Harmful Red Tide Research Articles

Degradation of Cochlodinium polykrikoides using photocatalytic reactor with TiO2-coated alumina

Cochlodinium polykrikoides (C. polykrikoides) is one of the most harmful red tide dinoflagellates due to its great economic damage and compromising recreational opportunity and public health. Titanium dioxide (TiO2) is known to be used as a photocatalyst for the control of the aquatic invasive algae under natural and artificial light. The purpose of this study was to design a highly efficient continuous photocatalytic reactor with TiO2-coated alumina for the demonstrated efficient degradation of C. polykrikoides. TiO2 photocatalyst beads were prepared by sol-gel dip-coating method using titanium tetra iso-propoxide on alumina. After 40 min of ultraviolet illumination time, the reduction of C. polykrikoides cell number was more than 80%. The degree of degradation of C. polykrikoides increased in a time-dependent manner in this novel reactor. The degradation begins with photocatalytic action by the oxidative species of TiO2 on the protective cell structures of C. polykrikoides.

Statistical optimization of medium components for the production of prodigiosin by Hahella chejuensis KCTC 2396.

Prodigiosin is a natural red pigment with algicidal activity against Cochlodinium polykrikoides, a major harmful redtide microalga. To increase the yield of prodigiosin production by Hahella chejuensis KCTC 2396, significant medium components were determined using a two-level Plackett- Burman statistical design technique. Among 12 components included in basal medium, NaHCO3, Na2SiO3, NH4NO3, Na2SO4, and CaCl2 were determined to be important for prodigiosin production. The medium formulation was finally optimized using a Box-Behnken design as follows: 1% sucrose; 0.4% peptone; 0.1% yeast extract; and (g/l): NaCl, 20.0; Na2SO4, 9.0; CaCl2, 1.71; KCl, 0.4; and (mg/l): H3BO3, 10.0; KBr, 50.0; NaF, 2.0; NaHCO3, 45.0; Na2SiO3, 4.5; NH4NO3, 4.5. The predicted maximum yield of prodigiosin in the optimized medium was 1.198 g/l by the Box-Behnken design, whereas the practical production was 1.495 g/l, which was three times higher than the basal medium (0.492 g/l).

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton, Alexandrium tamarense

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel-filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid-Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N-acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide-based compound. The toxin showed cytotoxic effects on various cell lines in a concentration-dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide-based toxin isolated from red tide phytoplankton can induce apoptotic cell death.

Taxonomy and identification of the armored dinoflagellate genus Heterocapsa (Peridiniales, Dinophyceae)

The armored dinoflagellate genus Heterocapsa is composed of relatively small species, including a species responsible for harmful red tides, H. circularisquama. Some Heterocapsa species such as H. rotundata and H. triquetra have been well documented as red tide-forming species, but are not recognized as causing harmful effects. Following sequential shellfish mass mortalities in the coastal waters of western Japan due to H. circularisquama red tides, this species has attracted considerable interest. Many properties of this species, such as its distribution and growth charac- teristics, have been investigated to better understand the mechanisms involved in harmful red tide formation. Related to the need for unambiguous identification of H. circularisquama, several unidentified (or undescribed) Heterocapsa species, which must be distinguished from the harmful taxon, have been detected sympatrically in regions where H. cir- cularisquama blooms occur. However, the taxonomic affiliation of these Heterocapsa species have not yet been deter- mined because several characteristics of Heterocapsa (e.g. body scale ultrastructure) have not yet been reported from all described species due to the changing taxonomic criteria. After the taxonomic ambiguities in this genus were re- solved, cellular and body scale morphology of Heterocapsa were reinvestigated and several new species were described. In the present paper, the taxonomic history and morphological characters of the genus Heterocapsa are summarized.

