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Spatial and Habitat Distribution of Anopheles gambiae and Anopheles arabiensis (Diptera: Culicidae) in Banambani Village, Mali FRANCES E. EDILLO,1 YEYA T. TOURE´,2 GREGORY C. LANZARO,3 GUIMOGO DOLO,2 CHARLES E. TAYLOR1

AND

J. Med. Entomol. 39(1): 70Ð77 (2002)

ABSTRACT We studied the larval distribution and composition of Anopheles arabiensis Patton, An. gambiae s.s. Giles, and its forms, among local habitats; and their association with the adults between these habitats in Banambani village, Mali during the mid-rainy seasons of 1997Ð1999. For species and form identiÞcation we used polymerase chain reaction (PCR) and PCR-restriction fragment-length polymorphism (RFLP). Differences among species in the distribution of larvae were observed in 1998, but not in 1997 or 1999, although they were on the borderline of statistical signiÞcance. Differences among the M and S molecular forms were statistically signiÞcant in 1999 when rainfall was high, but not in the two prior, drier sampling periods. Combining all information into the Fisher multiple comparisons test, there were statistically signiÞcant differences between species and molecular forms during the 3-yr study period. Hybrid larvae between the M and S forms were observed (0.57%), the Þrst such observation to our knowledge. In spite of differences among larval distribution, no differences of adult species composition were observed among habitats. Factors that inßuence the distributions of An. gambiae larval populations are discussed. KEY WORDS Anopheles gambiae, Anopheles arabiensis, Anopheles gambiae s.s., Mopti and Savannah, habitat differences, hybrid

Anopheles gambiae s.s. Giles and An. arabiensis Patton, sibling species in the An. gambiae Giles complex, are found together through much of their home ranges. Macrogeographic trends are marked, with An. arabiensis tending to be more frequent where it is hot and dry (Coluzzi et al. 1979; Toure´ et al. 1998). On a microgeographic scale the trends are less clear. White (1974) reports that in East Africa An. arabiensis tends to be more zoophilic whereas An. gambiae s.s. is anthropophilic, though in Mali, West Africa both species seem to be, for all practical purposes, completely anthropophilic (Toure´ 1985, Toure´ et al. 1986). Further, in mark-release-recapture (MRR) studies conducted over several years at Banambani, Mali, Toure´ et al. (1998a) found no difference in distribution or dispersal pattern between adults of the two species. Accordingly, if the species do occupy different niches when sympatric, then we would expect to Þnd differences between them during other parts of the life cycle, possibly the larval stages. In early investigations of this possibility, Service (1970) and White and Rosen (1973) searched for, but were unable to discern, any microgeographic differ1 Department of Organismic Biology, Ecology and Evolution, University of California at Los Angles, CA 90095Ð1606. 2 Malaria Research and Training Center, De´ partement dÕ Epide´ miologie des Affections Parasitaires, Ecole Nationale de Me´ decine de Pharmacie et dÕ Odonto-Stomatologie, Bamako, B.P. 1805, Mali. 3 Department of Pathology and Center for Tropical Diseases, University of Texas Medical Branch, Galveston, TX 77555Ð 0609.

ence in larval distributions at study sites in Kenya and in Nigeria. Because larvae are morphologically indistinguishable, the accuracy of these studies was necessarily limited (Gillies and De Meillon 1968, Gillies and Coetzee 1987). Since that time, much better methods for discriminating the larvae have been developed, most notably a PCR-based diagnostic test published by Scott et al. (1993). Using this probe, Charlwood and Edoh (1996) observed microgeographic differences in the distribution of larvae around the town of Ifakara, Tanzania. The differences appeared to be linked to location rather than types of breeding substrate. In that region An. arabiensis was much more zoophilic than was An. gambiae s.s., and larvae of An. arabiensis were relatively more common in pools close to cattle. They attributed the differences in distribution of immatures to distance from adult feeding sources. Similarly, Minakawa et al. (1999) sampled larvae from different sites throughout the Suba District in Kenya. They, too, found signiÞcant spatial heterogeneity in species composition, but were unable to identify the key environmental factors that determined species occurrence and abundance. We inquired whether such differences in microgeographic distribution occur at our focal study site, Banambani Village, in Mali, West Africa. Here both species are almost entirely anthropophilic (Toure´ 1979, 1985), so the explanation put forth by Charlwood and Edoh (1996) seems unlikely. In addition to differences between species, we also inquired about the

