Mosquito Species Identification via Genetic Barcoding

A subset morphologically identified mosquitoes (n = 2040) from the collections above (Table 1) were sequenced at ITS2 and/or COI loci for species identification. DNA was extracted from individual specimens using a CTAB technique. Briefly, samples were individually homogenized in CTAB extraction buffer, DNA extracted with phenol and isopropanol and precipitated with ethanol. The ribosomal DNA internal transcribed spacer region 2 (rDNA ITS2) region was isolated by PCR using primers developed for differentiating other Anopheles species complexes32 (link). The ITS2 region was amplified from genomic DNA using the ITS2A and ITS2B primers32 (link). The primer sequences were as follows: ITS2A 5′-TGTGAACTGCAGGACACAT-3′ and ITS2B 5′-TATGCTTAAATTCAGGGGGT-3′. The PCR mixture contained 2.5 μl of 10X buffer, 200 μM of each dNTP, 0.5 units of Taq DNA polymerase, 0.75 μl of 10 pmol/μl each of forward and reverse primers, and 2 μl of DNA template prepared as above. The thermocycling conditions were as follows: 94 °C for 5 minutes, 30 cycles of denaturation at 94 °C for 1 minute, annealing at 52 °C for 1 minute, and extension at 72 °C for 2 minutes, with a final extension at 72 °C for 5 minutes.
The mitochondrial DNA cytochrome c oxidase subunit 1 (COI) gene was amplified using LCO and HCO primers55 (link). The primers used were LCO 1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO 2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′). The 25 μl PCR mixture contained 2.5 μl of 10X buffer, 0.2 mM of each dNTP, 1.2 mM MgCl₂, 0.5 units of Taq DNA polymerase, 0.75 μl of 10 pmol/μl each of forward and reverse primers, and 1 μl of DNA template prepared as above. The thermocycling conditions were as follows: 94 °C for 5 min, 5 cycles of denaturation at 94 °C for 40 s, annealing at 45 °C for 1 min, and extension at 72 °C for 1.5 min; then 30 cycles of denaturation at 94 °C for 40 s, annealing at 51 °C for 1 min, and extension at 72 °C for 1.5 min; with a final extension at 72 °C for 5 min.
All specimens were initially amplified with ITS2 primers. Those specimens that did not amplify with ITS2 primers, and specimens corresponding to every novel ITS2 sequence were amplified with COI primers. The amplified fragments were visualized by electrophoresis on a 1% agarose gel. The PCR product was purified using an enzyme cleanup: 2U of Exonuclease 1 (USB Corporation, Cleveland, OH), 1U of Shrimp Alkaline Phosphatase (USB), and 1.8 μl of ddH20 were added to 8 μl of PCR product. This mixture was incubated at 37 °C for 15 min, followed by 15 min at 80 °C to inactivate the enzymes. The PCR products were sequenced directly (with one of the PCR primers) using Sanger sequencing on ABI 3730xl DNA Analyzer platform (PE Applied Biosystems, Warrington, England).