Morphological and Molecular Characterization Using Genitalia and CoxI Barcode Sequence Analysis of Afrotropical Mosquitoes with Arbovirus Vector Potential

Potential arboviral Afrotropical mosquito vectors are underrepresented in public databases of CoxI barcode sequences. Furthermore, available CoxI sequences for many species are often not associated with voucher specimens to match the corresponding fine morphological characterization of specimens. Hence, this study focused on the characterization of Culicine mosquitoes from South Africa, Mozambique, and Angola and their classification using a complementary approach including a morphological analysis of specimens' genitalia and phylogenetic study based on the analysis of CoxI barcode sequences using maximum likelihood and Bayesian phylogenetic inference methods, alongside Median-Joining Network and PCOORD analyses. Overall, 800 mosquitoes (652 males and 148 females) from 67 species, were analyzed. Genitalia from 663 specimens allowed the identification of 55 species of 10 genera. A total of 247 CoxI partial gene sequences corresponding to 65 species were obtained, 11 of which (Aedes capensis, Ae. mucidus, Culex andersoni, Cx. telesilla, Cx. inconspicuosus, Eretmapodites subsimplicipes, Er. quinquevittatus, Ficalbia uniformis, Mimomyia hispida, Uranotaenia alboabdominalis, and Ur. mashonaensis) are, to the best of our knowledge, provided here for the first time. The presence of Cx. pipiens ecotypes molestus and pipiens and their hybrids, as well as Cx. infula, is newly reported in the Afrotropical region. The rates of correct sequence identification using BOLD and BLASTn (>= 95% identity) were 64% and 53%, respectively. Phylogenetic analysis revealed that, except for subgenus Eumelanomyia of Culex, there was support for tribes Aedini, Culicini, Ficalbiini, and Mansoniini. A divergence >2% was observed in conspecific sequences, e.g., Aedeomyia africana, Ae. cumminsii, Ae. unilineatus, Ae. metallicus, Ae. furcifer, Ae. caballus, and Mansonia uniformis. Conversely, sequences from groups and species complexes, namely, Ae. simpsoni, Ae. mcintoshi, Cx. bitaeniorhynchus, Cx. simpsoni, and Cx. pipiens were insufficiently separated. A contribution has been made to the barcode library of Afrotropical mosquitoes with associated genitalia morphological identifications.