Gymnodinium Mikimotoi Research Articles

Purification and characterisation of a novel alkalophilic α-D-mannosidase from Pseudomonas fluorescens

An extracellular alkaline α-D-mannosidase in the cell culture of a marine bacterium Pseudomonas fluorescens JK-02 was purified to homogeneity with a 30.7-fold by ammonium sulphate fractionation, anion-exchange chromatography and gel-filtration chromatography. The molecular weight of the purified enzyme was estimated to be 50.5 kDa based on the sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature of the purified enzyme were 8.5 and 30°C. The Km and Vmax values of the purified enzyme towards p-nitrophenyl-α-D-mannopyranoside were determined to be 77 µM and 0.23 µM min−1mg−1 of protein, respectively. The α-D-mannosidase showed higher substrate specificity to α-1,3-mannobiose than other isomeric substrates such as α-1,2- and α-1,6-mannobiose. In addition, molecular characterisation of this enzyme reveals that it belongs to a class II α-mannosidase from the glycosyl hydrolase family 38. To the best of our knowledge, this is the first report on the alkalophilic α-1,3 D-mannosidase of Pseudomonas species, which has selective algal-lytic activity against Alexandrium tamarense, Akashiwo sanguine, Gymnodinium catenatum, Gymnodinium mikimotoi and Prorocentrum dentatum.

Comparative study of hydrographic conditions for algal bloom formation in the coastal waters of east and west of Hong Kong during 1998

Phytoplankton abundance was found to be positively correlated with seasonal changes of seawater temperature in Port Shelter and Lamma Channel, Hong Kong in 1998. Rising water temperature from around 20°C to 25°C coincided with an increase in phytoplankton abundance at both locations. Heavy rains from June to September reduced salinity from 30 to 20, but the decrease in salinity was not correlated with a decline in phytoplankton abundance. In spring 1998, over 0.6×106 cells dm−3 and 0.1×106 cells dm−3 of the dinoflagellate, Gymnodinium mikimotoi Miyake et Kominami ex Oda occurred in the coastal waters of Port Shelter and Lamma Channel, respectively. High abundance of the dinoflagellate Ceratium furca (Ehr.) Claparede et Lachmann (>1×106 cells dm−3) produced long-lasting blooms in the waters of Port Shelter from September to October in 1998. The abundances of both diatoms and dinoflagellates were significantly lower in the waters of Lamma Channel than those in Port Shelter due to the less frequent blooms in 1998. Hydrographic conditions such as stable water masses and water column stratification were the main reasons for the differences in the algal abundance and bloom frequency found between the two locations since neither of the two areas appeared to be nutrient-limited. This type water condition for the formation of algal bloom in Port Shelter has not been reported previously and it is not a general case for many bays along China’s coast where algal bloom occurs as well.

Divalent cation distribution in dinoflagellate chromosomes imaged by high-resolution ion probe mass spectrometry

From a variety of analytical electron microscopy experiments, the chromosomes of dinoflagellates are known to contain sizeable amounts of cations, the latter thought to contribute to the neutralization of the negative charge carried by the phosphate groups in the DNA backbone. From previous Ca and Mg chelation experiments, it is also known that these cations are necessary for the compaction and preservation of the chromosome architecture. Similar conclusions have been recently presented by our group concerning mammalian mitotic chromosomes, in studies based on secondary ion mass spectrometry (SIMS) carried out with the University of Chicago high-resolution scanning ion microprobe (UC-SIM). We have now applied this instrument to image the distribution of DNA-bound Ca 2+ and Mg 2+ in dinoflagellate chromosomes, a goal that could not be attained earlier by analytical electron microscopy. Analyzed quantitatively and imaged here by SIMS for the first time, through their cation content, are the chromosomes of the dinoflagellates Prorocentrum micans, Gymnodinium mikimotoi and Gymnodinium dorsum. The cell nuclei were isolated and prepared for SIMS analysis with a minimal protocol (mechanical fractionation in culture medium followed by ethanol drying), which did not expose the samples to artifact-creating, alien chemical agents. By this approach, we have confirmed the earlier findings by several authors, and contributed new structural information provided by our ion probe capability to erode the sample surface layer by layer (SIMS tomography). Dinoflagellates, due to the absence of histones, represent an ideal model system where cations may bind directly with DNA, allowing comparisons to be made with recently reported X-ray crystallography results at atomic resolution. Such comparisons yielded quantitative confirmation that the Ca 2++Mg 2+ concentrations found for e.g. P. micans are consistent with those anticipated to provide complete charge neutralization of naked DNA by cations, also resulting in maximal DNA compaction.

