Protist Cells Research Articles

The vampyrellid amoeba Strigomyxa ruptor gen. et sp. nov. and its remarkable strategy to acquire algal cell contents.

The vampire amoebae (Vampyrellida, Rhizaria) inhabit freshwater, marine, and terrestrial ecosystems and consume a wide range of eukaryotic prey. This includes diverse microalgae, fungi, and microscopic animals. One of the most captivating aspects of the vampyrellids is their ability to extract the cell contents of other eukaryotes after local dissolution of the prey cell wall, a feeding strategy that occurs in several vampyrellid families, but is best studied in Vampyrella species that attack zygnematophyte green algae. Here, we report two new vampyrellid strains from temperate moorlands in Germany with a yet-undescribed feeding strategy: internal protoplast extraction and cell wall regurgitation. This feeding strategy involves the phagocytosis of whole desmid cells (genus Closterium, Zygnematophyceae), internal cleavage of the algal cell wall, extraction of the cell contents, and subsequent exocytosis of bundled empty cell walls. The large primary food vacuole formed during the process has exceptional functions, as it forms internal feeding pseudopodia, packages algal cell contents into smaller secondary vacuoles, and transforms into a "waste vacuole" with cell wall remnants. The new feeding strategy, which - in the widest sense - is reminiscent of the pellet casting of owls, reveals a stunningly sophisticated behavior of single protist cells. Based on morphological, phylogenetic, and autecological data, both vampyrellid strains are nearly identical and here assigned to a new and quite unique vampyrellid taxon, Strigomyxa ruptor gen. et sp. nov. (Leptophryidae, Vampyrellida).

Responses of diatom assemblages and life cycle to sea ice variation in the eastern Indian sector of the Southern Ocean during austral summer 2018/2019

In the Southern Ocean, rapid environmental changes (warming, freshening, and poleward shifts of the physiological fronts) are underway. These changes affect the distribution, abundance, and life cycle of protists. However, the spatial distribution of protists is not well understood in the eastern Indian sector of the Southern Ocean. We, therefore, evaluated the effects of environmental factors at the community level (abundance and species composition) and species-specific level (life cycle in diatoms) based on field sampling during the austral summer of 2018/2019. High chlorophyll levels and protist cell densities were observed in the eastern area from 120°E, in contrast to the distribution reported in the 1996 BROKE (The Baseline Research on Oceanography, Krill and the Environment) survey. This inter-annual variation was caused by differences in the sampling periods, nutrients, and zooplankton grazing pressures. North–south variation in protist abundance was well explained by silicate distribution, whereas sea ice variation did not influence it explicitly. The clustering of protist groups was well associated with nutrients, but locally, offshore groups were extended southward by currents and eddies. The cell size of the two dominant Fragilariopsis curta and F. kerguelensis exhibited similar significant relationships with sea ice variation and decreased 60 days after sea ice melted. Our findings demonstrate that sea ice changes do not clearly affect the protist community level but potentially affect the life cycle level of diatoms.

First Molecular Characterization of the Elusive Marine Protist Meteora sporadica

Meteora sporadica is a protist species first described by Hausmann et al. (2002) in deep-sea sediments from the Sporades Basin, Eastern Mediterranean Sea. Due to its unusual morphology and locomotion, very different from those of other high-rank eukaryotic taxa, it was classified as an incertae sedis species. Unfortunately, its morphological characterization was not accompanied by the generation of molecular data, preventing its placement in molecular phylogenetic trees including other protist lineages. Here, we report the observation of protist cells in sediments from a shallow marine lagoon in the Mediterranean Sea with morphological characteristics indistinguishable from those of Meteora sporadica. Given this similarity and the geographical proximity to the type location, we consider that the organism that we observed likely corresponded to the type species, M. sporadica, which seems to be a benthic predator spanning from shallow to deep-sea habitats. We determined the 18S rRNA gene sequence of M. sporadica from micromanipulated cells. Searches in sequence databases did not yield closely related hits, suggesting that Meteora is a rare organism. Phylogenetic analyses did not show any close affinity with other eukaryotic groups, supporting its initial incertae sedis status and suggesting that it may define a new high-rank level eukaryotic lineage.

