Diplonemids are highly diverse and abundant marine plankton with significant ecological importance. However, little is known about their biology, even in the model diplonemid Paradiplonema papillatum whose genome sequence is available. Examining the subcellular localization of proteins using fluorescence microscopy is a powerful approach to infer their putative function. Here, we report a plasmid-based method that enables YFP-tagging of a gene at the endogenous locus. By examining the localization of proteins whose homologs are involved in chromosome organization or segregation in other eukaryotes, we discovered several notable features in mitotically dividing P. papillatum cells. Cohesin is enriched on condensed interphase chromatin. During mitosis, chromosomes organize into two rings (termed mitotic rings herein) that surround the elongating nucleolus and align on a bipolar spindle. Homologs of chromosomal passenger complex components (INCENP, two Aurora kinases and KIN-A), a CLK1 kinase, meiotic chromosome axis protein SYCP2L1, spindle checkpoint protein Mad1 and microtubule regulator XMAP215 localize in between the two mitotic rings. In contrast, a Mad2 homolog localizes near basal bodies as in trypanosomes. By representing the first molecular characterization of mitotic mechanisms in P. papillatum and raising many questions, this study forms the foundation for dissecting mitotic mechanisms in diplonemids.

• Klíčová slova
• Euglenozoa, chromosome, diplonemid, kinetochore, kinetoplastid,
• MeSH
• aparát dělícího vřeténka metabolismus   MeSH
• chromozomální proteiny, nehistonové metabolismus   MeSH
• chromozomy metabolismus   MeSH
• Dinoflagellata * genetika   metabolismus   cytologie   MeSH
• mitóza * MeSH
• proteiny buněčného cyklu metabolismus   MeSH
• segregace chromozomů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• proteiny buněčného cyklu MeSH

Telonemia are one of the oldest identified marine protists that for most part of their history have been recognized as a distinct incertae sedis lineage. Today, their evolutionary proximity to the SAR supergroup (Stramenopiles, Alveolates, and Rhizaria) is firmly established. However, their ecological distribution and importance as a natural predatory flagellate, especially in freshwater food webs, still remain unclear. To unravel the distribution and diversity of the phylum Telonemia in freshwater habitats, we examined over a thousand freshwater metagenomes from all over the world. In addition, to directly quantify absolute abundances, we analyzed 407 samples from 97 lakes and reservoirs using Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH). We recovered Telonemia 18S rRNA gene sequences from hundreds of metagenomic samples from a wide variety of habitats, indicating a global distribution of this phylum. However, even after this extensive sampling, our phylogenetic analysis did not reveal any new major clades, suggesting current molecular surveys are near to capturing the full diversity within this group. We observed excellent concordance between CARD-FISH analyses and estimates of abundances from metagenomes. Both approaches suggest that Telonemia are largely absent from shallow lakes and prefer to inhabit the colder hypolimnion of lakes and reservoirs in the Northern Hemisphere, where they frequently bloom, reaching 10%-20% of the total heterotrophic flagellate population, making them important predatory flagellates in the freshwater food web.

• Klíčová slova
• CARD-FISH, Telonemia, freshwater lakes, metagenomics, microbial food webs, predatory flagellate,
• MeSH
• biodiverzita MeSH
• fylogeneze * MeSH
• hybridizace in situ fluorescenční * MeSH
• jezera mikrobiologie   parazitologie   MeSH
• metagenom MeSH
• metagenomika MeSH
• RNA ribozomální 18S * genetika   MeSH
• sladká voda * mikrobiologie   parazitologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• RNA ribozomální 18S * MeSH

The knowledge of cell biology of a eukaryotic group is essential for correct interpretation of ecological and molecular data. Although diplonemid protists are one of the most species-rich lineages of marine eukaryotes, only very fragmentary information is available about the cellular architecture of this taxonomically diverse group. Here, a large serial block-face scanning electron microscopy data set complemented with light and fluorescence microscopy allowed the first detailed three-dimensional reconstruction of a diplonemid species. We describe numerous previously unknown peculiarities of the cellular architecture and cell division characteristic for diplonemid flagellates, and illustrate the obtained results with multiple three-dimensional models, comprehensible for non-specialists in protist ultrastructure.

