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.

Spatiotemporal Variations in Antarctic Protistan Communities Highlight Phytoplankton Diversity and Seasonal Dominance by a Novel Cryptophyte Lineage.

The Andvord fjord in the West Antarctic Peninsula (WAP) is known for its productivity and abundant megafauna. Nevertheless, seasonal patterns of the molecular diversity and abundance of protistan community members underpinning WAP productivity remain poorly resolved. We performed spring and fall expeditions pursuing protistan diversity, abundance of photosynthetic taxa, and the connection to changing conditions. 18S rRNA amplicon sequence variant (ASV) profiles revealed diverse predatory protists spanning multiple eukaryotic supergroups, alongside enigmatic heterotrophs like the Picozoa. Among photosynthetic protists, cryptophyte contributions were notable. Analysis of plastid-derived 16S rRNA ASVs supported 18S ASV results, including a dichotomy between cryptophytes and diatom contributions previously reported in other Antarctic regions. We demonstrate that stramenopile and cryptophyte community structures have distinct attributes. Photosynthetic stramenopiles exhibit high diversity, with the polar diatom Fragilariopsis cylindrus, unidentified Chaetoceros species, and others being prominent. Conversely, ASV analyses followed by environmental full-length rRNA gene sequencing, electron microscopy, and flow cytometry revealed that a novel alga dominates the cryptophytes. Phylogenetic analyses established that TPG clade VII, as named here, is evolutionarily distinct from cultivated cryptophyte lineages. Additionally, cryptophyte cell abundance correlated with increased water temperature. Analyses of global data sets showed that clade VII dominates cryptophyte ASVs at Southern Ocean sites and appears to be endemic, whereas in the Arctic and elsewhere, Teleaulax amphioxeia and Plagioselmis prolonga dominate, although both were undetected in Antarctic waters. Collectively, our studies provide baseline data against which future change can be assessed, identify different diversification patterns between stramenopiles and cryptophytes, and highlight an evolutionarily distinct cryptophyte clade that thrives under conditions enhanced by warming. IMPORTANCE The climate-sensitive waters of the West Antarctic Peninsula (WAP), including its many fjords, are hot spots of productivity that support multiple marine mammal species. Here, we profiled protistan molecular diversity in a WAP fjord known for high productivity and found distinct spatiotemporal patterns across protistan groups. Alongside first insights to seasonal changes in community structure, we discovered a novel phytoplankton species with proliferation patterns linked to temperature shifts. We then examined evolutionary relationships between this novel lineage and other algae and their patterns in global ocean survey data. This established that Arctic and Antarctic cryptophyte communities have different species composition, with the newly identified lineage being endemic to Antarctic waters. Our research provides critical knowledge on how specific phytoplankton at the base of Antarctic food webs respond to warming, as well as information on overall diversity and community structure in this changing polar environment.

Two new Nothophytophthora species from streams in Ireland and Northern Ireland: Nothophytophthora irlandica and N. lirii sp. nov.

Slow growing oomycete isolates with morphological resemblance to Phytophthora were obtained from forest streams during routine monitoring for the EU quarantine forest pathogen Phytophthora ramorum in Ireland and Northern Ireland. Internal Transcribed Spacer (ITS) sequence analysis indicated that they belonged to two previously unknown species of Nothophytophthora, a recently erected sister genus of Phytophthora. Morphological and temperature-growth studies were carried out to characterise both new species. In addition, Bayesian and Maximum-Likelihood analyses of nuclear 5-loci and mitochondrial 3-loci datasets were performed to resolve the phylogenetic positions of the two new species. Both species were sterile, formed chlamydospores and partially caducous nonpapillate sporangia, and showed slower growth than any of the six known Nothophytophthora species. In all phylogenetic analyses both species formed distinct, strongly supported clades, closely related to N. chlamydospora and N. valdiviana from Chile. Based on their unique combination of morphological and physiological characters and their distinct phylogenetic positions the two new species are described as Nothophytophthora irlandica sp. nov. and N. lirii sp. nov. Their potential lifestyle and geographic origin are discussed.

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.

Assessing the response of micro-eukaryotic diversity to the Great Acceleration using lake sedimentary DNA.