January 2002

EDILLO ET AL.: DISTRIBUTION OF Anopheles gambiae S.L. IN MALI

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Fig. 1. Map of Banambani village, Mali, West Africa (Arc View 3.2). Encircled numbers are the breeding sites (1Ð3: rock pools, 4Ð5: swamps, 6Ð8: road puddles). Number 35 is the central compound. Letters A-D are the four regions. Shaded circles are the compounds; shaded squares are landmarks.

forms of An. gambiae s.s. In Mali there are three chromosomal forms that can be distinguished from their gene arrangementÑMopti, Bamako, and Savanna. These chromosomal forms are postulated to be something between subpopulations and species (Toure´ et al. 1998). Favia et al. (1997) developed PCR-based methods to distinguish between certain of these forms (Mopti versus Bamako and Savanna). Recognizing that the criteria may not coincide perfectly, they are sometimes termed the M- and S-“molecular forms”(Lanzaro et al. 1995). In particular, the Mopti form tends to predominate in hot, dry areas, like An. arabiensis. We hypothesized that these species and forms might also differ in their utilization of larval substrates within localities. Materials and Methods Study Site and Time. The study was conducted in Banambani Village, ⬇20 km northeast of Bamako, in the Northern Sudan Savanna of Mali at 12o 48Õ N and 8o 03Õ W (Fig. 1). It contains 700 inhabitants occupying ⬇250 huts grouped into seventy compounds (Taylor Table 1.

et al. 1993; Lanzaro et al. 1998; Toure´ et al. 1998). Nearly all of the yearly precipitation of 500 Ð1,000 mm comes during the rainy season from mid-May to early October, and it is only at this time that anophelines are abundant. Our study was conducted during the midrainy season in 1997Ð1999 (see Table 1 for sampling dates). Banambani has been surveyed for mosquitoes most years since 1979 (Toure´ 1979, Toure´ et al. 1998). Anopheline larvae have been observed in three types of habitatsÑpermanent rock pools (sites 1Ð3), swamps (site 4 Ð5), and road puddles (sites 6 Ð 8). Site numbers refer to Fig. 1 and Table 2. Photos of rock pools and road puddles have been published in Toure´ et al. 1998. Despite thorough searches, no other larval habitats were observed within or near the village. Each habitat was characterized according to the protocol suggested by WHO (1975a). Weather Data. The average monthly temperature, relative humidity, amount of rainfall and number of hours of rain in each yearÕs sampling period were taken from the Malian Weather Bureau, Bamako station (Table 1). The amount of rainfall was calculated

Weather data in Banambani Village, Mali, during the sampling periods and rainy seasons (1997-1999)

Sampling period/ rainy season

Amt of rainfall, mm

No. of h of rain

Avg monthly temp, ⬚C

% avg monthly RH, %

1997 27 July-6 Aug. MayÐOct.

104.87 1,011.00

7.61 86.00

27.50 28.33

81.15 73.33

1998 5 Aug.-27 Aug. MayÐOct

113.75 830.30

11.50 81.00

26.40 28.76

79.66 68.20

156.90 1,093.60

12.40 89.00

26.00 28.40

87.00 74.00

1999 2Ð17 Aug. MayÐOct.

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Table 2. Characteristics of the larval habitats of An. gambiae complex in Banambani village, Mali (values are median and range for 1997, 1998 and 1999) Site no.

Physical Properties No.

