Vickermania gen. nov., trypanosomatids that use two joined flagella to resist midgut peristaltic flow within the fly host.

BACKGROUND: The family Trypanosomatidae encompasses parasitic flagellates, some of which cause serious vector-transmitted diseases of humans and domestic animals. However, insect-restricted parasites represent the ancestral and most diverse group within the family. They display a range of unusual features and their study can provide insights into the biology of human pathogens. Here we describe Vickermania, a new genus of fly midgut-dwelling parasites that bear two flagella in contrast to other trypanosomatids, which are unambiguously uniflagellate. RESULTS: Vickermania has an odd cell cycle, in which shortly after the division the uniflagellate cell starts growing a new flagellum attached to the old one and preserves their contact until the late cytokinesis. The flagella connect to each other throughout their whole length and carry a peculiar seizing structure with a paddle-like apex and two lateral extensions at their tip. In contrast to typical trypanosomatids, which attach to the insect host's intestinal wall, Vickermania is separated from it by a continuous peritrophic membrane and resides freely in the fly midgut lumen. CONCLUSIONS: We propose that Vickermania developed a survival strategy that relies on constant movement preventing discharge from the host gut due to intestinal peristalsis. Since these parasites cannot attach to the midgut wall, they were forced to shorten the period of impaired motility when two separate flagella in dividing cells interfere with each other. The connection between the flagella ensures their coordinate movement until the separation of the daughter cells. We propose that Trypanosoma brucei, a severe human pathogen, during its development in the tsetse fly midgut faces the same conditions and follows the same strategy as Vickermania by employing an analogous adaptation, the flagellar connector.

Annual Cycles of Two Cyanobacterial Mat Communities in Hydro-Terrestrial Habitats of the High Arctic.

Cyanobacteria form extensive macroscopic mats in shallow freshwater environments in the High Arctic and Antarctic. In these habitats, the communities are exposed to seasonal freezing and desiccation as well as to freeze-thawing and drying-rewetting cycles. Here, we characterized the annual cycles of two Phormidium communities in very shallow seepages located in central Svalbard. We observed the structure of the communities and the morphology, ultrastructure, metabolic activity, and viability of filaments and single cells. The communities overwintered as frozen mats, which were formed by long filaments enclosed in thick multilayered polysaccharide sheaths. No morphologically and/or ultrastructurally distinct spore-like cells were produced for surviving the winter, and the winter survival of the communities was not provided by a few resistant cells, which did not undergo visible morphological and ultrastructural transformations. Instead, a high proportion of cells in samples (85%) remained viable after prolonged freezing. The sheaths were the only morphological adaption, which seemed to protect the trichomes from damage due to freezing and freeze-associated dehydration. The cells in the overwintering communities were not dormant, as all viable cells rapidly resumed respiration after thawing, and their nucleoids were not condensed. During the whole vegetative season, defined by the presence of water in a liquid state, the communities were constantly metabolically active and contained <1% of dead and injured cells. The morphology and ultrastructure of the cells remained unaltered during observations throughout the year, except for light-induced changes in thylakoids. The dissemination events are likely to occur in spring as most of the trichomes were split into short fragments (hormogonia), a substantial proportion of which were released into the environment by gliding out of their sheaths, as well as by cracking and dissolving their sheaths. The short fragments subsequently grew longer and gradually produced new polysaccharide sheaths.

Ultrastructure and 3D reconstruction of a diplonemid protist (Diplonemea) and its novel membranous organelle.

IMPORTANCE: 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.

Massive Accumulation of Strontium and Barium in Diplonemid Protists.

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.

A single cryptomonad cell harbors a complex community of organelles, bacteria, a phage, and selfish elements.

Symbiosis between prokaryotes and microbial eukaryotes (protists) has broadly impacted both evolution and ecology. Endosymbiosis led to mitochondria and plastids, the latter spreading across the tree of eukaryotes by subsequent rounds of endosymbiosis. Present-day endosymbionts in protists remain both common and diverse, although what function they serve is often unknown. Here, we describe a highly complex community of endosymbionts and a bacteriophage (phage) within a single cryptomonad cell. Cryptomonads are a model for organelle evolution because their secondary plastid retains a relict endosymbiont nucleus, but only one previously unidentified Cryptomonas strain (SAG 25.80) is known to harbor bacterial endosymbionts. We carried out electron microscopy and FISH imaging as well as genomic sequencing on Cryptomonas SAG 25.80, which revealed a stable, complex community even after over 50 years in continuous cultivation. We identified the host strain as Cryptomonas gyropyrenoidosa, and sequenced genomes from its mitochondria, plastid, and nucleomorph (and partially its nucleus), as well as two symbionts, Megaira polyxenophila and Grellia numerosa, and one phage (MAnkyphage) infecting M. polyxenophila. Comparing closely related endosymbionts from other hosts revealed similar metabolic and genomic features, with the exception of abundant transposons and genome plasticity in M. polyxenophila from Cryptomonas. We found an abundance of eukaryote-interacting genes as well as many toxin-antitoxin systems, including in the MAnkyphage genome that also encodes several eukaryotic-like proteins. Overall, the Cryptomonas cell is an endosymbiotic conglomeration with seven distinct evolving genomes that all show evidence of inter-lineage conflict but nevertheless remain stable, even after more than 4,000 generations in culture.

