Identification of microsatellite markers in Plasmodium mexicanum,a lizard malaria parasite that infects nucleated erythrocytes

Reptile and bird hosts of malaria parasites (Plasmodium) have nucleated erythrocytes. Infected blood thus contains a mix of abundant host and scant parasite DNA which has prevented identification of Plasmodium microsatellites. We developed a protocol for isolation of microsatellite markers for Plasmodium mexicanum, a parasite of lizards. The ATT repeat was common in the genome of P. mexicanum, but most (87%) of these repeats were exceptionally long (50–206 + repeats). Seven microsatellite markers with polymerase chain reaction primers are described. The protocol should allow discovery of microsatellites of malaria parasites (with AT‐rich genomes) infecting bird and reptile hosts.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles funestus

Screening of the Anopheles funestus genomic DNA library detected 18 new sequences with dinucleotide tandem repeats. Primers were designed to amplify the loci and 14 out of 18 gave a repeatable and scorable amplification. Deviations from Hardy–Weinberg expectations were tested for each locus in a sample of 30 wild Anopheles funestus females. No heterozygote deficiency was detected for 11 loci of 14, thus revealing the absence of null alleles. The number of alleles per locus ranged from 5 to 15, and observed heterozygosity from 0.13 to 0.85.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles maculipennis

The Anopheles maculipennis complex includes the most important malaria vectors of the western Palearctic. Anopheles maculipennis s.s., one member of this complex, is a reported vector in the Middle East. Here we describe the isolation of 15 microsatellite polymorphic loci from the An. maculipennis s.s. genome, displaying a high among individual diversity (0.37–0.77) in a sample from France. Three loci displayed a significant departure from Hardy–Weinberg proportions, suggesting a substantial frequency of null alleles. The remaining 12 loci are good candidates for further genetic studies in this species.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles sacharovi

The mosquito Anopheles sacharovi, a member of the A. maculipennis complex, is an important malaria vector in the Middle East. Here we describe the isolation of 15 microsatellite polymorphic loci from the A. sacharovi genome, displaying a high among‐individual diversity (0.30–0.92) in a sample from Turkey. Seven loci displayed a significant departure from Hardy–Weinberg proportions, suggesting a substantial frequency of null alleles. The remaining eight loci are good candidates for further genetic studies in this species.     

Isolation and characterization of microsatellite DNA markers in the malaria vector Anopheles nili

The mosquito Anopheles nili is widespread across tropical Africa and appears to be the major vector of malaria in some rural forested areas of central Africa. Here we describe the isolation of 11 microsatellite polymorphic loci from the A. nili genome, displaying a high among‐individual diversity (0.58–0.96) in samples from west Africa. Two loci displayed a significant departure from Hardy–Weinberg proportions across all samples, suggesting a substantial frequency of null alleles. The remaining nine loci are good candidates for further genetic studies in this species.     

Isolation of polymorphic microsatellite markers from the malaria vector Anopheles darlingi

High molecular weight DNA was extracted from the primary Neotropical malaria vector, Anopheles darlingi from Capanema, Pará, Brazil, to create a small insert genomic library, and then a phagemid library. Enriched sublibraries were constructed from the phagemid library using a microsatellite oligo primed second strand synthesis protocol. The resulting 242 760 individual clones were screened. The mean clone size of the positive clones was 302 bp. Flanking primers were designed for each suitable microsatellite sequence. Eight polymorphic loci were optimized and characterized. The allele size ranges are based on 253 samples of A. darlingi from eastern Amazonian and central Brazil.     

Ten polymorphic microsatellite DNA markers for the platypus, Ornithorhynchus anatinus

We identified and optimized 10 microsatellite loci for the platypus, Ornithorhynchus anatinus (Monotremata: Ornithorhynchidae), and screened 21 individuals from the southern tablelands area of New South Wales, Australia. Each polymorphic locus possessed between two and 12 alleles with observed heterozygosities between 0.118 and 0.950. The intent of this effort was to provide informative loci for studies on the population genetics of this species.     

