Microgeographical structure in the major Neotropical malaria vector Anopheles darlingi using microsatellites and SNP markers.
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ABSTRACT: In recent decades, throughout the Amazon Basin, landscape modification contributing to profound ecological change has proceeded at an unprecedented rate. Deforestation that accompanies human activities can significantly change aspects of anopheline biology, though this may be site-specific. Such local changes in anopheline biology could have a great impact on malaria transmission. The aim of this study was to investigate population genetics of the main malaria vector in Brazil, Anopheles darlingi, from a microgeographical perspective.Microsatellites and ddRADseq-derived single nucleotide polymorphisms (SNPs) were used to assess levels of population genetic structuring among mosquito populations from two ecologically distinctive agricultural settlements (~60 km apart) and a population from a distant (~700 km) urban setting in the western Amazon region of Brazil.Significant microgeographical population differentiation was observed among Anopheles darlingi populations via both model- and non-model-based analysis only with the SNP dataset. Microsatellites detected moderate differentiation at the greatest distances, but were unable to differentiate populations from the two agricultural settlements. Both markers showed low polymorphism levels in the most human impacted sites.At a microgeographical scale, signatures of genetic heterogeneity and population divergence were evident in Anopheles darlingi, possibly related to local environmental anthropic modification. This divergence was observed only when using high coverage SNP markers.
<h4>Background</h4>In recent decades, throughout the Amazon Basin, landscape modification contributing to profound ecological change has proceeded at an unprecedented rate. Deforestation that accompanies human activities can significantly change aspects of anopheline biology, though this may be site-specific. Such local changes in anopheline biology could have a great impact on malaria transmission. The aim of this study was to investigate population genetics of the main malaria vector in Brazil ...[more]
Project description:BACKGROUND: Anopheles darlingi is the main malaria mosquito vector in the Amazonia region. In spite of being considered a riverine, forest-dwelling species, this mosquito is becoming more abundant in peri-urban areas, increasing malaria risk. This has been associated with human-driven environmental changes such as deforestation. METHODS: Microsatellites were used to characterize A. darlingi from seven localities along the Madeira River, Rondônia (Brazil), collected in the early and late periods of the rainy season. RESULTS: Two genetically distinct subpopulations were detected: one (subpopulation A) was associated with the late rainfall period and seems to be ecologically closer to the typical forest A. darlingi; the other (subpopulation B) was associated with the early rainfall period and is probably more adapted to drier conditions by exploiting permanent anthropogenic breeding sites. Results suggest also a pattern of asymmetric introgression, with more subpopulation A alleles introgressed into subpopulation B. Both subpopulations (and admixed mosquitoes) presented similar malaria infection rates, highlighting the potential for perennial malaria transmission in the region. CONCLUSIONS: The co-occurrence of two genetically distinct subpopulations of A. darlingi adapted to different periods of rainfall may promote a more perennial transmission of malaria throughout the year. These findings, in a context of strong environmental impact due to deforestation and dam construction, have serious implications for malaria epidemiology and control in the Amazonian region.
Project description:Anopheles darlingi is the principal neotropical malaria vector, responsible for more than a million cases of malaria per year on the American continent. Anopheles darlingi diverged from the African and Asian malaria vectors ?100 million years ago (mya) and successfully adapted to the New World environment. Here we present an annotated reference A. darlingi genome, sequenced from a wild population of males and females collected in the Brazilian Amazon. A total of 10 481 predicted protein-coding genes were annotated, 72% of which have their closest counterpart in Anopheles gambiae and 21% have highest similarity with other mosquito species. In spite of a long period of divergent evolution, conserved gene synteny was observed between A. darlingi and A. gambiae. More than 10 million single nucleotide polymorphisms and short indels with potential use as genetic markers were identified. Transposable elements correspond to 2.3% of the A. darlingi genome. Genes associated with hematophagy, immunity and insecticide resistance, directly involved in vector-human and vector-parasite interactions, were identified and discussed. This study represents the first effort to sequence the genome of a neotropical malaria vector, and opens a new window through which we can contemplate the evolutionary history of anopheline mosquitoes. It also provides valuable information that may lead to novel strategies to reduce malaria transmission on the South American continent. The A. darlingi genome is accessible at www.labinfo.lncc.br/index.php/anopheles-darlingi.
