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1.
Front Cell Infect Microbiol ; 13: 1182567, 2023.
Article in English | MEDLINE | ID: mdl-37600946

ABSTRACT

Introduction: Various sequencing based approaches are used to identify and characterize the activities of cis-regulatory elements in a genome-wide fashion. Some of these techniques rely on indirect markers such as histone modifications (ChIP-seq with histone antibodies) or chromatin accessibility (ATAC-seq, DNase-seq, FAIRE-seq), while other techniques use direct measures such as episomal assays measuring the enhancer properties of DNA sequences (STARR-seq) and direct measurement of the binding of transcription factors (ChIP-seq with transcription factor-specific antibodies). The activities of cis-regulatory elements such as enhancers, promoters, and repressors are determined by their sequence and secondary processes such as chromatin accessibility, DNA methylation, and bound histone markers. Methods: Here, machine learning models are employed to evaluate the accuracy with which cis-regulatory elements identified by various commonly used sequencing techniques can be predicted by their underlying sequence alone to distinguish between cis-regulatory activity that is reflective of sequence content versus secondary processes. Results and discussion: Models trained and evaluated on D. melanogaster sequences identified through DNase-seq and STARR-seq are significantly more accurate than models trained on sequences identified by H3K4me1, H3K4me3, and H3K27ac ChIP-seq, FAIRE-seq, and ATAC-seq. These results suggest that the activity detected by DNase-seq and STARR-seq can be largely explained by underlying DNA sequence, independent of secondary processes. Experimentally, a subset of DNase-seq and H3K4me1 ChIP-seq sequences were tested for enhancer activity using luciferase assays and compared with previous tests performed on STARR-seq sequences. The experimental data indicated that STARR-seq sequences are substantially enriched for enhancer-specific activity, while the DNase-seq and H3K4me1 ChIP-seq sequences are not. Taken together, these results indicate that the DNase-seq approach identifies a broad class of regulatory elements of which enhancers are a subset and the associated data are appropriate for training models for detecting regulatory activity from sequence alone, STARR-seq data are best for training enhancer-specific sequence models, and H3K4me1 ChIP-seq data are not well suited for training and evaluating sequence-based models for cis-regulatory element prediction.


Subject(s)
Drosophila melanogaster , Histones , Animals , Histones/genetics , Sequence Analysis, DNA , Chromatin/genetics , Deoxyribonucleases
2.
Parasit Vectors ; 15(1): 465, 2022 Dec 13.
Article in English | MEDLINE | ID: mdl-36514125

ABSTRACT

BACKGROUND: Anopheles cell lines are used in a variety of ways to better understand the major vectors of malaria in sub-Saharan Africa. Despite this, commonly used cell lines are not well characterized, and no tools are available for cell line identification and authentication. METHODS: Utilizing whole genome sequencing, genomes of 4a-3A and 4a-3B 'hemocyte-like' cell lines were characterized for insertions and deletions (indels) and SNP variation. Genomic locations of distinguishing sequence variation and species origin of the cell lines were also examined. Unique indels were targeted to develop a PCR-based cell line authentication assay. Mitotic chromosomes were examined to survey the cytogenetic landscape for chromosome structure and copy number in the cell lines. RESULTS: The 4a-3A and 4a-3B cell lines are female in origin and primarily of Anopheles coluzzii ancestry. Cytogenetic analysis indicates that the two cell lines are essentially diploid, with some relatively minor chromosome structural rearrangements. Whole-genome sequence was generated, and analysis indicated that SNPs and indels which differentiate the cell lines are clustered on the 2R chromosome in the regions of the 2Rb, 2Rc and 2Ru chromosomal inversions. A PCR-based authentication assay was developed to fingerprint three indels unique to each cell line. The assay distinguishes between 4a-3A and 4a-3B cells and also uniquely identifies two additional An. coluzzii cell lines tested, Ag55 and Sua4.0. The assay has the specificity to distinguish four cell lines and also has the sensitivity to detect cellular contamination within a sample of cultured cells. CONCLUSIONS: Genomic characterization of the 4a-3A and 4a-3B Anopheles cell lines was used to develop a simple diagnostic assay that can distinguish these cell lines within and across research laboratories. A cytogenetic survey indicated that the 4a-3A and Sua4.0 cell lines carry essentially normal diploid chromosomes, which makes them amenable to CRISPR/Cas9 genome editing. The presented simple authentication assay, coupled with screening for mycoplasma, will allow validation of the integrity of experimental resources and will promote greater experimental reproducibility of results.


