Sequence-based identification of Anopheles species in eastern Ethiopia

Abstract

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.

Keywords: Anopheles arabiensis; Cytochrome oxidase subunit I; Horn of Africa; Internal transcribed spacer 2; Malaria; Phylogenetics.

Fig. 1

Maximum-likelihood tree of Anopheles ITS2 sequences. Analysis based on a 465 base pair sequence of the locus. The taxon in red is a representative specimen collected in Ethiopia from the present study (all Ethiopian ITS2 sequences were identical). Tree includes An. gambiae complex sequences taken from NCBI’s Genbank. Bootstrap values 70 and higher are shown. Outgroup (Anopheles christyi) not shown. Final ML Optimization Likelihood: − 1110.705351

Fig. 2

Maximum-likelihood tree of Anopheles CO1 sequences. Analysis is based on a 611 base pair sequence of the gene. Taxa in bold are the Anopheles arabiensis specimens collected in Ethiopia (species confirmed with ITS2 sequences) from the present study. Taxa in blue were collected in the Harewe locality and red in Meki. Tree includes An. gambiae complex and non-An. gambiae complex sequences taken from NCBI’s Genbank. Bootstrap values 70 and higher are shown. Final ML Optimization Likelihood: − 2668.816013