Spatial distribution of the protist community in the southern part of the Okhotsk Sea off Hokkaido during summer

The southern part of the Okhotsk Sea off Hokkaido has rich fishery resources supported by high primary production. The phytoplankton community has been described by several studies based on size-fractionated chlorophyll a concentration and primary production; however, the details of the species composition of nano- and micro-protist communities have not been well investigated. We evaluated the spatial distribution and relationship with hydrography of nano- and micro-protist communities in the southern region of the Okhotsk Sea off Hokkaido during the summer of 2019. Forty-two species of diatoms from 19 genera, dinoflagellates from 7 genera, tintinnid ciliates, oligotrich ciliates, and 1 genus of silicoflagellates were identified. Their spatial distribution showed distinct differences among the taxa. Cluster analysis based on the protist cell density identified six groups with geographically well-distinguished distributions. Diatom-dominant communities were distributed in the Cold Water Belt and Soya warm current along the coastal area of Hokkaido. Nanoflagellate-dominated communities were distributed in the offshore area. Some harmful species of diatoms (Pseudonitzschia pseudodelicatissima) and dinoflagellates (Alexandrium spp.) were dominant in the Cold Water Belt off Wakkanai. We suggest that the upwelling of cold water could increase the abundance of harmful species, with immanent negative impacts on marine ecosystems or fishery resources. In summary, the species composition and abundance of the summer protist community was strongly related to different water masses in the southern part of the Okhotsk Sea off Hokkaido.

The Bacterial Diversity Lurking in Protist Cell Cultures

Laboratory cultures of heterotrophic protists are often xenic, meaning that the culture contains more than one microbial organism. In this study, we analyzed genome-assembly data from cultures of four marine protist flagellates—the marine malawimonad Imasa heleensis, the undescribed mantamonad strain SRT-306, the discobid Ophirina amphinema, and the cryptist Palpitomonas bilix—specifically to search for genomes of cocultured bacteria. As no external bacteria have been added to the protist stock cultures, it is probable that the cocultured bacteria came from the original water samples from which the protists were isolated. At least some of these bacteria are consumed as a food source by the protists, all of which are obligate heterotrophs. From four separate metagenomic de novo assemblies for these mixed cultures, we identified 28 scaffolds, which BUSCO analyses suggest represent complete or near-complete bacterial genomes. These scaffolds range in length from 3,139,436 to 6,090,282 bp and encode 2873 to 5666 genes. Only eight of the 28 scaffolds corresponded to entries in the NCBI genome database, meaning that 20 of these scaffolds represent genomes from putatively novel bacterial species. Our findings highlight that data like these, which are often discarded or overlooked, can be a source of novel genomes and/or species.

Composition and temporal dynamics of sand-dwelling dinoflagellate communities from three Mediterranean beaches

Compared to plankton, benthic communities are thought to be heterogeneous among sites and more stable through time; however, benthic protists in general, and sand-dwelling dinoflagellates in particular, have been scarcely studied, and their diversity remains mostly unknown. To test those claims, we studied the diversity, structure and temporal dynamics of benthic dinoflagellate communities in sandy sediments obtained from 3 NW Mediterranean Sea coastal locations during temperate spring and warm summer months. After separating protist cells from the substrate using the seawater-ice method, high-throughput amplicon sequencing of the V4 18S rRNA gene region followed by the analysis of amplicon sequence variants (ASVs) showed similar richness, alpha-diversity, and community composition among sand-dwelling dinoflagellates at the 3 locations. Dinoflagellates affiliating with the Thoracosphaeraceae or Gymnodiniales sensu stricto were highly represented. In contrast, other well-known dinoflagellate taxa, such as Gonyaulacales or Dinophysales, were poorly represented or absent. Dinoflagellate communities showed significant differences between the 2 studied seasons, mainly driven by water temperature. Samples from temperate months had higher diversity and were more dissimilar, while samples from warmer months showed lower diversity and higher similarity. Some species were always present in the community, others only appeared sporadically, and some others showed clear differences between seasons. Our results suggest that large-scale changes (pointing to seasonality) prevail over spatial heterogeneity in shaping community composition. The 3 studied locations had a similar dinoflagellate community composed of sand-dwelling taxa, with many representatives of undescribed species, highlighting the need to further study the diversity of the benthic compartment.