Extensive microsatellite diversity in the human malaria parasite Plasmodium vivax

The population structure of Plasmodium vivax remains elusive. The markers of choice for large-scale population genetic studies of eukaryotes, short tandem repeats known as microsatellites, have been recently reported to be less polymorphic in P. vivax. Here we investigate the microsatellite diversity and geographic structure in P. vivax, at both local and global levels, using 14 new markers consisting of tri- or tetranucleotide repeats. The local-level analysis, which involved 50 field isolates from Sri Lanka, revealed unexpectedly high diversity (average virtual heterozygosity [H(E)], 0.807) and significant multilocus linkage disequilibrium in this region of low malaria endemicity. Multiple-clone infections occurred in 60% of isolates sampled in 2005. The global-level analysis of field isolates or monkey-adapted strains identified 150 unique haplotypes among 164 parasites from four continents. Individual P. vivax isolates could not be unambiguously assigned to geographic populations. For example, we found relatively low divergence among parasites from Central America, Africa, Southeast Asia and Oceania, but substantial differentiation between parasites from the same continent (South Asia and Southeast Asia) or even from the same country (Brazil). Parasite relapses, which may extend the duration of P. vivax carriage in humans, are suggested to facilitate the spread of strains across continents, breaking down any pre-existing geographic structure.     

Isolation and characterization of polymorphic microsatellite markers from Asian malaria mosquito Anopheles sinensis (Diptera: Culicidae)

Microsatellite-containing region were isolated and characterized in Anopheles sinensis, a primary vector of malaria parasites in Asia. An enrichment protocol yielded 252 microsatellite sequences. We designed primers to amplify 20 unique microsatellites, 14 of which amplify cleanly and were polymorphic. A survey of 24 individuals showed that 12 loci are highly variable with the number of alleles ranging from two to 11, and expected heterozygosity ranging from 0.116 to 0.903. These markers will be useful for population genetic studies and genome mapping in A. sinensis.     

Clonal diversity of a lizard malaria parasite, Plasmodium mexicanum, in its vertebrate host, the western fence lizard: role of variation in transmission intensity over time and space

Within the vertebrate host, infections of a malaria parasite (Plasmodium) could include a single genotype of cells (single-clone infections) or two to several genotypes (multiclone infections). Clonal diversity of infection plays an important role in the biology of the parasite, including its life history, virulence, and transmission. We determined the clonal diversity of Plasmodium mexicanum, a lizard malaria parasite at a study region in northern California, using variable microsatellite markers, the first such study for any malaria parasite of lizards or birds (the most common hosts for Plasmodium species). Multiclonal infections are common (50-88% of infections among samples), and measures of genetic diversity for the metapopulation (expected heterozygosity, number of alleles per locus, allele length variation, and effective population size) all indicated a substantial overall genetic diversity. Comparing years with high prevalence (1996-1998 = 25-32% lizards infected), and years with low prevalence (2001-2005 = 6-12%) found fewer alleles in samples taken from the low-prevalence years, but no reduction in overall diversity (H = 0.64-0.90 among loci). In most cases, rare alleles appeared to be lost as prevalence declined. For sites chronically experiencing low transmission intensity (prevalence approximately 1%), overall diversity was also high (H = 0.79-0.91), but there were fewer multiclonal infections. Theory predicts an apparent excess in expected heterozygosity follows a genetic bottleneck. Evidence for such a distortion in genetic diversity was observed after the drop in parasite prevalence under the infinite alleles mutation model but not for the stepwise mutation model. The results are similar to those reported for the human malaria parasite, Plasmodium falciparum, worldwide, and support the conclusion that malaria parasites maintain high genetic diversity in host populations despite the potential for loss in alleles during the transmission cycle or during periods/locations when transmission intensity is low.     

Isolation and characterization of fourteen polymorphic microsatellite markers in the viperine snake Natrix maura

Nineteen polymorphic microsatellite loci were identified and developed for Natrix maura. Polymorphism was assessed for 120 individuals sampled across four sampling sites from the French Pyrenees Mountains. The number of alleles per locus ranged from 3 to 15, and expected heterozygosity per locus ranged from 0.227 to 0.863. We tested for deviation from Hardy–Weinberg equilibrium and linkage disequilibrium and assessed the presence of null alleles for all loci, resulting in a selection of 14 high‐quality polymorphic markers. These markers will be extremely useful in identifying fine‐scale genetic structures and providing insight into conservation management plans of this species.     