A neo-functionalized homolog of host transmembrane protein controls localization of bacterial endosymbionts in the trypanosomatid Novymonas esmeraldas.

The stability of endosymbiotic associations between eukaryotes and bacteria depends on a reliable mechanism ensuring vertical inheritance of the latter. Here, we demonstrate that a host-encoded protein, located at the interface between the endoplasmic reticulum of the trypanosomatid Novymonas esmeraldas and its endosymbiotic bacterium Ca. Pandoraea novymonadis, regulates such a process. This protein, named TMP18e, is a product of duplication and neo-functionalization of the ubiquitous transmembrane protein 18 (TMEM18). Its expression level is increased at the proliferative stage of the host life cycle correlating with the confinement of bacteria to the nuclear vicinity. This is important for the proper segregation of bacteria into the daughter host cells as evidenced from the TMP18e ablation, which disrupts the nucleus-endosymbiont association and leads to greater variability of bacterial cell numbers, including an elevated proportion of aposymbiotic cells. Thus, we conclude that TMP18e is necessary for the reliable vertical inheritance of endosymbionts.

Gene Transfer Agents in Bacterial Endosymbionts of Microbial Eukaryotes.

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.

Diplonemids - A Review on "New" Flagellates on the Oceanic Block.

Diplonemids are a group of flagellate protists, that belong to the phylum Euglenozoa alongside euglenids, symbiontids and kinetoplastids. They primarily inhabit marine environments, though are also found in freshwater lakes. Diplonemids have been considered as rare and unimportant eukaryotes for over a century, with only a handful of species described until recently. However, thanks to their unprecedented diversity and abundance in the world oceans, diplonemids now attract increased attention. Recent improvements in isolation and cultivation have enabled characterization of several new genera, warranting a re-examination of all available knowledge gathered so far. Here we summarize available data on diplonemids, focusing on the recent advances in the fields of diversity, ecology, genomics, metabolism, and endosymbionts. We illustrate the life stages of cultivated genera, and summarise all reported interspecies associations, which in turn suggest lifestyles of predation and parasitism. This review also includes the latest classification of diplonemids, with a taxonomic revision of the genus Diplonema. Ongoing efforts to sequence various diplonemids suggest the presence of large and complex genomes, which correlate with the metabolic versatility observed in the model species Paradiplonema papillatum. Finally, we highlight its successful transformation into one of few genetically tractable marine protists.

Bacterial and archaeal symbioses with protists.

Most of the genetic, cellular, and biochemical diversity of life rests within single-celled organisms - the prokaryotes (bacteria and archaea) and microbial eukaryotes (protists). Very close interactions, or symbioses, between protists and prokaryotes are ubiquitous, ecologically significant, and date back at least two billion years ago to the origin of mitochondria. However, most of our knowledge about the evolution and functions of eukaryotic symbioses comes from the study of animal hosts, which represent only a small subset of eukaryotic diversity. Here, we take a broad view of bacterial and archaeal symbioses with protist hosts, focusing on their evolution, ecology, and cell biology, and also explore what functions (if any) the symbionts provide to their hosts. With the immense diversity of protist symbioses starting to come into focus, we can now begin to see how these systems will impact symbiosis theory more broadly.

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.

Highly Reduced Genomes of Protist Endosymbionts Show Evolutionary Convergence.

