Free radical production by the red tide alga, Chattonella antiqua.

Summary The red tide alga,Chattonella antiqua, was found to show a strong chemiluminescence, using luminol as the reagent, when exposed to ultraviolet irradiation. This luminescence was completely inhibited by ascorbate or catalase, suggesting that hydrogen peroxide was generated by the plankton. Red tide cells exposed to fish gill mucus from young yellowtail resulted in the release of a large number of mucocysts and a weak luminosity, and showed a strong reduction of cytochromec in the medium. Therefore, the discharge of mucocysts from the red tide, induced by the presence of gill mucus, may be accompanied by the release of active oxygen species. The active oxygen may be involved in depolymerization of mucus glycoproteins from the gill lamellae.     

Inhibitory effect of red alga lectin and skipjack fat on the growth of the red tide plankton Chattonella antiqua

Lectin prepared from Gracilaria verrucosa and fish fat extracted from skipjack muscle were investigated for their activities against the growth of the red tide plankton Chattonella antiqua. Addition of the seaweed lectin at 50 μg/ml altered the morphology of the plankton cells, which became shrunken form, and their growth was completely suppressed. Skipjack fat not only inhibited microalgal growth but also destroyed the cells within 5 min at 20 μg/ml. These marine materials are promising candidates for restraining the action of red tide planktons.     

Sterols and fatty acids of the red tide flagellates Heterosigma akashiwo and Chattonella antiqua (Raphidophyceae)

The fatty acids and sterols of the raphidophyte flagellates, Heterosigma akashiwo (Australian and Plymouth strains) and Chattonella antiqua (Japanese strain) are reported. The major sterol of both species is 24-ethylcholesterol, which is more commonly associated with higher plants and has rarely been reported in unicellular algae. C. antiqua also contained 24-dihydrozymosterol [cholest-8(9)-en-3β-ol], which is also uncommon in marine algae. The major fatty acids in both raphidophytes are 16:0, 18:4ω3, 20:5ω3, 16:1ω7 and 14:0. Polyunsaturated fatty acids accounted for 46–50% of the total fatty acids in both species. The fatty acid 18:5ω3 was detected in H. akashiwo, but not in C. antiqua. This acid is found in some dinoflagellates and Prymnesiophycean algae, but this is the first report of its presence in the Raphidophyceae. The lipid distributions obtained for H. akashiwo and C. antiqua provide unique signature profiles for use in taxonomic, food-web and organic geochemical studies. The fatty acid and sterol distributions of these two raphidophytes justify their assignment to a separate class within the ‘brown algal’ line.     

Phosphate metabolism during diel vertical migration in the raphidophycean alga, Chattonella antiqua

The ecological advantage of diel vertical migration on the nutrition and accumulation of Chattonella antiqua, which is one of the dominant red-tide forming phytoplankton species in the Seto Inland Sea of Japan, was examined using a large axenic culture tank, in which vertical stratification of salinity, temperature and nutrients was maintained, analogous to natural conditions observed when red tides occur. C. antiqua was capable of migrating through very sharp salinity and temperature gradients. At night the species migrated to the deep nutrient-rich water and assimilated nutrients. During the daytime it migrated to the nutrient-depleted surface water and used the accumulated nutrients for photosynthesis. Nitrogen uptake was synchronized with phosphate uptake. ³¹P-NMR spectroscopy during the migration experiment revealed that C. antiqua has the capability of nocturnal phosphate uptake in the deep nutrient-rich water, but no capability of synthesizing polyphosphate, which was considered to be the intracellular phosphate pool. These findings were compared with those reported for another raphidophycean, Heterosigma akashiwo. Although both species carry out vertical migration and nocturnal nutrient uptake, only H. akashiwo has the capability of making an intracellular polyphosphate pool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Occurrence of the polyamines caldopentamine and homocaldopentamine in axenic cultures of the red tide flagellates Chattonella antiqua and Heterosigma akashiwo (Raphidophyceae)

The polyamines caldopentamine and homocaldopentamine were detected in axenic strains of Chattonella antiqua and Heterosigma akashiwo ( Raphidophyceae ), respectively, as well as spermidine, the most abundant polyamine in both phytoplankton species. Trace amounts of putrescine, diaminopropane and norspermine were also detected in both species. Spermine was detected only from C. antiqua . These long linear polyamines are characteristic components of thermophilic bacteria. The detection from two species of Raphidophyceae indicates that the occurrence of long linear polyamines is not restricted to thermophilic microorganisms.     