• Klíčová slova
• 3-dimensional reconstruction, Euglenozoa, SBF-SEM, cell division, diplonemid, ultrastructure,
• MeSH
• Eukaryota * MeSH
• mikroskopie elektronová rastrovací MeSH
• organely MeSH
• zobrazování trojrozměrné * metody   MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: Diplonemid flagellates are among the most abundant and species-rich of known marine microeukaryotes, colonizing all habitats, depths, and geographic regions of the world ocean. However, little is known about their genomes, biology, and ecological role. RESULTS: We present the first nuclear genome sequence from a diplonemid, the type species Diplonema papillatum. The ~ 280-Mb genome assembly contains about 32,000 protein-coding genes, likely co-transcribed in groups of up to 100. Gene clusters are separated by long repetitive regions that include numerous transposable elements, which also reside within introns. Analysis of gene-family evolution reveals that the last common diplonemid ancestor underwent considerable metabolic expansion. D. papillatum-specific gains of carbohydrate-degradation capability were apparently acquired via horizontal gene transfer. The predicted breakdown of polysaccharides including pectin and xylan is at odds with reports of peptides being the predominant carbon source of this organism. Secretome analysis together with feeding experiments suggest that D. papillatum is predatory, able to degrade cell walls of live microeukaryotes, macroalgae, and water plants, not only for protoplast feeding but also for metabolizing cell-wall carbohydrates as an energy source. The analysis of environmental barcode samples shows that D. papillatum is confined to temperate coastal waters, presumably acting in bioremediation of eutrophication. CONCLUSIONS: Nuclear genome information will allow systematic functional and cell-biology studies in D. papillatum. It will also serve as a reference for the highly diverse diplonemids and provide a point of comparison for studying gene complement evolution in the sister group of Kinetoplastida, including human-pathogenic taxa.

• Klíčová slova
• CAZymes, Ecological distribution, Feeding strategy, Gene-family evolution, Genome, Geographical distribution, Lateral gene transfer, Paradiplonema papillatum, Proteome, Protists, Transcriptome,
• MeSH
• Euglenozoa genetika   MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• Kinetoplastida * genetika   MeSH
• lidé MeSH
• multigenová rodina MeSH
• profáze meiózy I MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Barium and strontium are often used as proxies of marine productivity in palaeoceanographic reconstructions of global climate. However, long-searched biological drivers for such correlations remain unknown. Here, we report that taxa within one of the most abundant groups of marine planktonic protists, diplonemids (Euglenozoa), are potent accumulators of intracellular barite (BaSO4), celestite (SrSO4), and strontiobarite (Ba,Sr)SO4. In culture, Namystinia karyoxenos accumulates Ba2+ and Sr2+ 42,000 and 10,000 times higher than the surrounding medium, forming barite and celestite representing 90% of the dry weight, the greatest concentration in biomass known to date. As heterotrophs, diplonemids are not restricted to the photic zone, and they are widespread in the oceans in astonishing abundance and diversity, as their distribution correlates with environmental particulate barite and celestite, prevailing in the mesopelagic zone. We found diplonemid predators, the filter-feeding zooplankton that produces fecal pellets containing the undigested celestite from diplonemids, facilitating its deposition on the seafloor. To the best of our knowledge, evidence for diplonemid biomineralization presents the strongest explanation for the occurrence of particulate barite and celestite in the marine environment. Both structures of the crystals and their variable chemical compositions found in diplonemids fit the properties of environmentally sampled particulate barite and celestite. Finally, we propose that diplonemids, which emerged during the Neoproterozoic era, qualify as impactful players in Ba2+/Sr2+ cycling in the ocean that has possibly contributed to sedimentary rock formation over long geological periods. IMPORTANCE We have identified that diplonemids, an abundant group of marine planktonic protists, accumulate conspicuous amounts of Sr2+ and Ba2+ in the form of intracellular barite and celestite crystals, in concentrations that greatly exceed those of the most efficient Ba/Sr-accumulating organisms known to date. We propose that diplonemids are potential players in Ba2+/Sr2+ cycling in the ocean and have possibly contributed to sedimentary rock formation over long geological periods. These organisms emerged during the Neoproterozoic era (590 to 900 million years ago), prior to known coccolithophore carbonate biomineralization (~200 million years ago). Based on reported data, the distribution of diplonemids in the oceans is correlated with the occurrence of particulate barite and celestite. Finally, diplonemids may provide new insights into the long-questioned biogenic origin of particulate barite and celestite and bring more understanding of the observed spatial-temporal correlation of the minerals with marine productivity used in reconstructions of past global climate.

