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.

The ecology of West Nile virus (WNV) in the Danube Delta Biosphere Reserve (Romania) was investigated by combining studies on the virus genetics, phylogeography, xenosurveillance and host-feeding patterns of mosquitoes. Between 2014 and 2016, 655,667 unfed and 3842 engorged mosquito females were collected from four sampling sites. Blood-fed mosquitoes were negative for WNV-RNA, but two pools of unfed Culex pipiens s.l./torrentium collected in 2014 were tested positive. Our results suggest that Romania experienced at least two separate WNV lineage 2 introductions: from Africa into Danube Delta and from Greece into south-eastern Romania in the 1990s and early 2000s, respectively. The genetic diversity of WNV in Romania is primarily shaped by in situ evolution. WNV-specific antibodies were detected for 19 blood-meals from dogs and horses, but not from birds or humans. The hosts of mosquitoes were dominated by non-human mammals (19 species), followed by human and birds (23 species). Thereby, the catholic host-feeding pattern of Culex pipiens s.l./torrentium with a relatively high proportion of birds indicates the species’ importance as a potential bridge vector. The low virus prevalence in combination with WNV-specific antibodies indicate continuous, but low activity of WNV in the Danube Delta during the study period.

During the last two decades, Belarus faces an increase of human cases of Dirofilaria (Nematoda, Spirurida, Onchocercidae) infections. However, comprehensive analyses explaining this development and the identification of mosquito vector species are missing. Here, we present results using temperature data from Belarus and show that the annual number of human Dirofilaria cases is significantly correlated with the yearly average temperatures (Spearman's rho = 0.49, p < 0.05) and the average sum of potential Dirofilaria transmission days (Spearman's rho = 0.46, p < 0.05), suggesting that autochthonous transmission is at least in part responsible for the increasing number of clinical Dirofilaria cases in the country. In addition, 467 female mosquitoes were collected from different sampling sites in the regions of Brest and Minsk, which were analyzed by molecular methods for the presence of Dirofilaria repens and Dirofilaria immitis DNA, respectively. Two pools (5.56 %) were tested positive for Dirofilaria (estimated infection rate per 100 specimens = 0.44, 95 % confidence interval = 0.08-1.43), comprising one Anopheles claviger s.l. pool that was positive for D. repens and one Culex pipiens s.l./Culex torrentium pool positive for D. immitis DNA. This, to our knowledge, is the first molecular evidence for the presence of Dirofilaria in mosquitoes from Belarus, suggesting a high probability of autochthonous Dirofilaria transmission in the country.

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BackgroundMosquito-borne viruses (moboviruses) are of growing importance in many countries of Europe. In Romania and especially in the Danube Delta Biosphere Reserve (DDBR), mosquito and mobovirus surveillance are not performed on a regular basis. However, this type of study is crucially needed to evaluate the risk of pathogen transmission, to understand the ecology of emerging moboviruses, or to plan vector control programmes.MethodsWe initiated a longitudinal mosquito surveillance study with carbon dioxide-baited Heavy Duty Encephalitis Vector Survey traps at four sampling sites to analyse the spatio-temporal pattern of the (i) mosquito species composition and diversity, (ii) functional groups of mosquitoes (oviposition sites, overwintering stage, and number of generations), and (iii) the occurrence of potential West Nile virus (WNV) vectors.ResultsDuring 2014, a total of 240,546 female mosquitoes were collected. All species were identified using morphological characteristics and further confirmed by mitochondrial cytochrome c oxidase subunit I (COI) gene analysis of selected specimens. The two most common taxa were Coquilettidia richiardii (40.9 %) and Anopheles hyrcanus (34.1 %), followed by Culex pipiens (sensu lato) (s.l.)/Cx. torrentium (7.7 %), Aedes caspius (5.7 %), Cx. modestus (4.0 %), An. maculipennis (s.l.) (3.9 %), and Ae. vexans (3.0 %). A further seven species were less common in the area studied, including two new records for Romania: An. algeriensis and Ae. hungaricus. Phylogenetic analysis of COI gene demonstrated the evolutionary relatedness of most species with specimens of the same species collected in other European regions, except Ae. detritus and An. algeriensis, which exhibited high genetic diversity. Due to the dominance of Cq. richiardii and An. hyrcanus (75 % of all collected specimens), the overall phenology and temporal pattern of functional groups basically followed the phenology of both species. A huge proportion of the mosquito population in the course of the entire sampling period can be classified as potential WNV vectors. With 40 % of all collected specimens, the most frequent species Cq. richiardii is probably the most important vector of WNV in the DDBR.ConclusionThis is the first DNA-barcoding supported analysis of the mosquito fauna in the DDBR. The detection of two new species highlights the lack of knowledge about the mosquito fauna in Romania and in the DDBR in particular. The results provide detailed insights into the spatial-temporal mosquito species composition, which might lead to a better understanding of mobovirus activity in Romania and thus, can be used for the development of vector control programs.