Marine Heterotrophic Flagellates Research Articles

Evolution of exploitation and replication of giant viruses and virophages.

Tripartite biotic interactions are inherently complex, and the strong interdependence of species and often one-sided exploitation can make these systems vulnerable to extinction. The persistence of species depends then on the balance between exploitation and avoidance of exploitation beyond the point where sustainable resource use is no longer possible. We used this general prediction to test the potential role of trait evolution for persistence in a tripartite microbial system consisting of a marine heterotrophic flagellate preyed upon by a giant virus, which in turn is parasitized by a virophage. Host and virophage may benefit from this interaction because the virophage reduces the harmful effects of the giant virus on the host population and the virophage can persist integrated into the host genome when giant viruses are scarce. We grew hosts and virus in the presence and absence of the virophage over ∼280 host generations and tested whether levels of exploitation and replication in the giant virus and/or virophage population evolved over the course of the experiment, and whether the changes were such that they could avoid overexploitation and extinction. We found that the giant virus evolved toward lower levels of replication and the virophage evolved toward increased replication but decreased exploitation of the giant virus. These changes reduced overall host exploitation by the virus and virus exploitation by the virophage and are predicted to facilitate persistence.

Isolation and Identification of a Large Green Alga Virus (Chlorella Virus XW01) of Mimiviridae and Its Virophage (Chlorella Virus Virophage SW01) by Using Unicellular Green Algal Cultures.

Virophages are a group of small double-stranded DNA viruses that infect protist hosts and parasitize the viral factory of host giant/large viruses to propagate. Here, we discover a novel cell-virus-virophage (CVv) tripartite interaction system by using unicellular micro-green algae (Chlorella sp.) as eukaryotic hosts for the first time. Viral particles, resembling known virophages and large alga viruses, are detected in culture supernatants and inside algal cells. Complete genomic sequences of the virophage (Chlorella virus virophage SW01 [CVv-SW01]; 24,744 bp) and large virus (Chlorella virus XW01 [CV-XW01]; 407,612 bp) are obtained from the cocultures. Both genomic and phylogenetic analyses show that CVv-SW01 is closely related to virophages previously found in Dishui Lake. CV-XW01 shares the greatest number of homologous genes (n = 82) with Cafeteria roenbergensis virus (CroV) and phylogenetically represents the closest relative to CroV. This is the first report of a large green alga virus being affiliated with a heterotrophic zooplankton-infecting Cafeteriavirus of the family Mimiviridae. Moreover, the codon usage preferences of CV-XW01 and CVv-SW01 are highly similar to those of CroV and its virophage Mavirus, respectively. The discovery of such a novel CVv system with the green alga Chlorella sp. as the single cellular eukaryotic host paves a way to further investigate the potential interaction mechanism of CVv and its significance in the ecology of green algae and the evolution of large/giant viruses and their parasitic viruses. IMPORTANCE Parasitic virophages are small unicellular eukaryotic dsDNA viruses that rely on the viral factories of coinfecting giant/large dsDNA viruses for propagation. Presently, the identified eukaryotic hosts of isolated virophages were restricted to a free-living amoeba, Acanthamoeba polyphaga, and a widespread marine heterotrophic flagellate, Cafeteria roenbergensis. In this study, we successfully discovered and identified a novel tripartite interaction system comprised of a micro-green alga (Chlorella sp.), Mimiviridae large green alga virus, and virophage at the coculture level, with Chlorella sp. as the eukaryotic host, based on combination analysis of infection, morphotype, genome, and phylogeny. The large green alga virus CV-XW01 represents the closest relative to the Mimiviridae giant virus Cafeteria roenbergensis virus, host virus of the virophage Mavirus, as well as a novel large virus of Mimiviridae that infects a non-protozoan protist host. The virophage CVv-SW01 highly resembles Mavirus in its codon usage frequency and preference, although they are phylogenetically distantly related. These findings give novel insights into the diversity of large/giant viruses and their virophages.

Dominant marine heterotrophic flagellates are adapted to natural planktonic bacterial abundances.