Long-term time series have provided evidence that anthropogenic pressures can threaten lakes. Yet it remains unclear how and the extent to which lake biodiversity has changed during the Anthropocene, in particular for microbes. Here, we used DNA preserved in sediments to compare modern micro-eukaryotic communities with those from the end of the 19th century, i.e., before acceleration of the human imprint on ecosystems. Our results obtained for 48 lakes indicate drastic changes in the composition of microbial communities, coupled with a homogenization of their diversity between lakes. Remote high elevation lakes were globally less impacted than lowland lakes affected by local human activity. All functional groups (micro-algae, parasites, saprotrophs and consumers) underwent significant changes in diversity. However, we show that the effects of anthropogenic changes have benefited in particular phototrophic and mixotrophic species, which is consistent with the hypothesis of a global increase of primary productivity in lakes.

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.

A Facile and Sensitive DNA Sensing of Harmful Algal Blooms Based on Graphene Oxide Nanosheets.

Gene detection has important applications in biology, biomedical engineering, clinical, environmental, and marine fields. Rapid, sensitive, and selective recognition of specific genes is essential in practical applications. In this study, we describe a facile and sensitive DNA sensing platform for specific and quantitative detection of Heterosigma akashiwo, which is one of the causative agents of red tides. Fast and sensitive detection is achieved by using chemically synthesized graphene oxide (GO) nanosheets. Probe DNA is designed according to the specific DNA fragments of harmful algae and labeled with fluorescent molecules FAM (fluorescein-based dye). GO nanosheet solution is made, in which the strong interaction between FAM-labeled probe and GO nanosheets keeps them in close proximity, facilitating the fluorescence quenching of the fluorophore by GO nanosheets. In the presence of a complementary target DNA, the FAM-labeled DNA probe and the target DNA hybridize and desorb from the surface of GO nanosheets, resulting in restoration of fluorophore fluorescence. The concentration of target DNA fragments is analyzed by the fluorescence intensity at ~ 520 nm with emission wavelength of 480 nm. The sensitive detecting platform achieved stable measurement of 1 pM specific genes from Heterosigma akashiwo. Our GO nanosheet-based DNA-sensing platform performs fast and sensitive detection of trace amount of DNA, and enables quantitative recognition of harmful algae, which has promising applications in real-time monitoring in the marine environment of red tide generative dynamics, allowing effective control, particularly in relation to marine aquaculture.

Antarctic thraustochytrids: Producers of long-chain omega-3 polyunsaturated fatty acids.

Thraustochytrids have been isolated from different aquatic systems; however, few studies have reported their occurrence in Antarctica. In this study, 13 strains close to strains belonging to the genera Oblongichytrium, Thraustochytrium, and Aurantiochytrium were isolated from seawater samples collected near the Antarctic Base Professor Julio Escudero (S 62°12'57' E 58°57'35″). Docosahexaenoic acid (DHA) was found in the total lipids of all the isolates; DHA content of the biomass (dry weight) varied between 3.3 and 33 mg/g under the growth conditions for isolation. Five of the Antarctic thraustochytrids were able to accumulate lipids at levels higher than 20% w/w. Two strains, RT2316-7 and RT2316-13, were selected to test the effect of the incubation temperature (at 5°C for 14 days and at 15°C for 5 days). Incubation temperature had little effect on the lipid content and biomass yield; however, its effect on the fatty acid composition was significant (p < .05). The low incubation temperature favored the accumulation of eicosapentaenoic acid (EPA), palmitic acid and stearic acid in the total lipids of RT2316-7. Percentage of EPA, DHA and the omega-6 fatty acid dihomo-γ-linolenic acid of total fatty acids of RT2316-13 was higher at the low incubation temperature. RT2316-13 accumulated the highest lipid content (30.0 ± 0.5%) with a carbon to nitrogen mass ratio equal to 16.9. On the contrary, lipid accumulation in RT2316-7 occurred at high concentration of the nitrogen sources (monosodium glutamate or yeast extract). The capability to accumulate lipids with a fatty acid profile that can be tuned through cultivation temperature make the Antarctic thraustochytrid RT2316-13 a candidate for the production of lipids with different uses.

Marked changes in diversity and relative activity of picoeukaryotes with depth in the world ocean.