Competing for blood: the ecology of parasite resource competition in human malaria–helminth co‐infections

Ecological theory suggests that co‐infecting parasite species can interact within hosts directly, via host immunity and/or via resource competition. In mice, competition for red blood cells (RBCs) between malaria and bloodsucking helminths can regulate malaria population dynamics, but the importance of RBC competition in human hosts was unknown. We analysed infection density (i.e. the concentration of parasites in infected hosts), from a 2‐year deworming study of over 4000 human subjects. After accounting for resource‐use differences among parasites, we find evidence of resource competition, priority effects and a competitive hierarchy within co‐infected individuals. For example reducing competition via deworming increased Plasmodium vivax densities 2.8‐fold, and this effect is limited to bloodsucking hookworms. Our ecological, resource‐based perspective sheds new light into decades of conflicting outcomes of malaria–helminth co‐infection studies with significant health and transmission consequences. Beyond blood, investigating within‐human resource competition may bring new insights for improving human health.     

Isolation and characterization of polymorphic microsatellite markers from the mosquito Anopheles moucheti,malaria vector in Africa

Anopheles moucheti is a major human malaria vector in Equatorial Africa. The screening of an Anopheles moucheti genomic microsatellite library allowed us to select 36 sequences with AC/GT dinucleotide tandem repeats. Primer pairs were designed to amplify the loci and 25 out of 36 gave a repeatable and scorable amplification. In total, 17 loci were selected for their high degree of polymorphism (the number of alleles per locus ranged from four to 16, and observed heterozygosity from 0.43 to 0.87) and suspicion of absence of null alleles, using 30 wild females from South‐Cameroon. No linkage disequilibrium was found between the loci.     

Isolation and characterization of polymorphic microsatellite markers in the migratory freshwater fish Prochilodus costatus

We isolated six polymorphic microsatellite loci in the migratory freshwater fish Prochilodus costatus, which is an endemic species and important fisheries resource from São Francisco river basin, Brazil. We have evaluated the applicability of these loci to study genetic variation in wild population of this fish. Thus, based on the genotypes of 32–48 individuals, we detected two to 21 alleles per locus, observed and expected heterozygosities ranging from 0.19 to 0.89 and from 0.17 to 0.92, respectively. These polymorphic markers should provide efficient tools to study population genetic structure of this fish.     

Novel polymorphic microsatellite markers in section Caulorrhizae (Arachis,Fabaceae)

This work reports the characterization of 11 polymorphic microsatellite loci in section Caulorrhizae. The primer pairs were designed from Arachis pintoi and showed full transferability to Arachis repens species. These new markers were used to evaluate the genetic diversity in germplasm (accessions and cultivars) of section Caulorrhizae. This new set of markers detected greater gene diversity than morphological and molecular markers such as AFLP (amplified fragment length polymorphism) and RAPD (rapid analysis of polymorphic DNA) previously used in this germplasm.     

Comparison of naturally acquired antibody responses against the C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 under low transmission and unstable malaria conditions in Sri Lanka

