Tree-based delimitation of morphologically ambiguous taxa: a study of the lizard malaria parasites on the Caribbean island of Hispaniola

Malaria parasites in the genus Plasmodium have been classified primarily on the basis of differences in morphology. These single-celled organisms often lack distinguishing morphological features, and this can encumber both species delimitation and identification. Six saurian malaria parasites have been described from the Caribbean island of Hispaniola. All six infect lizards in the genus Anolis, but only two of these parasites can be distinguished using morphology. The remaining four species overlap in morphology and geography, and cannot be consistently identified using traditional methods. We compared a morphological approach with a molecular phylogenetic approach for assessing the taxonomy of these parasites. We surveyed for blood parasites from 677 Anolis lizards, representing 26 Anolis spp. from a total of 52 sites across Hispaniola. Fifty-five of these lizards were infected with Plasmodium spp., representing several new host records, but only 24 of these infections could be matched to previously described species using traditional morphological criteria. We then estimated the phylogeny of these parasites using both mitochondrial (cytb and coxI) and nuclear (EF2) genes, and included carefully selected GenBank sequences to confirm identities for certain species. Our molecular results unambiguously corroborated our morphology-based species identifications for only the two species previously judged to be morphologically distinctive. The remaining infections fell into two well-supported and reciprocally monophyletic clades, which contained the morphological variation previously reported for all four of the morphologically ambiguous species. One of these clades was identified as Plasmodium floridense and the other as Plasmodium fairchildi hispaniolae. We elevate the latter to Plasmodium hispaniolae comb. nov. because it is polyphyletic with the mainland species Plasmodium fairchildifairchildi and we contribute additional morphological and molecular characters for future species delimitation. Our phylogenetic hypotheses indicate that two currently recognised taxa, Plasmodium minasense anolisi and Plasmodium tropiduri caribbense, are not valid on Hispaniola. These results illustrate that molecular data can improve taxonomic hypotheses in Plasmodium when reliable morphological characters are lacking.     

Protein disulfide isomerase assisted protein folding in malaria parasites

In eukaryotes, the formation of protein disulfide bonds among cysteine residues is mediated by protein disulfide isomerases and occurs in the highly oxidised environment of the endoplasmic reticulum. This process is poorly understood in malaria parasites. In this paper, we report the gene isolation, sequence and phylogenetic comparisons, protein structure and thioredoxin-domain analyses of nine protein disulfide isomerases-like molecules from five species of malaria parasites including Plasmodium falciparum and Plasmodium vivax (human), Plasmodium knowlesi (simian) and Plasmodium berghei and Plasmodium yoelii (murine). Four of the studied protein disulfide isomerases belong to P. falciparum malaria and have been named PfPDI-8, PfPDI-9, PfPDI-11 and PfPDI-14, based on their chromosomal location. Among these, PfPDI-8 bears the closest similarity to a prototype PDI molecule with two thioredoxin domains (containing CGHC active sites) and a C-terminal Endoplasmic reticulum retrieval signal, SEEL. PfPDI-8 is expressed during all stages of parasite life cycle and is highly conserved (82-96% identity at amino acid level) in the other four Plasmodium species studied. Detailed biochemical analysis of PfPDI-8 revealed that this molecule is a potent oxido-reductase enzyme that facilitated the disulfide-dependent conformational folding of EBA-175, a leading malaria vaccine candidate. These studies open the avenues to understand the process of protein folding and secretory pathway in malaria parasites that in turn might aid in the production of superior recombinant vaccines and provide novel drug targets.     

The genome of model malaria parasites, and comparative genomics

The field of comparative genomics of malaria parasites has recently come of age with the completion of the whole genome sequences of the human malaria parasite Plasmodium falciparum and a rodent malaria model, Plasmodium yoelii yoelii. With several other genome sequencing projects of different model and human malaria parasite species underway, comparing genomes from multiple species has necessitated the development of improved informatics tools and analyses. Results from initial comparative analyses reveal striking conservation of gene synteny between malaria species within conserved chromosome cores, in contrast to reduced homology within subtelomeric regions, in line with previous findings on a smaller scale. Genes that elicit a host immune response are frequently found to be species-specific, although a large variant multigene family is common to many rodent malaria species and Plasmodium vivax. Sequence alignment of syntenic regions from multiple species has revealed the similarity between species in coding regions to be high relative to non-coding regions, and phylogenetic footprinting studies promise to reveal conserved motifs in the latter. Comparison of non-synonymous substitution rates between orthologous genes is proving a powerful technique for identifying genes under selection pressure, and may be useful for vaccine design. This is a stimulating time for comparative genomics of model and human malaria parasites, which promises to produce useful results for the development of antimalarial drugs and vaccines.     

