title: Direct PCR of indigenous and invasive mosquito species: a time- and cost-effective technique of mosquito barcoding.
abstract: Millions of people die each year as a result of pathogens transmitted by mosquitoes. However, the morphological identification of mosquito species can be difficult even for experts. The identification of morphologically indistinguishable species, such as members of the Anopheles maculipennis complex (Diptera: Culicidae), and possible hybrids, such as Culex pipiens pipiens/Culex pipiens molestus (Diptera: Culicidae), presents a major problem. In addition, the detection and discrimination of newly introduced species can be challenging, particularly to researchers without previous experience. Because of their medical importance, the clear identification of all relevant mosquito species is essential. Using the direct polymerase chain reaction (PCR) method described here, DNA amplification without prior DNA extraction is possible and thus species identification after sequencing can be achieved. Different amounts of tissue (leg, head; larvae or adult) as well as different storage conditions (dry, ethanol, -20 and -80 °C) and storage times were successfully applied and showed positive results after amplification and gel electrophoresis. Overall, 28 different indigenous and non-indigenous mosquito species were analysed using a gene fragment of the COX1 gene for species differentiation and identification by sequencing this 658-bp fragment. Compared with standard PCR, this method is time- and cost-effective and could thus improve existing surveillance and control programmes.
authors.affiliation: Institute for Ecology, Evolution and Diversity, Goethe University (GU); Senckenberg Biodiversity and Climate Research Centre (BiK-F); Senckenberg Gesellschaft für Naturforschung (SGN), Frankfurt am Main, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Werblow
authors.affiliation: Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: E
authors.initials: E
authors.lastname: Flechl
authors.affiliation: Institute for Ecology, Evolution and Diversity, Goethe University (GU); Senckenberg Biodiversity and Climate Research Centre (BiK-F); Senckenberg Gesellschaft für Naturforschung (SGN), Frankfurt am Main, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Klimpel
authors.affiliation: Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: C
authors.initials: C
authors.lastname: Zittra
authors.affiliation: Institute for Veterinary Public Health, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: K
authors.initials: K
authors.lastname: Lebl
authors.affiliation: Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: K
authors.initials: K
authors.lastname: Kieser
authors.affiliation: Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: A
authors.initials: A
authors.lastname: Laciny
authors.affiliation: Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: K
authors.initials: K
authors.lastname: Silbermayr
authors.affiliation: Institute for Ecology, Evolution and Diversity, Goethe University (GU); Senckenberg Biodiversity and Climate Research Centre (BiK-F); Senckenberg Gesellschaft für Naturforschung (SGN), Frankfurt am Main, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Melaun
authors.affiliation: Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine Vienna, Vienna, Austria.
authors.forename: H-P
authors.initials: HP
authors.lastname: Fuehrer
mesh_terms: D000818:Animals; D009033:Culicidae / Q000235:genetics* / Q000254:growth & development; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D003576:Electron Transport Complex IV / Q000235:genetics; D005260:Female; D019476:Insect Proteins / Q000235:genetics; D058865:Introduced Species; D007814:Larva / Q000235:genetics / Q000254:growth & development; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013048:Specimen Handling
chemical_list: D019476:Insect Proteins; D003576:Electron Transport Complex IV
keywords: Barcoding; direct PCR; molecular differentiation; molecular identification; mosquitoes; vector-borne diseases