Molecular identification of (Diptera: Culicidae) using internal transcribed spacer 2.

Background: is a widespread mosquito species in sub-Saharan Africa. It is a potential vector for human malaria parasites and has been found naturally infected with and . Morphological identification is challenging even with pristine specimens and current molecular methods such as the use of the internal transcribed spacer 2 (ITS2) polymerase chain reaction (PCR) cannot distinguish from morphologically similar .

Methods: Multiple alignments of previously published ITS2 contig sequences in NCBI from and . species 11 and 15, were used to identify candidate ITS2 regions for primer design. We evaluated six sets of primers overall for specificity of species identification. The one set with species-specific amplification was tested using 78 specimens from Zambia and South Africa.

Results: A new assay consisting of a forward (ITS2-ASQ-R10, 5'-CCC TCG AAG GGT GCT GTG-3') and reverse (ITS2-ASQ-R10 5'-AAT CCA CGG TGT GAT GGC-3') primer reliably (> 94.8%) amplified an ITS2 fragment of 301bp length for . The -specific primer set can be multiplexed with existing ITS2 assays frequently used for anopheline species identification.

Conclusions: The development of this robust PCR assay for is vital to accurate identification of this species in malaria vector surveillance efforts. Improved understanding of the anopheline community composition will lead to better targeted methods of vector eradication and malaria prevention. In addition, investigating host association and malaria transmission can be facilitated with this assay by correctly identifying . Applying genomic tools to correctly identified anopheline species may lead to the discovery of genetic factors that influence its behavior and new innovations in malaria elimination.