High Throughput Multiplex Assay for Species Identification of Papua New Guinea Malaria Vectors Members of the Anopheles punctulatus (Diptera Culicidae) Species Group

Malaria and filariasis are transmitted in the Southwest Pacific region by Anopheles punctulatus sibling species including An punctulatus An koliensis, the An farautt complex 1-8 (includes An hinesorum [An farautt 2] An torresiensis [An farauti 3]) Distinguishing these species from each other requires molecular diagnostic methods We developed a multiplex polymerase chain reaction (PCR)-based assay specific for known species specific nucleotide differences in the internal transcribed spacer 2 region and identified the five species most frequently implicated in transmitting disease (An punctulatus An koliensis, An farautt 1, An hinesorum, and An farauti 4) A set of 340 individual mosquitoes obtained from seven Papua New Guinea provinces representing a variety of habitats were analyzed by using this multiplex assay Concordance between molecular and morphological diagnosis was 56 4% for An punctulatus, 85 3% for An koliensis, and 88 9% for An Pram Among 158 mosquitoes morphologically designated as An farauti, 33 were re-classified by PCR as An punctulatus, 4 as An koliensis, 26 as An farautt 1, 49 as An hinesorum and 46 as An farautt 4 Misclassification results from variable coloration of the proboscis and overlap of An punctulatus, An koliensis, the An farauti 4 This multiplex technology enables further mosquito strain identification and simultaneous detection of microbial pathogens