Genome comparison of human and non-human malaria parasites reveals species subset-specific genes potentially linked to human disease

Genes underlying important phenotypic differences between Plasmodium species, the causative agents of malaria, are frequently found in only a subset of species and cluster at dynamically evolving subtelomeric regions of chromosomes. We hypothesized that chromosome-internal regions of Plasmodium genomes harbour additional species subset-specific genes that underlie differences in human pathogenicity, human-to-human transmissibility, and human virulence. We combined sequence similarity searches with synteny block analyses to identify species subset-specific genes in chromosome-internal regions of six published Plasmodium genomes, including Plasmodium falciparum, Plasmodium vivax, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. To improve comparative analysis, we first revised incorrectly annotated gene models using homology-based gene finders and examined putative subset-specific genes within syntenic contexts. Confirmed subset-specific genes were then analyzed for their role in biological pathways and examined for molecular functions using publicly available databases. We identified 16 genes that are well conserved in the three primate parasites but not found in rodent parasites, including three key enzymes of the thiamine (vitamin B1) biosynthesis pathway. Thirteen genes were found to be present in both human parasites but absent in the monkey parasite P. knowlesi, including genes specifically upregulated in sporozoites or gametocytes that could be linked to parasite transmission success between humans. Furthermore, we propose 15 chromosome-internal P. falciparum-specific genes as new candidate genes underlying increased human virulence and detected a currently uncharacterized cluster of P. vivax-specific genes on chromosome 6 likely involved in erythrocyte invasion. In conclusion, Plasmodium species harbour many chromosome-internal differences in the form of protein-coding genes, some of which are potentially linked to human disease and thus promising leads for future laboratory research.     

The Plasmodium Apicoplast Genome: Conserved Structure and Close Relationship of P. ovale to Rodent Malaria Parasites

Apicoplast, a nonphotosynthetic plastid derived from secondary symbiotic origin, is essential for the survival of malaria parasites of the genus Plasmodium. Elucidation of the evolution of the apicoplast genome in Plasmodium species is important to better understand the functions of the organelle. However, the complete apicoplast genome is available for only the most virulent human malaria parasite, Plasmodium falciparum. Here, we obtained the near-complete apicoplast genome sequences from eight Plasmodium species that infect a wide variety of vertebrate hosts and performed structural and phylogenetic analyses. We found that gene repertoire, gene arrangement, and other structural attributes were highly conserved. Phylogenetic reconstruction using 30 protein-coding genes of the apicoplast genome inferred, for the first time, a close relationship between P. ovale and rodent parasites. This close relatedness was robustly supported using multiple evolutionary assumptions and models. The finding suggests that an ancestral host switch occurred between rodent and human Plasmodium parasites.     

A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): evolution of life-history traits and host switches

Phylogenetic analysis of genomic data allows insights into the evolutionary history of pathogens, especially the events leading to host switching and diversification, as well as alterations of the life cycle (life-history traits). Hundreds, perhaps thousands, of malaria parasite species exploit squamate reptiles, birds, and mammals as vertebrate hosts as well as many genera of dipteran vectors, but the evolutionary and ecological events that led to this diversification and success remain unresolved. For a century, systematic parasitologists classified malaria parasites into genera based on morphology, life cycle, and vertebrate and insect host taxa. Molecular systematic studies based on single genes challenged the phylogenetic significance of these characters, but several significant nodes were not well supported. We recovered the first well resolved large phylogeny of Plasmodium and related haemosporidian parasites using sequence data for four genes from the parasites' three genomes by combining all data, correcting for variable rates of substitution by gene and site, and using both Bayesian and maximum parsimony analyses. Major clades are associated with vector shifts into different dipteran families, with other characters used in traditional parasitological studies, such as morphology and life-history traits, having variable phylogenetic significance. The common parasites of birds now placed into the genus Haemoproteus are found in two divergent clades, and the genus Plasmodium is paraphyletic with respect to Hepatocystis, a group of species with very different life history and morphology. The Plasmodium of mammal hosts form a well supported clade (including Plasmodium falciparum, the most important human malaria parasite), and this clade is associated with specialization to Anopheles mosquito vectors. The Plasmodium of birds and squamate reptiles all fall within a single clade, with evidence for repeated switching between birds and squamate hosts.     

