Louse flies (Hippoboscidae, Diptera) are hematophagous ectoparasites of birds and mammals. These obligatory parasites spend their adult life in the fur, among feathers, or in the nests of their hosts, where they feed on their blood (Baker, 1967). They express a high degree of unique morphological and physiological adaptations to their ectoparasitic lifestyle, and their life cycles are finely tuned to the host phenology (Lehikoinen et al., 2021). Louse flies are characterized by various degrees of host specificity, ranging from polyxeny which is observed in a number of species, including Ornithomya avicularia (one of the most widespread louse flies parasitizing birds in the Palearctic region (Oboňa et al., 2019), to monoxeny characteristic for Melophagus ovinus, a parasite of sheep (Boucheikhchoukh et al., 2019; Liu et al., 2016). All louse flies are parasites of homeothermic animals, the majority of 213 species described so far feed on avian blood. Among the three subfamilies of Hippoboscidae, the members of Ornithomyinae (171 species) are generally bird parasites, Lipopteninae (34 species) are mammal parasites, while Hippoboscinae (8 species) are dominated by mammal parasites, except for a single bird specialist parasite (Dick, 2006).

Hippoboscids feeding on mammal blood classified in the subfamily Lipopteninae are known as keds. They have long been considered important agents affecting condition of farm animals by direct bites, leading to blood loss and local infections, but even more importantly as vectors of bloodborne pathogens, including Trypanosoma melophagium (Pfeiffer, 1905), Megatrypanum trypanosomes (Böse and Petersen, 1991) and Bartonella sp. (Bartosik et al., 2021). Louse flies parasitizing wild mammals have recently gained significant scientific attention as vectors spreading potentially zoonotic pathogens. Lipoptena cervi (Buss et al., 2016; Werszko et al., 2020) or Hippobosca longipennis (Rani et al., 2011) can transmit various species of Trypanosoma, Acanthocheilonema, Anaplasma phagocytophilum, and Borrelia burgdorferi (Burakova, 1998). Lipoptena cervi has also been widely reported to be infected with Bartonella spp. (Peña-Espinoza et al., 2023; Regier et al., 2018; Szewczyk et al., 2017). Bird-specialized louse flies have also been reported to transmit epidemiologically important pathogens, but they seem highly understudied in this context. Ornithomya avicularia was found to be a vector of Trypanosoma avium (Baker, 1967), and the West Nile virus was detected in Icosta americana retrieved from birds of prey (Farajollahi et al., 2005). Evidence for the presence of Babesia spp., Wolbachia spp., and Trypanosoma corvi was recently reported in Ornithomya avicularia and Ornithomya biloba (Čisovská Bazsalovicsová et al., 2023). Transmission of flies most often occurs in the nest, where they can parasite on nestlings and their parents, but it can also occur in places where adult birds aggregate, e.g., during communal feeding and roosting on migration stopover sites (Nartshuk et al., 2022). Also, louse flies parasitizing long-distance migrants can travel long distances with their avian hosts, facilitating large-scale pathogen dispersal across landscapes and geographic regions (Bartos et al., 2020).

Considering the high diversity of louse flies parasitizing a wide range of bird hosts, their direct negative effect on host survival (Bartos et al., 2020), and a high potential to transmit bloodborne pathogens even along bird migratory routes (Herman, 1945), it is surprising that our knowledge about the ecology of louse flies is rather modest and fragmentary. Implementation of genetic approaches in molecular ecology research in louse flies is hampered by a lack of molecular tools. In general, louse flies have rarely been studied with molecular methods. Mitochondrial DNA barcoding was used in the identification of several species, including Icosta americana, Ornithomya anchineuria, O. avicularia, O. bequaerti, O. metallica, O. fringillina, Ornithophila gestroi, Crataerina hirundinis, and C. pallida (Lehikoinen et al., 2019; Gutiérrez-López, 2015; Levesque-Beaudin and Sinclair, 2021). Some of these studies focused on the comparative phylogeography of host species and their parasites. Implementation of nuclear molecular markers, such as microsatellites, in louse flies has been even more limited. In fact, we are aware of only one study using microsatellite genotyping in louse flies to compare genetic population structure of the fly Basilia nana and its host, the Bechstein's bat (Myotis bechsteinii) (Van Schaik et al., 2018). The results provided evidence for contrasting patterns between the parasite and host, revealing significant genetic divergence of bat populations within the geographical range in Europe, but no geographic divergence in B. nana (Van Schaik et al., 2018).

In this study, we focused on two Ornithomya species: O. avicularia and O. chloropus. Both species are among the most common European louse flies, and both have a wide range of hosts, including birds from 10 orders and over 65 genera (Maa, 1969; Borowiec, 1984; Eeva and Klemola, 2013). The study aimed to develop a new molecular tool based on microsatellite markers, which is expected to be highly useful in molecular ecology and populations genetics research in the genus Ornithomya.

For this purpose, we first conducted a genome-wide scan of putative microsatellite markers in O. avicularia. Second, we selected a panel of markers to quantify population-wide polymorphism in O. avicularia and to assess cross-amplification rate in a congeneric species, O. chloropus.