Population Structure of Anopheles gambiae in Africa

The population structure of Anopheles gambiae in Africa was studied using 11 microsatellite loci in 16 samples from 10 countries. All loci are located outside polymorphic inversions. Heterogeneity among loci was detected and two putative outlier loci were removed from analyses aimed at capturing genome-wide patterns. Two main divisions of the gene pool were separated by high differentiation (F(ST) > 0.1). The northwestern (NW) division included populations from Senegal, Ghana, Nigeria, Cameroon, Gabon, Democratic Republic of Congo (DRC), and western Kenya. The southeastern (SE) division included populations from eastern Kenya, Tanzania, Malawi, and Zambia. Inhospitable environments for A. gambiae along the Rift Valley partly separate these divisions. Reduced genetic diversity in the SE division and results of an analysis based on private alleles support the hypothesis that a recent bottleneck, followed by colonization from the NW populations shaped this structure. In the NW division, populations possessing the M rDNA genotype appeared to form a monophyletic clade. Although genetic distance increased with geographic distance, discontinuities were suggested between certain sets of populations. The absence of heterozygotes between sympatric M and S populations in the DRC and the high differentiation in locus 678 (F(ST)>0.28) contrasted with low differentiation in all other loci (-0.02相似文献   

The Population Genomics of Trans-Specific Inversion Polymorphisms in Anopheles gambiae

In the malaria mosquito Anopheles gambiae polymorphic chromosomal inversions may play an important role in adaptation to environmental variation. Recently, we used microarray-based divergence mapping combined with targeted resequencing to map nucleotide differentiation between alternative arrangements of the 2La inversion. Here, we applied the same technique to four different polymorphic inversions on the 2R chromosome of An. gambiae. Surprisingly, divergence was much lower between alternative arrangements for all 2R inversions when compared to the 2La inversion. For one of the rearrangements, 2Ru, we successfully mapped a very small region (∼100 kb) of elevated divergence. For the other three rearrangements, we did not identify any regions of significantly high divergence, despite ample independent evidence from natural populations of geographic clines and seasonal cycling, and stable heterotic polymorphisms in laboratory populations. If these inversions are the targets of selection as hypothesized, we suggest that divergence between rearrangements may have escaped detection due to retained ancestral polymorphism in the case of the youngest 2R rearrangements and to extensive gene flux in the older 2R inversion systems that segregate in both An. gambiae and its sibling species An. arabiensis.MORE than 70 years ago Dobzhansky and Sturtevant (1938) first discovered polymorphic inversion arrangements carried by various Drosophila pseudoobscura populations. After observing correlations between environmental conditions and inversion frequencies, Dobzhansky proposed that inversions are under strong selection due to their role in promoting local adaptation to the heterogeneous conditions a species encounters both spatially and temporally (Dobzhansky 1944, 1948; Powell 1997). More recent studies have implicated chromosomal inversions in the adaptation of a diversity of eukaryotes including humans (Coluzzi et al. 1979; Feder et al. 2003; Hoffmann et al. 2004; Stefansson et al. 2005). Long known to be common in dipteran insects, more recent HapMap data suggest that polymorphic inversions may be numerous in human populations and by extension other mammals (Bansal et al. 2007). Given their potential importance in facilitating adaptation, surprisingly little is known about the mechanism(s) or the genes responsible for maintaining inversion polymorphisms in natural populations.Gene exchange between inverted and standard arrangements, although reduced, can still occur through gene flux: the action of gene conversion and multiple crossovers in inversion heterozygotes (heterokaryotypes) (Chovnick 1973; Navarro et al. 1997; Schaeffer and Anderson 2005). Over time allelic variation unrelated to ecological adaptation should become homogenized between arrangements, while alleles which are under divergent selection pressures should remain in linkage disequilibrium with each other and with the inversion itself, leading to heightened differentiation between standard and inverted arrangements at and near the target loci. In principle, this process allows the identification of specific loci involved in adaptive divergence (Schaeffer et al. 2003; Schaeffer and Anderson 2005; Storz 2005). Consistent with this model, previous low-resolution studies of Drosophila inversions revealed heterogeneous patterns of nucleotide diversity relative to divergence, as well as the interspersion of regions of high and low genetic association potentially due to the interaction of selection and gene flux (Schaeffer et al. 2003; Kennington et al. 2006; but see Munte et al. 2005). The application of high-resolution tools flowing from completely sequenced genomes will facilitate the mapping of genes that are the targets of divergent natural selection within gene arrangements.Although Drosophila has been the favored model, the African malaria vector Anopheles gambiae sensu stricto also provides an excellent system for studying the maintenance of inversion polymorphisms, not only within a species but across speciation events of different ages in the An. gambiae sibling species complex. The nominal species An. gambiae s.s. (hereafter, An. gambiae) is synanthropic: almost exclusively biting humans, resting indoors, and exploiting anthropogenic larval habitats (Coluzzi 1999). This close association with humans, vital to making An. gambiae one of the most proficient vectors of malaria, is likely to have been facilitated by chromosomal inversions thought to confer adaptive benefits in heterogeneous climatic and ecological settings in Africa. Seven common polymorphic inversions exist on the second chromosome. Six of these are located on the right arm (2R): j, b, c, u, d, and k, while 2La is the only inversion on the left arm (Coluzzi et al. 2002). Facilitated by the sequenced reference genome (Holt et al. 2002), some of the breakpoints for these polymorphic inversions have been localized to small genomic regions (Sharakhov et al. 2006; Coulibaly et al. 2007; Sangare 2007). Most of these inversions appear to be the targets of strong selection. Five of the inversions (2La and 2Rb, -c, -d, and -u) are nonrandomly associated with degree of aridity; each cycles seasonally with rainfall, and all except 2Ru form stable geographic clines in frequency from mesic forest to xeric regions bordering the Sahara (Coluzzi et al. 1979; Toure et al. 1994, 1998; Powell et al. 1999). Inversion 2Rj is not clinal, but its distribution in Mali is consistent with adaptation to novel rockpool niches (Coluzzi et al. 1985; Manoukis et al. 2008).In the An. gambiae species complex, inversion polymorphisms can be maintained across the boundaries of emerging and even full species. An. gambiae and its sibling An. arabiensis, strictly sympatric throughout most of their extensive ranges in sub-Saharan Africa, differ by multiple fixed chromosomal rearrangements on the X but share three chromosome 2 inversions: 2La, fixed in An. arabiensis and polymorphic in An. gambiae; and 2Rb and -c, polymorphic in both species (Coluzzi et al. 1979, 2002). Moreover, these same inversions and all other common An. gambiae inversions with the exception of 2Rj are shared and polymorphic in two lineages apparently undergoing ecological speciation within An. gambiae—the assortatively mating M and S molecular forms (della Torre et al. 2002, 2005). Inversion frequencies are correlated with climatic and ecological conditions in parallel in both lineages (Costantini et al. 2009; Simard et al. 2009). Unlike the full species, the M and S incipient species are not distinguished by any fixed inversion differences. Indeed, genomewide divergence mapping between the M and S forms revealed that significant differentiation was confined to two small low-recombination regions adjacent to the centromeres of 2L and X which are distant from any inversions (Turner et al. 2005). Thus, in distinction to models of speciation invoking inversions as facilitating the persistence of hybridizing species (Noor et al. 2001; Rieseberg 2001; Ortiz-Barrientos et al. 2002; Navarro and Barton 2003), the An. gambiae data suggest that chromosome 2 inversions are not directly responsible for reproductive isolation. Instead, the same chromosome 2 inversion polymorphisms appear to confer similar ecological benefits, within and across species boundaries. A long-term research goal is to identify the mechanisms and the genes controlling these processes.Previously we conducted the first high-density genomic scan of divergence across a chromosomal inversion (2La) in An. gambiae (White et al. 2007). By hybridizing genomic DNA from S form mosquitoes homokaryotypic for alternate gene arrangements on chromosome 2L (2La or 2L+^a) to oligonucleotide microarrays we were able to measure divergence across the 22-Mb inversion at nearly 14,000 markers. Differentiation in the rearranged region was significantly higher than in collinear portions of chromosome 2L. Between breakpoints the pattern of differentiation was heterogeneous: two genomic clusters of significantly higher divergence were identified near but not adjacent to the breakpoints. Directed resequencing within the S form confirmed these results and suggested that both clusters contained genes targeted by selection. Observed levels of linkage disequilibrium between the 2La breakpoints and markers in the clusters are highly unlikely under a neutral scenario, in light of known recombination rates and plausible estimates of the age of the inversion.The present study characterizes the patterns of genetic variation in polymorphic rearrangements on the opposite (right) arm of chromosome 2: 2Rj, -b, -c, and -u. With the goal of identifying candidate genes maintaining these inversions in natural populations, we applied microarray-based divergence mapping to measure differentiation between alternative 2R arrangements. Because three of four inversions have taxonomic distributions that span incipient and/or completed speciation events, we validated the microarray findings by targeted sequencing in multiple taxa: sympatric Malian populations of An. gambiae M and S forms, and the sibling species An. arabiensis.     

