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ObjectivesTo assess vector behaviour and phenotypic resistance for effective vector control programming.DesignThis was a cross-sectional study.SettingThis study was conducted in the urban and periurban areas of Ndola district, Zambia.Participants/study unitsA total of 166 houses were selected for adult mosquito collection, and an additional 60 collection efforts were made for larval collection from potential larval habitats.Primary and secondary outcome measuresThe primary outcome was the behaviour of the malaria vectors, and the secondary outcome was their phenotypic resistance status.ResultsThe main breeding sites identified were irrigation trenches (4.67 larvae/dip) and garden ponds (2.72 larvae/dip) created from extensive urban agriculture practices. Anopheles funestus sensu stricto (An. funestus s.s) and Anopheles gambiae sensu stricto (An. gambiae s.s) were found to coexist in all the four sites, with An. funestus s.s identified as the most dominant malaria vector. Densities of An. gambiae s.s, seeking a blood meal (χ2=12.566, df=3, p=0.001) and resting indoors (Z=56.5, p=0.019), were found to be higher in urban than periurban sites compared with An. funestus s.s, which had similar distribution across the study sites. Sprayed houses were significantly associated with reduced mosquito numbers (B=−0.956, incidence rate ratio=0.384, p=0.001). Anopheles gambiae was fully susceptible to organophosphates and neonicotinoids but highly resistant to pyrethroids, carbamates and organochlorines.ConclusionsThe emergence of An. funestus s.s in an area previously dominated by An. gambiae s.s and its coexistence with An. gambiae s.s in the dry season pose a risk of sustaining malaria transmission all year round. Agricultural practices in urban areas resulted in highly productive mosquito breeding sites; thus, there is a need for targeted vector control.

The zoophilic and exophilic traits of outdoor-biting Anopheles have led to this group largely being overlooked for their role in malaria transmission, despite several species now recognized as locally important in regions of sub-Saharan Africa. Given the current limitations with identification of these understudied species, it is crucial to accurately correlate morphological features to molecular data. Here, we produced high quality reference sequence data for representative understudied anopheline species to better understand the phylogenetic relationships between under- and well-studied vectors of malaria. For mitochondrial genome assembly, shallow shotgun sequencing was implemented on single mosquito specimens and phylogenetic analyses were performed on the concatenated protein coding genes of the mitogenomes using a Bayesian approach. This study generated 10 complete mitogenomes focusing on less studied taxa with an average length 15,380 bp and A-T content of 77.4% consistent with other anophelines containing 37 genes. Bayesian inference analysis yielded four main clades with molecular dating indicating that well-studied malaria vectors diverged from outdoor-biting species more than 63 million years ago. These findings support the taxonomic grouping of mosquitoes belonging to the Anopheles genus based on morphological characteristics and can provide molecular diagnostics for species identification enabling more precise and adept interventions for malaria control.

Background Mosquito species belonging to the Anopheles coustani group have been implicated in driving residual malaria transmission in sub-Saharan Africa and are regarded as an established primary vector in Madagascar. The morphological identification of mosquitoes in this group is challenging due to similarity of features and their molecular confirmation is difficult due to a paucity of reference sequence data representing all members of the group. Conventional molecular barcoding with the cytochrome oxidase I (COI) gene and the internal transcribed spacer 2 (ITS2) region targets is limited in their discrimination and conclusive identification of members of species complexes. In contrast, complete mitochondrial genomes (mitogenomes) have demonstrated much improved power over barcodes to be useful in rectifying taxonomic discrepancies in Culicidae. The goal of this study was to characterize the phylogenetic taxonomy of Zambian members of the An. coustani group by generating and then using complete mitochondrial genomes for phylogenetic rectification. Methods A genome skimming approach was utilized via shallow shotgun sequencing on individual mosquito specimens to generate sequence reads for mitogenome assembly. Bayesian inferred phylogenies and molecular dating estimations were perfomed on the concatenated protein coding genes using the Bayesian Evolutionary Analysis by Sampling Trees 2 (BEAST 2) platform. Divergence estimates were calibrated for members of the An. coustani group based on published calucations for Anopheles - Aedes . Results This study generated 17 new complete mitogenomes which were comprable to reference An. coustani mitogenomes in the GenBank repository by having 13 protein coding, 22 transfer RNA and 2 ribosomal RNA genes, with an average length of 15,400 bp and AT content of 78.3%. Bayesian inference using the concatenated protein coding genes from the generated and publicly available mitogenomes yielded six clades: one for each of the four taxa targeted in this study, the GenBank references, and a currently unknown species. Divergence times estimated that the An. coustani group separated from the Anopheles gambiae complex approximately 110 million years ago (MYA), and members within the complex diverged at times points ranging from ~ 34 MYA to as recent as ~ 7 MYA. Conclusions These findings demonstrate the value of mitochondrial genomes in differentiating cryptic taxa and help to confirm morphological identities of An. coustani sensu stricto, Anopheles paludis , Anopheles zeimanni and Anopheles tenebrosus . Divergence estimates within the An. coustani group are similar when compared to species with morphologically distinct features. These analyses also highlight the likely presence of other cryptic An. coustani group members circulating in Zambia.