CRISPR-mediated knock-in of transgenes into the malaria vector Anopheles funestus.
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ABSTRACT:
SUBMITTER: Quinn C
PROVIDER: S-EPMC8496255 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BACKGROUND:Anopheles funestus is among the major malaria vectors in Kenya and sub-Saharan Africa and has been recently implicated in persistent malaria transmission. However, its ecology and genetic diversity remain poorly understood in Kenya. METHODS:Using 16 microsatellite loci, we examined the genetic structure of An. funestus sampled from 11 locations (n = 426 individuals) across a wide geographical range in Kenya spanning coastal, western and Rift Valley areas. RESULTS:Kenyan An. funestus resolved as three genetically distinct clusters. The largest cluster (FUN1) broadly included samples from western and Rift Valley areas of Kenya with two clusters identified from coastal Kenya (FUN2 and FUN3), not previously reported. Geographical distance had no effect on population differentiation of An. funestus. We found a significant variation in the mean Plasmodium infectivity between the clusters (?2 = 12.1, df = 2, P = 0.002) and proportional to the malaria prevalence in the different risk zones of Kenya. Notably, there was variation in estimated effective population sizes between the clusters, suggesting possible differential impact of anti-vector interventions in represented areas. CONCLUSIONS:Heterogeneity among Kenyan populations of An. funestus will impact malaria vector control with practical implications for the development of gene-drive technologies. The difference in Plasmodium infectivity and effective population size between the clusters could suggest potential variation in phenotypic characteristics relating to competence or insecticide resistance. This is worth examining in future studies.
| S-EPMC6323828 | biostudies-other
Project description:Chromosomal polymorphisms, such as inversions, are presumably involved in the rapid adaptation of populations to local environmental conditions. Reduced recombination between alternative arrangements in heterozygotes may protect sets of locally adapted genes, promoting ecological divergence and potentially leading to reproductive isolation and speciation. Through a comparative analysis of chromosomal inversions and microsatellite marker polymorphisms, we hereby present biological evidence that strengthens this view in the mosquito Anopheles funestus s.s, one of the most important and widespread malaria vectors in Africa. Specimens were collected across a wide range of geographical, ecological, and climatic conditions in Cameroon. We observed a sharp contrast between population structure measured at neutral microsatellite markers and at chromosomal inversions. Microsatellite data detected only a weak signal for population structuring among geographical zones (F(ST) < 0.013, P < 0.01). By contrast, strong differentiation among ecological zones was revealed by chromosomal inversions (F(ST) > 0.190, P < 0.01). Using standardized estimates of F(ST), we show that inversions behave at odds with neutral expectations strongly suggesting a role of environmental selection in shaping their distribution. We further demonstrate through canonical correspondence analysis that heterogeneity in eco-geographical variables measured at specimen sampling sites explained 89% of chromosomal variance in A. funestus. These results are in agreement with a role of chromosomal inversions in ecotypic adaptation in this species. We argue that this widespread mosquito represents an interesting model system for the study of chromosomal speciation mechanisms and should provide ample opportunity for comparative studies on the evolution of reproductive isolation and speciation in major human malaria vectors.
| S-EPMC3002248 | biostudies-literature
Project description:We have constructed a genetic map of the major African malaria vector, Anopheles funestus, using genetic markers segregating in F(2) progeny from crosses between two strains colonized from different field sites. Genotyping was performed on 174 progeny from three families using 33 microsatellite markers, a single RFLP, and 15 single nucleotide polymorphism (SNP) loci. Four linkage groups were resolved and these were anchored to chromosomes X and 2 and chromosomal arms 3R and 3L by comparison with a physical map of this species. Five markers were linked to the X chromosome, 16 markers to chromosome 2, and 10 and 11 markers to chromosomal arms 3R and 3L, respectively. This significantly increases the number of chromosomally defined genetic markers for this species and will facilitate the identification of genes controlling epidemiologically important traits such as resistance to insecticides or vector competence.
| S-EPMC1456103 | biostudies-literature
Project description:BackgroundAnopheles 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.FindingsHere 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.ConclusionThis 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.