Population structure, speciation, and introgression in the Anopheles gambiae complex

We review here what is known about the population structure and evolutionary dynamics of members of the Anopheles gambiae complex with emphasis on the situation in West Africa. First, the importance of the 2nd chromosome inversion polymorphism is demonstrated especially in adaptation to levels of aridity, a major environmental variable in Africa. This affects the distribution of karyotypes on both a macro- and micro-geographic scale as well as temporally. Such differentiation leads to karyotypes being differentially effective transmitters of malaria and differentially susceptible to indoor residual spraying of insecticides. Second, we review the evidence that cryptic taxa, especially in An. gambiae s.s., exist. This observation stems from both karyotype studies and molecular studies. It is abundantly clear that West African populations of An. gambiae s.s. are often not panmictic units, with premating factors evidently acting to maintain distinct genetic forms. Third, we review phylogenetic studies that have revealed the presence of introgression between the two most important vectors, An. gambiae and An. arabiensis. This is most evident for the 2nd chromosome inversions. This interpretation of phylogenetic data is consistent with a direct laboratory study indicating inversions in this chromosome are stably maintained in back-crossed populations. All of this information has led to the view that members of the An. gambiae complex are highly variable with an abundance of adaptive genetic variation. This presents a significant challenge to vector control programs designed to reduce malaria in sub-Saharan Africa.     

Landscape Movements of Anopheles gambiae Malaria Vector Mosquitoes in Rural Gambia

Background

For malaria control in Africa it is crucial to characterise the dispersal of its most efficient vector, Anopheles gambiae, in order to target interventions and assess their impact spatially. Our study is, we believe, the first to present a statistical model of dispersal probability against distance from breeding habitat to human settlements for this important disease vector.

Methods/Principal Findings

We undertook post-hoc analyses of mosquito catches made in The Gambia to derive statistical dispersal functions for An. gambiae sensu lato collected in 48 villages at varying distances to alluvial larval habitat along the River Gambia. The proportion dispersing declined exponentially with distance, and we estimated that 90% of movements were within 1.7 km. Although a ‘heavy-tailed’ distribution is considered biologically more plausible due to active dispersal by mosquitoes seeking blood meals, there was no statistical basis for choosing it over a negative exponential distribution. Using a simple random walk model with daily survival and movements previously recorded in Burkina Faso, we were able to reproduce the dispersal probabilities observed in The Gambia.

