Accumulation of docosapentaenoic acid (n-3 DPA) in a novel isolate of the marine ichthyosporean Sphaeroforma arctica.

OBJECTIVE: Currently, there is lack of a consistent and highly enriched source for docosapentaenoic acid (n-3 DPA, C22:5), and this work report the isolation of microorganism that naturally produces n-3 DPA. RESULTS: In this work, we screened microorganisms in our culture collections with the goal to isolate a strain with high levels of n-3 DPA. We isolated a strain of Sphaeroforma arctica that produces up to 11% n-3 DPA in total fatty acid and has a high n-3 DPA to DHA/EPA ratio. The cell growth of the isolated strain was characterized using microscopy imaging and flow cytometer technologies to confirm the coenocytic pattern of cell divisions previously described in S. arctica. Our novel isolate of S. arctica grew more robustly and produced significantly more n-3 DPA compared to previously isolated and described strains indicating the uniqueness of the discovered strain. CONCLUSION: Overall, this work reports a first isolate n-3 DPA producing microorganism and establishes the foundation for future strain improvement and elucidation of the physiological function of this LC-PUFA for human nutrition and health.

Development of a novel nannochloropsis strain with enhanced violaxanthin yield for large-scale production.

BACKGROUND: Nannochloropsis is a marine microalga that has been extensively studied. The major carotenoid produced by this group of microalgae is violaxanthin, which exhibits anti-inflammatory, anti-photoaging, and antiproliferative activities. Therefore, it has a wide range of potential applications. However, large-scale production of this pigment has not been much studied, thereby limiting its industrial application. RESULTS: To develop a novel strain producing high amount of violaxanthin, various Nannochloropsis species were isolated from seawater samples and their violaxanthin production potential were compared. Of the strains tested, N. oceanica WS-1 exhibited the highest violaxanthin productivity; to further enhance the violaxanthin yield of WS-1, we performed gamma-ray-mediated random mutagenesis followed by colorimetric screening. As a result, Mutant M1 was selected because of its significant higher violaxanthin content and biomass productivity than WS-1 (5.21 ± 0.33 mg g- 1 and 0.2101 g L- 1 d- 1, respectively). Subsequently, we employed a 10 L-scale bioreactor to confirm the large-scale production potential of M1, and the results indicated a 43.54 % increase in violaxanthin production compared with WS-1. In addition, comparative transcriptomic analysis performed under normal light condition identified possible mechanisms associated with remediating photo-inhibitory damage and other key responses in M1, which seemed to at least partially explain enhanced violaxanthin content and delayed growth. CONCLUSIONS: Nannochloropsis oceanica mutant (M1) with enhanced violaxanthin content was developed and its physiological characteristics were investigated. In addition, enhanced production of violaxanthin was demonstrated in the large-scale cultivation. Key transcriptomic responses that are seemingly associated with different physiological responses of M1 were elucidated under normal light condition, the details of which would guide ongoing efforts to further maximize the industrial potential of violaxanthin producing strains.

Shotgun metagenomics reveal a diverse assemblage of protists in a model Antarctic soil ecosystem.

The soils of the McMurdo Dry Valleys (MDV) of Antarctica are established models for understanding fundamental processes in soil ecosystem functioning (e.g. ecological tipping points, community structuring and nutrient cycling) because the extreme physical environment drastically reduces biodiversity and ecological complexity. Understanding the functioning of MDV soils requires in-depth knowledge of the diversity of MDV soil species. Protists, which contribute significantly to soil ecosystem functioning worldwide, remain poorly characterized in the MDV. To better assess the diversity of MDV protists, we performed shotgun metagenomics on 18 sites representing a variety of landscape features and edaphic variables. Our results show MDV soil protists are diverse at both the genus (155 of 281 eukaryote genera) and family (120) levels, but comprise only 6% of eukaryotic reads. Protists are structured by moisture, total N and distance from the local coast and possess limited richness in arid (< 5% moisture) and at high elevation sites, known drivers of communities in the MDV. High relative diversity and broad distribution of protists in our study promotes these organisms as key members of MDV soil microbiomes and the MDV as a useful system for understanding the contribution of soil protists to the structure of soil microbiomes.

