Molecular identification of Anopheles moghulensis (Diptera; Culicidae) in Iran: overcoming morphological misidentification

Phylogenetic relationships within Anopheles species are critical for accurate classification and vector surveillance. Anopheles superpictus typically serves as a secondary vector across its distribution range and has been morphologically recognized as a single species, despite differences observed in larval and adult female characteristics. However, its close similarity to Anopheles moghulensis has led to frequent misidentifications. This study introduces a molecular species diagnostic tool based on ribosomal DNA internal transcribed spacer 2 (rDNA-ITS2) sequences for differentiating An. moghulensis from An. superpictus. Using newly designed species-specific PCR primers, we identified a misclassification wherein An. moghulensis sequences were erroneously submitted as An. superpictus. Comparative ITS2 sequence analysis revealed that An. moghulensis shares 100% similarity with An. superpictus B, with distinct ITS2 size differences: 373 bp in An. moghulensis vs. 352 bp in An. superpictus. This study represents the first molecular identification of An. moghulensis and its taxonomic placement within Iranian and global Anopheles fauna. Despite advances in species identification, inconsistencies in GenBank accession records remain a significant challenge, necessitating taxonomic reassessment of Anopheles species.