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.     

Chemotaxonomic survey of sterols and fatty acids in six marine raphidophyte algae

Fatty acid and sterol profiles allowed for clear discrimination betweentheraphidophyte genera Chattonella,Heterosigma, Fibrocapsa andOlisthodiscus, but exhibited little differentiation forindividual Chattonella species(C.marina, C. antiqua and C.subsalsa). Sterol and fatty acid profiles do not support theseparation of Chattonella antiqua and C.marina as distinct species. Ecophenotypic variations in lipidprofiles were also observed between Chattonella strainsfromdifferent geographic locations. Sterol signatures which may be useful aschemotaxonomic markers were: the absence of C₂₇ sterols (cholesteroland 24-dihydrozymosterol) in Heterosigma akashiwo; thepresence of isofucosterol in Chattonella; and theoccurrence of brassicasterol, poriferasterol and fucosterol inOlisthodiscus luteus. High levels of eicosapentaenoic acid(EPA; 17-27% of fatty acids) were present in all raphidophyte species. Lipidcomposition correlated more closely to recent molecular classification ofraphidophytes than carotenoid pigments.     

Life cycle of Chattonella marina (Raphidophyceae) inferred from analysis of microsatellite marker genotypes

Red tides of Chattonella spp. have caused continuous damage to Japanese aquaculture, however, the life cycle of this organism remains incompletely understood. To further investigate this matter, we assessed genotypes at 14 microsatellite markers in three varieties of Chattonella marina, viz., C. marina var. antiqua, C. marina var. marina, and C. marina var. ovata, to establish whether Chattonella undergoes asexual diploidization or sexual reproduction. After genotyping 287 strains of C. marina, all but one of these strains was shown to be heterozygous for at least some loci, and thus, in the diploid state, suggesting that Chattonella strains undergo sexual reproduction. In addition, we performed single‐cell amplification on ‘small cells’ that are derived from vegetative cells under dark and low‐nutrient conditions. The results indicated the existence of two types of small cells. The ‘Small cell Type 1’ was found to be heterozygous, genotypically equivalent to the vegetative cells, and is therefore diploid. These small cells may change to resting cells (cysts) directly. The ‘Small cell Type 2’ was homozygous at all analyzed loci, suggesting that these small cells are haploid and may be derived by meiosis. As fusion between small cells has previously been observed, the ‘Small cell Type 2’ may be the gamete of Chattonella. We present a construct of the full life cycle of Chattonella marina based on our own and previous results.     

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL‐TIME PCR ASSAYS1

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real‐time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small‐subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.     

Morphological and genetic characterization of bloom‐forming Raphidophyceae from Brazilian coast

Massive fish kills caused by bloom‐forming species of the Raphidophyceae occur in many marine coastal areas and often cause significant economic losses. The ultrastructure and phylogeny of marine raphidophytes from the Brazilian coast have not been fully analyzed. Here, we present the first combined morphological and genetic characterization of raphidophyte strains from the Brazilian coast. Ten strains of four raphidophyte species (Chattonella subsalsa, C. antiqua, Heterosigma akashiwo, and Fibrocapsa japonica) were characterized based on morphology (including ultrastructure) and LSU rDNA sequences. Chattonella subsalsa and C. antiqua formed two distinct genetic clades. We found that the cell size is the only phenotypic feature separating C. subsalsa and C. antiqua strains from Brazil, whereas traditional characteristics used for species separation in the genus Chattonella (i.e., tail size, chloroplast presence in the tail, ‘oboe‐shaped’ mucocysts, and presence of thylakoids in the pyrenoid matrix) were not sufficiently discriminative, due to their overlapping in the two taxa. The phylogenetic analysis indicated intra‐specific geographic differences among C. subsalsa sequences, with two subclades: one formed by isolates from Brazil, USA, and Iran, and another by a sequence from the Adriatic Sea (Italy). Fibrocapsa japonica also showed intra‐specific geographic differences, with a sequence from a Brazilian strain grouped with strains from Japan, Australia, and Germany, all of them distinct from the Italian isolates. This is the first combined morphological and phylogenetic analysis of raphidophytes from the South Atlantic. Our findings broaden knowledge of the biodiversity of this important bloom‐forming algal group.     

