Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses.

Euglenozoa is a species-rich group of protists, which have extremely diverse lifestyles and a range of features that distinguish them from other eukaryotes. They are composed of free-living and parasitic kinetoplastids, mostly free-living diplonemids, heterotrophic and photosynthetic euglenids, as well as deep-sea symbiontids. Although they form a well-supported monophyletic group, these morphologically rather distinct groups are almost never treated together in a comparative manner, as attempted here. We present an updated taxonomy, complemented by photos of representative species, with notes on diversity, distribution and biology of euglenozoans. For kinetoplastids, we propose a significantly modified taxonomy that reflects the latest findings. Finally, we summarize what is known about viruses infecting euglenozoans, as well as their relationships with ecto- and endosymbiotic bacteria.

Ectosymbiotic bacteria at the origin of magnetoreception in a marine protist.

Mutualistic symbioses are often a source of evolutionary innovation and drivers of biological diversification1. Widely distributed in the microbial world, particularly in anoxic settings2,3, they often rely on metabolic exchanges and syntrophy2,4. Here, we report a mutualistic symbiosis observed in marine anoxic sediments between excavate protists (Symbiontida, Euglenozoa)5 and ectosymbiotic Deltaproteobacteria biomineralizing ferrimagnetic nanoparticles. Light and electron microscopy observations as well as genomic data support a multi-layered mutualism based on collective magnetotactic motility with division of labour and interspecies hydrogen-transfer-based syntrophy6. The guided motility of the consortia along the geomagnetic field is allowed by the magnetic moment of the non-motile ectosymbiotic bacteria combined with the protist motor activity, which is a unique example of eukaryotic magnetoreception7 acquired by symbiosis. The nearly complete deltaproteobacterial genome assembled from a single consortium contains a full magnetosome gene set8, but shows signs of reduction, with the probable loss of flagellar genes. Based on the metabolic gene content, the ectosymbiotic bacteria are anaerobic sulfate-reducing chemolithoautotrophs that likely reduce sulfate with hydrogen produced by hydrogenosome-like organelles6 underlying the plasma membrane of the protist. In addition to being necessary hydrogen sinks, ectosymbionts may provide organics to the protist by diffusion and predation, as shown by magnetosome-containing digestive vacuoles. Phylogenetic analyses of 16S and 18S ribosomal RNA genes from magnetotactic consortia in marine sediments across the Northern and Southern hemispheres indicate a host-ectosymbiont specificity and co-evolution. This suggests a historical acquisition of magnetoreception by a euglenozoan ancestor from Deltaproteobacteria followed by subsequent diversification. It also supports the cosmopolitan nature of this type of symbiosis in marine anoxic sediments.

Transformation of Diplonema papillatum, the type species of the highly diverse and abundant marine microeukaryotes Diplonemida (Euglenozoa).

Diplonema papillatum is the type species of diplonemids, which are among the most abundant and diverse heterotrophic microeukaryotes in the world's oceans. Diplonemids are also known for a unique form of post-transcriptional processing in mitochondria. However, the lack of reverse genetics methodologies in these protists has hampered elucidation of their cellular and molecular biology. Here we report a protocol for D. papillatum transformation. We have identified several antibiotics to which D. papillatum is sensitive and thus are suitable selectable markers, and focus in particular on puromycin. Constructs were designed encoding antibiotic resistance markers, fluorescent tags, and additional genomic sequences from D. papillatum to facilitate vector integration into chromosomes. We established conditions for effective electroporation, and demonstrate that electroporated constructs can be stably integrated in the D. papillatum nuclear genome. In D. papillatum transformants, the heterologous puromycin resistance gene is transcribed into mRNA and translated into protein, as determined by Southern hybridization, reverse transcription, and Western blot analyses. This is the first documented case of transformation in a euglenozoan protist outside the well-studied kinetoplastids, making D. papillatum a genetically tractable organism and potentially a model system for marine microeukaryotes.

Euglenoid pellicle morphogenesis and evolution in light of comparative ultrastructure and trypanosomatid biology: Semi-conservative microtubule/strip duplication, strip shaping and transformation.