Relative Incidence of Blood Parasites in Robins of Central New York and of the High Rockies

SUMMARY. A total of 60 robins, nearly equally divided among eastern and western species ( Turdus migratorius migratorius and T. m. propinquus respectively), and also almost evenly divided between juvenile and adult birds, has been studied for the relative incidence of blood parasites. Malaria of four species was found among the eastern robins, in most instances caused by Plasmodium vaughani. P. relictum (especially of the variety known as matutinum ) was next in frequency. Leucocytozoon was the next most common parasite. Among western robins only one case of malaria ( P. vaughani ) was found, but other blood parasites were much more common than in the eastern series, especially Leucocytozoon , which occurred in well over half the birds. Juvenile birds, even nestlings, seldom found infected with any of these parasites in the east, were very frequently infected in the western group. The explanation of these differences in incidence is believed to be chiefly differences in the distribution of still unknown vectors.     

Parasites in a biodiversity hotspot: a survey of hematozoa and a molecular phylogenetic analysis of Plasmodium in New Guinea skinks

A sample of 204 skinks (Squamata: Scincidae) from 10 genera representing 24 species were collected from 10 different localities in New Guinea and examined for blood parasites. Hemogregarines, trypanosomes, microfilarial worms, and 8 infections showing 2 distinct morphological types of malaria parasites (Plasmodium sp.) were observed. Molecular sequence data, in the form of mitochondrial cytochrome b sequences from the Plasmodium infections, showed 2 distinct clades of parasites, 1 in Sphenomorphus jobiense hosts and 1 in Emoia spp., which correspond to the 2 morphotypes. There was substantial genetic variation between the 2 clades, as well as within the clade of Emoia parasites. Nearly half of the skinks sampled had green blood pigmentation, resulting from the presence of biliverdin in the plasma; however, only 1 of these lizards was infected with Plasmodium sp. and only 2 had any blood parasites. These preliminary results suggest a high degree of phylogenetic diversity but a very low prevalence of Plasmodium spp. infections in the skinks of this globally important biodiversity hot spot.     

Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds

A fragment of the mitochondrial cytochrome b gene of avian malaria (genera Haemoproteus and Plasmodium) was amplified from blood samples of 12 species of passerine birds from the genera Acrocephalus, Phylloscopus and Parus. By sequencing 478 nucleotides of the obtained fragments, we found 17 different mitochondrial haplotypes of Haemoproteus or Plasmodium among the 12 bird species investigated. Only one out of the 17 haplotypes was found in more than one host species, this exception being a haplotype detected in both blue tits (Parus caeruleus) and great tits (Parus major). The phylogenetic tree which was constructed grouped the sequences into two clades, most probably representing Haemoproteus and Plasmodium, respectively. We found two to four different parasite mitochondrial DNA (mtDNA) haplotypes in four bird species. The phylogenetic tree obtained from the mtDNA of the parasites matched the phylogenetic tree of the bird hosts poorly. For example, the two tit species and the willow warbler (Phylloscopus trochilus) carried parasites differing by only 0.6% sequence divergence, suggesting that Haemoproteus shift both between species within the same genus and also between species in different families. Hence, host shifts seem to have occurred repeatedly in this parasite host system. We discuss this in terms of the possible evolutionary consequences for these bird species.     

Coamplification of Leucocytozoon by PCR diagnostic tests for avian malaria: a cautionary note

A number of PCR assays have now been described for detecting species of the avian malaria parasites Plasmodium and Haemoproteus from blood samples. The published protocols amplify both genera simultaneously, owing to the high degree of sequence similarity between them in target genes. However, the potential for coamplification in these assays of a third, closely related hematozoan parasite, Leucocytozoon spp. has been largely overlooked. In this paper, we highlight the importance of this issue, showing that coamplification of Leucocytozoon spp. occurs in several of the protocols designed to amplify avian malaria parasites. This leads not only to scoring of false positives but, in cases of mixed Leucocytozoon/malaria infections, may also lead to scoring of false negatives. We, therefore, advocate the use of a post-PCR diagnostic step, such as RFLP analysis or sequencing, to assess the contribution of Leucocytozoon spp. to overall prevalence.     

Studies on enzyme variation in the murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi by starch gel electrophoresis.