FLUORESCENCE IN SITU HYBRIDIZATION USING rRNA‐TARGETED PROBES FOR SIMPLE AND RAPID IDENTIFICATION OF THE TOXIC DINOFLAGELLATES ALEXANDRIUM TAMARENSE AND ALEXANDRIUM CATENELLA1

The toxic marine dinoflagellates Alexandrium tamarense (Lebor) Balech and A. catenella (Whedon and Kofoid) Taylor have been mainly responsible for paralytic shellfish poisoning in Japan. Rapid and precise identification of these algae has been difficult because this genus contains many morphologically similar toxic and nontoxic species. Here, we report a rapid, precise, and quantitative identification method using three fluorescent, rRNA‐targeted, oligonucleotide probes for A. tamarense (Atm1), A. catenella (Act1), and the nontoxic A. affine (Inoue et Fukuyo; Aaf1). Each probe was species specific when applied using fluorescence in situ hybridization (FISH). None of the probes reacted with three other Alexandrium spp., A. lusitanicum Balech, A. ostenfeldii (Paulsen) Balech & Tangen, and A. insuetum Balech, or with eight other microalgae, including Gymnodinium mikimotoi Miyake et Kominami ex Oda and Heterosigma akashiwo (Hada) Hara et Chihara, suggesting that the species specificity of each probe was very high. Cells labeled with fluorescein 5‐isothiocyanate–conjugated probes showed strong green fluorescence throughout the whole cell except for the nucleus. FISH could be completed within 1 h and largely eliminated the need for identifying species based on key morphological criteria. More than 80% of targeted cells of both species could be identified by microscopy and quantified during growth up to the early stationary phase; more than 70% of cells could be detected in the late stationary phase. The established FISH protocol was found to be a specific, rapid, precise, and quantitative method that might prove to be a useful tool to distinguish and quantify Alexandrium cells collected from Japanese coastal waters.

Production of Superoxide Anion and Hydrogen Peroxide by the Red Tide Dinoflagellate Karenia mikimotoi

We found that the red tide dinoflagellate Karenia mikimotoi ( Gymnodinium mikimotoi ) generates reactive oxygen species (ROS). In chemiluminescence analysis using an O 2 − specific probe, a slightly lower but significant level of O 2 − was detected in a K. mikimotoi cell suspension as compared to one containing Chattonella marina , a well-known ROS-producing red tide phytoplankton. Interestingly, the chemiluminescence response pattern caused by K. mikimotoi was different from that of C. marina , although both responses were almost completely abolished in the presence of superoxide dismutase (SOD). By fluorescence spectrophotometric analysis, H 2 O 2 was also detected in the K. mikimotoi cell suspension, but the level of H 2 O 2 was approximately 10% of that in the C. marina suspension based on the cell number. No significant increase in O 2 − generation by K. mikimotoi was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus substances prepared from the skin and gills of yellowtail, whereas C. marina generated an increased level of O 2 − in response to these stimuli. Furthermore, O 2 − production in C. marina , but not in K. mikimotoi , was inhibited by treatment with proteinase K.

Gymnocin‐A, a Cytotoxic Polyether from the Notorious Red Tide Dinoflagellate, Gymnodinium mikimotoi.

Abstract A new cytotoxic polyether, gymnocin-A, was isolated from the notorious red tide dinoflagellate, Gymnodinium mikimotoi, and its structure consisting of 14 contiguous ether rings and a 2-methyl-2-butenal side-chain was determined by NMR and CID MS/MS experiments.

