Blackflies (Diptera: Simuliidae) as vectors of avian blood parasites, with the first record of two new vector species in Lithuania

Blackflies (Diptera: Simuliidae) are notable for their painful bites and are significant vectors for various pathogens, posing risks to human and animal health. They are known vectors of several avian parasites such as Leucocytozoon Berestnev (Haemosporida: Leucocytozoidae), Trypanosoma Gruby (Trypanosomatida: Trypanosomatidae) and filarioid nematodes (Nematoda: Onchocercidae). However, the prevalence of infections and vector competence for different species remain poorly understood. In this study, we investigated wild-caught blackflies across various sites in Lithuania for their role as vectors of avian blood parasites, specifically Leucocytozoon, Trypanosoma, and filarioid nematodes, using microscopical analysis and molecular methods. A total of 229 female blackflies were collected, representing at least 10 species, with Simulium lineatum Meigen, S. equinum L. and S. maculatum Meigen being the most prevalent. Notably, S. cryophilum Rubtsov and S. rubzovianum Petrova were reported for the first time in Lithuania. Salivary glands or midgut of eight individuals were microscopically positive for parasites; Leucocytozoon sporozoites were the most common parasite stage (n=7), but trypanosomatid parasites were also detected (n=3). PCR analysis confirmed nine samples positive for at least one parasite, with Leucocytozoon detected in six samples, indicating the potential vector competence of S. cryophilum, S. rubzovianum, and S. aureum Fries. Trypanosoma avium Danilewsky and Trypanosoma theileri group Laveran, as well as monoxenous trypanosomatid Crithidia brevicula Frolov & Malysheva (Trypanosomatida: Trypanosomatidae), were detected. Splendidofilaria mavis Leiper (Nematoda: Onchocercidae) was also detected in blackflies. One specimen of S. cryophilum was positive for all three parasite genera, indicating the importance of this blackfly species in the transmission of avian parasites. This study adds new insights into the transmission dynamics of avian parasites among blackflies in Lithuania, highlighting the need for further research to explore ecological factors influencing vector competence and the epidemiology of avian blood parasites. These findings contribute to the understanding of blackfly-parasite relationships and underscore the necessity for targeted monitoring of avian blood parasites in changing ecological landscapes.