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.

Quantification of Plasmodium falciparum sporozoites by ribosomal RNA detection.

We used sequences specific to the small subunit ribosomal RNA (SSU rRNA) of the sporogonic stages of Plasmodium falciparum to design a reserve transcriptase-polymerase chain reaction (RT-PCR) assay that can detect 0.1 sporozoites in total RNA purified from potentially infected mosquitoes. We made a synthetic RNA that is amplified in the RT-PCR by the same primers as the parasite SSU rRNA and that serves as an internal control and competitive quantitation standard. We calibrated the assay for quantitation of sporozoites by making a standard curve with RNA from purified and counted sporozoites. The assay accurately measured sporozoite number with a linear range of at least three orders of magnitude in a single reaction. Some application and limitations of the assay are discussed.

Short report: simultaneous detection by polymerase chain reaction of mosquito species and Plasmodium falciparum infection in Anopheles gambiae sensu lato.

Total RNA purified from Anopheles gambiae mosquitoes can be used for detection of both 1) infection by Plasmodium falciparum using a reverse transcriptase-polymerase chain reaction (RT-PCR) assay specific for P. falciparum ribosomal RNA (rRNA) of sporogonic stage parasites, and 2) mosquito species using a PCR assay that distinguishes members of the Anopheles gambiae complex.

Gametocyte infectivity by direct mosquito feeds in an area of seasonal malaria transmission: implications for Bancoumana, Mali as a transmission-blocking vaccine site.

Infectivity of gametocytemic volunteers living in Bancoumana, a village 60 km from Bamako, Mali, was determined by direct feeds of laboratory-reared Anopheles gambiae s. l. Gametocytemic adolescents (10-18 years old) were as infectious to mosquitoes as younger volunteers and appear to be a more suitable population for testing transmission-blocking efficacy as compared with adults (> 18 years old). To begin to validate the membrane-feeding assay, sera collected from these same volunteers were subjected to a standard membrane-feeding assay. The data suggest that areas with intense but seasonal transmission might be feasible sites for testing transmission-blocking vaccines because of the high gametocytemic rates, high mosquito infectivity rates, and lack of pre-existing humoral-mediated transmission-blocking activity. The differences observed between field-based direct mosquito feeds and laboratory-based membrane feeding assays suggests that caution be used in interpreting Phase I study results in which laboratory-based membrane-feeding assays are used as a surrogate for vaccine efficacy.

The current state of studies of malaria vectors and the antivectorial campaign in west Africa.

Most malaria transmission in West Africa is by Anopheles funestus and An. gambiae s.l. An. funestus is not very polymorphic and is generally susceptible to currently used insecticides. An. gambiae in contrast is very polymorphic. On the basis of chromosomal polymorphism An. gambiae can be divided into forest and savannah forms. The savannah forms in turn exist as several types, viz. Bissau, Mopti and Bamako. Insecticide susceptibility does not correlate with chromosomal type in most cases; nevertheless, correct identification of vectors is necessary if a successful antimalarial campaign is to be developed. It is particularly important to determine which of the mosquitoes in the region under study are the important vectors of malaria. In the light of the difficulties encountered in mass vector control programmes, individual control measures such as home spraying and use of impregnated bed nets and curtains may be considered for malaria control.

The phenology of malaria mosquitoes in irrigated rice fields in Mali.

A field study was carried out in the large-scale rice irrigation scheme of the Office du Niger in Mali to investigate the relation between anopheline mosquito larval development and small-scale differences in irrigation practices, such as water level, irrigation application and irrigation frequency. The objective of the study was to find out if water management can be used as a tool for vector control to reduce the malaria transmission risk. Larvae of Anopheles gambiae s.s.,; the main malaria vector in the study area, developed mostly in the first 6 weeks after transplanting the rice. During rice development, a succession of anopheline species was observed. This was associated with a marked decrease in light intensity reaching the water surface as plant height increased. Minor differences in water management resulted in noticeable variations in larval densities and species composition. A. gambiae s.s. larvae were most abundant during the early growing stages and almost absent in a closed rice crop. Due to improper drainage after harvest, A. gambiae s.s. breeding was soon re-established in fields where small pools of water were retained. The results suggest that larval mosquito habitats in the Office du Niger can be significantly reduced by water management, simultaneous planting and harvesting and proper drainage of fallow fields.

Morphometric multivariate analysis of field samples of adult Anopheles arabiensis and An. gambiae s.s. (Diptera: Culicidae).

