Multiplex Assay for Species Identification and Monitoring of Insecticide Resistance in Anopheles punctulatus Group Populations of Papua New Guinea

Cara N. Henry-Halldin 0 1 2 Kogulan Nadesakumaran 0 1 2 John Bosco Keven 0 1 2 Allison M. Zimmerman 0 1 2 Peter Siba 0 1 2 Ivo Mueller 0 1 2 Manuel W. Hetzel manuel.hetzel@pngimr 0 1 2 James W. Kazura 0 1 2 Edward Thomsen 0 1 2 Lisa J. Reimer 0 1 2 Peter A. Zimmerman 0 1 2 0 Health and Diseases, Case Western Reserve University School of Medicine , 10900 Euclid Avenue, Cleveland, OH 44106-7286 1 Center for Global Health and Diseases, Case Western Reserve University School of Medicine , Cleveland, Ohio ; Papua New Guinea Institute of Medical Research , Madang, 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, Australia 2 Authors' addresses: Cara N. Henry-Halldin, Kogulan Nadesakumaran, Allison M. Zimmerman, James W. Kazura, and Peter A. Zimmerman, Center for Global Health and Diseases, Case Western Reserve University School of Medicine , Cleveland, OH Anopheles punctulatus sibling species (An. punctulatus s.s., Anopheles koliensis, and Anopheles farauti species complex [eight cryptic species]) are principal vectors of malaria and filariasis in the Southwest Pacific. Given significant effort to reduce malaria and filariasis transmission through insecticide-treated net distribution in the region, effective strategies to monitor evolution of insecticide resistance among An. punctulatus sibling species is essential. Mutations in the voltage-gated sodium channel (VGSC) gene have been associated with knock-down resistance (kdr) to pyrethroids and DDT in malarious regions. By examining VGSC sequence polymorphism we developed a multiplex assay to differentiate wild-type versus kdr alleles and query intron-based polymorphisms that enable simultaneous species identification. A survey including mosquitoes from seven Papua New Guinea Provinces detected no kdr alleles in any An. punctulatus species. Absence of VGSC sequence introgression between species and evidence of geographic separation within species suggests that kdr must be monitored in each An. punctulatus species independently. - Malaria vectors in the Southwest Pacific region belong to the Anopheles punctulatus group. The species group range extends from Moluccas to Vanuatu, including New Guinea and islands of the Bismarck Archipelago, the Solomon Islands, and northern Australia.1 Thirteen sibling species (An. punctulatus sensu stricto [s.s.], An. species near punctulatus, morphologically indistinguishable Anopheles farauti 18 [Farauti complex; former An. farauti 1, 2, 3, and 7 now An. farauti s.s., Anopheles hinesorum, Anopheles torresiensis, and Anopheles irenicus, respectively], Anopheles koliensis, Anopheles clowi, and Anopheles renellensis)15 are known to comprise the species group. Blood feeding behaviors and breeding habitats of the Punctulatus group members have been characterized as unspecialized.6 Distribution of individual sibling species is heterogeneous, displaying unique and occasional overlapping patterns of dispersal.6 In Papua New Guinea (PNG), studies have specifically implicated An. farauti s.s., An. hinesorum, An. farauti 4, An. koliensis, and An. punctulatus s.s. as the primary vectors of malaria and filariasis.712 As evidence accumulates to verify the reproductive independence of these sibling species, it becomes increasingly important to collect evidence regarding their species-specific susceptibility to vector control strategies and competence as malaria vectors. The importance of malaria transmission by An. punctulatus group mosquitoes was first recognized by Heydon and others in the 1920s.13,14 Despite establishing this connection over 90 years ago, mosquito and therefore malaria control in New Guinea has been inconsistently supported and pursued. As malaria casualties during WWII consistently outnumbered those from combat, military activities in New Guinea prompted intense United States and Australian interest in malaria control from 1942 to 1945.1517 During this time troop protection included military discipline, efforts of malaria control and survey units beginning in 1943, atabrine prophylaxis and treatment of closed environments with bug bomb (pyrethrum).1517 Perhaps the most effective means for controlling mosquito populations, DDT (dichloro-diphenyltrichloroethane), was not available to troops in the Southwest Pacific area until late 1944, when military operations in New Guinea were drawing to a close. DDT has been of greatest relevance to mosquito population management in New Guinea because it changed World Health Organization (WHO) strategies from malaria control to eradication.18,19 During the 194050s DDT mixed in oilbased solvents and sprayed on larval breeding sites showed short-term effectiveness against malaria transmission.2022 However, DDT indoor residual spraying (IRS) was associated with reduced exposure to An. punctulatus group mosquitoes in New Guinea2326 and remained active for 6 months after application. These combined features suggested that DDT IRS would enable deployment of time-efficient attack phase strategies by limited numbers of disease control specialists and unskilled workers.19,25,27 These findings encouraged DDT IRS activities from the late 1950s to 1970s,2730 to cover ~50% of the population in 1972.29 Limited WHO insecticide susceptibility tests performed in the early 1970s showed high levels of DDT susceptibility among An. punctulatus group species in surveyed parts of PNG and the Solomon Islands.28,31 In 1972 the WHO Malaria Eradication Program in PNG ceased because of operational failure,32 however DDT IRS remained an important control strategy through provincial government activities.33,34 Further attempts at National Program IRS coverage in the late 1970s appeared to reduce anopheline mosquito and Plasmodium falciparum prevalence, but by 1983 insufficient resources and strained community relations brought this effort to a close.3537 In the 1980s, the PNG Institute of Medical Research and Ministry of Health began to assess the effect of low-cost bednets. Although the studies were conducted in two different endemic sites north of the PNG Central ranges, results showed that both untreated and permethrin-treated nets (Wosera38,39 and Madang,40 respectively) were associated with protection from malaria exposure and prevalence. Additionally, studies showed that untreated bednets were associated with reduced transmission of lymphatic filariasis on Bagabag Island north of Madang.41 Presently, the Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM) has provided support for the distribution of over 2.5 million long-lasting insecticide-treated nets (LLIN; deltamethrin) at no cost to individual families.42 Introduction of LLINs to all of PNGs 20 Provinces represents the first attempt at a comprehensive plan for mosquito-based malaria control. Of importance, our recently published study observed no phenotypic evidenc (...truncated)