PhD Student, Case Western Reserve University
Assay that detects malaria, mosquito sp., and mosquito host from a smashed mosquito.
This post-PCR assay grants epidemiologists and medical entomologists the capacity to quickly power through large numbers of mosquito samples to detect malaria parasites (responsible for hundreds of thousands of deaths per year), the species of mosquito (which are hard to identify), and what the mosquito fed on (be it cow, human, dog, or some combination thereof). Mosquito sampling often involves working with hundreds to thousands of samples, making this assay a powerful tool for moving through these large volumes at a fast pace with a significant degree of cost efficiency.
Abstract: 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.
Pub.: 08 Jan '11, Pinned: 28 Jun '17
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.
Pub.: 11 Jan '12, Pinned: 28 Jun '17
Abstract: Understanding mosquito host choice is important for assessing vector competence or identifying disease reservoirs. Unfortunately, the availability of an unbiased method for comprehensively evaluating the composition of insect blood meals is very limited, as most current molecular assays only test for the presence of a few pre-selected species. These approaches also have limited ability to identify the presence of multiple mammalian hosts in a single blood meal. Here, we describe a novel high-throughput sequencing method that enables analysis of 96 mosquitoes simultaneously and provides a comprehensive and quantitative perspective on the composition of each blood meal. We validated in silico that universal primers targeting the mammalian mitochondrial 16S ribosomal RNA genes (16S rRNA) should amplify more than 95% of the mammalian 16S rRNA sequences present in the NCBI nucleotide database. We applied this method to 442 female Anopheles punctulatus s. l. mosquitoes collected in Papua New Guinea (PNG). While human (52.9%), dog (15.8%) and pig (29.2%) were the most common hosts identified in our study, we also detected DNA from mice, one marsupial species and two bat species. Our analyses also revealed that 16.3% of the mosquitoes fed on more than one host. Analysis of the human mitochondrial hypervariable region I in 102 human blood meals showed that 5 (4.9%) of the mosquitoes unambiguously fed on more than one person. Overall, analysis of PNG mosquitoes illustrates the potential of this approach to identify unsuspected hosts and characterize mixed blood meals, and shows how this approach can be adapted to evaluate inter-individual variations among human blood meals. Furthermore, this approach can be applied to any disease-transmitting arthropod and can be easily customized to investigate non-mammalian host sources.
Pub.: 11 Mar '16, Pinned: 28 Jun '17