Centromere-proximal differentiation and speciation in Anopheles gambiae

The M and S molecular forms of Anopheles gambiae are undergoing speciation as they adapt to heterogeneities in the environment, spreading malaria in the process. We hypothesized that their divergence despite gene flow is facilitated by reduced recombination at the centromeric (proximal) end of the X chromosome. We sequenced introns from 22 X chromosome genes in M and S from two locations of West Africa where the forms are sympatric. Generally, in both forms nucleotide diversity was high distally, lower proximally, and very low nearest the centromere. Conversely, differentiation between the forms was virtually zero distally and very high proximally. Pairwise comparisons to a close relative, the sibling species Anopheles arabiensis, demonstrated uniformly high divergence regardless of position along the X chromosome, suggesting that this pattern is not purely mechanical. Instead, the pattern observed for M and S suggests the action of divergent natural selection countering gene flow only at the proximal end of the X chromosome, where recombination is reduced. Comparison of sites with fixed differences between M and S to the corresponding sites in A. arabiensis revealed that derived substitutions had been fixed in both forms, further supporting the hypothesis that both have been under selection. These derived substitutions are fixed in the two West African samples and in samples of S from western and coastal Kenya, suggesting that selection occurred before the forms expanded to their current ranges. Our findings are consistent with a role for suppressed genetic recombination in speciation of A. gambiae.     

Molecular phylogeny of the Anopheles gambiae complex suggests genetic introgression between principal malaria vectors.

The six Afrotropical species of mosquitoes comprising the Anopheles gambiae complex include the most efficient vectors of malaria in the world as well as a nonvector species. The accepted interpretation of evolutionary relationships among these species is based on chromosomal inversions and suggests that the two principal vectors, A. gambiae and Anopheles arabiensis, are on distant branches of the phylogenetic tree. However, DNA sequence data indicate that these two species are sister taxa and suggest gene flow between them. These results have important implications for malaria control strategies involving the replacement of vector with nonvector populations.     

Intraspecific chromosomal polymorphism in the Anopheles gambiae complex as a factor affecting malaria transmission in the Kisumu area of Kenya.

The paracentric inversion polymorphisms of Anopheles gambiae and An. arabiensis populations in the Kisumu area of western Kenya were studied in relation to parameters of Plasmodium falciparum transmission. Anopheles gambiae (n = 1,387) was polymorphic for inversions b on chromosomal arm 2R and a on arm 2L, with frequencies of the inverted arrangements of 17% and 43%, respectively. Anopheles arabiensis (n = 484) was polymorphic for inversion b on chromosomal arm 2R and a on 3R, with frequencies of the inverted arrangements of 58% and 5%, respectively. Observed karyotypic frequencies did not deviate from Hardy-Weinberg equilibrium, indicating a condition of panmixia (i.e., random mating) for both species. The overall degree of intraspecific polymorphism was low, confirming findings from other zones of East Africa. No significant differences in inversion frequencies of either An. gambiae or An. arabiensis were observed, either between collecting sites or between similar sampling periods of consecutive years. At the same time, a stable, significant two-fold difference in Plasmodium infection rates was detected among An. gambiae carriers of different inversion karyotypes on chromosome 2. A significant non-uniform distribution of human- and bovid-fed specimens was also detected among the carriers of different 2Rb inversion karyotypes in indoor resting An. arabiensis. Relationships among inversion karyotypes of the two major malaria vectors in the An. gambiae complex and key factors affecting malaria transmission intensity emphasize that intraspecific variation could contribute significantly to the diversity and stability of malaria vectorial systems in Africa.     