Studies Toward the Total Synthesis of Gymnocin A, a Cytotoxic Polyether: A Highly Convergent Entry to the F—N Ring Fragment.

An efficient and highly convergent synthesis of the FGHIJKLMN ring fragment of gymnocin A, a cyctotoxic polycyclic ether isolated from the notorious red-tide forming dinoflagellate Gymnodinium mikimotoi, has been achieved. The present synthesis relied on extensive use of the B-alkyl Suzuki−Miyaura coupling reaction.

Identification of marine algicidal Flavobacterium sp. 5 N-3 using multiple probes and whole-cell hybridization

: We designed five 16S rRNA-targeted oligonucleotide probes (Sp probes) specific for Flavobacterium sp. 5 N-3, which inhibits the growth of red tide phytoplankton Gymnodinium mikimotoi (Dinophyceae). These probes were evaluated by whole-cell hybridization against 5 N-3 cells incubated under laboratory conditions. The fluorescence signal from the cell detected with Sp probe mix5, a mixture of the five probes, was 8.4-fold higher than that obtained with only one Sp probe (Sp01RF). The signal obtained by this method was strong enough to recognize 5 N-3 cells and count them under the epifluorescence microscope, while the signal was often undetectable when a single probe was used. Fluorescence intensities of cells at stationary phases and of ‘starved’ cells in sterile seawater using Sp probe mix5 were low but still sufficient for enumeration. These Sp probes did not hybridize with 11 strains from the Cytophaga/Flavobacteria/Bacteroides phylum and did react with strain 5 N-3 following whole-cell hybridization. These results show that 5 N-3 cells cultivated under our laboratory conditions can be detected by whole-cell hybridization with the five designed probes. These data also suggest that this technique may be useful for detection of an algicidal bacterium 5 N-3 in the natural environment.

Gymnocin-A, a cytotoxic polyether from the notorious red tide dinoflagellate, Gymnodinium mikimotoi

A new cytotoxic polyether, gymnocin-A, was isolated from the notorious red tide dinoflagellate, Gymnodinium mikimotoi, and its structure consisting of 14 contiguous ether rings and a 2-methyl-2-butenal side-chain was determined by NMR and CID MS/MS experiments.

Studies toward the total synthesis of gymnocin A, a cytotoxic polyether: a highly convergent entry to the F-N ring fragment.

[structure: see text] An efficient and highly convergent synthesis of the FGHIJKLMN ring fragment of gymnocin A, a cyctotoxic polycyclic ether isolated from the notorious red-tide forming dinoflagellate Gymnodinium mikimotoi, has been achieved. The present synthesis relied on extensive use of the B-alkyl Suzuki-Miyaura coupling reaction.

Effect of marine animal excretions on differential growth of phytoplankton species

Five phytoplankton species were cultivated in the presence of different marine animal excretions, and growth rates were compared. Growth of Chaetoceros gracilis was stimulated by excretion from the oyster (Crassostrea gigas) and inhibited by excretion from the sea bass (Dicentrarchus labrax). Growth of Heterosigma akashiwo was stimulated by excreta from the mussel (Mytilus chilensis) inhibited by excreta from sea bass and salmon (Salmo salar), and unaffected by oyster excretions. Growth of Gymnodinium mikimotoi was also inhibited by excreta from sea bass and unaffected by oyster excreta. Growth of Alexandrium catenella and A. minutum was not affected by animal excreta under our experimental conditions. The results indicate that the organic components of dissolved excreta were responsible for the observed effects: stimulators when excreted by shellfish, and inhibitors when excreted by finfish. 2001 International Council for the Exploration of the Sea

Polymerase chain reaction in detection of Gymnodinium mikimotoi and Alexandrium minutum in field samples from southwest India.