Phylogenetic relationships in the harmful dinoflagellate Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) inferred from LSU rDNA sequences

Phylogenetic relationships among chain-forming Cochlodinium species, including the harmful red tide forming dinoflagellate Cochlodinium polykrikoides, were investigated using specimens collected from coastal waters of Canada, Hong Kong, Japan, Korea, Malaysia, México, Philippines, Puerto Rico, and USA. The phylogenetic tree inferred from partial (D1–D6 regions) large subunit ribosomal RNA gene (LSU rDNA) sequences clearly differentiated between C. polykrikoides and a recently described species, Cochlodinium fulvescens. Two samples collected from the Pacific coasts of North America (British Columbia, Canada and California, USA) having typical morphological characters of C. fulvescens such as the sulcus located in the intermediate region of the cingulum, were closely related to C. fulvescens from western Japan in the phylogenetic tree. Cochlodinium polykrikoides formed a monophyletic group positioned as a sister group of the C. fulvescens clade with three well-supported sub-clades. These three clades were composed of (1) East Asian, including specimens collected from Hong Kong, western Japan, and southern Korea, (2) Philippines, from Manila Bay, Philippines and Omura Bay, Japan, and (3) American/Malaysian, from the Atlantic coasts of USA, the Pacific coast of México, Puerto Rico, and Borneo Island, Malaysia. Each of these clades is considered to be a so-called “ribotype” representing the population inhabiting each region, which is distinguished based on ribosomal RNA gene sequences in the species despite similarities in their morphological characters.

Phylogenetic analyses of the dinoflagellate Noctiluca scintillans based on β-tubulin and Hsp90 genes

The noctilucid dinoflagellate Noctiluca scintillans is an unarmed heterotrophic protist that inhabits the world's oceans and is sometimes responsible for harmful red tides. The phylogenetic position of the noctilucids has been widely disputed because of two alternative views based on morphological characters and phylogenetic analyses using SSU rDNA. Specifically, noctilucids are either placed in a basal position within the dinoflagellates or they are seen as evolutionarily recent derivations descended from unarmored dinoflagellates in the order Gymnodiniales. Thus, the precise relationship of noctilucids to other dinoflagellates is still uncertain. In this study, we isolated β-tubulin and heat shock protein 90 genes from N. scintillans to examine this relationship further. The deduced amino acid sequences share commonly substituted amino acids and a deletion with other dinoflagellates, but not with Perkinsus marinus or other alveolates. Although Hsp90 analysis did not give robust support, β-tubulin analysis including an AU test, as well as combined analysis of these two amino acid sequences showed that N. scintillans is the next earliest branch after Oxyrrhis marina, within the dinoflagellates. Given the phylogenetic position of N. scintillans, its extremely specialized diploid trophont, and the primitive dinoflagellate-like characteristics of its haploid zoospore, we propose that noctilucids are a possible evolutionary link between ancestral diploid dinoflagellates and haploid core dinoflagellates. This implies that the transition from diploidy to haploidy in trophonts probably occurred via neoteny of a noctilucid-like zoospore.

Cochlodinium fulvescens sp. nov. (Gymnodiniales, Dinophyceae), a new chain‐forming unarmored dinoflagellate from Asian coasts

SUMMARYCellular morphology and the phylogenetic position of a new unarmored photosynthetic dinoflagellate Cochlodinium fulvescens Iwataki, Kawami et Matsuoka sp. nov. were examined by light microscopy and molecular phylogenetic analyses based on partial large subunit ribosomal DNA (LSU rDNA) and small subunit ribosomal DNA (SSU rDNA) sequences. The cells of C. fulvescens closely resemble C. polykrikoides, one of the most harmful red tide forming dinoflagellates, due to it possessing a cingulum encircling the cell approximately twice, a spherical nucleus positioned in the anterior part of the cell and an eyespot‐like orange pigmented body located in the dorsal side of the epicone, as well as formation of cell‐chains. However, this species is clearly distinguished from C. polykrikoides based on several morphological characteristics, namely, cell size, shape of chloroplasts and the position of narrow sulcus situated in the cell surface. The sulcus of C. fulvescens is located at the intermediate position of the cingulum in the dorsal side, whereas that of C. polykrikoides is situated immediately beneath the cingulum. LSU rDNA phylogenies indicated that C. fulvescens is clearly distinct from, but closely related to C. polykrikoides among dinoflagellates.

