Design of species-specific molecular markers for PCR-based differentiation of bird louse flies Ornithomya avicularia, Ornithomya biloba and Ornithomya chloropus (Diptera: Hippoboscidae)

Louse flies of the genus Ornithomya (Hippoboscidae) are widespread avian ectoparasites of ecological and epidemiological importance, acting as blood-feeding parasites and potential vectors of avian and zoonotic pathogens. Traditional taxonomic identification can be challenging due to overlapping phenotypic traits, broad host associations, and the frequent occurrence of damaged specimens. This study aimed to develop a reliable, species-specific PCR-based molecular tool for differentiating three European louse fly species: Ornithomya avicularia , Ornithomya biloba, and Ornithomya chloropus . Mitochondrial cytochrome c oxidase subunit 1 gene ( cox 1) sequences from different geographical populations of the studied species, both newly sequenced and retrieved from GenBank, were analysed to assess intraspecific variation and interspecific divergence. The cox 1 regions characterised by high interspecific polymorphism and no intraspecific variability were selected for the design and validation of nine species-specific primer sets. Four of these produced PCR products exclusively for their target species, demonstrating their effectiveness for species identification. This new single-step PCR-based approach for accurate and cost-effective molecular identification can efficiently supplement taxonomy methods based solely on morphology. The findings highlight the necessity of combined approaches integrating morphological and DNA-based methods to resolve the taxonomy of louse flies and underscore the importance of developing novel molecular tools to improve species delimitation. • cox 1 sequences of different geographical populations of three species of Ornithomya were analysed. • Species-specific primers were designed for Ornithomya avicularia , Ornithomya biloba , and Ornithomya chloropus. • A single-step PCR-based approach for differentiation of these Ornithomya spp. was developed. • The novel molecular approach supplements the identification based on morphology.