Conclusions/Significance

Our results provide an important quantification of the probability of An. gambiae s.l. dispersal in a rural African setting typical of many parts of the continent. However, dispersal will be landscape specific and in order to generalise to other spatial configurations of habitat and hosts it will be necessary to produce tractable models of mosquito movements for operational use. We show that simple random walk models have potential. Consequently, there is a pressing need for new empirical studies of An. gambiae survival and movements in different settings to drive this development.     

Pyrethroid Resistance in Anopheles gambiae,in Bomi County,Liberia, Compromises Malaria Vector Control

Background

Long Lasting Insecticidal Nets (LLIN) and Indoor Residual Spraying (IRS) have both proven to be effective malaria vector control strategies in Africa and the new technology of insecticide treated durable wall lining (DL) is being evaluated. Sustaining these interventions at high coverage levels is logistically challenging and, furthermore, the increase in insecticide resistance in African malaria vectors may reduce the efficacy of these chemical based interventions. Monitoring of vector populations and evaluation of the efficacy of insecticide based control approaches should be integral components of malaria control programmes. This study reports on entomological survey conducted in 2011 in Bomi County, Liberia.

Methods

Anopheles gambiae larvae were collected from four sites in Bomi, Liberia, and reared in a field insectary. Two to five days old female adult An gambiae s.l. were tested using WHO tube (n = 2027) and cone (n = 580) bioassays in houses treated with DL or IRS. A sample of mosquitoes (n = 169) were identified to species/molecular form and screened for the presence of knock down resistance (kdr) alleles associated with pyrethroid resistance.

Results

Anopheles gambiae s.l tested were resistant to deltamethrin but fully susceptible to bendiocarb and fenithrothion. The corrected mortality of local mosquitoes exposed to houses treated with deltamethrin either via IRS or DL was 12% and 59% respectively, suggesting that resistance may affect the efficacy of these interventions. The presence of pyrethroid resistance was associated with a high frequency of the 1014F kdr allele (90.5%) although this mutation alone cannot explain the resistance levels observed.

Conclusion

High prevalence of resistance to deltamethrin in Bomi County may reduce the efficacy of malaria strategies relying on this class of insecticide. The findings highlight the urgent need to expand and sustain monitoring of insecticide resistance in Liberian malaria vectors, evaluate the effectiveness of existing interventions and develop appropriate resistance management strategies.     

Population dynamics of Plasmodium falciparum sporogony in laboratory-infected Anopheles gambiae.

The population dynamics of cultured Plasmodium falciparum parasites was examined during their sporogonic development in Anopheles gambiae mosquitoes. Estimates of absolute densities were determined for each life stage, and life tables were constructed for each of 38 experimental infections. Macrogametocyte and ookinete mortalities contributed equally to the overall mortality. On average, there was a 40-fold decrease in parasite numbers in the transition from the macrogametocyte to the ookinete stage, a 69-fold decrease in the transition from ookinete to oocyst stages, and a total net decrease in parasite numbers from macrogametocyte to oocyst stage of 2,754-fold (i.e., multiplicative). There was no relationship between macrogametocyte and ookinete densities due to the inherent variability in fertility among different gametocyte cultures. There was a curvilinear relationship (r2 = 0.66) between ookinete and oocyst densities. Above a threshold of about 30 ookinetes/mosquito, the oocyst yield per ookinete became increasingly greater with increasing ookinete density. There was a linear relationship (r2 = 0.73) between oocyst and sporozoite densities, with an average of 663 salivary gland sporozoites produced per oocyst. Sporozoite production per oocyst was not affected by oocyst density and virtually all oocyst infections resulted in sporozoite infections of the salivery glands. This quantitative study indicates that the sporogony of cultured P. falciparum in laboratory-infected A. gambiae is an inefficient process and that the ookinete is the key transitional stage affecting the probability of vector infectivity.     

Gene flow among populations of the malaria vector, Anopheles gambiae, in Mali, West Africa

The population structure of the Anopheles gambiae complex is unusual, with several sibling species often occupying a single area and, in one of these species, An. gambiae sensu stricto, as many as three "chromosomal forms" occurring together. The chromosomal forms are thought to be intermediate between populations and species, distinguishable by patterns of chromosome gene arrangements. The extent of reproductive isolation among these forms has been debated. To better characterize this structure we measured effective population size, N(e), and migration rates, m, or their product by both direct and indirect means. Gene flow among villages within each chromosomal form was found to be large (N(e)m > 40), was intermediate between chromosomal forms (N(e)m approximately 3-30), and was low between species (N(e)m approximately 0.17-1.3). A recently developed means for distinguishing among certain of the forms using PCR indicated rates of gene flow consistent with those observed using the other genetic markers.     