A new simple screening method for the detection of cytotoxic substances produced by harmful red tide phytoplankton

A highly sensitive, but simple and quantitative, cytotoxic assay method for the detection of toxic substances produced by red tide phytoplankton was developed by utilizing Vero cells which were the most resistant to seawater among the six cell lines tested. Heterocapsa circularisquama, which is known to be highly toxic to shellfish, showed cytotoxicity to Vero cells in a cell-density dependent manner when Vero cells were directly exposed to the cell suspension of H. circularisquama in seawater-based plankton culture medium, whereas Heterocapsa triquetra, which is morphologically similar to H. circularisquama but non-toxic to shellfish, showed no cytotoxic effect. Since the potent cytotoxicity was also detected in the cell-free culture supernatant of H. circularisquama, it was suggested that a certain cytotoxic substance is extracellularly secreted by H. circularisquama. Furthermore, by this direct exposure method, we found that Alexandrium fraterculus, Alexandrium tamiyavanichii, Alexandrium tamarense, and Alexandrium affine but not Alexandrium taylorii and Alexandrium catenella cause toxic effect on Vero cells with different extent depending on species. By gel-filtration and subsequent two cytotoxicity assays using Vero and mouse neuroblastoma cell line (Neuro-2a), we found that high molecular weight cytotoxic substance distinct from paralytic shellfish poisoning toxins is present in the aqueous extract of A. tamarense. These results suggest that our 96-well microplate cytotoxicity assay using Vero cells is useful not only as a primary screening assay for the detection of potential toxic activity of harmful phytoplankton but also as a quantitative routine toxicity assay for following the active substances during the extraction and purification processes.

Presence of the distinct systems responsible for superoxide anion and hydrogen peroxide generation in red tide phytoplankton Chattonella marina and Chattonella ovata

Raphidophycean flagellates, Chattonella marina and C. ovata, are harmful red tide phytoplankters; blooms of these phytoplankters often cause severe damage to fish farming. Previous studies have demonstrated that C. marina and C. ovata continuously produce reactive oxygen species (ROS) such as superoxide anion (O2−) hydrogen peroxide (H2O2) under normal growth conditions, and an ROS-mediated toxic mechanism against fish and other marine organisms has been proposed. Although the exact mechanism of ROS generation in these phytoplankters still remains to be clarified, our previous study suggested that NADPH oxidase-like enzyme located on the cell surface of C. marina may be involved in O2− generation. To investigate the localization of O2− and H2O2 generation in C. marina and C. ovata, we employed 2-methyl-6(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one and 5-(and-6)-carboxy-2′,7′-dichlorodihydrodihydrofluorescein dictate, acetyl ester, which are specific fluorescent probe for detecting O2− and H2O2, respectively. Observation by fluorescence microscopy of live phytoplankters incubated with each probe revealed that O2− is mainly generated on the cell surface, whereas H2O2 is generated in the intracellular compartment in these phytoplankters. When the cells were ruptured by ultrasonic treatment, O2− levels of C. marina and C. ovata decreased significantly, whereas a few times higher levels of H2O2 were detected in the ruptured cell suspensions when compared with the levels of the live cell suspension. In immunoblotting analysis, the protein recognized by anti-human gp91 phox was detected in both species. These results suggest that, in both phytoplankters, the underlying mechanisms of O2− and H2O2 generation may be distinct and such systems are independently operating in the cells.

Nitric oxide synthase-like enzyme mediated nitric oxide generation by harmful red tide phytoplankton, Chattonella marina

The unicellular marine phytoplankton Chattonella marina is known to exhibit potent fish-killing activity. Previous studies have demonstrated that C. marina produces reactive oxygen species (ROS), and ROS-mediated ichthyotoxic mechanism has been postulated. However, the exact toxic mechanism is still controversial. In this study, we obtained evidence that C. marina produces nitric oxide (NO) under normal growth conditions. We utilized chemiluminescence (CL) reaction between NO and luminol–H2O2 to detect NO in C. marina cell suspensions. In this assay, significant CL was observed in C. marina in a cell-number-dependent manner, and this was diminished by the addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), a specific NO scavenger. The NO generation by C. marina was also confirmed by a spectrophotometric assay based on the measurement of the diazo-reaction-positive substances (NOx) and by fluorometric assay using highly specific fluorescent indicator of NO. The NO level in C. marina was significantly decreased by NG-nitro-l-arginine methyl ester (l-NAME), a specific NO synthase (NOS) inhibitor. The addition of l-arginine resulted in the increased NO level, whereas NaNO2 had no effect. These results suggest that a NOS-like enzyme is mainly responsible for NO generation in C. marina.

