Linear and Spatial Organization of Polytene Chromosomes of the African Malaria Mosquito Anopheles funestus

Corresponding author: Guiyun Yan, Department of Biological Sciences, State University of New York, Buffalo, NY 14260., gyan{at}acsu.buffalo.edu (E-mail)

Communicating editor: G. B. GOLDING

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Anopheles funestus Giles is one of the major malaria vectors in Africa, but little is known about its genetics. Lack of a cytogenetic map characterized by regions has hindered the progress of genetic research with this important species. This study developed a cytogenetic map of An. funestus using ovarian nurse cell polytene chromosomes. We demonstrate an important application with the cytogenetic map for characterizing various chromosomal inversions for specimens collected from coastal Kenya. The linear and spatial organization of An. funestus polytene chromosomes was compared with the best-studied malaria mosquito, An. gambiae Giles. Comparisons of chromosome morphology between the two species have revealed that the most extensive chromosomal rearrangement occurs in pericentromeric heterochromatin of autosomes. Differences in pericentromeric heterochromatin types correlate with nuclear organization differences between An. funestus and An. gambiae. Attachments of chromosomes to the nuclear envelope strongly depend on the presence of diffusive ß-heterochromatin. Thus, An. funestus and An. gambiae exhibit species-specific characteristics in chromosome-linear and -spatial organizations.

ANOPHELES funestus Giles is an important malaria mosquito vector in Africa. It occupies a wide-range of ecological niches throughout the Afrotropical region, is highly anthropophilic, and is susceptible to the human malaria parasites (GILLIES and DE MEILLON 1968 ; GILLIES and COETZEE 1987 ; MBOGO et al. 1999 ). Its activity spans the dry season when another major malaria vector species, An. gambiae, is usually inactive (FONTENILLE et al. 1997 ). Previous studies with various mosquito species have demonstrated that mosquito host feeding behaviors and vector competence to malaria parasites are under genetic control (COLUZZI et al. 1977 ; COLLINS et al. 1999 ). Anopheline mosquitoes, like Drosophila, are renowned for the presence of polytene chromosomes and chromosomal inversions (COLUZZI et al. 1979 ). Localization of genes can be accomplished through linkage analysis with previously mapped genetic markers, and a physical map is critical for map-based gene cloning. Similarly, studying population genetic structure requires information on the location of genetic markers because markers in different genome regions exhibit different levels of gene flow. Reduced recombination and selection can influence loci within or near inversions, resulting in gene flow being lower relative to loci elsewhere in the genome (LANZARO et al. 1998 ). Development of a cytogenetic map represents the first step for these important tasks. For example, progress in An. gambiae genetic research in areas such as species identification, identification of chromosomal forms, and genome mapping has been facilitated by the availability of a cytogenetic map (COLUZZI and SABATINI 1967 ; COLUZZI et al. 1979 ; DELLA TORRE et al. 1996 ; TOURE et al. 1998 ).

The aims of this study are (1) to develop a cytogenetic map for An. funestus using polytene chromosomes and to define inversion breakpoints and determine inversion frequencies for specimens from coastal Kenya and (2) to compare linear and spatial organization of polytene chromosomes between An. funestus and another well-studied African malaria vector, An. gambiae Giles. GREEN and HUNT 1980 published a photomap of An. funestus and identified several polymorphic inversions for specimens from several African countries. So far, a total of 14 polymorphic inversions have been identified: 10 on chromosomal arm 2R [a, b, c, d, e, and h (GREEN and HUNT 1980 ), s, t, and u (BOCCOLINI et al. 1998 ), and z (LOCHOUARN et al. 1998 )], 2 on arm 3R [a and b (GREEN and HUNT 1980 )], and 2 on arm 3L [a and b (GREEN and HUNT 1980 )]. However, the photomap by GREEN and HUNT 1980 did not divide polytene chromosomes by regions and thus the breakpoints of these inversions could not be graphically defined. Thus, development of a standard cytogenetic map should prove to be useful for population comparisons by different investigators.