The distribution and inversion polymorphism of chromosomally recognized taxa of the Anopheles gambiae complex in Mali, West Africa

Data from polytene chromosome studies on the Anopheles gambiae complex in Mali were reviewed. The banding pattern was successfully scored in 17,705 specimens from 76 sampling sites representing the main ecological strata of the country. Two members of the complex, namely An. arabiensis and An. gambiae, were found widespread and frequently sympatric, with the latter prevalent in most localities. Population genetic analysis of the inversion polymorphisms indicated the existence of panmictic conditions for An. arabiensis only, whereas the parallel study of An. gambiae supported its splitting into at least three reproductive units, characterized by different 2R chromosome arrangements, designated Bamako, Mopti and Savanna. The chromosomal evidence was consistent with the hypothesis of complete reproductive isolation between Bamako and Mopti. Partial isolation between these two taxa and Savanna was suggested by the scoring of hypothetical hybrid 2R heterokaryotypes in various samples, but the actual hybrid origin of these specimens was not confirmed. Different patterns of geographical and seasonal distribution were shown as follows. An. arabiensis prevails in arid savannas (Sahel and Northern Sudan savanna) out of the flooded or irrigated zones; it is able to withstand the most arid conditions of Saharan localities and its breeding might extend throughout the dry season. An. gambiae Savanna and Bamako prevail in relatively humid savannas (Southern Sudan savanna) and their breeding generally occurs only during the rainy season. The Savanna taxon was almost absent in flooded or irrigated zones and in riverine localities; the Bamako taxon is distributed along the upper river Niger and its tributaries. An. gambiae Mopti extends its range in all ecological zones present in Mali including the Sahel and predesertic areas, showing high relative frequencies up to absolute dominance in flooded or irrigated areas; its breeding is highly successful also during the dry season. Rainfall at the sampling sites was found to correlate positively with the frequency of Savanna and negatively with the frequency of Mopti. The remarkable ecological flexibility of the latter was found associated with wide seasonal and geographical variations in its 2R inversion polymorphism bc/u. Higher frequencies of the bc arrangement were recorded both in the Southern localities during the dry season and in the Northern more arid localities during the rainy season. The absence or scarcity of An. arabiensis and An. gambiae Savanna in most flooded or irrigated zones suggests their competitive exclusion by An. gambiae Mopti.     

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).     

Insights from the Genome Annotation of Elizabethkingia anophelis from the Malaria Vector Anopheles gambiae

Elizabethkingia anophelis is a dominant bacterial species in the gut ecosystem of the malaria vector mosquito Anopheles gambiae. We recently sequenced the genomes of two strains of E. anophelis, R26^T and Ag1, isolated from different strains of A. gambiae. The two bacterial strains are identical with a few exceptions. Phylogenetically, Elizabethkingia is closer to Chryseobacterium and Riemerella than to Flavobacterium. In line with other Bacteroidetes known to utilize various polymers in their ecological niches, the E. anophelis genome contains numerous TonB dependent transporters with various substrate specificities. In addition, several genes belonging to the polysaccharide utilization system and the glycoside hydrolase family were identified that could potentially be of benefit for the mosquito carbohydrate metabolism. In agreement with previous reports of broad antibiotic resistance in E. anophelis, a large number of genes encoding efflux pumps and β-lactamases are present in the genome. The component genes of resistance-nodulation-division type efflux pumps were found to be syntenic and conserved in different taxa of Bacteroidetes. The bacterium also displays hemolytic activity and encodes several hemolysins that may participate in the digestion of erythrocytes in the mosquito gut. At the same time, the OxyR regulon and antioxidant genes could provide defense against the oxidative stress that is associated with blood digestion. The genome annotation and comparative genomic analysis revealed functional characteristics associated with the symbiotic relationship with the mosquito host.     

Population genetics of Plasmodium resistance genes in Anopheles gambiae: no evidence for strong selection

Anopheles mosquitoes are the primary vectors for malaria in Africa, transmitting the disease to more than 100 million people annually. Recent functional studies have revealed mosquito genes that are crucial for Plasmodium development, but there is presently little understanding of which genes mediate vector competence in the wild, or evolve in response to parasite-mediated selection. Here, we use population genetic approaches to study the strength and mode of natural selection on a suite of mosquito immune system genes, CTL4, CTLMA2, LRIM1, and APL2 (LRRD7), which have been shown to affect Plasmodium development in functional studies. We sampled these genes from two African populations of An. gambiae s.s., along with several closely related species, and conclude that there is no evidence for either strong directional or balancing selection on these genes. We highlight a number of challenges that need to be met in order to apply population genetic tests for selection in Anopheles mosquitoes; in particular the dearth of suitable outgroup species and the potential difficulties that arise when working within a closely-related species complex.     

The Virulent Wolbachia Strain wMelPop Efficiently Establishes Somatic Infections in the Malaria Vector Anopheles gambiae