Purification and characterization of photosensitizing hemolytic toxin from harmful red tide phytoplankton, Heterocapsa circularisquama

We have previously found that the methanol extract prepared from Heterocapsa circularisquama, a harmful red rid dinoflagellate, showed light-dependent hemolytic activity toward rabbit erythrocytes. Interestingly, the cytotoxicity of the extract against HeLa cells was also strictly light-dependent, and no significant toxic effect was observed even at very high concentration in the dark. In this study, the hemolytic agents present in the extract were purified by several chromatographic procedures. Final HPLC analysis revealed that there are at least two hemolytic toxins with different retention times (H2-a and H3-a), which have similar absorption spectra. Although H3-a fraction was contaminated by a trace amount of H2-a, H2-a fraction gave a single peak on a three-dimensional chromatogram. The results of fast atom bombardment (FAB)–mass spectrometry (MS) and electrospray ionization quadrupole time-of-flight (ESI Q-TOF) mass spectrometry analyses suggested that the molecular weight of H2-a was 566. Since nuclear magnetic resonance analysis indicated the presence of a pyrrole ring in H2-a molecule, H2-a may be a porphyrin derivative with similar structure of pyropheophorbide a methyl ester, a well-known photosensitizing hemolytic compound. However, some structural and physicochemical differences were observed between H2-a and pyropheophorbide a methyl ester. Since H2-a showed potent photosensitizing cytotoxicity toward HeLa cells in a concentration-dependent manner as well as hemolytic activity, H2-a may be a novel porphyrin derivative with photosensitizing hemolytic cytotoxicity.

Effects of temperature, salinity and irradiance on the growth of the harmful red tide dinoflagellate Cochlodinium polykrikoides Margalef (Dinophyceae)

The effects of temperature, salinity and irradiance on the growth of the harmful red tide dinoflagellate Cochlodinium polykrikoides were examined in the laboratory. From 60 different combinations of temperature (10-30°C) and salinity (10-40) under saturated irradiance, C. polykrikoides exhibited its maximum specific growth rate of 0.41 day -1 at a combination of 25°C and salinity of 34. Optimum growth rates of >0.3 day -1 were observed at temperatures ranging from 21 to 26° C and at salinities from 30 to 36. The organism did not grow at temperatures ≤10°C and only grew at salinities >30 if the temperature was >15°C. It was able to grow in temperatures ranging from 15 to 30° C and at salinities from 20 to 36. These values closely resembled those observed for this species in situ. It appears as if C. polykrikoides is a stenohaline organism that prefers high salinities, indicative of offshore waters. Temperature had the greatest influence on the growth rate, followed by salinity, and then the interaction between temperature and salinity. The optimum irradiance for growth was >90 μmol m -2 s -1 . Photoinhibition did not occur at 230 μmol m -2 s -1 , which was the maximum irradiance used in this study.

Infection of Ceratium furca by the parasitic dinoflagellate Amoebophyra ceratii (Amoebophrydae) in the Mexican Pacific

