Strong Positive Selection in Aedes aegypti and the Rapid Evolution of Insecticide Resistance.

Aedes aegypti vectors the pathogens that cause dengue, yellow fever, Zika virus, and chikungunya and is a serious threat to public health in tropical regions. Decades of work has illuminated many aspects of Ae. aegypti's biology and global population structure and has identified insecticide resistance genes; however, the size and repetitive nature of the Ae. aegypti genome have limited our ability to detect positive selection in this mosquito. Combining new whole genome sequences from Colombia with publicly available data from Africa and the Americas, we identify multiple strong candidate selective sweeps in Ae. aegypti, many of which overlap genes linked to or implicated in insecticide resistance. We examine the voltage-gated sodium channel gene in three American cohorts and find evidence for successive selective sweeps in Colombia. The most recent sweep encompasses an intermediate-frequency haplotype containing four candidate insecticide resistance mutations that are in near-perfect linkage disequilibrium with one another in the Colombian sample. We hypothesize that this haplotype may continue to rapidly increase in frequency and perhaps spread geographically in the coming years. These results extend our knowledge of how insecticide resistance has evolved in this species and add to a growing body of evidence suggesting that Ae. aegypti has an extensive genomic capacity to rapidly adapt to insecticide-based vector control.

Evaluation of DISCOVAR de novo using a mosquito sample for cost-effective short-read genome assembly.

BACKGROUND: De novo reference assemblies that are affordable, practical to produce, and of sufficient quality for most downstream applications, remain an unattained goal for many taxa. Insects, which may yield too little DNA from individual specimens for long-read sequencing library construction and often have highly heterozygous genomes, can be particularly hard to assemble using inexpensive short-read sequencing data. The large number of insect species with medical or economic importance makes this a critical problem to address. RESULTS: Using the assembler DISCOVAR de novo, we assembled the genome of the African malaria mosquito Anopheles arabiensis using 250 bp reads from a single library. The resulting assembly had a contig N50 of 22,433 bp, and recovered the gene set nearly as well as the ALLPATHS-LG AaraD1 An. arabiensis assembly produced with reads from three sequencing libraries and much greater resources. DISCOVAR de novo appeared to perform better than ALLPATHS-LG in regions of low complexity. CONCLUSIONS: DISCOVAR de novo performed well assembling the genome of an insect of medical importance, using simpler sequencing input than previous anopheline assemblies. We have shown that this program is a viable tool for cost-effective assembly of a modestly-sized insect genome.

Chromosomal inversions and ecotypic differentiation in Anopheles gambiae: the perspective from whole-genome sequencing.

The molecular mechanisms and genetic architecture that facilitate adaptive radiation of lineages remain elusive. Polymorphic chromosomal inversions, due to their recombination-reducing effect, are proposed instruments of ecotypic differentiation. Here, we study an ecologically diversifying lineage of Anopheles gambiae, known as the Bamako chromosomal form based on its unique complement of three chromosomal inversions, to explore the impact of these inversions on ecotypic differentiation. We used pooled and individual genome sequencing of Bamako, typical (non-Bamako) An. gambiae and the sister species Anopheles coluzzii to investigate evolutionary relationships and genomewide patterns of nucleotide diversity and differentiation among lineages. Despite extensive shared polymorphism and limited differentiation from the other taxa, Bamako clusters apart from the other taxa, and forms a maximally supported clade in neighbour-joining trees based on whole-genome data (including inversions) or solely on collinear regions. Nevertheless, FST outlier analysis reveals that the majority of differentiated regions between Bamako and typical An. gambiae are located inside chromosomal inversions, consistent with their role in the ecological isolation of Bamako. Exceptionally differentiated genomic regions were enriched for genes implicated in nervous system development and signalling. Candidate genes associated with a selective sweep unique to Bamako contain substitutions not observed in sympatric samples of the other taxa, and several insecticide resistance gene alleles shared between Bamako and other taxa segregate at sharply different frequencies in these samples. Bamako represents a useful window into the initial stages of ecological and genomic differentiation from sympatric populations in this important group of malaria vectors.

A novel tetra-primer ARMS-PCR approach for the molecular karyotyping of chromosomal inversion 2Ru in the main malaria vectors Anopheles gambiae and Anopheles coluzzii.

BACKGROUND: Chromosomal 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. METHODS: We 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. RESULTS: A 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. CONCLUSIONS: The 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.

High-Throughput Genotyping of Common Chromosomal Inversions in the Afrotropical Malaria Mosquito Anopheles Funestus.