When genetic distance matters: measuring genetic differentiation at microsatellite loci in whole-genome scans of recent and incipient mosquito species

Genetic distance measurements are an important tool to differentiate field populations of disease vectors such as the mosquito vectors of malaria. Here, we have measured the genetic differentiation between Anopheles arabiensis and Anopheles gambiae, as well as between proposed emerging species of the latter taxon, in whole genome scans by using 23-25 microsatellite loci. In doing so, we have reviewed and evaluated the advantages and disadvantages of standard parameters of genetic distance, F(ST), R(ST), (delta mu)(2), and D. Further, we have introduced new parameters, D' and D(K), which have well defined statistical significance tests and complement the standard parameters to advantage. D' is a modification of D, whereas D(K) is a measure of covariance based on Pearson's correlation coefficient. We find that A. gambiae and A. arabiensis are closely related at most autosomal loci but appear to be distantly related on the basis of X-linked chromosomal loci within the chromosomal Xag inversion. The M and S molecular forms of A. gambiae are practically indistinguishable but differ significantly at two microsatellite loci from the proximal region of the X, outside the Xag inversion. At one of these loci, both M and S molecular forms differ significantly from A. arabiensis, but remarkably, at the other locus, A. arabiensis is indistinguishable from the M molecular form of A. gambiae. These data support the recent proposal of genetically differentiated M and S molecular forms of A. gambiae.     

Dry season refugia of malaria-transmitting mosquitoes in a dry savannah zone of east Africa

Dry season survival of Anopheles funestus, Anopheles gambiae and Anopheles arabiensis in the Kilombero valley a dry savannah zone of east Africa, was investigated with over 400 collections from 23 areas, covering 300 sq km of the valley. Anopheles gambiae was found only in association with humans, in forested areas of high annual rainfall, while An. funestus occurred at high densities at the valley edge where large non-moving bodies of water remained. A large population of An. arabiensis was present along the river system throughout the middle of the valley, and mosquitoes probably derived from this population were occasionally caught in villages bordering the valley. No evidence was obtained of aestivation in any mosquito species. Anopheles gambiae was the most long lived, 6.3% compared to 2.0% of the An. arabiensis and 4% of the An. funestus surviving for four or more gonotrophic cycles, the approximate duration of the extrinsic cycle of most malaria parasites. Oocysts of malaria parasites were found in 5.4% of An. funestus and 2.3% of An. arabiensis from villages. Oocyst rates in An. funestus differed significantly between areas but not between houses within areas. Anopheles funestus is the most important dry season malaria vector in the valley, and remains in foci closely associated with groups of houses. All three species survive at high densities but as otherwise hidden refugia populations.     

Physiology of desiccation resistance in Anopheles gambiae and Anopheles arabiensis

Desiccation resistance and water balance were studied in the adult female mosquitoes Anopheles arabiensis and Anopheles gambiae sensu stricto. When the two species were reared from egg to adult under identical conditions, An. arabiensis had significantly higher desiccation resistance than did An. gambiae. Data are presented that indicate that this difference in desiccation resistance is associated with a higher body water content prior to desiccation in An. arabiensis. No differences in rate of water loss during desiccation or water content at death were observed. Measurements of metabolic rate and respiratory pattern also showed no statistically significant differences between the species. This study provides the first physiologic measurements of desiccation resistance in adults of these species and offers insights into the physiologic differences associated with differential resistance to desiccation stress.     

Variation in recombination rate across the X chromosome of Anopheles gambiae

The M and S molecular forms of Anopheles gambiae are considered to be incipient species, despite residual gene exchange. Of the three small genome regions that are strongly differentiated between the molecular forms ("speciation islands"), two are located near centromeres, on the left arm of chromosome 2 and the X chromosome. To test the prediction of reduced recombination in these islands, we estimated recombination rates between microsatellite loci on the X chromosome using two M-form strains. Across most of the chromosome, recombination occurred at approximately 1 centimorgan per megabase (cM Mb(-1)), a value closely matching the genome-wide average estimated for A. gambiae and for other eukaryotes. Recombination was much higher at the telomeric end, > 7 cM Mb(-1). In the speciation island at the centromeric end, recombination was sharply reduced to approximately 0.2 cM Mb(-1), consistent with a role for reduced recombination in maintaining differentiation between nascent species despite gene flow.     

