- Short report
- Open Access
Aedes vittatus in Spain: current distribution, barcoding characterization and potential role as a vector of human diseases
© The Author(s). 2018
- Received: 28 November 2017
- Accepted: 29 April 2018
- Published: 18 May 2018
Aedes vittatus is currently found in Africa, Asia and Europe, where it acts as a vector of pathogens causing animal and human diseases (e.g. chikungunya, Zika and dengue). Like other Aedes species, Ae. vittatus is able to breed in artificial containers. The ECDC has recently highlighted the need for molecular tools (i.e. barcoding characterization) that enable Aedes species to be identified in entomological surveys.
We sampled mosquito larvae and adults in southern Spain and used a molecular approach to amplify and sequence a fragment of the cytochrome c oxidase subunit 1 gene (barcoding region) of the mosquitoes. The blast comparison of the mosquito sequences isolated from Spain with those deposited in public databases provided a ≥ 99% similarity with sequences for two Aedes mosquitoes, Ae. vittatus and Ae. cogilli, while similarities with other Aedes species were ≤ 94%. Aedes cogilli is only present in India and there are no records of this species from Europe.
Due to the low genetic differences between Ae. vittatus and Ae. cogilli, the barcoding region should not be used as the only method for identifying Ae. vittatus, especially in areas where both of these Aedes species are present. This type of analysis should thus be combined with morphological identification using available keys and/or the characterization of other molecular markers. In addition, further entomological surveys should be conducted in order to identify the fine-scale distribution of this mosquito species in Europe.
- DNA barcoding
- Aedes mosquitoes
- Vector-borne diseases
Vector-borne pathogens are a global health concern in which mosquitoes play a central role as vectors of pathogens . In Europe both native and invasive species of Aedes mosquitoes are involved in the transmission of pathogens including viruses (e.g. dengue and chikungunya ) and parasites (e.g. Dirofilaria ). Of these mosquitoes, the invasive Aedes albopictus has received much attention in recent decades due to its role in the transmission of dengue  and chikungunya  in Europe. Certain Aedes species, including Ae. albopictus, are able to breed in artificial containers and it is important to develop accurate identification protocols for differentiating native and invasive Aedes species that breed in the same area [6–8]. The identification of mosquito species through the characterization of a fragment of the cytochrome c oxidase subunit 1 (cox1) gene is a useful tool for monitoring the presence of species [9, 10], above all given the difficulties in identifying mosquitoes in larval stages and the current scarcity of trained taxonomists . However, this method requires a previous genetic characterization of the species . This is an important limitation in the case of Aedes mosquitoes as this information is not available for most of the species of this genus that breed in Europe , despite their importance in pathogen transmission .
The aim of this study was to update the current distribution of Ae. vittatus and provide the first genetic characterization of the barcoding region of specimens of this species from Europe. Hitherto, sequences from this species were only available from China , India  and Kenya . In addition, we review here available information on the potential role of this species in the transmission of virus of public health concern.
Three mosquitoes (one male and two females) from Seville Province were selected for molecular characterization of the barcoding region and to confirm the morphological identification of the species. A fragment of the right hind-leg of each mosquito was cut-off using a sterile blade and placed on a Petri dish. Genomic DNA was extracted using the Maxwell 16 LEV Blood DNA Kit (Promega, Madison, WI, USA) following the manufacture’s instructions. PCR reactions were performed using the primer pair LCO1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and HCO2198 (5'-TAA ACTT CAG GGT GAC CAA AAA ATC A-3')  following Whiteman et al.  to amplify a 658 bp fragment of the cox1 gene (excluding primers) (see ). The presence of amplicons was verified on 1.8% agarose gels. Sequences were resolved in both directions by Macrogen sequencing service (Macrogen Inc., the Netherlands). Sequences were edited using the SequencherTM v4.9 software (Gene Codes Corp., Ann Arbor, MI, USA) and compared with sequences deposited in the GenBank DNA sequence database (National Center for Biotechnology Information) and the Barcode of Life Data Systems (BOLD).