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 phytoplanktersoften cause severe damage to fish farming. Previous studieshave demonstrated that C. marina and C. ovata continuously producereactive oxygen species (ROS) such as superoxide anion (O₂^–)hydrogen peroxide (H₂O₂) under normal growth conditions, andan ROS-mediated toxic mechanism against fish and other marineorganisms has been proposed. Although the exact mechanism ofROS generation in these phytoplankters still remains to be clarified,our previous study suggested that NADPH oxidase-like enzymelocated on the cell surface of C. marina may be involved inO₂^– 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-oneand 5-(and-6)-carboxy-2',7'-dichlorodihydrodihydrofluoresceindictate, acetyl ester, which are specific fluorescent probefor detecting O₂^– and H₂O₂, respectively. Observationby fluorescence microscopy of live phytoplankters incubatedwith each probe revealed that O₂^– is mainly generatedon the cell surface, whereas H₂O₂ is generated in the intracellularcompartment in these phytoplankters. When the cells were rupturedby ultrasonic treatment, O₂^– levels of C. marina and C.ovata decreased significantly, whereas a few times higher levelsof H₂O₂ were detected in the ruptured cell suspensions whencompared with the levels of the live cell suspension. In immunoblottinganalysis, the protein recognized by anti-human gp91 phox wasdetected in both species. These results suggest that, in bothphytoplankters, the underlying mechanisms of O₂^– and H₂O₂generation may be distinct and such systems are independentlyoperating in the cells.     

Diazo-reaction positive substance observed in the cortex of Chattonella antiqua

In order to investigate the causative factors responsible for removal of mucous coat from the gill lamellae of young yellowtail, Seriola quinqueradiata by red tide, diazo-reactions were employed for planktons and their media. The concentration of NO2- in the medium containing the raphidophyceae, Chatonella antiqua (ca 2000 cells/ml), was 0.70 +/- 0.05 (mu g/ml +/- SEM). In addition, diazo-reaction positive substances (NOx) which may degenerate the mucous, was highly concentrated in the cortex (perikaryon) of Chattonella antiqua. Morphologically, mucocysts, and chloroplats were likewise present in the cortex. Mucocysts were packed with fine fibrous content. Histochemically, the mucocysts were stained with PAS and had an abundance of nitrogen oxides (NOx). We observed discharge of the fibrous material from the mucocysts. These results suggest that when Chattonella antiqua is passing between the gill lamellae, NOx discharged from the mucocysts may act on the mucous, leading to the degeneration and concomitant removal of the mucous coat from gill lamellae.     

Life cycle,physiology, ecology and red tide occurrences of the fish-killing raphidophyte Chattonella