Spirotrichonymphea (Parabasalia) symbionts of the termite Paraneotermes simplicicornis

The desert dampwood termite Paraneotermes simplicicornis harbors several species of obligately symbiotic protists that support its nutrition by fermenting lignocellulose. Among them are three morphotypes with the dexiotropic spiraling flagellar bands characteristic of Spirotrichonymphea (Parabasalia). The largest morphotype, characterized by an elongated cell apex with axial columella and internally positioned spiraling flagellar bands, was previously described as Spirotrichonympha polygyra. A smaller morphotype, with similarly internalized flagellar bands but a more rounded posterior without a protruding axostyle, was previously reported but not named. The smallest morphotype has surface flagellar bands and can attach to other protist cells by its apex. In this study, we combine light microscopy of live specimens and 18S rRNA gene sequencing of individually isolated cells to better understand the diversity of symbionts in P. simplicicornis. We found that S. polygyra branches distantly from true Spirotrichonympha, which are associated with Reticulitermes termites. Thus, we propose the new genus Cuppa to accommodate C. polygyra n. comb. (type species) and the similar but smaller morphotype Cuppa taenia n. sp. The undescribed smallest morphotype can be excluded from all previously described Spirotrichonymphea genera by molecular and behavioral evidence, so we propose Fraterculus simplicicornis n. gen., n. sp., to accommodate this organism.

An efficient single-cell transcriptomics workflow for microbial eukaryotes benchmarked on Giardia intestinalis cells

BackgroundMost diversity in the eukaryotic tree of life is represented by microbial eukaryotes, which is a polyphyletic group also referred to as protists. Among the protists, currently sequenced genomes and transcriptomes give a biased view of the actual diversity. This biased view is partly caused by the scientific community, which has prioritized certain microbes of biomedical and agricultural importance. Additionally, some protists remain difficult to maintain in cultures, which further influences what has been studied. It is now possible to bypass the time-consuming process of cultivation and directly analyze the gene content of single protist cells. Single-cell genomics was used in the first experiments where individual protists cells were genomically explored. Unfortunately, single-cell genomics for protists is often associated with low genome recovery and the assembly process can be complicated because of repetitive intergenic regions. Sequencing repetitive sequences can be avoided if single-cell transcriptomics is used, which only targets the part of the genome that is transcribed.ResultsIn this study we test different modifications of Smart-seq2, a single-cell RNA sequencing protocol originally developed for mammalian cells, to establish a robust and more cost-efficient workflow for protists. The diplomonad Giardia intestinalis was used in all experiments and the available genome for this species allowed us to benchmark our results. We could observe increased transcript recovery when freeze-thaw cycles were added as an extra step to the Smart-seq2 protocol. Further we reduced the reaction volume and purified the amplified cDNA with alternative beads to test different cost-reducing changes of Smart-seq2. Neither improved the procedure, and reducing the volumes by half led to significantly fewer genes detected. We also added a 5′ biotin modification to our primers and reduced the concentration of oligo-dT, to potentially reduce generation of artifacts. Except adding freeze-thaw cycles and reducing the volume, no other modifications lead to a significant change in gene detection. Therefore, we suggest adding freeze-thaw cycles to Smart-seq2 when working with protists and further consider our other modification described to improve cost and time-efficiency.ConclusionsThe presented single-cell RNA sequencing workflow represents an efficient method to explore the diversity and cell biology of individual protist cells.

Single-cell amplicon sequencing reveals community structures and transmission trends of protist-associatedbacteria in atermite host.

