Molecular Identification of Chrysomya megacephala (Diptera: Calliphoridae) from Carrion Using COI Gene Barcoding

Abstract

Species identification with DNA barcodes has been proven to be effective on different organisms and, particularly, has become a routinely used and quite accurate tool in forensic entomology to study necrophagous Diptera species.  Accurate identification of cadaver-associated insects is fundamental in forensic entomology, particularly for reliable post-mortem interval (PMI) estimation. Traditional morphological identification of blowflies is sometimes limited sdue to damaged specimens or immature stages, especially eggs and early instar larvae. DNA barcoding using the mitochondrial COI gene has emerged as a reliable tool for accurate species-level identification. In the present study, mitochondrial cytochrome oxidase subunit I (COI) gene–based DNA barcoding was employed to molecularly identify a necrophagous insect specimen (F1-Cow) associated with a forensic case from central India. Genomic DNA extraction yielded a high-quality concentration of 124 ng/µl, suitable for downstream molecular analysis. The COI gene was successfully amplified using mitochondrial-specific primers and sequenced using the Sanger method, generating a high-quality 656 bp fragment. Sequence alignment and BLAST analysis revealed 99.09% similarity with reference sequences of Chrysomya megacephala deposited in the NCBI GenBank database. Phylogenetic reconstruction using the Neighbour-Joining method with the Jukes–Cantor distance model placed the F1-Cow sequence within a well-supported C. megacephala clade, confirming species identity. The concordance of DNA quantification, PCR amplification, BLAST similarity, and phylogenetic clustering validates the robustness of the molecular workflow. The study highlights the effectiveness of COI-based DNA barcoding as a reliable and complementary tool to morphological identification, particularly in forensic cases involving damaged, decomposed, or developmentally immature insect specimens.