Polymerase chain reaction (PCR) primers were constructed for the detection of two toxic dinoflagellate species, Gymnodinium mikimotoi and Alexandrium minutum. The primers amplified a product of expected size from cultured cells of G. mikimotoi and A. minutum. The species-specific primers targeting G. mikimotoi did not yield any product with a wide range of other cultured algae used as negative controls. Primers designed for A. minutum were species-group-specific since it PCR yielded a product from the closely related species A. ostenfeldii and A. andersonii, but not from other species of this genus tested. The confirmation of PCR products was performed by digestion of the products with restriction enzymes. Sensitivity analyses of the primers on DNA template from cultured cells was positive by PCR at a DNA template concentration of 1.5 x 10(-4) ng/microl (0.3 cells/L) for A. minutum, and at a DNA concentration of 2.5 x 10(-2) ng/microl (697 cells/L) for G. mikimotoi. The PCR method for detection of G. mikimotoi and A. minutum was applied on field samples collected with a plankton net. Gymnodinium mikimotoi could be detected in 11 field samples by microscopy, and all these field samples were positive by PCR. The cell counts of G. mikimotoi in simultaneously collected water samples ranged from 306 to 2077/L. Alexandrium minutum could be detected by microscopy in 3 different field samples. The cell counts in water samples collected at the same time as the net samples ranged from 115 to 1115 cells/L. Alexandrium minutum was detected by PCR in these field samples, with the exception of the sample displaying the lowest cell count (115 cells/L). Plankton samples that were negative by microscopy for any of the two target species were also negative by PCR. All the PCR products from field samples were confirmed by restriction enzyme digestion. The application of PCR-based detection of harmful algal bloom species for aquaculture and monitoring purposes in natural field samples is discussed.

Modelling of Gymnodinium mikimotoï blooms along the French Atlantic coast: geographical and vertical distributions

In order to study the occurrence of Gymnodinium mikimotoi bloom on the French Atlantic coast, a three-dimensional model of primary production was set up. The biological model was coupled to a hydrodynamic model previously developed at Ifremer, by the intermediary of transport, diffusion and heat fluxes. The cycles of three limiting elements for the phytoplankton growth were modelised: nitrogen, phosphorus and silicon. The second phase of the study aimed at reproducing the summer dinoflagellate blooms. In this new approach a model of the species Gymnodinium mikimotoi was coupled with the previous biomass model which provides the physical and chemical environments. The study focuses on the results obtained by this specific sub-model. The parametrisation was based on some physiological studies reported from laboratory cultures and on parameters existing in the literature. Consistent with the observations, the model reproduces the sub-surface cell concentrations, in the zone of minimal turbulence. The formulation retained for the temperature effect on the growth rate leads to a strong influence of temperature on the onset of the bloom and the mortality rate controls the vertical distribution. Nevertheless, as formulated here, the model underestimates the cell density.

HETEROGENEITY OF CHLOROPLAST GAPDH GENES IN THE DINOPHYCEAE

Dinoflagellates provide valuable models for studying endosymbiosis and chloroplast evolution. While many species are heterotrophic, photosynthetic species containing plastids of different evolutionary origin have been isolated, indicating that dinoflagellates acquired chloroplasts multiple times during evolution. Furthermore, dinoflagellates contain nuclear genes encoding chloroplast proteins that are not typically found in eukaryotes. These include the type II form of RuBisCO, and a eubacterial form of glyceraldehyde‐3‐phosphate dehydrogenase (GAPCp) that we have recently characterized from Gonyaulax polyedra. The evolutionary origin of this GAPCp isoform is unclear. It is phylogenetically distant from the Gonyaulax cytosolic form (GAPC) and from the chloroplast forms (GAPA/B) of cyanobacterial origin typically found in photosynthetic eukaryotes. It has previously been described only from cryptomonads, raising the possibility that Gonyaulax acquired the gene through lateral transfer. We have addressed this issue by examining the phylogeny of GAPDH genes from other dinoflagellates. We have isolated genes from Gymnodinium mikimotoi and Pyrocystis lunula. The predicted protein sequence of the G. mikimotoi gene is 47% similar to Gonyaulax GAPC and GAPCp and phylogenetic analysis places them in the same eubacterial clade. The predicted amino acid sequence of the P. lunula gene is 41% and 37% similar to Gonyaulax GAPCp and GAPC isoforms, respectively. However, phylogenetic analysis groups it within the clade comprising cyanobacterial and eukaryotic GAPA/B isoforms and shows that it is closely related to the chloroplast form of Euglena gracilis. Thus, within the Dinophyceae, GAPDH genes of different evolutionary origin have been recruited to participate in chloroplast metabolism.