Surface area, m2

Rock pools Site 1 47 0.549 (0.04Ð3.2)

Depth, m

Water Properties Light exposure

0.155 (0.07Ð0.95) Exposed

Quiet/ running Quiet

24 0.133 (0.0003Ð2.45) 0.054 (0.02Ð1.3) Semi-shady/ Quiet exposed

Site 3

14 0.525 (0.13Ð78.5)

Swamp Site 4

Site 5

Puddles Site 6

26 ⬇50 (32Ð100)

5 5.03 (1.6Ð5.7)

23

0.7 (0.01Ð0.1) Semi-shady/ Quiet/slight exposed ripples 0.1 (0.06Ð2)

7.5 (0.0007Ð19.6) 0.045 (0.01Ð5)

Grasses*

Very turbid

7.3 (6.8Ð7.8) Grasses* Green algae** Trees & herbs*** 7.18 (6.9Ð8.4) Trees, herbs, shrubs, grasses***

Turbid

Exposed

Quiet

Clear/turbid 7.18 (6.8Ð12.5) Grasses* Green algae** Very turbid 7.32 (6.5Ð8.9) Grasses* Green algae** Turbid 7.1 (7.2Ð7.5) None

9 0.07 (0.002Ð0.78)

0.04 (0.03Ð0.07) Exposed

Quiet

Site 8

5 0.24 (0.2Ð0.4)

0.06 (0.05Ð0.7) Exposed

Quiet

b

Clear/slightly 7.3 (6.5Ð15) turbid

Typea

Semi-shady Quiet/slight ripples

Site 7

a

pH

Green algae** Clear/slightly 7.9 (6.8Ð8.2) Grasses* turbid Green algae** Quiet/slightly Clear/slightly 7.6 (6.9Ð8.1) Grasses* running turbid Green algae**

Site 2

0.09 (0.03Ð1.5) Exposed

Clear/turbid

Vegetation Densityb

⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹ ⫹ ⫹⫹⫹ ⫹⫹⫹ ⫹⫹⫹

⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫺

*, emergent and edges; **, submersed; ***, around pools. ⫹, few; ⫹⫹, abundant; ⫹⫹⫹, very abundant.

by multiplying the mean daily rainfall by the number of sampling days. Precipitation was highest in 1999 (156.9 mm) during the sampling period and less during 1997 (104.87 mm) and 1998 (113.75 mm). Sampling. We examined all previously identiÞed larval habitats and searched for others each year of our study. Larval collections were attempted whenever weather permitted during the sampling periods. Collections were made following the protocols described in WHO (1975b), sampling from each site for 1.5 h. For each sample, we took 30 Ð 40 dips using a standard Bioquip mosquito larvae dipper per habitat. Larvae were stored in plastic bags containing water from the habitat and brought to the laboratory in Bamako. If no larvae were seen after 30 min, the site was considered negative. DNA Extraction. In the laboratory, larvae were poured from plastic bags into trays and pipetted individually into 1.5-ml microcentrifuge tubes. These were stored at ⫺80⬚C until processed for DNA extraction. DNA was extracted using a procedure modiÞed from that described by Lanzaro et al. (1995). Samples in each tube were homogenized with a pestle in 50 ␮l of Bender buffer with 1% DEPC and an additional 50 ␮l of Bender buffer was then added. They were then incubated at 65⬚C for 1 h, at which time 15 ␮l of cold 8M potassium acetate was added and incubated on ice for 45 min. The samples were then centrifuged for 10 min at 14,000 ⫻ g. The supernatant was transferred to a new microcentrifuge tube and 250 ␮l of 100% ethanol

was added and incubated at room temperature for 5 min. It was Þnally centrifuged for 15 min at 14,000 x g.The supernatant was again discarded and the dry pellet containing DNA was stored for shipment to the laboratory at the University of Texas Medical Branch at Galveston, where further processing was done. PCR. DNA templates were ampliÞed with primers speciÞc for An. gambiae s.s. and An. arabiensis as described by Scott et al. (1993). Aliquots from individuals identiÞed to be An. gambiae s.s. were then taken and further processed to identify Mopti from Savanna in the manner of Favia et al. (1997; 2001), using the primers by Lancrofratti et al. (1998). Mark-Release-Recapture Experiments. MRR studies were performed in 1997 and 1998 simultaneously with the larval collections in the manner described by Toure´ et al. (1998); the only difference was that we used a single release location for these studies, compound 35 (Fig. 1).