ANTIOXIDANT ENZYME RESPONSE AND REACTIVE OXYGEN SPECIES PRODUCTION IN MARINE RAPHIDOPHYTES1

Raphidophytes (class Raphidophyceae) produce high levels of reactive oxygen species (ROS), yet little is known regarding cellular scavenging mechanisms needed for protection against these radicals. Enzymatic activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in conjunction with the production of superoxide (O₂^??) and hydrogen peroxide (H₂O₂) in batch cultures of five different raphidophytes species during early exponential, late‐exponential, and stationary growth phases. The greatest concentrations of O₂^?? per cell were detected during exponential growth with reduced levels in stationary phases in raphidophytes Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara, Chattonella marina (Subrahman.) Y. Hara et Chihara, and Chattonella antiqua (Hada) Ono (strain 18). Decreasing trends from exponential to stationary phases for SOD activity and H₂O₂ per cell were observed in all species tested. Significant correlations between O₂^?? per cell and SOD activity per cell over growth phase were only observed in three raphidophytes (Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua strain 18), likely due to different cellular locations of externally released O₂^?? radicals and intracellular SOD enzymes measured in this study. CAT activity was greatest at early exponential phase for several raphidophytes, but correlations between H₂O₂ per cell and CAT activity per cell were only observed for Fibrocapsa japonica Toriumi et Takano, Chattonella antiqua (strain 18), and Chattonella subsalsa Biecheler. Our results suggest that SOD and CAT play important protective roles against ROS during exponential growth of several raphidophytes, while other antioxidant pathways may play a larger role for scavenging ROS during later growth.     

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 effects on Heterosigma akashiwo and Chattonella marina (Raphidophyceae), and toxic effects on natural plankton assemblages by a thiazolidinedione derivative TD49 in a microcosm

The algicidal effects of the thiazolidinedione derivative TD49 on Heterosigma akashiwo and Chattonella marina (Raphidophyceae) were assessed, and the response of the planktonic community and environment to the algicide was evaluated in a microcosm, quantifying 12 L. The abundance of over 80 % of H. akashiwo and C. marina declined in a day significantly in microcosms to which TD49 was added (final concentration 2 μM), and this was correlated with an abrupt decline in the culture pH. The number of protists (i.e., ciliates) other than H. akashiwo and C. marina gradually increased with time in the TD49 treatments, implying that the decline in numbers of H. akashiwo and C. marina cells resulting from TD49 treatment was a major factor in the growth of the other organisms. However, TD49 may be toxic to aquatic zooplankton communities, even though it is a highly selective algicide for harmful algae bloom species. The study indicates that TD49 is an effective agent for the control for H. akashiwo and C. marina blooms in enclosed and eutrophic water bodies.     

A novel thiazolidinedione derivative TD118 showing selective algicidal effects for red tide control

Thiazolidinedione (TD) derivatives have been found to have an algicidal effect on harmful algal bloom microalgae. In this study, 75 TD derivatives were synthesized and analyzed for algicidal activity. Among these synthetic TDs, 18 TD derivatives showed specific algicidal activity on two strains belonging to the classes Raphidophyceae (Chattonella marina and Heterosigma akashiwo) and Dinophyceae (Cochlodinium polykrikoides). Two strains belonging to Bacillariophyceae (Navicula pelliculosa and Phaeodactylum EPV), one strain belonging to Dinophyceae (Amphidinium sp.), and a Eustigmatophycean microalga (Nannochloropsis oculata) showed less sensitivity to the TD derivatives than the other two phyla. The most reactive TD derivative, compound 2 (TD118), was selected and tested for morphological and physiological changes. TD118 effectively damaged the cell membrane of C. marina, H. akashiwo and C. polykrikoides. The O₂ evolution and photosystem II efficiency (F _v /F _m ) of C. marina, H. akashiwo and C. polykrikoides were also severely reduced by TD118 treatment. Amphidinium sp., N. pelliculosa, Phaeodactylum EPV and N. oculata showed less reduction of O₂ evolution and the F _v /F _m by TD118. These results imply that the species-specific TD structure relationship may be due to structural and/or physiological differences among microalgal species.     

Utilization of dissolved organic phosphorus by different groups of phytoplankton taxa

