Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution.

BACKGROUND: The class Diphyllatea belongs to a group of enigmatic unicellular eukaryotes that play a key role in reconstructing the morphological innovation and diversification of early eukaryotic evolution. Despite its evolutionary significance, very little is known about the phylogeny and species diversity of Diphyllatea. Only three species have described morphology, being taxonomically divided by flagella number, two or four, and cell size. Currently, one 18S rRNA Diphyllatea sequence is available, with environmental sequencing surveys reporting only a single partial sequence from a Diphyllatea-like organism. Accordingly, geographical distribution of Diphyllatea based on molecular data is limited, despite morphological data suggesting the class has a global distribution. We here present a first attempt to understand species distribution, diversity and higher order structure of Diphyllatea. RESULTS: We cultured 11 new strains, characterised these morphologically and amplified their rRNA for a combined 18S-28S rRNA phylogeny. We sampled environmental DNA from multiple sites and designed new Diphyllatea-specific PCR primers for long-read PacBio RSII technology. Near full-length 18S rRNA sequences from environmental DNA, in addition to supplementary Diphyllatea sequence data mined from public databases, resolved the phylogeny into three deeply branching and distinct clades (Diphy I - III). Of these, the Diphy III clade is entirely novel, and in congruence with Diphy II, composed of species morphologically consistent with the earlier described Collodictyon triciliatum. The phylogenetic split between the Diphy I and Diphy II + III clades corresponds with a morphological division of Diphyllatea into bi- and quadriflagellate cell forms. CONCLUSIONS: This altered flagella composition must have occurred early in the diversification of Diphyllatea and may represent one of the earliest known morphological transitions among eukaryotes. Further, the substantial increase in molecular data presented here confirms Diphyllatea has a global distribution, seemingly restricted to freshwater habitats. Altogether, the results reveal the advantage of combining a group-specific PCR approach and long-read high-throughput amplicon sequencing in surveying enigmatic eukaryote lineages. Lastly, our study shows the capacity of PacBio RS when targeting a protist class for increasing phylogenetic resolution.

Correction to: Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution.

In the original publication of this article [1] there was an error in an author name. In this correction article the correct and incorrect name are indicated.

Identification of a Major Lipid Droplet Protein in a Marine Diatom Phaeodactylum tricornutum.

Various kinds of organisms, including microalgae, accumulate neutral lipids in distinct intracellular compartments called lipid droplets. Generally, lipid droplets are generated from the endoplasmic reticulum, and particular proteins localize on their surface. Some of these proteins function as structural proteins to prevent fusion between the lipid droplets, and the others could have an enzymatic role or might be involved in intracellular membrane trafficking. However, information about lipid droplet proteins in microalgae is scarce as compared with that in animals and land plants. We focused on the oil-producing, marine, pennate diatom Phaeodactylum tricornutum that forms lipid droplets during nitrogen deprivation and we investigated the proteins located on the lipid droplets. After 6 d of cultivation in a nitrate-deficient medium, the mature lipid droplets were isolated by sucrose density gradient centrifugation. Proteomic analyses revealed five proteins, with Stramenopile-type lipid droplet protein (StLDP) being the most abundant protein in the lipid droplet fraction. Although the primary sequence of StLDP did not have homology to any known lipid droplet proteins, StLDP had a central hydrophobic domain. This structural feature is also detected in oleosin of land plants and in lipid droplet surface protein (LDSP) of Nannochloropsis species. As a proline knot motif of oleosin, conservative proline residues existed in the hydrophobic domain. StLDP was up-regulated during nitrate deprivation, and fluctuations of StLDP expression levels corresponded to the size of the lipid droplets.

Therapeutic Potential of Seaweed-Derived Laminaran: Attenuation of Clinical Drug Cytotoxicity and Reactive Oxygen Species Scavenging.