Parasitism within dinoflagellates is a widespread and well-documented phenomenon. Parasitic dinoflagellates of the genus Amoebophrya commonly infect free-living toxic, and non-toxic dinoflagellates species which may cause harmful red tides. Infections of Ceratium furca by A. ceratii were observed in red tides samples collected in the northwest coast of Baja California between 30°01'05'' N, 115°51'16'' W and 31°09'33'' N, 116°31'09'' W. This is the first record of this particular parasitic dinoflagellate in Mexican Pacific waters. There were mainly three dinoflagellate species causing this particular seawater discoloration: a Gymnodinium-like dinoflagellate, Ceratium furca, and Akashiwo sanguinea. These reached concentrations as high as 560 000, 762 600, and 395 400 cells L-1, respectively. During the bloom, surface water temperature ranged between 13 and 17°C. Seawater salinity ranged from 33.2 to 33.8 psu. About 1.5% of the individuals of C. furca observed were infected by the intracellular parasite dinoflagellate Amoebophrya ceratii. This parasite was observed mainly inside specimens of Ceratium furca and very few specimens of Ceratium macroceros. In general, individuals of C. furca were partially or totally deformed. Infections by A. ceratii could delay or inhibit the dinoflagellate blooms as infected dinoflagellates become reproductively incompetent.

Cardiac output during exposure to Chattonella marina and environmental hypoxia in yellowtail (Seriola quinqueradiata)

The experiments were undertaken to measure, for the first time, cardiac output in yellowtail (Seriola quinqueradiata) during exposure to a harmful red tide flagellate (Chattonella marina). The responses were compared with those during exposure to environmental hypoxia to evaluate the significance of the drop of arterial oxygen partial pressure (PaO2) in the fish-kill mechan- isms by C. marina. PaO2 immediately decreased, whereas heart rate (HR) was maintained until shortly before death during exposure to C. marina. Suffocation developing during the exposure resulted from a decrease in blood oxygen content, but not from lowered blood flow to the tissue. Although exposure to both C. marina and hypoxia immediately decreased PaO2, arterial oxygen content (CaO2) and pH (pHa) were significantly lower, whereas HR and cardiac output ( _ Q remained significantly higher, for the C. marina-exposed fish than for hypoxia-exposed fish. Although the drop in PaO2 appears to play a pivotal role in the mechanisms of fish death by C. marina, other physiological response(s) should also be considered.

Proteomic study of a model causative agent of harmful red tide, Prorocentrum triestinum I: Optimization of sample preparation methodologies for analyzing with two-dimensional electrophoresis.

A comprehensive study to find the optimal sample preparation conditions for two-dimensional electrophoresis (2-DE) analysis of Prorocentrum triestinum, a model causative agent of harmful algal blooms (HABs) was carried out. The four major sample preparation steps for 2-DE: (a) cell disruption: i.e. sonication and homogenization with glass beads; (b) protein extraction : i.e. sequential and independent extraction procedures; (c) pre-electrophoretic treatment: these included (i) treatment with RNAase/DNAase or benzonase; (ii) ultracentrifugation to sediment large macromolecules such as DNA; (iii) desalting and concentration by ultrafiltration through a Microcon centrifugal filter device (MWCO: 3000 daltons); and (iv) desalting by a micro BioSpin chromatography column (MWCO: 6000 daltons); and (d) rehydration buffers, reducing agents and sample application in the first dimension isoelectric focussing were studied. Our results showed that sonication is easy to perform and resulted in a higher protein yield. Among the four extraction buffers, the urea containing buffers resulted in the extraction of the highest amount of protein while tris(hydroxymethyl)aminomethane buffers and trichloroacetic acid (TCA)/acetone precipitation allowed detection of a higher number of protein species (i.e. protein spots). Desalting by BioSpin and ultrafiltration have improved the 2-DE resolution of the water soluble fraction but have less effect on urea containing fractions. TCA/acetone precipitation was able to desalt all protein fractions independent of the extraction media, however extended exposure to this low pH medium has caused protein modification. Introduction of either DNase/RNase or benzonase treatment did not improve the discriminatory power of the 2-DE but this treatment did yield 2-DE with the clearest background. Proteolytic digestion was inhibited by addition of a protease inhibitor cocktail. Taken overall, a combination of sequential extraction and desalting by BioSpin chromatography for sample treatment before first dimension of 2-DE gave best results based on its simplicity and minimal protein loss. Finally, triscarboxyethylphosphine (TCEP) has performed well as a reducing agent in both the rehydration and equilibration buffers. The rehydration buffer found to be best in this study was 8.0 M urea, 2% 3-[(3-cholamidoprphyldimethylamino]-1-propanesulfonate, 4 mM TCEP and 1% immobilized pH gradient buffer. Subsequently, we applied this finding and performed 2-DE analysis on the soluble protein fractions extracted from light-starved cultured algal cells (nonblooming) and cultured cells grown under optimal conditions (blooming). 2-DE maps of these algal cultures were visibly different and many differentially expressed proteins were found.