The anthropophilic feeding preference of Ae. vittatus females was confirmed by the fact that four mosquitoes (57.1%) fed on a human arm after < 5 min of exposure.
We characterized for the first time in Europe the barcoding region of Ae. vittatus. A BLAST comparison of this sequence with those deposited in public databases provided a ≥ 99% similarity with sequences of two Aedes mosquitoes, Ae. vittatus and Ae. cogilli. However, Ae. cogilli, is only present in India and is not found in Europe . The other Aedes sequences on GenBank differed by about 6% from the Ae. vittatus sequence isolated here. Although varying between taxa, interspecific differences in the barcoding region are established at 0–2% . Based on the low interspecific differences found between Ae. vittatus and Ae. cogilli, our results do not support the use of the cox1 region as a method for separating these species where they coincide; rather, this method should be combined with morphological identification using available keys or the characterization of other molecular markers. Based on the morphological characteristics of the specimens captured here, we conclude that the mosquitoes we captured belong to the species Ae. vittatus .
Main viruses causing diseases transmitted by Ae. vittatus with information of the potential hosts and known distribution of the diseases
Family Togaviridae (Alphavirus)
Humans, birds, domestic animals, monkeys, rodents
Africa, America, Asia, Europe
Humans, domestic animals
Semliki Forest virus
Humans, birds, domestic animals, non-human primates, rodents
Africa, Asia, Europe
Family Flaviviridae (Flavivirus)
Humans, non-human primates
Africa, South America
Humans, domestic animals, monkeys
Yellow fever virus
Humans, non-human primates
Africa, South America
Humans, bats, birds, domestic animals, non-human primates
Africa, America, Asia
Family Bunyaviridae (Bunyavirus)
When identifying Ae. vittatus in areas where its distribution overlaps with that of the related Asian species Ae. cogilli, the identification of the barcoding region should be combined with morphological identification and/or the characterization of other molecular markers. However, in Europe, molecular tools may allow for the accurate identification of this species due to the great genetic difference (6%) found between Spanish Ae. vittatus and other Aedes species. Further entomological studies should be conducted in order to identify the fine-scale distribution of Ae. vittatus in European countries, where it could play a role in the transmission of viruses with public health relevance.
We thank Esmeralda Pérez, Juana Moreno Fernández and Ernesto García for their help in the fieldwork and Isabel Martín and Laura Gómez for help during laboratory work. David Aragonés and Isabel Afán (LAST-EBD) provided advice necessary to generate the maps. Members of the bank of tissues and DNA of the MNCN-CSIC helped in the deposition of samples. Two anonymous reviewers provided valuable comments in a previous version of the manuscript. Michael Lockwood revised the English text.
This study was funded by the CGL2015-65055-P project from the Spanish Ministerio de Economía y Competitividad and European Regional Development’s funds (FEDER). ADF and RGL were supported by Severo-Ochoa grant (SVP-2014-068571) and a FPI grant (BES-2013-065274), respectively. JMP was partially supported by a 2017 Leonardo Grant for Researchers and Cultural Creators, BBVA Foundation. The Foundation accepts no responsibility for the opinions, statements and contents included in the project and/or the results thereof, which are entirely the responsibility of the authors. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).
Availability of data and materials
Sequences generated in this study were deposited in the GenBank database under the accession number MF429950 Mosquitoes were deposited in the collection of the Museo Nacional de Ciencias Naturales (MNCN-CSIC), Madrid, Spain, under the accession numbers MNCN/ADN 86743 and 86744.
All authors designed the study. ADF, RGL and SR collected and morphologically identified the mosquitoes. ADF, RGL and JMP conducted the molecular analyses. ADF and JMP drafted the first version of the manuscript. All authors read and approved the final manuscript.
All experimental procedures were approved by the CSIC Ethics Committee and Animal Health authorities, and complied with Spanish laws.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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