ß-glucan has been shown to be effective for several diseases such as immune regulation and blood pressure suppression. Seaweed contains a ß-1,3/1,6-glucan called laminaran. The present commercial source of ß-glucan is black yeast; however, a fermentation process using organic carbon substrates makes production unsustainable, whereas macroalgae provide a sustainable alternative with the use of CO2 and seawater as growth substrates. However, bioactivity studies on laminaran are limited. We aimed to evaluate whether laminaran can scavenge reactive oxygen species (ROS) and attenuate cytotoxicity caused by clinical drugs such as indomethacin (Ind) and dabigatran (Dab). Electron spin resonance assay revealed that laminaran scavenged singlet oxygen (1O2) and superoxide anions (O2•-) directly but did not scavenge hydroxyl radicals (•OH). Mitochondrial ROS detection dye showed that laminaran scavenged mitochondrial O2•- produced upon administration of Ind or Dab. Moreover, significant reductions in •OH and peroxynitrate (ONOO-) levels were observed. Since •OH and ONOO- are generated from O2•- in the cells, laminaran could indirectly suppress the generation of •OH and ONOO- via the removal of O2•-. Both Ind and Dab induce cell injury via ROS production. Laminaran attenuated the cytotoxicity derived from these drugs and may represent a functional food with anti-aging and disease prevention properties.

Thraustochytrid Aurantiochytrium sp. 18W-13a accummulates high amounts of squalene.

Here we report on the 18W-13a strain of Aurantiochytrium sp., which accumulates very high amounts of squalene. The squalene contents and production at 4 d of culture were 198 mg/g and 1.29 ± 0.13 g/L, respectively, exceptionally high values compared to previous reports.

A Novel Polyculture Growth Model of Native Microalgal Communities to Estimate Biomass Productivity for Biofuel Production.

Native polyculture microalgae is a promising scheme to produce microalgal biomass as biofuel feedstock in an open raceway pond. However, predicting biomass productivity of native polycultures microalgae is incredibly complicated. Therefore, developing polyculture growth model to forecast biomass yield is indispensable for commercial-scale production. This research aims to develop a polyculture growth model for native microalgal communities in the Minamisoma algae plant and to estimate biomass and biocrude oil productivity in a semi-continuous open raceway pond. The model was built based on monoculture growth of polyculture species and it is later formulated using species growth, polyculture factor (k value ), initial concentration, light intensity, and temperature. In order to calculate species growth, a simplified Monod model was applied. In the simulation, 115 samples of the 2014-2015 field dataset were used for model training, and 70 samples of the 2017 field dataset were used for model validation. The model simulation on biomass concentration showed that the polyculture growth model with k value had a root-mean-square error of 0.12, whereas model validation provided a better result with a root-mean-square error of 0.08. Biomass productivity forecast showed maximum productivity of 18.87 g/m2 /d in June with an annual average of 13.59 g/m2 /d. Biocrude oil yield forecast indicated that hydrothermal liquefaction process was more suitable with a maximum productivity of 0.59 g/m2 /d compared with solvent extraction which was only 0.19 g/m2 /d. With satisfactory root mean square errors less than 0.3, this polyculture growth model can be applied to forecast the productivity of native microalgae. This article is protected by copyright. All rights reserved.

Deciphering functional biomolecule potential of marine diatoms through complex network approach.

Marine diatoms are unique reservoirs of bioactive compounds having enormous applications in therapeutics. But high-throughput screening methods are needed to elucidate the interaction between numerous biomolecules and their targets, facilitating rapid screening for novel drug molecules. So, in the present study chemical constituents were extracted from five marine diatoms using un-targeted metabolite profiling and in-silico virtual screening bioinformatics was employed to predict their bioactivity and molecular targets. A total of 17 chemical constituents out of 51 showed interactions with 76 protein targets associated with 213 pathways. Ingredient-target-pathway network revealed oleic acid, linoleic acid and cholest-5-en-3-ol as major active constituents. Core subnetwork and protein association network showed involvement of these compounds in key metabolic pathways related to cell signaling, cell growth and metabolism of xenobiotics. Thus, the present study for the first time revealed the main active ingredients and their associated pathways from marine diatoms using complex network approach.

Characteristic terahertz absorption spectra of paramylon and paramylon-ester compounds.

