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Investigations of host feeding behaviour in haematophagous insects are critical to assess transmission routes of vector‐borne diseases. Understanding if a certain species has ornithophilic or mammalophilic feeding behaviour can facilitate future studies focused on pathogens transmission to and from certain host species. Culicoides Latreille (Diptera: Ceratopogonidae) are vectors of several pathogens, which include arboviruses, bacteria and parasites to a considerable diversity of vertebrate hosts. However, most of the studies focused on feeding habits target Culicoides species that could transmit the Bluetongue virus, consequently with a mammalophilic feeding behaviour, leaving aside the Culicoides species that are involved in the transmission of vector‐borne parasites to birds, such as Haemoproteus Kruse (Haemosporida: Haemoproteidae). This study aimed to investigate the source of blood meals of wild‐caught Culicoides using molecular‐based methods and to correlate our findings with the reports of Haemoproteus parasites in Culicoides species. Engorged Culicoides females were collected using ultraviolet (UV)‐light traps at seven different localities in Lithuania in 2021–2023. Biting midges were dissected, and the abdomens of engorged females were used for molecular investigation of the blood meal source. A polymerase chain reaction (PCR) protocol that amplifies a fragment of the Cytochrome B gene of vertebrates was used. Obtained sequences were compared to available information in GenBank database to confirm the source of the blood meal. In total, 258 engorged Culicoides females, representing nine different species, were analysed. The source of blood meal was identified in 29.1% of them with most of the insects having fed on birds (74.7%) and the remaining on mammals (25.3%). Culicoides segnis Campbell, Pelham‐Clinton was the only species to feed exclusively on birds; Culicoides from the Obsoletus group, C. pallidicornis Kieffer and C. punctatus Latreille were found to feed exclusively on mammals; C. festivipennis Kieffer, C. kibunensis Tokunaga and C. pictipennis Staeger had an opportunistic feeding behaviour, with the first two preferably feeding on birds. Due to their feeding behaviour and the presence of Haemoproteus parasites reported in the literature, C. festivipennis, C. kibunensis, C. pictipennis, and C. segnis play an important role in the transmission of those avian vector‐borne parasite in the wild. These Culicoides species were already confirmed as being able to support the development of several Haemoproteus species and lineages. Future studies focused on understanding the epidemiology of avian pathogens transmitted by Culicoides should target these species. [ABSTRACT FROM AUTHOR]

Simple Summary: Haemoproteus parasites are one of the most studied avian blood parasites; however, their natural vectors, Culicoides biting midges, have been identified for only a small portion of them. The main reason for that might be due to the existence of a few research groups working with an integrative approach that allows not only the identification of parasite DNA in the insects, but also confirms the presence of the parasite infective stage (sporozoites) using microscopy. In this study, we aimed to identify the natural vectors of Haemoproteus parasites and to determine their prevalence in Culicoides biting midges in four different localities in Lithuania. Almost 2000 parous Culicoides females belonging to 11 species previously registered in the country were processed. The most abundant one was C. pictipennis. Parasite DNA was found in 7.9% of the analyzed biting midges, and sporozoites were present in almost 30% of these insects. The Botanical Garden was the locality with the highest abundance of biting midges and parasite species, as well as the place with the highest number of biting midges containing sporozoites. One new Haemoproteus vector (C. reconditus) was confirmed, and 12 new interactions between Haemoproteus parasite lineages and Culicoides biting midges of different species were identified. This study helps to understand the relationship between Haemoproteus parasites and Culicoides biting midges in the wild. Haemoproteus parasites are the most diverse among Haemosporida. However, their natural vectors (Culicoides) are still poorly investigated and were identified for only a few parasite species and lineages. The application of an integrative approach (insect dissection, microscopic analysis, and molecular-based methods) is necessary in these studies, which have been carried out by a few research groups, mainly in Europe. The aim of this study was (i) to determine the Culicoides species that are naturally infected by Haemoproteus parasites, and which can support its complete sporogonic development, and (ii) to investigate the prevalence of Culicoides species and Haemoproteus parasite lineages in different study sites. In total, 1953 parous Culicoides females, from 11 species, were collected in four different localities in Lithuania and were dissected and analyzed using an integrative approach. The most abundant was C. pictipennis (30.3%). Parasite DNA was found in 7.9% of all investigated Culicoides, of which ~30% had sporozoites in their salivary glands, confirming their vector competence for these parasites. The Botanical Garden presented the highest number of Culicoides parous females, Culicoides species, and parasite lineages, as well as the highest positivity for sporozoites. Culicoides reconditus was confirmed as a natural vector of Haemoproteus parasites, sporozoites of six Haemoproteus lineages were reported for the first time, and 12 new interactions between Haemoproteus parasite lineages and Culicoides species were identified. Haemoproteus parasites seem to be transmitted by a high number of Culicoides species, with C. kibunensis, C. pictipennis, and C. segnis being the most important vectors. [ABSTRACT FROM AUTHOR]