Unveiling protist diversity associated with the Pacific oyster Crassostrea gigas using blocking and excluding primers.

BACKGROUND: Microbiome of macroorganisms might directly or indirectly influence host development and homeostasis. Many studies focused on the diversity and distribution of prokaryotes within these assemblages, but the eukaryotic microbial compartment remains underexplored so far. RESULTS: To tackle this issue, we compared blocking and excluding primers to analyze microeukaryotic communities associated with Crassostrea gigas oysters. High-throughput sequencing of 18S rRNA genes variable loops revealed that excluding primers performed better by not amplifying oyster DNA, whereas the blocking primer did not totally prevent host contaminations. However, blocking and excluding primers showed similar pattern of alpha and beta diversities when protist communities were sequenced using metabarcoding. Alveolata, Stramenopiles and Archaeplastida were the main protist phyla associated with oysters. In particular, Codonellopsis, Cyclotella, Gymnodinium, Polarella, Trichodina, and Woloszynskia were the dominant genera. The potential pathogen Alexandrium was also found in high abundances within some samples. CONCLUSIONS: Our study revealed the main protist taxa within oysters as well as the occurrence of potential oyster pathogens. These new primer sets are promising tools to better understand oyster homeostasis and disease development, such as the Pacific Oyster Mortality Syndrome (POMS) targeting juveniles.

Dynamic flux balance analysis of biomass and lipid production by Antarctic thraustochytrid Oblongichytrium sp. RT2316-13.

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.

Protist Biodiversity and Biogeography in Lakes From Four Brazilian River-Floodplain Systems.

The biodiversity and biogeography of protists inhabiting many ecosystems have been intensely studied using different sequencing approaches, but tropical ecosystems are relatively under-studied. Here, we sampled planktonic waters from 32 lakes associated with four different river-floodplains systems in Brazil, and sequenced the DNA using a metabarcoding approach with general eukaryotic primers. The lakes were dominated by the largely free-living Discoba (mostly the Euglenida), Ciliophora, and Ochrophyta. There was low community similarity between lakes even within the same river-floodplain. The protists inhabiting these floodplain systems comprise part of the large and relatively undiscovered diversity in the tropics.

Molecular Detection and Spatiotemporal Characterization of Labyrinthulomycete Protist Diversity in the Coastal Waters Along the Pearl River Delta.

The heterotrophic labyrinthulomycete protists have long been known to play an important role in the nutrient cycling of coastal seawater. Yet, their spatiotemporal abundance and diversity in polluted coastal waters remain poorly discussed, due in part to the paucity of a rapid detection method. To this end, we developed a qPCR detection method based on a newly designed primer pair targeting their 18S rRNA gene. Using this method, we studied the population dynamics of labyrinthulomycete protists in nutrient-rich (Shenzhen Bay) and low-nutrient (Daya) coastal habitats along the Pearl River Delta. We found a significantly (P < 0.05) higher abundance of Labyrinthulomycetes in the Shenzhen bay (average 3455 gene copies mL-1) than that in Daya Bay (average 378 gene copies mL-1). Their abundance gradient positively correlated (P < 0.05) with the levels of inorganic nitrogen and phosphates. Further characterization of the molecular diversity of these protists in Shenzhen Bay using different primer sets revealed the presence of several genera besides a large number of unclassified OTUs. Regardless of the primer biases, our results show significant (P < 0.05) spatiotemporal changes in the molecular abundance and diversity of these heterotrophic protists. Overall, this study provides a rapid molecular detection tool for Labyrinthulomycetes and expands our current understanding of their dynamics controlled by physicochemical gradients in coastal waters.

Isolation, Characterization and Biotechnological Potentials of Thraustochytrids from Icelandic Waters.

