Axenic culture of Gyrodinium impudicum strain KG03, a marine red-tide microalga that produces exopolysaccharide

An exopolysaccharide-producing microalgal dinoflagellate was isolated from a red-tide bloom and designated strain KG03. A bacteria-free culture of strain KG03 was achieved using a modified wash with phototaxis and antibiotic treatment. Combined treatment with neomycin and cephalosporin was the most effective for eliminating the bacteria associated with the microalgae. Strain KG03 was identified as Gyrodinium impudicum by analyzing the ITS regions of the 5.8S rDNA, 18S rDNA, morphological phenotype and fatty acid composition. The exopolysaccharide production and cell growth in a 300-ml photobioreactor were increased 2.7- and 2.4-fold, respectively, compared with that in a flask culture at the first isolation step.     

Optimal conditions for the production of sulfated polysaccharide by marine microalga Gyrodinium impudicum strain KG03

A marine microalga Gyrodinium impudicum strain KG03 produced sulfated exopolysaccharide designated as p-KG03, which showed a strong antiviral activity against encephalomyocarditis virus (EMCV). To optimize culture conditions for the production of p-KG03, mineral salts, vitamins, plant growth hormones, temperature, pH and light conditions were examined. From this study, M-KG03 medium for the maximum production of p-KG03 was suggested as follows; NH(4)Cl 75 microM, NaH(3)PO(4) 200 microM, NaHCO(3) 50 microM, Na(2)SO(4) 10 microM, FeCl(2) x 6H(2)O 10 microM, MnCl(2) x 4H(2)O 0.1 microM, vitamin B(12) 0.75 microg, naphthalene acetic acid (NAA) 7.5 microg and myo-inositol 200 mg per liter of aged sea water. The optimal temperature and pH were 22.5 degrees C and 8.0, respectively. The optimal light conditions of intensity and period were 150 microE m(-2) s(-1) and 16:8 h light:dark cycle. Finally, the cell growth and p-KG03 production were measured in one liter of M-KG03 medium with 1% CO(2) and 50 ml min(-1) of airflow using two liters airlift balloon type photobioreactor (ABTPR). At these optimal conditions, p-KG03 production and cell growth were 134.6+/-5.9 mg l(-1) and 123,076+/-1,597 cells ml(-1), respectively, representing a 7.7 and 5.1 times compared with f/2 medium with Erlenmeyer flask culture (p-KG03 production 17.5+/-1.3 mg l(-1) and cell growth 24,311+/-1,291 cells ml(-1)).     

Novel sulfated polysaccharide derived from red-tide microalga Gyrodinium impudicum strain KG03 with immunostimulating activity in vivo

The high-sulfate-containing exopolysaccharide p-KG03 is produced by the red-tide microalga Gyrodinium impudicum strain KG03. The immunostimulatory effects of this sulfated exopolysaccharide were investigated by isolating peritoneal macrophages from mice 10 or 20 days after they had received a single dose of p-KG03 (100 or 200 mg/kg body weight). The cytotoxicity of the isolated macrophages for B16 tumor cells was tested, as B16 tumor cells are sensitive to tumor necrosis factor α (TNF-α) and nitric oxide. The activities of natural killer cells from the p-KG03-treated mice against YAC-1 mouse lymphoma cells were also tested. The nonspecific immune functions mediated by natural killer cells and macrophages were increased by treatment with p-KG03 in vivo. These results suggest that p-KG03 has immunostimulatory effects and enhances the tumoricidal activities of macrophages and NK cells in vivo. In addition, p-KG03 treatment increased the plaque-forming cell response to sheep red blood cells, as well as the levels of IgM and IgG Exposure to p-KG03 also increased the production by macrophages of cytokines, such as interleukins -1β and -6, and TNF-α. This is the first report of a marine microalgal sulfated polysaccharide having immunostimulatory activities. The p-KG03 polysaccharide may be useful for the development of biotechnological and pharmaceutical products that incorporate bioactive marine exopolysaccharides.     