The Afrotropical complex of sibling species Anopheles gambiae Giles includes the most efficient vectors of human malaria south of the Sahara. Anopheles arabiensis Patton and An. gambiae s.s. Giles are the members of the complex more adapted to the human environment. They are sympatric and synchronic over most of their distribution range; however, they show a different involvement in malaria transmission, with An. gambiae being more anthropophilic and endophilic than An. arabiensis. Discriminating between them is essential for a correct assessment of epidemiological parameters. The identification is currently achieved through recognition of species-specific chromosomal inversions or by molecular biology techniques. Both methods require considerable technical resources, not always available in the field. We carried out a morphometric analysis of field and laboratory samples of An. arabiensis and An. gambiae s.s. from sites in Madagascar, Burkina Faso, Mali, and Liberia to evaluate the degree of morphological differentiation. We examined 17 morphometric characters in samples representing each of the geographic sites. All of the measures were significantly larger for An. arabiensis (regardless of the collection site), demonstrating an intrinsic greater body size of this species. To assess the reliability associated with the multivariate statistic, we applied the discriminant function analysis, which provided a method for predicting to which group a new case will most likely be assigned. In a blind experiment, the morphometric method correctly identified approximately 85% of field-collected An. arabiensis and An. gambiae s.s., which provided a relatively simple method to approximate the relative frequencies of the 2 species in areas in which their concurrent presence was already known. The influence of laboratory conditions on the morphometrics of the 2 species was also analyzed.

Microsatellite DNA polymorphism and heterozygosity among field and laboratory populations of Anopheles gambiae ss (Diptera: Culicidae).

We compared microsatellite polymorphism at nine loci located on chromosome 3 among two colonies and a field population of Anopheles gambiae sensu stricto Giles mosquitoes. Numbers of microsatellite alleles observed at each locus and mean heterozygosities were drastically reduced among laboratory colonies. Genetic analysis of the field population used in this study revealed an unprecedented frequency of rare alleles (<0.05). In contrast, colony samples revealed large numbers of alleles with frequencies >0.50. Partitioning of field data to assess the impact of rare alleles, null alleles, and sample size on estimates of mean heterozygosity revealed the plasticity of this measurement and suggests that heterozygosity may be reliably estimated from relatively small collections using microsatellites.

Midgut bacteria in Anopheles gambiae and An. funestus (Diptera: Culicidae) from Kenya and Mali.

Field studies in Kenya and Mali investigated the prevalence of bacteria in the midguts of malaria vectors, and the potential relationship between gram-negative bacteria species and Plasmodium falciparum sporozoites. Midguts were dissected from 2,430 mosquitoes: 863 Anopheles funestus Giles and 1,037 An. gambiae s.l. Giles from Kenya, and 530 An. gambiae s.l. from Mali. An. funestus had a higher prevalence of gram-negative bacteria (28.5%) compared with An. gambiae collected in Kenya and Mali (15.4 and 12.5%, respectively). Twenty different genera of bacteria were identified by gas chromatography from 73 bacterial isolates from mosquito midguts. Pantoea agglomerans (Enterobacter agglomerans) was the most common species identified. There was no association between gram-negative bacteria in the midgut and P. falciparum sporozoites in field-collected An. gambiae s.l. and An. funestus. However, An. funestus females that harbored gram positive bacteria were more likely to be infected with sporozoites compared with those with no cultivable bacteria or gram negative bacteria in their midguts. Habitat-related variation in the prevalence of diverse types of bacteria in mosquitoes could influence malaria parasite development in mosquitoes and corresponding sporozoite prevalence.

[Sensitivity of Anopheles gambiae s.l. to insecticides in the Selingue dam area]. / Sensibilité d'Anopheles gambiae s.l. Aux insecticides dans la zone du barrage de Sélingué.

In the Selingue hydroelectric dam area the An. gambiae complex members are susceptible to DDT, resistant to dieldrin (the adults mainly) and also susceptible to temephos, chlorpyrifos, fenthion, fenitrothion and malathion. The LC50 of the larvae for fenthion is 0.00325 ppm. Due to the abundance of An. gambiae s.s. in this area (nearly 98%), it is probable that these data refer mainly to this species. Recent cytogenetic studies have shown that this species is composed of three chromosomal types (Bamako, Savanna and Mopti) with partially reproductive isolation. In the case of resistance of members of a species complex to an insecticide, it will be of great interest to know how this resistance is distributed within the complex. This can be particularly important for An. gambiae s.s. and An. arabiensis, and also for the chromosomal forms Bamako and Mopti of An. gambiae s.s. which are reproductively well isolated.

The malaria challenge in the 21st century: perspectives for Africa.

Malaria control has had little success in Africa despite the achievements in malaria research. It is time to put more emphasis on sustainable control measures through local commitment to diagnose and treat malaria in order to prevent illness and death. This goal can be best achieved through basic health care centers, schools and safe water supplies to rural areas. Complementary actions through research and international support will be strongly needed.

[Mosquito net barriers for sample collection of zoophilic Culicidae]. / Barrière de tulle moustiquaire pour l'échantillonnage des Culicidés zoophiles.

Mosquito net fences 2.30-2.50 m high placed around cowsheds at their night resting sites have been successfully utilized to obtain large samples of malaria vectors of the Anopheles gambiae complex and of other mosquito species. Collections of blood-fed mosquitoes were carried out during the night by inspecting regularly the net side facing the animal enclosure. This sampling procedure has important advantages over alternative procedures based on direct collection on animal or on the use of animal-baited traps.

Field observations on the use of anti-sporozoite monoclonal antibodies for determination of infection rates in malaria vectors.

