Mosaic genome architecture of the Anopheles gambiae species complex

Background

Attempts over the last three decades to reconstruct the phylogenetic history of the Anopheles gambiae species complex have been important for developing better strategies to control malaria transmission.

Methodology

We used fingerprint genotyping data from 414 field-collected female mosquitoes at 42 microsatellite loci to infer the evolutionary relationships of four species in the A. gambiae complex, the two major malaria vectors A. gambiae sensu stricto (A. gambiae s.s.) and A. arabiensis, as well as two minor vectors, A. merus and A. melas.

Principal Findings

We identify six taxonomic units, including a clear separation of West and East Africa A. gambiae s.s. S molecular forms. We show that the phylogenetic relationships vary widely between different genomic regions, thus demonstrating the mosaic nature of the genome of these species. The two major malaria vectors are closely related and closer to A. merus than to A. melas at the genome-wide level, which is also true if only autosomes are considered. However, within the Xag inversion region of the X chromosome, the M and two S molecular forms are most similar to A. merus. Near the X centromere, outside the Xag region, the two S forms are highly dissimilar to the other taxa. Furthermore, our data suggest that the centromeric region of chromosome 3 is a strong discriminator between the major and minor malaria vectors.

Conclusions

Although further studies are needed to elucidate the basis of the phylogenetic variation among the different regions of the genome, the preponderance of sympatric admixtures among taxa strongly favor introgression of different genomic regions between species, rather than lineage sorting of ancestral polymorphism, as a possible mechanism.     

DNA probes for species identification of mosquitoes in the Anopheles gambiae complex

Abstract. Identification of species within the Anopheles gambiae Giles species complex is essential for the correct evaluation of malaria vector ecology studies and control programmes. The development of DNA probes to distinguish species of the An.gambiae complex is described. Genomic libraries were prepared for four members of the An.gambiae complex. These were screened using radiolabeled DNA from different species of An. gambiae sensu lato and a number of clones selected on the basis of their species specificity. These clones could be divided into two groups, each containing homologous sequences. Sequences homologous to group 1 inserts are highly reiterated in the genomes of Anopheles arabiensis Patton and Anopheles merus Dönitz, present in low copy number in Anopheles melas Theobald, but were not detected in Anopheles gambiae sensu stricto. Studies on the organization of this sequence in the genome of An.arabiensis show that homologous sequences are male specific and interspersed within the chromatin. Sequences homologous to group 2 inserts are highly repeated in the genomes of An.merus and An.melas, but present in low copy number in An.gambiae s.s. and An.arabiensis. Group 2 homologous sequences are not sex-specific in the species tested and appear to be tandemly repeated. When used as hybridization probes, these sequences provide a sensitive means for the identification of species within the Anopheles gambiae complex.     

Larvae of cryptic species of Anopheles gambiae respond differently to cues of predation risk

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Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.     

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.     

Intraspecific polymorphism of sex chromosome heterochromatin in two species of the Anopheles gambiae complex

The Hoechst 33258 banding pattern of the mitotic chromosomes of several laboratory and natural populations of the sibling species A. gambiae and A. arabiensis has been analyzed. A clear intraspecific polymorphism of sex chromosome heterochromatin has been observed. Nevertheless in each species heterochromatic variations fall within a characteristic species-specific pattern. Moreover, while laboratory populations tend to be monomorphic for a given heterochromatic variant, natural populations exhibit a high degree of intrapopulation polymorphism. The possible role of sex chromosome heterochromatin in controlling fertility and mating behaviour of Anopheles mosquitoes is discussed.     

Laboratory colonization of Anopheles quadriannulatus from sympatry with other sibling species of the Anopheles gambiae complex in Zimbabwe

Abstract. A laboratory colony of the mosquito Anopheles quadriannulatus was established from a wild population occurring sympatrically with An.arabiensis in Zimbabwe. These sibling species are members of the An.gambiae Giles complex and were distinguished primarily by means of their specific polytene chromosome banding patterns. By using an ox-baited trap, we sampled selectively for the more zoophilic An.quadriannulatus. It was confirmed that An.quadriannulatus has the diagnostic slow allozyme of aspartate aminotransferase (AAT^95/95). In a mixed population under laboratory conditions, An.arabiensis displaced An.quadriannulatus within eight generations, without introgression. Colonization of An.quadriannulatus was facilitated by pooling the progeny from wild-caught mothers of confirmed identity and by using a specially adapted cage to promote mating.     