Anopheles species of the mount Cameroon region: biting habits,feeding behaviour and entomological inoculation rates

There is a lack of data on the Anopheles fauna, its biology and the roles played by different vector species in the transmission of malaria in the mount Cameroon region. The biting habits, feeding behaviour and entomological inoculation rates of different Anopheles species during the dry and rainy season were investigated. A total of 2165 Anopheles was collected, 805 in the rainy season and 1360 in the dry season. Five Anopheles species were identified: Anopheles gambiae s.l., An. funestus, An. hancocki, An. moucheti and An. nili. An. gambiae, An. funestus and An. hancocki, recorded during both seasons, were the main vectors of malaria in the region. An. gambiae s.s. was the only member of the An. gambiae (Giles) complex. These three species had their peak activity between 1 and 2 am. A human blood index (HBI) of 98.29% was recorded for fed Anopheles. The sporozoite rate, for all vectors together, was significantly higher in the rainy season (9.4%) than in the dry season (4.2%) with all the species infected by Plasmodium falciparum. The average inoculation rate was 0.44 infective bites per man per night, which adds up to 161 infective bites per year in this study area. Analyses of relative abundance and infection rate of malaria vectors at different sites situated along a transect of 20 km during the dry season showed high heterogeneity in biting and sporozoite rates. No malaria vector was caught at 1200 m a.s.l. The mount Cameroon region should be considered an area of high malaria transmission intensity.     

Population genetic structure of Plasmodium falciparum in the two main African vectors, Anopheles gambiae and Anopheles funestus

We investigated patterns of genetic diversity of Plasmodium falciparum associated with its two main African vectors: Anopheles gambiae and Anopheles funestus. We dissected 10,296 wild-caught mosquitoes from three tropical sites, two in Cameroon (Simbock and Tibati, separated by 320 km) and one in Kenya (Rota, >2,000 km from the other two sites). We assayed seven microsatellite loci in 746 oocysts from 183 infected mosquito guts. Genetic polymorphism was very high in parasites isolated from both vector species. The expected heterozygosity (H(E)) was 0.79 in both species; the observed heterozygosities (H(O)) were 0.32 in A. funestus and 0.42 in A. gambiae, indicating considerable inbreeding within both vector species. Mean selfing (s) between genetically identical gametes was s = 0.33. Differences in the rate of inbreeding were statistically insignificant among sites and between the two vector species. As expected, because of the high rate of inbreeding, linkage disequilibrium was very high; it was significant for all 21 loci pairs in A. gambiae and for 15 of 21 pairs in A. funestus, although only two pairwise comparisons were between loci on the same chromosome. Overall, the genetic population structure of P. falciparum, as evaluated by F statistics, was predominantly clonal rather than panmictic, a population structure that facilitates the spread of antimalarial drug and vaccine resistance and thus may impair the effectiveness of malaria control efforts.     

Differential introgression of chromsomal regions between Anopheles gambiae and An. arabiensis

Evidence for introgression between Anopheles gambiae and An. arabiensis has accumulated for some time. We examined the fate of introgressed DNA directly, using microsatellite markers located throughout the genome. Introgressed X chromosomes were removed within two generations. Furthermore, substantial differences in introgressive capacity between the two autosomes were found. After introgression from An. arabiensis into An. gambiae, most introgressed alleles at third chromosome markers, particularly those on 3R, decreased steadily, indicating selection against them. No such pattern was observed for 2L markers and several 2R markers. The frequency of introgressed alleles on 2L were close to the original frequency even after 19 generations, whereas only two 2R markers showed a modest decrease. Even though limited information was available on the reciprocal cross, the pattern appears to be identical. Although the decrease in frequency of the introgressed X chromosome can be attributed to the presence of sterility and inviability effects, the variation in introgressive capacity of the autosomes does not appear to be explained by the presence of inversion polymorphisms, or regions causing hybrid sterility and inviability. These results can have some important implications for the spread of insecticide resistance and the control of these vector populations via the release of transgenic mosquitoes.     

