Evidence for X-linked introgression between molecular forms of Anopheles gambiae from Angola

The M and S molecular forms of the African malaria vector Anopheles gambiae (Diptera: Culicidae) are morphologically identical incipient species in which reproductive isolation is incomplete, enabling low-level gene flow between forms. In an attempt to find differences between the M and S forms, sequence variation was studied at loci along the X chromosome in adult female An. gambiae from Angola. A high proportion of M form specimens from Angola (79% of the 456 X chromosomes sampled) were found to contain a 16-bp insertion in intron 4 of the X-linked GPRCCK1 locus, relative to the AgamP3 release of the An. gambiae PEST genome sequence. The insertion was in Hardy-Weinberg equilibrium in Angolan M form populations. The same insertion was found in all S form specimens examined, regardless of where in Africa they were sampled, but was absent from a sample of M form specimens collected in Ghana, Bioko and Mali. In M form specimens from Angola, there was an association between alleles at the GPRCCK1 locus and those at a microsatellite locus, AGXH678, close to the centromere of the X chromosome, with significant linkage disequilibrium between loci separated by 0.472 Mbp (P < 0.033). We show that the insertion results from introgression from the S form into the M form, rather than from the retention of an ancestral character. Gene flow from the S to M form could allow genes of adaptive value to be transferred, including those conferring insecticide resistance and others influencing ecology and behaviour, and thus malaria transmission and control. We discuss factors that may have led to this introgression event.     

Spatial swarm segregation and reproductive isolation between the molecular forms of Anopheles gambiae

Anopheles gambiae, the major malaria vector in Africa, can be divided into two subgroups based on genetic and ecological criteria. These two subgroups, termed the M and S molecular forms, are believed to be incipient species. Although they display differences in the ecological niches they occupy in the field, they are often sympatric and readily hybridize in the laboratory to produce viable and fertile offspring. Evidence for assortative mating in the field was recently reported, but the underlying mechanisms awaited discovery. We studied swarming behaviour of the molecular forms and investigated the role of swarm segregation in mediating assortative mating. Molecular identification of 1145 males collected from 68 swarms in Donéguébougou, Mali, over 2 years revealed a strict pattern of spatial segregation, resulting in almost exclusively monotypic swarms with respect to molecular form. We found evidence of clustering of swarms composed of individuals of a single molecular form within the village. Tethered M and S females were introduced into natural swarms of the M form to verify the existence of possible mate recognition operating within-swarm. Both M and S females were inseminated regardless of their form under these conditions, suggesting no within-mate recognition. We argue that our results provide evidence that swarm spatial segregation strongly contributes to reproductive isolation between the molecular forms in Mali. However this does not exclude the possibility of additional mate recognition operating across the range distribution of the forms. We discuss the importance of spatial segregation in the context of possible geographic variation in mechanisms of reproductive isolation.     

Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR-RFLP

For differential identification of sibling species in the Anopheles gambiae Giles complex (Diptera: Culicidae), including simultaneous separation of M and S molecular forms within An. gambiae Giles sensu stricto, we describe a PCR-RFLP method. This procedure is more efficient, faster and cheaper than those used before, so is recommended for large-scale processing of field-collected larval and adult specimens to be identified in malaria vector studies.     

On the distribution and genetic differentiation of Anopheles gambiae s.s. molecular forms

This paper summarises published and unpublished data on the spatial and temporal distribution, and on the genetic characterisation of molecular forms M and S of Anopheles gambiae s.s. The two forms are characterised by a high level of gene-flow restriction, by a largely overlapping geographical and temporal distribution, and by a low degree of genetic differentiation. Floating paracentric inversions on chromosome-2 are shown to be shared by the two forms, although with very different frequencies of alternative arrangements, confirming that these inversions are most probably involved in ecotypic adaptation, rather than in the building of reproductive barriers. Further studies and tools are needed to throw light on the genetic and biological differentiation of M and S to improve the knowledge of the real composition of the vector system, of its demography, population genetics and dynamics, also in view of the possible consequences on the transmission of human pathogens in sub-Saharan Africa. Preliminary results and perspectives of the use of transposable element insertion sites as markers of genetic differentiation and tools for population genetic studies are discussed.     

