M. Coluzzi

Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex

Speciation in the Anopheles gambiae complex is reviewed and discussed with emphasis on the patterns of chromosomal differentiation, particularly at the intraspecific level. The significance of inversion polymorphism in gambiae and arabiensis (the two species of greatest medical importance) is evaluated with reference to recent field investigations carried out in Nigeria. In both sibling species some of the inversions show clinical geographical changes in frequencies, with evident correlations with climatic conditions and vegetation zones. Microgeographical variations in species distribution and in intraspecific inversion frequencies are also present, which appear mostly related to man-made environmental contrasts. Parallel indoor-/outdoor collections of samples from polymorphic populations of arabiensis and gambiae show that adult mosquitoes carrying certain inversion karyotypes do not distribute at random in relation to the human environment, being significantly more frequent in outdoor than in indoor samples, or vice-versa. Optimal habitat choice appears to be involved in such variations of indoor resting behaviour, since the chromosomal types carried by less endophilic individuals are those more adapted to humid climates, i.e. those which tend to avoid the higher nocturnal saturation deficit of the indoor environment. This phenomenon, producing non-uniform exposure of the vector population to residual insecticides sprayed in houses, might explain the mediocrity of the results of malaria control projects based on house-spraying against endophilic vectors in the African savannas.

Chromosomal inversion intergradation and incipient speciation in Anopheles gambiae

Abstract Polytene chromosome studies on the Afrotropical mosquitoes of the Anopheles gambiae complex show that the rearrangements of the banding pattern are all based on paracentric inversions. Some of these occur at the homozygous state only (fixed inversions) and are used for cytotaxonomic identification of the six sibling species. Others occur as intraspecific chromosomal polymorphisms which are frequent particularly in An. gambiae and An. arabiensis, the two taxa showing the widest distribution and the closest association with man and man-made breeding places. A particularly high level of chromosome differentiation has been recorded in Anpoheles gambiae (the most anthropophilic taxon of the complex and possibly the one most recently evolved) which can be split into forest and savanna chromosomal forms. The forest form seems chromosomally uniform, being characterized in all the rain forest areas by the chromosome-2 standard arrangement, probably one of the most primitive in the complex. The forest form...

Haemoglobin C protects against clinical Plasmodium falciparum malaria.

Haemoglobin C (HbC; β6Glu → Lys) is common in malarious areas of West Africa, especially in Burkina Faso. Conclusive evidence exists on the protective role against severe malaria of haemoglobin S (HbS; β6Glu → Val) heterozygosity, whereas conflicting results for the HbC trait have been reported and no epidemiological data exist on the possible role of the HbCC genotype. In vitro studies suggested that HbCC erythrocytes fail to support the growth of P. falciparum but HbC homozygotes with high P. falciparum parasitaemias have been observed. Here we show, in a large case–control study performed in Burkina Faso on 4,348 Mossi subjects, that HbC is associated with a 29% reduction in risk of clinical malaria in HbAC heterozygotes (P = 0.0008) and of 93% in HbCC homozygotes (P = 0.0011). These findings, together with the limited pathology of HbAC and HbCC compared to the severely disadvantaged HbSS and HbSC genotypes and the low βS gene frequency in the geographic epicentre of βC, support the hypothesis that, in the long term and in the absence of malaria control, HbC would replace HbS in central West Africa.

Molecular evidence of incipient speciation within Anopheles gambiae s.s. in West Africa

We karyotyped and identified by polymerase chain reaction restriction fragment length polymorphism (PCR‐RFLP) analysis Anopheles gambiae s.s. samples collected in several African countries. The data show the existence of two non‐panmictic molecular forms, named S and M, whose distribution extended from forest to savannahs. Mosquitoes of the S and M forms are homosequential standard for chromosome‐2 inversions in forest areas. In dry savannahs, S is characterized mainly by inversion polymorphisms typical of Savanna and Bamako chromosomal forms, while M shows chromosome‐2 arrangements typical of Mopti and/or Savanna and/or Bissau, depending on its geographical origin. Chromosome‐2 inversions therefore seem to be involved in ecotypic adaptation rather than in mate‐recognition systems. Strong support for the reproductive isolation of S and M in Ivory Coast comes from the observation that the kdr allele is found at high frequencies in S specimens and not at all in chromosomal identical M specimens. However, the kdr allele does not segregate with molecular forms in Benin.

Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation

Three chromosomal forms of Anopheles gambiae s.s., designated as Bamako, Mopti and Savanna, were studied for diagnostic PCR assays based on the analysis of the X‐linked ribosomal DNA (rDNA). The study was performed on a 1.3 kb fragment containing part of the 28S coding region and part of the intergenic spacer region. The amplified material was cut with fourteen restriction enzymes to detect Restriction Fragment Length Polymorphisms (RFLPs). The enzymes Tru9I and HhaI produced patterns of DNA bands which differentiated Mopti from Savanna and Bamako; moreover, a distinct ‘hybrid’ pattern was recognized in the F1 female progeny from the cross of Mopti with either one of the other two chromosomal forms. The diagnostic significance of the PCR‐RFLP assay was verified on 203 karyotyped females from field samples collected in two villages in Mali and one village in Burkina Faso. Agreement was observed between the chromosomal and the molecular identifications. No ‘hybrid’ molecular patterns were detected even among carriers of rare heterokaryotypes hypothetically produced by crosses between Mopti and Savanna. The results confirm previous observations indicating barriers to gene flow within An. gambiae s.s. and supporting the specific status of the taxonomic units proposed on cytogenetic ground.

An updated catalogue of salivary gland transcripts in the adult female mosquito, Anopheles gambiae

SUMMARY Salivary glands of blood-sucking arthropods contain a variety of compounds that prevent platelet and clotting functions and modify inflammatory and immunological reactions in the vertebrate host. In mosquitoes, only the adult female takes blood meals, while both sexes take sugar meals. With the recent description of the Anopheles gambiae genome, and with a set of∼ 3000 expressed sequence tags from a salivary gland cDNA library from adult female mosquitoes, we attempted a comprehensive description of the salivary transcriptome of this most important vector of malaria transmission. In addition to many transcripts associated with housekeeping functions, we found an active transposable element, a set of Wolbachia-like proteins, several transcription factors, including Forkhead, Hairy and doublesex, extracellular matrix components and 71 genes coding for putative secreted proteins. Fourteen of these 71 proteins had matching Edman degradation sequences obtained from SDS-PAGE experiments. Overall, 33 transcripts are reported for the first time as coding for salivary proteins. The tissue and sex specificity of these protein-coding transcripts were analyzed by RT–PCR and microarray experiments for insight into their possible function. Notably, two gene products appeared to be differentially spliced in the adult female salivary glands, whereas 13 contigs matched predicted intronic regions and may include additional alternatively spliced transcripts. Most An. gambiae salivary proteins represent novel protein families of unknown function, potentially coding for pharmacologically or microbiologically active substances. Supplemental data to this work can be found at http://www.ncbi.nlm.nih.gov/projects/omes/index.html#Ag2.

Density, survival and dispersal of Anopheles gambiae complex mosquitoes in a west African Sudan savanna village.

Abstract. To obtain information on adult populations of Afrotropical malaria vector mosquitoes, mark‐release‐recapture experiments were performed with Anopheles females collected from indoor resting‐sites in a savanna area near Ouagadougou, Burkina Faso, during September 1991 and 1992. Results were used to estimate the absolute population densities, daily survival rates, and dispersal parameters of malaria vectors in that area.

The pyrethroid knock‐down resistance gene in the Anopheles gambiae complex in Mali and further indication of incipient speciation within An. gambiae s.s.