Electrophoretic variation of the enzymes glucose phosphate isomerase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and glutamate dehydrogenase (NADP-dependent) has been studied in the African murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi and their subspecies. Horizontal starch gel electrophoresis was used throughout. The number of isolates examined in each subspecies varied from 1 (P. y. nigeriensis) to 24 (P. c. chabaudi). Extensive enzyme variation was found among isolates of most of the subspecies from which more than two such isolates were available for study. It is clear that the phenomenon of enzyme polymorphism is of common occurrence among malaria parasites. With the exception of P. berghei and P. yoelii, of which all isolates share an identical electrophoretic form of lactate dehydrogenase, no enzyme forms are shared between any of the 4 species of murine plasmodia. By contrast, within each species common enzyme forms are shared among each of the subspecies. The subspecies are nevertheless, distinguished from each other by the electrophoretic forms of at least one enzyme. The distribution and reassortment of enzyme variation among isolates of a single subspecies is in accordance with the concept of malaria parasites as sexually reproducing organisms. The study of variation among parasites present in individual wild-caught rodent hosts demonstrates that natural malarial infections usually comprise genetically heterogeneous populations of parasites. Nevertheless, the number of genetically distinct types of parasite of any one species present in a single infected host appears to be small. Generally not more than 2 or 3 clones of parasite of distinct genetic constitution are present in a single infected animal.     

Phylogeny of nuclear small subunit rRNA genes of hemogregarines amplified with specific primers

Hemogregarines, apicomplexan intracellular blood parasites, are cosmopolitan in distribution and infect a broad range of vertebrate and invertebrate hosts. Molecular phylogenetic studies have been hampered by lack of hemogregarine-specific polymerase chain reaction primers that would allow amplification of parasite, but not host, DNA. A novel method for separating parasite and host 18S rRNA genes has been developed, and new primers that are specific for hemogregarine rRNA genes have been designed. These primers were used to obtain sequences from 4 isolates of hemogregarines of lizards from California, the Caribbean island of Grenada, eastern Australia, and Israel. Combining these results with already published sequences, a preliminary phylogeny of hemogregarines and several other apicomplexan taxa has been created. The hemogregarines form a monophyletic group and appear to be more closely related to coccidia than to Plasmodium species. The difficulty of using 18S genes that have multiple copies in some apicomplexan parasites was explored for systematic studies.     

Evolutionary analysis of circumsporozoite surface protein and merozoite surface protein-1 (CSP and MSP-1) sequences of malaria parasites

Malaria, one of the world''s most common diseases, is caused by the intracellular protozoan parasite known as Plasmodium. In this study, we have determined the evolutionary relationship of two single-copy proteins, circumsporozoite protein (CSP) and merozoite surface protein-1 (MSP-1), among Plasmodium species using various bioinformatics tools and softwares. These two proteins are major blood stage antigens of Plasmodium species. This study demonstrates that the circumsporozoite protein of Plasmodium falciparum shows similarity with Plasmodium cynomolgi and Plasmodium knowlesi. The merozoite surface protein-1 of Plasmodium coatneyi forms a monophyletic group with Plasmodium knowlesi, demonstrating their close relationship and these two species also reveal similarity between the human malaria Plasmodium vivax. This Plasmodium phylogenetic arrangement is evidently crucial to identify shared derived characters as well as particular adaptation of plasmodium species from inside and between monophyletic groups.     

Phylogeography of Caribbean lizard malaria: tracing the history of vector‐borne parasites

The Anolis lizards of the eastern Caribbean islands are parasitized by several species of malaria parasites (Plasmodium). Here I focus on two species of Plasmodium, using molecular data (mitochondrial cytochrome b sequences) to recover the phylogeography of the parasites throughout the Lesser Antilles and Puerto Rico. The two parasites were originally described as a single species, P. azurophilum, which infects both red and white blood cells. Here the two species are termed P. azurophilum Red and P. azurophilum White based on their host cell type. Six haplotypes were found in 100 infections sequenced of P. azurophilum Red and six in 45 infections of P. azurophilum White. Nested clade analysis revealed a significant association of geographical location and clades as well as a pattern of past fragmentation of parasite populations. This is consistent with the hypothesis that vector‐borne parasites such as malaria may be subject to frequent local extinctions and recolonizations. Comparison of the phylogeography of the lizard and parasites shows only weak concordance; that is, the parasites colonized the lizards in the islands, but dispersal events between islands via vectors or failed lizard colonizations were present. The two parasites had different histories, P. azurophilum Red colonized the islands from both the north and south, and P. azurophilum White originated in the central Lesser Antilles, probably from P. azurophilum Red, then moved to both north and south. This is the first study to examine the biogeography of a pair of sibling species of vector‐borne parasites within an island archipelago system.     