Subject(s)
Anopheles , Animals , Female , Male , Anopheles/genetics , Hemocytes , Reproducibility of Results , Mosquito Vectors/genetics , Cell Line
3.
Sci Rep ; 12(1): 6315, 2022 04 15.
Article in English | MEDLINE | ID: mdl-35428783

ABSTRACT

Entomopathogenic fungi have been explored as a potential biopesticide to counteract the insecticide resistance issue in mosquitoes. However, little is known about the possibility that genetic resistance to fungal biopesticides could evolve in mosquito populations. Here, we detected an important genetic component underlying Anopheles coluzzii survival after exposure to the entomopathogenic fungus Metarhizium anisopliae. A familiality study detected variation for survival among wild mosquito isofemale pedigrees, and genetic mapping identified two loci that significantly influence mosquito survival after fungus exposure. One locus overlaps with a previously reported locus for Anopheles susceptibility to the human malaria parasite Plasmodium falciparum. Candidate gene studies revealed that two LRR proteins encoded by APL1C and LRIM1 genes in this newly mapped locus are required for protection of female A. coluzzii from M. anisopliae, as is the complement-like factor Tep1. These results indicate that natural Anopheles populations already segregate frequent genetic variation for differential mosquito survival after fungal challenge and suggest a similarity in Anopheles protective responses against fungus and Plasmodium. However, this immune similarity raises the possibility that fungus-resistant mosquitoes could also display enhanced resistance to Plasmodium, suggesting an advantage of selecting for fungus resistance in vector populations to promote naturally diminished malaria vector competence.


Subject(s)
Anopheles , Malaria , Metarhizium , Plasmodium , Animals , Anopheles/parasitology , Female , Humans , Metarhizium/genetics , Mosquito Vectors/genetics
5.
Front Microbiol ; 12: 635772, 2021.
Article in English | MEDLINE | ID: mdl-34054746

ABSTRACT

Exposure of mosquitoes to numerous eukaryotic and prokaryotic microbes in their associated microbiomes has probably helped drive the evolution of the innate immune system. To our knowledge, a metagenomic catalog of the eukaryotic microbiome has not been reported from any insect. Here we employ a novel approach to preferentially deplete host 18S ribosomal RNA gene amplicons to reveal the composition of the eukaryotic microbial communities of Anopheles larvae sampled in Kenya, Burkina Faso and Republic of Guinea (Conakry). We identified 453 eukaryotic operational taxonomic units (OTUs) associated with Anopheles larvae in nature, but an average of 45% of the 18S rRNA sequences clustered into OTUs that lacked a taxonomic assignment in the Silva database. Thus, the Anopheles microbiome contains a striking proportion of novel eukaryotic taxa. Using sequence similarity matching and de novo phylogenetic placement, the fraction of unassigned sequences was reduced to an average of 4%, and many unclassified OTUs were assigned as relatives of known taxa. A novel taxon of the genus Ophryocystis in the phylum Apicomplexa (which also includes Plasmodium) is widespread in Anopheles larvae from East and West Africa. Notably, Ophryocystis is present at fluctuating abundance among larval breeding sites, consistent with the expected pattern of an epidemic pathogen. Species richness of the eukaryotic microbiome was not significantly different across sites from East to West Africa, while species richness of the prokaryotic microbiome was significantly lower in West Africa. Laboratory colonies of Anopheles coluzzii harbor 26 eukaryotic OTUs, of which 38% (n = 10) are shared with wild populations, while 16 OTUs are unique to the laboratory colonies. Genetically distinct An. coluzzii colonies co-housed in the same facility maintain different prokaryotic microbiome profiles, suggesting a persistent host genetic influence on microbiome composition. These results provide a foundation to understand the role of the Anopheles eukaryotic microbiome in vector immunity and pathogen transmission. We hypothesize that prevalent apicomplexans such as Ophryocystis associated with Anopheles could induce interference or competition against Plasmodium within the vector. This and other members of the eukaryotic microbiome may offer candidates for new vector control tools.