Project description:Malaria transmission from humans to mosquitoes is modulated by human host immune factors. Understanding mechanisms by which the human host response may impair parasite infectivity for mosquitoes has direct implications for the development of transmission-blocking vaccines. We hypothesized that despite a low transmission intensity of malaria in the Peruvian Amazon region of Iquitos, transmission-blocking immunity against Plasmodium vivax might be common, given an unexpectedly high proportion of asymptomatic parasitemic individuals in this region. To test this hypothesis, the ability of symptomatic P. vivax malaria patients to experimentally infect wild-caught outbred Anopheles darlingi mosquitoes was tested using the indirect membrane feeding technique. Only half (52/102) of P. vivax parasitemic patients successfully infected mosquitoes. Transmitters were more likely to have gametocytes (OR 6.35, P = 0.003), high parasitemia (OR 3.79, P = 0.024), and, in terms of basic clinical parameters, a slower pulse rate (mean +/- SD: 82.3 +/- 12.3 versus 88.7 +/- 13.5, P = 0.016) than non-transmitters. Log(10) gametocytemia and log(10) real-time reverse transcriptase Pvs25 PCR quantifying gametocytes were significantly and positively correlated with oocyst counts (correlation coefficient 0.505, R2 = 0.26, P = 0.001). These experiments are the first to establish a system of determining transmission patterns in experimental infection of outbred natural neotropical malaria vectors in the Amazon region. Patients with P. vivax inefficiently infect outbred An. darlingi mosquitoes, raising the possibility that some degree of naturally occurring transmission-blocking immunity is present on a population basis in the Peruvian Amazon, an area of low intensity of malaria transmission.
Project description:BackgroundMosquito saliva, consisting of a mixture of dozens of proteins affecting vertebrate hemostasis and having sugar digestive and antimicrobial properties, helps both blood and sugar meal feeding. Culicine and anopheline mosquitoes diverged ~150 MYA, and within the anophelines, the New World species diverged from those of the Old World ~95 MYA. While the sialotranscriptome (from the Greek sialo, saliva) of several species of the Cellia subgenus of Anopheles has been described thoroughly, no detailed analysis of any New World anopheline has been done to date. Here we present and analyze data from a comprehensive salivary gland (SG) transcriptome of the neotropical malaria vector Anopheles darlingi (subgenus Nyssorhynchus).ResultsA total of 2,371 clones randomly selected from an adult female An. darlingi SG cDNA library were sequenced and used to assemble a database that yielded 966 clusters of related sequences, 739 of which were singletons. Primer extension experiments were performed in selected clones to further extend sequence coverage, allowing for the identification of 183 protein sequences, 114 of which code for putative secreted proteins.ConclusionComparative analysis of sialotranscriptomes of An. darlingi and An. gambiae reveals significant divergence of salivary proteins. On average, salivary proteins are only 53% identical, while housekeeping proteins are 86% identical between the two species. Furthermore, An. darlingi proteins were found that match culicine but not anopheline proteins, indicating loss or rapid evolution of these proteins in the old world Cellia subgenus. On the other hand, several well represented salivary protein families in old world anophelines are not expressed in An. darlingi.
Project description:Anopheles darlingi is an important vector of Plasmodium spp. in several malaria-endemic regions of Colombia. This study was conducted to test genetic variation of An. darlingi at a microgeographic scale (approximately 100 km) from localities in Córdoba and Antioquia states, in western Colombia, to better understand the potential contribution of population genetics to local malaria control programs. Microsatellite loci: nuclear white and cytochrome oxidase subunit I (COI) gene sequences were analyzed. The northern white gene lineage was exclusively distributed in Córdoba and Antioquia and shared COI haplotypes were highly represented in mosquitoes from both states. COI analyses showed these An. darlingi are genetically closer to Central American populations than southern South American populations. Overall microsatellites and COI analysis showed low to moderate genetic differentiation among populations in northwestern Colombia. Given the existence of high gene flow between An. darlingi populations of Córdoba and Antioquia, integrated vector control strategies could be developed in this region of Colombia.