| S-EPMC6545970 | biostudies-literature
Project description:BackgroundThe malaria vector Anopheles funestus is increasingly recognized as a dominant vector of residual transmission in many African settings. Efforts to better understand its biology and control are significantly impeded by the difficulties of colonizing it under laboratory conditions. To identify key bottlenecks in colonization, this study compared the development and fitness characteristics of wild An. funestus from Tanzania (FUTAZ) and their F1 offspring during colonization attempts. The demography and reproductive success of wild FUTAZ offspring were compared to that of individuals from one of the only An. funestus strains that has been successfully colonized (FUMOZ, from Mozambique) under similar laboratory conditions.MethodsWild An. funestus (FUTAZ) were collected from three Tanzanian villages and maintained inside an insectary at 70-85% RH, 25-27 °C and 12 h:12 h photoperiod. Eggs from these females were used to establish three replicate F1 laboratory generations. Larval development, survival, fecundity, mating success, percentage pupation and wing length were measured in the F1 -FUTAZ offspring and compared with wild FUTAZ and FUMOZ mosquitoes.ResultsWild FUTAZ laid fewer eggs (64.1; 95% CI [63.2, 65.0]) than FUMOZ females (76.1; 95% CI [73.3, 79.1]). Survival of F1-FUTAZ larvae under laboratory conditions was low, with an egg-to-pupae conversion rate of only 5.9% compared to 27.4% in FUMOZ. The median lifespan of F1-FUTAZ females (32 days) and males (33 days) was lower than FUMOZ (52 and 49 for females and males respectively). The proportion of female F1-FUTAZ inseminated under laboratory conditions (9%) was considerably lower than either FUMOZ (72%) or wild-caught FUTAZ females (92%). This resulted in nearly zero viable F2-FUTAZ eggs produced. Wild FUTAZ wings appear to be larger compared to the lab reared F1-FUTAZ and FUMOZ.ConclusionsThis study indicates that poor larval survival, mating success, low fecundity and shorter survival under laboratory conditions all contribute to difficulties in colonizing of An. funestus. Future studies should focus on enhancing these aspects of An. funestus fitness in the laboratory, with the biggest barrier likely to be poor mating.
| S-EPMC7955623 | biostudies-literature
Project description:BackgroundMalaria affects large parts of the developing world and is responsible for almost 800,000 deaths annually. As climates change, concerns have arisen as to how this vector-borne disease will be impacted by changing rainfall patterns and warming temperatures. Despite the importance and controversy surrounding the impact of climate change on the potential spread of this disease, little information exists on the tolerances of several of the vector species themselves.MethodsUsing a ramping protocol (to assess critical thermal limits--CT) and plunge protocol (to assess lethal temperature limits--LT) information on the thermal tolerance of two of Africa's important malaria vectors, Anopheles arabiensis and Anopheles funestus was collected. The effects of age, thermal acclimation treatment, sex and strain (laboratory versus wild adults) were investigated for CT determinations for each species. The effects of age and sex for adults and life stage (larvae, pupae, adults) were investigated for LT determinations.ResultsIn both species, females are more tolerant to low and high temperatures than males; larvae and pupae have higher upper lethal limits than do adults. Thermal acclimation of adults has large effects in some instances but small effects in others. Younger adults tend to be more tolerant of low or high temperatures than older age groups. Long-standing laboratory colonies are sufficiently similar in thermal tolerance to field-collected animals to provide reasonable surrogates when making inferences about wild population responses. Differences between these two vectors in their thermal tolerances, especially in larvae and pupae, are plausibly a consequence of different habitat utilization.ConclusionsLimited plasticity is characteristic of the adults of these vector species relative to others examined to date, suggesting limited scope for within-generation change in thermal tolerance. These findings and the greater tolerance of females to thermal extremes may have significant implications for future malaria transmission, especially in areas of current seasonal transmission and in areas on the boundaries of current vector distribution.
| S-EPMC3507762 | biostudies-literature
Project description:Background: The insecticide susceptibility status of Anopheles funestus, one of the main malaria vectors in the Afrotropical regions, remains under-studied due to the difficulty of working with this mosquito species. Collecting their larvae in natural breeding sites, rearing and maintaining them in normal laboratory conditions have been a difficult task. Forced-egg laying technique has been a very good tool to generate eggs from adult mosquitoes collected from the wild but rearing these eggs to obtain satisfying portion as adults has always been the problem. In this study, we optimized the development of mosquito species larvae under standard laboratory conditions for desired production of adult mosquitoes that can be useful for insecticide susceptibility tests. Methods: A forced-egg laying technique was used to obtain eggs from gravid female Anopheles funestus collected from Kpome locality in Benin. Eggs were reared in three different water samples (water from the borehole,and two mineral water namely FIFA and Possotômè) and larvae were fed with TetraMin baby fish food. The physico-chemical parameters of the waters were investigated prior to use for egg incubation. Results:In contrast to mineral water that had no contamination, the borehole water source was contaminated with lead (2.5mg/L) and nitrate (118.8mg/L). Egg hatching rates ranged as 91.9 ± 4.4%, 89.1 ± 2.5% and 87.9 ± 2.6% in FIFA, Possotômè and borehole water respectively. High emergence of larvae to adult mosquitoes was recorded as in FIFA (74.3%) and Possotômè(79.5%) water. No adult mosquito was obtained from larvae reared in borehole water. Conclusions: This study gave insight on the water sources that could be good for rearing to mass produce An. funestus in the laboratory. More analysis with other local mineral water sources in our environments could be considered in the future, hopefully giving better outputs.