6.
Sci Rep ; 11(1): 6458, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33742030

ABSTRACT

In the Greater Mekong Subregion, malaria cases have significantly decreased but little is known about the vectors or mechanisms responsible for residual malaria transmission. We analysed a total of 3920 Anopheles mosquitoes collected during the rainy and dry seasons from four ecological settings in Cambodia (villages, forested areas near villages, rubber tree plantations and forest sites). Using odor-baited traps, 81% of the total samples across all sites were collected in cow baited traps, although 67% of the samples attracted by human baited traps were collected in forest sites. Overall, 20% of collected Anopheles were active during the day, with increased day biting during the dry season. 3131 samples were identified morphologically as 14 different species, and a subset was also identified by DNA amplicon sequencing allowing determination of 29 Anopheles species. The investigation of well characterized insecticide mutations (ace-1, kdr, and rdl genes) indicated that individuals carried mutations associated with response to all the different classes of insecticides. There also appeared to be a non-random association between mosquito species and insecticide resistance with Anopheles peditaeniatus exhibiting nearly fixed mutations. Molecular screening for Plasmodium sp. presence indicated that 3.6% of collected Anopheles were positive, most for P. vivax followed by P. falciparum. These results highlight some of the key mechanisms driving residual human malaria transmission in Cambodia, and illustrate the importance of diverse collection methods, sites and seasons to avoid bias and better characterize Anopheles mosquito ecology in Southeast Asia.


Subject(s)
Anopheles/physiology , Malaria/transmission , Mosquito Vectors/physiology , Animals , Anopheles/classification , Anopheles/genetics , Cambodia , Forests , Humans , Insect Proteins/genetics , Insecticide Resistance/genetics , Mosquito Vectors/classification , Mosquito Vectors/genetics , Mutation , Seasons
7.
Insects ; 12(2)2021 Feb 22.
Article in English | MEDLINE | ID: mdl-33671692

ABSTRACT

The portion of the mosquito genome that does not code for proteins contains regulatory elements that likely underlie variation for important phenotypes including resistance and susceptibility to infection with arboviruses and Apicomplexan parasites. Filtering the non-coding genome to uncover these functional elements is an expanding area of research, though identification of non-coding regulatory elements is challenging due to the lack of an amino acid-like code for the non-coding genome and a lack of sequence conservation across species. This review focuses on three types of non-coding regulatory elements: (1) microRNAs (miRNAs), (2) long non-coding RNAs (lncRNAs), and (3) enhancers, and summarizes current advances in technical and analytical approaches for measurement of each of these elements on a genome-wide scale. The review also summarizes and highlights novel findings following application of these techniques in mosquito-borne disease research. Looking beyond the protein-coding genome is essential for understanding the complexities that underlie differential gene expression in response to arboviral or parasite infection in mosquito disease vectors. A comprehensive understanding of the regulation of gene and protein expression will inform transgenic and other vector control methods rooted in naturally segregating genetic variation.

8.
Front Genet ; 12: 785934, 2021.
Article in English | MEDLINE | ID: mdl-35082832

ABSTRACT

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences. Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

9.
Front Microbiol ; 11: 306, 2020.
Article in English | MEDLINE | ID: mdl-32174902

ABSTRACT

The commensal gut microbiome is contained by the enteric epithelial barrier, but little is known about the degree of specificity of host immune barrier interactions for particular bacterial taxa. Here, we show that depletion of leucine-rich repeat immune factor APL1 in the Asian malaria mosquito Anopheles stephensi is associated with higher midgut abundance of just the family Enterobacteraceae, and not generalized dysbiosis of the microbiome. The effect is explained by the response of a narrow clade containing two main taxa related to Klebsiella and Cedecea. Analysis of field samples indicate that these two taxa are recurrent members of the wild Anopheles microbiome. Triangulation using sequence and functional data incriminated relatives of C. neteri and Cedecea NFIX57 as candidates for the Cedecea component, and K. michiganensis, K. oxytoca, and K.sp. LTGPAF-6F as candidates for the Klebsiella component. APL1 presence is associated with host ability to specifically constrain the abundance of a narrow microbiome clade of the Enterobacteraceae, and the immune factor may promote homeostasis of this clade in the enteric microbiome for host benefit.