An. funestus represents a group and subgroup of species with slight morphological differences. The An. funestus group includes An. brucei Service, An. confusus Evans and Leeson, An. fuscivenosus Leeson, An. rivulorum Leeson and the four members of An. funestus subgroup, An. parensis Gillies, An. funestus Giles, An. vaneedeni Gillies and Coetzee, and An. aruni Sobti (GILLIES and COETZEE 1987 ). The An. funestus group and An. gambiae species complex belong to two different series (Pyretophorus and Myzomyia) of the same subgenus, Cellia (GILLIES and DE MEILLON 1968 ). Comparisons of polytene chromosome morphology and DNA sequences between the two species may help reveal conserved and variable genomic blocks, and thus the information would be valuable for comparative mapping. Spatial organization of chromosomes is also an important consideration because chromosome-spatial organization, in addition to gene-linear order, may have changed during evolution. For example, homosequential species within the An. maculipennis complex can be discriminated on the basis of the spatial localization and morphology of the chromosomal regions to which the nuclear envelope is attached (STEGNII 1987 ).

This article presents a cytogenetic map for An. funestus using ovarian nurse cell polytene chromosomes. First, we divided the polytene chromosomes into 46 regions according to banding patterns. Next, we identified differences in chromosome-linear and -spatial organization between An. funestus and An. gambiae. Finally, we characterized inversions for specimens collected from Kenya.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mosquito collection and preservation:
Despite keen interest and several attempts at An. funestus colonization, this species has not been successfully colonized until very recently (R. H. HUNT, personal communication). Thus construction of a cytogenetic map had to use field-collected specimens. Half-gravid An. funestus females were collected from the Kenyan coast using the standard indoor pyrethrum spray catch method (WORLD HEALTH ORGANIZATION 1975). Mosquitoes were preserved in Carnoy's fixative solution (3 ethanol:1 glacial acetic acid by volume) and stored at -20° until use. Half-gravid An. gambiae (strain 4arr) females were used as a control in the chromosome-spatial organization and in situ hybridization experiments.

Species identification:
Collected specimens were identified as An. funestus in a field following the morphological keys of GILLIES and DE MEILLON (1968) and GILLIES and COETZEE 1987 .The An. funestus subgroup includes four species: An. parensis Gillies, An. funestus Giles (or An. funestus sensu stricto), An. vaneedeni Gillies and Coetzee (formerly An. aruni?), and An. aruni Sobti (GILLIES and DE MEILLON 1968 ). An. parensis and An. funestus differ by two fixed inversions, 2Rg and 3Rc, while An. vaneedeni is homosequential with An. funestus, but differs in the frequency of floating inversions, 2Ra and 2Rd (GREEN and HUNT 1980 ). The identity of specimens used in this study was determined on the basis of the patterns of fixed and floating inversions, and only An. funestus s.s. were used for cytogenetic map construction. Recently, KOEKEMOER et al. 1999 developed a molecular method using rDNA PCR single-strand conformation polymorphism to distinguish four members of the An. funestus group. We used this method to confirm that the specimens used for this study were An. funestus s.s.

Chromosome and nucleus preparations and analysis:
We followed the protocol of DELLA TORRE 1997 for polytene chromosome preparation with An. funestus ovarian nurse cells. Briefly, ovaries from half-gravid females prefixed in Carnoy's fixative solution (3 ethanol:1 glacial acetic acid by volume) were dissected in 50% propionic acid and stained with 2% lacto-orcein solution. Nurse cells were then washed in 50% propionic acid to remove the excess stain. A cover slide was placed on the nurse cells and gently pressed to squash the cells. To prevent infiltration of air into the nurse cells, a small amount of lactic acid was added to the edge of a coverslip. The banding pattern of polytene chromosomes was examined under a Zeiss phase-contrast microscope (x1000). Good An. funestus chromosome preparations are notoriously difficult to obtain due to often-blurred banding patterns; it is, therefore, important to have a large sample size for cytogenetic studies. We chose the 6 best chromosome preparations from a total of 60 preparations for cytogenetic map construction. Thus, the cytogenetic map presented in this article is a composite image from six specimens. We used An. gambiae cytogenetic maps (available at http://konops.imbb.forth.gr/AnoDB/Cytomap/Photos/phototable.html) for chromosome morphology comparisons.

