2.2. DNA Barcoding

Some species collected in the “Mosquitos da Caatinga” project had their CO1 gene studied for taxonomic purposes. Five representative specimens of each species of interest were processed. DNA was extracted from whole adult specimens and dried over silica gel, at −4 °C, using the DNeasy Blood & Tissue (Qiagen^®, Valencia, CA, USA) kit. The products were stored at −20 °C in the Entomological Collection at the Federal University of Sergipe, in Aracaju (Brazil) prior to the amplification of a fragment of the Cytochrome c oxidase subunit 1 (CO1) gene. The primers LCO1490 and HCO2198 [29] were used to amplify a ~658 bp fragment, located at the 5’ end of the COI sequence, which was trimmed to 609 bps. Both the PCR and the sequencing reaction were run using the Big Dye PCR Direct (Applied Biosystems^®, Foster City, CA, USA) kit, according to the manufacturer’s instructions. For the three Wyemoyia (Phoniomyia) morphotypes the ITS2 ribossomal fragments were amplified using 5.8SF5’ and 28SR5’ universal primers [30].

The PCR products were electrophoresed in 1.5% agarose gel stained with GelRed™ Nucleic Acid Gel stain (Biotium Inc, Hayward, CA, USA). The products were sequenced in both directions with the same set of primers and analyzed in an ABI 3130 DNA Analyser (PE Applied Biosystems, Warrington, UK). The samples were sequenced with bidirectional primers and the quality of the chromatograms was verified visually in Chromas v2.6.5 (Technelysium Pty Ltd, Brisbane, Australia). Homologies, insertions/deletions (indels), and frameshifts were identified in MUSCLE v3.8.31 [31]. The sequences, obtained in FASTA format, were aligned in ClustalW [32]. The sequence from each specimen was compared to barcode sequences from GenBank using ‘Blast’ and assigned to mosquito species in BOLD using the ‘Identification Request’ function. The CO1 sequences were deposited in the EMBL nucleotide sequence database.