NMR and crystallographic structures of the FK506 binding domain of human malarial parasite Plasmodium vivax FKBP35

The emergence of drug‐resistant malaria parasites is the major threat to effective malaria control, prompting a search for novel compounds with mechanisms of action that are different from the traditionally used drugs. The immunosuppressive drug FK506 shows an antimalarial activity. The mechanism of the drug action involves the molecular interaction with the parasite target proteins PfFKBP35 and PvFKBP35, which are novel FK506 binding protein family (FKBP) members from Plasmodium falciparum and Plasmodium vivax, respectively. Currently, molecular mechanisms of the FKBP family proteins in the parasites still remain elusive. To understand their functions, here we have determined the structures of the FK506 binding domain of Plasmodium vivax (PvFKBD) in unliganded form by NMR spectroscopy and in complex with FK506 by X‐ray crystallography. We found out that PvFKBP35 exhibits a canonical FKBD fold and shares kinetic profiles similar to those of PfFKBP35, the homologous protein in P. falciparum, indicating that the parasite FKBP family members play similar biological roles in their life cycles. Despite the similarity, differences were observed in the ligand binding modes between PvFKBD and HsFKBP12, a human FKBP homolog, which could provide insightful information into designing selective antimalarial drug against the parasites.     

Malaria parasites (Apicomplexa,Haematozoea) and their relationships with their hosts: is there an evolutionary cost for the specialization?

Parasite–host specialization is frequently considered to be a derived state such that it represents an 'evolutionary dead end' that strongly limits further evolution. In this study, it was tested whether this theory is applicable to the relationship of malaria parasites and their vertebrate hosts. For this, we revisited Perkins and Schall (2002) analysis of the phylogenetic relationships of the malaria parasites (belonging to the genera Plasmodium , Haemoproteus and Hepatocystis ) based on the mitochondrial Cytochrome b gene sequence, and inferred, using a maximum likelihood (ML) approach, the putative ancestral vertebrate hosts. As the topology in this study presents several unresolved branches and is slightly different from that of Perkins and Schall, a Shimodaira and Hasegawa (SH; 1999) test has been performed in order to properly consider several alternative topologies. The results of this study suggest that the common ancestor of all these malaria parasites was a reptile (more specific of the order Squamata), and that the host switches from Squamata to Aves and vice versa were quite frequent along the evolution of these parasites. On the contrary, a strong evidence that the host shift from Squamata to Mammalia had occurred only once during the evolution of these organisms was found. This evidence (added to the current knowledge about the association of the malaria parasites with their vertebrate hosts) allows us to suggest, at least considering the species included in this study, that the adaptation in mammals had required a high level of specialization. Hence, the acquisition of this host class had culminated in an evolutionary dead end for the mammalian malaria parasites.     

Mitochondrial genome sequences support ancient population expansion in Plasmodium vivax

Examination of nucleotide diversity in 106 mitochondrial genomes of the most geographically widespread human malaria parasite, Plasmodium vivax, revealed a level of diversity similar to, but slightly higher than, that seen in the virulent human malaria parasite Plasmodium falciparum. The pairwise distribution of nucleotide differences among mitochondrial genome sequences supported the hypothesis that both these parasites underwent ancient population expansions. We estimated the age of the most recent common ancestor (MRCA) of the mitochondrial genomes of both P. vivax and P. falciparum at around 200,000-300,000 years ago. This is close to the previous estimates of the time of the human mitochondrial MRCA and the origin of modern Homo sapiens, consistent with the hypothesis that both these Plasmodium species were parasites of the hominid lineage before the origin of modern H. sapiens and that their population expansion coincided with the population expansion of their host.     

Recent advances in the study of avian malaria: an overview with an emphasis on the distribution of Plasmodium spp in Brazil

Avian malaria parasites (Plasmodium) have a worldwide distribution except for Antarctica. They are transmitted exclusively by mosquito vectors (Diptera: Culicidae) and are of particular interest to health care research due to their phylogenetic relationship with human plasmodia and their ability to cause avian malaria, which is frequently lethal in non-adapted avian hosts. However, different features of avian Plasmodium spp, including their taxonomy and aspects of their life-history traits, need to be examined in more detail. Over the last 10 years, ecologists, evolutionary biologists and wildlife researchers have recognized the importance of studying avian malaria parasites and other related haemosporidians, which are the largest group of the order Haemosporida by number of species. These studies have included understanding the ecological, behavioral and evolutionary aspects that arise in this wildlife host-parasite system. Molecular tools have provided new and exiting opportunities for such research. This review discusses several emerging topics related to the current research of avian Plasmodium spp and some related avian haemosporidians. We also summarize some important discoveries in this field and emphasize the value of using both polymerase chain reaction-based and microscopy-based methods in parallel for wildlife studies. We will focus on the genus Plasmodium, with an emphasis on the distribution and pathogenicity of these parasites in wild birds in Brazil.