Genome evolution in bacterial endosymbionts is notoriously extreme: the combined effects of strong genetic drift and unique selective pressures result in highly reduced genomes with distinctive adaptations to hosts [1-4]. These processes are mostly known from animal endosymbionts, where nutritional endosymbioses represent the best-studied systems. However, eukaryotic microbes, or protists, also harbor diverse bacterial endosymbionts, but their genome reduction and functional relationships with their hosts are largely unexplored [5-7]. We sequenced the genomes of four bacterial endosymbionts from three species of diplonemids, poorly studied but abundant and diverse heterotrophic protists [8-12]. The endosymbionts come from two bacterial families, Rickettsiaceae and Holosporaceae, that have invaded two families of diplonemids, and their genomes have converged on an extremely small size (605-632 kilobase pairs [kbp]), similar gene content (e.g., metabolite transporters and secretion systems), and reduced metabolic potential (e.g., loss of energy metabolism). These characteristics are generally found in both families, but the diplonemid endosymbionts have evolved greater extremes in parallel. They possess modified type VI secretion systems that could function in manipulating host metabolism or other intracellular interactions. Finally, modified cellular machinery like the ATP synthase without oxidative phosphorylation, and the reduced flagellar apparatus present in some diplonemid endosymbionts and nutritional animal endosymbionts, indicates that intracellular mechanisms have converged in bacterial endosymbionts with various functions and from different eukaryotic hosts across the tree of life.

Morphological, Ultrastructural, Motility and Evolutionary Characterization of Two New Hemistasiidae Species.

Until now, Hemistasia phaeocysticola was the only representative of the monogeneric family Hemistasiidae available in culture. Here we describe two new axenized hemistasiids isolated from Tokyo Bay, Japan. Like in other diplonemids, cellular organization of these heterotrophic protists is characterized by a distinct apical papilla, a tubular cytopharynx contiguous with a deep flagellar pocket, and a highly branched mitochondrion with lamellar cristae. Both hemistasiids also bear a prominent digestive vacuole, peripheral lacunae, and paraflagellar rods, are highly motile and exhibit diverse morphologies in culture. We argue that significant differences in molecular phylogenetics and ultrastructure between these new species and H. phaeocysticola are on the generic level. Therefore, we have established two new genera within Hemistasiidae - Artemidia gen. n. and Namystynia gen. n. to accommodate Artemidia motanka, sp. n. and Namystynia karyoxenos, sp. n., respectively. A. motanka permanently carries tubular extrusomes, while in N. karyoxenos, they are present only in starving cells. An additional remarkable feature of the latter species is the presence, in both the cytoplasm and the nucleus, of the endosymbiotic rickettsiid Candidatus Sneabacter namystus.

Life Cycle, Ultrastructure, and Phylogeny of New Diplonemids and Their Endosymbiotic Bacteria.

Diplonemids represent a hyperdiverse and abundant yet poorly studied group of marine protists. Here we describe two new members of the genus Diplonema (Diplonemea, Euglenozoa), Diplonema japonicum sp. nov. and Diplonema aggregatum sp. nov., based on life cycle, morphology, and 18S rRNA gene sequences. Along with euglenozoan apomorphies, they contain several unique features. Their life cycle is complex, consisting of a trophic stage that is, following the depletion of nutrients, transformed into a sessile stage and subsequently into a swimming stage. The latter two stages are characterized by the presence of tubular extrusomes and the emergence of a paraflagellar rod, the supportive structure of the flagellum, which is prominently lacking in the trophic stage. These two stages also differ dramatically in motility and flagellar size. Both diplonemid species host endosymbiotic bacteria that are closely related to each other and constitute a novel branch within Holosporales, for which a new genus, "Candidatus Cytomitobacter" gen. nov., has been established. Remarkably, the number of endosymbionts in the cytoplasm varies significantly, as does their localization within the cell, where they seem to penetrate the mitochondrion, a rare occurrence.IMPORTANCE We describe the morphology, behavior, and life cycle of two new Diplonema species that established a relationship with two Holospora-like bacteria in the first report of an endosymbiosis in diplonemids. Both endosymbionts reside in the cytoplasm and the mitochondrion, which establishes an extremely rare case. Within their life cycle, the diplonemids undergo transformation from a trophic to a sessile and eventually a highly motile swimming stage. These stages differ in several features, such as the presence or absence of tubular extrusomes and a paraflagellar rod, along with the length of the flagella. These morphological and behavioral interstage differences possibly reflect distinct functions in dispersion and invasion of the host and/or prey and may provide novel insight into the virtually unknown function of diplonemids in the oceanic ecosystem.

Phylogeny and Morphology of New Diplonemids from Japan.

