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

Abstract

Abstract. 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. 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 PNG's 20 Provinces represents the first attempt at a comprehensive plan for mosquito-based malaria control. Of importance, our recently published study observed no phenotypic evidence of resistance to the synthetic pyrethroid insecticides among Anopheles mosquitoes collected at five different PNG malaria-endemic sites. 43 In response to intensive insecticide exposure, malaria vectors in other regions of the world have developed resistance to a number of different classes of insecticide compounds. Because several insect species have shown evidence for development of specific polymorphisms in the voltage-gated sodium channel ( VGSC ) after prolonged exposure to DDT and pyrethroid insecticides, 44, 45 a number of studies have focused on sequence encoding the S4-S6 region of VGSC domain II. 46,47 In Anopheles mosquitoes, knockdown resistance ( kdr ) has been associated with an A → T mutation (L1014F) 46 or a T → C mutation (L1014S) 48 in VGSC . These single nucleotide polymorphisms (SNPs) have been found in African, Indian, and Asian malaria vectors, 49-54 and most recently in neighboring Indonesia, 55 showing the capacity of these mutations to develop independently in different Anopheles species . Another point mutation in VGSC associated with very high levels of DDT resistance, super-kdr (M918T), has been described in many insects, however this point mutation has not yet been detected in Anopheles . 44,45,56 To date, Southwest Pacific malaria vectors have not been surveyed for any mutations associated with insecticide resistance. Assuming validity of An. punctulatus sibling species definitions, and the hetero...

NovelKdrmutations (K964R and A943V) in pyrethroid‐resistant populations ofTriatoma mazzottiiandTriatoma longipennisfrom Mexico and detoxifying enzymes

“…() observed that T . infestans‐ resistant populations from Gran Chaco (ecoregion covering part of Argentina, Bolivia and Paraguay) have the kdr mutation at similar frequencies but have very different resistance ratios (Henry‐Halldin et al ., ). These data suggest that other mechanisms could be involved in triatomine insecticide resistance.…”

Mitochondrial genome sequences reveal deep divergences among Anopheles punctulatus sibling species in Papua New Guinea

“…For example, control of malaria by releasing genetically modified-, sterile-, male mosquitoes as was recently proposed [59] would require, in PNG, independent engineering of mosquitoes from, at least, five highly divergent species to significantly impact the populations of the main malaria vectors. In addition, if AP sibling species are reproductively isolated from each other, insecticide resistance arising in one species is unlikely to spread quickly across all AP mosquitoes, but instead resistance mutations would have to occur independently in each species [29]. Further investigations are required to definitively rule out the existence of gene flow among most AP sibling species, as well as to confirm the observation of putative mt genome introgression between An .…”

“…farauti 4 - have been described as competent vectors of malaria [23,24]. Phylogenetic studies of this group have focused on DNA sequences of ribosomal RNAs [25,26], mitochondrial genes [27,28], ribosomal ITS2 [19], and voltage-gated sodium channel gene [29]. However, the genetic information generated in these studies has not allowed robust determination of the AP group phylogeny and has often yielded conflicting results [19,27].…”

Whole‐genome sequencing reveals absence of recent gene flow and separate demographic histories for Anopheles punctulatus mosquitoes in Papua New Guinea

“…punctulatus mosquitoes have shown limited fine‐scale population stratification in mainland PNG (Henry‐Halldin et al . ; Logue et al . ; Seah et al .…”

“…Wild‐caught mosquitoes were collected in the Madang province by the Entomology unit of the Papua New Guinea Institute of Medical Research (PNGIMR) as previously described (Henry‐Halldin et al . , ). We extracted genomic DNA from individual mosquitoes with DNeasy blood and tissue kits (Qiagen ® ) according to the supplemental protocol for purification of insect DNA with one modification: each mosquito was initially placed in a 2 mL tube with one 5‐mm steel bead and 180 μL of PBS and homogenized by high‐speed shaking at 15 Hz for 90 s with a Qiagen TissueLyser II instrument.…”