The marine fish-killing raphidophytes of the genus Chattonella currently consist of five species, i.e. C. antiqua, C. marina, C. minima, C. ovata and C. subsalasa. The distribution of Chattonella species was confirmed in tropical, subtropical and temperate regions in the world accompanying mass mortalities of fishes in nature and in aquaculture. The fish-killing mechanisms are still unclear, but suffocation is the ultimate cause of fish death. Increasing evidence is pointing towards the generation of reactive oxygen species (ROS, e.g. superoxide), which are responsible for the gill tissue injury and mucus production that leads to death of fishes. A taxonomic revision was proposed based on morphology and genetic diversity that Chattonella antiqua and Chattonella ovata should be varieties of Chattonella marina possessing nomenclatural priority. Optimum temperatures for growth are 25 °C for C. antiqua and C. marina, 25–30 °C for C. ovata and 20–30 °C for Chattonella subsalsa. Adequate ranges of salinity for growth were about 20–30 for Chattonella species. Chattonella cells generally divide once a day. Laboratory culture experiments with artificial synthetic medium demonstrated that C. antiqua, C. marina and C. ovata used only Fe chelated with EDTA for growth, although tested diatoms and dinoflagellates used rather many kinds of chelated Fe. A suitable concentration of humic acid supplied with iron also had enhancing effects on the growth of C. antiqua. Diel vertical migration was observed in Chattonella, and the cells reached 7.5 m deep at night in the case of C. antiqua demonstrated by a mesocosm experiment in the Seto Inland Sea. Chattonella species have diplontic life history and have haploid cyst stage in their life cycle. Encystment was observed through formation of pre-encystment small cells after the depletion of nitrogen, and the small cells sink to the sea bottom to complete cyst formation by attachment to the solid surface such as diatom frustules and sand grains. Newly formed cysts are in the state of spontaneous dormancy and they need cold temperature period of four months or longer for maturation (acquisition of germination ability). Cysts germinate in early summer and resultant vegetative cells play an important role as seed populations in blooming in the summer season. However, relatively small part of cyst populations actually germinate from bottom sediments, and success of red tide formation is dependent on the growth in water columns. Since red tides of Chattonella were observed when diatoms were scarce in seawater, diatoms appear to have a key for the predominance of Chattonella in water columns. Diatom resting stages in sediments need light for germination/rejuvenation, whereas Chattonella cysts can germinate even in the dark, implying the selective germination of Chattonella cysts at the sea bottom under calm oceanographic conditions which contribute to bloom formation of Chattonella. As a mechanism of red tide occurrences of Chattonella in coastal sea, “diatom resting hypothesis” was presented. Biological control using diatoms is proposed through the germination/rejuvenation of resting stages suspending from bottom sediments to euphotic layer by sediment perturbation with submarine tractors or fishing trawling gears. Since diatoms have much higher growth rates, and newly joined diatom vegetative cells grow faster and prevent occurrence of Chattonella red tides as a result. As another prevention strategy for Chattonella red tides, algicidal bacteria inhabiting in seaweed beds and seagrass beds are presented. Co-culture of fish and seaweeds in aquaculture areas, and the developments of seaweed- and seagrass-beds would be practical and ultimately environment-friendly strategies for the prevention of harmful red tides of Chattonella by virtue of natural algicidal bacteria supplied from seaweeds and leaves of seagrass.     

Development of compound microsatellite markers in the harmful red tide species Chattonella ovata (Raphidophyceae)

We isolated 12 polymorphic microsatellites from the noxious red‐tide‐causing alga Chattonella ovata. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from four to 12, and the observed and expected heterozygosities ranged from 0.238 to 0.850 and from 0.310 to 0.889, respectively. These loci are useful for revealing the genetic structure of and gene flow among C. ovata populations.     

Galacturonic-acid-induced increase of superoxide production in red tide phytoplankton Chattonella marina and Heterosigma akashiwo

Red tide phytoplankton, Chattonella marina and Heterosigma akashiwo, are known to generate superoxide anion (O2-). We found that galacturonic acid (GaLUA) stimulated C. marina and H. akashiwo to generate increased amounts of O2-. Since such effect was not observed in any other monosaccharides tested, our results suggest that the binding of GalUA to specific sites on the flagellate cell surface may induce the increase of 02- production.     

Isolation and characterization of light-dependent hemolytic cytotoxin from harmful red tide phytoplankton Chattonella marina

Chattonella marina (C. marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C. marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.     

Photocontrol of Nuclear DNA Replication in Chattonella antiqua (Raphidophyceae)

The timing of nuclear DNA replication was examined in a synchronizedcell population of a red-tide flagellate, Chattonella antiqua,using fluorescence microspectrophotometry with a DNA-specificfluorochrome, 4',6-diamidino-2-phenylindole (DAPI). Under alternating12-h periods of light and dark (12L12D), nuclear DNA began toincrease synchronously ca. 10 h after the onset of light irradiation.Even when the light-off timing of the light period or the wholespan of the 12-h light period was shifted after synchronizationunder 12L12D cycles, the timing of the beginning of nuclearDNA replication was invariably ca. 10 h from the onset of lightirradiation. When irradiation was not given, there was no increaseof nuclear DNA. The conclusion reached was that light irradiationis necessary for nuclear DNA replication in Chattonella antiquaand that the timing of the replication is dependent upon onlythe timing of the onset of the last irradiation. In other words,a light-on signal induces the transition of cell nuclei fromthe G₁ into the S phase and also determines the timing of thisevent. When not irradiated, cells are arrested in the G₁ phase. ³ Present address: Department of Biology, Faculty of Science,University of Tokyo, Hongo, Tokyo 113, Japan. ⁴ Present address: Frontier Research Programs, RIKEN, Wako-city,Saitama 351-01, Japan. (Received February 28, 1987; Accepted June 5, 1987)     