Results Larval Habitats. Ecological parameters of these larval habitats are shown in Table 2. All road puddles and most of the rock pools were exposed to the sun, while the swamps were semishady because of surrounding trees. Water was very turbid in swamps, slightly turbid in road puddles, and clear to slightly turbid in rock pools. All larval habitats had shallow water (⬍21 cm


January 2002

Table 3. Statistical analysis of the larval distribution of An. arabiensis, An. gambiae s. s., and its forms (M and S) in Banambani village (1997–1999) Year

df

1997 1998 1999 FisherÕs test

4 4 4 6

Species

Form

␹2

P

␹2

P

8.214 9.516 8.606 16.256

0.084 0.049* 0.072 0.012*

2.543 7.097 28.959 28.450

0.637 0.131 ⬍0.001*** 0.001***

deep at the deepest place) except for site 3, where the river became deeper and in the lower swamp where water was usually deep. In these habitats, larvae were sampled only near the edges. Stagnant water predominated in all road road puddles, lower swamp, and some rock pools toward the banks of the river. Those rock pools, particularly at site 3, had running water from time to time due to the adjacent river. The upper swamp could hold rainwater only brießy, at most a day during our sampling, before the water evaporated or seeped down toward lower elevations. Larvae were collected in water with wide pH values ranging from acidic to basic (6.5Ð15 in rock pools, 6.8 Ð 8.4 in swamp, and 6.5Ð12.5 in road puddles). Larval Distribution. Larvae were relatively abundant in sites 1Ð3 (rock pools), 4 (lower swamp), and 6 (road puddles) in both 1997 and 1998, and in sites 1, 2, 4, 6, 8 (road puddles) in 1999 (Fig. 2). The upper swamp (site 5) contained immatures during 1997 and 1998, but not in 1999. In 1998 and 1999 there were An. gambiae s.l. larvae in road puddles (site 7), but in 1997 these contained Culex tigripes Grandpre` & Charmoy which prey upon them (WHO 1975a) and were presumably responsible for their absence. There were few larvae recovered at only one sampling out of the many we did from site 5 (upper swamp) (1 in 1997, 54 in 1998, and 0 in 1999), and site 7 (road puddle) (0 in 1997, 20 in 1998, and 33 in 1999). We could not be conÞdent that they were the offspring from more than one or two females because an average of 100 eggs are oviposited by one female, so these were excluded from further analysis. Rainfall was very high during the collection period in 1999, leading to strong currents in the river adjacent to site 3 and precluding the presence of larvae there. At the same time, new puddles (site 8) were created in the roads, providing new habitats for larvae. We identiÞed 1,034 larvae to species and molecular forms in 1997Ð1999 (332 in 1997, 262 in 1998, and 440 in 1999). A few samples could not be identiÞed to their form because of poor conditions of DNA extracts or because they were hybrids between molecular forms. In total, there was a total of 0.57% hybrids (1/212 in 1997, 0/182 in 1998, and 3/307 in 1999). These hybrids were found in 1997 in rock pools (site 2), whereas in 1999 they were found in a different rock pool (site 1) and in road puddles (sites 6 and 8). Species Distribution. Anopheles gambiae s.s. were more numerous than An. arabiensis in all sites that contained both species. A 5 (sites) ⫻ 2 (species) contingency table was constructed for each year and