Wolbachia pipientis bacteria are maternally inherited endosymbionts that are of interest to control the Anopheles mosquito vectors of malaria. Wolbachia does not infect Anopheles mosquitoes in nature, although cultured Anopheles cells can be infected. Here, we show that the virulent Wolbachia strain wMelPop can survive and replicate when injected into female Anopheles gambiae adults, but the somatic infections established are avirulent. These in vivo data suggest that stable Wolbachia infections of Anopheles may be possible.Infecting up to 500 million people per year (with almost 3 million annual deaths), malaria is the most important vector-borne disease in the world (8, 30, 31, 32). The Plasmodium parasites that cause the disease are transmitted to humans by the bite of Anopheles mosquitoes, with Anopheles gambiae being the principle vector in sub-Saharan Africa (6). Malaria control is limited by the lack of a vaccine and by parasite and mosquito evolution of drug and insecticide resistance (9, 28, 31). In light of these problems, there has been a recent concerted effort to develop innovative methods for malaria control based on the genetic modification of Anopheles mosquitoes (transgenesis) or their associated symbiotic microorganisms (paratransgenesis) (5, 10, 11, 13, 15, 23, 25, 27, 36, 37).In many mosquitoes, Wolbachia pipientis symbionts are the causative agents of cytoplasmic incompatibility, a phenomenon where matings between uninfected females and infected males have reduced egg hatch, while matings in the reciprocal cross are fertile. In a mixed population, infected females have a reproductive advantage which can allow Wolbachia to increase rapidly in frequency due to maternal inheritance. The propensity of Wolbachia to “drive” through populations has been investigated using mathematical models and has been validated by both laboratory and field investigations (20, 21, 33, 34, 35, 36).There are three scenarios currently envisioned to use Wolbachia as part of a malaria control strategy: (i) use Wolbachia spread to “drive” refractory transgenes into Anopheles populations, converting the mosquito population into one that cannot maintain transmission of the malaria parasites (18, 21, 24, 29, 33, 36); (ii) release Wolbachia-infected males into uninfected Anopheles populations to reduce population sizes through cytoplasmic incompatibility, similar to the sterile insect technique but without exposing males to radiation or chemical sterilants that could lower their fitness (2, 4, 37); and (iii) release mosquitoes infected with pathogenic or virulent Wolbachia strains that shorten mosquito life span. Pathogens must pass through an extrinsic incubation period in the vector before they are able to be transmitted. By shortening mosquito life span, it is theoretically possible to reduce the number of mosquitoes that live through the extrinsic incubation period and become infectious (14, 15, 17, 20, 24).All of the above strategies require the stable transfer of Wolbachia into Anopheles. Wolbachia symbionts are common in mosquitoes, but no infections have ever been identified in any species of Anopheles (12, 22, 26). The negative infection status of natural Anopheles populations offers good potential for Wolbachia-based malaria control strategies, since preexisting infections can complicate the behavior of introduced infections (33). However, the absence of natural infections in anophelines has led some to suggest that Anopheles mosquitoes may be physiologically/genetically incapable of harboring Wolbachia infections (1, 29). If this hypothesis is true, then Wolbachia-based malaria control strategies are likely doomed to fail. In vitro studies demonstrated that cultured immunocompetent Anopheles gambiae cell lines (Sua5B and Moss55) are fully competent to harbor infections of distinct Wolbachia strains (the “A” supergroup strains wRi and wMelPop from Drosophila simulans and Drosophila melanogaster and the “B” supergroup strain wAlbB from Aedes albopictus) (16, 18). Some cultured infections reached very high levels where 100% of cells were infected at extremely high levels (wAlbB in Sua5B and wMelPop in Moss55) (16, 18), while other strains were eventually eliminated from the cells (wRi in Sua5B cells) (24). The combined results of these experiments, using multiple Wolbachia strains and multiple Anopheles cell lines, indicate that there is no intrinsic genetic block to Wolbachia infection in Anopheles cells, although certain strains of Wolbachia may be more likely to colonize Anopheles than others.In this study, we investigated the establishment of in vivo Wolbachia infections in Anopheles gambiae (Keele strain) mosquitoes by injection of the virulent Wolbachia strain wMelPop into the hemolymph of adult female mosquitoes. Approximately 200 adult mosquitoes were reared in 30-cm cube cages in a walk-in insectary held at 28°C and 80% relative humidity on a 12:12 h light/dark cycle. Mosquitoes were allowed access to a cotton wick soaked in 10% sucrose as a carbohydrate source. Adults were allowed to blood feed on an anesthetized mouse 5 days postemergence according to JHU animal use protocol MO-03H210. Two days after blood feeding, an oviposition substrate (consisting of a filter paper cone inside a 50-ml beaker half filled with water) was introduced into cages and removed the next day for egg collection. Approximately 250 eggs were placed into a 41- by 34- by 6-cm rearing tray half filled with distilled water and one pellet of dry cat food, with one additional pellet added to each tray daily after day 3. Larvae were removed, and tray water changed if polluted. Pupae were picked with an eyedropper, placed in a cup, and introduced into cages (∼200 pupae/cage) to begin the next generation.wMelPop was cultured in Anopheles gambiae Moss55 cells (16), purified, and assessed for viability as described previously (18, 19). Purified Wolbachia cells were suspended in culture medium and adjusted to a final concentration of 10⁸ bacteria per ml. Using a calibrated glass capillary needle, amounts of 100 to 200 nl suspended Wolbachia cells were injected into the thorax of 2-day-old, cold-immobilized adult Anopheles gambiae females. Injected mosquitoes were held at 18°C for 5 days and then transferred to the 28°C insectary. Mosquitoes were allowed to blood feed on a mouse twice per week.Mosquito genomic DNA was extracted by salt extraction/ethanol precipitation as described previously (21), quantified using a NanoDrop spectrophotometer, and adjusted to 20 ng/μl. Wolbachia infections in individual mosquitoes were detected by PCR amplification of a fragment of the Wolbachia 16S rRNA gene (440 bp) using primers WspecF and WspecR (18). As a control, we amplified a 400-bp fragment from the Anopheles mitochondrial NADH dehydrogenase subunit 4 gene (ND4) (18). Mosquitoes were assayed for Wolbachia infection by PCR at 6, 10, 20, or 30 days postinjection (p.i.). In a second experiment, mosquitoes were assayed at 0, 3, 8, 13, 15, and 21 days p.i. Amplified fragments were separated by 1% agarose gel electrophoresis, stained with ethidium bromide, and viewed under UV light. Template DNA from infected