Anopheles Gambiae Species Complex Research Articles

Variation in spatial population structure in the Anopheles gambiae species complex.

Anopheles gambiae, Anopheles coluzzii, and Anopheles arabiensis are three of the most widespread vectors of malaria parasites, with geographical ranges stretching across wide swaths of Africa. Understanding the population structure of these closely related species, including the extent to which populations are connected by gene flow, is essential for understanding how vector control implemented in one location might indirectly affect vector populations in other locations. Here, we assessed the population structure of each species based on a combined data set of publicly available and newly processed whole-genome sequences. The data set included single nucleotide polymorphisms from whole genomes of 2,410 individual mosquitoes sampled from 128 locations across 19 African countries. We found that A. gambiae sampled from several countries in West and Central Africa showed low genetic differentiation from each other according to principal components analysis (PCA) and ADMIXTURE modeling. Using Estimated Effective Migration Surfaces (EEMS), we showed that this low genetic differentiation indicates high effective migration rates for A. gambiae across this region. Outside of this region, we found eight groups of sampling locations from Central, East, and Southern Africa for which A. gambiae showed higher genetic differentiation, and lower effective migration rates, between each other and the West/Central Africa group. These results indicate that the barriers to and corridors for migration between populations of A. gambiae differ across the geographical range of this malaria vector species. Using the same methods, we found higher genetic differentiation and lower migration rates between populations of A. coluzzii in West and Central Africa than for A. gambiae in the same region. In contrast, we found lower genetic differentiation and higher migration rates between populations of A. arabiensis in Tanzania, compared to A. gambiae in the same region. These differences between A. gambiae, A. coluzzii, and A. arabiensis indicate that migration barriers and corridors may vary, even between very closely related species. Overall, our results demonstrate that migration rates vary both within and between species of Anopheles mosquitoes, presumably based on species-specific responses to the ecological or environmental conditions that may impede or facilitate migration, and the geographical patterns of these conditions across the landscape. Together with previous findings, this study provides robust evidence that migration rates between populations of malaria vectors depend on the ecological context, which should be considered when planning surveillance of vector populations, monitoring for insecticide resistance, and evaluating interventions.

Wolbachia endosymbionts in two Anopheles species indicates independent acquisitions and lack of prophage elements

Wolbachia is a genus of obligate bacterial endosymbionts that infect a diverse range of arthropod species as well as filarial nematodes, with its single described species, Wolbachia pipientis , divided into several ‘supergroups’ based on multilocus sequence typing. Wolbachia strains in mosquitoes have been shown to inhibit the transmission of human pathogens, including Plasmodium malaria parasites and arboviruses. Despite their large host range, Wolbachia strains within the major malaria vectors of the Anopheles gambiae and Anopheles funestus complexes appear at low density, established solely on PCR-based methods. Questions have been raised as to whether this represents a true endosymbiotic relationship. However, recent definitive evidence for two distinct, high-density strains of supergroup B Wolbachia within Anopheles demeilloni and Anopheles moucheti has opened exciting possibilities to explore naturally occurring Wolbachia endosymbionts in Anopheles for biocontrol strategies to block Plasmodium transmission. Here, we utilize genomic analyses to demonstrate that both Wolbachia strains have retained all key metabolic and transport pathways despite their smaller genome size, with this reduction potentially attributable to degenerated prophage regions. Even with this reduction, we confirmed the presence of cytoplasmic incompatibility (CI) factor genes within both strains, with wAnD maintaining intact copies of these genes while the cifB gene was interrupted in wAnM, so functional analysis is required to determine whether wAnM can induce CI. Additionally, phylogenetic analysis indicates that these Wolbachia strains may have been introduced into these two Anopheles species via horizontal transmission events, rather than by ancestral acquisition and subsequent loss events in the Anopheles gambiae species complex. These are the first Wolbachia genomes, to our knowledge, that enable us to study the relationship between natural strain Plasmodium malaria parasites and their anopheline hosts.

Horizontal Transmission of the Symbiont Microsporidia MB in Anopheles arabiensis.