For comparative analysis of chromosome spatial organization between An. funestus and An. gambiae, unsquashed nucleus preparations were made from both species and examined under a Zeiss phase-contrast microscope (x1000). We examined a total of 56 nuclei: 30 An. funestus nuclei from six field-collected mosquitoes, 21 An. gambiae nuclei from eight laboratory mosquitoes (strain 4arr), and 5 An. gambiae nuclei from two mosquitoes collected from coastal Kenya. Data analysis on chromosome arm associations was based on 161 nuclei from 44 squashed chromosome preparations. All images were taken using a Micromax CCD camera (Princeton Instruments, Trenton, NJ).

In situ hybridization:
This experiment examined molecular homology in the telomeric region between An. gambiae and An. funestus. An 826-bp An. gambiae subtelomeric satellite DNA of arm 2L [ BIESSMANN et al. 1996 ; GenBank accession no. U34306] was used as a probe for in situ hybridization to An. funestus chromosomes. This marker was located in region 28D of the arm 2L of An. gambiae. The satellite DNA was labeled with biotin using the GIBCO BRL BioNick labeling system (Life Technologies, Gaithersburg, MD). An. funestus chromosomal preparations were made according to KUMAR et al. 1997 . Ovaries were gently pressed with a cover slide in 50% propionic acid, dipped in liquid nitrogen, and then dehydrated in 50, 70, 95, and 100% ethanol. The in situ hybridization was performed with the GIBCO BRL in situ Hybridization and detection system, using the manufacturer's recommended protocol. We used An. gambiae chromosome preparation as a positive control.

Examination of inversion frequencies:
Half-gravid An. funestus females collected from a village in Kwale, coastal Kenya, were used. A total of 124 chromosome preparations were made, but only 67 preparations were readable. Homozygous and heterozygous inversions were scored using the chromosomal map presented in this article. Inversion frequency was calculated, and a ² test was used to examine whether inversion genotype frequencies were in Hardy-Weinberg equilibrium.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cytogenetic map for An. funestus Giles:
We divided An. funestus polytene chromosomes into 46 regions on the basis of its banding patterns. Landmarks for arm recognition were the following:

Chromosome X: Chromosome X can be easily distinguished from other chromosomes by having the shortest length (Fig 1).

View larger version (63K): In this window In a new window Download PPT slide   Figure 1. Photomap of An. funestus Giles polytene chromosomes. Brackets represent inversions found in the specimens from coastal Kenya. Stars indicate centromeric regions.

Arm 2R: Arm 2R is the longest among the five arms, and its telomeric end in region 7A is also the widest. The most unique character of this arm is near the centromeric region (region 19). In particular, there is a wide light zone in region 19C and densely stained thick bands in region 19D (Fig 1).

Arm 2L: Arm 2L can be easily recognized by its lightly stained telomere and a dark band in region 28C (Fig 1).

Arm 3R: Arm 3R has the biggest heterochromatic block in the pericentromeric region 37D and a unique pattern of three thin well-stained bands in the telomeric region 29A (Fig 1).

Arm 3L: Regions 38 and 39 are excellent landmarks for arm 3L. There are two areas of diffusive heterochromatin separated by the euchromatic region 38C–39A (Fig 1). The telomeric region 46D has a typical fan-like shape.

Chromosome arm associations:
Pericentromeric heterochromatin of the polytene chromosomes in An. funestus provides fusion of all centromeric regions in one chromocenter. To reveal the arm pairs with prevalent association we have analyzed the squashed chromosome preparations with a broken chromocenter. The analysis revealed eight types of arm associations in An. funestus polytene chromosomes in ovarian nurse cells, including two- (2R + 2L, 2R + 3R, 2R + 3L, 3R + 2L, and 3R + 3L), three- (2R + 3R + 3L), four- (2R + 2L + 3R + 3L), and five-arm associations (Fig 2). Arm (2L + 3L) association has not been observed. A ² test indicates that (2R + 2L)- and (3R + 3L)-arm associations are significantly more prevalent than random chance (² = 82.3, d.f. = 4, P < 0.001; Table 1). This is consistent with the findings of GREEN and HUNT 1980 and GREEN 1982 that An. funestus and An. gambiae differ significantly in chromosome arm associations and supports the notion that translocation events have occurred during anopheline mosquito speciation. We determined the correspondence between An. funestus and An. gambiae chromosomal arms on the basis of their relative length and morphology. Accordingly, chromosome X of An. funestus corresponds to chromosome X of An. gambiae, 2R = 2R, 2L = 3R, 3R = 2L, and 3L = 3L.