Geographic and ecologic distributions of the Anopheles gambiae complex predicted using a genetic algorithm

The distribution of the Anopheles gambiae complex of malaria vectors in Africa is uncertain due to under-sampling of vast regions. We use ecologic niche modeling to predict the potential distribution of three members of the complex (A. gambiae, A. arabiensis, and A. quadriannulatus) and demonstrate the statistical significance of the models. Predictions correspond well to previous estimates, but provide detail regarding spatial discontinuities in the distribution of A. gambiae s.s. that are consistent with population genetic studies. Our predictions also identify large areas of Africa where the presence of A. arabiensis is predicted, but few specimens have been obtained, suggesting under-sampling of the species. Finally, we project models developed from African distribution data for the late 1900s into the past and to South America to determine retrospectively whether the deadly 1929 introduction of A. gambiae sensu lato into Brazil was more likely that of A. gambiae sensu stricto or A. arabiensis.     

Pupal habitat productivity of Anopheles gambiae complex mosquitoes in a rural village in western Kenya

The productivity of larval habitats of the malaria vector Anopheles gambiae for pupae (the stage preceding adult metamorphosis) is poorly known, yet adult emergence from habitats is the primary determinant of vector density. To assess it, we used absolute sampling methods in four studies involving daily sampling for 25 days in 6 habitat types in a village in western Kenya. Anopheles gambiae s.s. comprised 82.5% of emergent adults and Anopheles arabiensis the remainder. Pupal production occurred from a subset of habitats, primarily soil burrow pits, and was discontinuous in time, even when larvae occupied all habitats continuously. Habitat stability was positively associated with pupal productivity. In a dry season, pupal productivity was distributed between burrow pits and pools in streambeds. Overall, these data support the notion that source reduction measures against recognizably productive habitats would be a useful component of an integrated management program for An. gambiae in villages.     

Application of a biochemical key to study transmission of malaria and Bancroftian filariasis in sibling species of the Anopheles gambiae complex in north-eastern Tanzania

A biochemical key was applied in order to study transmission of malaria and Bancroftian filariasis in Anopheles gambiae sensu stricto, An. arabiensis and An. merus in different localities in north-eastern Tanzania. The technique was found to be a useful additional taxonomic tool for field entomologists. Significant differences between species in the rate of infection with Bancroftian filariasis were obtained between An. gambiae s.s. and An. funestus (P less than 0.005) and between An. funestus and An. arabiensis (P less than 0.0001). There were also significant differences between most of the investigated localities in the rate of filarial infection. However, there were no significant differences between the three species or between localities with respect to malaria sporozoite rates. Possible reasons for the observed variation between species and localities with respect to vectorial activity for Bancroftian filariasis are discussed.     

Comparison of DNA probe and cytogenetic methods for identifying field collected Anopheles gambiae complex mosquitoes

A recently developed DNA probe method was compared with the standard cytogenetic method for identifying the species of individual mosquitoes in the Anopheles gambiae complex. The complex consists of 6 morphologically indistinguishable sibling species that include the major African malaria vectors. Half-gravid, field collected mosquitoes were split into 2 portions: the abdomen was preserved for ovarian nurse cell cytotaxonomy and the head/thorax portion was desiccated for DNA extraction. Cytogenetic examination of the Kenya specimens showed 88 An. gambiae and 108 An. arabiensis. The Zimbabwe specimens consisted of 6 An. gambiae and 55 An. Quadriannulatus. All samples of the 3 species were polymorphic for the major chromosomal inversions previously recorded in field specimens from eastern and southern Africa, indicating that the collections reflected natural levels of intraspecific variation in the field populations sampled. Approximately 97% of the cytologically identified mosquitoes were also identified to species by the DNA probe method, and in every case the DNA probe and cytogenetic methods of species identification produced concordant results.     

