Phylogenetic relationships of trypanosomatids parasitising true bugs (Insecta: Heteroptera) in sub-Saharan Africa

Three hundred and eighty-six heteropteran specimens belonging to more than 90 species captured in Ghana, Kenya and Ethiopia were examined for the presence of trypanosomatid flagellates. Of those, 100 (26%) specimens were positive for trypanosomatids and the spliced leader RNA gene sequence was obtained from 81 (80%) of the infected bugs. Its sequence-based analysis placed all examined flagellates in 28 typing units. Among 19 newly described typing units, 16 are restricted to sub-Saharan Africa, three belong to previously described species and six to typing units found on other continents. This result was corroborated by the analysis of the ssrRNA gene, sequenced for at least one representative of each major spliced leader RNA-based clade. In all trees obtained, flagellates originating from sub-Saharan Africa were intermingled with those isolated from American, Asian and European hosts, revealing a lack of geographic correlation. They are dispersed throughout most of the known diversity of monoxenous trypanosomatids. However, a complex picture emerged when co-evolution with their heteropteran hosts was taken into account, since some clades are specific for a single host clade, family or even species, whereas other flagellates display a very low host specificity, with a capacity to parasitise heteropteran bugs belonging to different genera/families. The family Reduviidae contains the widest spectrum of trypanosomatids, most likely a consequence of their predatory feeding behaviour, leading to an accumulation of a variety of flagellates from their prey. The plant pathogenic genus Phytomonas is reported here from Africa, to our knowledge for the first time. Finding the same typing units in hosts belonging to different heteropteran families and coming from different continents strongly indicates that the global diversity of the insect trypanosomatids is most likely lower than was predicted on the basis of the "one host-one parasite" paradigm. The analysis presented significantly extends the known diversity of monoxenous insect trypanosomatids and will be instrumental in building a new taxonomy that reflects their true phylogenetic relationships.     

Use of Monoclonal Antibodies for Differentiation of Different Isolates of Phytomonas (Plant Trypanosomatids)

The Phytomonas genus was created arbitrarily to designate plant trypanosomes. A serological study with polyclonal and monoclonal antibodies was carried out to situate these trypanosomatids with respect to other trypanosomatids - Herpetomonas, Crithidia, Trypanosoma - and to compare different plant trypanosome strains with each other. The use of monoclonal antibodies directed against two different isolates makes it possible to distinguish plant trypanosomatids according to their geographical origin and to separate clearly the plant trypanosomatids from the South of France from other lower trypanosomatids, which seems to justify creating the Phytomonas genus.     

Telomere biology of trypanosomatids: beginning to answer some questions

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.     

The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants

Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.     

Cytokinesis in trypanosomatids

The process of cytokinesis, where the cytoplasm of one cell is divided to produce two daughter cells, is intricate in trypanosomatids because of the requirement to replicate and segregate a number of single copy organelles, including the nucleus, kinetoplast, Golgi apparatus, and flagellum. Identifying regulators of the three stages of cytokinesis, initiation, furrow ingression, and abscission is complicated by the fact that cell division in trypanosomatids is easily perturbed and aberrant cells are readily produced during functional characterization of gene products. In this review, we discuss direct and indirect effects on cytokinesis, using Trypanosoma brucei as a model.     

Molecular evidence for trypanosomatids in Culex mosquitoes collected during a West Nile virus survey

Adult mosquitoes were previously collected and tested for West Nile virus during an intense WNV outbreak in 2003-2004 along the Cache la Poudre River in Colorado, USA. A subset of these mosquitoes was also tested for infection with trypanosomatids using nested PCR to amplify 18S rRNA. Of the 69 pools of Culex pipiens that were screened for both pathogens, 4.3% were positive for WNV and 11.6% tested positive for trypanosomes; no pools were found to be co-infected with both pathogens. One hundred and forty-three pools of Culex tarsalis, considered to be the principal WNV vector in this area, were tested in the same manner. 7.7% were positive for WNV and 20.3% of these pools tested positive for trypanosomes. Five pools of C. tarsalis were found to be co-infected with both pathogens, which was approximately 2.2 times more frequent than would be expected if these pathogens are independent of each other. Sequencing and maximum parsimony analysis of 18S rRNA revealed that four of the isolates arise in or near clades of described avian trypanosomes, likely indicating that these are vectored pathogens between birds and mosquitoes. Unexpectedly, the majority (24/28, 86%) of our positive samples form their own separate clade within the order Trypanosomatida with 100% bootstrap support. We have identified a potential new clade of trypanosomatids that exist within important mosquito vectors and discuss the potential ecological connections between these trypanosomes, arboviruses and mosquitoes.     