High genetic differentiation between the M and S molecular forms of Anopheles gambiae in Africa

Background

Anopheles gambiae, a major vector of malaria, is widely distributed throughout sub-Saharan Africa. In an attempt to eliminate infective mosquitoes, researchers are trying to develop transgenic strains that are refractory to the Plasmodium parasite. Before any release of transgenic mosquitoes can be envisaged, we need an accurate picture of the differentiation between the two molecular forms of An. gambiae, termed M and S, which are of uncertain taxonomic status.

Methodology/Principal Findings

Insertion patterns of three transposable elements (TEs) were determined in populations from Benin, Burkina Faso, Cameroon, Ghana, Ivory Coast, Madagascar, Mali, Mozambique, Niger, and Tanzania, using Transposon Display, a TE-anchored strategy based on Amplified Fragment Length Polymorphism. The results reveal a clear differentiation between the M and S forms, whatever their geographical origin, suggesting an incipient speciation process.

Conclusions/Significance

Any attempt to control the transmission of malaria by An. gambiae using either conventional or novel technologies must take the M/S genetic differentiation into account. In addition, we localized three TE insertion sites that were present either in every individual or at a high frequency in the M molecular form. These sites were found to be located outside the chromosomal regions that are suspected of involvement in the speciation event between the two forms. This suggests that these chromosomal regions are either larger than previously thought, or there are additional differentiated genomic regions interspersed with undifferentiated regions.     

Molecular identification of chromosomal forms of Anopheles gambiae sensu stricto

The Afrotropical malaria vectors Anopheles gambiae sensu stricto and An. arabiensis, responsible for more than 3/4 of the world Plasmodium falciparum inoculations, are members of the Anopheles gambiae complex, a group consisting of six sibling species. The nominal species (An. gambiae s.s.) is by far the most anthropophilic vector and its adaptation to man and his environment involves further ongoing speciation processes. This fact is shown by the existence of a number of incipient taxonomic units characterised by different chromosomal arrangements derived from the presence of polymorphic paracentric inversions. This speciation process is centered in West Africa, where five so-called 'chromosomal forms' have been described, designated with a non-Linnean nomenclature: Forest, Bissau, Savanna, Bamako, and Mopti. Studies on the distribution and the ecology of these incipient species have highlighted specific adaptations to eco-ethological parameters, which might reflect on their relative efficiency as malaria vectors. Cytogenetic analysis, in spite of some inherent difficulties, has proved to be a powerful tool for the identification of An. gambiae sibling species and the individual chromosomal forms. Yet, modern molecular tools are now available, providing alternative faster low-cost technologies, and we discuss here their relative merits.     

DNA analysis of transferred sperm reveals significant levels of gene flow between molecular forms of Anopheles gambiae

Anopheles gambiae populations in west Africa are complex, being composed of multiple, sympatric subpopulations. Recent studies have failed to reveal significant genetic differences among subpopulations, stimulating a debate regarding the levels of gene flow among them. The observed homogeneity may be the consequence of substantial contemporary gene flow or it may be that reproductive isolation is complete, but too recent for the accumulation of significant levels of genic divergence. Here, we report the results of a study estimating contemporary levels of gene flow between An. gambiae subpopulations by analysing females and transferred sperm removed from their reproductive systems. A total of 251 female and associated sperm extracts was analysed from a single site in Mali. Two molecular forms of An. gambiae, the M- and S-forms, occurred in sympatry at this site. Overall, we found very strong positive assortative mating within forms, however, we did observe significant hybridization between forms. In the M subpopulation 2/195 females (1.03%) contained sperm from S-form males and in 55 S-form females we found one female containing M-form sperm (1.82%). We also identified a mated M xS hybrid adult female. From mating frequencies, we estimate the Nem between the M- and S-form at 16.8, and from the adult hybrid frequency at 5.6. These values are consistent with our earlier estimate, based on FST for 21 microsatellite loci in which Nem = 5.8. We conclude that the general lack of genetic divergence between the M and S subpopulations of An. gambiae can be explained entirely by contemporary gene flow.     