and uninfected Moss55 cells was included as positive and negative controls.Quantitative PCR (qPCR) was used to determine if Wolbachia could survive and replicate in Anopheles by comparing normalized Wolbachia levels in individual mosquitoes at day 6 and day 30 p.i. The relative abundance of wMelPop bacteria in each mosquito was assessed by comparing the abundance of the single-copy Wolbachia ankyrin repeat gene WD_0550 (16) to that of the single-copy Anopheles gambiae ribosomal S7 gene (forward, 5′-TCC-TGG-AGC-TGG-AGA-TGA-AC-3′, and reverse, 5′-GAC-GGG-TCT-GTA-CCT-TCT-GG-3′). For each time point, 14 mosquitoes (biological replicates) were examined. Duplicate reactions were performed for every mosquito, and the results differed by less than 3%, demonstrating consistency of the assay. qPCR was performed using an ABI Prism 7300 detection system (Applied Biosystems) with a QuantiTect SYBR green PCR kit (Qiagen). Determinations of relative abundance of wMelPop in each mosquito and relative changes in wMelPop levels between time points, confidence interval estimation, and statistical analyses were carried out as described by Yuan et al. (38).To test for virulence of wMelPop, 2-day-old adult female mosquitoes were injected with either wMelPop purified from cell culture or filtered lysate of uninfected Moss55 cells (control) and held at 18°C for 2 days as described above. Injected mosquitoes were held an additional 3 days at 28°C to control for mortality due to the injection procedure. At day 5 p.i., mosquitoes were moved into pint-sized cup cages for life table experiments. Approximately 25 mosquitoes were placed in each cage (two replicate control cages and three replicate Wolbachia treatment cages) and the entire experiment replicated two times, for a total of four control cages and six Wolbachia treatment cages. Mosquitoes were provided a cotton pad soaked in 10% sucrose but were not given access to blood. Dead mosquitoes were removed from each cage approximately every other day. For each experiment, mortality data were used to construct treatment-specific cohort life tables (3). Because the data did not conform to parametric assumptions, they were analyzed by the Mann-Whitney U test using STATVIEW (SAS Corporation).In injected mosquitoes, Wolbachia bacteria were detectable by PCR at all time points, as follows [infection frequency (95% exact binomial confidence interval)]: day 6, 0.875 (0.617 to 0.985; n = 16); day 10, 0.75 (0.401 to 0.968; n = 8); day 20, 0.722 (0.465 to 0.903; n = 18); and day 30, 1.0 (0.794 to 1.0; n = 13). In further experiments, Wolbachia bacteria were easily detectable through day 3 p.i. but were weak or not detectable by conventional PCR by day 8 p.i. After 13 days p.i., Wolbachia bacteria were easily detectable again, and the bands increased in intensity for the remainder of the time series experiment (Fig. ​(Fig.1).1). This initial decrease, followed by an increase, in the apparent infection rate is possibly due to initial clearance of some of the injected bacteria and then establishment of infection and bacterial replication. By qPCR, a highly statistically significant 42-fold increase in the normalized Wolbachia level was observed: on day 6 p.i., there were 23.7 Wolbachia genomes per host genome (95% confidence interval, 10.6 to 52.7; n = 14), and on day 30 p.i., there were 992 Wolbachia genomes per host genome (95% confidence interval, 433.6 to 2,267.4; n = 14) (Mann-Whitney U test, tied Z value = −4.319; P < 0.0001). Since Wolbachia cannot replicate in the extracellular environment (19), these results confirm that injected bacteria are able to infect cells, survive, and replicate in Anopheles gambiae in vivo.Open in a separate windowFIG. 1.Typical results using conventional PCR, showing changes in Wolbachia levels in injected adult Anopheles gambiae females at sequential time points postinjection. Results for mt control (host ND4 mitochondrial gene) indicate that PCR efficiency was approximately equal for all samples. M, 100-bp marker; d, days.wMelPop is a virulent Wolbachia strain that reduces the life span of its host by approximately 50%. While originally found and characterized in a laboratory colony of Drosophila melanogaster, it has similar pathogenic effects when artificially transferred into Drosophila simulans (14), and recently, the yellow fever mosquito Aedes aegypti (15). However, there was no statistically significant difference in survival trajectories between Anopheles gambiae mosquitoes injected with wMelPop and mosquitoes injected with filtered uninfected cell lysate (Mann-Whitney U test, tied Z value = −1.799; P = 0.702) (Fig. ​(Fig.2).2). Although wMelPop replicates to high levels in injected Anopheles (approximately 1,000 bacterial genomes per host genome), these levels do not seem to be associated with virulence. It is possible that the virulence of wMelPop has been attenuated during its culture in Moss55 cells, although during long-term culture in an Aedes aegypti cell line, wMelPop retained its virulent phenotype when reintroduced into either Drosophila or Aedes aegypti in vivo (15, 16). The specific mechanism of wMelPop virulence is not completely understood, but it seems that increased host mortality is not simply due to overreplication and high infection levels but rather to overreplication in and damage to specific host tissues, such as the brain and central nervous system (14, 15, 17). Investigation into the tissue localization of Wolbachia in injected Anopheles mosquitoes is currently ongoing, but in light of these results, it is reasonable to hypothesize that when injected into the hemolymph, Wolbachia bacteria reach high levels in some mosquito tissues but either do not infect or do not replicate in the Anopheles central nervous system. It remains to be seen whether vertically acquired infections will show virulence in Anopheles gambiae.Open in a separate windowFIG. 2.Mean survival trajectories of wMelPop-injected versus cell lysate-injected Anopheles gambiae adult females. Survival trajectories do not differ significantly. Error bars show standard deviations.Previous studies showed that cultured Anopheles gambiae cells can be infected with Wolbachia (16, 18), but no data were available to assess whether the in vitro results could be extrapolated to Anopheles mosquitoes in vivo. The experiments outlined in this paper demonstrate that Wolbachia can infect Anopheles mosquitoes in vivo. However, for a Wolbachia-based malaria control strategy to be effective, simply infecting Anopheles by injection is not sufficient—the infection must be transmitted vertically to offspring. Stable (100%) vertical transmission of Wolbachia after injection into adults has been reported for Drosophila melanogaster (7). A similar phenomenon has been reported for Aedes aegypti, but transmission was unstable (approximately 40%) (27). Experiments to determine whether Wolbachia bacteria injected into the hemolymph of adult Anopheles will be transmitted vertically to offspring are ongoing, and if efficient vertical transmission of the symbionts can be established, Wolbachia-based strategies for malaria control should be possible.     