The following study reports on the first thraustochytrid isolates identified from Iceland. They were collected from three different locations off the northern coast of the country (Location A, Skagaströnd; Location B, Hveravík; and Location C, Eyjafjörður). Using 18S rDNA sequence analysis, isolates from Locations A and B were identified within the Thraustochytrium kinnei species while other isolates within the Sicyoidochytrium minutum species when compared to other known strains. Cells isolated from Locations A ( 2 . 10 ± 0 . 70 g/L) and B ( 1 . 54 ± 0 . 17 g/L) produced more biomass than the ones isolated from Location C ( 0 . 43 ± 0 . 02 g/L). This study offers the first-time examination of the utility of byproducts from fisheries as a nitrogen source in media formulation for thraustochytrids. Experiments showed that isolates produced more biomass (per unit of substrate) when cultured on nitrogen of marine ( 2 . 55 ± 0 . 74 g/L) as compared to of commercial origin (  1 . 06 ± 0 . 57 g/L). Glycerol ( 2 . 43 ± 0 . 56 g/L) was a better carbon source than glucose ( 1 . 84 ± 0 . 57 g/L) in growth studies. Fatty acid (FA) profiles showed that the isolates from Location C (S. minutum) had low ratios of monounsaturated ( 4 . 21 ± 2 . 96 % ) and omega-6 ( 0 . 68 ± 0 . 59 % ) FAs. However, the isolates also had high ratios of docosahexaenoic acid (DHA; 35 . 65 ± 1 . 73 % ) and total omega-3 FAs ( 40 . 39 ± 2 . 39 % ), indicating that they could serve as a source of marine oils for human consumption and in aquaculture feeds. The T. kinnei isolates from Location A could be used in biodiesel production due to their high ratios of monounsaturated ( 18 . 38 ± 6 . 27 % ) long chain ( 57 . 43 ± 8 . 27 % ) FAs.

New Species of Saprobic Labyrinthulea (=Labyrinthulomycota) and the Erection of a gen. nov. to Resolve Molecular Polyphyly within the Aplanochytrids.

A culture of a unicellular heterotrophic eukaryote was established from pollen-baited seawater acquired from the nearshore environment in Tromsø, Norway. Light microscopy revealed the production of ectoplasmic nets and reproduction by biflagellated zoospores, as well as binary division. After culturing and subsequent nucleotide extraction, database queries of the isolate's 18S small ribosomal subunit coding region identified closest molecular affinity to Aplanochytrium haliotidis, a pathogen of abalone. Testing of phylogenetic hypotheses consistently grouped our unknown isolate and A. haliotidis among the homoplasious thraustochytrids. Transmission electron microscopy revealed complex cell walls comprised of electron-dense lamella that formed protuberances, some associated with bothrosomes. Co-culturing experiments with the marine fungus Penicillium brevicompactum revealed prolonged interactions with hyphal strands. Based on the combined information acquired from electron microscopy, life history information, and phylogenetic testing, we describe our unknown isolate as a novel species. To resolve molecular polyphyly within the aplanochytrids, we erect a gen. nov. that circumscribes our novel isolate and the former A. haliotidis within the thraustochytrids.

Photosynthetic Picoeukaryotes in the Land-Fast Ice of the White Sea, Russia.

The White Sea is a unique marine environment combining features of temperate and Arctic seas. The composition and abundance of photosynthetic picoeukaryotes (PPEs) were investigated in the land-fast ice of the White Sea, Russia, in March 2013 and 2014. High-throughput tag sequencing (Illumina MiSeq system) of the V4 region of the 18S rRNA gene was used to reveal the diversity of PPE ice community. The integrated PPE abundance varied from 11 × 106 cells/m2 to 364 × 106 cells/m2; the integrated biomass ranged from 0.02 to 0.26 mg С/m2. The composition of sea-ice PPEs was represented by 16 algae genera belonging to eight classes and three super-groups. Chlorophyta, especially Mamiellophyceae, dominated among ice PPEs. The detailed analysis revealed the latent diversity of Micromonas and Mantоniella. Micromonas clade E2 revealed in the subarctic White Sea ice indicates that the area of distribution of this species is wider than previously thought. We suppose there exists a new Micromonas clade F. Micromonas clade C and Minutocellulus polymorphus were first discovered in the ice and extend the modern concept of sympagic communities' diversity generally and highlights the importance of further targeting subarctic sea ice for microbial study.