In Mali the Anopheles gambiae complex consists of An. arabiensis and Mopti, Savanna and Bamako chromosomal forms of An. gambiae s.s. Previous chromosomal data suggests a complete reproductive isolation among these forms. Sequence analysis of rDNA regions led to the characterization of two molecular forms of An. gambiae, named M‐form and S‐form, which in Mali correspond to Mopti and to Savanna/Bamako, respectively, while it has failed so far to show any molecular difference between Savanna and Bamako. The population structure of An. gambiae s.l. was analysed in three villages in the Bamako and Sikasso areas of Mali and the frequency of pyrethroid resistance of the knock‐down resistance (kdr) type was calculated. The results show that the kdr allele is associated only with the Savanna form populations and absent in sympatric and synchronous populations of Bamako, Mopti and An. arabiensis. This is the first molecular indication of barriers to gene flow between the Bamako and Savanna chromosomal forms. Moreover, analyses of specimens collected in the Bamako area in 1987 show that the kdr allele was already present in the Savanna population at that time, and that the frequency of this allele has gradually increased since then.

Functional deficit of T regulatory cells in Fulani, an ethnic group with low susceptibility to Plasmodium falciparum malaria

Previous interethnic comparative studies on the susceptibility to malaria performed in West Africa showed that Fulani are more resistant to Plasmodium falciparum malaria than are sympatric ethnic groups. This lower susceptibility is not associated to classic malaria-resistance genes, and the analysis of the immune response to P. falciparum sporozoite and blood stage antigens, as well as non-malaria antigens, revealed higher immune reactivity in Fulani. In the present study we compared the expression profile of a panel of genes involved in immune response in peripheral blood mononuclear cells (PBMC) from Fulani and sympatric Mossi from Burkina Faso. An increased expression of T helper 1 (TH1)-related genes (IL-18, IFNγ, and TBX21) and TH2-related genes (IL-4 and GATA3) and a reduced expression of genes distinctive of T regulatory activity (CTLA4 and FOXP3) were observed in Fulani. Microarray analysis on RNA from CD4+CD25+ (T regulatory) cells, performed with a panel of cDNA probes specific for 96 genes involved in immune modulation, indicated obvious differences between the two ethnic groups with 23% of genes, including TGFβ, TGFβRs, CTLA4, and FOXP3, less expressed in Fulani compared with Mossi and European donors not exposed to malaria. As further indications of a low T regulatory cell activity, Fulani showed lower serum levels of TGFβ and higher concentrations of the proinflammatory chemokines CXCL10 and CCL22 compared with Mossi; moreover, the proliferative response of Fulani to malaria antigens was not affected by the depletion of CD25+ regulatory cells whereas that of Mossi was significantly increased. The results suggest that the higher resistance to malaria of the Fulani could derive from a functional deficit of T regulatory cells.

Ecological genetic studies in the chromosomal form Mopti ofAnopheles gambiae s.str. in Mali, West Africa

Among the sibling species of the AfrotropicalAnopheles gambiae complex, the nominal taxon (An. gambiae s.str.) is the major malaria vector. Its bionomics suggest a man-dependent speciation process which involves, in West Africa, various incipient species chromosomally recognized by different combinations of 2R paracentric inversions. One of the most recent evolutionary steps of such a speciation process appears to be the chromosomal form Mopti, which is associated with dry season irrigation in arid zones, and is characterized by a remarkable ecological flexibility related to three 2R alternative arrangements, namelybc, u and +, whose expected karyotypes are found in Hardy-Weinberg equilibrium. The study of this chromosomal polymorphism in samples from a 16-locality transect in Mali shows wide variations and highly significant correlation with both temporal and spatial climatic differences. Mosquitoes homokaryotypic for 2Rbc are the actual dry season and arid areas breeders. The regular rise of 2Rbc frequency, up to fixation, during each dry season, corresponds to the South-North clinal increase of the same arrangement along the transect, from about 30% in the humid savanna to near fixation in the South-Saharan zone. This coherent ecological genetics case provides full support to the hypothesis of the adaptive nature of paracentric inversions. Moreover, the very peculiar system of combinations of contiguous 2R inversions, utilized by Mopti as well as by other chromosomal forms ofAn. gambiae, suggests a process of polygenic reorganization based on linkage disequilibria and involving the inversions as driving selection units.