Cyst morphology of a chain-forming unarmored dinoflagellate Gyrodinium impudicum Fraga et Bravo

Cysts of a chain‐forming dinoflagellate Gyrodinium impudicum Fraga et Bravo (Gymnodiniales) were found in surface sediments of Harima‐Nada and Nakaumi, western Japan. The detailed morphology of living and empty cysts is described. The living cysts are roundish to ellipsoidal in polar view, and hemispherical in lateral view. Among three empty cysts obtained, two different archeopyles were observed; either a long slit with an operculum, or a hole with irregular zigzag outline. The living cysts of Gyro. impudicum are morphologically similar to those of the genus Chattonella antiqua (Hada) Ono and Chattonella marina (Subrahmanyan) Y. Hara et Chihara (Raphidophyceae), except cyst color and contents. The living cysts of Gyro. impudicum were rarely encountered, and their density was always less than 1 cell in 1 cm^?3 in the present samples.     

Zooxanthellamide B, a novel large polyhydroxy metabolite from a marine dinoflagellate of Symbiodinium sp

Zooxanthellamide B, C(128)H(220)N(2)O(53)S(2), a polyhydroxy secondary metabolite, was isolated from a cultured marine dinoflagellate of the genus Symbiodinium. A detailed 2D NMR analysis revealed the chemical structure as a delta-lactone analogue of zooxanthellamide A, which had previously been isolated from the same dinoflagellate by us. The relative configuration of the delta-lactone moiety was determined by NOE experiments and a coupling constant analysis, and that of other ring systems was found to be the same as zooxanthellamide A by the chemical correlation between zooxanthellamides A and B.     

Gyrodinium undulans Hulburt, a marine dinoflagellate feeding on the bloom-forming diatomOdontella aurita, and on copepod and rotifer eggs

The marine dinoflagellateGyrodinium undulans was discovered as a feeder on the planktonic diatomOdontella aurita. Every year, during winter and early spring, a certain percentage of cells of this bloom-forming diatom, in the Wadden Sea along the North Sea coast, was regularly found affected by the flagellate. Supplied with the food diatomO. aurita the dinoflagellate could be maintained successfully in clonal culture. The vegetative life cylce was studied, mainly by light microscopy on live material, with special regard to the mode of food uptake. Food is taken up by a so-called phagopod, emerging from the antapex of the flagellate. Only fluid or tiny prey material could be transported through the phagopod. Larger organelles like the chloroplasts ofOdontella are not ingested and are left behind in the diatom cell. Thereafter, the detached dinoflagellate reproduces by cell division, occasionally followed by a second division. As yet, stages of sexual reproduction and possible formation of resting cysts could not be recognized, neither from wild material nor from laboratory cultures. Palmelloid stages (sometimes with a delicate wall) occurring in ageing cultures may at least partly function as temporary resting stages. The winter speciesG. undulans strongly resemblesSyltodinium listii, a summer species feeding on copepod and rotifer eggs. Surprisingly, in a few cases this prey material was accepted byG. undulans as well, at least under culture conditions. When fed with copepod eggs, the dinoflagellate developed into a large trophont, giving rise thereafter by repeated binary fission to 4, 8 or 16 flagellates, as a result of a single feeding act. A re-examination of both species under simultaneous culture conditions is planned.     

Predation by a photosynthetic dinoflagellate Gyrodinium instriatum on loricated ciliates

Feeding of a naked photosynthetic dinoflagellate, Gyrodiniuminstriatum, on loricated ciliates was investigated. Gyrodiniuminstriatum preyed on Favella azorica and Eutintinnus tubulosusby engulfment through the posterior end of the sulcus. In thecase of E.tubulosus, G.instriatum preyed on this small ciliatekeeping the original gymnodinioid cell shape. On the other hand,G.instriatum preyed on Favella taraikaensis by absorbing thecell contents of this large ciliate, which resulted in a balloonlike inflation of its body size. It seemed that G.instriatumcan change its feeding style according to the size of prey.Thus, the present study shows, by using direct observationson the feeding of G.instriatum on loricated ciliates, a reversalof energy flow processes in the food chain in which photosyntheticorganisms eat primary consumers. The growth of G.instriatumafter feeding on F.taraikaensis and E.tubulosus is also describedbriefly.     

