Abstract
BackgroundChromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species’ spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping.MethodsWe designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony.ResultsFor three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed.ConclusionsThe availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology.
Highlights
Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments
The three main malaria vector species belonging to the Afrotropical Anopheles gambiae complex, An. coluzzii, An. gambiae and An. arabiensis, are characterized by extensive paracentric inversion polymorphisms mostly involving the right arm of chromosome 2 [1, 2]
Inversion polymorphisms are implicated in the efficient exploitation of anthropogenic environmental modifications and ecological disturbances such as irrigation and deforestation [1, 2, 9,10,11,12,13], helping to promote a strong association between these mosquitoes and humans
Summary
Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the efficient exploitation of anthropogenic environmental modifications and ecological disturbances such as irrigation and deforestation [1, 2, 9,10,11,12,13], helping to promote a strong association between these mosquitoes and humans This has extended their spatial and temporal distribution and helped transform these species into the most efficient malaria vectors worldwide. The adaptive value of inversion polymorphisms is evident in the case of the 2La arrangement in An. coluzzii and An. gambiae, whose temporal and spatial distribution is strongly correlated with degree of aridity [2, 14,15,16] This strong correlation was first shown 40 years ago based on the demanding cytological karyotyping of thousands of polytene chromosome slides obtained from ovarian nurse cells of females at the half-gravid stage after blood meal - the only adult stage with sufficient chromosome polytenization to allow examination of the banding pattern [17]. Future applications of this assay and others under development for additional inversions will foster a deeper understanding of already recognized or suspected phenotypic associations, and boost the discovery and dissection of unsuspected physiological and behavioural traits of epidemiological and ecological relevance determined by inversions
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