Inductive and Inhibitory Effects of Light on Cell Division in Chattonella antiqua

The cell division of a red tide flagellate, Chattonella antiqua,was synchronously induced under light and dark regimes of 10L14D(a light period, L, for 10 h followed by a dark period, D, for14 h), 12L12D and l4L10D. In all regimes cell number began toincrease ca. 14 h after the onset of L and almost doubled duringone LD cycle. When the light-off timing of the last L was changedor the whole L was shifted, cells that had been synchronizedunder 12L12D invariably began to divide ca. 14 h after the onsetof L. This shows that the timing of cell division was determinedby the time of the onset of L. When cells were continuously exposed to light after a cell division,the subsequent cell division was inhibited. This effect waslimited to cells that had been synchronized under short-dayconditions. Thus it can be concluded that light has both inductive and inhibitoryeffects on cell division in this alga, the latter effect dependingupon the previously given light and dark regimes. (Received December 21, 1984; Accepted February 28, 1985)     

Growth response of the marine blue-green alga, Gomphosphaeria aponina, to inorganic nutrients and significance to management of Florida red tide.

A bioactive isolate from the blue-green alga Gomphosphaeria aponina is cytolytic towards the dinoflagellate, Gymnodinium breve, Florida's red tide organism. Batch and continuous cultures of G. aponina were used to determine nutrient limitation and to optimize mass-culture conditions. Iron and inorganic carbon were growth limiting; first-order saturation kinetics were observed for both substrates. For Fe3+, kinetic parameters were: Ks = 62 +/- 9 microgram 1(-1), and Ke max = 2.14 days-1. Maximum growth was observed at 150 micrograms Fe3+1(-1), with minimal growth below 10 microgram 1(-1). Cells colonized with increasing Fe3+ supplements, and time to reach maximum culture populations was inversely related to the concentration. For HCO3-,Ks = 62 +/- 4 mg1(-1) and Ke max = 1.3 day-1. Additions of NH4+ up to 200 micrograms 1(-1) were not stimulatory, whereas at 1.0 mg 1(-1) levels, Ke was 50% greater than for NO3- enriched medium. Concentrations greater than 25 micrograms PO4(3-) 1(-1) were stimulatory. However, at 1 mg1(-1), growth was less than in controls. Comparison of similar data available for G. breve would suggest that the inorganic nutrient requirements of G. aponina were minimal. Potential for natural control of G. breve by G. aponina is perhaps related to the efficiency of contact of the two organisms.     

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

The unicellular marine phytoplankton Chattonella marina is knownto exhibit potent fish-killing activity. Previous studies havedemonstrated that C. marina produces reactive oxygen species(ROS), and ROS-mediated ichthyotoxic mechanism has been postulated.However, the exact toxic mechanism is still controversial. Inthis study, we obtained evidence that C. marina produces nitricoxide (NO) under normal growth conditions. We utilized chemiluminescence(CL) reaction between NO and luminol–H₂O₂ to detect NOin C. marina cell suspensions. In this assay, significant CLwas 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 byC. marina was also confirmed by a spectrophotometric assay basedon the measurement of the diazo-reaction-positive substances(NO_x) and by fluorometric assay using highly specific fluorescentindicator of NO. The NO level in C. marina was significantlydecreased by N^G-nitro-L-arginine methyl ester (L-NAME), a specificNO synthase (NOS) inhibitor. The addition of L-arginine resultedin the increased NO level, whereas NaNO₂ had no effect. Theseresults suggest that a NOS-like enzyme is mainly responsiblefor NO generation in C. marina.     