Observations on pigments and morphology of Gyrodinium aureolum Hulburt, a marine dinoflagellate containing 19' -hexanoyloxyfucoxanthin as the main carotenoid

Gyrodinium aureolum, a common "red tide" dinoflagellate in Europeanwaters often associated with fish mortality, was isolated fromthe Oslofjord, Norway, and analysed for chlorophylls and carotenoids.Besides chlorophyll a and c the following carotenoids were characterizedby thin-layer chromatography, visible light spectrophotometryand mass spectrometry: ß,-carotene, ß,ß-carotene,djatoxanthin, diadinoxanthin, 19'-hexanoyloxyfucoxanthin and3 xanthophylls which could not be correlated with hitherto structurallyknown carotenoids from dinoflagellates. G. aureolum deviatesfrom most dinoflagellates by the lack of peridinin, but showsaffinity with Gyrodinium sp.-A by the possession of 19'-hexanoyloxyfucoxanthin. Preliminary light microscopical observations on the internalstructure indicate that G. aureolum is uni-nucleate with a typicaldinokaryotic nucleus containing continually condensed chromosomes.The chloroplasts seem to possess an internal pyrenoid like someother dinoflagellates with deviating carotenoid pigmentation.The similarity in carotenoid pigmentation and chloroplast structureof Emiliania huxleyi (Prymnesiophyceae) and Gyrodinium sp.-Aand G.aureolum (Dinophyceae) is pointed out. The potential chemotaxonomicvalue of the carotenoid composition in establishing identitywith morphologically similar and ichthyotoxic dinoflagellatesis briefly discussed.     

Lingshuiol, a novel polyhydroxyl compound with strongly cytotoxic activity from the marine dinoflagellate Amphidinium sp

A novel polyhydroxy compound with a linear carbon-chain, lingshuiol (1), had been isolated from the cultured marine dinoflagellate Amphidinium sp. Its structure was elucidated by extensive analysis of 2D NMR spectral data. Lingshuiol possessed a powerful cytotoxic activity against A-549 and HL-60 cells in vitro with the IC(50) of 0.21 and 0.23 microM, respectively.     

The Characterization of Scintillons : Bioluminescent particles from the marine dinoflagellate, Gonyaulax polyedra

A new type of biological particle, isolated from the marine dinoflagellate Gonyaulax polyedra, has been partially purified and characterized. When the pH is lowered, the particle emits light in vitro in a fashion closely mimicking the flash of the living cell, and it is referred to as a scintillon (flashing unit). Scintillons are obtained by breaking the cells in buffer at pH 8.2 and purifying by differential and sucrose density gradient centrifugation. The particle has a density of about 1.23 g cc^-1, and activity is quantitatively correlated with the number of crystal-like birhombohedral structures. These have been found to contain guanine, but since the density of authentic guanine is about 1.73 g cc^-1, the scintillon is believed to comprise additional but as yet unidentified components. The properties of the scintillon and the effects of various physical and chemical treatments are described. The reasons for believing that this particle is responsible for the flash of the intact cell are discussed.     

The circadian rhythm of a behavioral photoresponse in the dinoflagellate Gyrodinium dorsum

Summary The circadian rhythm of the photoresponse to blue light in the dinoflagellate Gyrodinium dorsum Kofoid was investigated by the use of a closed circuit television system. The initial cessation of movement upon stimulation (stop-response) was used as the index of light reception. Under constant dark conditions cells grown on a 12L:12D regime show an endogenous circadian rhythm in their stop-response with maximum responsiveness occurring approximately one hour before the beginning of the expected light phase. This rhythmic response was only observed if the cells were irradiated with red light (620 nm) prior to stimulation with blue light. After preirradiation both far-red reversibility and the shift in the stop-response action spectrum from 470 nm to 490 nm could also be demonstrated. These findings may be related to the diurnal migration of marine dinoflagellates.This study was supported by National Science Foundation grant GB 5137.     

