The protist cultural renaissance.

Protists are key players in the biosphere. Here, we provide a perspective on integrating protist culturing with omics approaches, imaging, and high-throughput single-cell manipulation strategies, concluding with actions required for a successful return of the golden age of protist culturing.

Circadian rhythms and circadian clock gene homologs of complex alga Chromera velia.

Most organisms on Earth are affected by periodic changes in their environment. The circadian clock is an endogenous device that synchronizes behavior, physiology, or biochemical processes to an approximately 24-hour cycle, allowing organisms to anticipate the periodic changes of day and night. Although circadian clocks are widespread in organisms, the actual molecular components differ remarkably among the clocks of plants, animals, fungi, and prokaryotes. Chromera velia is the closest known photosynthetic relative of apicomplexan parasites. Formation of its motile stage, zoospores, has been described as associated with the light part of the day. We examined the effects on the periodic release of the zoospores under different light conditions and investigated the influence of the spectral composition on zoosporogenesis. We performed a genomic search for homologs of known circadian clock genes. Our results demonstrate the presence of an almost 24-hour free-running cycle of zoosporogenesis. We also identified the blue light spectra as the essential compound for zoosporogenesis. Further, we developed a new and effective method for zoospore separation from the culture and estimated the average motility speed and lifespan of the C. velia zoospores. Our genomic search identified six cryptochrome-like genes, two genes possibly related to Arabidopsis thaliana CCA/LHY, whereas no homolog of an animal, cyanobacterial, or fungal circadian clock gene was found. Our results suggest that C. velia has a functional circadian clock, probably based mainly on a yet undefined mechanism.

Short-term acidification promotes diverse iron acquisition and conservation mechanisms in upwelling-associated phytoplankton.

Coastal upwelling regions are among the most productive marine ecosystems but may be threatened by amplified ocean acidification. Increased acidification is hypothesized to reduce iron bioavailability for phytoplankton thereby expanding iron limitation and impacting primary production. Here we show from community to molecular levels that phytoplankton in an upwelling region respond to short-term acidification exposure with iron uptake pathways and strategies that reduce cellular iron demand. A combined physiological and multi-omics approach was applied to trace metal clean incubations that introduced 1200 ppm CO2 for up to four days. Although variable, molecular-level responses indicate a prioritization of iron uptake pathways that are less hindered by acidification and reductions in iron utilization. Growth, nutrient uptake, and community compositions remained largely unaffected suggesting that these mechanisms may confer short-term resistance to acidification; however, we speculate that cellular iron demand is only temporarily satisfied, and longer-term acidification exposure without increased iron inputs may result in increased iron stress.

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.

Intragenomic diversity of the V9 hypervariable domain in eukaryotes has little effect on metabarcoding.

Metabarcoding revolutionized our understanding of diversity and ecology of microorganisms in different habitats. However, it is also associated with several inherent biases, one of which is associated with intragenomic diversity of a molecular barcode. Here, we compare intragenomic variability of the V9 region of the 18S rRNA gene in 19 eukaryotic phyla abundant in marine plankton. The level of intragenomic variability is comparable across all the phyla, and in most genomes and transcriptomes one V9 sequence and one OTU is predominant. However, most of the variability observed at the barcode level is probably caused by sequencing errors and is mitigated by using a denoising tool, DADA2. The SWARM algorithm commonly used in metabarcoding studies is not optimal for collapsing genuine and erroneous sequences into a single OTU, leading to an overestimation of diversity in metabarcoding data. For an unknown reason, SWARM inflates diversity of eupelagonemids more than that of other eukaryotes.

Water masses shape pico-nano eukaryotic communities of the Weddell Sea.

Polar oceans belong to the most productive and rapidly changing environments, yet our understanding of this fragile ecosystem remains limited. Here we present an analysis of a unique set of DNA metabarcoding samples from the western Weddell Sea sampled throughout the whole water column and across five water masses with different characteristics and different origin. We focus on factors affecting the distribution of planktonic pico-nano eukaryotes and observe an ecological succession of eukaryotic communities as the water masses move away from the surface and as oxygen becomes depleted with time. At the beginning of this succession, in the photic zone, algae, bacteriovores, and predators of small eukaryotes dominate the community, while another community develops as the water sinks deeper, mostly composed of parasitoids (syndinians), mesoplankton predators (radiolarians), and diplonemids. The strongly correlated distribution of syndinians and diplonemids along the depth and oxygen gradients suggests their close ecological link and moves us closer to understanding the biological role of the latter group in the ocean ecosystem.

Genomic Confirmation of Borrelia garinii, United States.

Lyme disease is a multisystem disorder primarily caused by Borrelia burgdorferi sensu lato. However, B. garinii, which has been identified on islands off the coast of Newfoundland and Labrador, Canada, is a cause of Lyme disease in Eurasia. We report isolation and whole-genome nucleotide sequencing of a B. garinii isolate from a cotton mouse (Peromyscus gossypinus) in South Carolina, USA. We identified a second B. garinii isolate from the same repository. Phylogenetic analysis does not associate these isolates with the previously described isolates of B. garinii from Canada.