The hindgut protists of wood-feeding termites are usually colonized by prokaryotic symbionts. Many of the hurdles that have prevented a better understanding of these symbionts arise from variation among protist and termite host species and the inability to maintain prominent community members in culture. These issues have made it difficult to study the fidelity, acquisition, and differences in colonization of protists by bacterial symbionts. In this study, we use high throughput amplicon sequencing of the V4 region of 16S rRNA genes to determine the composition of bacterial communities associated with single protist cells of six protist species, from the genera Pyrsonympha, Dinenympha, and Trichonympha that are present in the hindgut of the termite Reticulitermes flavipes. By analyzing amplicon sequence variants (ASVs), the diversity and distribution of protist-associated bacteria was compared within and across these six different protist species. ASV analysis showed that, in general, each protist genus associated with a distinct community of bacterial symbionts which were conserved across different termite colonies. However, some ASVs corresponding to ectosymbionts (Spirochaetes) were shared between different Dinenympha species and to a lesser extent with Pyrsonympha and Trichonympha hosts. This suggested that certain bacterial symbionts may be cosmopolitan to some degree and perhaps acquired by horizontal transmission. Using a fluorescence-based cell assay, we could observe the horizontal acquisition of surface-bound bacteria. This acquisition was shown to be time-dependent, involve active processes, and was non-random with respect to binding locations on some protists.

Ballast water management system certification testing requirements for protists are a poor fit for the Great Lakes

Challenge condition requirements for testing of ballast water management systems (BWMS) are a poor fit with regard to protection of the Laurentian Great Lakes from aquatic invasive species, particularly with respect to protists. Though protists are abundant in the Great Lakes, required densities of cells (1000 cells/mL) meeting the 10–50 µm (“protist”) regulatory size class of the ballast water discharge standard (BWDS) are rarely achievable under ambient conditions. This deficiency drives certification testing to aquatic systems dissimilar to the Great Lakes or necessitates manipulation of intake water during testing. This requirement is unnecessary because: (1) protist cells both within and smaller than the regulatory size class are largely equivalent in their challenge to BWMS performance and their threat to ecosystems; and (2) lower densities of cells in challenge water can meet regulatory requirements; i.e. at least 100 live cells/mL in untreated discharge (control) water are required for test validity. We describe how current requirements for high densities of protists within the regulatory size class as a challenge condition in certification testing unnecessarily undermine vetting of BWMS performance and operation. We posit a range of alternatives and identify approaches to modifying challenge requirements to alleviate problems while protecting test rigor and relevancy to the BWDS. Without a change to these requirements there will be no certification testing in freshwater resources like the Great Lakes without substantial intake stream manipulation during testing, and therefore, little way to confirm whether a BWMS will perform in the Great Lakes and other freshwater systems.

Distribution and relative abundance of three protist genera within the Zootermopsis nevadensis nuttingi hindgut

The termite Zootermopsis nevadensis nuttingi, which inhabits coastal forests of the western United States, plays an important ecological role in the breakdown and digestion of wood. Vital to this role are symbiotic protists residing in the termite’s hindgut. Five protist genera of varying size and morphology make up this gut community, but very little is known about their spatial organization within the hindgut or the number of protist cells per host. To resolve this issue, we used light microscopy and a hemocytometer to determine the distribution of the larger protist genera across hindgut segments. We found that Streblomastix were the most abundant cells in all three segments, but especially in the anterior hindgut. Trichomitopsis cells were significantly more abundant in the posterior hindgut. Trichonympha were the least abundant overall, often reaching their highest abundance in the middle segment but with a more variable distribution. Understanding the distribution of different protists within the hindgut may improve our understanding of the ecological relationships among protists, as well as their individual roles in lignocellulose digestion, contributing to a better understanding of the symbiotic system as a whole.