Antiviral Effects of Sulfated Exopolysaccharide from the Marine Microalga <Emphasis Type="Italic">Gyrodinium impudicum</Emphasis> Strain KG03

The sulfated exopolysaccharide p-KG03, which is produced by the marine microalga Gyrodinium impudicum strain KG03, exhibited impressive antiviral activity in vitro (EC₅₀ = 26.9 µg/ml) against the encephalomyocarditis virus (EMCV). Depending on the p-KG03 concentration, the development of cytopathic effects in EMCV-infected HeLa cells was either inhibited completely or slowed. Moreover, p-KG03 did not show any cytotoxic effects on HeLa cells, even at concentrations up to 1000 µg/ml. The polysaccharide was purified by repeated precipitation in ethanol, followed by gel filtration. The p-KG03 polysaccharide had a molecular weight of 1.87 × 10⁷, and was characterized as a homopolysaccharide of galactose with uronic acid (2.96% wt/wt) and sulfate groups (10.32% wt/wt). The biological activities of p-KG03 suggest that sulfated metabolites from marine organisms are a rich source of antiviral agents. This is the first reported marine source of antiviral sulfated polysaccharides against EMCV. The p-KG03 polysaccharide may be useful in the development of marine bioactive exopolysaccharide for biotechnological and pharmaceutical products.     

Characterization of a bioflocculant produced by the marine myxobacterium Nannocystis sp. NU-2

The marine myxobacterium strain NU-2, which can grow on high concentrations (up to 7%) of NaCl, was isolated from a salt soil sample collected from the coast of the Huanghai Sea, China. Morphological properties and 16S rDNA sequence analysis indicated that the isolate is a novel species related to the genus Nannocystis. Nannocystis sp. NU-2 produced a new kind of flocculating substance in a starch medium with a yield of 14.8 g l^–1. The NU-2 flocculant was composed of 40.3% proteins and 56.5% polysaccharides, of which glucose, mannose and glucuronic acid were the principal constituents in the relative proportions of 5:4:1. The flocculation activity of the NU-2 flocculant depends strongly on cations such as Fe³⁺ and Al³⁺. When a 30 mg l^–1 FeCl₃ solution is present in kaolin clay suspension, 30 mg l^–1of the flocculant produced a high flocculating activity value of 90%, which remained unchanged over an extensive pH range (pH 2.0–13.0). The flocculant was tested for its ability to bleach dyeing liquors, and the bleaching activities were 98.2% for acid red in 100 mg l^–1of the flocculant and 99.0% for direct emerald blue in 50 mg l^–1of the flocculant under test conditions. Use of the flocculant to bleach basic pink and cation emerald blue liquors was not effective. Electronic Publication     

Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate

Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, ²³Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na⁺ flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism.     

Membrane-permeabilizing activities of amphidinol 3, polyene-polyhydroxy antifungal from a marine dinoflagellate

Amphidinols, which are polyene-polyhydroxy metabolites produced by the marine dinoflagellate Amphidinium klebsii, possess potent antifungal and hemolytic activities. The membrane permeabilizing actions of amphidinol 3, the most potent homologue, were compared with those of polyene antibiotics, amphotericin B (AmB) and filipin, in hemolytic tests, 23Na nuclear magnetic resonance (NMR)-based membrane permeabilizing assays, and UV spectroscopy for liposome-bound forms. In Na+ flux experiments using large unilamellar vesicles (LUVs), ion efflux by amphidinol 3 was inhibited by cholesterol or ergosterol, which was opposed to previous results [J. Mar. Biotechnol., 5 (1997) 124]. When the effect of the agents on the size of vesicles was examined by light scattering experiments, amphidinol 3 did not significantly alter their size while filipin and synthetic detergent Triton X-100 did. The observations implied that the activity of amphidinol 3 was mainly due to formation of large pores/lesions in liposomes rather than detergent-like disruption of membrane. The pore/lesion size was estimated to be 2.0-2.9 nm in diameter on the basis of osmotic protection experiments using blood cells. The UV spectra in liposomes, which revealed the close interaction of polyene moieties in a lipid bilayer, further implied that the membrane activity of amphidinol 3 is caused by the molecular assemblage formed in biomembrane. These results disclose that amphidinol 3 is one of few non-ionic compounds that possess potent membrane permeabilizing activity with non-detergent mechanism.     