Karenia digitata sp. nov. (Gymnodiniales, Dinophyceae), a new harmful algal bloom species from the coastal waters of west Japan and Hong Kong

A new unarmoured dinoflagellate, Karenia digitata Yang, Takayama, Matsuoka & Hodgkiss sp. nov., is described on the basis of light and electron microscopy. It has bloomed in Hong Kong and Japan, and has caused significant fish kills and financial loss. High ichthyotoxicity persisted for approximately one week after the blooms had subsided. Karenia digitata cells are 10–26.25 μm long and 10–22.5 μm wide. The epicone is hemispherical or broadly conical; the hypocone is hemispherical and is not concave at the antapex. The girdle displacement varies but is more than 20% of the body length. The sulcus continues into the epicone as small finger-like extensions; it does not reach the antapex. At its centre it is displaced by the girdle. Chloroplasts are numerous, irregular in shape and randomly distributed, though usually clumped. The nucleus is spherical to ovoid and centrally to posteriorly located. The cell has an apical groove, which lies to the right of the sulcal axis, extends in a straight line over the apex to about one-third to half-way down the dorsal epicone, and gradually narrows near its centre. Karenia digitata may be confused with Gymnodinium pulchellum based on size, but the latter has a sigmoid apical groove. Karenia digitata shares features with Gyrodinium aureolum, Gymnodinium mikimotoi and Gymnodinium brevisulcatum [recently transferred to the genera Gymnodinium, Karenia and Karenia, respectively, by Daugbjerg et al. (2000) (Phycologia 39: 302–317)], but differs from them in aspects such as overall appearance, cell thickness, size, the apical groove, sulcus arrangement and the shape and location of the nucleus. It closely resembles Gymnodinium vitiligo and Gymnodinium veneficum (both now transferred to the genus Karlodinium), except that these species are smaller and have a sulcus that extends onto the epicone.

Origin of the plastid in the anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta) as inferred from phylogenetic analysis based on the gene encoding the large subunit of form I-type RuBisCO

SUMMARY The dinoflagellate Gymnodinium mikimotoi Miyake et Kominami ex Oda possesses an anomalously pigmented plastid which contains 19′-hexanoyloxyfucoxanthin, 19′-butanoyloxyfucoxanthin and fucoxanthin instead of peridinin as the major carotenoids. Previously, we have shown that the plastid of G. mikimotoi belongs to the rhodoplast lineage as inferred from phylogenetic analyses based on the amino acid sequences deduced from psbA and psaA and the nucleotide sequence of the plastid small subunit ribosomal RNA. Furthermore, in the present study, we cloned and sequenced an additional representative plastid gene, rbcL, encoding the large subunit of ribulose 1–5 bisphosphate carboxylase/oxygenase (RuBisCO LSU) from G. mikimotoi. The amino acid sequence deduced from the rbcL gene of G. mikimotoi apparently revealed the conventional form I RuBisCO LSU, which is present in most photosynthetic organisms, and not the divergent form II existing in typically pigmented dinofl age Nates with plastids containing peridinin as the main carotenoid. This finding supports the hypothesis that the origins of the plastids in G. mikimotoi and peridinin-type dinoflagellates are not related to each other. Molecular phylogenetic analysis based on the amino acid sequence deduced from the rbcL gene further showed that the plastid of G. mikimotoi belongs to the rhodoplast lineage. In particular, G. mikimotoi clustered with haptophytes in the phylogenetic tree. From this result, two hypotheses with respect to the origin of the plastid in G. mikimotoi can be proposed: G. mikimotoi may have engulfed a haptophyte-like cell (tertiary symbiosis) or englulfed a rhodophyte-like cell that was closely related to the origin of the plastid in the haptophyte (secondary symbiosis).