SummaryGrazing controls bacterial abundances and composition in many ecosystems. In marine systems, heterotrophic flagellates (HFs) are important predators. Assemblages of HFs are primarily formed by species still uncultured; therefore, many aspects of their trophic behaviour are poorly known. Here, we assessed the functional response of the whole assemblage and of four taxa grown in an unamended seawater incubation. We used fluorescently labelled bacteria to create a prey gradient of two orders of magnitude in abundance and estimated ingestion rates. Natural HFs had a half‐saturation constant of 6.7 × 105 prey ml−1, a value lower than that of cultured flagellates and within the range of marine planktonic bacterial abundances. Minorisa minuta was well adapted to low prey abundances and very efficient in ingesting bacteria. MAST‐4 and MAST‐7 were also well adapted to the typical marine abundances but less voracious. In contrast, Paraphysomonas imperforata, a typical cultured species, did not achieve ingestion rate saturation even at the highest prey concentration assayed. Our study, beside to set the basis for the fundamental differences between cultured and uncultured bacterial grazers, indicate that the examined predator taxa have different functional responses, suggesting that they occupy distinct ecological niches according to their grazing strategies and prey preferences.

Gene expression during bacterivorous growth of a widespread marine heterotrophic flagellate

Phagocytosis is a fundamental process in marine ecosystems by which prey organisms are consumed and their biomass incorporated in food webs or remineralized. However, studies searching for the genes underlying this key ecological process in free-living phagocytizing protists are still scarce, in part due to the lack of appropriate ecological models. Our reanalysis of recent molecular datasets revealed that the cultured heterotrophic flagellate Cafeteria burkhardae is widespread in the global oceans, which prompted us to design a transcriptomics study with this species, grown with the cultured flavobacterium Dokdonia sp. We compared the gene expression between exponential and stationary phases, which were complemented with three starvation by dilution phases that appeared as intermediate states. We found distinct expression profiles in each condition and identified 2056 differentially expressed genes between exponential and stationary samples. Upregulated genes at the exponential phase were related to DNA duplication, transcription and translational machinery, protein remodeling, respiration and phagocytosis, whereas upregulated genes in the stationary phase were involved in signal transduction, cell adhesion, and lipid metabolism. We identified a few highly expressed phagocytosis genes, like peptidases and proton pumps, which could be used to target this ecologically relevant process in marine ecosystems.

Expression of genes involved in phagocytosis in uncultured heterotrophic flagellates

AbstractEnvironmental molecular sequencing has revealed an abundance of microorganisms that were previously unknown, mainly because most had not been cultured in the laboratory. Within this novel diversity, there are the uncultured MAST clades (MArine STramenopiles), which are major components of marine heterotrophic flagellates (HFs) thought to be active bacterial grazers. In this study, we investigated the gene expression of natural HFs in a mixed community where bacterivory was promoted. Using fluorescence in situ hybridization and 18S rDNA derived from metatranscriptomics, we followed the taxonomic dynamics during the incubation, and confirmed the increase in relative abundance of different MAST lineages. We then used single cell genomes of several MAST species to gain an insight into their most expressed genes, with a particular focus on genes related to phagocytosis. The genomes of MAST‐4A and MAST‐4B were the most represented in the metatranscriptomes, and we identified highly expressed genes of these two species involved in motility and cytoskeleton remodeling, as well as many lysosomal enzymes. Particularly relevant were the cathepsins, which are characteristic digestive enzymes of the phagolysosome and the rhodopsins, perhaps used for vacuole acidification. The combination of single cell genomics and metatranscriptomics gives insights on the phagocytic capacity of uncultured and ecologically relevant HF species.

Three Newly Recorded Marine Heterotrophic Flagellates (Protist),Neometanema parovale, Stephanopogon pattersoni and Thaumatomastix sp. from South Korea

Three marine heterotrophic flagellates from intertidal sediments of Gwang-Am beach and Garorim Bay, Korea were identified as Neometanema parovale Lee and Patterson 2014, Stephanopogon pattersoni Lee et al. 2014 and Thaumatomastix sp. These species are reported taxonomically for the first time from Korea, and are described with illustrations and micrographs. Diagnostics of these species are as follows. Neometanema parovale (Euglenozoa): size in vivo, 10~23 μm long with 22 pellicular strips, ingestion apparatus barely visible by light microscopy and two flagella pointed in different directions when moving. Stephanopogon pattersoni (Percolozoa): size in vivo, 20~33 μm long, with 6 ventral and 1 ventro-lateral ciliary rows, and three barbs. Thaumatomastix sp. (Cercozoa): size in vivo, 14~17 μm with body scales and spines, and two flagella with one naked and one scaled.