10.
PLoS Negl Trop Dis ; 14(2): e0008059, 2020 02.
Article in English | MEDLINE | ID: mdl-32032359

ABSTRACT

During a blood meal, female Anopheles mosquitoes are potentially exposed to diverse microbes in addition to the malaria parasite, Plasmodium. Human and animal African trypanosomiases are frequently co-endemic with malaria in Africa. It is not known whether exposure of Anopheles to trypanosomes influences their fitness or ability to transmit Plasmodium. Using cell and molecular biology approaches, we found that Trypanosoma brucei brucei parasites survive for at least 48h after infectious blood meal in the midgut of the major malaria vector, Anopheles coluzzii before being cleared. This transient survival of trypanosomes in the midgut is correlated with a dysbiosis, an alteration in the abundance of the enteric bacterial flora in Anopheles coluzzii. Using a developmental biology approach, we found that the presence of live trypanosomes in mosquito midguts also reduces their reproductive fitness, as it impairs the viability of laid eggs by affecting their hatching. Furthermore, we found that Anopheles exposure to trypanosomes enhances their vector competence for Plasmodium, as it increases their infection prevalence. A transcriptomic analysis revealed that expression of only two Anopheles immune genes are modulated during trypanosome exposure and that the increased susceptibility to Plasmodium was microbiome-dependent, while the reproductive fitness cost was dependent only on the presence of live trypanosomes but was microbiome independent. Taken together, these results demonstrate multiple effects upon Anopheles vector competence for Plasmodium caused by eukaryotic microbes interacting with the host and its microbiome, which may in turn have implications for malaria control strategies in co-endemic areas.


Subject(s)
Anopheles/parasitology , Malaria/parasitology , Plasmodium yoelii/physiology , Trypanosoma/physiology , Animals , Coinfection , Host-Parasite Interactions , Mice , Polymerase Chain Reaction , Reproduction
11.
Parasit Vectors ; 13(1): 18, 2020 Jan 13.
Article in English | MEDLINE | ID: mdl-31931885

ABSTRACT

BACKGROUND: The 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. METHODS: The 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. RESULTS: APL1 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. CONCLUSIONS: APL1 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.


Subject(s)
Anopheles/genetics , Chaperonin 60/genetics , Immunologic Factors/genetics , Peptide Fragments/genetics , Animals , Anopheles/immunology , Evolution, Molecular , Gene Dosage , Insect Proteins/genetics , Insect Vectors/genetics , Longevity/genetics , Malaria/immunology , Malaria/transmission , Phylogeny
12.
Sci Rep ; 9(1): 15275, 2019 10 24.
Article in English | MEDLINE | ID: mdl-31649293

ABSTRACT

Enhancers are cis-regulatory elements that control most of the developmental and spatial gene expression in eukaryotes. Genetic variation of enhancer sequences is known to influence phenotypes, but the effect of enhancer variation upon enhancer functional activity and downstream phenotypes has barely been examined in any species. In the African malaria vector, Anopheles coluzzii, we identified candidate enhancers in the proximity of genes relevant for immunity, insecticide resistance, and development. The candidate enhancers were functionally validated using luciferase reporter assays, and their activity was found to be essentially independent of their physical orientation, a typical property of enhancers. All of the enhancers segregated genetically polymorphic alleles, which displayed significantly different levels of functional activity. Deletion mutagenesis and functional testing revealed a fine structure of positive and negative regulatory elements that modulate activity of the enhancer core. Enhancer polymorphisms segregate in wild A. coluzzii populations in West Africa. Thus, enhancer variants that modify target gene expression leading to likely phenotypic consequences are frequent in nature. These results demonstrate the existence of naturally polymorphic A. coluzzii enhancers, which may help explain important differences between individuals or populations for malaria transmission efficiency and vector adaptation to the environment.


Subject(s)
Anopheles/genetics , Enhancer Elements, Genetic , Insecticide Resistance/genetics , Mosquito Vectors/genetics , Polymorphism, Genetic , Animals , Anopheles/drug effects , Insecticides/pharmacology , Malaria/transmission , Mosquito Control/methods , Mosquito Vectors/drug effects
13.
Sci Rep ; 7(1): 3241, 2017 06 12.
Article in English | MEDLINE | ID: mdl-28607435