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examined for statistical independence of species and sites (Table 3). The distribution of larvae of the two species differed statistically among sites in 1998, but not in 1997 and 1999; all bordered statistical signiÞcance (P ⬍ 0.05). The Fisher test for multiple independent comparison (Fisher 1954) showed an overall statistically signiÞcant heterogeneous distribution of species among habitats (P ⫽ 0.012) over the 3-yr mid-rainy season data. Chromosomal Form Distribution. M form larvae comprised 28.17% of all An. gambiae s.s. in 1997, 9.89% in 1998, and 10.97% in 1999 (Fig. 2). Analyzing the data as above, the M and S distribution behaved somewhat differently than those for species. Their frequency and distribution in breeding sites was highly signiÞcant in 1999 (P ⬍ 0.001), but not in the previous two years. The Fisher test for multiple independent comparison (Fisher 1954) revealed that the overall differences were highly signiÞcant (P ⬍ 0.001). Distribution of Adult Females. The distribution of unmarked female mosquitoes from the 1997 and 1998 MRR studies are summarized in Table 4 and analyzed in Table 5 (Sokal and Rohlf 1995). It was necessary to pool mosquito larval sites for statistical analysis. We expected females from regions A, B, and C to preferentially oviposit in the swamp and puddle habitats closest to this area. Females from region D were closer to the rock pools and would seem more likely to breed there. The numbers of each species across year and area (Table 5) were viewed as a 2 ⫻ 2 ⫻ 2 contingency table and analyzed statistically with a log-linear model to test for association using the CATMOD procedure of SAS (SAS Institute 1999). The results of such a comparison can be interpreted essentially as an analysis of variance (ANOVA). The “intercept” terms, which indicate whether the two species were observed in a 50:50 proportion, were in all cases statistically signiÞcant. But in no case was there an effect for life stage, indicating that these larvae and adult stages behaved similarly; nor was there an effect of habitats in designated regions in the village, when the several sites were pooled. Discussion We found statistically signiÞcant differences in the larval distribution of An. gambiae s.s. and An. arabiensis among the three habitats where larvae could be identiÞed (rock pools, swamp, and temporary road pools) in Banambani village, Mali. We were unable to identify which habitat was preferred by either species or by either form of An. gambiae s.s. This Þnding is consistent with a similar study of Minakawa et al. (1999) in Suba District, Kenya. They too noted species differences, and were unable to identify which key environmental factors were associated with one species or the other. This inability to link speciÞc habitats to either An. gambiae s.s. or An. arabiensis in Banambani village invites further questions. It is generally believed that if two species are to coexist, they must have some divergence in niche or other part of their life history

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Fig. 2. Larval frequency distribution of An. gambiae s.s. Mopti (hatched) and Bamako-Savanna (shaded) and An. arabiensis (solid dark) sampled from different breeding sites (1Ð3: rock pools, 4Ð5: swamps, 6Ð8: road puddles) during the mid-rainy seasons of 1997Ð1999.

(see, e.g., Hutchinson 1978). Several possibilities suggest themselves: Þrst, faster development of An. arabiensis larvae, at least in laboratory conditions Table 4.

(Schneider et al. 2000), may explain its tendency to precede An. gambiae s.s. when rains follow drought, whereas An. gambiae s.s. tends to more numerous

Composition of An. gambiae s. l. unmarked females and larvae, 1997 and 1998 Proportion of An. gambiae s.s.a

Region D A, B, C a

Habitat Rock pools Swamps & road puddles

Numbers are in parentheses.

1997

1998

乆

Larvae

乆

Larvae

0.672 (39/19) 0.660 (31/16)

0.564 (75/58) 0.692 (137/61)

0.739 (65/23) 0.669 (93/46)

0.689 (91/41) 0.700 (91/39)

January 2002 Table 5.


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Maximum-likelihood ANOVA of Table 4

Source

df/yr

Intercept Life stage (adult/larvae) Habitat ([D/ABC]/habitat) Life stage ⫻ habitat

1 1 1 1

1997

1998

␹2

Probability

␹2

Probability

26.34 0.43 1.08 1.64

0.001 0.509 0.299 0.201

70.24 0.06 0.50 0.91

0.001 0.808 0.481 0.341

during the rainy season (White et al. 1972). Second, if the species with the included fundamental niche is to survive, then it must be competitively superior within at least part of that niche (Hutchinson 1978). It appears that An. arabiensis may occupy a more specialized niche than An. gambiae s.s. (Charlwood and Edoh 1996), and thereby enjoys superiority in hot dry environments. Whether the two species have different requirements from their larval habitats, the possibility examined here is less clear. Charlwood and Edoh (1996) suggested that the larval requirements of both species are similar, and that their distribution may be largely explained by location of breeding site and host rather by habitat availability. In contrast, Minakawa et al. (1999) postulated they have different larval habitat requirements but were unable to demonstrate that explicitly. Coluzzi (1999) and Powell et al. (1999) have stressed the importance of historical changes in humanÐmosquito associations. They also suggest that the two species may not be at a long-term equilibrium, but are rather in a transient, historically contingent balance in an evolutionary competition, where now one species is advantaged, then the other. They suggest the 2La chromosomal inversion plays a key role in this, as it is closely tied to arid environmental adaptation in An. arabiensis (Powell et al. 1999). If so, then we may be observing the evolution of An. gambiae s.s. Mopti form vying for that part of the niche space where An. arabiensis is currently able to exclude the other forms of An. gambiae s.s. We found a very signiÞcant difference in the larval distribution of the M and S molecular forms in 1999, but not in the previous two years. This is possibly tied to the high rainfall at that time, where we observed much more frequent destruction of larval breeding sites than in 1997 or 1998 (Table 1). This suggests that here, too, transient events might determine overall evolutionary advantages. The detection of 0.57% putative larval hybrids between M and S Þeld-collected samples is important for inference of gene ßow between the forms of An. gambiae s.s. It is consistent with the recent DNA analysis of transferred sperm (Tripet et al. 2001) which revealed signiÞcant hybridization between forms. Taylor et al. (2001) used the observations reported here to estimate that gene ßow between forms exceeded the threshold generally thought required for maintaining species boundaries. While we were able to discern only small differences in distribution, it remains possible that the larval distribution of these species and forms are actually