The hemolymph proteome of Anopheles gambiae

We used two-dimensional SDS-PAGE and microsequencing or peptide mass fingerprinting to identify major proteins in the hemolymph of Anopheles gambiae. We found approximately 280 protein spots in hemolymph and identified 28 spots, representing 26 individual proteins. Most of these proteins have known or predicted functions in immunity, iron transport, or lipid biology. Many of the proteins have been found in hemolymph in other insects but one protein is novel: a new member of the ML family (involved in lipid recognition). Three of the identified proteins increased in spot intensity or appeared de novo following bacterial injection: a phenoloxidase, and two chitinase-like proteins. A subset of proteins decreased following bacterial injections: these included the light and heavy chains of ferritin. Several proteins appeared in hemolymph following any wound or injection. Most of these are metabolic enzymes lacking signal peptides that are likely to be released as a result of damage to muscles and other tissues by injury. The map will provide a useful tool for examining changes in hemolymph proteins following blood feeding and infection by parasites.     

Gene flow-dependent genomic divergence between Anopheles gambiae M and S forms

Anopheles gambiae sensu stricto exists as two often-sympatric races termed the M and S molecular forms, characterized by fixed differences at an X-linked marker. Extreme divergence between M and S forms at pericentromeric "genomic islands" suggested that selection on variants therein could be driving interform divergence in the presence of ongoing gene flow, but recent work has detected much more widespread genomic differentiation. Whether such genomic islands are important in reproductive isolation or represent ancestral differentiation preserved by low recombination is currently unclear. A critical test of these competing hypotheses could be provided by comparing genomic divergence when rates of recent introgression vary. We genotyped 871 single nucleotide polymorphisms (SNPs) in A. gambiae sensu stricto from locations of M and S sympatry and allopatry, encompassing the full range of observed hybridization rates (0-25%). M and S forms were readily partitioned based on genomewide SNP variation in spite of evidence for ongoing introgression that qualitatively reflects hybridization rates. Yet both the level and the heterogeneity of genomic divergence varied markedly in line with levels of introgression. A few genomic regions of differentiation between M and S were common to each sampling location, the most pronounced being two centromere-proximal speciation islands identified previously but with at least one additional region outside of areas expected to exhibit reduced recombination. Our results demonstrate that extreme divergence at genomic islands does not simply represent segregating ancestral polymorphism in regions of low recombination and can be resilient to substantial gene flow. This highlights the potential for islands comprising a relatively small fraction of the genome to play an important role in early-stage speciation when reproductive isolation is limited.     

Insertion polymorphism of transposable elements and population structure of Anopheles gambiae M and S molecular forms in Cameroon

The insertion polymorphism of five transposable element (TE) families was studied by Southern blots in several populations of the M and S molecular forms of the mosquito Anopheles gambiae sensu stricto from southern Cameroon. We showed that the mean TE insertion site number and the within-population insertion site polymorphism globally differed between the M and S molecular forms. The comparison of the TE insertion profiles of the populations revealed a significant differentiation between these two molecular forms (0.163 < Phi(ST) < 0.371). We cloned several insertions of a non-LTR retrotransposon (Aara8) that were fixed in one form and absent in the other one. The only insertion that could be clearly located on a chromosome arm mapped to cytological division 6 of chromosome X, confirming the importance of this region in the ongoing speciation between the M and S molecular forms.     

Anopheles gambiae innate immunity

The successful development of Plasmodium in Anopheles mosquitoes is governed by complex molecular and cellular interactions that we are just beginning to understand. Anopheles immune system has received particular attention as genetic evidence points clearly to its critical role in eliminating the majority of parasites invading the midgut epithelium. Several factors regulating Plasmodium development have been identified and tentatively assigned to the individual steps leading to mosquito immune reactions; non-self-recognition, signal modulation, signal transduction and effector mechanisms. Detailed knowledge of these steps and their underlying molecular mechanisms may offer novel perspectives to abort Plasmodium development in the vector. Here, we summarize our current knowledge of mosquito innate immunity highlighting both, recent advances and areas where additional research is required.     