The recently discovered Anopheles symbiont, Microsporidia MB, has a strong malaria transmission-blocking phenotype in Anopheles arabiensis, the predominant Anopheles gambiae species complex member in many active transmission areas in eastern Africa. The ability of Microsporidia MB to block Plasmodium transmission together with vertical transmission and avirulence makes it a candidate for the development of a symbiont-based malaria transmission blocking strategy. We investigate the characteristics and efficiencies of Microsporidia MB transmission between An. arabiensis mosquitoes. We show that Microsporidia MB is not transmitted between larvae but is effectively transmitted horizontally between adult mosquitoes. Notably, Microsporidia MB was only found to be transmitted between male and female An. arabiensis, suggesting sexual horizontal transmission. In addition, Microsporidia MB cells were observed infecting the An. arabiensis ejaculatory duct. Female An. arabiensis that acquire Microsporidia MB horizontally are able to transmit the symbiont vertically to their offspring. We also investigate the possibility that Microsporidia MB can infect alternate hosts that live in the same habitats as their An. arabiensis hosts, but find no other non-anopheline hosts. Notably, Microsporidia MB infections were found in another primary malaria African vector, Anopheles funestus s.s. The finding that Microsporidia MB can be transmitted horizontally is relevant for the development of dissemination strategies to control malaria that are based on the targeted release of Microsporidia MB infected Anopheles mosquitoes.

The Genetic Architecture of Post-Zygotic Reproductive Isolation Between Anopheles coluzzii and An. quadriannulatus.

The Anopheles gambiae complex is comprised of eight morphologically indistinguishable species and has emerged as a model system for the study of speciation genetics due to the rapid radiation of its member species over the past two million years. Male hybrids between most An. gambiae complex species pairs are sterile, and some genotype combinations in hybrid males cause inviability. We investigated the genetic basis of hybrid male inviability and sterility between An. coluzzii and An. quadriannulatus by measuring segregation distortion and performing a QTL analysis of sterility in a backcross population. Hybrid males were inviable if they inherited the An. coluzzii X chromosome and were homozygous at one or more loci in 18.9 Mb region of chromosome 3. The An. coluzzii X chromosome has a disproportionately large effect on hybrid sterility when introgressed into an An. quadriannulatus genetic background. Additionally, an epistatic interaction between the An. coluzzii X and a 1.12 Mb, pericentric region of the An. quadriannulatus 3L chromosome arm has a statistically significant contribution to the hybrid sterility phenotype. This same epistatic interaction occurs when the An. coluzzii X is introgressed into the genetic background of An. arabiensis, the sister species of An. quadriannulatus, suggesting that this may represent one of the first Dobzhansky–Muller incompatibilities to evolve early in the radiation of the Anopheles gambiae species complex. We describe the additive effects of each sterility QTL, epistatic interactions between them, and genes within QTL with protein functions related to mating behavior, reproduction, spermatogenesis, and microtubule morphogenesis, whose divergence may contribute to post-zygotic reproductive isolation between An. coluzzii and An. quadriannulatus.

The Impact of Cross-Species Gene Flow on Species Tree Estimation.

Recent analyses of genomic sequence data suggest cross-species gene flow is common in both plants and animals, posing challenges to species tree estimation. We examine the levels of gene flow needed to mislead species tree estimation with three species and either episodic introgressive hybridization or continuous migration between an outgroup and one ingroup species. Several species tree estimation methods are examined, including the majority-vote method based on the most common gene tree topology (with either the true or reconstructed gene trees used), the UPGMA method based on the average sequence distances (or average coalescent times) between species, and the full-likelihood method based on multilocus sequence data. Our results suggest that the majority-vote method based on gene tree topologies is more robust to gene flow than the UPGMA method based on coalescent times and both are more robust than likelihood assuming a multispecies coalescent (MSC) model with no cross-species gene flow. Comparison of the continuous migration model with the episodic introgression model suggests that a small amount of gene flow per generation can cause drastic changes to the genetic history of the species and mislead species tree methods, especially if the species diverged through radiative speciation events. Estimates of parameters under the MSC with gene flow suggest that African mosquito species in the Anopheles gambiae species complex constitute such an example of extreme impact of gene flow on species phylogeny. [IM; introgression; migration; MSci; multispecies coalescent; species tree.].

