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. 2014 Dec 12:7:569.
doi: 10.1186/s13071-014-0569-4.

DNA barcoding: complementing morphological identification of mosquito species in Singapore

Abigail Chan  1 , Lee-Pei Chiang  2 , Hapuarachchige C Hapuarachchi  3 , Cheong-Huat Tan  4 , Sook-Cheng Pang  5 , Ruth Lee  6 , Kim-Sung Lee  7 , Lee-Ching Ng  8 , Sai-Gek Lam-Phua  9
Affiliations

DNA barcoding: complementing morphological identification of mosquito species in Singapore

Abigail Chan et al. Parasit Vectors. .

Abstract

Background: Taxonomy that utilizes morphological characteristics has been the gold standard method to identify mosquito species. However, morphological identification is challenging when the expertise is limited and external characters are damaged because of improper specimen handling. Therefore, we explored the applicability of mitochondrial cytochrome C oxidase subunit 1 (COI) gene-based DNA barcoding as an alternative tool to identify mosquito species. In the present study, we compared the morphological identification of mosquito specimens with their differentiation based on COI barcode, in order to establish a more reliable identification system for mosquito species found in Singapore.

Methods: We analysed 128 adult mosquito specimens, belonging to 45 species of 13 genera. Phylogenetic trees were constructed for Aedes, Anopheles, Culex and other genera of mosquitoes and the distinctive clustering of different species was compared with their taxonomic identity.

Results: The COI-based DNA barcoding achieved a 100% success rate in identifying the mosquito species. We also report COI barcode sequences of 16 mosquito species which were not available previously in sequence databases.

Conclusions: Our study utilised for the first time DNA barcoding to identify mosquito species in Singapore. COI-based DNA barcoding is a useful tool to complement taxonomy-based identification of mosquito species.

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Figures

Figure 1
Figure 1
Phylogenetic tree based on COI sequences of Aedes and Verrallina spp. mosquitoes. An alignment of COI gene sequences (440 bp) was used to construct the neighbour joining tree in MEGA 6.06 software. Numbers displayed on branches are the bootstrap support obtained through 1000 replications. GenBank sequences are shown with accession numbers. Sequences starting with “EHI” were generated during this study and are highlighted in blue.
Figure 2
Figure 2
Phylogenetic tree based on COI sequences of Culex and Lutzia spp. mosquitoes. A 432 bp-region of the COI gene was used to construct the neighbour joining tree in MEGA 6.06 software. Numbers displayed on branches are the bootstrap support obtained through 1000 replications. GenBank sequences are shown with accession numbers. Sequences starting with “EHI” were generated during this study and are highlighted in blue.
Figure 3
Figure 3
Phylogenetic tree based on COI sequences of Anopheles spp. mosquitoes. A 452 bp-region of the COI gene was used to construct the neighbour joining tree in MEGA 6.06 software. Numbers displayed on branches are the bootstrap support obtained through 1000 replications. GenBank sequences are shown with accession numbers. Sequences starting with “EHI” were generated during this study and are highlighted in blue.
Figure 4
Figure 4
Phylogenetic tree based on COI sequences of other genera of mosquitoes. A 447 bp-region of the COI gene was used to construct the neighbour joining tree in MEGA 6.06 software. Numbers displayed on branches are the bootstrap support obtained through 1000 replications. GenBank sequences are shown with accession numbers. Sequences starting with “EHI” were generated during this study and are highlighted in blue.
Figure 5
Figure 5
Morphological comparison of the abdominal terga of Ae. aegypti queenslandensis and Ae. aegypti aegypti. (a): Ae. aegypti queenslandensis: unbroken median stripes of pale scales along the abdominal terga. (b): Ae. aegypti aegypti: no median stripes of pale scales from terga II to VII.
Figure 6
Figure 6
Morphological comparison of the hind tarsa of Ae. vexans and Ae. vigilax . (a): Ae. vexans: pale basal bands in less than a quarter of the length of hind tarsomeres. (b): Ae. vigilax: pale basal bands covering more than a quarter of the length of hind tarsomeres.
Figure 7
Figure 7
Morphological comparison of the hind femur of Cx. vishnui and Cx. pseudovishnui. (a): Cx. vishnui: hind femur with apical dark band not well contrasted with pale scales on the hind femur. (b): Cx. pseudovishnui: hind femur with apical dark band well contrasted with pale scales on the hind femur.
Figure 8
Figure 8
Morphological comparison of the abdominal terga of Lt. fuscana and Lt. halifaxii. (a): Lt. fuscana: yellowish scales on abdominal terga. (b): Lt. halifaxii: entirely dark abdominal terga.
Figure 9
Figure 9
Morphological comparison of the abdominal terga of Ae. albopictus and Ae. malayensis . (a): Ae. albopictus: dorsal white bands separated from the lateral spots on abdominal terga. (b): Ae. malayensis: dorsal white bands connected to the lateral pale patches on abdominal terga.
Figure 10
Figure 10
Morphological comparison of the thorax of Ae. albopictus and Ae. malayensis. (a): Ae. albopictus: pale patches of scales do not extended towards the scutellum on the thorax. (b): Ae. malayensis: pale scales extended towards the scutellum on the thorax.
Figure 11
Figure 11
Morphological comparison of the vein CuA of An. sinensis. (a): Pale fringe spot at vein CuA. (b): Dark fringe spot at vein CuA.
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