Diplonemids were recently found to be the most species-rich group of marine planktonic protists. Based on phylogenetic analysis of 18S rRNA gene sequences and morphological observations, we report the description of new members of the genus Rhynchopus - R. humris sp. n. and R. serpens sp. n., and the establishment of two new genera - Lacrimia gen. n. and Sulcionema gen. n., represented by L. lanifica sp. n. and S. specki sp. n., respectively. In addition, we describe the organism formerly designated as Diplonema sp. 2 (ATCC 50224) as Flectonema neradi gen. n., sp. n. The newly described diplonemids share a common set of traits. Cells are sac-like but variable in shape and size, highly metabolic, and surrounded by a naked cell membrane, which is supported by a tightly packed corset of microtubules. They carry a single highly reticulated peripheral mitochondrion containing a large amount of mitochondrial DNA, with lamellar cristae. The cytopharyngeal complex and flagellar pocket are contiguous and have separate openings. Two parallel flagella are inserted sub-apically into a pronounced flagellar pocket. Rhynchopus species have their flagella concealed in trophic stages and fully developed in swimming stages, while they permanently protrude in all other known diplonemid species.

Genome of Ca. Pandoraea novymonadis, an Endosymbiotic Bacterium of the Trypanosomatid Novymonas esmeraldas.

We have sequenced, annotated, and analyzed the genome of Ca. Pandoraea novymonadis, a recently described bacterial endosymbiont of the trypanosomatid Novymonas esmeraldas. When compared with genomes of its free-living relatives, it has all the hallmarks of the endosymbionts' genomes, such as significantly reduced size, extensive gene loss, low GC content, numerous gene rearrangements, and low codon usage bias. In addition, Ca. P. novymonadis lacks mobile elements, has a strikingly low number of pseudogenes, and almost all genes are single copied. This suggests that it already passed the intensive period of host adaptation, which still can be observed in the genome of Polynucleobacter necessarius, a certainly recent endosymbiont. Phylogenetically, Ca. P. novymonadis is more related to P. necessarius, an intracytoplasmic bacterium of free-living ciliates, than to Ca. Kinetoplastibacterium spp., the only other known endosymbionts of trypanosomatid flagellates. As judged by the extent of the overall genome reduction and the loss of particular metabolic abilities correlating with the increasing dependence of the symbiont on its host, Ca. P. novymonadis occupies an intermediate position P. necessarius and Ca. Kinetoplastibacterium spp. We conclude that the relationships between Ca. P. novymonadis and N. esmeraldas are well-established, although not as fine-tuned as in the case of Strigomonadinae and their endosymbionts.

Effect of nitrogen starvation on desiccation tolerance of Arctic Microcoleus strains (cyanobacteria).

Although desiccation tolerance of Microcoleus species is a well-known phenomenon, there is very little information about their limits of desiccation tolerance in terms of cellular water content, the survival rate of their cells, and the environmental factors inducing their resistance to drying. We have discovered that three Microcoleus strains, isolated from terrestrial habitats of the High Arctic, survived extensive dehydration (to 0.23 g water g(-1) dry mass), but did not tolerate complete desiccation (to 0.03 g water g(-1) dry mass) regardless of pre-desiccation treatments. However, these treatments were critical for the survival of incomplete desiccation: cultures grown under optimal conditions failed to survive even incomplete desiccation; a low temperature enabled only 0-15% of cells to survive, while 39.8-65.9% of cells remained alive and intact after nitrogen starvation. Unlike Nostoc, which co-exists with Microcoleus in Arctic terrestrial habitats, Microcoleus strains are not truly anhydrobiotic and do not possess constitutive desiccation tolerance. Instead, it seems that the survival strategy of Microcoleus in periodically dry habitats involves avoidance of complete desiccation, but tolerance to milder desiccation stress, which is induced by suboptimal conditions (e.g., nitrogen starvation).

A novel staining protocol for multiparameter assessment of cell heterogeneity in Phormidium populations (cyanobacteria) employing fluorescent dyes.

Bacterial populations display high heterogeneity in viability and physiological activity at the single-cell level, especially under stressful conditions. We demonstrate a novel staining protocol for multiparameter assessment of individual cells in physiologically heterogeneous populations of cyanobacteria. The protocol employs fluorescent probes, i.e., redox dye 5-cyano-2,3-ditolyl tetrazolium chloride, 'dead cell' nucleic acid stain SYTOX Green, and DNA-specific fluorochrome 4',6-diamidino-2-phenylindole, combined with microscopy image analysis. Our method allows simultaneous estimates of cellular respiration activity, membrane and nucleoid integrity, and allows the detection of photosynthetic pigments fluorescence along with morphological observations. The staining protocol has been adjusted for, both, laboratory and natural populations of the genus Phormidium (Oscillatoriales), and tested on 4 field-collected samples and 12 laboratory strains of cyanobacteria. Based on the mentioned cellular functions we suggest classification of cells in cyanobacterial populations into four categories: (i) active and intact; (ii) injured but active; (iii) metabolically inactive but intact; (iv) inactive and injured, or dead.