View larger version (169K): In this window In a new window Download PPT slide   Figure 2. Polytene chromosome arm associations in the ovarian nurse cells of An. funestus. Arrows point at connections between proximal ends of the arms. (A) (2R + 2L)-arm association. (B) (2R + 3R)-arm association. (C) Three-arm association (2R + 3R + 3L). (D) Four-arm association (2L + 2R + 3R + 3L). (E) Five-arm association (X + 2R + 2L + 3R + 3L). Bar, 10 µm.

Localization of inversion breakpoints and inversion frequencies:
Five inversions were found in our An. funestus specimens from the Kenyan coast, including two inversions on arm 2R, two on arm 3R, and one on arm 3L (Fig 3). No inversion was found on chromosomes X and 2L. The breakpoints of inversion a of arm 2R are in regions 13D and 15C. Three double bands in regions 13E, 14A, and 14B can serve as a landmark for discrimination of standard and inverted variants of inversion a (Fig 1). In the case of the standard (+/+) chromosome, the region 13E–14B appears in the distal part of the inversion, while in the case of homozygous inversion (a/a) this region is near the proximal part. The breakpoints of inversion h of arm 2R are in regions 14D and 16A. This inversion partially overlaps with inversion a. The inversion h can be recognized by the position of a puff-like light zone with two bands in region 14E–15A (Fig 1). The inversion breakpoints of arm 3R are in regions 29C and 32C for inversion a and in regions 34D and 36B for inversion b. Homozygous inversion a of arm 3R can be recognized by a series of dense bands in region 31A–32B, and the wide diffuse zone in region 35EF can be used to identify homozygous inversion b (Fig 1). Breakpoints of inversion a on arm 3L are in regions 40C and 45B, and the homozygous inversion can be recognized by the position of the four distinctive bands in region 41D–42A (Fig 1).

View larger version (87K): In this window In a new window Download PPT slide   Figure 3. Heterozygous inversions of An. funestus collected from Kenya. (A) Inversion h of arm 2R. (B) Inversions a and b of arm 3R and inversion a of arm 3L.

Frequencies of the five inversions were estimated. For all five inversions, the frequency of the inverted variants was less than the standard variants (Table 2). No significant departure from the Hardy-Weinberg equilibrium was detected in our samples (Table 2), suggesting that random mating occurred in our population.

View this table: In this window In a new window   Table 2. Inversion frequency and 2 test statistics for Hardy-Weinberg equilibrium for an A. funestus population from coastal Kenya

Morphological differences between chromosomes of An. gambiae and An. funestus:
One of the conspicuous differences is between the An. funestus telomeric region 29A of arm 3R and the An. gambiae telomeric region 28D of arm 2L. An. gambiae has one weakly stained band while An. funestus has three well-stained bands (Fig 4A). This raises the possibility that there may be no correspondence in the telomeric region between the two species. To examine this possibility, we conducted an in situ hybridization to An. funestus polytene chromosomes using An. gambiae chromosome 2L subtelomeric satellite DNA as a probe. The satellite probe hybridized to the An. funestus arm 3R telomeric tip (Fig 4B), suggesting that DNA homology exists between the two species in the telomeric tips despite substantial difference in polytene chromosome banding patterns.

View larger version (77K): In this window In a new window Download PPT slide   Figure 4. (A) Comparison of linear structure in the telomeric region between An. gambiae arm 2L and An. funestus arm 3R. (B) Localization of An. gambiae 2L telomeric satellite in the polytene chromosome of An. funestus arm 3R (arrow). Bar, 10 µm.

An. funestus and An. gambiae also exhibit remarkable difference in pericentromeric heterochromatin. Using the classification method of HEITZ 1934 for heterochromatin, we define dense, compact heterochromatin as -type and diffuse, granular heterochromatin as ß-type. An. funestus has only -heterochromatin in the centromeric region of arms 2R (region 19E, Fig 5A), 2L (region 20A, Fig 5B), and 3L (region 38A, Fig 5C), while An. gambiae consists of only ß-heterochromatin. The heterochromatic block in the region 37D of An. funestus arm 3R is also more compact than heterochromatin in An. gambiae region 20A of arm 2L (Fig 5D). Finally, regions 38C and 39A contain more diffuse and reticular chromatin for An. funestus, than for An. gambiae (Fig 5C).