Gene copy number and function of the APL1 immune factor changed during Anopheles evolution

BackgroundThe recent reference genome assembly and annotation of the Asian malaria vector Anopheles stephensi detected only one gene encoding the leucine-rich repeat immune factor APL1, while in the Anopheles gambiae and sibling Anopheles coluzzii, APL1 factors are encoded by a family of three paralogs. The phylogeny and biological function of the unique APL1 gene in An. stephensi have not yet been specifically examined.MethodsThe APL1 locus was manually annotated to confirm the computationally predicted single APL1 gene in An. stephensi. APL1 evolution within Anopheles was explored by phylogenomic analysis. The single or paralogous APL1 genes were silenced in An. stephensi and An. coluzzii, respectively, followed by mosquito survival analysis, experimental infection with Plasmodium and expression analysis.ResultsAPL1 is present as a single ancestral gene in most Anopheles including An. stephensi but has expanded to three paralogs in an African lineage that includes only the Anopheles gambiae species complex and Anopheles christyi. Silencing of the unique APL1 copy in An. stephensi results in significant mosquito mortality. Elevated mortality of APL1-depleted An. stephensi is rescued by antibiotic treatment, suggesting that pathology due to bacteria is the cause of mortality, and indicating that the unique APL1 gene is essential for host survival. Successful Plasmodium development in An. stephensi depends upon APL1 activity for protection from high host mortality due to bacteria. In contrast, silencing of all three APL1 paralogs in An. coluzzii does not result in elevated mortality, either with or without Plasmodium infection. Expression of the single An. stephensi APL1 gene is regulated by both the Imd and Toll immune pathways, while the two signaling pathways regulate different APL1 paralogs in the expanded APL1 locus.ConclusionsAPL1 underwent loss and gain of functions concomitant with expansion from a single ancestral gene to three paralogs in one lineage of African Anopheles. We infer that activity of the unique APL1 gene promotes longevity in An. stephensi by conferring protection from or tolerance to an effect of bacterial pathology. The evolution of an expanded APL1 gene family could be a factor contributing to the exceptional levels of malaria transmission mediated by human-feeding members of the An. gambiae species complex in Africa.

Fine-Mapping Complex Inversion Breakpoints and Investigating Somatic Pairing in the Anopheles gambiae Species Complex Using Proximity-Ligation Sequencing.

Chromosomal inversions are fundamental drivers of genome evolution. In the main Afrotropical malaria vector species, belonging to the Anopheles gambiae species complex, inversions play an important role in local adaptation and have a rich history of cytological study. Despite the importance and ubiquity of some chromosomal inversions across the species complex, inversion breakpoints are often challenging to map molecularly due to the presence of large repetitive regions. Here, we develop an approach that uses Hi-C sequencing data to molecularly fine-map the breakpoints of inversions. We demonstrate that this approach is robust and likely to be widely applicable for both identification and fine-mapping inversion breakpoints in species whose inversions have heretofore been challenging to characterize. We apply our method to interrogate the previously unknown inversion breakpoints of 2Rbc and 2Rd in An. coluzzii We found that inversion breakpoints occur in large repetitive regions, and, strikingly, among three inversions analyzed, two breakpoints appear to be reused in two separate inversions. These breakpoint-adjacent regions are strongly enriched for the presence of a 30 bp satellite repeat sequence. Because low frequency inversion breakpoints are not correlated with genomic regions containing this satellite, we suggest that interrupting this particular repeat may result in arrangements with higher relative fitness. Additionally, we use heterozygous individuals to quantitatively investigate the impacts of somatic pairing in the regions immediately surrounding inversion breakpoints. Finally, we discuss important considerations for possible applications of this approach for inversion breakpoint identification in a range of organisms.

Analysis-ready datasets for insecticide resistance phenotype and genotype frequency in African malaria vectors

The impact of insecticide resistance in malaria vectors is poorly understood and quantified. Here a series of geospatial datasets for insecticide resistance in malaria vectors are provided, so that trends in resistance in time and space can be quantified, and the impact of resistance found in wild populations on malaria transmission in Africa can be assessed. Specifically, data have been collated and geopositioned for the prevalence of insecticide resistance, as measured by standard bioassays, in representative samples of individual species or species complexes. Data are provided for the Anopheles gambiae species complex, the Anopheles funestus subgroup, and for nine individual vector species. Data are also given for common genetic markers of resistance to support analyses of whether these markers can improve the ability to monitor resistance in low resource settings. Allele frequencies for known resistance-associated markers in the Voltage-gated sodium channel (Vgsc) are provided. In total, eight analysis-ready, standardised, geopositioned datasets encompassing over 20,000 African mosquito collections between 1957 and 2017 are released.