The utilization of nine dissolved organic phosphorus (DOP) compounds by five bloom-causing phytoplankton species was studied under batch culture conditions. The DOP compounds included were adenosine 5-triphosphate (ATP), adenosine 5-monophosphate (AMP), cytidine 5-monophosphate (CMP), guanosine 5-monophosphate (GMP), uridine 5-monophosphate (UMP), glucose-6-phosphate (G6P), sodium glycerophosphate (GYP), 4-nitrophenyl phosphate (NPP), and triethyl phosphate (TEP), and the phytoplankton taxa were Skeletonema costatum, Prorocentrum micans, Alexandrium tamarense, Chattonella marina, and Heterosigma akashiwo. The four flagellate taxa, P. micans, A. tamarense, C. marina, and H. akashiwo, grew well under various DOP regimes. P. micans and C. marina were the most capable of using DOP compounds, sustaining better growth on a majority of nucleotides (ATP, AMP, CMP, GMP, and UMP) and phosphomonoesters (G6P and GYP) than in inorganic phosphorus (P) controls. A. tamarense and H. akashiwo showed equivalent growth in most organic and inorganic P cultures, while the diatom species, S. costatum, could only utilize AMP and GMP. Furthermore, A. tamarense and C. marina could endure N, P-depleted conditions. Among the nine DOP compounds tested, the nucleotide compounds had the highest nutritional value for algal cell growth, while TEP could not sustain growth as the sole source of P. These results suggest that enhanced DOP utilization and the endurance of nutrient-limitation by harmful flagellate taxa offer their competitive advantages, which may account for the frequent occurrence of their blooms in coastal waters.     

Lethal effects of ichthyotoxic raphidophytes,Chattonella marina,C. antiqua,and Heterosigma akashiwo,on post-embryonic stages of the Japanese pearl oyster,Pinctada fucata martensii

The inimical effects of the ichthyotoxic harmful algal bloom (HAB)-forming raphidophytes Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua on the early-life stages of the Japanese pearl oyster Pinctada fucata martensii were studied. Fertilized eggs and developing embryos were not affected following exposure to the harmful raphidophytes; however, all three algal species severely affected trochophores and D-larvae, early-stage D-larvae, and late-stage pre-settling larvae. Exposure to C. marina (5 × 10² cells ml⁻¹), C. antiqua (10³ cells ml⁻¹), and H. akashiwo (5 × 10³ cells ml⁻¹) resulted in decreased success of metamorphosis to the trochophore stage. A complete inhibition of trochophore metamorphosis was observed following exposure to C. antiqua at 5 × 10³ cells ml⁻¹ and C. marina at 8 × 10³ cells ml⁻¹. In all experiments, more than 80% of newly formed trochophores were anomalous, and in the case of exposure to H. akashiwo at 10⁵ cells ml⁻¹ more than 70% of D-larvae were anomalous. The activity rates of D-larvae (1-day-old) were significantly reduced following exposure to C. antiqua (8 × 10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (10⁴ cells ml⁻¹, 24 h). The activity rates of pre-settling larvae (21-day-old) were also significantly reduced following exposure to C. antiqua (10³ cells ml⁻¹, 24 h), C. marina (8 × 10³ cells ml⁻¹, 24 h), and H. akashiwo (5 × 10⁴ cells ml⁻¹, 24 h). Significant mortalities of both larval stages were induced by all three raphidophytes, with higher mortality rates registered for pre-settling larvae than D-larvae, especially following exposure to C. marina (5 × 10²–8 × 10³ cells ml⁻¹, 48–86 h) and C. antiqua (10³–8 × 10³ cells ml⁻¹, 72–86 h). Contact between raphidophyte cells and newly metamorphosed trochophores and D-larvae, 1-day-old D-larvae, and 21-day-old larvae resulted in microscopic changes in the raphidophytes, and then, in the motile early-life stages of pearl oysters. Upon contact and physical disturbance of their cells by larval cilia, H. akashiwo, C. marina and C. antiqua became immotile and shed their glycocalyx. The trochophores and larvae were observed trapped in a conglomerate of glycocalyx and mucus, most probably a mixture of larval mucous and raphidophyte tricosyts and mucocytes. All motile stages of pearl oyster larvae showed a typical escape behavior translating into increased swimming in an effort to release themselves from the sticky mucous traps. The larvae subsequently became exhausted, entrapped in more heavy mucous, lost their larval cilia, sank, become immotile, and died. Although other toxic mediators could have been involved, the results of the present study indicate that all three raphidophytes were harmful only for motile stages of pearl oysters, and that the physical disturbance of their cells upon contact with the ciliary structures of pearl oyster larvae initiated the harmful mechanism. The present study is the first report of lethal effects of harmful Chattonella spp. towards larvae of a bivalve mollusc. Blooms of H. akashiwo, C. antiqua and C. marina occur in all major cultivation areas of P. fucata martensii during the developmental period of their larvae. Therefore, exposure of the motile early-life stages of Japanese pearl oysters could adversely affect their population recruitment. In addition, the present study shows that further research with early-life development of pearl oysters and other bivalves could contribute to improving the understanding of the controversial harmful mechanisms of raphidophytes in marine organisms.     