Uniquely in eukaryotes, euglenoid pellicles comprise longitudinal proteinaceous, epiplasmic strips underlain by microtubules. Contradictory interpretations of pellicle microtubule duplication and segregation assumed opposite microtubule polarity from kinetoplastid Euglenozoa and conservative microtubule segregation. Distigma shows new pellicle microtubules nucleating posteriorly as in trypanosomatids, unifying euglenoid and kinetoplastid pellicle morphogenesis, but strip-growth is unpolarised. Epiplasmic insertion and cutting make new strip junctions between alternating wide and narrow daughter strips that grow intussusceptively. Nanotubules, overlooked epiplasm-associated components, define strip edges. At strip heel/toe junctions all euglenoids have a morphogenetic centre microtubule mt2/3 pair. Arguably, proteolysis, epiplasmic growth, and toe-nanotubule-associated epiplasmic scission initiate daughter strips, separating old mts2/3; new mt2/3/bridge-B assembly, sub-heel scission, nanotubule-bridge-A assembly complete duplication. Only mt2/3 pair fully enters the canal, one master microtubule also the reservoir, other pellicle microtubules terminating near canal rims. A related cytokinesis model involving ciliary attachment zone duplication explains near-universally even spirocute strip number. I consider Serpenomonas and Entosiphon alternating heteromorphic strips developmental stages of 'strip transformation'; explain intergroup diversity of strip morphology and dorsoventral strip differentiation causally by specific pellicle-complex components; propose centrin-based mechanisms for strip shaping and euglenoid movement; unify pellicle cytokinetic microtubule segregation across Euglenozoa; and discuss origin and diversification of pellicle complexes.

Evolution: Plumbing the Depths of Diplonemid Diversity.

Environmental molecular sequence surveys have opened a window on the hidden riches of the microbial biosphere. Recent genetic 'barcoding' and single-cell genomics studies have provided a snapshot of the biology of diplonemids - abundant, diverse, marine heterotrophic protists whose ecological roles are becoming clearer.

Diplonemids.

Lukes et al. introduce an enigmatic group of unicellular eukaryotes called the diplonemids, which according to recent surveys may be widespread in marine ecosystems.

Reconstruction of the feeding apparatus in Postgaardi mariagerensis provides evidence for character evolution within the Symbiontida (Euglenozoa).

Microbial eukaryotes living in low oxygen environments often have novel physiological and morphological features that facilitate symbiotic relationships with bacteria and other means for acquiring nutrients. Comparative studies of these features provide evidence for phylogenetic relationships and evolutionary history. Postgaardi mariagerensis, for instance, is a euglenozoan that lives in low oxygen environments and is enveloped by episymbiotic bacteria. The general ultrastructure of P. mariagerensis was described more than a decade ago and no further studies have been carried out since, mainly because these cells are difficult to obtain. Postgaardi lacks the diagnostic features found in other major euglenozoan lineages (e.g., pellicle strips and kinetoplast-like mitochondrial inclusions) and no molecular data are available, so the phylogenetic position of this genus within the Euglenozoa remains unclear. We re-examined and reconstructed the ultrastructural organization of the feeding apparatus in Postgaardi by serial sectioning an existing block of resin-embedded cells. Postgaardi possesses distinctive finger-like projections within the feeding apparatus; this system has only been found in one other highly distinctive flagellate, namely the symbiontid Calkinsia. Detailed comparisons of the cytoskeleton in Postgaardi and in two symbiontids, Calkinsia and Bihospites, provided new evidence for phylogenetic relationships and character evolution in all three genera.

Morphostasis in a novel eukaryote illuminates the evolutionary transition from phagotrophy to phototrophy: description of Rapaza viridis n. gen. et sp. (Euglenozoa, Euglenida).