Generation of superoxide anion radicals by the marine phytoplankton organism, Chattonella antiqua

The generation of superoxide anion radical (O₂^–, believedto be a causative factor in the killing of fish by the phytoplanktonChattonella antiqua, has been examined using several methods:electrochemical technique, reduction of ferricytochrome c andfluorescent laser microscope. Evidence is presented to suggestthat these organisms release superoxide continuously while theyare living, even in the resting state. Additional generationof O₂^– accompanies the discharge of mucocysts, and istriggered when they are exposed to mucus from the gill lamellaeof fish. Such instantaneous generation of O₂^– is alsoinduced when the organisms are in contact with an electrodepoised at a potential of +0.1 V versus Ag/AgCI, which is positiveenough to oxidize O₂^– to O₂.     

Comparative studies on the fish-killing activities of Chattonella marina isolated in 1985 and Chattonella antiqua isolated in 2010, and their possible toxic factors

Chattonella antiqua isolated in 2010 showed extremely more potent fish-killing activities against red sea bream, Japanese horse mackerel, and blue damselfish than those of Chattonella marina isolated in 1985. Chemiluminescence and electron spin resonance (ESR) analyses suggested greater reactive oxygen species (ROS)-producing activity of C. antiqua than that of C. marina. Sodium benzoate, a hydroxyl radical scavenger, significantly suppressed the fish-killing activity of C. antiqua on blue damselfish. The chlorophyll level in the gill tissue of blue damselfish exposed to flagellate cells increased along with the exposure time, and the cell count of gill-associated C. antiqua estimated with chlorophyll level was higher than that of C. marina. These results suggest that the ROS-producing activity and affinity of Chattonella cells to the gill surface may be important factors influencing the fish-killing activity of Chattonella species.     

Algicidal activity of glycerolipids from brown alga Ishige sinicola toward red tide microalgae

Bioassay-guided fractionation of a methanol extract of the brown alga, Ishige sinicola, led to the isolation of five algicidal compounds. Their structures were determined to be α-monoglycerides of eicosa-5Z,8Z,11Z,14Z-tetraenoic (arachidonic) acid, octadeca-6Z,9Z,12Z,15Z-tetraenoic acid, linoleic acid and oleic acid, and 1-O-palmitoyl-3-O-(6-sulfo-α-D-quinovopyranosyl)-sn-glycerol on the basis of spectroscopic data and a comparison with the data in the literature. These glycerolipids showed moderate-to-high cell lysis activity against the red tide microalgal species, Heterosigma akashiwo, Karenia mikimotoi and Alexandrium catenella, at a concentration of 20 μg/mL.     

Some characteristics of photosynthetic inorganic carbon uptake of a marine macrophytic red alga

Abstract Some characteristics of photosynthetic inorganic carbon uptake by Palmaria palmata, a marine red macroalga, have been measured under physiological conditions in artificial seawater. The apparent affinity of thallus for CO₂ [K_1/2(CO₂)] at pH 8.0 and 15°C was 21.4±3.0mmol m^?3 CO₂ under air, and 25.7±70mmol m^?3 CO₂ under N₂. The corresponding values of V_max were 2.98 ± 0.42 and 3.65±0.87 mmol O₂ evolved g Chr^?1 s^?l. The apparent K_m(CO₂) of isolated ribulose bisphosphate carboxylase was determined at pH 8.0 and 30 °C to be 30.2 mmol m^?3 CO₂, and the corresponding value of V_max was 19.67 μniol CO₂ g protein^?1 s^?1. The CO₂ compensation points of the thallus were measured in artificial seawater at pH 8.0 under air and N₂, using a gas-chromatographic method. The values were relatively low, rising from 10 cm³ m^?3 at 15°C, to 35 cm³ m^?3 at 25°C, but were not affected by the O₂ concentration. The lack of an effect of O₂ on photosynthesis and on compensation point indicates that there is little photorespiratory CO₂ loss in this macroalga. The high affinity of the thallus for CO₂, and the low CO₂ compensation concentrations, are consistent with the occurrence of bicarbonate uptake in this alga.