The impact of uniform and mixed species blood meals on the fitness of the mosquito vector Anopheles gambiae s.s: does a specialist pay for diversifying its host species diet?

We investigated the fitness consequences of specialization in an organism whose host choice has an immense impact on human health: the African malaria vector Anopheles gambiae s.s. We tested whether this mosquito's specialism on humans can be attributed to the relative fitness benefits of specialist vs. generalist feeding strategies by contrasting their fecundity and survival on human-only and mixed host diets consisting of blood meals from humans and animals. When given only one blood meal, An. gambiae s.s. survived significantly longer on human and bovine blood, than on canine or avian blood. However, when blood fed repeatedly, there was no evidence that the fitness of An. gambiae s.s. fed a human-only diet was greater than those fed generalist diets. This suggests that the adoption of generalist host feeding strategies in An. gambiae s.s. is not constrained by intraspecific variation in the resource quality of blood from other available host species.

Morphological evidence for proliferative regeneration of the Anopheles stephensi midgut epithelium following Plasmodium falciparum ookinete invasion.

Ookinetes are motile invasive stages of the malaria parasite that enter the midgut epithelium of the mosquito vector via an intracellular route. Ookinetes often migrate through multiple adjacent midgut epithelial cells, which subsequently undergo apoptosis/necrosis and are extruded from the midgut epithelium into the midgut lumen. Hundreds of ookinetes may simultaneously invade the midgut epithelium, causing destruction of an appreciable proportion of the total number of midgut epithelial cells. However, there is little evidence that ookinete invasion of the midgut epithelium per se is detrimental to the survival of the mosquito vector implying that efficient mechanisms exist to restore the damaged midgut epithelium following malaria parasite infection. Proliferation and differentiation of precursor stem cells could replace the midgut epithelial cells destroyed and lost as a consequence of ookinete invasion. Although the existence of so-called "regenerative" cells within the mosquito midgut epithelium has long been recognized, there has been no previously published evidence for proliferation/differentiation of these putative precursor midgut epithelial cells in mature adult female mosquitoes. In the current study, examination of Giemsa-stained histological sections from Anopheles stephensi mosquito midguts infected with the human malaria parasite Plasmodium falciparum provided morphological evidence that regenerative cells undergo division and subsequent differentiation into normal columnar midgut epithelial cells. Furthermore, the number of these putatively proliferating/differentiating regenerative cells was significantly higher in P. falciparum-infected compared to uninfected mosquitoes, and was positively correlated with both the level of malaria parasite infection and midgut epithelial cell destruction. The loss of invaded midgut epithelial cells associated with intracellular migration by ookinetes, therefore, appears to trigger, and to be compensated by, proliferative regeneration of the mosquito midgut epithelium.

A real-time PCR assay for quantifying Plasmodium falciparum infections in the mosquito vector.

Transmission-blocking vaccines prevent the development of Plasmodium parasite within the mosquito vector, thereby thwarting the spread of malaria through a community. The gold standard for determining the efficacy of a transmission-blocking vaccine is the standard membrane feeding assay. This assay requires the dissection of mosquitoes and microscopic counting of oocysts present on the mosquito mid-gut, typically at 7-10 days p.i. Here we describe a real-time quantitative PCR assay that is rapid, target-specific and robust, with a sensitive detection threshold and which may be employed earlier p.i. than the standard membrane feeding assay and is applicable to preserved material. The real-time PCR assay utilises the LightCycler platform and SYBR Green I detection system to amplify 180 bp of the asexual form of the Plasmodium falciparum rRNA gene. It has a quantitative range of greater than four orders of magnitude and a detection threshold of 10 parasites. Validation experiments using a monoclonal antibody of known blocking activity revealed the real-time PCR assay to give equivalent results to the standard membrane feeding assay. In addition, the PCR assay can establish the effect of such a monoclonal antibody on the parasites' development within the oocyst and on the sporozoite (the transmissible stage) yield, providing a more pertinent assessment of transmission blocking activity than is possible by the standard membrane feeding assay. This assay may also be employed to monitor the sporogonic development of P. falciparum parasites within the mosquito vector.

Plasmodium falciparum ookinete invasion of the midgut epithelium of Anopheles stephensi is consistent with the Time Bomb model.