| S-EPMC5721565 | biostudies-literature
Project description:BackgroundSingle nucleotide polymorphisms (SNPs) are the most common source of genetic variation in eukaryotic species and have become an important marker for genetic studies. The mosquito Anopheles funestus is one of the major malaria vectors in Africa and yet, prior to this study, no SNPs have been described for this species. Here we report a genome-wide set of SNP markers for use in genetic studies on this important human disease vector.ResultsDNA fragments from 50 genes were amplified and sequenced from 21 specimens of An. funestus. A third of specimens were field collected in Malawi, a third from a colony of Mozambican origin and a third form a colony of Angolan origin. A total of 494 SNPs including 303 within the coding regions of genes and 5 indels were identified. The physical positions of these SNPs in the genome are known. There were on average 7 SNPs per kilobase similar to that observed in An. gambiae and Drosophila melanogaster. Transitions outnumbered transversions, at a ratio of 2:1. The increased frequency of transition substitutions in coding regions is likely due to the structure of the genetic code and selective constraints. Synonymous sites within coding regions showed a higher polymorphism rate than non-coding introns or 3' and 5'flanking DNA with most of the substitutions in coding regions being observed at the 3rd codon position. A positive correlation in the level of polymorphism was observed between coding and non-coding regions within a gene. By genotyping a subset of 30 SNPs, we confirmed the validity of the SNPs identified during this study.ConclusionThis set of SNP markers represents a useful tool for genetic studies in An. funestus, and will be useful in identifying candidate genes that affect diverse ranges of phenotypes that impact on vector control, such as resistance insecticide, mosquito behavior and vector competence.
| S-EPMC1781065 | biostudies-literature
Project description:BackgroundAnopheles funestus is one of the major malaria vectors in Africa and yet there are few genomic tools available for this species compared to An. gambiae. To start to close this knowledge gap, we sequenced the An. funestus transcriptome using cDNA libraries developed from a pyrethroid resistant laboratory strain and a pyrethroid susceptible field strain from Mali.ResultsUsing a pool of life stages (pupae, larvae, adults: females and males) for each strain, 454 sequencing generated 375,619 reads (average length of 182 bp). De novo assembly generated 18,103 contigs with average length of 253 bp. The average depth of coverage of these contigs was 8.3. In total 20.8% of all reads were novel when compared to reference databases. The sequencing of the field strain generated 204,758 reads compared to 170,861 from the insecticide resistant laboratory strain. The contigs most differentially represented in the resistant strain belong to the P450 gene family and cuticular genes which correlates with previous studies implicating both of these gene families in pyrethroid resistance. qPCR carried out on six contigs indicates that these ESTs could be suitable for gene expression studies such as microarray. 31,000 sites were estimated to contain Single Nucleotide Polymorphisms (SNPs) and analysis of SNPs from 20 contigs suggested that most of these SNPs are likely to be true SNPs. Gene conservation analysis confirmed the close phylogenetic relationship between An. funestus and An. gambiae.ConclusionThis study represents a significant advance for the genetics and genomics of An. funestus since it provides an extensive set of both Expressed Sequence Tags (ESTs) and SNPs which can be readily adopted for the design of new genomic tools such as microarray or SNP platforms.
| S-EPMC3045460 | biostudies-literature
Project description:Pyrethroid resistance in Anopheles funestus is a potential obstacle to malaria control in Africa. Tools are needed to detect resistance in field populations. We have been using a positional cloning approach to identify the major genes conferring pyrethroid resistance in this vector. A quantitative trait locus (QTL) named rp1 explains 87% of the genetic variance in pyrethroid susceptibility in two families from reciprocal crosses between susceptible and resistant strains. Two additional QTLs of minor effect, rp2 and rp3, were also detected. We sequenced a 120-kb BAC clone spanning the rp1 QTL and identified 14 protein-coding genes and one putative pseudogene. Ten of the 14 genes encoded cytochrome P450s, and expression analysis indicated that four of these P450s were differentially expressed between susceptible and resistant strains. Furthermore, two of these genes, CYP6P9 and CYP6P4, which are 25 and 51 times overexpressed in resistant females, are tandemly duplicated in the BAC clone as well as in laboratory and field samples, suggesting that P450 gene duplication could contribute to pyrethroid resistance in An. funestus. Single nucleotide polymorphisms (SNPs) were identified within CYP6P9 and CYP6P4, and genotyping of the progeny of the genetic crosses revealed a maximum penetrance value f(2) = 1, confirming that these SNPs are valid resistance markers in the laboratory strains. This serves as proof of principle that a DNA-based diagnostic test could be designed to trace metabolic resistance in field populations. This will be a major advance for insecticide resistance management in malaria vectors, which requires the early detection of resistance alleles.
| S-EPMC2661802 | biostudies-literature