Paramylon is a long-chain polysaccharide, composed of glucose units connected via ß-(1,3) glycosidic bonds, that spontaneously forms a three-strand helical bundle. Paramylon-esters can be made by partially or fully replacing saccharide chain hydroxide groups with carboxylic functional groups, such as stearoyl (CH3(CH2)16CO) and palmitoyl (CH3(CH2)24CO). The paramylon-ester with carboxylic acids has superior characteristics, including high thermal resistance, stability and transparency under visible light, which are necessary for thermoplastic applications. In this study, the absorption coefficient α(ν) and absorbance spectra of paramylons and paramylon-esters were measured in the 0.3-8.0 THz range and compared with the corresponding spectra of glucose and cellulose. Paramylon and paramylon-ester molecules were found to exhibit unique, so-called fingerprint, α(ν)peaks at 4.0, 6.0 and 8.0 THz, and 2.5 and 5.0 THz, respectively. We speculate that the spectral features observed are owing to intermolecular interaction modes of the weakly coupled polysaccharide chains. The paramylons with different molecular weights show very similar absorption features in the low-frequency side, both in spectral shapes and intensities, indicating that absorption is independent of molecular size. The paramylon-esters with varying degrees of substitution (DS) are similar spectral shapes but different intensities. A linear correlation between α(ν) peak intensity and the DS of paramylon-esters was established with the R2 value above 0.99. This behavior can be used for the detection and identification of novel paramylon-ester molecules.

Molecular vibration and Boson peak analysis of glucose polymers and ester via terahertz spectroscopy.

Complex permittivity spectra were obtained herein by performing broadband terahertz (THz) spectroscopy on cellulose, paramylon, and paramylon ester. Absorption peaks observed for cellulose and paramylon at approximately 3 THz are attributed to hydrogen bonds. In addition, a broad absorption peak around 2 THz was observed for all the polymers, demonstrating a general feature of polymer glasses derived from weak interatomic van der Waals forces. The boson peak was observed for cellulose and paramylon ester. The boson peak frequency for cellulose nearly equaled that for glassy glucose-a unit structure of the cellulose polymer. Additionally, the insensitivity of cellulose to the polymerization degree was consistent with recent results obtained via molecular dynamics simulations. In contrast, the boson peak frequency of paramylon ester was markedly smaller than that of cellulose. These results demonstrate the importance of hydrogen bonds as determinants of the boson peak frequency.

Recombination, cryptic clades and neutral molecular divergence of the microcystin synthetase (mcy) genes of toxic cyanobacterium Microcystis aeruginosa.

BACKGROUND: The water-bloom-forming cyanobacterium Microcystis aeruginosa is a known producer of various kinds of toxic and bioactive chemicals. Of these, hepatotoxic cyclic heptapeptides microcystins have been studied most intensively due to increasing concerns for human health risks and environmental damage. More than 70 variants of microcystins are known, and a single microcystin synthetase (mcy) gene cluster consisting of 10 genes (mcyA to mcyJ) has been identified to be responsible for the production of all known variants of microcystins. Our previous multilocus sequence typing (MLST) analysis of the seven housekeeping genes indicated that microcystin-producing strains of M. aeruginosa are classified into two phylogenetic groups. RESULTS: To investigate whether the mcy genes are genetically structured similarly as in MLST analysis of the housekeeping genes and to identify the evolutionary forces responsible for the genetic divergence of these genes, we used 118 mcy-positive isolates to perform phylogenetic and population genetic analyses of mcy genes based on three mcy loci within the mcy gene cluster (mcyD, mcyG, and mcyJ), none of which is involved in the production of different microcystin variants. Both individual phylogenetic analysis and multilocus genealogical analysis of the mcy genes divided our isolates into two clades, consistent with the MLST phylogeny based on seven housekeeping loci. No shared characteristics within each clade are known, and microcystin analyses did not identify any compositional trend specific to each clade. Statistical analyses for recombination indicated that recombination among the mcy genes is much more frequent within clades than between, suggesting that recombination has been an important force maintaining the cryptic divergence of mcy genes. On the other hand, a series of statistical tests provided no strong evidence for selection to explain the deep divergence of the mcy genes. Furthermore, analysis of molecular variance (AMOVA) indicated a low level of geographic structuring in the genetic diversity of mcy. CONCLUSION: Our phylogenetic analyses suggest that the mcy genes of M. aeruginosa are subdivided into two cryptic clades, consistent with the phylogeny determined by MLST. Population genetic analyses suggest that these two clades have primarily been maintained as a result of homology-dependent recombination and neutral genetic drift.

