Anopheline Mosquito Species Identification

Each anopheline specimen was morphologically identified to species, using a taxonomic key [36 ]. For molecular confirmation of species, genomic DNA was extracted from the whole body specimens using a sodium hydroxide extraction method [37 ]. Molecular identification was determined by sequencing the internal transcribed spacer region 2 (ITS2) and the cytochrome oxidase subunit 1 (CO1) loci. The ribosomal ITS2 [38 (link)–40 (link)] and mitochondrial CO1 [41 (link)] loci were amplified by PCR, and Sanger sequenced on ABI 3730xl DNA analyzer platform (PE Applied Biosystems). ITS2 and CO1 sequences were aligned with a minimum match percentage of 98% and 95%, respectively, using Seqman Pro assembly software (Lasergene v 12.3.1). Contiguous sequence assemblies were trimmed and examined manually for quality, any contaminated and poor-quality sequences were removed from the analysis. Nucleotide BLASTs (NCBI) of sequence assemblies was used for final species determination.