Coalescent Analysis of Phylogenomic Data Confidently Resolves the Species Relationships in the Anopheles gambiae Species Complex.

Deep coalescence and introgression make it challenging to infer phylogenetic relationships among closely related species that arose through radiative speciation events. Despite numerous phylogenetic analyses and the availability of whole genomes, the phylogeny in the Anopheles gambiae species complex has not been confidently resolved. Here we extract over 80, 000 coding and noncoding short segments (called loci) from the genomes of six members of the species complex and use a Bayesian method under the multispecies coalescent model to infer the species tree, which takes into account genealogical heterogeneity across the genome and uncertainty in the gene trees. We obtained a robust estimate of the species tree from the distal region of the X chromosome: (A. merus, ((A. melas, (A. arabiensis, A. quadriannulatus)), (A. gambiae, A. coluzzii))), with A. merus to be the earliest branching species. This species tree agrees with the chromosome inversion phylogeny and provides a parsimonious interpretation of inversion and introgression events. Simulation informed by the real data suggest that the coalescent approach is reliable while the sliding-window analysis used in a previous phylogenomic study generates artifactual species trees. Likelihood ratio test of gene flow revealed strong evidence of autosomal introgression from A. arabiensis into A. gambiae (at the average rate of migrants per generation), but not in the opposite direction, and introgression of the 3 L chromosomal region from A. merus into A. quadriannulatus. Our results highlight the importance of accommodating incomplete lineage sorting and introgression in phylogenomic analyses of species that arose through recent radiative speciation events.

The Effect of Hybridization on Dosage Compensation in Member Species of the Anopheles gambiae Species Complex.

Dosage compensation has evolved in concert with Y-chromosome degeneration in many taxa that exhibit heterogametic sex chromosomes. Dosage compensation overcomes the biological challenge of a “half dose” of X chromosome gene transcripts in the heterogametic sex. The need to equalize gene expression of a hemizygous X with that of autosomes arises from the fact that the X chromosomes retain hundreds of functional genes that are actively transcribed in both sexes and interact with genes expressed on the autosomes. Sex determination and heterogametic sex chromosomes have evolved multiple times in Diptera, and in each case the genetic control of dosage compensation is tightly linked to sex determination. In the Anopheles gambiae species complex (Culicidae), maleness is conferred by the Y-chromosome gene Yob, which despite its conserved role between species is polymorphic in its copy number between them. Previous work demonstrated that male An. gambiae s.s. males exhibit complete dosage compensation in pupal and adult stages. In the present study, we have extended this analysis to three sister species in the An. gambiae complex: An. coluzzii, An. arabiensis, and An. quadriannulatus. In addition, we analyzed dosage compensation in bi-directional F1 hybrids between these species to determine if hybridization results in the mis-regulation and disruption of dosage compensation. Our results confirm that dosage compensation operates in the An. gambiae species complex through the hypertranscription of the male X chromosome. Additionally, dosage compensation in hybrid males does not differ from parental males, indicating that hybridization does not result in the mis-regulation of dosage compensation.

The Anopheles gambiae 2La chromosome inversion is associated with susceptibility to Plasmodium falciparum in Africa.

Chromosome inversions suppress genetic recombination and establish co-adapted gene complexes, or supergenes. The 2La inversion is a widespread polymorphism in the Anopheles gambiae species complex, the major African mosquito vectors of human malaria. Here we show that alleles of the 2La inversion are associated with natural malaria infection levels in wild-captured vectors from West and East Africa. Mosquitoes carrying the more-susceptible allele (2L+a) are also behaviorally less likely to be found inside houses. Vector control tools that target indoor-resting mosquitoes, such as bednets and insecticides, are currently the cornerstone of malaria control in Africa. Populations with high levels of the 2L+a allele may form reservoirs of persistent outdoor malaria transmission requiring novel measures for surveillance and control. The 2La inversion is a major and previously unappreciated component of the natural malaria transmission system in Africa, influencing both malaria susceptibility and vector behavior.