A survey of Anopheles gambiae (species A) and An. arabiensis (species B) of the An. gambiae Giles complex in the Kisumu area of Kenya following insecticidal spraying with OMS-43 (Fenitrothion)

After village huts in the Kisumu area of Kenya had been sprayed with a nominal 2 g/m2 of OMS-43 (fenitrothion) as part of an insecticidal evaluation programme 964 adults of the Anopheles gambiae complex collected from huts were identified cytogenetically as either An. gambiae (species A) or An. arabiensis (species B). Similarly, cytogenetic methods were used to identify 349 adults collected from granaries and artificial pit-shelters. In addition, 2203 larvae of the gambiae complex collected from different types of habitats were specifically identified cytogenetically. As in a previous survey made prior to insecticidal spraying, adults of An. arabiensis predominated in outdoor collections while An. gambiae was highly endophilic. However, it appeared that spraying with fenitrothion had resulted in an increase in both the degree of exophily in An. arabiensis and also its relative numbers in respect to An. gambiae, although the overall pooulation of both species was greatly reduced by spraying. Insecticidal spraying may have diverted both species from feeding on man to cattle. Except for one village, where larvae of An. arabiensis were commoner than expected in cattle hoof-prints, there was little difference between the selection of different types of larval habitats by the two sibling species. Chromosomal inversions were more common in adults of An. arabiensis than in An. gambiae.     

The effects of rainfall and evapotranspiration on the temporal dynamics of Anopheles gambiae s.s. and Anopheles arabiensis in a Kenyan village

The population dynamics of the larval and adult life stages of the malaria vector Anopheles gambiae Giles were studied in Miwani, western Kenya, in relation to meteorological conditions. Larval density within a habitat, the number of larval habitats and sibling species composition were investigated as determinants of larval population dynamics. Female vector densities inside local houses and sibling species composition were investigated as determinants of adult population dynamics. Larval densities were estimated using a modified area-sampling method. Within the habitats, all instars showed a highly aggregated distribution, with the exception of second instars. A longitudinal study on the larval populations of A. gambiae s.l. in two different types of habitat (dirt track and ditch) was carried out, using a novel sampling procedure. A. gambiae s.s. and Anopheles arabiensis, the two sibling species occurring sympatrically in the study area, showed some spatial segregation between the two types of habitat. Rainfall was significantly correlated with the number of A. gambiae s.l. larval habitats during the first 6 weeks of study taking 1 week time lag into account, while over the entire 5-month study period correlations were less clear. With 1 week time lag, rainfall was also significantly correlated with the number of female A. gambiae s.l. collected from CDC-light traps in the study houses. Both larval and adult populations showed a significant increase in the proportion of A. gambiae s.s. within the mixed population of A. gambiae s.s. and A. arabiensis over time. Although not significantly correlated, the ratio of rainfall over precipitation/potential evapotranspiration (P/PE), indicative of the humidity conditions in the area, was probably the driving force of this increase.     

Anopheles gambiae s.s. breeding in polluted water bodies in urban Lagos, southwestern Nigeria