5S ribosomal RNA gene repeat sequences define at least eight groups of plant trypanosomatids (Phytomonas spp.): phloem-restricted pathogens form a distinct section

Trypanosomatids isolated from plants have been assigned typically into the genus Phytomonas. Such designations do not reflect the biology of the diverse isolates; confusion may arise due to the transient presence in plants of monogenetic (insect) trypanosomatids deposited by phytophagous bugs. To develop further molecular markers for the plant kinetoplastids, we have obtained the DNA sequence of the 5S ribosomal RNA gene from 24 isolates harvested from phloem, latex, and fruit. Small, distinct sequence differences were found at the 3'-ends of the transcribed regions; substantial sequence and size differences were found in the non-transcribed regions. Alignment of the gene sequences from all the isolates suggested the presence of eight groupings. While six groups contained isolates from single plant tissues, groups C and A contained isolates from both fruit and latex. The DNA sequences of the 10 phloem-restricted pathogenic isolates from South America and the Carribean were highly conserved and thus comprised a single group (H). The conserved nature of the 5S ribosomal RNA genes in these plant pathogens supports the proposal that they be considered as a distinct section, the phloemicola.     

The more insect trypanosomatids under study-the more diverse Trypanosomatidae appears

From 10 trypanosomatids genera six comprise monogenetic parasites of insects and for the rest of four genera insects may serve as vectors. The invertebrate host is an essential element of trypanosomatids life cycle, but from more than 900 recognised vertebrate hosts only about 500 species of insects have been discovered to be the hosts of homoxenous trypanosomatids. Nothing or very little is known about insect trypanosomatids in many extensive areas such as South East Asia, Australia, Japan and some others. Each new region explored brings many new findings. Recently flagellates were found in new insect species and families. The border of parasites distribution was expanded till Central Asia, Far East and North over the Polar Circle. As paleogeographical events are now under contemplating in trypanosomatids phylogeny researches so northern insect trypanosomatids may attract some attention as the elements of postglacial fauna which is definitely young. Very broad host specificity of insect trypanosomatids and high probability to isolate non-specific parasite show causes that only the investigation of a culture may solve the question 'what parasite was really isolated?'. Examination of cell morphotypes in the host has clearly demonstrated that they are not sufficient for classification and may lead us to be mistaken. The number of insect trypanosomatid cultures is inadequate for characterisation of the diversity of insects trypanosomatids. Trypanosoma is actually the only trypanosomatid genus which is out of questions. Insect trypanosomatids comprise the most diversified part of trypanosomatids evolutionary tree. Recent ssrRNA phylogenetic analysis and morphological data show that three insect isolates represent new lineages on trypanosomatid evolutionary tree, as well as dendrograms derived from PCR data demonstrated some new groups of isolates. Therefore, the more insect trypanosomatids are involved in laboratory investigations--the more new clusters or/and new lineages are appearing on the tree.     

Concepts of species in trypanosomatids

This paper is a commentary on "Species concepts for trypanosomes: from morphological to molecular definitions?" by Wendy Gibson published in this journal [1]. Taxonomy has been traditionally based on expert opinion which is influenced among other factors by the philosophical and educational background of the expert concerned. This has resulted in widely different criteria for species among the trypanosomatids when compared to the actual genetic diversity involved. Gibson's paper presents an example of this within the trypanosome sub-genera. Although attempts have been made to put taxonomy on a more objective basis expert opinion still appears to dominate in the actual classifications in use.     

The karyotype of Trypanosoma cruzi Dm 28c: comparison with other T. cruzi strains and trypanosomatids

Chromosome-sized DNA molecules from Trypanosoma cruzi clone Dm 28c were analyzed and compared with other T. cruzi strains and monogenetic trypanosomatids by orthogonal field alteration gel electrophoresis. The results showed that T. cruzi Dm 28c displays at least 18 chromosomes ranging from 550 to more than 1500 kb and that in general the trypanosomatids have smaller chromosomes distributed in the size range from 300 to 1500 kb. With the exception of T. cruzi strain G49, there is no evidence of minichromosomes, suggesting they are not widely distributed among different isolates of the parasite. The hybridization of T. cruzi chromosomal Southern blots with probes for T. cruzi-specific genes showed that their location can change from one strain to another, supporting the idea of the plasticity of the parasite genome. Furthermore, the chromosome pattern is strictly conserved during the transformation of T. cruzi Dm 28c epimastigotes to metacyclic trypomastigotes, suggesting that extensive chromosomal rearrangements do not occur during at least part of the life cycle of the parasite.     