BACKGROUND: Morphostasis of traits in different species is necessary for reconstructing the evolutionary history of complex characters. Studies that place these species into a molecular phylogenetic context test hypotheses about the transitional stages that link divergent character states. For instance, the transition from a phagotrophic mode of nutrition to a phototrophic lifestyle has occurred several times independently across the tree of eukaryotes; one of these events took place within the Euglenida, a large group of flagellates with diverse modes of nutrition. Phototrophic euglenids form a clade that is nested within lineages of phagotrophic euglenids and that originated through a secondary endosymbiosis with green algae. Although it is clear that phototrophic euglenids evolved from phagotrophic ancestors, the morphological disparity between species representing these different nutritional modes remains substantial. RESULTS: We cultivated a novel marine euglenid, Rapaza viridis n. gen. et sp. ("green grasper"), and a green alga, Tetraselmis sp., from the same environment. Cells of R. viridis were comprehensively characterized with light microscopy, SEM, TEM, and molecular phylogenetic analysis of small subunit rDNA sequences. Ultrastructural and behavioral observations demonstrated that this isolate habitually consumes a specific strain of Tetraselmis prey cells and possesses a functional chloroplast that is homologous with other phototrophic euglenids. A novel feeding apparatus consisting of a reduced rod of microtubules facilitated this first and only example of mixotrophy among euglenids. R. viridis also possessed a robust photoreception apparatus, two flagella of unequal length, euglenoid movement, and a pellicle consisting of 16 strips and one (square-shaped) whorl of posterior strip reduction. The molecular phylogenetic data demonstrated that R. viridis branches as the nearest sister lineage to phototrophic euglenids. CONCLUSIONS: The unusual combination of features in R. viridis combined with its molecular phylogenetic position completely conforms to the expected transitional stage that occurred during the early evolution of phototrophic euglenids from phagotrophic ancestors. The marine mixotrophic mode of nutrition, the preference for green algal prey cells, the structure of the feeding apparatus, and the organization of the pellicle are outstanding examples of morphostasis that clarify pivotal stages in the evolutionary history of this diverse group of microbial eukaryotes.

Modulation of mammalian apoptotic pathways by intracellular protozoan parasites.

During intracellular parasitic infections, pathogens and host cells take part in a complex web of events that are crucial for the outcome of the infection. Modulation of host cell apoptosis by pathogens attracted the attention of scientists during the last decade. Apoptosis is an efficient mechanism used by the host to control infection and limit pathogen multiplication and dissemination. In order to ensure completion of their complex life cycles and to guarantee transmission between different hosts, intracellular parasites have developed mechanisms to block apoptosis and sustain the viability of their host cells. Here, we review how some of the most prominent intracellular protozoan parasites modulate the main mammalian apoptotic pathways by emphasizing the advances from the last decade, which have begun to dissect this dynamic and complex interaction.

Species richness and trophic diversity increase decomposition in a co-evolved food web.

Ecological communities show great variation in species richness, composition and food web structure across similar and diverse ecosystems. Knowledge of how this biodiversity relates to ecosystem functioning is important for understanding the maintenance of diversity and the potential effects of species losses and gains on ecosystems. While research often focuses on how variation in species richness influences ecosystem processes, assessing species richness in a food web context can provide further insight into the relationship between diversity and ecosystem functioning and elucidate potential mechanisms underpinning this relationship. Here, we assessed how species richness and trophic diversity affect decomposition rates in a complete aquatic food web: the five trophic level web that occurs within water-filled leaves of the northern pitcher plant, Sarracenia purpurea. We identified a trophic cascade in which top-predators--larvae of the pitcher-plant mosquito--indirectly increased bacterial decomposition by preying on bactivorous protozoa. Our data also revealed a facultative relationship in which larvae of the pitcher-plant midge increased bacterial decomposition by shredding detritus. These important interactions occur only in food webs with high trophic diversity, which in turn only occur in food webs with high species richness. We show that species richness and trophic diversity underlie strong linkages between food web structure and dynamics that influence ecosystem functioning. The importance of trophic diversity and species interactions in determining how biodiversity relates to ecosystem functioning suggests that simply focusing on species richness does not give a complete picture as to how ecosystems may change with the loss or gain of species.

Compartmentalization of a glycolytic enzyme in Diplonema, a non-kinetoplastid euglenozoan.

Glycosomes are peroxisome-related organelles containing glycolytic enzymes that have been found only in kinetoplastids. We show here that a glycolytic enzyme is compartmentalized in diplonemids, the sister group of kinetoplastids. We found that, similar to kinetoplastid aldolases, the fructose 1,6-bisphosphate aldolase of Diplonema papillatum possesses a type 2-peroxisomal targeting signal. Western blotting showed that this aldolase was present predominantly in the membrane/organellar fraction. Immunofluorescence analysis showed that this aldolase had a scattered distribution in the cytosol, suggesting its compartmentalization. In contrast, orotidine-5'-monophosphate decarboxylase, a non-glycolytic glycosomal enzyme in kinetoplastids, was shown to be a cytosolic enzyme in D. papillatum. Since euglenoids, the earliest diverging branch of Euglenozoa, do not possess glycolytic compartments, these findings suggest that the routing of glycolytic enzymes into peroxisomes may have occurred in a common ancestor of diplonemids and kinetoplastids, followed by diversification of these newly established organelles in each of these euglenozoan lineages.