Enhanced production of carotenoids using a Thraustochytrid microalgal strain containing high levels of docosahexaenoic acid-rich oil.

Results to date suggest that microalgal Thraustochytrids family strains can be used to produce high-functional omega-3 rich oil (~ 30-70% of dry cell weight) and carotenoid-based antioxidant pigments simultaneously with value-added bioactive potential. In the present study, we describe the isolation and characterization of a new Thraustochytrid Schizochytrium sp. from the west coastal area of Korea. This newly isolated Thraustochytrid, identified as Schizochytrium sp. through 18S rRNA analysis and named SH104, simultaneously produces high levels of DHA and carotenoid-based antioxidant pigments. An improved Schizochytrium mutant, named SHG104, was obtained from the original host strain by γ-irradiation-induced mutagenesis. Under combined temperature-shift cultivation conditions employing white-light LEDs (light-emitting diodes), Schizochytrium sp. SHG104 yielded 10.8 g L-1 of biomass comprising 45.8% total lipids (32.1% DHA) and 4.6 mg L-1 of astaxanthin. In addition to DHA, the main fatty acids produced by Schizochytrium sp. SHG104 were palmitic acid and a trace of other long-chain fatty acids. The carotenoid profile of SH104 and SHG104 was ß-carotene, astaxanthin, canthaxanthin, pheonicoxanthin and echinenone, which analyzed by HPLC and LC/APCI-MS. Furthermore, genomic analysis of Schizochytrium and Aurantiochytrium microalgae confirmed that the presence of carotenogenesis pathway enzymes and genes including geranylgeranyl diphosphate, phytoene synthase, lycopene cyclase, and cytochrome P450 hydroxylase that necessary for the production of antioxidants via a complete biosynthetic KEGG synthesis pathway. This newly isolated Schizochytrium microalga potentially have wide application as a source of antioxidants for astaxanthin-containing pigments, commercial omega-3 lipids and feed additives, such as nutritional supplements for aquaculture.

Differential Gene Expression in Five Isolates of the Clam Pathogen, Quahog Parasite Unknown (QPX).

Quahog parasite unknown (QPX) is a thraustochytrid protist that infects the hard clam, Mercenaria mercenaria, causing significant economic losses along the northeastern coast of North America. Previous investigations noted differences in growth dynamics and virulence in QPX cells from different geographic locations. In order to probe the molecular determinants for these variations, we investigated the transcriptomic profiles of five geographically distinct QPX isolates using custom 15k 60-mer oligonucleotide arrays. A total of 1,263 transcripts were differentially expressed (DE) among the five QPX isolates. The hierarchical clustering of gene expression profiles showed that the QPX isolates from Raritan Bay (RB, NY) and from Provincetown Harbor (MA) were more similar to each other and diverged from QPX isolates from Peconic Bay (PB, NY) and Old Plantation Creek (VA), which had more similar gene expression profiles. The most prominent difference was based on 78 transcripts coding for heat shock proteins DE between the five QPX isolates. The study generated contrasting transcriptomic profiles for QPX isolated from northern (MA) and deeper (RB, NY) locations as compared to southern (VA) and shallower (PB, NY) areas, suggesting the adaptation of the parasite to local environmental, in particular temperature, conditions.

The First Isolation and Characterisation of the Protist Labyrinthula sp. in Southeastern Australia.