Project description:BackgroundOutdoor malaria transmission hinders malaria elimination efforts in the Amazon region and novel vector control tools are needed. Ivermectin mass drug administration (MDA) to humans kills wild Anopheles, targets outdoor-feeding vectors, and can suppress malaria parasite transmission. Laboratory investigations were performed to determine ivermectin susceptibility, sporontocidal effect and inhibition of time to re-feed for the primary Amazonian malaria vector, Anopheles darlingi.MethodsTo assess ivermectin susceptibility, various concentrations of ivermectin were mixed in human blood and fed to An. darlingi. Mosquito survival was monitored daily for 7 days and a non-linear mixed effects model with Probit analysis was used to calculate lethal concentrations of ivermectin that killed 50% (LC50), 25% (LC25) and 5% (LC5) of mosquitoes. To examine ivermectin sporonticidal effect, Plasmodium vivax blood samples were collected from malaria patients and offered to mosquitoes without or with ivermectin at the LC50, LC25 or LC5. To assess ivermectin inhibition of mosquito time to re-feed, concentrations of ivermectin predicted to occur after a single oral dose of 200 μg/kg ivermectin were fed to An. darlingi. Every day for 12 days thereafter, individual mosquitoes were given the opportunity to re-feed on a volunteer. Any mosquitoes that re-blood fed or died were removed from the study.ResultsIvermectin significantly reduced An. darlingi survivorship: 7-day-LC50 = 43.2 ng/ml [37.5, 48.6], -LC25 = 27.8 ng/ml [20.4, 32.9] and -LC5 = 14.8 ng/ml [7.9, 20.2]. Ivermectin compound was sporontocidal to P. vivax in An. darlingi at the LC50 and LC25 concentrations reducing prevalence by 22.6 and 17.1%, respectively, but not at the LC5. Oocyst intensity was not altered at any concentration. Ivermectin significantly delayed time to re-feed at the 4-h (48.7 ng/ml) and 12-h (26.9 ng/ml) concentrations but not 36-h (10.6 ng/ml) or 60-h (6.3 ng/ml).ConclusionsIvermectin is lethal to An. darlingi, modestly inhibits sporogony of P. vivax, and delays time to re-feed at concentrations found in humans up to 12 h post drug ingestion. The LC50 value suggests that a higher than standard dose (400-μg/kg) is necessary to target An. darlingi. These results suggest that ivermectin MDA has potential in the Amazon region to aid malaria elimination efforts.
Project description:Anopheles stephensi is the key vector of malaria throughout the Indian subcontinent and Middle East and an emerging model for molecular and genetic studies of mosquito-parasite interactions. The type form of the species is responsible for the majority of urban malaria transmission across its range.Here, we report the genome sequence and annotation of the Indian strain of the type form of An. stephensi. The 221 Mb genome assembly represents more than 92% of the entire genome and was produced using a combination of 454, Illumina, and PacBio sequencing. Physical mapping assigned 62% of the genome onto chromosomes, enabling chromosome-based analysis. Comparisons between An. stephensi and An. gambiae reveal that the rate of gene order reshuffling on the X chromosome was three times higher than that on the autosomes. An. stephensi has more heterochromatin in pericentric regions but less repetitive DNA in chromosome arms than An. gambiae. We also identify a number of Y-chromosome contigs and BACs. Interspersed repeats constitute 7.1% of the assembled genome while LTR retrotransposons alone comprise more than 49% of the Y contigs. RNA-seq analyses provide new insights into mosquito innate immunity, development, and sexual dimorphism.The genome analysis described in this manuscript provides a resource and platform for fundamental and translational research into a major urban malaria vector. Chromosome-based investigations provide unique perspectives on Anopheles chromosome evolution. RNA-seq analysis and studies of immunity genes offer new insights into mosquito biology and mosquito-parasite interactions.