Polymorphic chromosomal inversions have been implicated in local adaptation. In anopheline mosquitoes, inversions also contribute to epidemiologically relevant phenotypes such as resting behavior. Progress in understanding these phenotypes and their mechanistic basis has been hindered because the only available method for inversion genotyping relies on traditional cytogenetic karyotyping, a rate-limiting and technically difficult approach that is possible only for the fraction of the adult female population at the correct gonotrophic stage. Here, we focus on an understudied malaria vector of major importance in sub-Saharan Africa, Anopheles funestus. We ascertain and validate tag single nucleotide polymorphisms (SNPs) using high throughput molecular assays that allow rapid inversion genotyping of the three most common An. funestus inversions at scale, overcoming the cytogenetic karyotyping barrier. These same inversions are the only available markers for distinguishing two An. funestus ecotypes that differ in indoor resting behavior, Folonzo and Kiribina. Our new inversion genotyping tools will facilitate studies of ecotypic differentiation in An. funestus and provide a means to improve our understanding of the roles of Folonzo and Kiribina in malaria transmission.

Inversion Genotyping in the Anopheles gambiae Complex Using High-Throughput Array and Sequencing Platforms.

Chromosomal inversion polymorphisms have special importance in the Anopheles gambiae complex of malaria vector mosquitoes, due to their role in local adaptation and range expansion. The study of inversions in natural populations is reliant on polytene chromosome analysis by expert cytogeneticists, a process that is limited by the rarity of trained specialists, low throughput, and restrictive sampling requirements. To overcome this barrier, we ascertained tag single nucleotide polymorphisms (SNPs) that are highly correlated with inversion status (inverted or standard orientation). We compared the performance of the tag SNPs using two alternative high throughput molecular genotyping approaches vs. traditional cytogenetic karyotyping of the same 960 individual An. gambiae and An. coluzzii mosquitoes sampled from Burkina Faso, West Africa. We show that both molecular approaches yield comparable results, and that either one performs as well or better than cytogenetics in terms of genotyping accuracy. Given the ability of molecular genotyping approaches to be conducted at scale and at relatively low cost without restriction on mosquito sex or developmental stage, molecular genotyping via tag SNPs has the potential to revitalize research into the role of chromosomal inversions in the behavior and ongoing adaptation of An. gambiae and An. coluzzii to environmental heterogeneities.

In Silico Karyotyping of Chromosomally Polymorphic Malaria Mosquitoes in the Anopheles gambiae Complex.

Chromosomal inversion polymorphisms play an important role in adaptation to environmental heterogeneities. For mosquito species in the Anopheles gambiae complex that are significant vectors of human malaria, paracentric inversion polymorphisms are abundant and are associated with ecologically and epidemiologically important phenotypes. Improved understanding of these traits relies on determining mosquito karyotype, which currently depends upon laborious cytogenetic methods whose application is limited both by the requirement for specialized expertise and for properly preserved adult females at specific gonotrophic stages. To overcome this limitation, we developed sets of tag single nucleotide polymorphisms (SNPs) inside inversions whose biallelic genotype is strongly correlated with inversion genotype. We leveraged 1,347 fully sequenced An. gambiae and Anopheles coluzzii genomes in the Ag1000G database of natural variation. Beginning with principal components analysis (PCA) of population samples, applied to windows of the genome containing individual chromosomal rearrangements, we classified samples into three inversion genotypes, distinguishing homozygous inverted and homozygous uninverted groups by inclusion of the small subset of specimens in Ag1000G that are associated with cytogenetic metadata. We then assessed the correlation between candidate tag SNP genotypes and PCA-based inversion genotypes in our training sets, selecting those candidates with >80% agreement. Our initial tests both in held-back validation samples from Ag1000G and in data independent of Ag1000G suggest that when used for in silico inversion genotyping of sequenced mosquitoes, these tags perform better than traditional cytogenetics, even for specimens where only a small subset of the tag SNPs can be successfully ascertained.

Mosquito genomics. Extensive introgression in a malaria vector species complex revealed by phylogenomics.

Introgressive hybridization is now recognized as a widespread phenomenon, but its role in evolution remains contested. Here, we use newly available reference genome assemblies to investigate phylogenetic relationships and introgression in a medically important group of Afrotropical mosquito sibling species. We have identified the correct species branching order to resolve a contentious phylogeny and show that lineages leading to the principal vectors of human malaria were among the first to split. Pervasive autosomal introgression between these malaria vectors means that only a small fraction of the genome, mainly on the X chromosome, has not crossed species boundaries. Our results suggest that traits enhancing vectorial capacity may be gained through interspecific gene flow, including between nonsister species.