Evidence for subdivision within the M molecular form of Anopheles gambiae

The principal vector of malaria in sub-Saharan Africa, Anopheles gambiae is subdivided into two molecular forms M and S. Additionally, several chromosomal forms, characterized by the presence of various inversion polymorphisms, have been described. The molecular forms M and S each contain several chromosomal forms, including the Savanna, Mopti and Forest forms. The M and S molecular forms are now considered to be the reproductive units within A. gambiae and it has recently been argued that a low recombination rate in the centromeric region of the X chromosome has facilitated isolation between these forms. The status of the chromosomal forms remains unclear however. Therefore, we studied genetic differentiation between Savanna S, Forest S, Forest M and Mopti M populations using microsatellites. Genetic differentiation between Savanna S and Forest S populations is very low (F(ST) = 0.0053 +/- 0.0049), even across large distances. In comparison, the Mopti M and Forest M populations show a relatively high degree of genetic differentiation (F(ST) = 0.0406 +/- 0.0054) indicating that the M molecular form may not be a single entity, but could be subdivided into at least two distinct chromosomal forms. Previously it was proposed that inversions have played a role in the origin of species within the A. gambiae complex. We argue that a possible subdivision within the M molecular form could be understood through this process, with the acquisition of inversions leading to the expansion of the M molecular form into new habitat, dividing it into two distinct chromosomal forms.     

The Anopheles gambiae genome: an update

As a result of an international collaborative effort, the first draft of the Anopheles gambiae genome sequence and its preliminary annotation were published in October 2002. Since then, the assembly, annotation and means of accession of the An. gambiae genome have been under continuous development. This article reviews progress and considers limitations in the current sequence assembly and gene annotation, as well as approaches to address these problems and outstanding issues that users of the data must bear in mind.     

The Anopheles gambiae complex in Guinea Bissau

Three species of the Anopheles gambiae complex were identified in Guinea Bissau (West Africa) by chromosomal analysis. They were An. melas, An. arabiensis and An. gambiae s.s. An melas was observed in coastal and insular zones of the study area as well as in areas where the rivers are tidal and brackish and bordered by mangroves. For this reason, the species occurs also in inland riverine localities such as Farim and Bissorà. An. arabiensis apparently occurs only in low numbers in a very limited inland area during the dry season. An gambiae s.s. was observed nearly everywhere in the study area. In the samples of An. melas three inversion polymorphisms occurred: one on the chromosomal arm 2R (2Rn) and two on the arm 3R (3Rc and 3Re). It was observed that the frequencies of the inverted arrangements 2Rn and 3Re were significantly higher in the islands as compared to the continental sampling localities. The An. arabiensis sample was characterized by the presence of three inversion polymorphisms: 2Ra, 2Rb and 3Ra. A very high degree of polymorphism was shown by the An. gambiae s.s. samples. Four inversion polymorphisms were observed (three on chromosomal arm 2R and one on arm 2L) with very different frequencies of the alternative arrangements in different zones of the study area. From these data it seemed possible to split the species into three populations, each of them apparently linked with a peculiar ecological situation. The first population, characterized by high frequencies of 2Rd arrangement, is present on the coastal zones and in the islands; the second one is present in the northern inland areas particularly during the dry season and it is characterized by high frequencies of 2Rb and 2La arrangements. The third population is present only in the inland zones and it is characterized by high frequencies of 2Rjb, 2Rjd and 2Rjbd arrangements.     

Physiology and development of the M and S molecular forms of Anopheles gambiae in Burkina Faso (West Africa)

In West Africa, M and S molecular forms of Anopheles gambiae sensu stricto (Diptera: Culicidae) Giles, frequently occur together, although with different population bionomics. The S form typically breeds in rain‐dependant water collections and is present during the rainy season only whereas the M form can thrive all year long in areas with permanent breeding opportunities. In the present study, we explored physiological and developmental trade‐offs at play in laboratory colonies and field populations of the M and S forms that originated from an area of sympatry in Burkina Faso, where M and S larvae exhibit such habitat segregation. In the laboratory, larvae of the M form developed slower than the S form (mean values 9.51 and 8.85 days, respectively, Wilcoxon's test, P < 0.001). Although wing length and dry weight at emergence showed large variations, M females were on average 8% heavier than S females of similar wing length. Higher nutritional reserves (proteins and lipids) in teneral adults explained part of this weight difference, reflecting a better ability of the M form to garner resources at the larval stage. Furthermore, a higher rate of ovarian maturation was observed in the M form after a single bloodmeal. The relevance of these findings for parasite transmission is discussed.     