Possible factors responsible for the toxicity of Cochlodinium polykrikoides, a red tide phytoplankton

Cochlodinium polykrikoides, a harmful red tide dinoflagellate, is highly toxic to fish, but the toxic mechanism is still unknown. Recent study has suggested that C. polykrikoides generates reactive oxygen species (ROS) such as superoxide anion (O 2 −) and hydrogen peroxide (H 2O 2), and the ROS-mediated ichthyotoxicity has been proposed. In this study, we found that the levels of O 2 − and H 2O 2 detected in C. polykrikoides were trace levels as compared with those of Chattonella marina which is well-known to produce ROS. Furthermore, no significant increase in O 2 − generation by C. polykrikoides was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus prepared from skin and gill of yellowtail, whereas C. marina generated increased level of O 2 − responding to these stimuli. Interestingly, the cell-free aqueous extract prepared from C. polykrikoides showed toxic effect on the HeLa cells, but the extract of C. marina had no significant effect. Furthermore, gradual accumulation of polysaccharides in the medium was observed during the growth of C. polykrikoides, and the medium gradually became viscous, but no such changes were observed in the medium of C. marina. These results suggest that multiple factors may be responsible for the toxic mechanism of C. polykrikoides.

Ciliate grazing on the parasite Amoebophrya sp. decreases infection of the red-tide dinoflagellate Akashiwo sanguinea

Parasitic dinoflagellates of the genus Amoebophrya commonly infect free-living dino- flagellates, some of which cause toxic or otherwise harmful red tides. These parasites prevent repro- duction of their hosts and kill infected cells on a time scale of days. Thus, epidemic outbreaks of Amoebophrya spp. are thought to facilitate the decline of red tides by causing mass mortality of host taxa. However, little is known about biotic and abiotic factors that regulate epidemic occurrence of Amoebophrya spp. in nature. We addressed the hypothesis that grazing by ciliate microzooplankton on the infective stage of Amoebophrya sp. can regulate parasite prevalence in the bloom-forming dinoflagellate Akashiwo sanguinea. In culture, the choreotrich ciliate Strobilidium sp. rapidly in- gested and digested infective dinospores of Amoebophrya sp. ex A. sanguinea. Laboratory experi- ments also showed that grazing by Strobilidium sp. could decrease infection of A. sanguinea by 70 to 80% relative to controls. Field experiments using plankton assemblages from Chesapeake Bay, USA, indicated that grazing by natural populations of ciliates may contribute to the regulation of parasitism in A. sanguinea. Thus, grazing by ciliates and other microzooplankton may indirectly influence the occurrence of red tides by limiting the spread of parasites like Amoebophrya sp.

Fluctuations of the red tide flagellates Chattonella spp. (Raphidophyceae) and the algicidal bacterium Cytophaga sp. in the Seto Inland Sea, Japan

A marine algicidal gliding bacterium Cytophaga sp. strain J18/M01 was isolated in 1990 from a station in northern Harima-Nada, the Seto Inland Sea, Japan, using the harmful red tide alga Chattonella antiqua (Hada) Ono as a susceptible organism. The bacterium can prey upon various species of microalgae. Temporal fluctuations of this bacterium and Chattonella spp. [C. antiqua and C. marina (Subrahmanyan) Hara et Chihara] were investigated weekly at the above station in the summer of 1997 and 1998, using immunofluorescence assay employing highly specific polyclonal antibodies for the bacterium. In the summer of 1997, the cell density of Chattonella spp. showed a maximum value (70 cells ml−1) on 8 July, and decreased thereafter. The bacterium Cytophaga sp. J18/M01 was commonly detected around a few hundreds of cells per milliliter or less. The number of Cytophaga sp. J18/M01 increased after the peak of Chattonella spp., and the maximum cell number of the bacterium was 1350 ml−1. This algicidal bacterium also followed the changes of total amounts of microalgal biomass (chlorophyll a+pheophytin) when Chattonella spp. were absent. In the summer of 1998, Chattonella spp. were relatively less abundant (maximum 21 cells ml−1), and the algicidal bacterium Cytophaga sp. J18/M01 showed a close relationship with the change of total microalgal biomass. The present study suggests that the algicidal bacterium Cytophaga sp. J18/M01 preyed upon, not only harmful red tide microalgae, but also other common microalgae such as diatoms, and the bacterium presumably plays an important role in regulating microalgal biomass in natural marine environments.