An analysis of trypanosomatids kDNA minicircle by absolute dinucleotide frequency

Trypanosomatid mitochondrial DNA (kDNA) possesses thousands of copies of small circular molecules called minicircles. Due to a high level of nucleotide polymorphism among copies, sequence alignment for species or strain characterization is not appropriate. In this work we report dinucleotide absolute frequency as a method to analyze minicircle sequences heterogeneity in trypanosomatids. Using Trypanosoma rangeli and Leishmania guyanensis minicircles as example of sequence length heterogeneity, we show that dinucleotide frequency of minicircles whose length variation is less than to 10% is relatively constant. Dinucleotide frequencies in Leishmania genus point out three clusters of predominant dinucleotide profiles: GG/TT/TG for Old World species; ii) TT/AA/TA for New World species and iii) TT/GG(AA) TA(AT) for Sauroleishmania. Trypanosoma species displayed broad range composition and the highest frequency values. Their dinucleotide profile appears to be species specific, except for African trypanosomes which exhibit similar composition. The low number of sequences from Crithidia, Herpetomonas, Phytomonas and Wallaceina did not allow a generalized analysis, however some species present highly similar compositional profile, e.g., Wallaceina species. Distinct signatures for Trypanosomatidae family members can be generated by using values of absolute frequencies, range and composition of most/least frequent dinucleotides from minicircles. Each species can be graphically represented by a diagram of frequencies along with a box plot of summary statistics.     

Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts

We comparatively examined the nutritional, molecular and optical and electron microscopical characteristics of reference species and new isolates of trypanosomatids harboring bacterial endosymbionts. Sequencing of the V7V8 region of the small subunit of the ribosomal RNA (SSU rRNA) gene distinguished six major genotypes among the 13 isolates examined. The entire sequences of the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes were obtained for phylogenetic analyses. In the resulting phylogenetic trees, the symbiont-harboring species clustered as a major clade comprising two subclades that corresponded to the proposed genera Angomonas and Strigomonas. The genus Angomonas comprised 10 flagellates including former Crithidia deanei and C. desouzai plus a new species. The genus Strigomonas included former Crithidia oncopelti and Blastocrithidia culicis plus a new species. Sequences from the internal transcribed spacer of ribosomal DNA (ITS rDNA) and size polymorphism of kinetoplast DNA (kDNA) minicircles revealed considerable genetic heterogeneity within the genera Angomonas and Strigomonas. Phylogenetic analyses based on 16S rDNA and ITS rDNA sequences demonstrated that all of the endosymbionts belonged to the Betaproteobacteria and revealed three new species. The congruence of the phylogenetic trees of trypanosomatids and their symbionts support a co-divergent host-symbiont evolutionary history.     

From a mono to a digenetic life-cycle: how was the jump for flagellates of the family Trypanosomatidae?

It has been found that species of the digenetic genus Trypanosoma as well as species of monogenetic trypanosomatids of insects can grow in the lumen of the scent glands of opossums reproducing the cycle they perform in the intestinal tract of their insect hosts. Based on these findings, speculations are made on the incompletely known cycles of many mammalian trypanosomes and on the evolution of the family Trypanosomatidae.     

Metabolic functions of glycosomes in trypanosomatids

Protozoan Kinetoplastida, including the pathogenic trypanosomatids of the genera Trypanosoma and Leishmania, compartmentalize several important metabolic systems in their peroxisomes which are designated glycosomes. The enzymatic content of these organelles may vary considerably during the life-cycle of most trypanosomatid parasites which often are transmitted between their mammalian hosts by insects. The glycosomes of the Trypanosoma brucei form living in the mammalian bloodstream display the highest level of specialization; 90% of their protein content is made up of glycolytic enzymes. The compartmentation of glycolysis in these organelles appears essential for the regulation of this process and enables the cells to overcome short periods of anaerobiosis. Glycosomes of all other trypanosomatid forms studied contain an extended glycolytic pathway catalyzing the aerobic fermentation of glucose to succinate. In addition, these organelles contain enzymes for several other processes such as the pentose-phosphate pathway, beta-oxidation of fatty acids, purine salvage, and biosynthetic pathways for pyrimidines, ether-lipids and squalenes. The enzymatic content of glycosomes is rapidly changed during differentiation of mammalian bloodstream-form trypanosomes to the forms living in the insect midgut. Autophagy appears to play an important role in trypanosomatid differentiation, and several lines of evidence indicate that it is then also involved in the degradation of old glycosomes, while a population of new organelles containing different enzymes is synthesized. The compartmentation of environment-sensitive parts of the metabolic network within glycosomes would, through this way of organelle renewal, enable the parasites to adapt rapidly and efficiently to the new conditions.     