Microbial eukaryotes are key components of the ocean plankton. Yet, our understanding of their community composition and activity in different water layers of the ocean is limited, particularly for picoeukaryotes (0.2-3 µm cell size). Here, we examined the picoeukaryotic communities inhabiting different vertical zones of the tropical and subtropical global ocean: surface, deep chlorophyll maximum, mesopelagic (including the deep scattering layer and oxygen minimum zones), and bathypelagic. Communities were analysed by high-tthroughput sequencing of the 18S rRNA gene (V4 region) as represented by DNA (community structure) and RNA (metabolism), followed by delineation of Operational Taxonomic Units (OTUs) at 99% similarity. We found a stratification of the picoeukaryotic communities along the water column, with assemblages corresponding to the sunlit and dark ocean. Specific taxonomic groups either increased (e.g., Chrysophyceae or Bicosoecida) or decreased (e.g., Dinoflagellata or MAST-3) in abundance with depth. We used the rRNA:rDNA ratio of each OTU as a proxy of metabolic activity. The highest relative activity was found in the mesopelagic layer for most taxonomic groups, and the lowest in the bathypelagic. Altogether, we characterize the change in community structure and metabolic activity of picoeukaryotes with depth in the global ocean, suggesting a hotspot of activity in the mesopelagic.

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.

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.

Flow Cytometry for Rapid Enumeration and Biomass Quantification of Thraustochytrids in Coastal Seawaters.

Marine fungus-like eukaryotic unicellular protists (thraustochytrids) are considered to play an important role in the marine microbial food web. However, their abundance, distribution, and relative biomass in coastal waters have not yet been examined in detail. By using a flow cytometry method (FCM) for the rapid enumeration of thraustochytrids in nearshore and offshore stations along the Gulf of Bohai, China, we herein expanded current knowledge on their ecological significance. The FCM method allows for the rapid detection and quantification of prokaryotic and eukaryotic cells, but is rarely applied to the enumeration of small eukaryotic protists. Epifluorescence microscopy (EpiM) has been commonly used for the direct detection and enumeration of thraustochytrids; however, this method is time-consuming and inapplicable to a large-scale analysis of complex seawater samples. There is no available FCM method to track the abundance and biomass of thraustochytrids in marine habitats. The FCM enumeration of thraustochytrids in seawater samples ranged between 400 and 4,080 cells mL-1 with a biomass range of 8.15-83.96 µg C L-1. The thraustochytrid biomass contributed 10.9% to 98.1% of the total biomass of the heterotrophic microbial community comprising bacterioplankton and thraustochytrids. Their overall abundance in nearshore stations was significantly different from that in offshore stations (P<0.5). The present results provide an optimized method for the rapid detection and enumeration of thraustochytrids in seawater and facilitate large-scale studies of the ecological role of thraustochytrids in the microbial food web of coastal waters.

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.

Development of a qPCR assay to detect and quantify ichthyotoxic flagellates along the Norwegian coast, and the first Norwegian record of Fibrocapsa japonica (Raphidophyceae).

Blooms of ichthyotoxic microalgae pose a great challenge to the aquaculture industry world-wide, and there is a need for fast and specific methods for their detection and quantification in monitoring programs. In this study, quantitative real-time PCR (qPCR) assays for the detection and enumeration of three ichthyotoxic flagellates: the dinoflagellate Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup and the two raphidophytes Heterosigma akashiwo (Hada) Hada ex Hara & Chihara and Fibrocapsa japonica Toriumi & Takano were developed. Further, a previously published qPCR assay for the dinoflagellate Karlodinium veneficum (Ballantine) Larsen was used. Monthly samples collected for three years (Aug 2009-Jun 2012) in outer Oslofjorden, Norway were analysed, and the results compared with light microscopy cell counts. The results indicate a higher sensitivity and a lower detection limit (down to 1 cell L-1) for both qPCR assays. Qualitative and semi-quantitative results were further compared with those obtained by environmental 454 high throughput sequencing (HTS, metabarcoding) and scanning electron microscopy (SEM) examination from the same samplings. All four species were detected by qPCR and HTS and/or SEM in outer Oslofjorden (Aug 2009-Jun 2012); Karlodinium veneficum was present year-round, whereas Karenia mikimotoi, Heterosigma akashiwo and Fibrocapsa japonica appeared mainly during the autumn in all three years. This is the first observation of Fibrocapsa japonica in Norwegian coastal waters. This species has previously been recorded off the Swedish west coast and German Bight, which may suggest a northward dispersal.