distinct, but simply differ along environmental axes we did not recognize or measure. Alternatively, the abundance of these larvae may be determined by many habitat variables, each contributing a small effect as suggested by Minakawa et al. (1999). Sampling bias might also have an inßuence; it seems more difÞcult to obtain unbiased samples of mosquito immatures than adults since they have a highly aggregated distribution (Lakhani and Service 1974, Service 1985). A careful evaluation of location of the larvae within breeding sites might reveal unsuspected differences in microhabitat use between species (Charlwood and Edoh 1996). We are currently exploring these possibilities in detail. We found no signiÞcant association between species of female adult mosquitoes in different regions and larval composition in the habitats adjoining them, for either the 1997 or 1998 mid-rainy seasons. Previous MRR experiments conducted in the village during the wet seasons of 1993Ð1994 found apparently identical dispersal for the two species and among forms of An. gambiae s.s. (Toure´ et al. 1998). This was also observed in Kenya by Minakawa et al. (1999). At another location, An. arabiensis females were more common near cattle, e.g., Ouagadougou, Burkina Faso (Costantini et al. 1996) and in Ifakara, Tanzania, leading to the suggestion by Charlwood and Edoh (1996) that differences in their distribution were due to host availability. The overall view emerging from these studies is that An. gambiae s.l. species differ from place to place in their association with humans and their domestic animalsÑand quite possibly in their niche overlap as a result. It seems that historical contingencies, ongoing evolutionary changes, year-to-year ßuctuation in weather, and spatially varying host specialization all contribute to the dynamics of niche overlap and competition between these mosquitoes. Along with the numerous chromosome polymorphisms of these species, such environments would seem to epitomize conditions favorable to Sewall WrightÕs “shifting balance” theory of evolution (Wright 1978; Provine 1986).

Acknowledgments We are grateful to Adama Dao, Bakary Sissoko, Fah Niare´ , and the village of Banambani who have been very helpful to us for undertaking the MRR studies and for the regular larval collections. Also to Douglas Norris and Frederick Tripet for sharing their knowledge of the molecular techniques used here. The studies beneÞted from the technical support of Richard Sakai and Robert W. Gwadz. This research was

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supported by grants from the National Institutes of Health, from the John Sealy Memorial Endowment Fund, and from a WHO/TDR partnership grant. Special mention to IIEFulbright is made for granting the Þrst authorÕs full scholarship while undertaking this project (SY 1996 Ð1999). We also express our sincere thanks to two reviewers for their valuable comments on an early draft of the manuscript.