Geographical distributions of African malaria vector sibling species and evidence for insecticide resistance

BackgroundMany of the mosquito species responsible for malaria transmission belong to a sibling complex; a taxonomic group of morphologically identical, closely related species. Sibling species often differ in several important factors that have the potential to impact malaria control, including their geographical distribution, resistance to insecticides, biting and resting locations, and host preference. The aim of this study was to define the geographical distributions of dominant malaria vector sibling species in Africa so these distributions can be coupled with data on key factors such as insecticide resistance to aid more focussed, species-selective vector control.ResultsWithin the Anopheles gambiae species complex and the Anopheles funestus subgroup, predicted geographical distributions for Anopheles coluzzii, An. gambiae (as now defined) and An. funestus (distinct from the subgroup) have been produced for the first time. Improved predicted geographical distributions for Anopheles arabiensis, Anopheles melas and Anopheles merus have been generated based on records that were confirmed using molecular identification methods and a model that addresses issues of sampling bias and past changes to the environment. The data available for insecticide resistance has been evaluated and differences between sibling species are apparent although further analysis is required to elucidate trends in resistance.ConclusionsSibling species display important variability in their geographical distributions and the most important malaria vector sibling species in Africa have been mapped here for the first time. This will allow geographical occurrence data to be coupled with species-specific data on important factors for vector control including insecticide resistance. Species-specific data on insecticide resistance is available for the most important malaria vectors in Africa, namely An. arabiensis, An. coluzzii, An. gambiae and An. funestus. Future work to combine these data with the geographical distributions mapped here will allow more focussed and resource-efficient vector control and provide information to greatly improve and inform existing malaria transmission models.

Circumsporozoite protein rates, blood-feeding pattern and frequency of knockdown resistance mutations in Anopheles spp. in two ecological zones of Mauritania.

BackgroundMosquitoes belonging to Anopheles gambiae species complex are the main malaria vector in Mauritania but data on their vector capacities, feeding habits and insecticide susceptibility are still scanty. The objectives of this study were to fill this gap.MethodsAdult Anopheles spp. mosquitoes were collected using pyrethrum spray catch method from two ecological zones of Mauritania: Nouakchott (Saharan zone) and Hodh Elgharbi region (Sahelian zone). Circumsporozoite proteins (CSP) for P. falciparum, P. vivax VK210 and P. vivax VK247 were detected by enzyme-linked immunosorbent assay (ELISA) from the female anopheline mosquitoes. To confirm CSP-ELISA results, polymerase chain reaction (PCR) was also performed. Blood meal identification was performed in all engorged females by partial sequencing of the mitochondrial cytochrome b gene. Molecular assessments of pyrethroid knockdown resistance (kdr) and insensitive acetylcholinesterase resistance (ace-1) were conducted.ResultsIn Nouakchott, the only species of Anopheles identified during the survey was Anopheles arabiensis (356 specimens). In Hodh Elgharbi, 1016 specimens of Anopheles were collected, including 578 (56.9 %) Anopheles rufipes, 410 (40.35 %) An. arabiensis, 20 (1.96 %) An. gambiae, 5 (0.5 %) An. pharoensis and 3 (0.3 %) An. funestus. Three of 186 female An. arabiensis collected in Nouakchott and tested by ELISA were found positive for Plasmodium vivax VK210, corresponding to a sporozoite rate of 1.6 %; however PCR confirmed infection by P. vivax sporozoite in only one of these. In Hodh Elgharbi, no mosquito was found positive for Plasmodium spp. infection. There was a statistically significant difference in the percentage of human blood-fed Anopheles spp. between Nouakchott (58.7 %, 47 of 80 blood-engorged An. arabiensis females) and Hodh Elgharbi (11.1 %, 2 of 18 blood-engorged mosquitoes). Analysis of the kdr polymorphisms showed 48.2 % (70/145) of East African kdr mutation (L1014S) in Nouakchott compared to 10 % (4/40) in Hodh Elgharbi region (P < 0.001). Nevertheless, West African kdr mutation (L1014F) was found only in An. gambiae populations (4/40, 10 %) from Hodh Elgharbi region. No ace-1 mutation was found in mosquito specimens from the two study zones.ConclusionsOverall, this study confirmed the autochthonous P. vivax malaria transmission in Nouakchott, involving An. arabiensis as the main vector. It also described for the first time the absence of ace-1 mutation, the co-occurrence of both West and East African kdr mutation in An. gambiae in Mauritania, and highlighted the regional variations in the prevalence and type of kdr mutations.