PCR assay for identification of Anopheles quadriannulatus species B from Ethiopia and other sibling species of the Anopheles gambiae complex

Sibling species A and B of Anopheles quadriannulatus (Theobald) are recognized as allopatric members of the Anopheles gambiae Giles complex of Afrotropical mosquitoes (Diptera: Culicidae). Species A represents An. quadriannulatus sensu stricto, widespread in southern Africa, whereas An. quadriannulatus species B occurs in Ethiopia. Because of difficulty of identification, distribution of An. quadriannulatus sensu lato remains poorly known. Cytotaxonomy and the standard DNA polymerase chain reaction (PCR) assay do not distinguish between species A and B of An. quadriannulatus. By optimizing the standard PCR assay (Scott et al., 1993) for identification of members of the An. gambiae complex, we identified two discriminant fragments of 153 bp and 900 bp from DNA of An. quadriannulatus species B, whereas only the 153 bp fragment was amplified for species A from South Africa. This modified PCR assay can therefore be used to distinguish between species A and B of An. quadriannulatus s.l. as well as other members of the An. gambiae complex.     

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.     

Epidemiological consequences of a newly discovered cryptic subgroup of Anopheles gambiae

A cryptic subgroup of Anopheles gambiae sensu stricto mosquitoes was recently discovered in West Africa. This 'GOUNDRY' subgroup has increased susceptibility to Plasmodium falciparum, the most deadly form of malaria. Unusual for this major malaria vector, GOUNDRY mosquitoes also seem to bite exclusively outdoors. A mathematical model is developed to assess the epidemiological implications of current vector control tools, bednets and indoor residual spray, preferentially suppressing the more typical indoor biting mosquitoes. It is demonstrated that even if the GOUNDRY mosquitoes have a decreased preference for human blood, vector controls which select for increased GOUNDRY abundance relative to their indoor biting counterparts risks intensifying malaria transmission. Given the widely observed phenomenon of outdoor biting by major malaria vectors, this behaviour should not be ignored in future modelling efforts and warrants serious consideration in control programme strategy.     

Use of the polymerase chain reaction to identify mosquito species of the Anopheles gambiae complex

A nonradiometric method has been developed for distinguishing between the sibling species Anopheles gambiae Giles and An. arabiensis Patton, two important Afrotropical vectors of malaria. DNA fragments of species diagnostic length are amplified by polymerase chain reaction (PCR) from a small amount of unknown DNA and three different PCR primers. All three PCR primers are based on ribosomal DNA (rDNA) sequences. A universal plus-strand primer (A0) is derived from a conserved region at the 3' end of the 28S rDNA coding region. Two species-specific minus-strand primers (Aa0.5 and Ag1.3) are derived from sequences in the intergenic spacers. The Ag1.3 sequence is approximately 1.3 kb downstream of A0; the Aa0.5 sequence is about 0.5 kb downstream of A0. When mosquito DNA is amplified in the presence of all three primers, a 1.3 kb fragment is produced if An. gambiae DNA is used as template, and a 0.5 kb fragment is produced if An. arabiensis DNA is used. Amplification of DNA from An.gambiae/An. arabiensis hybrids produces both the 1.3 kb and the 0.5 kb fragments. Neither diagnostic fragment is produced when DNA from other species in the An. gambiae complex is used as template.     

Distribution of the species of the Anopheles gambiae complex and first evidence of Anopheles merus as a malaria vector in Madagascar

BACKGROUND: Members of the Anopheles gambiae complex are amongst the best malaria vectors in the world, but their vectorial capacities vary between species and populations. A large-scale sampling of An. gambiae sensu lato was carried out in various bioclimatic domains of Madagascar. Local abundance of an unexpected member of this complex raised questions regarding its role in malaria transmission. METHODS: Sampling took place at 38 sites and 2,067 females were collected. Species assessment was performed using a PCR targeting a sequence in the IGS of the rDNA. Analysis focused on the relative prevalence of the species per site, bioclimatic domain and altitude. Infectivity of Anopheles merus was assessed using an ELISA to detect the presence of malarial circumsporozoite protein in the head-thorax. RESULTS: Three species were identified: An. gambiae, Anopheles arabiensis and An. merus. The distribution of each species is mainly a function of bioclimatic domains and, to a lesser extent, altitude. An. arabiensis is present in all bioclimatic domains with highest prevalence in sub-humid, dry and sub-arid domains. An. gambiae has its highest prevalence in the humid domain, is in the minority in dry areas, rare in sub-humid and absent in sub-arid domains. An. merus is restricted to the coastal fringe in the south and west; it was in the majority in one southern village. The majority of sites were sympatric for at least two of the species (21/38) and two sites harboured all three species.The role of An. merus as malaria vector was confirmed in the case of two human-biting females, which were ELISA-positive for Plasmodium falciparum. CONCLUSION: Despite the huge environmental (mainly man-made) changes in Madagascar, the distribution of An. gambiae and An. arabiensis appears unchanged for the past 35 years. The distribution of An. merus is wider than was previously known, and its effectiveness as a malaria vector has been shown for the first time; this species is now on the list of Malagasy malaria vectors.     