References Cited Charlwood, J. D., and D. Edoh. 1996. Polymerase chain reaction used to describe larval habitat use by Anopheles gambiae complex (Diptera: Culicidae) in the environs of Ifakara, Tanzania. J. Med. Entomol. 33(2): 202Ð204. Coluzzi, M. 1999. The clay feet of the malaria giant and its African roots: hypothesis and inferences about origin, spread and control of Plasmodium falciparum. Parassitologia 41: 277Ð283. Coluzzi, M., A. Sabatini, V. Petrarca, and M. A. Di Deco. 1979. Chromosomal differentiation and adaptation to human environments in the Anopheles gambiae complex. Trans. R. Soc. Trop. Med. Hyg. 73: 483Ð 497. Costantini, C., S. Li, A. della Torre, N’F. Sagnon, M. Coluzzi, and C. E. Taylor. 1996. Density, survival and dispersal of Anopheles gambiae complex mosquitoes in a West African Sudan savanna village. Med. Vet. Entomol. 10(3): 203Ð 219. Favia, G., A. della Torre, M. Bagayoko, A. Lanfrancotti, N’F. Sagnon, Y. T. Toure, and M. Coluzzi. 1997. Molecular identiÞcation of sympatric chromosomal forms of An. gambiae and further evidence of their reproductive isolation. Ins. Mol. Biol. 6(4): 377Ð383. Favia, G., A. Lanfrancotti, L. Spanos, I. Siden-Kiamos, and C. Louis. 2001. Molecular characterization of ribosome DNA polymorphisms discriminating among chromosomal forms of An. gambiae s.s. Insect Mol. Biol. 10(1): 19 Ð23. Fisher, R. A. 1954. Statistical Methods for Res. Workers, 12th edition. Oliver and Boyd, London. Gillies, M. T., and B. De Meillon. 1968. The Anophelinae of Africa South of the Sahara (Ethiopian Zoogeographical Region). No. 54. The South African Institute for Medical Research, Johannesburg. Gillies, M. T., and M. Coetzee. 1987. A Supplement to the Anophelinae of Africa South of the Sahara. No. 55. The South Africa Institute for Medical Research, Johannesburg. Hutchinson, E. G. 1978. What is a niche? pp. 152Ð219. In E. G. Hutchinson [ed.], An introduction to population ecology. Yale University Press, London. Lakhani, H., and M. W. Service. 1974. Estimated mortalities of mosquito preadults, pp. 185Ð201. In L. P. Lounibos, J. R. Rey, and J. H. Frank [eds], Ecology of mosquitoes: proceedings of a workshop. Florida Medical Entomology Laboratory, Vero Beach, FL. Lancrofratti, A., A. della Torre, and G. Favia. 1998. Improvement of a PCR-based assay for the identiÞcation of sympatric forms of Anopheles gambiae s.s. Parassitologia 40(suppl. 1): 87. Lanzaro, G. C., L. Zheng, Y. T. Toure´, S. F. Traore, F. C. Kafatos, and K. D. Vernick. 1995. Microsatellite DNA and isozyme variability in a west African population of An. gambiae. Insect Mol. Biol. 4(2): 105Ð112. Lanzaro, G. C., Y. T. Toure´, J. Carnahan, L. Zheng, G. Dolo, S. Traore, V. Petrarca, K. D. Vernick, and C. E. Taylor. 1998. Complexities in the genetic structure of Anopheles gambiae populations in west Africa as revealed by mic-