ABSTRACT

Anopheles mosquitoes are vectors of the human malaria parasite, Plasmodium falciparum. The vector microbiota is a likely factor influencing parasite transmission. The prokaryotic microbiota of mosquitoes is efficiently surveyed by sequencing of hypervariable regions of the 16s ribosomal RNA (rRNA) gene. However, identification of the eukaryotic microbiota by targeting the 18s rRNA gene is challenging due to simultaneous amplification of the abundant 18s rRNA gene target in the mosquito host. Consequently, the eukaryotic microbial diversity of mosquitoes is vastly underexplored. An efficient methodology is needed to identify this component of the microbiota, expected to include relatives of Plasmodium. Here, we use defined panels of Anopheles samples from West Africa to test two experimental PCR clamp approaches to maximize the specific amplification of 18s rRNA gene hypervariable regions from eukaryotic microbes: anneal-inhibiting blocking primers and peptide-nucleic acid (PNA) oligonucleotide blockers. Of the two, PNA blockers were the only efficient blocking strategy, allowing a reduction of mosquito 18s rRNA gene sequences by more than 80% for the V4 hypervariable region. These PNA blockers will facilitate taxonomic profiling of the eukaryotic microbiota of the A. gambiae species complex, and contribute to a better understanding of microbial influence upon immunity and pathogen infection.


Subject(s)
Anopheles/microbiology , Microbiota , Polymerase Chain Reaction/methods , RNA, Ribosomal, 18S , Africa, Western , Animals , DNA Primers , Eukaryota/genetics , Mammals/genetics , Mosquito Vectors/microbiology , Plasmodium
14.
Elife ; 62017 06 23.
Article in English | MEDLINE | ID: mdl-28643631

ABSTRACT

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.


Subject(s)
Anopheles/genetics , Anopheles/parasitology , Chromosome Inversion , Chromosomes, Insect , Mosquito Vectors/genetics , Mosquito Vectors/parasitology , Plasmodium falciparum/growth & development , Africa , Animals , Behavior, Animal , Host-Parasite Interactions , Humans , Malaria/transmission
16.
Malar J ; 15: 191, 2016 Apr 08.
Article in English | MEDLINE | ID: mdl-27059057

ABSTRACT

BACKGROUND: Malaria remains a pervasive public health problem in sub-Saharan West Africa. Here mosquito vector populations were explored across four sites in Mali and the Republic of Guinea (Guinea Conakry). The study samples the major ecological zones of malaria-endemic regions in West Africa within a relatively small distance. METHODS: Mosquito vectors were sampled from larval pools, adult indoor resting sites, and indoor and outdoor human-host seeking adults. Mosquitoes were collected at sites spanning 350 km that represented arid savannah, humid savannah, semi-forest and deep forest ecological zones, in areas where little was previously known about malaria vector populations. 1425 mosquito samples were analysed by molecular assays to determine species, genetic attributes, blood meal sources and Plasmodium infection status. RESULTS: Anopheles gambiae and Anopheles coluzzii were the major anophelines represented in all collections across the ecological zones, with A. coluzzii predominant in the arid savannah and A. gambiae in the more humid sites. The use of multiple collection methodologies across the sampling sites allows assessment of potential collection bias of the different methods. The L1014F kdr insecticide resistance mutation (kdr-w) is found at high frequency across all study sites. This mutation appears to have swept almost to fixation, from low frequencies 6 years earlier, despite the absence of widespread insecticide use for vector control. Rates of human feeding are very high across ecological zones, with only small fractions of animal derived blood meals in the arid and humid savannah. About 30 % of freshly blood-fed mosquitoes were positive for Plasmodium falciparum presence, while the rate of mosquitoes with established infections was an order of magnitude lower. CONCLUSIONS: The study represents detailed vector characterization from an understudied area in West Africa with endemic malaria transmission. The deep forest study site includes the epicenter of the 2014 Ebola virus epidemic. With new malaria control interventions planned in Guinea, these data provide a baseline measure and an opportunity to assess the outcome of future interventions.


Subject(s)
Anopheles/classification , Anopheles/growth & development , Insect Vectors , Plasmodium falciparum/isolation & purification , Animals , Anopheles/genetics , Gambia , Guinea , Humans , Mali
17.
Mol Ecol ; 25(7): 1494-510, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26846876