Cyclic AMP signaling in trypanosomatids

Curative interference with signal transduction pathways is a spectacularly successful concept in many domains of modern pharmacology; indeed, the 'wonder drug' Viagra is but a humble inhibitor of a cyclic GMP (cGMP)-specific phosphodiesterase and, thus, interferes with cGMP-signaling in a strategic organ. In fact, about half of the 100 most successful drugs currently on the market act through modulating cellular signal transduction. Despite these encouraging findings, signal transduction pathways as potential drug targets in trypanosomatids have remained largely unexplored. However, what little is known indicates that adenylyl cyclases of trypanosomatids, and probably other enzymes of the cyclic nucleotide signaling pathways, are significantly different from their mammalian counterparts. Here, Christina Naula and Thomas Seebeck summarize what is known about cAMP signal transduction in trypanosomatids.     

Horizontal gene transfer in trypanosomatids

Trypanosomes harbour a large number of structural and biochemical peculiarities. Kinetoplast DNA, mitochondrial RNA editing, the sequestration of glycolysis inside glycosomes and unique oxidative-stress protection mechanisms (to name but a few) are found only in the members of the order Kinetoplastida. Thus, it is not surprising that they have provoked much speculation about why and how such oddities have evolved in trypanosomes. However, the true reasons for their existence within the eukaryotic world are still far from clear. Here, Fred Opperdoes and Paul Michels argue that the trypanosome-specific evolution of novel processes and organization could only have been made possible by the acquisition of a large number of foreign genes, which entered a trypanosomatid ancestor through lateral gene transfer. Many different organisms must have served as donors. Some of them were viruses, and others were bacteria, such as cyanobacterial endosymbionts and non-phototrophic bacteria.     

The evolution and diversity of kinetoplastid flagellates

Five years ago, little was known about kinetoplastid evolution. Recent improvements in the taxon sampling for nuclear rRNA genes and several protein markers have transformed this understanding. Parasitism evolved at least four times in kinetoplastids. Obligate parasitic trypanosomatids are a relatively 'derived' group within kinetoplastids; their closest relative is likely to be the free-living Bodo saltans, and the ancestral trypanosomatids were probably parasites of insects. Although subject to recent controversy, trypanosomes (genus Trypanosoma) probably constitute a monophyletic group. Several unusual features of trypanosomatid genomes (e.g. trans-splicing, mitochondrial RNA editing and intron poverty) are common in kinetoplastids and pre-date the adoption of parasitism. The framework of relationships is becoming robust enough for real comparative approaches to be used to understand kinetoplastid biology.     

Diversity of insect trypanosomatids assessed from the spliced leader RNA and 5S rRNA genes and intergenic regions

We have determined the sequences of 5S rRNA and spliced leader (SL) RNA genes, and adjacent intergenic regions for representatives of all known trypanosomatid genera parasitizing insects. The genetic loci have been analyzed separately as well as by a combined approach. Several isolates, assigned by morphology to different genera (Leptomonas spp., Blastocrithidia spp.), seem to belong to a single species with an unexpectedly wide host and geographical range. An unnamed trypanosomatid isolated from rats in Egypt was found to belong to the genus Herpetomonas, so far associated with insect hosts only. It is closely related to Herpetomonas ztiplika, a parasite of a blood-sucking biting midge. Apparently several different trypanosomatid species can infect one insect species, as exemplified by Leptomonas sp. PL and Wallaceina sp. Wsd, which were isolated from different specimens of Salda littoralis on the same locality and day. However, since the same species of Leptomonas was obtained from insect hosts belonging to different genera, some insect trypanosomatids may have low host specificity. Our data revealed additional discrepancies between molecular phylogenetic data and cell morphology, rendering current trypanosomatid taxonomy unreliable.     

Distribution of carbohydrates recognized by the lectins Euonymus europaeus and concanavalin A in monoxenic and heteroxenic trypanosomatids.

We observed a wide distribution of the carbohydrate epitopes galactosyl alpha(1-3)galactose (gal alpha1-3 gal), alpha-glucoside and alpha-mannoside in mono- and heteroxenic trypanosomatids by using fluorescein-labelled lectins of Euonymus europaeus (EE) and Concanavalin A (Con A) as well as sera from acute chagasic patients who have very high levels of anti-gal alpha(1-3)gal antibodies. The direct fluorescence test for gal alpha1-3 gal with EE was positive at minimum concentrations of 6 micrograms/ml for heteroxenic trypanosomatids and 0.7 micrograms/ml for monoxenic ones and for the plant parasite, Phytomonas. On the other hand, heteroxenic trypanosomatids that infect vertebrates bound ten-fold more Con A than monoxenic flagellates and Phytomonas. These data were confirmed in ELISA and Western Blot assays carried out with peroxidase-labelled EE and Con A. Euonymus europaeus recognized several glycoproteins in all trypanosomatids that we tested. Con A, however, recognized a glycoprotein cluster in heteroxenic protozoa, which ranging from 60-120 kDa, seemed to lack monoxenic parasites and Phytomonas. These findings suggest that alpha-D-mannose and alpha-D-glucose might play an important role in the interaction between trypanosomatids and vertebrate hosts.