Samples of indoor-resting Anopheles gambiae s.1. from Mali and Burkina Faso (West Africa) were processed in order to compare Plasmodium falciparum sporozoite rates obtained by immunoradiometric assay (IRMA) with circumsporozoite (CS) monoclonal antibody and by microscope examination of salivary glands. The immunological method provided sporozoite rates always higher than those obtained by microscope examination. This result does not appear to be related to cross-reactions involving non-sporozoite antigens. A small fraction of IRMA-positive mosquitoes is necessarily negative by microscope, since these mosquitoes actually contain the CS antigen only in the abdomen, presumably in connection with the presence of fully mature oocysts. However, the frequency of these mosquitoes cannot explain in itself an average ratio of 1:2 between microscope and IRMA sporozoite rates. A more important source of difference appears to depend on the detection of positive mosquitoes with low sporozoite numbers which remain more frequently undetected by microscope examination. Failure of salivary gland penetration by sporozoites is also considered as a possible source of discrepancy between the two methods.

The ecology and behaviour of the forest form of Anopheles gambiae s.s.

Studies on the ecology of Anopheles gambiae s.s. and the transmission of malaria were undertaken in a high rainfall forested area in southern Sierra Leone. Anopheles gambiae s.s., identified by chromosomal techniques as the Forest form, was the only malaria vector in the study village. Surprisingly, rice fields or swamps were not favoured breeding places for this species; breeding mainly occurred in temporary pools. The mean annual sporozoite rate of An. gambiae s.s. determined by ELISA was 3.9%. Pyrethrum spray, human bait, and exit trap collections, as well as identification of mosquito blood-meals using the ELISA method, showed that the forest chromosomal form of An. gambiae s.s. was highly anthropophagic and exophilic.

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.

[Epidemiology of malaria in a village of Sudanese savannah area in Mali (Bancoumana). 2. Entomo-parasitological and clinical study ]. / Epidémiologie du paludisme dans un village de savane soudanienne du Mali (Bancoumana). 2. Etude entomo-parasitologique et clinique.

We carried out five cross sectional surveys between 1993 and 1994 to assess the epidemiology of malaria in the village of Bancoumana, located in the Sudanese savannah areas of Mali. Each survey included a collection of entomological, clinical, parasitological and immunological data. The study population involved 1600 children from six months to 9 years of age. The main vector was Anopheles gambiae s.l., man bite rate and entomological inoculation rate were maximum respectively in August (peak of the transmission season) and October (end of transmission season). Plasmodium. falciparum was the main parasite species observed. Spleen enlargement rate, parasite rate, gametocyte rate and parasite density varied significantly with age and season. The parasite rate, gametocyte rate and parasite density were significantly low in October 1994 compared with October 1993 while the entomologic parameter did not show any variation over the two years. This reduction of parasitologic index between 1993 and 1994 may be related to an increase of anti-malarial drug use in the population. Our results show that malaria is hyperendemic in the village of Bancoumana.

The spatial distribution of Anopheles gambiae sensu stricto and An. arabiensis (Diptera: Culicidae) in Mali.

Variations in the biology and ecology and the high level of genetic polymorphism of malaria vectors in Africa highlight the value of mapping their spatial distribution to enhance successful implementation of integrated vector management. The objective of this study was to collate data on the relative frequencies of Anopheles gambiae s.s. and An. arabiensis mosquitoes in Mali, to assess their association with climate and environmental covariates, and to produce maps of their spatial distribution. Bayesian geostatistical logistic regression models were fitted to identify environmental determinants of the relative frequencies of An. gambiae s.s. and An. arabiensis species and to produce smooth maps of their geographical distribution. The frequency of An. arabiensis was positively associated with the normalized difference vegetation index, the soil water storage index, the maximum temperature and the distance to water bodies. It was negatively associated with the minimum temperature and rainfall. The predicted map suggests that, in West Africa, An. arabiensis is concentrated in the drier savannah areas, while An. gambiae s.s. prefers the southern savannah and land along the rivers, particularly the inner delta of Niger. Because the insecticide knockdown resistance (kdr) gene is reported only in An. gambiae s.s. in Mali, the maps provide valuable information for vector control. They may also be useful for planning future implementation of malaria control by genetically manipulated mosquitoes.

Effective population size and persistence of Anopheles arabiensis during the dry season in west Africa.

The way that the malaria vectors Anopheles arabiensis and An.gambiae survive the dry season in sub-saharan Sahel or northern Savanna areas of Africa remains enigmatic. We examined this problem by calculating the effective sizes (Ne) of An.arabiensis populations for several locations in West Africa. An indirect/genetic procedure was used, comparing gene frequencies at several time intervals. The amount of drift which occurred provides an estimate of Ne. Most estimates of Ne were approximately 2000 individuals, probably close to the yearly minimum. This supports the hypothesis that populations of An.arabiensis in this region are continuous throughout the year, with many individuals surviving through the dry season, perhaps in a physiologically altered state, rather than extinction or severe bottlenecks during the dry season, followed by recolonization by a few individual survivors or immigrants in the subsequent rainy season.