Use of Cell Penetrating Peptides to Deliver Cargo in Protists

Algae and plankton are important both ecologically as a food chain base and economically as nutraceutical “factories”. Despite their importance, technologies to transfect genes or proteins are limited or nonexistent in many protist species of interest, probably because cell walls present a barrier to macromolecular passage. We are using a novel cell penetrating peptide (CPP) system as a means to overcome this barrier. CPPs are a class of short peptides that can cross the cell membrane and sometimes the cell wall in a variety of species. Membrane translocation by CPPs involves endocytosis as well as other mechanisms. CPPs can be linked in a variety of ways to desired cargos to cause their entry into cells. Microscopic imaging of protist cells treated with CPP linked to fluorescently labeled cargo demonstrates that CPPs can cause the association of cargo with cells while fluorescently labeled cargo‐only treatment does not cause extensive cargo association with cells. Using fluorescence microscopy, we are currently optimizing the type of CPP and the CPP‐cargo uptake procedure to find the best conditions for cargo delivery to protists. We are also attempting proteolytic treatment to eliminate cargo stuck to the cell exterior and allow flow cytometric measurements of true cargo entry. Success of our method will provide a new means for rapid introduction of macromolecules into protist cells, allowing both investigation of their cellular function and potential manipulation of economically important products.Support or Funding InformationMentor Protégé Research Program, College of Science and Mathematics, Kennesaw State University; Research Opportunity Award, Office of Research, Kennesaw State UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transduction and transfection of difficult-to-transfect cells: Systematic attempts for the transfection of protozoa Leishmania.

Cell-penetrating peptides (CPPs) are used to internalize different cargoes, including DNA, into live mammalian and plant cells. Despite many cells being easily transfected with this approach, other cells are rather "difficult" or "hard to transfect," including protist cells of the genus Leishmania. Based on our previous results in successfully internalizing proteins into Leishmania tarentolae cells, we used single CPPs and three different DNA-binding proteins to form protein-like complexes with plasmids covered with CPPs. We attempted magnetofection, electroporation, and transfection using a number of commercially available detergents. While complex formation with negatively charged DNA required substantially higher amounts of CPPs than those necessary for mostly neutral proteins, the cytotoxicity of the required amounts of CPPs and auxiliaries was thoroughly studied. We found that Leishmania cells were indeed susceptible to high concentrations of some CPPs and auxiliaries, although in a different manner compared with that for mammalian cells. The lack of successful transfections implies the necessity to accept certain general limitations regarding DNA internalization into difficult-to-transfect cells. Only electroporation allowed reproducible internalization of large and rigid plasmid DNA molecules through electrically disturbed extended membrane areas, known as permeable membrane macrodomains.

Discovery of ectosymbiotic Endomicrobium lineages associated with protists in the gut of stolotermitid termites.

The genus Endomicrobium is a dominant bacterial group in the gut of lower termites, and most phylotypes are intracellular symbionts of gut protists. Here we report the discovery of Endomicrobium ectosymbionts of termite gut protists. We found that bristle-like Endomicrobium cells attached to the surface of spirotrichosomid protist cells inhabiting the termite Stolotermes victoriensis. Transmission electron microscopy revealed that a putative Endomicrobium cell likely attached to the protist surface via a protrusion from the tip of the bacterium. A phylotype, sharing 98.9% 16S rRNA sequence identity with the Endomicrobium ectosymbionts of the spirotrichosomid protists, was also found on the cell surface of the protist Trichonympha magna in the gut of the termite Porotermes adamsoni. We propose the novel species 'Candidatus Endomicrobium superficiale' for these bacteria. T. magna simultaneously harboured another Endomicrobium ectosymbiont that shared 93.5-94.2% 16S rRNA sequence identities with 'Ca. Endomicrobium superficiale'. Furthermore, Spirotrichonympha-like protists in P. adamsoni guts were associated with an Endomicrobium phylotype that possibly attached to the host flagella. A phylogenetic analysis suggested that these ectosymbiotic lineages have evolved multiple times from free-living Endomicrobium lineages and are relatively distant from the endosymbionts. Our results provide novel insights into the ecology and evolution of the Endomicrobium.