As a result of anthropogenic influences and global climate change, emerging infectious marine diseases are thought to be increasingly more common and more severe than in the past. The aim of our investigation was to confirm the presence of Labyrinthula, the aetiological agent of the seagrass wasting disease, in Southeastern Australia and provide the first isolation and characterisation of this protist, in Australia. Colonies and individual cells were positively identified as Labyrinthula using published descriptions, diagrams, and photographs. Their identity was then confirmed using DNA barcoding of a region of the 18S rRNA gene. Species level identification of isolates was not possible as the taxonomy of the Labyrinthula is still poorly resolved. Still, a diversity of Labyrinthula was isolated from small sections of the southeast coast of Australia. The isolates were grouped into three haplotypes that are biogeographically restricted. These haplotypes are closely related to previously identified saprotrophic clades. The study highlights the need for further investigation into the global distribution of Labyrinthula, including phylogenetic pathogenicity and analysis of host-parasite interactions in response to stressors. Given the results of our analyses, it is prudent to continue research into disease and epidemic agents to better prepare researchers for potential future outbreaks.

Reference Tree and Environmental Sequence Diversity of Labyrinthulomycetes.

Labyrinthulomycetes are heterotrophic stramenopiles that are ubiquitous in a wide range of both marine and freshwater habitats and play important roles in decomposition of organic matter. The diversity and taxonomy of Labyrinthulomycetes has been studied for many years, but we nevertheless lack both a comprehensive reference database and up-to-date phylogeny including all known diversity, which hinders many global insights into their ecological distribution and the relative importance of various subgroups in different environments. Here, we present a curated reference database and a phylogenetic tree of Labyrinthulomycetes small subunit ribosomal RNA (SSU or 18S rRNA) data. Based on this created reference database, we analyzed high-throughput environmental sequencing data, revealing many previously unknown environmental clades and exploring the ecological distribution of various subgroups. Particularly, a number of newly identified environmental clades that are widespread in the open ocean. Comparing the manually curated reference database to existing tools for identification of environmental sequences (e.g. PR2 or SILVA databases) suggests that the curated database provides a higher degree of specificity and a lower frequency of misidentification. The phylogenetic framework and database will be a useful tool for future ecological and evolutionary studies.

Novel squalene-producing thraustochytrids found in mangrove water.

On extended screening of squalene-producing strains in Okinawa mangrove water, we identified 14 novel squalene-producing thraustochytrids from 172 unialgal clonal isolates. The novel thraustochytrids produced 13.9-7.54 mg squalene/g dry cell weight. Eight isolates were found to belong to potentially novel squalene-producing genera, forming a monophyletic cluster independent from any known thraustochytrids.

Sorodiplophrys stercorea: Another Novel Lineage of Sorocarpic Multicellularity.

Sorodiplophrys stercorea is a sorocarpic organism that utilizes filose pseudopodia for locomotion and absorptive nutrition. It has traditionally been considered to be a member of the Labyrinthulae based on its morphology. Its closest relatives were thought to be species in the taxon Diplophrys. Since the genus Diplophrys has been shown to be paraphyletic and S. stercorea has pseudopodia similar to some members of Rhizaria, we examined its relationship with other eukaryotes. We obtained four isolates from the dung of cow and horse, brought each into monoeukaryotic culture, and sequenced their SSU rRNA gene for phylogenetic analysis. All our isolates were shown to form a monophyletic group in the Labyrinthulae, nested in the Amphifiloidea clade. Our results demonstrate that Sorodiplophrys is more closely related to species of the genus Amphifila than to Diplophrys and represents an additional independent origin of sorocarpic multicellularity among eukaryotes. This study represents the first confirmed sorocarpic lifestyle in the Stramenopiles.

Spatial Distribution of Eukaryotic Communities Using High-Throughput Sequencing Along a Pollution Gradient in the Arsenic-Rich Creek Sediments of Carnoulès Mine, France.

Microscopic eukaryotes play a key role in ecosystem functioning, but their diversity remains largely unexplored in most environments. To advance our knowledge of eukaryotic microorganisms and the factors that structure their communities, high-throughput sequencing was used to characterize their diversity and spatial distribution along the pollution gradient of the acid mine drainage at Carnoulès (France). A total of 16,510 reads were retrieved leading to the identification of 323 OTUs after normalization. Phylogenetic analysis revealed a quite diverse eukaryotic community characterized by a total of eight high-level lineages including 37 classes. The majority of sequences were clustered in four main groups: Fungi, Stramenopiles, Alveolata and Viridiplantae. The Reigous sediments formed a succession of distinct ecosystems hosting contrasted eukaryotic communities whose structure appeared to be at least partially correlated with sediment mineralogy. The concentration of arsenic in the sediment was shown to be a significant factor driving the eukaryotic community structure along this continuum.

