Background: Culicoides biting midges are potential vectors of different pathogens. However, especially for eastern Europe, there is a lack of knowledge on the host-feeding patterns of this vector group. Therefore, this study aimed to identify Culicoides spp. and their vertebrate hosts collected in a wetland ecosystem. Methods: Culicoides spp. were collected weekly from May to August 2017, using Biogents traps with UV light at four sites in the Danube Delta Biosphere Reserve, Romania. Vectors and hosts were identified with a DNA barcoding approach. The mitochondrial cytochrome c oxidase subunit 1 was used to identify Culicoides spp., while vertebrate hosts were determined targeting cytochrome b or 16S rRNA gene fragments. A maximum likelihood phylogenetic tree was constructed to verify the biting midge identity against other conspecific Palaearctic Culicoides species. A set of unfed midges was used for morphological confirmation of species identification using slide-mounted wings. Results: Barcoding allowed the species identification and detection of corresponding hosts for 1040 (82.3%) of the 1264 analysed specimens. Eight Culicoides spp. were identified with Culicoides griseidorsum, Culicoides puncticollis and Culicoides submaritimus as new species records for Romania. For 39 specimens no similar sequences were found in GenBank. This group of unknown Culicoides showed a divergence of 15.6–16.3% from the closest identified species and clustered in a monophyletic clade, i.e. a novel species or a species without reference sequences in molecular libraries. For all Culicoides spp., nine mammalian and 24 avian species were detected as hosts. With the exception of C. riethi (n = 12), at least one avian host was detected for all Culicoides spp., but this host group only dominated for Culicoides kibunensis and the unknown Culicoides sp.. The most common host group were mammals (n = 993, 87.6% of all identified blood sources) dominated by cattle (n = 817, 70.6%). Conclusions: Most Culicoides spp. showed a broad host-feeding pattern making them potential bridge vectors. At the same time, new records of biting midge species for Romania, as well as a potentially unknown Culicoides species, highlight the lack of knowledge regarding the biting midge species and their genetic diversity in eastern Europe.

Culex spp. mosquitoes are important vectors of viruses, such as West Nile virus, Eastern equine encephalitis virus and Rift valley fever virus. However, their interactions with innate antiviral immunity, especially RNA interference (RNAi), are not well known. Most research on RNAi pathways in mosquitoes is focused on the tropical vector mosquito Aedes aegypti. Here, we investigated the production of arbovirus-specific small RNAs in Cx. quinquefasciatus-derived HSU cells. Furthermore, by silencing RNAi-related proteins, we investigated the antiviral role of these proteins for two different arboviruses: Semliki Forest virus (SFV) and Bunyamwera orthobunyavirus (BUNV). Our results showed an expansion of Ago2 and Piwi6 in Cx. quinquefasciatus compared to Ae. aegypti. While silencing Ago2a and Ago2b increased BUNV replication, only Ago2b showed antiviral activity against SFV. Our results suggest differences in the function of Cx. quinquefasciatus and Ae. aegypti RNAi proteins and highlight the virus-specific function of these proteins in Cx. quinquefasciatus.