Genome of \u2018Ca. Desulfovibrio trichonymphae\u2019, an H2-oxidizing bacterium in a tripartite symbiotic system within a protist cell in the termite gut

The cellulolytic protist Trichonympha agilis in the termite gut permanently hosts two symbiotic bacteria, ‘Candidatus Endomicrobium trichonymphae’ and ‘Candidatus Desulfovibrio trichonymphae’. The former is an intracellular symbiont, and the latter is almost intracellular but still connected to the outside via a small pore. The complete genome of ‘Ca. Endomicrobium trichonymphae’ has previously been reported, and we here present the complete genome of ‘Ca. Desulfovibrio trichonymphae’. The genome is small (1 410 056 bp), has many pseudogenes, and retains biosynthetic pathways for various amino acids and cofactors, which are partially complementary to those of ‘Ca. Endomicrobium trichonymphae’. An amino acid permease gene has apparently been transferred between the ancestors of these two symbionts; a lateral gene transfer has affected their metabolic capacity. Notably, ‘Ca. Desulfovibrio trichonymphae’ retains the complex system to oxidize hydrogen by sulfate and/or fumarate, while genes for utilizing other substrates common in desulfovibrios are pseudogenized or missing. Thus, ‘Ca. Desulfovibrio trichonymphae’ is specialized to consume hydrogen that may otherwise inhibit fermentation processes in both T. agilis and ‘Ca. Endomicrobium trichonymphae’. The small pore may be necessary to take up sulfate. This study depicts a genome-based model of a multipartite symbiotic system within a cellulolytic protist cell in the termite gut.

NMR Structural Studies of the C-Domain of Tcb2, A Calcium Binding Protein from Tetrahymena Thermophila

Many protist cells use an ATP-independent form of contractile motility, which results from the calcium-triggered contraction of filaments containing novel structural and/or calcium-binding proteins. We have previously identified several such proteins during proteomic analysis of contractile fibers isolated from the membrane-associated cytoskeleton of the ciliated protozoan Tetrahymena thermophila, including the putative calcium-binding protein Tcb2 and the filament-forming structural protein Epc1. To gain insight into the structure-function relationship of Tcb2, we have engineered genes for optimized bacterial expression of the full-length protein, as well as its isolated N- and C-terminal domains. Interestingly, a calcium-modulated contractile fabric can be reconstituted with purified full-length Tcb2. The full-length protein and its N-terminal domain are not amenable to high-resolution structure determination, as they tend to aggregate in the presence of calcium and/or upon concentration. However, the C-terminal domain (Tcb2-C) is highly soluble at both low and high calcium concentrations. Solution NMR HSQC spectra of 15N-labeled Tcb2-C indicate that the protein is well folded in the presence and absence of calcium and undergoes a dramatic conformational change upon calcium addition. We expressed and purified 15N, 13C-labeled Tcb2-C, and obtained nearly complete chemical shift assignments for the protein in both the presence and absence of calcium. The solution structure of calcium-free Tcb2-C was determined using NMR-derived experimental distance and torsion angle restraints. The current structural models reveal an architecture exhibited by other calcium-binding proteins, with paired EF-hand motifs connected by a flexible loop. NMR structure determination of calcium-bound Tcb2-C is currently underway. We are also using NMR spectroscopy to quantify the calcium-binding properties of the domain and investigate its conformational dynamics. These studies will establish a structural basis for elucidating the function and unique contractile properties of Tcb2.

Cryptic infection of a broad taxonomic and geographic diversity of tadpoles by Perkinsea protists

The decline of amphibian populations, particularly frogs, is often cited as an example in support of the claim that Earth is undergoing its sixth mass extinction event. Amphibians seem to be particularly sensitive to emerging diseases (e.g., fungal and viral pathogens), yet the diversity and geographic distribution of infectious agents are only starting to be investigated. Recent work has linked a previously undescribed protist with mass-mortality events in the United States, in which infected frog tadpoles have an abnormally enlarged yellowish liver filled with protist cells of a presumed parasite. Phylogenetic analyses revealed that this infectious agent was affiliated with the Perkinsea: a parasitic group within the alveolates exemplified by Perkinsus sp., a "marine" protist responsible for mass-mortality events in commercial shellfish populations. Using small subunit (SSU) ribosomal DNA (rDNA) sequencing, we developed a targeted PCR protocol for preferentially sampling a clade of the Perkinsea. We tested this protocol on freshwater environmental DNA, revealing a wide diversity of Perkinsea lineages in these environments. Then, we used the same protocol to test for Perkinsea-like lineages in livers of 182 tadpoles from multiple families of frogs. We identified a distinct Perkinsea clade, encompassing a low level of SSU rDNA variation different from the lineage previously associated with tadpole mass-mortality events. Members of this clade were present in 38 tadpoles sampled from 14 distinct genera/phylogroups, from five countries across three continents. These data provide, to our knowledge, the first evidence that Perkinsea-like protists infect tadpoles across a wide taxonomic range of frogs in tropical and temperate environments, including oceanic islands.