Identification and Characterization of Two Novel RNA Viruses from Anopheles gambiae Species Complex Mosquitoes

Mosquitoes of the Anopheles gambiae complex display strong preference for human bloodmeals and are major malaria vectors in Africa. However, their interaction with viruses or role in arbovirus transmission during epidemics has been little examined, with the exception of O’nyong-nyong virus, closely related to Chikungunya virus. Deep-sequencing has revealed different RNA viruses in natural insect viromes, but none have been previously described in the Anopheles gambiae species complex. Here, we describe two novel insect RNA viruses, a Dicistrovirus and a Cypovirus, found in laboratory colonies of An. gambiae taxa using small-RNA deep sequencing. Sequence analysis was done with Metavisitor, an open-source bioinformatic pipeline for virus discovery and de novo genome assembly. Wild-collected Anopheles from Senegal and Cambodia were positive for the Dicistrovirus and Cypovirus, displaying high sequence identity to the laboratory-derived virus. Thus, the Dicistrovirus (Anopheles C virus, AnCV) and Cypovirus (Anopheles Cypovirus, AnCPV) are components of the natural virome of at least some anopheline species. Their possible influence on mosquito immunity or transmission of other pathogens is unknown. These natural viruses could be developed as models for the study of Anopheles-RNA virus interactions in low security laboratory settings, in an analogous manner to the use of rodent malaria parasites for studies of mosquito anti-parasite immunity.

Reticulate evolutionary history and extensive introgression in mosquito species revealed by phylogenetic network analysis.

The role of hybridization and subsequent introgression has been demonstrated in an increasing number of species. Recently, Fontaine etal. (Science, 347, 2015, 1258524) conducted a phylogenomic analysis of six members of the Anopheles gambiae species complex. Their analysis revealed a reticulate evolutionary history and pointed to extensive introgression on all four autosomal arms. The study further highlighted the complex evolutionary signals that the co-occurrence of incomplete lineage sorting (ILS) and introgression can give rise to in phylogenomic analyses. While tree-based methodologies were used in the study, phylogenetic networks provide a more natural model to capture reticulate evolutionary histories. In this work, we reanalyse the Anopheles data using a recently devised framework that combines the multispecies coalescent with phylogenetic networks. This framework allows us to capture ILS and introgression simultaneously, and forms the basis for statistical methods for inferring reticulate evolutionary histories. The new analysis reveals a phylogenetic network with multiple hybridization events, some of which differ from those reported in the original study. To elucidate the extent and patterns of introgression across the genome, we devise a new method that quantifies the use of reticulation branches in the phylogenetic network by each genomic region. Applying the method to the mosquito data set reveals the evolutionary history of all the chromosomes. This study highlights the utility of 'network thinking' and the new insights it can uncover, in particular in phylogenomic analyses of large data sets with extensive gene tree incongruence.

Genetic Structure of a Local Population of the Anopheles gambiae Complex in Burkina Faso

Members of the Anopheles gambiae species complex are primary vectors of human malaria in Africa. Population heterogeneities for ecological and behavioral attributes expand and stabilize malaria transmission over space and time, and populations may change in response to vector control, urbanization and other factors. There is a need for approaches to comprehensively describe the structure and characteristics of a sympatric local mosquito population, because incomplete knowledge of vector population composition may hinder control efforts. To this end, we used a genome-wide custom SNP typing array to analyze a population collection from a single geographic region in West Africa. The combination of sample depth (n = 456) and marker density (n = 1536) unambiguously resolved population subgroups, which were also compared for their relative susceptibility to natural genotypes of Plasmodium falciparum malaria. The population subgroups display fluctuating patterns of differentiation or sharing across the genome. Analysis of linkage disequilibrium identified 19 new candidate genes for association with underlying population divergence between sister taxa, A. coluzzii (M-form) and A. gambiae (S-form).

Reticulate Speciation and Barriers to Introgression in the Anopheles gambiae Species Complex.

