Cox-1 Gene-Based Identification and Molecular Phylo-genetics of Horseflies

DOI: 10.22161/ijeab.104.2

Abstract:
The hematophagous flies of the family Tabanidae are involved in the transmission of various disease-causative agents such as protozoans, helminths, bacteria, and viruses. However, molecular research on this family is not conducted in Pakistan. This study seeks to investigate the molecular characterization of the horseflies, feeding on buffaloes of District Swat, Khyber Pakhtunkhwa, by targeting the mitochondri-al partial Cox1 gene for the nucleotide identity/diversity and phylogenetic analysis. The Cox1 gene se-quences were compared with NCBI databases using BLASTn. The analyses revealed two Tabanus species (Tabanus sp. 1 and Tabanus sp. 2) and one Atylotus species. Genetic comparisons found a close relation-ship: Tabanus sp. 1 (93.95%) to T. superjumentarius, Tabanus sp. 2 (94.55%) belong to T. bromius, and Atylotus sp. (93.97%) to A. agrestis. Species were identified at the genus level due to limited data availa-bility. The nucleotide identity among the collected species was Atylotus sp. vs. Tabanus sp. 1 (90.7%), Atylotus sp. vs. Tabanus sp. 2 (89.4%), and Tabanus sp. 1 vs. Tabanus sp. 2 (90.1%). As expected, Taba-nus sp. 2, clustered together with its congener, T. bromius forming a basal clade of all other Tabanus spp included in our phylogenetic analysis. This means that Tabanus sp. 2 and T. bromius have some similari-ties and both of them are from the Palaearctic region. However, Tabanus sp.1, though showing com-paratively higher sequence identity with T. superjumentarius (94.55%) placed on a separate branch as a sister clade of the clade containing T. fontinalis and T. rubidus. T. fontinalis and T. rubidus are native to North America and their sequence identity with Tabanus sp.1 were 93.8% and 93.3%, respectively. Simi-larly, the phylogenetic tree placed our sequenced Atylotus sp. on a separate branch inside the clade unit-ing all Atylotus spp. included in our analysis containing A. agrestis as well. From our study, we conclud-ed that the genus Atylotus is monophyletic while the Tabanus genus is may be polyphyletic or paraphylet-ic because as it does not include all descendants of a common ancestor. Molecular characterization of additional species of horseflies from Pakistani hosts will provide a clear image of their genetic inter-relationship and their phylogenetic affinities with other species recorded across the world. Such molecu-lar study will provide a base for determining the prevalence of respective species and the development of their effective management strategies.

Keywords:
horseflies, PCR, phylogenetic tree, nucleotide identity and Cox-1 gene.

Article Info:
Received: 23 May 2025; Received in revised form: 25 Jun 2025; Accepted: 30 Jun 2025; Available online: 04 Jul 2025

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