Plasmodium falciparum gametocytes grown in vitro were fed through membrane feeders to laboratory-reared Anopheles stephensi mosquitoes. Intact midguts, including entire bloodmeal contents, were removed between 24 and 48 h post-bloodfeeding. Giemsa-stained histological sections were prepared from the midguts and examined by light microscopy. Contrary to previous reports, ookinetes were clearly visible within midgut epithelial cells, demonstrating intracellular migration across the midgut wall. Ookinetes entered epithelial cells through the lateral apical membrane at sites where 3 adjacent cells converged. There was no evidence for the existence of a morphologically distinct group of epithelial cells preferentially invaded by ookinetes. However, ookinete penetration was associated with significant morphological changes to invaded cells, including differential staining, condensation and fragmentation of the nucleus, vacuolization, loss of microvilli and various degrees of extrusion into the midgut lumen. Epithelial cells completely separated from the midgut wall were found within the midgut lumen. These cells were associated with invading parasites suggesting that ookinete penetration resulted in complete ejection of invaded cells from the midgut wall. Small clusters of morphologically altered midgut cells and invading parasites spanning the membranes of adjacent abnormal epithelial cells were observed, consistent with intracellular movement of ookinetes between neighbouring midgut cells. Extruded epithelial cells were also observed rarely in uninfected midguts. Epithelial cell extrusion, therefore, may be a general mechanism of tissue repair through which damaged cells are removed from the midgut wall rather than a parasite-specific response. These observations demonstrate that human malaria parasite infection of mosquitoes is consistent with, and provides further support for, the Time Bomb model of ookinete invasion of the mosquito midgut epithelium previously proposed for rodent malaria parasites.

Critical comparison of molecular genotyping methods for detection of drug-resistant Plasmodium falciparum.

We have critically evaluated 3 techniques for the detection of mutations conferring drug resistance of Plasmodium falciparum, using samples containing known numbers of well-characterized parasites and artificial mixtures of these parasites at known proportions. We compared the sensitivity and specificity of mutation-specific polymerase chain reaction (MS-PCR), polymerase chain reaction followed by restriction enzyme digestion at polymorphic sites (PCR/RFLP), and a dot-blot/probe hybridization technique, for detection of point mutations at nucleotide 323 of the P. falciparum dihydrofolate reductase gene (dhfr) that confer resistance to the antimalarial drug pyrimethamine. We have also investigated the benefits in terms of sensitivity and reproducibility of the incorporation of radiolabelled nucleotides into the PCR/RFLP assay. We found that MS-PCR was very sensitive--at least 10 parasites could be detected in a sample--but non-specific amplification resulted in erroneous typing of some samples. PCR/RFLP was less sensitive; 10 parasites per sample could not always be detected, but the technique was specific. The addition of radiolabelled nucleotides to the assay did not markedly improve the sensitivity but the results were easier to read and there was less subjectivity in scoring the results. The dot-blot/probe hybridization technique was specific and sensitive, with similar levels of specificity and sensitivity to PCR/RFLP. On balance, the dot-blot/probe hybridization technique seems best suited to large-scale epidemiological surveys of genes associated with antimalarial drug resistance.

Plasmodium falciparum: gene mutations and amplification of dihydrofolate reductase genes in parasites grown in vitro in presence of pyrimethamine.

