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Proc Natl Acad Sci U S A. 2005 Nov 29; 102(48): 17388–17393.
Published online 2005 Nov 21. doi:  10.1073/pnas.0508871102
PMCID: PMC1297693
Evolution

“Clonal” population structure of the malaria agent Plasmodium falciparum in high-infection regions

Abstract

The population genetic structure of Plasmodium falciparum, the agent of malignant malaria, has been shown to be predominantly “clonal” (i.e., highly inbred) in regions of low infectivity; in high-infectivity regions, it is often thought to be panmictic, or nearly so, although there is little supporting evidence for this. The matter can be settled by investigating the parasite's genetic makeup in the midgut oocysts of the mosquito vector, where the products of meiosis can directly be observed. The developmental stages of P. falciparum are haploid, except in the oocysts of infected mosquito vectors, where two gametes fuse, diploidy occurs, and meiosis ensues. We have investigated genetic polymorphisms at seven microsatellite loci located on five chromosomes by assaying 613 oocysts in 145 mosquitoes sampled from 11 localities of Kenya, where malignant malaria is perennial and intense. There is considerable allelic variation, 16.3 ± 2.1 alleles per locus, and considerable inbreeding, ≈50% on the average. The inbreeding is caused by selfing (≈25%) and nonrandom genotype distribution of oocysts among mosquito guts (35%). The observed frequency of heterozygotes is 0.43 ± 0.03; the expected frequency, assuming random mating, is 0.80 ± 0.05. Linkage disequilibrium is statistically significant for all 21 pairwise comparisons between loci, even though 19 comparisons are between loci in different chromosomes, which is consistent with strong deviation from panmixia and the consequent reproduction of genomes as clones, without recombination between gene loci. This is of considerable evolutionary significance and of epidemiological consequence, concerning the spread of multilocus drug and vaccine resistance.

Keywords: epidemiology, evolutionary genetics, parasitism, population genetics

Malaria is a devastating disease, caused by four species of the parasitic protozoan Plasmodium, endemic in tropical regions of >100 countries and territories. Plasmodium falciparum, the agent of the most malignant form of malaria, accounts for 300–600 million episodes of clinical malaria (1) and 1.5–2.7 million deaths each year (2), mostly of African children (3). P. falciparum is transmitted by species of Anopheles mosquitoes. During the mosquito bloodmeal, male and female gametocytes are ingested, which rapidly mature into gametes that fuse to form an ookinete, which encysts in the mosquito gut, forming an oocyst where meiosis rapidly ensues (4). A single mosquito may carry several oocysts, each resulting from the fusion of two gametes. Oocysts are thus the only “diploid” stage in the life cycle of P. falciparum, because only oocysts contain the results of gamete mixing and the products of a single meiosis. Elsewhere in the mosquito, as everywhere in the human host, P. falciparum exists only as a haploid organism.

The population genetic structure of P. falciparum is generally ascertained by investigating the haploid stages encountered in infected human blood, where pairs of genomes derived each from a single meiosis cannot be distinguished from genomes derived from multiple infections, even in regions with low endemicity (57). The incidence of clonality or inbreeding versus outbreeding or panmixia is estimated indirectly by measuring genetic diversity, linkage disequilibrium, and the like (2, 6, 816). Hence the conflicting evidence favoring different levels of clonality or panmixia, although it is often accepted that P. falciparum displays different population structures in different places, ranging from clonality to panmixia in approximate correlation with the increasing intensity of transmission (2, 5, 6, 816).

Conflicting interpretations of the population structure of P. falciparum also arise because of a failure to distinguish between two meanings of the phrases “sexual reproduction” and, correspondingly, “clonal reproduction.” Sexual reproduction as a physiological phenomenon is predicated of individuals that each develop from a zygote formed by the fusion of gametes and that each experiences meiosis and produces gametes. As a genetic phenomenon, sexual reproduction refers to genetic recombination between loci, whereas clonal reproduction refers to the reproduction of whole genomes as units, without genetic recombination. It seems likely that physiological sexuality evolved as an adaptive mechanism for producing genetic recombination, but physiological and genetic sexuality have repeatedly become separated in the evolution of many organisms. In terms of long-term evolutionary consequences, it is genetic sexuality (i.e., genetic recombination between dissimilar genomes) that is of consequence (see ref. 17). To determine the genetic structure of a population, what must be ascertained is the extent to which reproduction departs from panmixia and approaches clonal reproduction of genomes. P. falciparum, physiologically, is an obligate sexual organism, because fusion of gametes, zygote formation, and meiosis must occur in the mosquito gut for the parasite's life cycle to be completed. But, with respect to its population structure and evolutionary pattern, what needs to be determined is the extent of the parasite's genetic clonality relative to panmixia, because genetic recombination impacts the genetic makeup of genomes; physiological sexuality, by itself, does not. The genetic composition and evolutionary change of a population is of great import to ascertain the ecophysiology of pathogens, host–pathogen interactions, and the evolution of a pathogen's drug resistance.

