidae has been understudied due to lack of expertise on morphological taxonomy and lack of genetic library. Materials and Methods: Blackflies were collected from eight distinct locations in the Central Himalayan region that are part of the West Bengal, India, districts of Kalimpong and Darjeeling. Various traps were used to collect the specimens, and they were kept it in 70% ethyl alcohol. Following the morphological identification of each fly specimen, genomic DNA was extracted from its dissected legs using the QIAmp DNA extraction kit (QIAGEN, Germany). The voucher specimen slide was deposited in the National Zoological collection, ZSI, Kolkata, India. Results: This is the first comprehensive DNA barcoding study of black flies (Feuerborni and Multistriatum species group) using mitochondrial cytochrome c oxidase subunit I (COI) gene sequences along with morphological identification from the Central Himalayan region of West Bengal involving four species: Simulium dentatum, Simulium digitatum, Simulium praelargum, and Simulium senile. DNA barcode approach through ML tree clearly distinguished all the species with supporting PTP, ASAP, and GMYC analysis. Interspecific genetic distances were also calculated where S. dentatum and S. digitatum showed minimum distances in the study area. Conclusion: Coupled with a robust morpho-taxonomic framework, the DNA barcodes generated here will help with accurate species identification, which will lead to better management and control strategies for these harmful vector species at the study site.