CHARACTERIZATION OF GYMNODINIUM MIKIMOTOI (DINOPHYCEAE) NUCLEI AND IDENTIFICATION OF THE MAJOR HISTONE-LIKE PROTEIN, HGm.

A simple and rapid method for isolation of nuclei from Gymnodinium mikimotoi Miyake et Kominami ex Oda is described along with chemical characterization of the nuclei. The isolated nuclei were completely free of whole cells, 99.96% free of cytoplasmic contamination, and were collected with a yield of 40% from harvested whole cells. Each nucleus contained 47 pg of DNA and the ratio of DNA to acid-soluble proteins to acid-insoluble proteins was 1:0.25:1.21, respectively. SDS electrophoresis of acid-extracted proteins showed one histone-like protein, which we termed HGm, with an apparent molecular mass of 12 kDa. V8 protease digestion analysis of HGm, the histone-like protein from Crypthecodinium cohnii (HCc), and two histone-like proteins from Gymnodinium dorsum, showed that the HGm digestion pattern was more similar to that of HCc than to that of either of the G. dorsum histone-like proteins.

Origin of the plastid in the anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta) as inferred from phylogenetic analysis based on the gene encoding the large subunit of form I-type RuBisCO

Origin of the plastid in the anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta) as inferred from phylogenetic analysis based on the gene encoding the large subunit of form I-type RuBisCO

Comparative study of Gymnodinium mikimotoi and Gymnodinium aureolum, comb. nov. (=Gyrodinium aureolum) based on morphology, pigment composition, and molecular data

Light and electron microscopy, nuclear‐encoded LSU rDNA sequences, and pigment analyses were performed on five geographically separate isolates of Gymnodinium mikimotoi. The morphological variation between the isolates equals that found within the isolates. The nuclear‐encoded LSU rDNA sequences were nearly identical in all isolates, and molecular analyses using maximum likelihood, parsimony, and neighbor joining showed the geographical isolates as an unresolved clade. Based on the available data it is concluded that the European isolates, formerly identified as Gyrodinium aureolum, Gyrodinium cf. aureolum, or Gymnodinium cf. nagasakiense, are conspecific with the Japanese Gymnodinium mikimotoi. An isolate from the Pettaquamscutt River, USA, is suggested to represent what Hulburt (1957) described as Gyrodinium aureolum. The LSU rDNA sequence data and ultrastructural characters in this isolate closely resemble those of Gymnodinium fuscum, the type species of Gymnodinium, and Gyrodinium aureolum Hulburt is therefore renamed Gymnodinium aureolum (Hulburt) G. Hansen, comb. nov.

Characteristics of virus-like growth suppression agents against phytoplankton obtained from seawater at the mouth of Funka Bay, Hokkaido, Japan

Eighteen samples of virus-like growth suppression agents against the marine phytoplankton Alexandrium catenella, Gymnodinium mikimotoi and Tetraselmis sp., obtained from the coastal waters at the mouth of Funka Bay, Hokkaido, Japan, from 1993 to 1995, were characterized on size estimation, heat stability, nuclease sensitivity, proteinase K sensitivity, stability under acidic condition, titration, diethyl ether sensitivity, and ultraviolet (UV) sensitivity. All agents were affected by heating at 50°C for 30 min, exposure to acidic conditions below pH 5.0, passing through a 0.05 μm filter, RNase treatment, and irradiation of UV dosage at 5 × 104μW/s per cm2. The growth suppression effects of 11 agents from Tetraselmis sp. and A. catenella disappeared after proteinase K treatment, however, seven agents from G. mikimotoi were unaffected by this treatment. Furthermore, 11 agents against Tetraselmis sp. and A. catenella were collected from the bottom fraction by ultracentrifugation, while seven agents against G. mikimotoi were collected from the upper fraction, not from the precipitated fraction. These results suggest that at least two types exist in the virus-like agents showing growth suppression for phytoplankton.