We have sought to unravel the population structure of P. falciparum by investigating directly the genetic makeup of oocysts, where diploid genotypes can be determined. We carry out our investigation in a region of intense infectivity, where panmixia is often assumed to prevail. Two previous population-genetic investigations of P. falciparum in mosquito oocysts have considered only two (18) or three (8) gene loci and have not directly analyzed the causes of inbreeding. Moreover, the loci studied are clearly subject to selection by the host; selectively neutral gene markers are more suitable for settling the issues at hand, namely the extent of random relative to nonrandom mating (19). Herein, we present the results of a study of genetic diversity and the extent and causes of inbreeding in seven microsatellite gene loci sampled from 613 oocysts from 145 Anopheles gambiae mosquitoes, collected in 2 consecutive years from 11 localities in western Kenya. We have found a high level of inbreeding in this African region, with perennial and intense malarial transmission.

Materials and Methods

Mosquito Collection. Resting bloodfed A. gambiae (sensu lato) females were collected with aspirators in indoor resting traps early in the morning during the main rainy seasons of 2002 and 2003, between mid-March and June. Mosquitoes were collected each year for 10 weeks in 11 villages in western Kenya, near Lake Victoria (Fig. 1 and Table 1). Fifteen to 20 houses per village were visited each week for 20 min. Mosquitoes were brought to an insectarium, fed with a sugar solution ad libitum, and maintained for 7 days in small pots.

Fig. 1.
The 11 sampling sites (in white ovals) from western Kenya.
Table 1.
A. gambiae samples from 11 localities in western Kenya during 2002 and 2003

Oocyst Collection. On the seventh day after capture, we dissected the mosquito midguts and checked them with a microscope for the presence of oocysts. The dissection tools were rinsed in alcohol and sterilized with a Bunsen burner for each mosquito. Infected midguts were stored in absolute ethyl alcohol.

Each oocyst was later isolated from each mosquito's midgut under a Leica (Deerfield, IL) DMIRB microscope. Dissected oocysts were individually preserved at –20°C in absolute ethyl alcohol until their DNA extraction. Oocyst DNA was isolated and purified by using the DNeasy Tissue Kit following the manufacturer's instructions (Qiagen, Valencia, CA).

Genotyping. We have investigated the seven microsatellite loci listed in Table 1 (20); PCR amplifications followed the protocol of ref. 5, using an MJ Research (Cambridge, MA) PTC100.96-well; fluorescence-labeled PCR products were sized on Applied Biosystems Prism310, with a Genescan500LIZ internal size standard. We obtained complete genotypes for 555 oocysts at the seven loci, 37 oocysts at six loci, and 21 oocysts at five loci.

Oocyst Distribution. We computed the mean (m) and variance (s2) of oocyst load per mosquito, as well as the aggregation coefficient β = s2/m. For a Poisson (random) distribution, the expected value of β is 1; but β < 1 if the distribution is underdispersed, and β > 1 if it is aggregated.

Genetic and Inbreeding Analyses. Expected heterozygosity (Hexp) was calculated by using the unbiased estimator of ref. 21, which corrects for small samples.

We estimated F statistics as defined by ref. 22. Here FIS measures the inbreeding of individuals resulting from deviations from panmixia (random union of gametes) within individual oocysts relative to gametes from different oocysts within the same mosquito. Its significance is tested by permutation of genes between oocysts within each individual mosquito. FST measures relatedness between individuals, that is, the deficiency of heterozygotes due to the nonrandom distribution of genotypes among mosquito guts; thus, FST quantifies the differentiation between subpopulations (mosquito guts) in the total population (locality). Finally, FIT measures overall inbreeding, resulting both from effects combined, i.e., deviations form panmixia among all oocysts from all mosquitoes in a given locality: FIT = 1 – (1 – FIS) (1 – FST).