Reproductive physiology of Anopheles gambiae and Anopheles atroparvus.

When exposed to a human host, Anopheles gambiae started probing 4 h post-eclosion, but 95% successfully blood-fed by 16-20 h with maximal blood volumes of 5- 10 microl per female. When fed sugar, the 95% feeding was not observed until 36-40 h post-eclosion; sugar meals appeared to interfere with blood meals. Similarly in An. atroparvus, maximum volumes were 10 microl when starved but only 6 microl when fed sugar. This species did not bite before 2 d, and 95% biting was by 4 d. Given single blood meals to water-kept An. gambiae, a threshold body size for oogenesis was detected. With wing lengths below 2.8 mm, eggs never matured, but when sugar-fed, females of all sizes matured eggs including the synthesis of maternal deposits. Although sugar feeding interfered with blood feeding, more lipid was transferred to the yolk. In water-kept An. atroparvus only 5% of the females produced eggs. When sugar-fed for 4 d, all females matured eggs, so in this species sugar feeding appeared to be essential for oogenesis. An. gambiae always took multiple blood meals, tested at any time after the first ones, leading to 120 mature eggs/female. Yolk composition was 3.9 mcal protein and 3.8 mcal lipid/oocyte when kept on water, but 2.8 meal protein and 4.3 mcal lipid/oocyte with intermittent sugar meals, thus marking a surprising flexibility in synthesis of yolk protein and lipid that strongly depends on additional carbohydrates sources. Only 80% of water-fed An. atroparvus re-fed 2 d after a first blood meal with small females taking three blood meals but they still showed reduced fecundity. Only the large water-fed females matured eggs, with blood volumes higher than 9-12 microl. When fed sugar, the blood meal input was reduced, but oogenesis was possible, whereas water-fed females required three blood meals to reach the caloric level comparable to pre-feeding sugar-fed females. Water-fedAn. gambiae could survive on daily blood meals alone, but survival was further extended by intermittent sugar meals. When offered a blood donor daily, there was a behavioral difference. Females maintained alone showed a more or less regular 3 d feeding and oviposition activity, while females kept in groups fed daily followed a daily oviposition pattern, suggesting gonotrophic discordance.     

Mapping distributions of chromosomal forms of Anopheles gambiae in West Africa using climate data

The mosquito Anopheles gambiae Giles sensu stricto (Diptera: Culicidae), the principal vector of malaria in West Africa, comprises several chromosomal forms (e.g. Bissau, Forest, Mopti, Savanna) associated with climatic zones. Here we show how climate data can be used to map the geographical distribution of these chromosomal forms. The climate at 144 sites surveyed for mosquitoes in West Africa between 1971 and 92 was determined using computerized climate surfaces. Forest and Bissau forms occurred at relatively wet sites: median annual precipitation 1325 mm and 1438 mm, respectively, interquartile ranges (IQR) 1144-1858 mm and 1052-1825 mm), whilst the Mopti form was found at dry sites (annual 938 mm, IQR 713-1047 mm) and the Savanna form at sites intermediate between the wet and dry forms (annual 1067 mm, IQR 916-1279). Logistic regression analyses of the climate variables were carried out on a stratified random sample of half the sites. The resulting models correctly classified over 80% of the sites for presence or absence of each chromosomal form. When these models were tested against excluded sites they were also correct at over 80% of sites. The combined data produced models that were correct at over 86% of sites. Mean annual precipitation, evapotranspiration, minimum temperature and maximum temperature were the most important climate variables correlated with the distribution of these forms of An. gambiae. We used the logistic models to map the distribution of each chromosomal form within the reported range for An. gambiae s.s. in West Africa employing a geographical information system. Our maps indicate that each chromosomal form favours particular climate envelopes in well-defined ecoclimatic zones, although these forms are sympatric at the edges of their ranges. This study demonstrates that climate can be used to map the distribution of chromosomal forms of insects across large areas.