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.

Deep Learning Analysis of Polish Electronic Health Records for Diagnosis Prediction in Patients with Cardiovascular Diseases.

Electronic health records naturally contain most of the medical information in the form of doctor's notes as unstructured or semi-structured texts. Current deep learning text analysis approaches allow researchers to reveal the inner semantics of text information and even identify hidden consequences that can offer extra decision support to doctors. In the presented article, we offer a new automated analysis of Polish summary texts of patient hospitalizations. The presented models were found to be able to predict the final diagnosis with almost 70% accuracy based just on the patient's medical history (only 132 words on average), with possible accuracy increases when adding further sentences from hospitalization results; even one sentence was found to improve the results by 4%, and the best accuracy of 78% was achieved with five extra sentences. In addition to detailed descriptions of the data and methodology, we present an evaluation of the analysis using more than 50,000 Polish cardiology patient texts and dive into a detailed error analysis of the approach. The results indicate that the deep analysis of just the medical history summary can suggest the direction of diagnosis with a high probability that can be further increased just by supplementing the records with further examination results.

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.

Trophic flexibility of marine diplonemids - switching from osmotrophy to bacterivory.

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.

Highly flexible metabolism of the marine euglenozoan protist Diplonema papillatum.

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, ß-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.

Olisthodiscus represents a new class of Ochrophyta.

The phylogenetic diversity of Ochrophyta, a diverse and ecologically important radiation of algae, is still incompletely understood even at the level of the principal lineages. One taxon that has eluded simple classification is the marine flagellate genus Olisthodiscus. We investigated Olisthodiscus luteus K-0444 and documented its morphological and genetic differences from the NIES-15 strain, which we described as Olisthodiscus tomasii sp. nov. Phylogenetic analyses of combined 18S and 28S rRNA sequences confirmed that Olisthodiscus constitutes a separate, deep, ochrophyte lineage, but its position could not be resolved. To overcome this problem, we sequenced the plastid genome of O. luteus K-0444 and used the new data in multigene phylogenetic analyses, which suggested that Olisthodiscus is a sister lineage of the class Pinguiophyceae within a broader clade additionally including Chrysophyceae, Synchromophyceae, and Eustigmatophyceae. Surprisingly, the Olisthodiscus plastid genome contained three genes, ycf80, cysT, and cysW, inherited from the rhodophyte ancestor of the ochrophyte plastid yet lost from all other ochrophyte groups studied so far. Combined with nuclear genes for CysA and Sbp proteins, Olisthodiscus is the only known ochrophyte possessing a plastidial sulfate transporter SulT. In addition, the finding of a cemA gene in the Olisthodiscus plastid genome and an updated phylogenetic analysis ruled out the previously proposed hypothesis invoking horizontal cemA transfer from a green algal plastid into Synurales. Altogether, Olisthodiscus clearly represents a novel phylogenetically distinct ochrophyte lineage, which we have proposed as a new class, Olisthodiscophyceae.

Proximity proteomics in a marine diatom reveals a putative cell surface-to-chloroplast iron trafficking pathway.

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed; however, proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

EukRef-excavates: seven curated SSU ribosomal RNA gene databases.

The small subunit ribosomal RNA (SSU rRNA) gene is a widely used molecular marker to study the diversity of life. Sequencing of SSU rRNA gene amplicons has become a standard approach for the investigation of the ecology and diversity of microbes. However, a well-curated database is necessary for correct classification of these data. While available for many groups of Bacteria and Archaea, such reference databases are absent for most eukaryotes. The primary goal of the EukRef project (eukref.org) is to close this gap and generate well-curated reference databases for major groups of eukaryotes, especially protists. Here we present a set of EukRef-curated databases for the excavate protists-a large assemblage that includes numerous taxa with divergent SSU rRNA gene sequences, which are prone to misclassification. We identified 6121 sequences, 625 of which were obtained from cultures, 3053 from cell isolations or enrichments and 2419 from environmental samples. We have corrected the classification for the majority of these curated sequences. The resulting publicly available databases will provide phylogenetically based standards for the improved identification of excavates in ecological and microbiome studies, as well as resources to classify new discoveries in excavate diversity.

Common origin of ornithine-urea cycle in opisthokonts and stramenopiles.

Eukaryotic complex phototrophs exhibit a colorful evolutionary history. At least three independent endosymbiotic events accompanied by the gene transfer from the endosymbiont to host assembled a complex genomic mosaic. Resulting patchwork may give rise to unique metabolic capabilities; on the other hand, it can also blur the reconstruction of phylogenetic relationships. The ornithine-urea cycle (OUC) belongs to the cornerstone of the metabolism of metazoans and, as found recently, also photosynthetic stramenopiles. We have analyzed the distribution and phylogenetic positions of genes encoding enzymes of the urea synthesis pathway in eukaryotes. We show here that metazoan and stramenopile OUC enzymes share common origins and that enzymes of the OUC found in primary algae (including plants) display different origins. The impact of this fact on the evolution of stramenopiles is discussed here.

