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Background Anopheles squamosus 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 Plasmodium falciparum and Plasmodium vivax . 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 An. squamosus from morphologically similar Anopheles species . Described in the following methods is the development and validation of a new PCR assay that will reliably identify An. squamosus . Methods Multiple alignments of previously published ITS2 contig sequences in NCBI from An. squamosus and An. species 11 and 15, were used to identify candidate ITS2 regions for primer design. Six sets of primers were evaluated overall for specificity of species identification. The one set with An. squamosus species-specific amplification was tested using 78 specimens morphologically identified 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 (> 94.9%) amplified an ITS2 fragment of 301 bp length for An. squamosus . The An. squamosus- 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 An. squamosus 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. To further the validation of this ITS2 PCR assay, more species of Anopheles should be compared in addition to An. squamosus collected in different regions. To refine and optimize the PCR process with these primers, touchdown PCR can be used to increase specificity. Applying genomic tools to correctly identify An. squamosus will allow for a better understanding of their role in malaria transmission and may lead to genomic insights into what influences their behaviour, thus leading to new innovations in malaria elimination.

With ongoing global efforts to eliminate malaria, several countries have entered a pre-elimination stage, in which populations of the primary mosquito vector are reduced and the rates of malaria transmission are low. Efforts to eliminate malaria have changed the dynamics of disease transmission, causing a shift in anopheline community composition in some regions. One of the most abundant species of Anopheles found in pre-elimination areas, such as southern Africa, is Anopheles squamosus (Theobald, 1901; Diptera: Culicidae). Although An. squamosus has been documented across the African continent, limited information is available about its biology. Knowing what, when, and where the malaria vector bites humans can help develop effective malaria control strategies. The aim of this review is to compile the information available on An. squamosus’s biology, ecology, distribution, behavior, and potential role in disease transmission. The review of current literature suggested that An. squamosus is abundant throughout the African continent. Studies indicate that this species occupies the same range and exhibits similar behaviors to primary malaria vectors in southern and eastern African countries, like Zambia. In conclusion, An. squamosus continues to be an understudied species that has circumvented disease control measures and further studies are needed to develop effective control strategies.