Culturing Bias in Marine Heterotrophic Flagellates Analyzed Through Seawater Enrichment Incubations

The diversity of heterotrophic flagellates is generally based on cultivated strains, on which ultrastructural, physiological, and molecular studies have been performed. However, the relevance of these cultured strains as models of the dominant heterotrophic flagellates in the marine planktonic environment is unclear. In fact, molecular surveys typically recover novel eukaryotic lineages that have refused cultivation so far. This study was designed to directly address the culturing bias in planktonic marine heterotrophic flagellates. Several microcosms were established adding increasing amounts and sources of organic matter to a confined natural microbial community pre-filtered by 3 μm. Growth dynamics were followed by epifluorescence microscopy and showed the expected higher yield of bacteria and heterotrophic flagellates at increased organic matter additions. Moreover, protist diversity analyzed by molecular tools showed a clear substitution in the community, which differed more and more from the initial sample as the organic matter increased. Within this gradient, there was also an increase of sequences related to cultured organisms as well as a decrease in diversity. Culturing bias is partly explained by the use of organic matter in the isolation process, which drives a shift in the community to conditions closer to laboratory cultures. An intensive culturing effort using alternative isolation methods is necessary to allow the access to the missing heterotrophic flagellates that constitute the abundant and active taxa in marine systems.

Biogeography of the uncultured marine picoeukaryote MAST-4: temperature-driven distribution patterns

The MAST-4 (marine stramenopile group 4) is a widespread uncultured picoeukaryote that makes up an important fraction of marine heterotrophic flagellates. This group has low genetic divergence and is composed of a small number of putative species. We combined ARISA (automated ribosomal intergenic spacer analysis) and ITS (Internal Transcribed Spacer) clone libraries to study the biogeography of this marine protist, examining both spatial and temporal trends in MAST-4 assemblages and associated environmental factors. The most represented MAST-4 clades appeared adapted to different temperature ranges, and their distributions did not suggest clear geographical barriers for dispersal. Distant samples sharing the same temperature had very similar assemblages, especially in cold temperatures, where only one clade, E1, dominated. The most highly represented clades, A and E1, showed very little differentiation between populations from distant geographical regions. Within a single site, temporal variation also followed patterns governed by temperature. Our results contribute to the general discussion on microbial biogeography by showing strong environmental selection for some picoeukaryotes in the marine environment.

The transcriptome of the novel dinoflagellate Oxyrrhis marina (Alveolata: Dinophyceae): response to salinity examined by 454 sequencing

BackgroundThe heterotrophic dinoflagellate Oxyrrhis marina is increasingly studied in experimental, ecological and evolutionary contexts. Its basal phylogenetic position within the dinoflagellates make O. marina useful for understanding the origin of numerous unusual features of the dinoflagellate lineage; its broad distribution has lent O. marina to the study of protist biogeography; and nutritive flexibility and eurytopy have made it a common lab rat for the investigation of physiological responses of marine heterotrophic flagellates. Nevertheless, genome-scale resources for O. marina are scarce. Here we present a 454-based transcriptome survey for this organism. In addition, we assess sequence read abundance, as a proxy for gene expression, in response to salinity, an environmental factor potentially important in determining O. marina spatial distributions.ResultsSequencing generated ~57 Mbp of data which assembled into 7, 398 contigs. Approximately 24% of contigs were nominally identified by BLAST. A further clustering of contigs (at ≥ 90% identity) revealed 164 transcript variant clusters, the largest of which (Phosphoribosylaminoimidazole-succinocarboxamide synthase) was composed of 28 variants displaying predominately synonymous variation. In a genomic context, a sample of 5 different genes were demonstrated to occur as tandem repeats, separated by short (~200-340 bp) inter-genic regions. For HSP90 several intergenic variants were detected suggesting a potentially complex genomic arrangement. In response to salinity, analysis of 454 read abundance highlighted 9 and 20 genes over or under expressed at 50 PSU, respectively. However, 454 read abundance and subsequent qPCR validation did not correlate well - suggesting that measures of gene expression via ad hoc analysis of sequence read abundance require careful interpretation.ConclusionHere we indicate that tandem gene arrangements and the occurrence of multiple transcribed gene variants are common and indicate potentially complex genomic arrangements in O. marina. Comparison of the reported data set with existing O. marina and other dinoflagellates ESTs indicates little sequence overlap likely as a result of the relatively limited extent of genome scale sequence data currently available for the dinoflagellates. This is one of the first 454-based transcriptome surveys of an ancestral dinoflagellate taxon and will undoubtedly prove useful for future comparative studies aimed at reconstructing the origin of novel features of the dinoflagellates.