A Comparative Study of the Harmful Dinoflagellates Cochlodinium polykrikoides and Gyrodinium impudicum Using Transmission Electron Microscopy, Fatty Acid Composition, Carotenoid Content, DNA Quantification and Gene Sequences

The massive growth of the dinoflagellate alga Cochlodinium polykrikoides in Korean coastal waters was first observed in 1982. Thereafter the blooms associated with massive fish kills have been occurring annually, and C. polykrikoides is now regarded a harmful red tide organism in Korea. Recently, a new chain-forming Gyrodinium impudicum, usually occurring with Cochlodinium polykrikoides, was observed. Thus, we need to identify the toxic C. polykrikoides from the non-toxic Gyrodinium impudicum. Because of a similarity in morphology, it is difficult to distinguish the two species under the light microscope. In this study, comparisons were made using fine-scale features of transverse sections and biochemical analysis including rDNA sequencing targeted ITS regions. The observation of cellular arrangement and structure by transmission electron microscopy showed a similar distribution of the main components in both species, but location of the nucleus and distribution of the chloroplasts were different. Fatty acid composition showed that the long-chain polyunsaturated fatty acid docosahexanoic acid was not present in Cochlodinium polykrikoides. These data suggest that fatty acid composition may be a useful biochemical indicator to distinguish the microalgae. The amount of total carotenoids in Gyrodinium impudicum was approximately 10 times higher than in Cochlodinium polykrikoides. The nuclear DNA content in Cochlodinium polykrikoides was twice as high as that in Gyrodinium impudicum (p < 0.06). Sequence analyses coding for the 5.8S rDNA and ITS regions showed quite different nucleotide alignments with only 55% similarity, the length of the ITS regions also differed. The genetic differences between the two microalgae will allow the development of species specific DNA probes for species differentiation.

韓国南海沿岸のＣｏｃｈｌｏｄｉｎｉｕｍ ｐｏｌｙｋｒｉｋｏｉｄｅｓ赤潮の出現特性

A red tide occurred over a small portion of they bay in the 1980s. And a red tide has been transported up the South, West and East seas since the middle of the 1990s. The most common cause of a red tide was flagellate algae since the 1990s. In 1995, accounted for 95% of the total occurrence of the red tide. The harmful red tide was caused by the Cochlodinium that affected fishery operations in 1993 and 1994. Furthermore, it spread to the southern part of the East sea. In late August, 1995, a Cochlodinium red tide occurred frequently in outside of the Narodo coast, and then moved to the western part of the South sea. It lasted a month and then went away at the beginning of October. In August, 1998, It first appeared off the Narodo coast, and then spread repeatedly to the West and East coasts until the 15 of September. By carefully studying water quality, three characteristics of a Cochodinium red tide were identified about a month before the appearance of the red tide, there by effectively preventing a loss to the fishing industry. As a result, the water temperature was high (about 24°C) at the beginning. The concentrations of dissolved nitrate and phosphate were low, especially in the Narodo. However, conditions were favourable for the multiplication of Cochlodinium polykrikoides. With regards to phytoplankton, the amount of flagellate algae went up rapidly in the middle of August. By the latter half of the month, it accounted for 98% of the plant life. With the increase of nitrate and phosphate, the tablated water temperature reached 24-26°C. Cochlodinium red tide occurred in the Narodo coast several days later.