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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.

Blood-meal analysis of Culicoides (Diptera: Ceratopogonidae) reveals a broad host range and new species records for Romania