Characterization of a planctomycete associated with the marine dinoflagellate Prorocentrum micans Her

During attempts to obtain axenic the cultures of the marine dinoflagellate Prorocentrum micans, a microorganism with peculiar features was isolated. This contaminant resisted the physical and antibiotic treatments performed. Subsequent characterization showed that in agar plates this microorganism develops round granular pink colonies. It is a salt-dependent mesophilic and chemoheterotrophic Gram negative bacterium with a rod to ovoid shape, presenting cell motility in young cultures. Cell division occurs by cell budding. The bacterium forms aggregates with a variable number of cells that are stacked by fibrillar glycoproteic material, the holdfast. A tuft of numerous short glycoproteic fimbriae emerges from one pole of the cell. Preeminent granular inclusions, also of glycoproteic nature, are present in the cytoplasm. Several structural and compositional aspects of the cell envelope and cytoplasm are provided. The production of fibrillar material and the existence of the polar appendages suggest that this microorganism should occur in aquatic environments bound to substrates and could be associated with P. micans in natural marine habitats. Based on the characteristics displayed, this microorganism is a member of the Planctomycetes, order Planctomycetales.     

Amphidinolide H, a novel type of actin-stabilizing agent isolated from dinoflagellate

The effect of novel cytotoxic marine macrolide, amphidinolide H (Amp-H), on actin dynamics was investigated in vitro. Amp-H attenuated actin depolymerization induced by diluting F-actin. This effect remained after washing out of unbound Amp-H by filtration. In the presence of either Amp-H or phalloidin, lag phase, which is the rate-limiting step of actin polymerization, was shortened. Phalloidin decreased the polymerization-rate whereas Amp-H did not. Meanwhile, the effects of both compounds were the same when barbed end of actin was capped by cytochalasin D. Quartz crystal microbalance system revealed interaction of Amp-H with G-actin and F-actin. Amp-H also enhanced the binding of phalloidin to F-actin. We concluded that Amp-H stabilizes actin in a different manner from that of phalloidin and serves as a novel pharmacological tool for analyzing actin-mediated cell function.     

Characterization of a novel thiosulfate dehydrogenase from a marine acidophilic sulfur-oxidizing bacterium,Acidithiobacillus thiooxidans strain SH

A marine acidophilic sulfur-oxidizing bacterium, Acidithiobacillus thiooxidans strain SH, was isolated to develop a bioleaching process for NaCl-containing sulfide minerals. Because the sulfur moiety of sulfide minerals is metabolized to sulfate via thiosulfate as an intermediate, we purified and characterized the thiosulfate dehydrogenase (TSD) from strain SH. The enzyme had an apparent molecular mass of 44 kDa and was purified 71-fold from the solubilized membrane fraction. Tetrathionate was the product of the TSD-oxidized thiosulfate and ferricyanide or ubiquinone was the electron acceptor. Maximum enzyme activity was observed at pH 4.0, 40 °C, and 200 mM NaCl. To our knowledge, this is the first report of NaCl-stimulated TSD activity. TSD was structurally different from the previously reported thiosulfate-oxidizing enzymes. In addition, TSD activity was strongly inhibited by 2-heptyl-4-hydroxy-quinoline N-oxide, suggesting that the TSD is a novel thiosulfate:quinone reductase.