Samples of three pyrimethamine-sensitive clones of Plasmodium falciparum were grown for periods of 22-46 weeks in media containing stepwise increases in pyrimethamine concentrations and were seen to develop up to 1000-fold increases in resistance to the drug. With clone T9/94RC17, the dihydrofolate reductase (DHFR) gene was sequenced from 10 uncloned populations and 29 pure clones, all having increased resistance to pyrimethamine, and these sequences were compared with the sequence of the original pyrimethamine-sensitive clone. No changes in amino acid sequence were found to have occurred. Some resistant clones obtained by this method were then examined by pulsed-field gel electrophoresis, and the results indicated that there had been an increase in the size of chromosome 4. This was confirmed by hybridization of Southern blots with a chromosome 4-specific probe, the vacuolar ATPase subunit B gene, and a probe to DHFR. Dot-blotting with an oligonucleotide probe to DHFR confirmed that there had been increases up to 44-fold in copy number of the DHFR gene in the resistant strains. Resistant clones obtained by this procedure were then grown in medium lacking pyrimethamine for a period of nearly 2 years, and reversion nearly to the level of pyrimethamine sensitivity of the original clone T9/94RC17 was found to occur after about 16 months. Correspondingly, the chromosome 4 of the reverted population reverted to a size like that of the original sensitive clone T9/94RC17. The procedure of growing parasites in stepwise increases of pyrimethamine concentration was repeated with two other pyrimethamine-sensitive clones: TM4CB8-2.2.3 and G112CB1.1. (The DHFR gene of these clones encodes serine at position 108, in place of threonine as in clone T9/94RC17, and it was thought that this difference might conceivably affect the rate of mutation to asparagine at this position). Clones TM4CB8-2.2.3 and G112CB1.1 also responded by developing gradually increased resistance to pyrimethamine. However, in clone TM4CB8-2.2.3 a single mutation from Ile to Met at position 164 in the DHFR gene sequence was identified, and in clone G112CB1.1 there was a single mutation from Ala to Ser at position 16, but no mutations at position 108 were obtained in any of the clones studied here. In addition, chromosome 4 of clone TM4CB8-2.2.3 increased in size, presumably due to amplification of the DHFR gene. No increase in size was seen in clone G112CB1.1. We conclude that whereas some mutations producing changes in the amino acid sequence of the DHFR molecule may occur occasionally in clones or populations of P. falciparum grown in vitro in the presence of pyrimethamine, amplification of the DHFR gene following adaptation to growth in medium containing pyrimethamine occurs as a regular feature. The bearing of these findings on the development of pyrimethamine-resistant forms of malaria parasites in endemic areas is discussed.

Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre study.

Genetic diversity of malaria parasites represents a major issue in understanding several aspects of malaria infection and disease. Genotyping of Plasmodium falciparum infections with polymerase chain reaction (PCR)-based methods has therefore been introduced in epidemiological studies. Polymorphic regions of the msp1, msp2 and glurp genes are the most frequently used markers for genotyping, but methods may differ. A multicentre study was therefore conducted to evaluate the comparability of results from different laboratories when the same samples were analysed. Analyses of laboratory-cloned lines revealed high specificity but varying sensitivity. Detection of low-density clones was hampered in multiclonal infections. Analyses of isolates from Tanzania and Papua New Guinea revealed similar positivity rates with the same allelic types identified. The number of alleles detected per isolate, however, varied systematically between the laboratories especially at high parasite densities. When the analyses were repeated within the laboratories, high agreement was found in getting positive or negative results but with a random variation in the number of alleles detected. The msp2 locus appeared to be the most informative single marker for analyses of multiplicity of infection. Genotyping by PCR is a powerful tool for studies on genetic diversity of P. falciparum but this study has revealed limitations in comparing results on multiplicity of infection derived from different laboratories and emphasizes the need for highly standardized laboratory protocols.

Commitment to sexual differentiation in the human malaria parasite, Plasmodium falciparum.

The differentiation of the two sexes in the gametocytogenesis of Plasmodium falciparum was investigated using a plaque assay and antibodies specific for various stages and sexes of gametocytes. Immunofluorescence assays on plaques of cultured parasites grown in monolayers of erythrocytes revealed that the merozoites released from a single sexually-committed schizont became either all male or all female gametocytes. Thus, the commitment of this species to differentiate into one sex or the other is likely to occur prior to the nuclear division of the sexually-committed schizont. The characteristic female-biased gametocyte sex ratio observed for many Plasmodium species is manifested in P. falciparum by a greater percentage of schizonts that produce female gametocytes (67-71%) than those that yield males. From the plaque assay, it was determined that the number of gametocytes produced per sexually-committed schizont was similar for both sexes, indicating that allocation of parasite resources was equal for each sex of gametocyte. The timing of sexual differentiation and features of the gametocyte sex ratio is discussed in relation to previous observations on P. falciparum and related malaria parasites.

Genetic structure and dynamics of Plasmodium falciparum infections in the Kilombero region of Tanzania.

Plasmodium falciparum parasites exist as genetically distinct haploid clones in infected people. In the Kilombero valley in south-east Tanzania, at least 85% of the inhabitants of Michenga village harbour more than one clone. Using 2 highly polymorphic unlinked markers, it has been estimated that each infected person harbours between one and 6 P. falciparum clones at any one time, with a mean of 3.5 clones. When mosquitoes acquire gametocytes of 2 different clones in a blood meal, crossing generates recombinant clones differing from their parental genotypes. The inbreeding coefficient of the parasite population has been estimated as 0.33.