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rosatellite DNA analysis. Proc. Natl. Acad. Sci. U.S.A. 95: 14260 Ð14265. Minakawa, N., C. M. Mutero, J. I. Githure, J. C. Beier, and G. Yan. 1999. Spatial distribution and habitat characterization of Anopheline mosquito larvae in Western Kenya. Am. J. Trop. Med. Hyg. 61(6): 1010 Ð1016. Powell, J. R., V. Petrarca, A. della Torre, A. Caccone, and M. Coluzzi. 1999. Population structure, speciation, and introgression in the Anopheles gambiae complex. Parassitologia 41: 101Ð113. Provine, W. B. 1986. WrightÕs shifting balance theory of evolution, pp. 277Ð326. In W.B. Provine: Sewall Wright and evolutionary biology. The University of Chicago Press, Chicago, IL. SAS Institute. 1999. SAS/STAT UserÕs Guide, Version 8. SAS Institute, Carey, NC. Schneider P., W. Takken, and P. J. McCall. 2000. InterspeciÞc competition between sibling species larvae of Anopheles arabiensis and An. gambiae. Med. Vet. Entomol. 14: 165Ð170. Scott, J. A., W. G. Brogdon, and F. H. Collins. 1993. IdentiÞcation of single specimens of the An. gambiae complex by the polymerase chain reaction. Am. J. Trop. Med. Hyg. 49: 520 Ð529. Service, M. W. 1970. Ecological notes on species A and B of the An. gambiae complex in the Kisumu area of Kenya. Bull. Entomol. Res. 60: 105Ð108. Service, M. W. 1985. Population dynamics and mortalities of mosquito preadults, pp. 185Ð201. In L. P. Lounibos, J. R. Rey, and J. H. Frank (eds.), Ecology of mosquitoes: proceedings of a workshop. Florida Medical Entomology Laboratory, Vero Beach, FL. Sokal, R. R., and F. J. Rohlf. 1995. Biometry. The principles and practice of statistics in biological research, 3rd ed. Freeman, New York. Taylor, C. E., Y. T. Toure´, J. Carnahan, D. E. Norris, G. Dolo, S. F. Traore, F. E. Edillo, and G. C. Lanzaro. 2001. Gene ßow among populations of the malaria vector, Anopheles gambiae, in Mali, West Africa. Genetics 157: 743Ð750. Tripet, F., Y. Toure´, C. E. Taylor, D. E. Norris, G. Dolo, and G. C. Lanzaro. 2001. DNA analysis of transferred sperm reveals signiÞcant levels of gene ßow between molecular forms of the Anopheles gambiae complex. Mol. Ecol. (in press). Toure´, Y. T. 1979. Bio-ecologie des Anopheles (Diptera: Culicidae) dansune zone rurale de savane soudanienne au Mali village de Banambani ÐArrondissement de Kati: Incidence sur la transmission du Paludisme et de la Filariose de Bancroft. Ph.D. dissertation, Centre Pedagogie Superieur, Bamako, Mali. Toure´, Y. T. 1985. Ge´ ne´ tique e´ cologique et capacite´ vetorielle des membres du complexe Anopheles gambiae au Mali. The´ se de Doctorat dÕEtat e´ s Sciences Naturelles. Universite´ de Droit, dÕEconomie et des Sciences AixMarseille III. Marseille, France. Toure´, Y. T., V. Petrarca, et M. Coluzzi. 1986. Bioe´ cologie et importance vectorielle des taxa du complexe Anopheles gambiae au Mali, pp. 552Ð589. In Proceedings, IVe Congre´ s sur la Protection de la Sante´ Humaine et des Cultures en Milieu Tropical, 2Ð 4 Juillet 1986, Marseille, France. Toure´, Y. T., V. Petrarca, S. F. Traore, A. Coulibaly, H. M. Maiga, O. Sankare, M. Sow, M. A. Di Deco, and M. Coluzzi. 1998. The distribution and inversion polymorphism of chromosomally recognized taxa of the Anopheles gambiae complex in Mali, West Africa. Rome. Parassitologia 40(4): 477Ð511. Toure´, Y. T., G. Dolo, V. Petrarca, S. F. Traore, M. Bouare, A. Dao, J. Carnahan, and C. E. Taylor. 1998a. Mark-

January 2002


release-recapture experiments with An. gambiae s.l. in Banambani village, Mali to determine population size and structure. Med. Vet. Entomol. 12(1): 74 Ð 83. White, G. B. 1974. Anopheles gambiae complex and disease transmission in Africa. Trans. R. Soc. Trop. Med. Hyg. 68: 278 Ð301. White, G. B., and P. Rosen. 1973. Comparative studies on sibling species of the An. gambiae Giles complex (Diptera: Culicidae). II. Ecology of species A and B in savanna around Kaduna, Nigeria, during transition from wet to dry season. Bull. Entomol. Res. 62: 613Ð 625. White, G. B., Magayuka, S. A., and Brocham, P.F.L. 1972. Comparative studies on sibling species of the Anopheles gambiae Giles complex (Diptera: Culicidae): bionomics and vectorial activity at Segera, Tanzania. Bull. Entomol. Res. 62: 295Ð317.

77

WHO. 1975a. Manual on Practical Entomology in Malaria. Part I. Vector Bionomics and Organization of Anti-malaria Activities. WHO Division of Malaria and other Parasitic Diseases, Geneva. WHO. 1975b. Manual on Practical Entomology in Malaria. Part II. Methods and Techniques. WHO Division of Malaria and Other Parasitic Diseases, Geneva. Wright, S. 1978. Evolution and the genetics of populations, vol. 4: Variability within and among natural populations. Chicago: University of Chicago Press.

Received for publication 28 December 2000; accepted 22 June 2001.