Multiplex PCR Assay for the Identification of Four Species of the Anopheles Leucosphyrus Sub-Group in Malaysia

SIMPLE SUMMARY: Plasmodium parasites cause malaria. The bites of infected female Anopheles mosquitoes, known as “malaria vectors,” transmit the parasites to people. To prevent the spread of malaria, precise mosquito species identification is essential. This study aims to develop a quick and accurate method for identifying the Anopheles species (An. introlatus, An. latens, An. cracens, and An. balabacensis), which have been incriminated as vectors for simian malaria in Malaysia. Overall, six primers targeting the internal transcribed spacer 2 (ITS2) region of each species were designed for this assay. This study is helpful for the researchers or vector-related field workers to correctly identify the mosquitoes for control activities. ABSTRACT: The Leucosphyrus Group of mosquitoes are the major simian malaria vectors in Malaysia. Accurate species identification is required to help in curbing the spread of simian malaria. The aim of the study is to provide an accurate molecular method for identifying the four important Anopheles vector species found in Malaysia. Mosquito specimens were collected from various localities in Malaysia, where simian malaria cases were reported. DNA from 122 mosquito specimens was tested to develop a multiplex polymerase chain reaction (PCR) assay. The specificity of this assay was tested against other mosquito species. Molecular identification of the species was further confirmed by analysing the internal transcribed spacer 2 (ITS2) DNA region of the specimens. Anopheles balabacensis and An. latens showed two distinct clades in the phylogenetic tree. The multiplex PCR assay was developed based on the ITS2 region for the identification of Anopheles introlatus (298–299 bp), Anopheles latens (197–198 bp), Anopheles cracens (421–426 bp), and Anopheles balabacensis (224–228 bp). This method will be useful to accurately identify the major Anopheles Leucosphyrus Group species in Malaysia, which are difficult to identify morphologically, to determine the correct vector as well as its geographical distribution.