Chloroquine increases Plasmodium falciparum gametocytogenesis in vitro.

Malaria parasites are capable of modulating the diversion of resources from asexual growth to the production of stages infective to mosquitoes (gametocytes). Increased rates of gametocytogenesis appear to be a general response to stress, both naturally encountered and novel. We have previously reported earlier and greater gametocytogenesis in response to subcurative antimalarial chemotherapy in the rodent malaria, Plasmodium chabaudi, in vivo. Using an immunofluorescent assay to detect parasites that had invaded red blood cell monolayers, we demonstrate a 5-fold increase in gametocytogenesis in the human malaria, P. falciparum, in vitro, in response to treatment with the antimalarial drug chloroquine. In all clones used, gametocytogenesis increased with increasing inhibition of asexual growth by chloroquine. Furthermore, there were clone differences in the relationship between stress and gametocyte production, implying the response was genetically variable. This was not, however, associated with chloroquine resistance. The epidemiological significance of these results is discussed.

Characteristics of Plasmodium falciparum parasites that survive the lengthy dry season in eastern Sudan where malaria transmission is markedly seasonal.

We have examined 83 inhabitants of Asar village in eastern Sudan, where malaria transmission lasts approximately 2-3 months each year, for the presence of Plasmodium falciparum during the prolonged dry season. All patients were treated with a standard dose of chloroquine following the first diagnosis, then examined by microscopy and the polymerase chain reaction (PCR) every two weeks for the first two months and subsequently once each month for the next 15 months throughout the dry season until the following transmission season. The PCR primers used amplified polymorphic regions of the merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein genes. Results show that subpatent and asymptomatic parasitemias persisted in some patients for several months throughout the dry season, often as genetically complex infections. Different genotypes could coexist together in a single infection and the proportions of each could fluctuate dramatically during this period. However, in some individuals, single genotypes appeared to persist for several months. Reappearance of clinical symptoms among patients with chronic infections was often associated with appearance of new alleles, indicating reinfections with parasites of novel genotypes.

Molecular analysis of recrudescent parasites in a Plasmodium falciparum drug efficacy trial in Gabon.

Recrudescent Plasmodium falciparum parasites were sampled from 108 children taking part in a drug efficacy trial in Gabon. A finger-prick blood sample was taken from each child before treatment, and a post-treatment sample taken of the recrudescent parasites. Sample deoxyribonucleic acid was amplified by the polymerase chain reaction using primers specific to the P. falciparum antigen genes MSP-1, MSP-2 and GLURP. Seventy-seven children had identical parasites in their pre- and post-treatment samples, indicating genuine recrudescences of resistant parasites. Fourteen children had completely different parasites in their pre- and post-treatment samples, indicating either a fresh infection from a mosquito or growth of a population of parasites not detected in the pre-treatment sample, perhaps due to sequestration. The remaining 17 children had a mixture of pre-treatment and new parasites in their post-treatment samples. This study demonstrated the use of polymorphic markers to confirm whether parasites in patients with clinical recrudescences after drug treatment are genuinely resistant.

Differential antibody recognition of FC27-like Plasmodium falciparum merozoite surface protein MSP2 antigens which lack 12 amino acid repeats.

Three alleles of the FC27-type allelic family of the MSP2 gene of the malaria parasite Plasmodium falciparum have been sequenced from parasites from the field (The Gambia and Tanzania). These alleles lack the 12 amino acid repeat units which are usual in this family of MSP2 alleles. We have investigated the recognition by sera from an endemic area (The Gambia) of three recombinant MSP2 proteins that have 5, 1 and no copies of this repeat region. Antibody recognition of these recombinant proteins varied according to the number of repeats present. High titre antibody levels were seen with most sera using the recombinant protein with 5 x 12-mer repeats, whereas only low responses were measured using proteins containing 1 or no 12-mer repeats. Several sera entirely failed to recognise the protein which lacked 12-mer repeats. The data suggest that variation in the number of tandem repeat sequences could allow the parasite to avoid high avidity antibody binding and this may allow escape from immune recognition.

Mating patterns in malaria parasite populations of Papua New Guinea.

