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Spontaneous mutation rate estimates for the principal malaria vectors Anopheles coluzzii and Anopheles stephensi.

ABSTRACT: Using high-depth whole genome sequencing of F0 mating pairs and multiple individual F1 offspring, we estimated the nuclear mutation rate per generation in the malaria vectors Anopheles coluzzii and Anopheles stephensi by detecting de novo genetic mutations. A purpose-built computer program was employed to filter actual mutations from a deep background of superficially similar artifacts resulting from read misalignment. Performance of filtering parameters was determined using software-simulated mutations, and the resulting estimate of false negative rate was used to correct final mutation rate estimates. Spontaneous mutation rates by base substitution were estimated at 1.00 × 10^-9 (95% confidence interval, 2.06 × 10^-10-2.91 × 10^-9) and 1.36 × 10^-9 (95% confidence interval, 4.42 × 10^-10-3.18 × 10^-9) per site per generation in A. coluzzii and A. stephensi respectively. Although similar studies have been performed on other insect species including dipterans, this is the first study to empirically measure mutation rates in the important genus Anopheles, and thus provides an estimate of µ that will be of utility for comparative evolutionary genomics, as well as for population genetic analysis of malaria vector mosquito species.

SUBMITTER: Rashid I

PROVIDER: S-EPMC8742016 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Identification of Plasmodium-resistance genes in malaria vectors remains an elusive goal despite the recent availability of high-quality genomes of several mosquito vectors. Anopheles stephensi, with its three distinctly-identifiable forms at the egg stage, correlating with varying vector competence, offers an ideal species to discover functional mosquito genes implicated in Plasmodium resistance. Recently, the genomes of several strains of An. stephensi of the type-form, known to display high vectorial capacity, were reported. Here, we report a chromosomal-level assembly of an intermediate-form of An. stephensi strain (IndInt), shown to have reduced vectorial capacity relative to a strain of type-form (IndCh). The contig level assembly with a L50 of 4 was scaffolded into chromosomes by using the genome of IndCh as the reference. The final assembly shows a heterozygous paracentric inversion, 3Li, involving 8 Mbp, which is syntenic to the extensively-studied 2La inversion implicated in Plasmodium resistance in An. gambiae involving 21 Mbp. Deep annotation of genes within the 3Li region in the IndInt assembly using the state-of-the-art protein-fold prediction and other annotation tools reveals the presence of a tumor necrosis factor-alpha (TNF-alpha) like gene, which is the homolog of the Eiger gene in Drosophila. Subsequent chromosome-wide searches revealed homologs of Wengen (Wgn) and Grindelwald (Grnd) genes, which are known to be the receptors for Eiger in Drosophila. We have identified all the genes in IndInt required for Eiger-mediated signaling by analogy to the TNF-alpha system, suggesting the presence of a functionally-active Eiger signaling pathway in IndInt. Comparative genomics of the three type-forms with that of IndInt, reveals structurally disruptive mutations in Eiger gene in all three strains of the type-form, suggesting compromised innate immunity in the type-form as the likely cause of high vectorial capacity in these strains. This is the first report of the presence of a homolog of Eiger in malaria vectors, known to be involved in cell death in Drosophila, within an inversion region in IndInt syntenic to an inversion associated with Plasmodium resistance in An. gambiae.

Project description:BackgroundChromosomal inversion polymorphisms have been associated with adaptive behavioral, physiological, morphological and life history traits in the two main Afrotropical malaria vectors, Anopheles coluzzii and Anopheles gambiae. The understanding of the adaptive value of chromosomal inversion systems is constrained by the feasibility of cytological karyotyping. In recent years in silico and molecular approaches have been developed for the genotyping of most widespread inversions (2La, 2Rb and 2Rc). The 2Ru inversion, spanning roughly 8% of chromosome 2R, is commonly polymorphic in West African populations of An. coluzzii and An. gambiae and shows clear increases in frequency with increasing rainfall seasonally and geographically. The aim of this work was to overcome the constraints of currently available cytological and high-throughput molecular assays by developing a simple PCR assay for genotyping the 2Ru inversion in individual specimens of both mosquito species.MethodsWe designed tetra-primer amplification refractory mutation system (ARMS)-PCR assays based on five tag single-nucleotide polymorphisms (SNPs) previously shown to be strongly correlated with 2Ru inversion orientation. The most promising assay was validated against laboratory and field samples of An. coluzzii and An. gambiae karyotyped either cytogenetically or molecularly using a genotyping-in-thousands by sequencing (GT-seq) high-throughput approach that employs targeted sequencing of multiplexed PCR amplicons.ResultsA successful assay was designed based on the tag SNP at position 2R, 31710303, which is highly predictive of the 2Ru genotype. The assay, which requires only one PCR, and no additional post-PCR processing other than electrophoresis, produced a clear banding pattern for 98.5% of the 454 specimens tested, which is a 96.7% agreement with established karyotyping methods. Sequences were obtained for nine of the An. coluzzii specimens manifesting 2Ru genotype discrepancies with GT-seq. Possible sources of these discordances are discussed.ConclusionsThe tetra-primer ARMS-PCR assay represents an accurate, streamlined and cost-effective method for the molecular karyotyping of the 2Ru inversion in An. coluzzii and An. gambiae. Together with approaches already available for the other common polymorphic inversions, 2La, 2Rb and 2Rc, this assay will allow investigations of the adaptive value of the complex set of inversion systems observed in the two major malaria vectors in the Afrotropical region.

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Spontaneous mutation rate estimates for the principal malaria vectors Anopheles coluzzii and Anopheles stephensi.

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