Freshwater protists do not go with the flow: population structure in Gonyostomum semen independent of connectivity among lakes.

Many recent studies have found genetically differentiated populations in microorganisms despite potentially high dispersal. We designed a study to specifically examine the importance of physical dispersal barriers, i.e. geographic distance and lack of hydrological connectivity, in restricting gene flow and enhancing divergence in limnic microorganisms. We focused on the nuisance microalga Gonyostomum semen, which has recently expanded in Northern Europe and differentiated into genetically distinct populations. G. semen was sampled from six lakes distributed in two adjacent watersheds, which thereby comprised, both connected and non-connected lakes. The individual isolates were genotyped by amplified fragment length polymorphism. Several lake populations were differentiated from each other, but connectivity within watersheds could not explain the observed population genetic pattern. However, isolation by distance was moderate and might limit the gene flow among distant populations. In addition, we found low, but significant linkage disequilibrium, which indicates regular sexual recombination in this species, despite its high degree of asexual reproduction. Therefore, we conclude that the genetic properties of microalgae with occasional sexual reproduction essentially mirror regularly recombining species. Furthermore, the data indicated bottlenecks supporting the hypothesized recent range expansion of this species.

Depth shapes α- and ß-diversities of microbial eukaryotes in surficial sediments of coastal ecosystems.

Little is known about the relative influence of historic processes and environmental gradients on shaping the diversity of single-celled eukaryotes in marine benthos. By combining pyrosequencing of 18S ribosomal RNA genes with data on multiple environmental factors, we investigated the diversity of microeukaryotes in surficial sediments of three basins of the Yellow Sea Large Marine Ecosystem. A considerable proportion (about 20%) of reads was affiliated with known parasitoid protists. Dinophyta and Ciliophora appeared dominant in terms of relative proportion of reads and operational taxonomic unit (OTU) richness. Overall, OTU richness of benthic microeukaryotes decreased with increasing water depth and decreasing pH. While community composition was significantly different among basins, partial Mantel tests indicated a depth-decay pattern of community similarity, whereby water depth, rather than geographic distance or environment, shaped ß-diversity of benthic microeukaryotes (including both the abundant and the rare biosphere) on a regional scale. Similar hydrographic and mineralogical factors contributed to the biogeography of both the abundant and the rare OTUs. The trace metal vanadium had a significant effect on the biogeography of the rare biosphere. Our study sheds new light on the composition, diversity patterns and underlying mechanisms of single-celled eukaryote distribution in surficial sediments of coastal oceans.

Diversity and potential sources of microbiota associated with snow on western portions of the Greenland Ice Sheet.

Snow overlays the majority of the Greenland Ice Sheet (GrIS). However, there is very little information available on the microbiological assemblages that are associated with this vast and climate-sensitive landscape. In this study, the structure and diversity of snow microbial assemblages from two regions of the western GrIS ice margin were investigated through the sequencing of small subunit ribosomal RNA genes. The origins of the microbiota were investigated by examining correlations to molecular data obtained from marine, soil, freshwater and atmospheric environments and geochemical analytes measured in the snow. Snow was found to contain a diverse assemblage of bacteria (Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria) and eukarya (Alveolata, Fungi, Stramenopiles and Chloroplastida). Phylotypes related to archaeal Thaumarchaeota and Euryarchaeota phyla were also identified. The snow microbial assemblages were more similar to communities characterized in soil than to those documented in marine ecosystems. Despite this, the chemical composition of snow samples was consistent with a marine contribution, and strong correlations existed between bacterial beta diversity and the concentration of Na(+) and Cl(-) . These results suggest that surface snow from western regions of Greenland contains exogenous microbiota that were likely aerosolized from more distant soil sources, transported in the atmosphere and co-precipitated with the snow.