Description of the genetic structure of malaria parasite populations is central to an understanding of the spread of multiple-locus drug and vaccine resistance. The Plasmodium falciparum mating patterns from madang, Papua New Guinea, where intense transmission of malaria occurs, are described here. A high degree of inbreeding occurs in the absence of detectable linkage disequilibrium. This contrasts with other studies, indicating that the genetic structure of malaria parasite populations is neither clonal nor panmictic but will vary according to the transmission characteristics of the region.

Estimation of inbreeding coefficients from genotypic data on multiple alleles, and application to estimation of clonality in malaria parasites.

Methods for estimating probability of identity by descent (f) are derived for data on numbers of genotypes at single loci and at pairs of loci with many alleles at each locus. The methods are general, but are specifically applied to data on genotype frequencies in zygotes of the malaria parasite sampled from its mosquito host in order to find the extent of outcrossing in the parasite and the degree of clonality in populations. It is assumed that zygotes are the outcome either of gametes of the same clone, in which they are identical at all loci, or are products of two random, unrelated clones. From the estimate of f an effective number of clones per human host can also be derived. For Plasmodium falciparum from a Tanzanian village, estimates of f are 0.33 from data on zygote frequencies at two multiallelic loci, indicating that two-thirds of zygotes produce recombinant type.

Random mating in a natural population of the malaria parasite Plasmodium falciparum.

The genetic structure of a population of the malaria parasite Plasmodium falciparum has been examined in a village in Tanzania. Seventeen alleles of the merozoite surface protein MSP-1 and 23 of MSP-2 were detected by the polymerase chain reaction (PCR) among the blood parasites of the inhabitants. Most infections contained mixtures of genetically distinct parasite clones. PCR was then used to examine individual P. falciparum oocysts, the products of fertilization events, in wild-caught mosquitoes. Forty-five out of 71 oocysts were heterozygous for one or both genes, showing that crossing between clones was taking place frequently, following uptake of mixtures of gametocytes by the mosquitoes. The frequency of heterozygous forms showed that random mating events probably occurred within mosquito bloodmeals between gametes belonging to different parasite clones.

Proof of intragenic recombination in Plasmodium falciparum.

Intragenic recombination in the merozoite surface protein MSP-1 of Plasmodium falciparum has been demonstrated in a cross between two cloned lines (3D7 and HB3) of this species. Following passage of a mixture of the clones through mosquitoes, uncloned progeny were examined by PCR for molecules containing sequences of both parent MSP-1 alleles. A recombinant molecule possessing both 3D7 and HB3 sequences has been obtained. Such molecules were not obtained from artificial mixtures of the blood forms of each clone. It is concluded that the novel allele was formed by a recombination event during meiosis of a hybrid 3D7/HB3 zygote.

Frequency of cross-fertilization in the human malaria parasite Plasmodium falciparum.

Two clones of the human malaria parasite Plasmodium falciparum, denoted 3D7 and HB3, were grown in vitro under conditions permitting the development of gametocytes. The two clones differ in their allelic forms of two antigen genes MSP1 and MSP2. The alleles can be distinguished as size differences of polymerase chain reaction (PCR) amplified fragments of repetitive regions of each gene. Mosquitoes (Anopheles stephensi) were fed on a mixture of these gametocytes. A total of 128 oocysts was isolated from the midguts of infected mosquitoes from 9 crossing experiments between the clones. DNA extracted from these oocysts was amplified by PCR. Oocysts which contained both alleles of each gene (MSP1 and MSP2) had developed from heterozygotes produced by cross-fertilization events between 3D7 and HB3 gametes. The remaining oocysts contained single alleles of each gene, in parent clone combinations, and these had developed from homozygotes formed by self-fertilizations. The results suggest that gametes in the original mixture fed to mosquitoes had undergone random mating.

Uniparental inheritance of the mitochondrial gene cytochrome b in Plasmodium falciparum.

The inheritance of an extrachromosomal 6-kb element has been examined in the human malaria parasite Plasmodium falciparum. A single base pair difference in the cytochrome b gene from the 6-kb element of two different cloned lines of the parasite was identified, and used as a marker in a cross in the mosquito stage of the life cycle. Analysis of 59 individual hybrid oocysts resulting from this cross clearly demonstrated that inheritance of the cytochrome b gene was uniparental. This observation makes it possible to investigate the inheritance and evolution of cytoplasmic traits, including certain forms of drug resistance, in natural populations of this parasite.