• Klíčová slova
• Euglenozoa, barite, biocrystallization, biogeochemical cycles, celestite,
• MeSH
• baryum MeSH
• minerály MeSH
• oceány a moře MeSH
• plankton MeSH
• síran barnatý * MeSH
• stroncium * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• oceány a moře MeSH
• Názvy látek
• baryum MeSH
• minerály MeSH
• síran barnatý * MeSH
• stroncium * MeSH

Gene transfer agents (GTAs) are virus-like structures that package and transfer prokaryotic DNA from donor to recipient prokaryotic cells. Here, we describe widespread GTA gene clusters in the highly reduced genomes of bacterial endosymbionts from microbial eukaryotes (protists). Homologs of the GTA capsid and portal complexes were initially found to be present in several highly reduced alphaproteobacterial endosymbionts of diplonemid protists (Rickettsiales and Rhodospirillales). Evidence of GTA expression was found in polyA-enriched metatranscriptomes of the diplonemid hosts and their endosymbionts, but due to biases in the polyA-enrichment methods, levels of GTA expression could not be determined. Examining the genomes of closely related bacteria revealed that the pattern of retained GTA head/capsid complexes with missing tail components was common across Rickettsiales and Holosporaceae (Rhodospirillales), all obligate symbionts with a wide variety of eukaryotic hosts. A dN/dS analysis of Rickettsiales and Holosporaceae symbionts revealed that purifying selection is likely the main driver of GTA evolution in symbionts, suggesting they remain functional, but the ecological function of GTAs in bacterial symbionts is unknown. In particular, it is unclear how increasing horizontal gene transfer in small, largely clonal endosymbiont populations can explain GTA retention, and, therefore, the structures may have been repurposed in endosymbionts for host interactions. Either way, their widespread retention and conservation in endosymbionts of diverse eukaryotes suggests an important role in symbiosis.

• Klíčová slova
• Holosporaceae, Rickettsiales, endosymbiosis, evolution, gene transfer agent, protist,
• MeSH
• Bacteria genetika   MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• přenos genů horizontální MeSH
• symbióza genetika   MeSH
• viry * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Diplonemids are one of the most abundant groups of heterotrophic planktonic microeukaryotes in the world ocean and, thus, are likely to play an essential role in marine ecosystems. So far, only few species have been introduced into a culture, allowing basic studies of diplonemid genetics, morphology, ultrastructure, metabolism, as well as endosymbionts. However, it remains unclear whether these heterotrophic flagellates are parasitic or free-living and what are their predominant dietary patterns and preferred food items. Here we show that cultured diplonemids, maintained in an organic-rich medium as osmotrophs, can gradually switch to bacterivory as a sole food resource, supporting positive growth of their population, even when fed with a low biovolume of bacteria. We further observed remarkable differences in species-specific feeding patterns, size-selective grazing preferences, and distinct feeding strategies. Diplonemids can discriminate between low-quality food items and inedible particles, such as latex beads, even after their ingestion, by discharging them in the form of large waste vacuoles. We also detected digestion-related endogenous autofluorescence emitted by lysosomes and the activity of a melanin-like material. We present the first evidence that these omnipresent protists possess an opportunistic lifestyle that provides a considerable advantage in the generally food resource-limited marine environments.

• MeSH
• Bacteria genetika   MeSH
• ekosystém * MeSH
• Eukaryota * MeSH
• plankton MeSH
• stravovací zvyklosti MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. RESULTS: We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. CONCLUSIONS: The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.

• Klíčová slova
• Adaptation, Diplonema, Euglenozoa, Hypoxia, Metabolism, Mitochondrion, Multiomics,
• MeSH
• Euglenozoa genetika   MeSH
• Eukaryota MeSH
• fylogeneze MeSH
• kyslík MeSH
• profáze meiózy I * MeSH
• proteomika * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kyslík MeSH

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.

• Klíčová slova
• Diplonemida, Euglenida, Kinetoplastida, microbial eukaryotes, phylogeny, systematics,
• MeSH
• ekosystém MeSH
• Euglenozoa klasifikace   genetika   fyziologie   virologie   MeSH
• fylogeneze MeSH
• Mimiviridae patogenita   MeSH
• symbióza MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

In this work, we studied the biochemical properties and evolutionary histories of catalase (CAT) and ascorbate peroxidase (APX), two central enzymes of reactive oxygen species detoxification, across the highly diverse clade Eugenozoa. This clade encompasses free-living phototrophic and heterotrophic flagellates, as well as obligate parasites of insects, vertebrates, and plants. We present evidence of several independent acquisitions of CAT by horizontal gene transfers and evolutionary novelties associated with the APX presence. We posit that Euglenozoa recruit these detoxifying enzymes for specific molecular tasks, such as photosynthesis in euglenids and membrane-bound peroxidase activity in kinetoplastids and some diplonemids.

• Klíčová slova
• Euglenozoa, ascorbate peroxidase, catalase, enzymatic activity, phylogeny,
• Publikační typ
• časopisecké články MeSH