Islands and Stepping-Stones: Comparative Population Structure of Anopheles gambiae sensu stricto and Anopheles arabiensis in Tanzania and Implications for the Spread of Insecticide Resistance

Population genetic structures of the two major malaria vectors Anopheles gambiae s.s. and An. arabiensis, differ markedly across Sub-Saharan Africa, which could reflect differences in historical demographies or in contemporary gene flow. Elucidation of the degree and cause of population structure is important for predicting the spread of genetic traits such as insecticide resistance genes or artificially engineered genes. Here the population genetics of An. gambiae s.s. and An. arabiensis in the central, eastern and island regions of Tanzania were compared. Microsatellite markers were screened in 33 collections of female An. gambiae s.l., originating from 22 geographical locations, four of which were sampled in two or three years between 2008 and 2010. An. gambiae were sampled from six sites, An. arabiensis from 14 sites, and both species from two sites, with an additional colonised insectary sample of each species. Frequencies of the knock-down resistance (kdr) alleles 1014S and 1014F were also determined. An. gambiae exhibited relatively high genetic differentiation (average pairwise F_ST = 0.131), significant even between nearby samples, but without clear geographical patterning. In contrast, An. arabiensis exhibited limited differentiation (average F_ST = 0.015), but strong isolation-by-distance (Mantel test r = 0.46, p = 0.0008). Most time-series samples of An. arabiensis were homogeneous, suggesting general temporal stability of the genetic structure. An. gambiae populations from Dar es Salaam and Bagamoyo were found to have high frequencies of kdr 1014S (around 70%), with almost 50% homozygote but was at much lower frequency on Unguja Island, with no. An. gambiae population genetic differentiation was consistent with an island model of genetic structuring with highly restricted gene flow, contrary to An. arabiensis which was consistent with a stepping-stone model of extensive, but geographically-restricted gene flow.     

The Effective Population Size of Malaria Mosquitoes: Large Impact of Vector Control

Malaria vectors in sub-Saharan Africa have proven themselves very difficult adversaries in the global struggle against malaria. Decades of anti-vector interventions have yielded mixed results—with successful reductions in transmission in some areas and limited impacts in others. These varying successes can be ascribed to a lack of universally effective vector control tools, as well as the development of insecticide resistance in mosquito populations. Understanding the impact of vector control on mosquito populations is crucial for planning new interventions and evaluating existing ones. However, estimates of population size changes in response to control efforts are often inaccurate because of limitations and biases in collection methods. Attempts to evaluate the impact of vector control on mosquito effective population size (N_e) have produced inconclusive results thus far. Therefore, we obtained data for 13–15 microsatellite markers for more than 1,500 mosquitoes representing multiple time points for seven populations of three important vector species—Anopheles gambiae, An. melas, and An. moucheti—in Equatorial Guinea. These populations were exposed to indoor residual spraying or long-lasting insecticidal nets in recent years. For comparison, we also analyzed data from two populations that have no history of organized vector control. We used Approximate Bayesian Computation to reconstruct their demographic history, allowing us to evaluate the impact of these interventions on the effective population size. In six of the seven study populations, vector control had a dramatic impact on the effective population size, reducing N_e between 55%–87%, the exception being a single An. melas population. In contrast, the two negative control populations did not experience a reduction in effective population size. This study is the first to conclusively link anti-vector intervention programs in Africa to sharply reduced effective population sizes of malaria vectors.     

How the Malaria Vector Anopheles gambiae Adapts to the Use of Insecticide-Treated Nets by African Populations

BackgroundInsecticide treated bed nets have been recommended and proven efficient as a measure to protect African populations from malaria mosquito vector Anopheles spp. This study evaluates the consequences of bed nets use on vectors resistance to insecticides, their feeding behavior and malaria transmission in Dielmo village, Senegal, were LLINs were offered to all villagers in July 2008.MethodsAdult mosquitoes were collected monthly from January 2006 to December 2011 by human landing catches (HLC) and by pyrethroid spray catches (PCS). A randomly selected sub-sample of 15–20% of An. gambiae s.l. collected each month was used to investigate the molecular forms of the An. gambiae complex, kdr mutations, and Plasmodium falciparum circumsporozoite (CSP) rate. Malaria prevalence and gametocytaemia in Dielmo villagers were measured quarterly.ResultsInsecticide susceptible mosquitoes (wild kdr genotype) presented a reduced lifespan after LLINs implementation but they rapidly adapted their feeding behavior, becoming more exophageous and zoophilic, and biting earlier during the night. In the meantime, insecticide-resistant specimens (kdr L1014F genotype) increased in frequency in the population, with an unchanged lifespan and feeding behaviour. P. falciparum prevalence and gametocyte rate in villagers decreased dramatically after LLINs deployment. Malaria infection rate tended to zero in susceptible mosquitoes whereas the infection rate increased markedly in the kdr homozygote mosquitoes.ConclusionDramatic changes in vector populations and their behavior occurred after the deployment of LLINs due to the extraordinary adaptative skills of An. gambiae s. l. mosquitoes. However, despite the increasing proportion of insecticide resistant mosquitoes and their almost exclusive responsibility in malaria transmission, the P. falciparum gametocyte reservoir continued to decrease three years after the deployment of LLINs.     

Flight performance of the malaria vectors Anopheles gambiae and Anopheles atroparvus.