Project description:BackgroundAnopheles darlingi is the most important malaria vector in the Neotropics. An understanding of A. darlingi's population structure and contemporary gene flow patterns is necessary if vector populations are to be successfully controlled. We assessed population genetic structure and levels of differentiation based on 1,376 samples from 31 localities throughout the Peruvian and Brazilian Amazon and Central America using 5-8 microsatellite loci.ResultsWe found high levels of polymorphism for all of the Amazonian populations (mean RS = 7.62, mean HO = 0.742), and low levels for the Belize and Guatemalan populations (mean RS = 4.3, mean HO = 0.457). The Bayesian clustering analysis revealed five population clusters: northeastern Amazonian Brazil, southeastern and central Amazonian Brazil, western and central Amazonian Brazil, Peruvian Amazon, and the Central American populations. Within Central America there was low non-significant differentiation, except for between the populations separated by the Maya Mountains. Within Amazonia there was a moderate level of significant differentiation attributed to isolation by distance. Within Peru there was no significant population structure and low differentiation, and some evidence of a population expansion. The pairwise estimates of genetic differentiation between Central America and Amazonian populations were all very high and highly significant (FST = 0.1859 - 0.3901, P < 0.05). Both the DA and FST distance-based trees illustrated the main division to be between Central America and Amazonia.ConclusionWe detected a large amount of population structure in Amazonia, with three population clusters within Brazil and one including the Peru populations. The considerable differences in Ne among the populations may have contributed to the observed genetic differentiation. All of the data suggest that the primary division within A. darlingi corresponds to two white gene genotypes between Amazonia (genotype 1) and Central America, parts of Colombia and Venezuela (genotype 2), and are in agreement with previously published mitochondrial COI gene sequences interpreted as incipient species. Overall, it appears that two main factors have contributed to the genetic differentiation between the population clusters: physical distance between the populations and the differences in effective population sizes among the subpopulations.
Project description:BACKGROUND:Anopheles funestus is one of the 3 most consequential and widespread vectors of human malaria in tropical Africa. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis in this important mosquito. FINDINGS:Here we present a new high-quality A. funestus reference genome (AfunF3) assembled using 240× coverage of long-read single-molecule sequencing for contigging, combined with 100× coverage of short-read Hi-C data for chromosome scaffolding. The assembled contigs total 446 Mbp of sequence and contain substantial duplication due to alternative alleles present in the sequenced pool of mosquitos from the FUMOZ colony. Using alignment and depth-of-coverage information, these contigs were deduplicated to a 211 Mbp primary assembly, which is closer to the expected haploid genome size of 250 Mbp. This primary assembly consists of 1,053 contigs organized into 3 chromosome-scale scaffolds with an N50 contig size of 632 kbp and an N50 scaffold size of 93.811 Mbp, representing a 100-fold improvement in continuity versus the current reference assembly, AfunF1. CONCLUSION:This highly contiguous and complete A. funestus reference genome assembly will serve as an improved basis for future studies of genomic variation and organization in this important disease vector.
Project description:Physical and genetic maps have been used for chromosomal localization of genes in vectors of infectious diseases. The availability of polytene chromosomes in malaria mosquitoes provides a unique opportunity to precisely map genes of interest. We report the physical mapping of two actin genes on polytene chromosomes of the major malaria vector in the Amazon, Anopheles darlingi (Diptera: Culicidae). Clones with actin gene sequences were obtained from a cDNA library constructed from RNA isolated from adult females and males of An. darlingi. Each of the two clones was mapped to a unique site on chromosomal arm 2L in subdivisions 21A (clone pl05-A04) and 23B (clone pl17-G06). The obtained results, together with previous mapping data, provide a suitable basis for comparative genomics and for establishing chromosomal homologies among major malaria vectors.