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

Abstract

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.

The monsoon-associated equine South African pointy mosquito ‘Aedes caballus’; the first comprehensive record from southeastern Iran with a description of ecological, morphological, and molecular aspects

“…therefore, our COI sequence data may eventually prove more valuable for phylogenetic inference, especially when applied to larger sample sizes. While intraspecific variation within the published sequences was low (≤ 0.5% ± 0.2) [ 63 ], our results suggest the presence of different haplotypes in Iran based on COI and ITS2 genes. Further exploration of ITS2, which exhibited higher overall divergence compared to COI, in Ae .…”

“…This research should encompass additional samples and locations, not limited to rural areas, and should include the temporal variation of specimens, with a particular focus on periods of heavy rainfall. Additionally, the divergence of ≥ 3.6% between South African samples [ 63 ] and our specimens emphasizes the need for continued investigation.…”

Metabarcoding mosquitoes: MinION sequencing of bulk samples gives accurate species profiles for vector surveillance (Culicidae)

“…The last step is particularly time-consuming, tedious, costly, and sometimes made impossible due to several reasons. Firstly, mosquito specimens are tiny, and identification of morphological characteristics under the microscope is often a laborious and timeintensive process (15,16). Additionally, mosquito scales and setae, which are crucial diagnostic features, are frequently damaged during specimen handling and storage (17)(18)(19)(20) thus impeding species identification.…”

“…There are also DNA barcode databases of mosquito species from all around the world, readily available online (such as NCBI GenBank and BOLD Systems), which one could use as references to attain species-level identities for some specimens. However, it is crucial to exercise caution, as misidentified species are prevalent in these databases and it is important to validate DNA barcodes for local mosquito specimens together with morphological documentation (16,30). Researchers bear the responsibility of diligently checking the publication record of all sequences sourced from such databases.…”

First report of Culex (Culex) camposi Dyar, 1925 and Culex (Carrollia) bonnei Dyar, 1921 via male genitalia and CoxI barcoding sequence analyses in Huanuco, Peru

“…species groups or complexes have been reported in previous studies(Demari et al, 2015;Laurito et al, 2013Laurito et al, , 2018Montalvo et al, 2022) Demari et al (2017). to differentiate the species Cx coronator and Cx usquatus, they used morphological data and nuclear and mitochondrial gene sequences (CoxI, NADH5, CAD, Hunchback), for his part,Vesgueiro et al (2011) used sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA, however, their results did not resolve a clear separation between these two species.…”