We report here, for the first time, a comparison of naturally acquired antibody responses to the 42 and 19 kDa C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 assayed by ELISA using p42 and p19 baculovirus-derived recombinant proteins, respectively. Test populations comprised patients with microscopy confirmed acute P. vivax infections from two regions endemic for vivax malaria where low transmission and unstable malaria conditions prevail, and a non-endemic urban area, in Sri Lanka. The antibody prevalence to the two proteins, both at the individual and population levels, tend to respond more to p42 than to p19 in all test areas, where >14% of individuals preferentially recognized p42, compared with <2% for p19. In patients with no previous exposure to malaria, 21% preferentially recognized p42, whereas none exclusively recognized p19. A significantly lower prevalence of anti-p19 IgM, but not anti-p42 IgM, was observed among residents from endemic areas compared with their non-endemic counterparts. Individuals from both endemic areas produced significantly less anti-p19 IgM compared with anti-p42 IgM. IgG1 was the predominant IgG isotype for both antigens in all individuals. With increasing exposure to malaria in both endemic areas, anti-p19 antibody responses were dominated by the functionally important IgG1 and IgG3 isotypes, with a concurrent reduction in IgM that was lacking in the non-endemic residents. This antibody switch was also reflected for PvAMA-1 as we previously reported with the identical battery of sera. In contrast, the antibody switch for p42 was restricted to endemic residents with more extensive exposure. These results suggest that an IgM-dominated antibody response against the p42 polymorphic region in endemic residents may interfere with the development of an IgG-dominated "protective" isotype shift to p19, that may complicate vaccine development.     

Cost‐effective development of highly polymorphic microsatellite in Japanese quail facilitated by next‐generation sequencing

Next‐generation sequencing technologies permit rapid and cost‐effective identification of numerous putative microsatellite loci. Here, from the genome sequences of Japanese quail, we developed microsatellite markers containing dinucleotide repeats and employed these for characterisation of genetic diversity and population structure. A total of 385 individuals from 12 experimental and one wild‐derived Japanese quail lines were genotyped with newly developed autosomal markers. The maximum number of alleles, expected heterozygosity and polymorphic information content (PIC) per locus were 10, 0.80 and 0.77 respectively. Approximately half of the markers were highly informative (PIC ≥ 0.50). The mean number of alleles per locus and observed heterozygosity within a line were in the range of 1.3–4.1 and 0.11–0.53 respectively. Compared with the wild‐derived line, genetic diversity levels were low in the experimental lines. Genetic differentiation (F_ST) between all pairs of the lines ranged from 0.13 to 0.83. Genetic clustering analyses based on multilocus genotypes of individuals showed that most individuals formed clearly defined clusters corresponding to the origins of the lines. These results suggest that Japanese quail experimental lines are highly structured. Microsatellite markers developed in this study may be effective for future genetic studies of Japanese quail.     

Characterization of polymorphic microsatellite markers for the endangered Malagasy radiated tortoise (Geochelone radiata)

The radiated tortoise (Geochelone radiata) is an endangered endemic species from Madagascar that inhabits the semiarid spiny forest of the southern part of the island. Habitat destruction and illegal harvesting greatly threaten this species, as attested by the significant reduction of its distribution area in the past 30 years. In order to undertake appropriate conservation actions, it is essential to acquire a better knowledge of its genetic structure. For this study, 145 blood samples were collected from three populations of radiated tortoises in southwestern Madagascar. Eight microsatellite loci were found to be polymorphic, and allelic diversity and observed heterozygosity were high for all markers.     

Within-host competition and drug resistance in the human malaria parasite Plasmodium falciparum

Infections with the malaria parasite Plasmodium falciparum typically comprise multiple strains, especially in high-transmission areas where infectious mosquito bites occur frequently. However, little is known about the dynamics of mixed-strain infections, particularly whether strains sharing a host compete or grow independently. Competition between drug-sensitive and drug-resistant strains, if it occurs, could be a crucial determinant of the spread of resistance. We analysed 1341 P. falciparum infections in children from Angola, Ghana and Tanzania and found compelling evidence for competition in mixed-strain infections: overall parasite density did not increase with additional strains, and densities of individual chloroquine-sensitive (CQS) and chloroquine-resistant (CQR) strains were reduced in the presence of competitors. We also found that CQR strains exhibited low densities compared with CQS strains (in the absence of chloroquine), which may underlie observed declines of chloroquine resistance in many countries following retirement of chloroquine as a first-line therapy. Our observations support a key role for within-host competition in the evolution of drug-resistant malaria. Malaria control and resistance-management efforts in high-transmission regions may be significantly aided or hindered by the effects of competition in mixed-strain infections. Consideration of within-host dynamics may spur development of novel strategies to minimize resistance while maximizing the benefits of control measures.