Linkage disequilibrium between pairs of loci is measured with the coefficient of correlation R generalized for the multiallelic case; the statistical significance of linkage disequilibrium is assessed with a randomization test. Genotypes at two loci are associated at random a number of times, and the log likelihood G test statistic is recalculated on the randomized data set. Because this procedure was repeated on all pairs of loci, we have applied a Bonferroni correction to the P values (P value times the number of tests). F statistics and linkage disequilibrium analyses are performed with fstat, version 2.9.4 (23).

Results

We captured 4,602 mosquitoes in 11 villages from western Kenya (Fig. 1) during 2002 and 2003, of which 181 (3.9%) mosquitoes were infected by P. falciparum oocysts, with 6.0 ± 0.7 oocysts per infected mosquito (Table 1), values similar to those obtained in previous studies of the same region (24, 25). The allele frequencies (Table 2) are based on 613 oocysts from 145 mosquitoes; some oocysts were lost during extraction from the mosquito guts.

Table 2.
Allele frequencies at seven microsatellite loci in P. falciparum oocysts from Kenya

The mean parasitic load is m = 0.24, with a variance s2 = 4.58, values that imply a highly aggregated distribution of the parasites as measured by the coefficient β = s2/m = 19. This positive aggregation reflects that most oocysts are contained in few mosquito individuals, whereas most mosquitoes remain uninfected.

Table 2 displays the allele frequencies at the seven microsatellite loci in the 11 Kenyan populations. The frequency data for 2002 and 2003 have been combined; there is no significant difference in allele frequencies between the 2 years. A total of 613 oocysts were genetically analyzed, obtained from 145 infected mosquitoes, an average of 4.23 oocysts per mosquito. This average is lower than the average of 6.0 oocysts, observed in 181 mosquitoes in the full sample (Table 1), because some oocysts were lost during dissection. In 60 of the 145 mosquitoes, only one oocyst was successfully analyzed (although some of the 60 mosquitoes harbored more than one oocyst).

The average number of alleles per locus is 16.3 ± 2.1, ranging from seven alleles (Pfg377) to 23 (POLYa) at each locus (Table 2). High allele diversity is typically observed at microsatellite loci in regions of perennial malaria transmission (5). The average observed heterozygosity (Hobs = 0.43 ± 0.03) per population (sites; Fig. 1) is much lower than expected (Hexp = 0.80 ± 0.05). This discrepancy is approximately similar for each locus separately and for each population with a sample of 10 or more oocysts. The large disparity between observed and expected heterozygosity indicates a considerable amount of inbreeding, even in this region of high infectivity.

We use F statistics for further exploring the pattern of inbreeding in these populations (Fig. 2). If we treat all oocysts as members of one population, the departure from panmixia is 0.46. Little of this departure can be attributed to the pooling of localities and years (Wahlund effect): the inbreeding within each village and year is FIT = 0.43 [95% confidence interval (CI) = 0.37–0.48], P < 10–4 for each locus separately, as well as for all loci. The inbreeding attributed to nonrandom distribution of genotypes among mosquitoes is also high, FST = 0.36 (CI = 0.33–0.38; P < 10–4). Finally, the rate of inbreeding within a mosquito gut is FIS = 0.147 (CI = 0.062–0.209, P < 10–4). We estimate the rate of selfing within each mosquito gut as s = 0.25 (CI = 0.16–0.32; P < 10–4), where s = 2FIS/(1 + FIS). This value is based on 553 oocysts from 85 mosquitoes (60 mosquitoes each with only one oocyst are not included in the calculation of FIS).

Fig. 2.
Inbreeding statistics for P. falciparum oocysts collected from A. gambiae mosquitoes. L1 to L7 refer to the seven microsatellite loci sequentially listed in Table 2. FIS measures the deficiency of heterozygous genotypes as a result of self fertilization ...

Linkage disequilibrium is significant for all 21 pairs of loci, R = 0.50–0.60 between any two loci, P < 0.05 for each of the 21 pairs, with the Bonferroni correction. This cannot be attributed to close physical linkage between loci, because the seven loci are distributed among five different chromosomes, so that all but two pairs among the 21 involve loci located on different chromosomes (moreover, the loci on the same chromosome are far apart, 45.8 cM for the pair on chromosome 12 and 36.5 cM for the pair on chromosome 6). Rather, linkage disequilibrium is a consequence of inbreeding, which reduces the effectiveness of recombination because of selfing and strong relatedness among oocysts within each mosquito. That is, multilocus genotypes within a given gamete are transmitted together even when loci are located on different chromosomes (26, 27).