Unveiling in situ interactions between marine protists and bacteria through single cell sequencing

Heterotrophic protists are a highly diverse and biogeochemically significant component of marine ecosystems, yet little is known about their species-specific prey preferences and symbiotic interactions in situ. Here we demonstrate how these previously unresolved questions can be addressed by sequencing the eukaryote and bacterial SSU rRNA genes from individual, uncultured protist cells collected from their natural marine environment and sorted by flow cytometry. We detected Pelagibacter ubique in association with a MAST-4 protist, an actinobacterium in association with a chrysophyte and three bacteroidetes in association with diverse protist groups. The presence of identical phylotypes among the putative prey and the free bacterioplankton in the same sample provides evidence for predator-prey interactions. Our results also suggest a discovery of novel symbionts, distantly related to Rickettsiales and the candidate divisions ZB3 and TG2, associated with Cercozoa and Chrysophyta cells. This study demonstrates the power of single cell sequencing to untangle ecological interactions between uncultured protists and prokaryotes.

Comparison of phosphate uptake rates by the smallest plastidic and aplastidic protists in the North Atlantic subtropical gyre

The smallest phototrophic protists (<3 μm) are important primary producers in oligotrophic subtropical gyres - the Earth's largest ecosystems. In order to elucidate how these protists meet their inorganic nutrient requirements, we compared the phosphate uptake rates of plastidic and aplastidic protists in the phosphate-depleted subtropical and tropical North Atlantic (4-29°N) using a combination of radiotracers and flow cytometric sorting on two Atlantic Meridional Transect cruises. Plastidic protists were divided into two groups according to their size (<2 and 2-3 μm). Both groups of plastidic protists showed higher phosphate uptake rates per cell than the aplastidic protists. Although the phosphate uptake rates of protist cells were on average seven times (P<0.001) higher than those of bacterioplankton, the biomass-specific phosphate uptake rates of protists were one fourth to one twentieth of an average bacterioplankton cell. The unsustainably low biomass-specific phosphate uptake by both plastidic and aplastidic protists suggests the existence of a common alternative means of phosphorus acquisition - predation on phosphorus-rich bacterioplankton cells.

Mode of action of membrane-disruptive lytic compounds from the marine dinoflagellate Alexandrium tamarense

Certain allelochemicals of the marine dinoflagellate Alexandrium tamarense cause lysis of a broad spectrum of target protist cells but the lytic mechanism is poorly defined. We first hypothesized that membrane sterols serve as molecular targets of these lytic compounds, and that differences in sterol composition among donor and target cells may cause insensitivity of Alexandrium and sensitivity of targets to lytic compounds. We investigated Ca2+ influx after application of lytic fractions to a model cell line PC12 derived from a pheochromocytoma of the rat adrenal medulla to establish how the lytic compounds affect ion flux associated with lysis of target membranes. The lytic compounds increased permeability of the cell membrane for Ca2+ ions even during blockade of Ca2+ channels with cadmium. Results of a liposome assay suggested that the lytic compounds did not lyse such target membranes non-specifically by means of detergent-like activity. Analysis of sterol composition of isolates of A. tamarense and of five target protistan species showed that both lytic and non-lytic A. tamarense strains contain cholesterol and dinosterol as major sterols, whereas none of the other tested species contain dinosterol. Adding sterols and phosphatidylcholine to a lysis bioassay with the cryptophyte Rhodomonas salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent high affinity for free sterols and phosphatidylcholine. Lysis of protistan target cells was dose-dependently reduced by adding various sterols or phosphatidylcholine. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. We conclude therefore that although the molecular targets of the lytic compounds are likely to involve sterol components of membranes, A. tamarense must have a complex self-protective mechanism that still needs to be addressed.