Anti-Inflammatory Effects of Aurantiochytrium limacinum 4W-1b Ethanol Extract on Murine Macrophage RAW264 Cells.

Aurantiochytrium limacinum 4W-1b (AL4W-1b) is a newly discovered microalgal strain with unique features. In the present study, we investigated the effects of ethanol extracts of AL4W-1b on lipopolysaccharide- (LPS-) induced inflammatory responses in RAW264 murine macrophage cells. Pretreatment of RAW264 cells with the AL4W-1b extract significantly reduced the production of LPS-induced nitric oxide (NO) and the expression of proinflammatory cytokine genes, including tumor necrosis factor α, interleukin- (IL-) 1ß, and IL-6. Treatment with the AL4W-1b extract also decreased the production of IL-1ß and IL-6. These results suggest that AL4W-1b might have anti-inflammatory effects in RAW264 cells. The NF-κB inhibitor, BAY 11-7082, synergistically prevented LPS-induced NO production after pretreatment with the AL4W-1b extract. Thus, the AL4W-1b extract may affect not only the NF-κB pathway but also other inflammatory pathways. To the best of our knowledge, this is the first study to report the anti-inflammatory effects of AL4W-1b extract and its mechanism of action in LPS-stimulated murine macrophage cells.

Origins of plastids and glyceraldehyde-3-phosphate dehydrogenase genes in the green-colored dinoflagellate Lepidodinium chlorophorum.

The dinoflagellate Lepidodinium chlorophorum possesses "green" plastids containing chlorophylls a and b (Chl a+b), unlike most dinoflagellate plastids with Chl a+c plus a carotenoid peridinin (peridinin-containing plastids). In the present study we determined 8 plastid-encoded genes from Lepidodinium to investigate the origin of the Chl a+b-containing dinoflagellate plastids. The plastid-encoded gene phylogeny clearly showed that Lepidodinium plastids were derived from a member of Chlorophyta, consistent with pigment composition. We also isolated three different glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes from Lepidodinium-one encoding the putative cytosolic "GapC" enzyme and the remaining two showing affinities to the "plastid-targeted GapC" genes. In a GAPDH phylogeny, one of the plastid-targeted GapC-like sequences robustly grouped with those of dinoflagellates bearing peridinin-containing plastids, while the other was nested in a clade of the homologues of haptophytes and dinoflagellate genera Karenia and Karlodinium bearing "haptophyte-derived" plastids. Since neither host nor plastid phylogeny suggested an evolutionary connection between Lepidodinium and Karenia/Karlodinium, a lateral transfer of a plastid-targeted GapC gene most likely took place from a haptophyte or a dinoflagellate with haptophyte-derived plastids to Lepidodinium. The plastid-targeted GapC data can be considered as an evidence for the single origin of plastids in haptophytes, cryptophytes, stramenopiles, and alveolates. However, in the light of Lepidodinium GAPDH data, we need to closely examine whether the monophyly of the plastids in the above lineages inferred from plastid-targeted GapC genes truly reflects that of the host lineages.

Draft Genome Sequence of "Candidatus Phycosocius bacilliformis," an Alphaproteobacterial Ectosymbiont of the Hydrocarbon-Producing Green Alga Botryococcus braunii.

"Candidatus Phycosocius bacilliformis" is an alphaproteobacterial ectosymbiont of the hydrocarbon-producing green alga Botryococcus braunii We sequenced the whole genome of "Ca. P. bacilliformis" BOTRYCO-2, isolated from a two-membered culture with B. braunii The genome contains approximately 3.3 Mb, with an average G+C content of 56.91% and 3,125 predicted protein-coding genes.

Adaptation of the Freshwater Bloom-Forming Cyanobacterium Microcystis aeruginosa to Brackish Water Is Driven by Recent Horizontal Transfer of Sucrose Genes.

