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

Cara N. Henry-Halldin 0 1 Lisa Reimer 0 1 Edward Thomsen 0 1 Gussy Koimbu 0 1 Allison Zimmerman 0 1 John B. Keven 0 1 Henry Dagoro 0 1 Manuel W. Hetzel 0 1 Ivo Mueller 0 1 Peter Siba 0 1 Peter A. Zimmerman 0 1 0 Center for Global Health and Diseases, Case Western Reserve University School of Medicine , Cleveland, Ohio; Papua New Guinea Institute of Medical Research , Madang , Papua New Guinea; Papua New Guinea Institute of Medical Research , Goroka, Eastern Highlands , Papua New Guinea; School of Population Health, University of Queensland , Brisbane, Queensland , Australia 1 Authors' addresses: Cara Henry-Halldin, Allison M. Zimmerman, and Peter A. Zimmerman, Center for Global Health and Diseases, Case Western Reserve University School of Medicine , Wolstein Research Building, Cleveland, OH Malaria and filariasis are transmitted in the Southwest Pacific region by Anopheles punctulatus sibling species including An. punctulatus, An. koliensis, the An. farauti complex 1-8 (includes An. hinesorum [An. farauti 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. farauti 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. farauti. Among 158 mosquitoes morphologically designated as An. farauti, 33 were re-classified by PCR as An. punctulatus, 4 as An. koliensis, 26 as An. farauti 1, 49 as An. hinesorum, and 46 as An. farauti 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. - Mosquitoes belonging to the Anopheles punctulatus group are sibling species found in the Southwest Pacific region ranging from the Weber Line and Moluccas (former Spice Islands) to Vanuatu, including New Guinea and islands of the Bismarck Archipelago, the Solomon Islands and northern Australia.1 The group includes 13 sibling species (An. punctulatus, An. species near punctulatus, morphologically indistinguishable An. farauti 18 [Farauti complex; former An. farauti 2 and 3, now An. hinesorum and An. torresiensis, respectively], An. koliensis, An. clowi, and An. rennellensis).15 Studies by Bryan and Foley and others initially described species diversity within the Farauti complex by cross-mating,6,7 and allozyme polymorphisms.5 Beebe and others, and Cooper and others have extended molecular characterization of the Punctulatus group by DNA sequence analyses of ribosomal RNA loci.4,812 Additional diversity has been suggested among isolates of An. koliensis between collection sites in East Sepik and Madang Provinces in Papua New Guinea.13 Although members of the Punctulatus group have been characterized as unspecialized in regard to blood feeding behaviors and breeding habitats,14 individual species within the group are not distributed ubiquitously throughout the region. In previous surveys, An. farauti s.s and An. hinesorum have been characterized as most widely distributed, throughout Papua New Guinea, northern Australia, the Solomon Islands, and Vanuatu. Anopheles farauti s.s. is found most often within 12 km of the coast. In contrast An. hinesorum is most commonly found in areas between 10 and 100 km from the coast. Anopheles koliensis and An. punctulatus are also widely distributed throughout lowlands in Papua New Guinea and the Solomon Islands. Anopheles torresiensis has been found in northern Australia and southwestern Papua New Guinea where climate has been characterized as monsoonal. Anopheles farauti 4 is found primarily in northern Papua New Guinea throughout the Sepik and Ramu River plains. Less widely distributed species include An. farauti 5 and 6, found in the Papua New Guinea highlands; An. farauti 7, found in the Solomon Islands and Vanuatu; and An. near species punctulatus, found in inland habitats of southern and northern Papua New Guinea.4,1416 Anopheles farauti s.l. larvae are found most frequently in coastal streams and brackish pools or swamps. Anopheles punctulatus larvae are found in sunlit rainwater pools made in tire tracks or drainage pools. Anopheles koliensis larvae are found in temporary grassland or forest edge pools more than 2 km from the coast.17 In Papua New Guinea, studies have most often implicated An. farauti s.s., An. hinesorum, An. farauti 4, An. koliensis, and An. punctulatus as the primary vectors of malaria and filarial parasites.13,18,19 However, given the heterogeneous distribution of An. punctulatus species complex members across Papua New Guinea, in addition to what is known about the variable breeding and biting preferences among species3,17,20 it will be important to understand if all members of the Punctulatus group transmit parasites, or if there is further heterogeneity among species in their competence to transmit parasites. Assessment and continuous monitoring of the vector species composition in disease-endemic regions is a necessary component of vector biology and consistent with the integrated vector management strategy of the World Health Organization.21 Although DNA probe hybridization and polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) methods have been developed for performing species-level classification of Punctulatus group sibling species,1113 currently, morphology remains the most common method of species identification. Morphological traits used to distinguish species (proboscis coloration) are unreliable because individual mosquitoes within single species display polymorphism, such that similar proboscis coloration is observed among species.1,4,13,22 This finding emphasizes that molecular diagnostic strategies will be required tools to enhance the capacity of entomologists for assessing vector capacity, insecticide resistance, and distribution patterns of insect species complexes in many infectious disease scenarios. Significant technical advances can now be incorporated into new diagnostic strategies to enable more efficient surveillance of these important human disease vectors. We describe a high-throughput multiplex strategy for molecular identification of the Punctulatus group sibling species that serve as primary vectors of malaria and filarial parasites in Papua New Guinea based on species-specific sequence differences in the internal transcribed spacer 2 (ITS2) (...truncated)