Abstract
BackgroundGenotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage. Here, we circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, particularly aridity.MethodsUsing a set of tag single-nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation, we identified those that overlapped restriction enzyme recognition sites and developed four polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) assays that distinguish alternative allelic states at the tag SNPs. We assessed the performance of these assays using mosquito population samples from Burkina Faso that had been cytogenetically karyotyped as well as genotyped, using two complementary high-throughput molecular methods based on tag SNPs. Further validation was performed using mosquito population samples from additional West African (Benin, Mali, Senegal) and Central African (Cameroon) countries.ResultsOf four assays tested, two were concordant with the 2Rc cytogenetic karyotype > 90% of the time in all samples. We recommend that these two assays be employed in tandem for reliable genotyping. By accepting only those genotypic assignments where both assays agree, > 99% of assignments are expected to be accurate.ConclusionsWe have developed tandem PCR-RFLP assays for the accurate genotyping of inversion 2Rc in An. coluzzii. Because this approach is simple, inexpensive, and requires only basic molecular biology equipment, it is widely accessible. These provide a crucial tool for probing the molecular basis of eco-phenotypes relevant to malaria epidemiology and vector control.Graphical
Highlights
Genotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes
The particular Burkina Faso population sample analyzed in this study (n = 463; Additional file 1: Table S1) had been previously genotyped for chromosomal inversions using each of three independent methodologies that collectively provide a robust basis for evaluating performance of individual polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays developed here: (i) classical cytogenetics based on phase contrast microscopy and two molecular approaches, (ii) amplicon sequencing (GT-Seq) and (iii) array hybridization (TaqMan Open Array), based on the detection of tag single nucleotide polymorphisms (SNPs) strongly correlated with inversion orientation [15]
Note that owing to distinct filtering requirements among molecular approaches, two of the four tag SNPs targeted by these assays differ from those employed in the respective sets of 23 and 11 tags developed for highthroughput amplicon sequencing and array hybridization genotyping of 2Rc [15], though the common principles underlying all 2Rc tags suggest that their performance should be similar
Summary
Genotyping of polymorphic chromosomal inversions in malaria vectors such as An. coluzzii Coetzee & Wilkerson is important, both because they cause cryptic population structure that can mislead vector analysis and control and because they influence epidemiologically relevant eco-phenotypes. The conventional cytogenetic method of genotyping is an impediment because it is labor intensive, requires specialized training, and can be applied only to one gender and developmental stage We circumvent these limitations by developing a simple and rapid molecular method of genotyping inversion 2Rc in An. coluzzii that is both economical and field-friendly. This inversion is strongly implicated in temporal and spatial adaptations to climatic and ecological variation, aridity. The central part of chromosome 2R that overlaps the 2Rc inversion in three taxa [An. gambiae (s.s.) (An. gambiae), An. coluzzii, and An. arabiensis] is disproportionately involved in both fixed and polymorphic inversions in the species complex, potentially implicating the 2Rc region in oviposition site specializations that distinguish taxa in this group [10]. In an extensive review of these findings, the authors conclude: “The evidence for consistent temporal and spatial adaptive changes in (2Rbc) inversion frequencies is unquestionable” [p.505, ref. 11]
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