Pilot longitudinal mosquito surveillance study in the Danube Delta Biosphere Reserve and the first reports of Anopheles algeriensis Theobald, 1903 and Aedes hungaricus Mihályi, 1955 for Romania

Török et al. Parasites & Vectors (2016) 9:196 DOI 10.1186/s13071-016-1484-7 RESEARCH Open Access Pilot longitudinal mosquito surveillance study in the Danube Delta Biosphere Reserve and the first reports of Anopheles algeriensis Theobald, 1903 and Aedes hungaricus Mihályi, 1955 for Romania Edina Török1,2,3†, Alexandru Tomazatos4†, Daniel Cadar4, Cintia Horváth1, Lujza Keresztes1, Stephanie Jansen4, Norbert Becker5,6, Achim Kaiser5, Octavian Popescu2,3, Jonas Schmidt-Chanasit4,7, Hanna Jöst4,7 and Renke Lühken4* Abstract Background: Mosquito-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. Methods: We 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. Results: During 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. (Continued on next page) * Correspondence: † Equal contributors 4 Bernhard Nocht Institute for Tropical Medicine, WHO Collaborating Centre for Arbovirus and Haemorrhagic Fever Reference and Research National Reference Centre for Tropical Infectious Diseases, Hamburg, Germany Full list of author information is available at the end of the article © 2016 Török et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Török et al. Parasites & Vectors (2016) 9:196 Page 2 of 13 (Continued from previous page) Conclusion: This 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. Keywords: Romania, Danube Delta Biosphere Reserve, Mosquito surveillance, Mitochondrial cytochrome c oxidase subunit I, Aedes hungaricus, Anopheles algeriensis Background In Europe, at least ten different mosquito-borne viruses (moboviruses) are circulating [1] and especially members of the family Flaviviridae, i.e. dengue virus, West Nile virus (WNV), and Usutu virus (USUV), are of growing public health and veterinary importance [2]. Although mosquito and pathogen surveillance in Romania is not performed on a regular basis, the presence of several moboviruses is well known (e.g. WNV, Sindbis virus, Tahyna virus, Lednice virus) [1]. Since the first large WNV outbreak in 1996, with several hundred human cases in Southern Romania [3], WNV has a high relevance for the country. In 2010, another WNV epidemic with more than 50 human cases demonstrated that the virus is widely distributed and established in the country [4]. The Danube Delta is situated in eastern Romania and was formed by Europe’s second largest river discharging into the Black Sea [5]. Under protection since 1991, the Danube Delta Biosphere Reserve (DDBR) covers 580,000 hectares in Romania and 4600 hectares in the Ukraine. The biological diversity in the DDBR is huge, comprising over 1800 species of flora and 3500 species of fauna [6]. Located halfway between the Equator and North Pole, the DDBR is an important hub for migratory birds from Africa and Asia. These circumstances strongly suggest a high risk of introduction of bird associated zoonotic pathogens such as WNV or USUV. Pathogens imported by migratory birds find a diverse mosquito fauna, which have excellent breeding habitats in this ecologically heterogeneous wetland. Covering more than 30 different ecosystems [5], the DDBR is characterized by vast natural marshes and fresh water bodies, mainly lakes and channels, providing excellent conditions for a diverse and very abundant mosquito fauna [7]. The checklist of the mosquitoes in the DDBR consist of 31 species [7], compromising 56.4 % of the 55 species known for Romania [8–12]. However, regular mosquito monitoring programmes are missing in Romania. As already highlighted by Prioteasa & Falcuta [7], in the DDBR, these types of studies are predominantly hampered by transportation problems, as many areas can only be reached by boat. However, a detailed knowledge on the species composition and phenology are crucially needed to evaluate the risk of pathogen transmission, plan vector control programmes, and to understand the ecology of circulating moboviruses. Therefore, this longitudinal mosquito surveillance study in the DDBR was conducted in order to evaluate the spatio-temporal pattern of t (...truncated)