Microcystis aeruginosa is a bloom-forming cyanobacterium found in eutrophic water bodies worldwide. M. aeruginosa blooms usually occur in freshwater; however, they have also been reported to occur in brackish water. Because M. aeruginosa often produces the cyanotoxin microcystin, they are a major concern to public health and environment. Despite this, the ecology, genomic basis, and evolutionary process underlying the M. aeruginosa bloom invasion from fresh to brackish water have been poorly investigated. Hence, in the present study, we have sequenced and characterized genomes of two newly discovered salt-tolerant M. aeruginosa strains obtained from Japanese brackish water lakes (Lakes Shinji and Tofutsu). Both genomes contain a set of genes for the synthesis of osmolyte sucrose (sppA, spsA, and susA), hitherto identified in only one strain (PCC 7806) of M. aeruginosa. Chemical and gene expression analyses confirmed sucrose accumulation induced by salt. A comprehensive genetic survey of >200 strains indicated that sucrose genes are extremely rare in M. aeruginosa. Most surprisingly, comparative genome analyses of the three strains indicated extremely low genetic diversity in the sucrose genes compared with other core genome genes, suggesting very recent acquisitions via horizontal transfer. Invasion of M. aeruginosa blooms into brackish water may be a recent event triggered by anthropogenic eutrophication of brackish water.

Ethanol Extract of Aurantiochytrium mangrovei 18W-13a Strain Possesses Anti-inflammatory Effects on Murine Macrophage RAW264 Cells.

In this study, the effects of an ethanolic extract of Aurantiochytrium mangrovei 18W-13a strain (AM18W-13a) on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264 murine macrophages were studied. Pre-treatment with the AM18W-13a extract significantly suppressed the LPS-induced production of nitric oxide and pro-inflammatory cytokines. RAW264 cells treated with the AM18W-13a extract for 1 and 24 h were subjected to DNA microarray analyses for detecting the differentially expressed genes. The treatment of RAW264 cells with the AM18W-13a extract for 24 h significantly suppressed the expression of several genes associated with inflammation or chemotaxis. Furthermore, treatment with the AM18W-13a extract for 1 h suppressed the expression of Pde4b, but induced the expression of Egr2 and Egr3 in RAW264 cells. Additionally, the AM18W-13a extract significantly enhanced the expression of certain anti-inflammatory mediators. This study is the first report of the anti-inflammatory effects of the AM18W-13a extract and its mechanism of action in LPS-stimulated murine macrophages.

Nutrient recycle from defatted microalgae (Aurantiochytrium) with hydrothermal treatment for microalgae cultivation.

Defatted heterotrophic microalgae (Aurantiochytrium limacinum SR21) was treated with high temperature water (175-350°C, 10-90min) to obtain nitrogen and phosphorous nutrients as a water soluble fraction (WS). Yields of nitrogen and phosphorous recovered in WS varied from 38 to 100% and from 57 to 99%, respectively. Maximum yields of nitrogen containing compounds in WS were proteins (43%), amino acids (12%) and ammonia (60%) at treatment temperatures of 175, 250 and 350°C, respectively. Maximum yield of phosphorous in WS was 99% at a treatment temperature of 250°C. Cultivation experiments of microalgae (A. limacinum SR21) using WS obtained at 200 and 250°C showed positive growth. Water soluble fractions from hydrothermal treatment of defatted microalgae are effective nitrogen and phosphorous nutrient sources for microalgae cultivation.

UV protection in the photosymbiotic ascidian Didemnum molle inhabiting different depths.