ABSTRACT

The recent discovery of a previously unknown genetic subgroup of Anopheles gambiae sensu lato underscores our incomplete understanding of complexities of vector population demographics in Anopheles. This subgroup, named GOUNDRY, does not rest indoors as adults and is highly susceptible to Plasmodium infection in the laboratory. Initial description of GOUNDRY suggested it differed from other known Anopheles taxa in surprising and sometimes contradictory ways, raising a number of questions about its age, population size and relationship to known subgroups. To address these questions, we sequenced the complete genomes of 12 wild-caught GOUNDRY specimens and compared these genomes to a panel of Anopheles genomes. We show that GOUNDRY is most closely related to Anopheles coluzzii, and the timing of cladogenesis is not recent, substantially predating the advent of agriculture. We find a large region of the X chromosome that has swept to fixation in GOUNDRY within the last 100 years, which may be an inversion that serves as a partial barrier to contemporary gene flow. Interestingly, we show that GOUNDRY has a history of inbreeding that is significantly associated with susceptibility to Plasmodium infection in the laboratory. Our results illuminate the genomic evolution of one of probably several cryptic, ecologically specialized subgroups of Anopheles and provide a potent example of how vector population dynamics may complicate efforts to control or eradicate malaria.


Subject(s)
Anopheles/genetics , Evolution, Molecular , Genome, Insect , Plasmodium falciparum , Animals , Anopheles/parasitology , Chromosome Inversion , Gene Flow , Genetic Speciation , Genetics, Population , Inbreeding , Insect Vectors/genetics , Insect Vectors/parasitology , Polymorphism, Single Nucleotide , Population Dynamics , Sequence Analysis, DNA , X Chromosome/genetics
18.
PLoS One ; 11(1): e0145308, 2016.
Article in English | MEDLINE | ID: mdl-26731649

ABSTRACT

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).


Subject(s)
Anopheles/genetics , Genetic Structures , Genome, Insect/genetics , Insect Vectors/genetics , Polymorphism, Single Nucleotide , Animals , Anopheles/classification , Burkina Faso/epidemiology , Genetics, Population/methods , Genotype , Geography , Humans , Insect Vectors/classification , Linkage Disequilibrium , Malaria, Falciparum/epidemiology , Malaria, Falciparum/parasitology , Phylogeny , Population Dynamics , Species Specificity
19.
PLoS Pathog ; 11(12): e1005306, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26633695

ABSTRACT

Nucleotide variation patterns across species are shaped by the processes of natural selection, including exposure to environmental pathogens. We examined patterns of genetic variation in two sister species, Anopheles gambiae and Anopheles coluzzii, both efficient natural vectors of human malaria in West Africa. We used the differentiation signature displayed by a known coordinate selective sweep of immune genes APL1 and TEP1 in A. coluzzii to design a population genetic screen trained on the sweep, classified a panel of 26 potential immune genes for concordance with the signature, and functionally tested their immune phenotypes. The screen results were strongly predictive for genes with protective immune phenotypes: genes meeting the screen criteria were significantly more likely to display a functional phenotype against malaria infection than genes not meeting the criteria (p = 0.0005). Thus, an evolution-based screen can efficiently prioritize candidate genes for labor-intensive downstream functional testing, and safely allow the elimination of genes not meeting the screen criteria. The suite of immune genes with characteristics similar to the APL1-TEP1 selective sweep appears to be more widespread in the A. coluzzii genome than previously recognized. The immune gene differentiation may be a consequence of adaptation of A. coluzzii to new pathogens encountered in its niche expansion during the separation from A. gambiae, although the role, if any of natural selection by Plasmodium is unknown. Application of the screen allowed identification of new functional immune factors, and assignment of new functions to known factors. We describe biochemical binding interactions between immune proteins that underlie functional activity for malaria infection, which highlights the interplay between pathogen specificity and the structure of immune complexes. We also find that most malaria-protective immune factors display phenotypes for either human or rodent malaria, with broad specificity a rarity.


Subject(s)
Anopheles/genetics , Anopheles/immunology , Insect Vectors/genetics , Insect Vectors/immunology , Animals , Base Sequence , Evolution, Molecular , Genes, Insect/immunology , Genetic Variation , Insect Proteins/genetics , Insect Proteins/immunology , Malaria/transmission , Mice , Molecular Sequence Data , Polymerase Chain Reaction
20.
Genome Biol Evol ; 7(11): 3116-31, 2015 Nov 28.
Article in English | MEDLINE | ID: mdl-26615027

ABSTRACT

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.


Subject(s)
Anopheles/genetics , Genetic Speciation , Genome, Insect , Animals , Anopheles/classification , Female , Gene Flow , Genetic Variation , Genetics, Population , Models, Genetic , Reproductive Isolation , Sequence Analysis, DNA , X Chromosome
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