Speciation as a process remains a central focus of evolutionary biology, but our understanding of the genomic architecture and prevalence of speciation in the face of gene flow remains incomplete. The Anopheles gambiae species complex of malaria mosquitoes is a radiation of ecologically diverse taxa. This complex is well-suited for testing for evidence of a speciation continuum and genomic barriers to introgression because its members exhibit partially overlapping geographic distributions as well as varying levels of divergence and reproductive isolation. We sequenced 20 genomes from wild A. gambiae s.s., Anopheles coluzzii, Anopheles arabiensis, and compared these with 12 genomes from the “GOUNDRY” subgroup of A. gambiae s.l. Amidst a backdrop of strong reproductive isolation, we find strong evidence for a speciation continuum with introgression of autosomal chromosomal regions among species and subgroups. The X chromosome, however, is strongly differentiated among all taxa, pointing to a disproportionately large effect of X chromosome genes in driving speciation among anophelines. Strikingly, we find that autosomal introgression has occurred from contemporary hybridization between A. gambiae and A. arabiensis despite strong divergence (∼5× higher than autosomal divergence) and isolation on the X chromosome. In addition to the X, we find strong evidence that lowly recombining autosomal regions, especially pericentromeric regions, serve as barriers to introgression secondarily to the X. We show that speciation with gene flow results in genomic mosaicism of divergence and introgression. Such a reticulate gene pool connecting vector taxa across the speciation continuum has important implications for malaria control efforts.

The kdr-bearing haplotype and susceptibility to Plasmodium falciparum in Anopheles gambiae: genetic correlation and functional testing.

BackgroundMembers of the Anophelesgambiae species complex are primary vectors of human malaria in Africa. It is known that a large haplotype shared between An. gambiae and Anophelescoluzzii by introgression carries point mutations of the voltage-gated sodium channel gene para, including the L1014F kdr mutation associated with insensitivity to pyrethroid insecticides. Carriage of L1014F kdr is also correlated with higher susceptibility to infection with Plasmodium falciparum. However, the genetic mechanism and causative gene(s) underlying the parasite susceptibility phenotype are not known.MethodsMosquitoes from the wild Burkina Faso population were challenged by feeding on natural P. falciparum gametocytes. Oocyst infection phenotypes were determined and were tested for association with SNP genotypes. Candidate genes in the detected locus were prioritized and RNAi-mediated gene silencing was used to functionally test for gene effects on P. falciparum susceptibility.ResultsA genetic locus, Pfin6, was identified that influences infection levels of P. falciparum in mosquitoes. The locus segregates as a ~3 Mb haplotype carrying 65 predicted genes including the para gene. The haplotype carrying the kdr allele of para is linked to increased parasite infection prevalence, but many single nucleotide polymorphisms on the haplotype are also equally linked to the infection phenotype. Candidate genes in the haplotype were prioritized and functionally tested. Silencing of para did not influence P. falciparum infection, while silencing of a predicted immune gene, serine protease ClipC9, allowed development of significantly increased parasite numbers.ConclusionsGenetic variation influencing Plasmodium infection in wild Anopheles is linked to a natural ~3 megabase haplotype on chromosome 2L that carries the kdr allele of the para gene. Evidence suggests that para gene function does not directly influence parasite susceptibility, and the association of kdr with infection may be due to tight linkage of kdr with other gene(s) on the haplotype. Further work will be required to determine if ClipC9 influences the outcome of P. falciparum infection in nature, as well as to confirm the absence of a direct influence by para.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0924-8) contains supplementary material, which is available to authorized users.

A multi-detection assay for malaria transmitting mosquitoes.

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Male motion coordination in anopheline mating swarms.

The Anopheles gambiae species complex comprises the primary vectors of malaria in much of sub-Saharan Africa. Most of the mating in these species occurs in swarms composed almost entirely of males. Intermittent, organized patterns in such swarms have been observed, but a detailed description of male-male interactions has not previously been available. We identify frequent, time-varying interactions characterized by periods of parallel flight in data from 8 swarms of Anopheles gambiae and 3 swarms of Anopheles coluzzii filmed in 2010 and 2011 in the village of Donéguébogou, Mali. We use the cross correlation of flight direction to quantify these interactions and to induce interaction graphs, which show that males form synchronized subgroups whose size and membership change rapidly. A swarming model with damped springs between each male and the swarm centroid shows good agreement with the correlation data, provided that local interactions represented by damping of relative velocity between males are included.