Abstract
BackgroundThe Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. Rapid identification of the two main species which vector malaria, Anopheles arabiensis and An. gambiae sensu stricto, from the non-vector species Anopheles quadriannulatus is often required as part of vector control programmes. Currently the most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences. While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. Recently, a high-throughput assay based on TaqMan single nucleotide polymorphism genotyping that detects and discriminates An. gambiae s.s and An. arabiensis has been reported.MethodsA new TaqMan assay was developed that distinguishes between the main malaria vectors (An. arabiensis and An. gambiae s.s.) and the non-vector An. quadriannulatus after it was found that the existing TaqMan assay incorrectly identified An. quadriannulatus, An. merus and An. melas as An. gambiae s.s. The performance of this new TaqMan assay was compared against the existing TaqMan assay and the standard PCR method in a blind species identification trial of over 450 samples using field collected specimens from a total of 13 countries in Sub-Saharan Africa.ResultsThe standard PCR method was found to be specific with a low number of incorrect scores (<1%), however when compared to the TaqMan assays it showed a significantly higher number of failed reactions (15%). Both the new vector-specific TaqMan assay and the exisiting TaqMan showed a very low number of incorrectly identified samples (0 and 0.54%) and failed reactions (1.25% and 2.96%). In tests of analytical sensitivity the new TaqMan assay showed a very low detection threshold and can consequently be used on a single leg from a fresh or silica-dried mosquito without the need to first extract DNA.ConclusionThis study describes a rapid and sensitive assay that very effectively identifies the two main malaria vectors of the An. gambiae species complex from the non-vector sibling species. The method is based on TaqMan SNP genotyping and can be used to screen single legs from dried specimens. In regions where An. merus/melas/bwambae, vectors with restricted distributions, are not present it can be used alone to discriminate vector from non-vector or in combination with the Walker TaqMan assay to distinguish An. arabiensis and An. gambiae s.s.
Highlights
The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable
Of the remaining members Anopheles quadriannulatus species A, which is widespread in southern Africa, and Anopheles quadriannulatus species B, found in Ethiopia, are considered to be zoophilic non-malaria vectors [1,5]
Samples were field collected from Cameroon, Ghana, Kenya, South Africa, Malawi, Sao Tome, La Reunion, Tanzania, Sudan, Angola, Burkina Faso, Gabon, and Mozambique. These samples had been initially identified to species at the time of collection using the standard PCR method and included 169 An. gambiae s.s., 173 An. arabiensis, 66 An. quadriannulatus, and 21 samples of An. melas and An. merus, the remaining samples were either undetermined or negative controls
Summary
The Anopheles gambiae sensu lato species complex comprises seven sibling species of mosquitoes that are morphologically indistinguishable. The most widely used method for species identification is a multiplex PCR protocol that targets species specific differences in ribosomal DNA sequences While this assay has proved to be reasonably robust in many studies, additional steps are required post-PCR making it time consuming. The Anopheles gambiae sensu lato (s.l.) species complex contains the most important mosquito vectors of malaria in sub-Saharan Africa. It comprises seven morphologically indistinguishable sibling species up to four of which may be sympatric [1,2,3,4]. The final named member of the complex Anopheles bwambae is restricted to a region close to the Buranga hot springs in Uganda [8]
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