Didemnum molle is a colonial ascidian that harbors the prokaryotic photosymbiont Prochloron in its cloacal cavity. Colonies occur over a relatively wide bathymetric range (approximately 0-30 m), and colony color is widely variable, partly depending on depth. Colonies in shallow sites are bright white, with densely distributed spicules, and often with brown or dark gray pigmentation, while colonies in deeper sites are less pigmented, with sparsely distributed spicules. Didemnum molle colonies contain mycosporine-like amino acids (MAAs) as UV-absorbing substances. These include mycosporine-glycine, shinorine, and porphyra-334. Among colonies from 5-, 10-, 15-, and 20-m depths, the concentration of total MAAs was significantly high at 10 m and low at 20 m. Colonies at 10 m need to maintain low spicule densities to have enough photosynthetically active radiation (PAR) to maintain the photosymbionts, and they probably concentrate MAAs to block UV radiation without attenuating PAR. Because high levels of PAR cause photoinhibition of photosynthesis, spicules and pigment cells would be more effective for photoprotection in shallow water. Colonies of D. molle may adjust the light conditions for photosymbionts by combining MAAs, spicules, and pigment cells in varying amounts.

Fibrophrys columna gen. nov., sp. nov: A member of the family Amphifilidae.

A novel Diplophrys-like organism, Fibrophrys columna, was isolated from Hiuchigaike Pond in Japan. F. columna showed a nearly orbicular or broadly elliptical cell shape and has fine filamentous, branching ectoplasmic elements emanating from both polar ends of the cell. Cells also contain orange, amber, or colorless lipid bodies. Although its whole cell morphology resembles that of the genus Diplophrys, Fibrophrys is clearly distinct from Diplophrys on the basis of 18S rDNA sequences. Molecular phylogenetic analysis showed a close relationship of F. columna with Amphifila marina, and its sequence is similar to many environmental stramenopile sequences. The cells of F. columna measured 5.0-8.3×5.6-10.3µm and sometimes possessed hernia-like prongs instead of filamentous ectoplasmic elements. An axis-like electron-dense body was observed in the mitochondria. We also studied the ultrastructure of another Fibrophrys strain, Fibrophrys sp. E-1, which is different from the type strain of F. columna. A ladder-like pattern was recognized in the outer part of unidentified cytoplasmic membranes connected with the mitochondria. The unidentified cytoplasmic membranes were connected to the nuclear, lipid body, and mitochondrial outer membranes. We propose a new genus, Fibrophrys, and a new species, F. columna, based on these ultrastructural and molecular features.

Evolutionary relationships among basal fungi (Chytridiomycota and Zygomycota): Insights from molecular phylogenetics.

Evolutionary relationships of the two basal fungal phyla Chytridiomycota and Zygomycota are reviewed in light of recent molecular phylogenetic investigation based on rDNA (nSSU, nLSU rDNA), entire mitochondrial genomes, and nuclear protein coding gene sequences (e.g., EF-1alpha, RPB1). Accumulated molecular evidence strongly suggests that the two basal fungal phyla are not monophyletic. For example, the chytridiomycete order Blastocladiales appears to be closely related to the zygomycete order Entomophthorales. Within the Zygomycota, a monophyletic clade, consisting of the Dimargaritales, Harpellales, and Kickxellales, which is characterized by a shared unique septal ultrastructure, was identified. Moreover, evidence for the exclusion of zygomycete orders Amoebidiales and Eccrinales from the Fungi, and their placement at the Animal-Fungi boundary has been clearly documented. Microsporidia, a group of amitochondriate organisms currently under intensive study, is not supported as derived within the Fungi, but a fungal affinity cannot be ruled out. Taking these molecular phylogenetic studies into account, we proposed a hypothetical evolutionary framework of basal fungi.

Catalytic gasification of oil-extracted residue biomass of Botryococcus braunii.

Catalytic gasification of the oil-extracted residue biomass of Botryococcus braunii was demonstrated in a laboratory-scale continuous feeding dual bed reactor. Steam gasification at 1023 K over Ni-Fe/Mg/Al catalyst can completely reform tar derived from pyrolysis of the residue biomass into C1 gases and hydrogen, and has achieved 91%-C conversion to gaseous product (CO+CO2+CH4). Composition of product gas has higher contents of CO and H2 with their ratio (H2/CO) of around 2.4 which is slightly H2-rich syngas. Maximum hydrogen yield of 74.7 mmol g-biomass(-1) obtained in this work is much higher than that from gasification of other algal biomass reported in literature. The residue biomass of B. braunii can be a superior renewable source of syngas or hydrogen.