View larger version (88K): In this window In a new window Download PPT slide   Figure 5. Comparison of autosome pericentomeric regions between An. gambiae and An. funestus. (A) Arm 2R of An. gambiae and arm 2R of An. Funestus. (B) An. gambiae arm 3R and An. funestus arm 2L. (C) An. gambiae arm 3L and An. funestus arm 3L. (D) An. gambiae arm 2L and An. funestus arm 3R.

Differences in spatial organization of ovarian nurse cell polytene chromosomes between An. gambiae and An. funestus:
As demonstrated in the An. maculipennis complex and Drosophila melanogaster subgroup, spatial organization of polytene chromosomes has species-specific characteristics (STEGNII 1987 ; STEGNII and VASSERLAUF 1994 ). Therefore, information on the arrangement of chromosomes in the nuclear space will increase our understanding of mosquito genome evolution and may lead to new tools for species identification. Our particular interest is in the structure of the chromocenter (fusion of the pericentromeric regions) and chromosome attachments to the nuclear envelope because these two traits are most approachable for investigation.

Examination of unsquashed nuclei found that An. gambiae or An. funestus has its own unique characteristics in the associations of polytene chromosomes in pericentromeric regions. The pericentromeric regions of different arms stay closer to each other in An. funestus than in An. gambiae (Fig 6). The pericentromeric regions were connected through compact -heterochromatin in An. funestus (Fig 6A), but through long ß-heterochromatic fibers in An. gambiae (Fig 6B). Chromosome X contacts with the periphery of the nucleus near region 6 in both species (not shown). However, as to autosomes, only region 38C–39A of arm 3L attaches to the nuclear envelope in An. funestus (Fig 6A). In contrast, all ß-heterochromatic pericentromeric regions of arms 2R, 2L, 3R, and 3L in An. gambiae directly touch the nuclear periphery (Fig 6B). This pattern was consistent among 30 nuclei of An. funestus and 21 nuclei of An. gambiae that we examined.

View larger version (145K): In this window In a new window Download PPT slide   Figure 6. Comparison of spatial organization of polytene chromosomes between An. funestus (A) and An. gambiae (B). Arrows show attachments of chromosomes to the nuclear envelope (NE). Arrowheads point to the chromocenter. The right graphs are schematic presentations of chromosomal regions that attach to the nuclear envelope. Bar, 10 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

This study constructed an annotated cytogenetic map for the African malaria mosquito, An. Funestus, on the basis of polytene chromosomes of ovarian nurse cells. For our map we adopted the system of An. gambiae for arm notation. It refers to the nomenclature proposed by GREEN and HUNT 1980 as the following: X = 1, 2R = 2, 2L = 4, 3R = 3, and 3L = 5. Although An. funestus is an important malaria vector in Africa and there is a keen interest in its ecology and genetics, little genetic research has been done with this species. In this article we presented a cytogenetic map using well-preserved, field-collected specimens. A noticeable feature of our cytogenetic map is that the polytene chromosomes are divided by regions according to the banding patterns, as for the An. gambiae complex (COLUZZI and SABATINI 1967 ). Thus, with our map, inversion breakpoints can be described in text and easily located on the map. We described several landmarks for recognition of chromosomal arms and inversions and characterized five inversions for specimens collected from coastal Kenya using the cytogenetic map. Finally, we compared linear and spatial organizations of An. funestus polytene chromosomes with another well-studied malaria mosquito, An. gambiae.

We are particularly interested in the comparison of linear organizations of polytene chromosomes between An. gambiae and An. funestus because it facilitates the development of the An. funestus physical map. For example, numerous An. gambiae genes have been cloned and mapped; these genes can be used as probes for identification of homologous or heterologous chromosome blocks in An. funestus. The most conspicuous difference between the two species was autosomal heterochromatin. That is, the proximal (pericentromeric) regions of An. gambiae polytene chromosomes are mostly diffuse ß-heterochromatin, but compact -heterochromatin in An. funestus. Other studies using mitotic chromosomes and polytene chromosomes have also observed a high level of variability in pericentromeric heterochromatin in the An. gambiae and An. maculipennis species complex (BONACCORSI et al. 1980 ; MARCHI and MEZZANOTTE 1990 ; SHARAKHOVA et al. 1997 ). Thus, during mosquito speciation, in addition to inversions and translocations, heterochromatin types and positions on chromosomes have also changed.