Discussion

Rich et al. (26) investigated the highly polymorphic circumsporozoite (Csp) gene in 25 DNA sequences from the worldwide distribution of P. falciparum and conjectured that the reproduction of the parasite is prevailingly clonal, that is, that haplotypes predominantly reproduce as units, with rare recombination events between loci. Conway et al. (28) concluded that meiotic recombination is frequent in African populations of P. falciparum because linkage disequilibrium in the large (5.1-kb) msp1 gene decreases with distance along the gene. This evidence, however, is far from conclusive, because msp1 contains several repetitive regions, including short DNA sequences repeated in tandem, where intragenic recombination occurs independently of meiosis, at frequencies orders of magnitude higher than the mutation rate; linkage disequilibrium between sites along the gene may arise as a consequence of intragenic recombination (16, 27). Further, the results do not imply that recombination is effectively breaking haplotype structure. Indeed, strong linkage disequilibrium has been observed between physically unlinked markers (29), as well as significant deficit of heterozygous oocysts in populations of Tanzania (18) and Papua New Guinea (8), where overall inbreeding coefficients have been estimated at 0.34 and 0.90, respectively (12). Anderson et al. (5) observed significant linkage disequilibrium among 12 microsatellite loci in all localities with low infectivity and in two of five populations from regions with high transmission frequencies. Other results have been interpreted as indicating that P. falciparum reproduction may be genetically clonal in low-infectivity regions but predominantly panmictic in high-infectivity regions (13, 14). Nevertheless, Durand et al. (30) found significant linkage disequilibrium in two African populations from the Republic of the Congo, where infectivity is high.

We sought to provide additional insight into the question whether recombination is effective in high-infectivity African populations by investigating the genetic makeup at seven microsatellite loci of P. falciparum in mosquito oocysts, the only stage of the parasite's life where diploidy and the immediate products of meiosis can be observed. The Kenya populations of P. falciparum harbor ample genetic variation, such as is typical for regions with intense perennial transmission of malignant malaria. The average number of alleles at the seven microsatellite loci is 16.3 ± 2.1, and the average expected heterozygosity per locus in a population is huge, 0.80 ± 0.05, with relatively little variation from one to another population among the eight populations for which at least 10 oocysts have been sampled. The observed heterozygosity, however, is much lower, 0.43 ± 0.03, indicating that there is a considerable amount of inbreeding. Evidence of inbreeding exists within each mosquito with two or more oocysts (FIS = 0.147 ± 0.031). There is considerable inbreeding as well when the oocysts from each mosquito gut are considered as a subpopulation (FST = 0.355 ± 0.013) and also within each locality (FIT = 0.425 ± 0.026). Quite relevant for our purposes is that linkage disequilibrium is significant for all 21 pairwise comparisons between seven genetic markers located in five different chromosomes. This implies that the reproduction of P. falciparum, even in this African region of perennial infection, is prevailingly clonal. A contributing factor is the observed high rate of self fertilization (s = 0.25).

Reduced genetic recombination is consistent with the observation that linkage disequilibrium is associated with the haplotypic spread of chloroquine resistance in diverse populations of P. falciparum from two foci in Asia and South America (31). Current models of the spread of resistance to antimalarial drugs have shown that a positive association between the intensity of transmission and the degree of the parasite's inbreeding may be involved (3234); low rates of inbreeding may lead to a slow spread of resistance in regions with high infectivity (33, 34). Be that as it may, accurate knowledge of the population genetic structure of P. falciparum is of great import for the successful control of malignant malaria. Although a variety of population structures may prevail in different regions, as a consequence at least in part of the rates of malaria transmission, it must be kept in mind that a clonal population structure of P. falciparum may prevail in regions of perennial transmission as well as in regions of low infectivity.

Acknowledgments

We thank Phil Agnew, Benjamin Roche, Mario Coluzzi, Steve Rich, Frank Prugnolle, and Michel Tibayrenc for useful comments for improving the manuscript. We thank l'Institut de Recherche pour le Développement and Centre National de la Recherche Scientifique for financial support. This project was supported by the program PAL+ of the French Ministry of Education and Research. We are grateful to the Kenya Medical Research Institute for their kind help.

Notes

Conflict of interest statement: No conflicts declared.

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