Environmental determinants of the distribution of planktonic diplonemids and kinetoplastids in the oceans.

We analysed a widely used barcode, the V9 region of the 18S rRNA gene, to study the effect of environmental conditions on the distribution of two related heterotrophic protistan lineages in marine plankton, kinetoplastids and diplonemids. We relied on a major published dataset (Tara Oceans) where samples from the mesopelagic zone were available from just 32 of 123 locations, and both groups are most abundant in this zone. To close sampling gaps and obtain more information from the deeper ocean, we collected 57 new samples targeting especially the mesopelagic zone. We sampled in three geographic regions: the Arctic, two depth transects in the Adriatic Sea, and the anoxic Cariaco Basin. In agreement with previous studies, both protist groups are most abundant and diverse in the mesopelagic zone. In addition to that, we found that their abundance, richness, and community structure also depend on geography, oxygen concentration, salinity, temperature, and other environmental variables reflecting the abundance of algae and nutrients. Both groups studied here demonstrated similar patterns, although some differences were also observed. Kinetoplastids and diplonemids prefer tropical regions and nutrient-rich conditions and avoid high oxygen concentration, high salinity, and high density of algae.

Evolution of metabolic capabilities and molecular features of diplonemids, kinetoplastids, and euglenids.

BACKGROUND: The Euglenozoa are a protist group with an especially rich history of evolutionary diversity. They include diplonemids, representing arguably the most species-rich clade of marine planktonic eukaryotes; trypanosomatids, which are notorious parasites of medical and veterinary importance; and free-living euglenids. These different lifestyles, and particularly the transition from free-living to parasitic, likely require different metabolic capabilities. We carried out a comparative genomic analysis across euglenozoan diversity to see how changing repertoires of enzymes and structural features correspond to major changes in lifestyles. RESULTS: We find a gradual loss of genes encoding enzymes in the evolution of kinetoplastids, rather than a sudden decrease in metabolic capabilities corresponding to the origin of parasitism, while diplonemids and euglenids maintain more metabolic versatility. Distinctive characteristics of molecular machines such as kinetochores and the pre-replication complex that were previously considered specific to parasitic kinetoplastids were also identified in their free-living relatives. Therefore, we argue that they represent an ancestral rather than a derived state, as thought until the present. We also found evidence of ancient redundancy in systems such as NADPH-dependent thiol-redox. Only the genus Euglena possesses the combination of trypanothione-, glutathione-, and thioredoxin-based systems supposedly present in the euglenozoan common ancestor, while other representatives of the phylum have lost one or two of these systems. Lastly, we identified convergent losses of specific metabolic capabilities between free-living kinetoplastids and ciliates. Although this observation requires further examination, it suggests that certain eukaryotic lineages are predisposed to such convergent losses of key enzymes or whole pathways. CONCLUSIONS: The loss of metabolic capabilities might not be associated with the switch to parasitic lifestyle in kinetoplastids, and the presence of a highly divergent (or unconventional) kinetochore machinery might not be restricted to this protist group. The data derived from the transcriptomes of free-living early branching prokinetoplastids suggests that the pre-replication complex of Trypanosomatidae is a highly divergent version of the conventional machinery. Our findings shed light on trends in the evolution of metabolism in protists in general and open multiple avenues for future research.

Complex Evolution of Insect Insulin Receptors and Homologous Decoy Receptors, and Functional Significance of Their Multiplicity.

Evidence accumulates that the functional plasticity of insulin and insulin-like growth factor signaling in insects could spring, among others, from the multiplicity of insulin receptors (InRs). Their multiple variants may be implemented in the control of insect polyphenism, such as wing or caste polyphenism. Here, we present a comprehensive phylogenetic analysis of insect InR sequences in 118 species from 23 orders and investigate the role of three InRs identified in the linden bug, Pyrrhocoris apterus, in wing polymorphism control. We identified two gene clusters (Clusters I and II) resulting from an ancestral duplication in a late ancestor of winged insects, which remained conserved in most lineages, only in some of them being subject to further duplications or losses. One remarkable yet neglected feature of InR evolution is the loss of the tyrosine kinase catalytic domain, giving rise to decoys of InR in both clusters. Within the Cluster I, we confirmed the presence of the secreted decoy of insulin receptor in all studied Muscomorpha. More importantly, we described a new tyrosine kinase-less gene (DR2) in the Cluster II, conserved in apical Holometabola for ∼300 My. We differentially silenced the three P. apterus InRs and confirmed their participation in wing polymorphism control. We observed a pattern of Cluster I and Cluster II InRs impact on wing development, which differed from that postulated in planthoppers, suggesting an independent establishment of insulin/insulin-like growth factor signaling control over wing development, leading to idiosyncrasies in the co-option of multiple InRs in polyphenism control in different taxa.

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.