BACKGROUND & OBJECTIVE: Urban malaria is on the rise in Nigeria due to rapid industrialisation and developmental activities. A study was undertaken in Lagos, Nigeria to study the Anopheles breeding in polluted water bodies. METHODS: Anopheles larval breeding habitats were surveyed and water samples from 24 larval breeding sites from four strategic areas in urban Lagos were analysed. The relationship between eight abiotic variables (pH, dissolved oxygen, conductivity, hardness, nitrate, total dissolved solids, turbidity and oil) and density of Anopheles larvae were investigated. The levels of heavy metals (Zn, Co, Cu, Pb, Mn, Fe, Hg and Ni) pollution were analysed by Atomic Absorption Spectrophotometry. RESULTS: Morphological and PCR analysis of 2358 anopheline larvae revealed only the presence of two members of the Anopheles gambiae complex consisting of 93.1% Anopheles gambiae s.s. and 6.9% An. arabiensis. Multiple regression analysis revealed a significant association between occurrence of An. arabiensis larvae and two independent variables: pH and turbidity but not for An. gambiae s.s. The levels of three heavy metals: Fe, Cu and Pb from more than half of the sites surveyed were three times higher than the values obtained in natural breeding sites of An. gambiae s.s. from a rural area of Lagos. Over 85% of An. gambiae s.s. larvae were found in water bodies characterised by low dissolved oxygen (<3 mg/L), high conductivity (>900 uS/cm), turbidity (>180 FAU), oil (>11 mg/L) and heavy metals: Fe, Cu, and Pb (>0.4 mg/L). INTERPRETATION & CONCLUSION: These results indicate that An. gambiae s.s. is adapting to a wide range of water pollution in this urban area. The survival of the mosquito in widespread polluted water bodies across Lagos metropolis could be responsible for the rise in the incidence of malaria.     

Differentiation between species of the Anopheles gambiae Giles complex (Diptera: Culicidae) by analysis of cuticular hydrocarbons

Lipids were extracted from 500 female Anopheles gambiae and An. arabiensis in n-hexane and the extracts examined by gas chromatographic analysis using a glass column packed with 3% OV-1 on a 100-200 mesh Gas Chrom Q which was maintained at 225 degrees C. Cuticular hydrocarbons consisted mostly of unbranched paraffins. There were differences between the distribution of compounds with 25-29 carbons. After adjusting the C27 peak to 100%, hydrocarbons C26 and C29 represent heights of 80 and 69% in An. gambiae compared with values of 49 and 95 % in An. arabiensis. Other small but consistent differences were also found. Investigations are being performed on extracts from smaller samples and on other species within the An. gambiae complex.     

A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex

A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gambiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns. Although all are important (and often sympatric) African malaria vectors, their relative roles in malaria transmission have thus far been difficult to assess. The probe, an EcoRI-SalI fragment from the 3' end of the 28S beta coding region of the cistron, is present in all three species, but the species differ uniquely with respect to the location of an EcoRI site in the nontranscribed spacer (NTS) downstream of the fragment. We have routinely used the probe to identify A. gambiae complex mosquitoes to species on the basis of genomic DNA extracted from individual air dried specimens. A single mosquito abdomen provides more than sufficient DNA for the assay, and neither eggs nor a bloodmeal in the abdomen interfere with DNA yield. Moreover, the DNA extraction procedure does not degrade the bloodmeal IgG, so the residual protein pellet can be used to identify the mosquito bloodmeal source. Since the rDNA cistron organization as detected by the probe does not differ between male and female mosquitoes, the probe can be used for either sex. Preliminary experiments show that the probe is equally useful for mosquito larvae and pupae.     

Breakpoint structure reveals the unique origin of an interspecific chromosomal inversion (2La) in the Anopheles gambiae complex

Paracentric chromosomal inversions are major architects of organismal evolution and have been associated with adaptations relevant to malaria transmission in anopheline mosquitoes. The processes responsible for their origin and maintenance, still poorly understood, can be illuminated by analysis of inversion breakpoint sequences. Here, we report the breakpoint structure of chromosomal inversion 2La from the principal malaria vector Anopheles gambiae and its relatives in the A. gambiae complex. The distal and proximal breakpoints of the standard (2L+a) arrangement contain gene duplications: full-length genes and their truncated copies at opposite ends. Intact genes without pseudogene copies in the alternative arrangement (2La) imply that 2L+a is derived and was viable despite damage to genes, because duplication preserved gene function. A unique origin for the interspecific 2La inversion was challenged previously by indirect genetic evidence, but breakpoint sequences determined from members of the A. gambiae complex strongly suggest their descent from a single event. The derived position of 2L+a, long considered ancestral in this medically important group, has significant implications for the phylogenetic history and the evolution of vectorial capacity in the A. gambiae complex.