DNA barcoding of morphologically characterized mosquitoes belonging to the subfamily Culicinae from Sri Lanka
BackgroundVectors of mosquito-borne diseases in Sri Lanka, except for malaria, belong to the subfamily Culicinae, which includes nearly 84% of the mosquito fauna of the country. Hence, accurate and precise species identification of culicine mosquitoes is a crucial factor in implementing effective vector control strategies. During the present study, a combined effort using morphology and DNA barcoding was made to characterize mosquitoes of the subfamily Culicinae for the first time from nine districts of Sri Lanka. Cytochrome c oxidase subunit 1 (cox1) gene from the mitochondrial genome and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization.ResultsAccording to morphological identification, the field collected adult mosquitoes belonged to 5 genera and 14 species, i.e. Aedes aegypti, Ae. albopictus, Ae. pallidostriatus, Aedes sp. 1, Armigeres sp. 1, Culex bitaeniorhynchus, Cx. fuscocephala, Cx. gelidus, Cx. pseudovishnui, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Cx. whitmorei, Mansonia uniformis and Mimomyia chamberlaini. Molecular analyses of 62 cox1 and 36 ITS2 sequences were exclusively comparable with the morphological identifications of all the species except for Ae. pallidostriatus and Aedes sp. 1. Although the species identification of Armigeres sp. 1 specimens using morphological features was not possible during this study, DNA barcodes of the specimens matched 100% with the publicly available Ar. subalbatus sequences, giving their species status. Analysis of all the cox1 sequences (14 clades supported by strong bootstrap value in the Neighbor-Joining tree and interspecific distances of > 3%) showed the presence of 14 different species. This is the first available DNA sequence in the GenBank records for morphologically identified Ae. pallidostriatus. Aedes sp. 1 could not be identified morphologically or by publicly available sequences. Aedes aegypti, Ae. albopictus and all Culex species reported during the current study are vectors of human diseases. All these vector species showed comparatively high diversity.ConclusionsThe current study reflects the significance of integrated systematic approach and use of cox1 and ITS genetic markers in mosquito taxonomy. Results of DNA barcoding were comparable with morphological identifications and, more importantly, DNA barcoding could accurately identify the species in the instances where the traditional morphological identification failed due to indistinguishable characters of damaged specimens and the presence of subspecies.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2810-z) contains supplementary material, which is available to authorized users.
[…] The trace files/chromatograms of cox1 and ITS2 sequences were manually edited using BioEdit software. Sequences of low quality were excluded during data analysis. Consensus sequences were aligned using Clustal W in BioEdit software. Once the alignment was completed, species identification was confirmed by comparison to publicly available sequence data in GenBank using Basic Logical Alignment Search Tool (BLAST) [https://blast.ncbi.nlm.nih.gov/Blast.cgi] and the Barcode of Life Database (BOLD) interface [www.boldsystems. org]. The number of parsimony informative sites, number of variable sites, number of haplotypes, haplotype diversity and GC content were analyzed using the DNA Sequences Polymorphism software (DnaSP, version 5.1.10). MEGA version 6.0 was used to calculate intraspecific and interspecific pairwise sequence divergence using the Kimura-2 parameter distance model . Neighbor-Joining (NJ) phylogenetic trees of cox1 and ITS2 sequences were constructed in MEGA 6.0 using Kumura-2 Parameter distances. Branch support of NJ trees were assessed by bootstrapping with 1000 replicates. Codon positions included 1st + 2nd + 3rd + noncoding regions. All the haplotype sequences of cox1 and ITS2 were deposited in the GenBank database (see below). […]
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