Sequence-based identification of Anopheles species in eastern Ethiopia

Background: The recent finding of a typically non-African Anopheles species in eastern Ethiopia emphasizes the need for detailed species identification and characterization for effective malaria vector surveillance. Molecular approaches increase the accuracy and interoperability of vector surveillance data. To develop effective molecular assays for Anopheles identification, it is important to evaluate different genetic loci for the ability to characterize species and population level variation. Here the utility of the internal transcribed spacer 2 (ITS2) and cytochrome oxidase I (COI) loci for detection of Anopheles species from understudied regions of eastern Ethiopia was investigated. Methods: Adult mosquitoes were collected from the Harewe locality (east) and Meki (east central) Ethiopia. PCR and Sanger sequencing were performed for portions of the ITS2 and COI loci. Both NCBI's Basic Local Alignment Search tool (BLAST) and phylogenetic analysis using a maximum-likelihood approach were performed to identify species of Anopheles specimens. Results: Two species from the east Ethiopian collection, Anopheles arabiensis and Anopheles pretoriensis were identified. Analyses of ITS2 locus resulted in delineation of both species. In contrast, analysis of COI locus could not be used to delineate An. arabiensis from other taxa in Anopheles gambiae complex, but could distinguish An. pretoriensis sequences from sister taxa. Conclusion: The lack of clarity from COI sequence analysis highlights potential challenges of species identification within species complexes. These results provide supporting data for the development of molecular assays for delineation of Anopheles in east Ethiopia.