In this study we demonstrated a major difference in chromosome spatial organizations between An. funestus and An. gambiae. The two species differ in the structural arrangement of the chromocenter. That is, the chromocenter of An. funestus is local, but spread in An. gambiae. The two species also differ in the autosome arms and regions that attach to the nuclear periphery. In An. gambiae, regions 19E (arm 2R), 20A (arm 2L), 37D (arm 3R), and 38A (arm 3L) attach to the nuclear envelope, while in An. funestus the region that attaches to the nuclear envelope is 38C–39A (arm 3L). Other studies with the An. maculipennis species complex and D. melanogaster subgroup also demonstrated reorganizations of the attachments and chromocenter structure during speciation (STEGNII 1987 ; STEGNII and VASSERLAUF 1994 ).

One potential explanation for the difference in chromosome spatial organization between An. funestus and An. gambiae is the difference in heterochromatin. In this study we found that the structure of the chromocenter (local or spread) and chromosome regions that attach to the nuclear envelope depends on the type of heterochromatin rather than on the position of heterochromatin on a chromosome. Studies with An. messeae found that connections of chromosomes with the nuclear envelope are through ß-heterochromatin, but not -heterochromatin (STEGNII and SHARAKHOVA 1991 ). The copy number of repetitive DNA can affect the local chromatin structure (CLARK et al. 1998 ). The chromosomal regions that attach to the nuclear envelope may also depend on the presence of specific DNA. For example, repetitive M/SAR (matrix/scaffold attachment region) DNA, specifically binding to the structural protein of the nuclear periphery, was localized in the attached regions of heterochromatin in D. melanogaster (BARICHEVA et al. 1996 ; SHARAKHOVA et al. 1997 ). It is noticeable that M/SAR DNA is severalfold richer in heterochromatin than in euchromatic regions (STRAUSBAUGH and WILLIAMS 1996 ). Thus, future investigations of An. gambiae and An. funestus heterochromatin need to show whether molecular changes can actually lead to chromosome-spatial reorganization.

Finally, we point out that chromosome homology between An. funestus and An. gambiae remains to be tested at the DNA sequence level. Visual comparison of the banding patterns alone does not allow drawing inferences about the homology of all chromosomal regions. For example, the telomeric region of arm 3R of An. funestus exhibits morphology very different than the telomeric region of arm 2L of An. gambiae. However, when we used an An. gambiae arm 2L subtelomeric satellite as a probe for in situ hybridization to An. funestus polytene chromosomes, the probe hybridized to the telomeric region of An. funestus arm 3R. Thus, molecular homology between the two species, at least for this satellite sequence, exists despite a clear interspecific difference in chromosome morphology. This phenomenon may not be unique to mosquitoes, but rather common for Dipteran insects. For example, D. melanogaster and D. subobscura belong to the D. melanogaster and D. obscura groups in subgenus Sophophora and show no structurally recognizable homology in some polytene chromosome loci, but substantial molecular homology was observed between the two species (CUENCA et al. 1998 ). We are currently examining the extent of chromosomal synteny between An. funestus and An. gambiae using An. gambiae cDNA clones that have been mapped as a probe for in situ hybridization to An. funestus polytene chromosomes.

	ACKNOWLEDGMENTS

We thank Harold Biessmann (University of California at Irvine) for providing an An. gambiae telomeric satellite DNA clone and Maureen J. Gorman and Susan M. Paskewitz (University of Wisconsin at Madison) for half-gravid An. gambiae 4arr strain females. Alan J. Siegel provided technical assistance. Maureen Coetzee, Richard H. Hunt (South African Institute for Medical Research), Vladimir Stegnii (Tomsk State University), and two anonymous reviewers provided helpful comments on the manuscript. This research was supported by National Institutes of Health grant R01 AI-50243 and the UNDP/WORLD BANK/WHO Special Program for Research and Training in Tropical Diseases (TDR).

Manuscript received March 7, 2001; Accepted for publication June 22, 2001.

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