Sex-linked differentiation between incipient species of Anopheles gambiae

Emerging species within the primary malaria vector Anopheles gambiae show different ecological preferences and significant prezygotic reproductive isolation. They are defined by fixed sequence differences in X-linked rDNA, but most previous studies have failed to detect large and significant differentiation between these taxa elsewhere in the genome, except at two other loci on the X chromosome near the rDNA locus. Hypothesizing that this pericentromeric region of the X chromosome may be accumulating differences faster than other regions of the genome, we explored the pattern and extent of differentiation between A. gambiae incipient species and a sibling species, A. arabiensis, from Burkina Faso, West Africa, at 17 microsatellite loci spanning the X chromosome. Interspecific differentiation was large and significant across the entire X chromosome. Among A. gambiae incipient species, we found some of the highest levels of differentiation recorded in a large region including eight independent loci near the centromere of the X chromosome. Outside of this region, no significant differentiation was detected. This pattern suggests that selection is playing a role in the emergence of A. gambiae incipient species. This process, associated with efficient exploitation of anthropogenic modifications to the environment, has public health implications as it fosters the spread of malaria transmission both spatially and temporally.     

Mosquito behavioural aspects of vector-human interactions in the Anopheles gambiae complex

The behaviour of two of the most anthropophilic malaria vectors in the world, Anopheles gambiae Giles and An. arabiensis Patton, is revisited with respect to recent studies on their host preferences and the chemical ecology of host-seeking. Issues are discussed in relation to the ways anthropophily may have arisen in the complex, and the opportunities the study of olfaction and host-seeking behaviour offers to malaria control in Africa.     

Cannibalism and predation among larvae of the Anopheles gambiae complex

Among the aquatic developmental stages of the Anopheles gambiae complex (Diptera: Culicidae), both inter- and intra-specific interactions influence the resulting densities of adult mosquito populations. For three members of the complex, An. arabiensis Patton, An. quadriannulatus (Theobald) and An. gambiae Giles sensu stricto, we investigated some aspects of this competition under laboratory conditions. First-instar larvae were consumed by fourth-instar larvae of the same species (cannibalism) and by fourth-instar larvae of other sibling species (predation). Even when larvae were not consumed, the presence of one fourth-instar larva caused a significant reduction in development rate of first-instar larvae. Possible implications of these effects for population dynamics of these malaria vector mosquitoes are discussed.     

Mapping members of the Anopheles gambiae complex using climate data

Abstract. Climate is the most important factor governing the distribution of insects over large areas. Warmth and moisture are essential for most insects' reproduction, development and survival. Here, it is shown that the principal vectors of malaria in Africa, members of the Anopheles gambiae complex, flourish within specific climate envelopes. By identifying these climatic conditions empirically, using climate or environmental databases, it is possible to map the distribution and relative abundance of mosquito species, and their chromosomal forms, at continental scales. Alternatively, mathematical models based on a fundamental understanding of how mosquitoes are affected by different climate factors, such as temperature and humidity, can also be employed to map distributions. Empirical or process‐driven models based on climate, or other environmental variables, provide simple tools for mapping the distribution and relative abundance of vectors at a coarse scale over large areas.     

Use of a male-specific DNA probe to distinguish female mosquitoes of the Anopheles gambiae species complex

Abstract. A method has been developed to distinguish between mated female individuals of the sibling species Anopheles gambiae Giles sensu stricto and Anopheles arabiensis Patton. The DNA probe pAnal, reported by Gale & Crampton (1987a) to be useful for the specific identification of An.arabiensis males, is here shown to be sufficiently sensitive to deduce the species identity of inseminated females from the identity of the sperm contained within the spermatheca.     

Polytene chromosome relationships of five species of the Anopheles dirus complex in Thailand.

Photographic maps and rearrangements of each salivary gland polytene chromosome arm of Anopheles nemophilous (species F) and of An. dirus species A, B, C, and D of the Dirus group from natural populations in Thailand are presented. Structural conformation of heterokaryotypes and comparison of chromosome banding sequences reveal 10 paracentric inversions. The data on fixed inversion of 3Rb and inversion polymorphism of the X chromosome shared by these species were used to construct a phylogeny of the five members of the An. dirus complex, thereby outlining their patterns of speciation through chromosomal rearrangements.