The flight potential and metabolism of two malaria vectors, Anopheles gambiae s.str. and An. atroparvus, were analyzed on flightmills. The flight distance, the flight time, and individual flight activities of females were recorded during 22 h flight trials. The glycogen and lipid before flight, after flight, and of unflown controls were measured for starved, sugar-, or blood-fed females. Maximal flight distances of An. gambiae were 9 km when sugar-fed and 10 km when blood-fed, while in starved females it was below 3 km and the average speed was around 1 km/h. In Anopheles atroparvus, the maximal flight distances were 10-12 km when sugar-fed, 4.5 km when blood-fed, and below 3.5 km when starved, with an average speed of 1.3 km/h. Flight performances consisted of 1-4 h intervals of continuous flights, but mainly of bouts shorter than one h, randomly distributed during the long flight trials in both species. An. gambiae utilized an average of 47% of its pre-flight carbohydrate reserves for survival and 38% for flight at a rate of 0.07 cal/h/female. After a blood meal they utilized 11% for survival and 61% for flight at a rate of 0.04 cal/h. At the same time, 25% of the pre-flight lipid was mobilized for flight at a rate of 0.09 cal/h when sugar-fed and 22% when blood-fed at a rate of 0.06 cal/h; lipid was barely mobilized for survival. An. atroparvus differed: carbohydrate mobilization was 28% for survival and 41% for flight at a rate of 0.15 cal/h when sugar-fed; lipid mobilization for flight was only 13% at a rate of 0.06 cal/h. After a blood meal only 2% of the pre-flight lipid was used (0.02 cal/h). The contribution of carbohydrate reserves for flight metabolism at the high rate of 0.21 cal/h could not be fully elucidated because its decrease coincided with a pronounced resynthesis from the blood meal. An. atroparvus always depended on sugar meals for its flight activities and barely utilized lipid reserves. An. gambiae was independent of sugar sources for strong flights due to its early blood feeding and because of its equicaloric lipid mobilization during flights. Strong evidence for lipid oxidation during its flight is discussed.     

Genetic differentiation of Anopheles gambiae s.s. populations in Mali,West Africa,using microsatellite loci

Anopheles gambiae sensu stricto is a principal vector of malaria through much of sub-Saharan Africa, where this disease is a major cause of morbidity and mortality in human populations. Accordingly, population sizes and gene flow in this species have received special attention, as these parameters are important in attempts to control malaria by impacting its mosquito vector. Past measures of genetic differentiation have sometimes yielded conflicting results, in some cases suggesting that gene flow is extensive over vast distances (6000 km) and is disrupted only by major geological disturbances and/or barriers. Using microsatellite DNA loci from populations in Mali, West Africa, we measured genetic differentiation over uniform habitats favorable to the species across distances ranging from 62 to 536 km. Gene flow was strongly correlated with distance (r(2) = 0.77), with no major differences among chromosomes. We conclude that in this part of Africa, at least, genetic differentiation for microsatellite DNA loci is consistent with traditional models of isolation by distance.     

Evidence for recent population expansion in the evolutionary history of the malaria vectors Anopheles arabiensis and Anopheles gambiae.

Gene flow in malaria vectors is usually estimated based on differentiation indices (e.g., F(ST)) in order to predict the contemporary spread of genes such as those conferring resistance to insecticides. This approach is reliant on a number of assumptions, the most crucial, and the one most likely to be violated in these species, being mutation-migration-drift equilibrium. Tests of this assumption for the African malaria vectors Anopheles gambiae and Anopheles arabiensis are the focus of this study. We analyzed variation at 18 microsatellite loci and the ND5 region of the mitochondrial genome in two populations of each species. Equilibrium was rejected by six of eight tests for the A. gambiae population from western Kenya and by three tests in eastern Kenya. In western Kenya, all departures from equilibrium were consistent with a recent population expansion, but in eastern Kenya, there were traces of a recent expansion and a bottleneck. Equilibrium was also rejected by two of the eight tests for both A. arabiensis populations; the departure from equilibrium was consistent with an expansion. These multiple-locus tests detected a genomewide effect and therefore a demographic event rather than a locus-specific effect, as would be caused by selection. Disequilibrium due to a recent expansion in these species implies that rates of gene flow, as inferred from differentiation indices, are overestimates as they include a historical component. We argue that the same effect applies to the majority of pest species due to the correlation of their demography with that of humans.     

Breakdown in the Process of Incipient Speciation in Anopheles gambiae

Understanding genetic causes and effects of speciation in sympatric populations of sexually reproducing eukaryotes is challenging, controversial, and of practical importance for controlling rapidly evolving pests and pathogens. The major African malaria vector mosquito Anopheles gambiae sensu stricto (s.s.) is considered to contain two incipient species with strong reproductive isolation, hybrids between the M and S molecular forms being very rare. Following recent observations of higher proportions of hybrid forms at a few sites in West Africa, we conducted new surveys of 12 sites in four contiguous countries (The Gambia, Senegal, Guinea-Bissau, and Republic of Guinea). Identification and genotyping of 3499 A. gambiae s.s. revealed high frequencies of M/S hybrid forms at each site, ranging from 5 to 42%, and a large spectrum of inbreeding coefficient values from 0.11 to 0.76, spanning most of the range expected between the alternative extremes of panmixia and assortative mating. Year-round sampling over 2 years at one of the sites in The Gambia showed that M/S hybrid forms had similar relative frequencies throughout periods of marked seasonal variation in mosquito breeding and abundance. Genome-wide scans with an Affymetrix high-density single-nucleotide polymorphism (SNP) microarray enabled replicate comparisons of pools of different molecular forms, in three separate populations. These showed strong differentiation between M and S forms only in the pericentromeric region of the X chromosome that contains the molecular form-specific marker locus, with only a few other loci showing minor differences. In the X chromosome, the M/S hybrid forms were more differentiated from M than from S forms, supporting a hypothesis of asymmetric introgression and backcrossing.