Molecular confirmation of Culex pipiens Linnaeus, 1758 (Diptera: Culicidae), Culex torrentium Martini, 1925 (Diptera: Culicidae), and Culiseta longiareolata Macquart, 1838 (Diptera: Culicidae) using ITS2 DNA barcoding

Purpose: This study aimed to identify Culex pipiens Linnaeus, 1758 (Diptera: Culicidae), Culex torrentium Martini, 1925 (Diptera: Culicidae), and Culiseta longiareolata Macquart, 1838 (Diptera: Culicidae) species using the internal transcribed spacer 2 (ITS2) region of ribosomal DNA and to provide molecular characterization of these species.Method: Mosquito samples collected in Kocaeli between 2017 and 2020 were used in the study. Genomic DNA isolated from the samples was amplified by PCR using suitable primers for ITS2. Agarose gel electrophoresis results of the ITS2 region revealed different band lengths among the species, consistent with the structure of ITS2 exhibiting length polymorphism. The ITS2 sequences obtained from DNA sequencing were submitted to GenBank and compared with reference sequences in the NCBI database. Phylogenetic analyses were conducted using MEGA X software with 1000 bootstrap replicates.Findings: Sequence analysis and phylogenetic tree construction confirmed that the analyzed samples belonged to Culex pipiens, Culex torrentium, and Culiseta longiareolata. The ITS2 region revealed length polymorphism across species on agarose gel and proved particularly effective in distinguishing between the morphologically similar species Culex pipiens and Culex torrentium.Conclusion: The ITS2 region of ribosomal DNA is a reliable molecular marker for the identification and characterization of these three mosquito species, especially for discriminating between Culex pipiens and Culex torrentium, which are difficult to differentiate based on morphology alone.