Isolation of High Carotenoid-producing Aurantiochytrium sp. Mutants and Improvement of Astaxanthin Productivity Using Metabolic Information.

The marine eukaryotic microheterotroph thraustochytrid genus Aurantiochytrium is a known producer of polyunsaturated fatty acids, carotenoids, and squalene. We previously constructed a lipid fermentation system for Aurantiochytrium sp. strains using underutilized biomass, such as canned syrup and brown macroalgae. To improve the productivity, in this study, Aurantiochytrium sp. RH-7A and RH-7A-7 that produced high levels of carotenoids, such as astaxanthin and canthaxanthin, were isolated through chemical mutagenesis. Moreover, metabolomic analysis of the strain RH-7A revealed that oxidative stress impacts carotenoid accumulation. Accordingly, the addition of ferrous ion (Fe2+), as an oxidative stress compound, to the culture medium significantly enhanced the production of astaxanthin by the mutants. These approaches improved the productivity of astaxanthin up to 9.5 mg/L/day at the flask scale using not only glucose but also fructose which is the main carbon source in fermentation systems with syrup and brown algae as the raw materials.

Comparison of loop-mediated isothermal amplification with hyperbranched rolling circle amplification as a simple detection method for Heterosigma akashiwo.

The fish-killing alga Heterosigma akashiwo is a globally distributed, toxic, and bloom-forming raphidophyte that has caused great losses to the fishing industry in many coastal countries. Therefore, rapid and sensitive detection methods should be developed to present timely warning of harmful algal blooms. In this study, hyperbranched rolling circle amplification (HRCA) was established for the detection of H. akashiwo and compared with loop-mediated isothermal amplification (LAMP) in terms of specificity and sensitivity. The partial D1-D2 sequence of the large subunit (LSU) of rDNA of H. akashiwo was used to design a specific padlock probe for HRCA and two pairs of specific primers for LAMP. The parameters for HRCA were optimized. Cross-reactivity tests showed that the specificity of the developed HRCA for H. akashiwo was greater than that of LAMP in this study. The sensitivities of HRCA and LAMP were comparable and were 10-fold higher than that of regular PCR. These methods also yielded a detection limit of 20 fg/µL for the recombinant plasmid containing the target LSU D1-D2 and 1 cell for target species. The test with the simulated field samples indicated that the developed HRCA obtained a detection limit of 5 cells mL-1, which was lower than the warning cell density (100 cells mL-1) of H. akashiwo. The visual detection of positive HRCA could be achieved via coloration reaction with the addition of fluorescent SYBR Green I dye to the amplification products. The developed HRCA was also efficient for field samples with target cell densities ranging from 10 cells mL-1 to 1000 cells mL-1. Therefore, the proposed HRCA detection protocols are possibly applicable to the field monitoring of H. akashiwo.

A Re-investigation of Sarcinochrysis marina (Sarcinochrysidales, Pelagophyceae) from its Type Locality and the Descriptions of Arachnochrysis, Pelagospilus, Sargassococcus and Sungminbooa genera nov.

Systematists increasingly use molecular markers to identify species; however, most microalgae were described before gene sequencing and type specimens were often ink drawings. Cryptic speciation and biogeographic isolation are other potential problems when anchoring an old species name with a modern gene sequence. Therefore when biological type material is absent, the best approach is to recollect the alga from the type locality and sequence genes. Sarcinochrysis marina, described in 1930 by Geitler from the Canary Islands, Spain, is the oldest Pelagophyceae genus. Geitler used two cultures in his study, but these cultures no longer exist. We re-isolated S. marina from the type locality near Las Palmas, Gran Canaria. Furthermore, we included additional Pelagophyceae strains that were isolated from natural habitats for this study or were obtained from culture collections. We produced 85 sequences, representing the nuclear-encoded SSU rRNA and the plastid-encoded rbcL, psaA, psaB, psbA, and psbC genes. The sequences were used to infer maximum likelihood phylogenetic trees. We anchored the name Sarcinochrysis marina using the Las Palmas isolate, and we described four new genera (Arachnochrysis, Pelagospilus, Sargassococcus, Sungminbooa) and nine new species in the Sarcinochrysidales. We also described a new family, Chrysocystaceae, based upon molecular phylogenetic analyses.