Effect of viruses on marine stramenopile (MAST) communities in an oligotrophic coastal marine system

Viruses are the most abundant biological entities in aquatic systems, infecting from bacteria to mammals. However, there has been little study so far on their impact on marine heterotrophic flagellates (HFs). For this reason, four experiments were carried out between April 2006 and February 2007 in the Blanes Bay Microbial Observatory (NW Mediterranean coast). We investigated whether viruses could affect HF communities, and specifically two uncultured marine stramenopile groups (MAST-4 and MAST-1C). For each experiment, four microcosms containing 12 L of 5-mm filtered seawater were prepared, two received active viruses and the other two received heat-inactivated viruses. Microcosms were then incubated for 48 h in order to measure changes in the abundance of the target groups. In three of the four experiments, both the growth rates of HF and MAST-4 cells and the percentage of MAST-4 cells with respect to HF after 48 h were higher in the heat-inactivated treatment compared with the active viruses treatment. These results indicate that viruses can negatively affect the HF community either directly via lysis of protists or indirectly via lysis of bacteria, and highlight the interactions between, virus, bacteria and protists.

Carbon and nitrogen densities of the cultured marine heterotrophic flagellate Paraphysomonas sp.

Cell carbon and nitrogen densities (fg μm −3) and C:N ratios of the heterotrophic (bacterivorous) flagellate Paraphysomonas sp. at different growth phases were studied in batch co-cultures with their bacterial food. Flagellate growth phase was observed during daily cell counts using image analyzed epifluorescence microscopy. At logarithmic and stationary growth phases, the flagellate-bacteria co-cultures were filtered onto GF/F filters for CHN analyses. Parallel cultures of bacteria alone were similarly analyzed to determine carbon and nitrogen relationships to bacterial cell volume. These bacterial C and N densities (range 393 to 1390 fg C μm −3 for C; 113 to 401 fg N μm −3 for N) were used to calculate C and N concentration of bacteria in the flagellate-bacteria cultures. The total flagellate C and N concentrations were determined by difference between the calculated C and N of bacteria alone and of mixed bacteria and flagellates. C and N densities of flagellates were determined by dividing the total flagellate biovolume (μm 3 ml −1) into the flagellate C and N concentration (μg ml −1). A one-tailed t-test showed a significant difference between C density of Paraphysomonas sp. at logarithmic and stationary growth phase at a significance level of 0.05. We suggest using cell C density of 430 fg C μm −3 for growing cells similar to Paraphysomonas sp. and 260 fg C μm −3 for stationary growth cells in nature. N density and C:N were not significantly different between logarithmic and stationary growth phase.

Significance of the Virus‐Rich 2–200 nm Size Fraction of Seawater for Heterotrophic Flagellates: I. Impact on Growth

Abstract. The effect of artificially changing the concentration of naturally occurring free virus‐like particles (VLPs) on growth of marine heterotrophic flagellates was tested in batch cultures using ultrafiltration technology. This manipulation influenced the course of flagellate growth markedly. During 1 week of incubation the growth of 4 tested flagellate strains (Oxyrrhis marina, Paraphysomonas imperforata, Petalomonas cantuscygni, Pteridomonas danica) was strongly favoured after increasing the VLP concentration. Bacteria, however, were repressed in these treatments beyond 50 h. In cultures with natural seawater microbiota, we both reduced and enriched the concentration of VLPs. In these cultures, reduction of material in the virus‐rich 2–200 nm size fraction led to a strong positive growth response of heterotrophic flagellates. In VLP‐enriched treatments of natural seawater microbiota a tendency toward growth stimulation was also found, although this was not significant. Enrichment with VLPs caused no recognizable mortality either in cultured flagellate strains or in naturally occurring flagellate communities. However, it is suggested that a highly bioactive component must be present in the virus‐rich 2–200 nm size fraction. Virus‐like particles are discussed as possible candidates influencing heterotrophic flagellate community successions.

Ultrastructure of Amastigomonas bermudensis ATCC 50234 sp. nov. : A new heterotrophic marine flagellate

The ultrastructure of a new marine heterotrophic flagellate is described. The cell is dorso-ventrally flattened and displays a steady gliding forward movement. A longitudinal groove whose lips are appressed to the substrate runs along the ventral aspect of the cell. The two heterodynamic flagella originate from the proximal end of a cytoplasmic sheath that enfolds most of the length of the anterior flagellum. The lips of this sheath are continuous with the margins of the ventral groove. The posterior trailing flagellum is held closely appressed to the cell body on one side of the ventral groove. Three bands of microtubules extend posteriorly into the cell body and are associated with the kinetosomes. The part of the surface membrane extending over the cell's dorsal and lateral aspect is five-layered, giving the semblance of two fused cell membranes. There is a fibrillar layer underneath these membranes. There is also a fibrous network whose arrays are oriented in different directions within the cytoplasm. Mitochondria have tubular cristae. Based on a comparison with previously described species of Amastigomonas, we establish the species, A. bermudensis n.sp. We further conclude that Thecamonas is a junior synonym of Amastigomonas and move the three nominal species to the latter genus.