Molecular Analysis of Ribosomal RNA Gene of Red Tide Algae Obtained from the Seto Inland Sea

Eleven clones from five species of the planktonic microalgae, (Chattonella antiqua, Chattonella marina, Heterosigma akashiwo, Alexandrium catenella, and Scrippsiella trochoidea), which were collected from the Seto Inland Sea in Japan and from Thailand, were subjected to nucleotide sequence analysis of the D1/D2 domain of the large subunit (LSU) of their ribosomal RNA genes. After amplification by polymerase chain reaction using degenerated primers, whole-nucleotide sequences for the D1/D2 domains of the LSU rRNA gene of 11 microalgae were analyzed. Phylogenic tree analysis using these nucleotide sequences showed each species located in a cluster corresponding to its morphological classification. The nucleotide sequence data for Chattonella spp. suggest that multiple clones of both Chattonella antiqua and Chattonella marina are present in the Seto Inland Sea and that red tide blooms of Chattonella spp. in different years may have contained different clones. Received September 6, 1999; accepted December 16, 1999.     

Genetic structure of Japanese Chattonella marina (Raphidophyceae) populations revealed using microsatellite markers

Chattonella marina var. antiqua and C. marina var. marina (Raphidophyceae) are red tide‐forming, harmful phytoplankton species. We investigated the genetic diversity and genetic relationship among the populations using microsatellite markers to identify putative sources of C. marina var. antiqua and C. marina var. marina in Japanese coastal populations. A positive correlation between genetic divergence and geographical distance (isolation by distance) was recognized for C. marina var. antiqua. The C. marina var. antiqua populations were established throughout a geological time scale, and genetic divergence had progressed in each population with gene flow depending on geographic distances. In contrast, isolation by distance was not observed for C. marina var. marina populations, and the genetic divergence among populations was extremely high. The Tokyo Bay population of C. marina var. marina, which was first recognized in 2008, had many private alleles but was related to the Kagoshima Bay population. The Tokyo Bay population may have been established by several invasions from the Kagoshima Bay population and other regions.     

Assessment of Microzooplankton Grazing on <Emphasis Type="Italic">Heterosigma akashiwo</Emphasis> Using a Species- Specific Approach Combining Quantitative Real-Time PCR (QPCR) and Dilution Methods

Delaware’s Inland Bays (DIB) are subject to numerous mixed blooms of harmful raphidophytes each year, and Heterosigma akashiwo is one of the consistently occurring species. Often, Chattonella subsalsa, C. cf. verruculosa, and Fibrocapsa japonica co-occur with H. akashiwo, indicating a dynamic consortium of raphidophyte species. In this study, microzooplankton grazing pressure was assessed as a top–down control mechanism on H. akashiwo populations in mixed communities. Quantitative real-time polymerase chain reaction (QPCR) with species-specific primers and probes were used in conjunction with the dilution method to assess grazing pressure on H. akashiwo and other raphidophytes. As a comparison, we measured changes in chlorophyll a (chl a) to determine whole community growth and mortality caused by grazing. We detected grazing on H. akashiwo using QPCR in samples where chl a analyses indicated little or no grazing on the total phytoplankton community. Overall, specific microzooplankton grazing pressure on H. akashiwo ranged from 0.88 to 1.88 day⁻¹ at various sites. Experiments conducted on larger sympatric raphidophytes (C. subsalsa, C. cf. verruculosa and F. japonica) demonstrated no significant microzooplankton grazing on these species. Grazing pressure on H. akashiwo may provide a competitive advantage to other raphidophytes such as Chattonella spp. that are too large to be consumed at high rates by microzooplankton and help to shape the dynamics of this harmful algal bloom consortium. Our results show that QPCR can be used in conjunction with the dilution method for evaluation of microzooplankton grazing pressure on specific phytoplankton species within a mixed community. An erratum to this article can be found at     

Relationships between dynamics of red tide-causing raphidophycean flagellates and algicidal micro-organisms in the coastal sea of Japan

Temporal fluctuations of algicidal micro-organisms against the red tide causing raphidophycean flagellates Chattonella antiqua (Hada) Ono and Heterosigma akashiwo (Hada) Hada ex Hara et Chihara were investigated using the microplate most probable number (MPN) method in northern Hiroshima Bay and Harima-Nada, the Seto Inland Sea, in 1992 and 1993. In Har-ima-Nada, both flagellates appeared at low levels (< 1 cell mL^?1), and killer micro-organisms against the two flagellates (C-killer for C. antiqua and H-killer for H. akashiwo) also appeared at low densities (< 2 mL^?1). In northern Hiroshima Bay, C. antiqua cells were scarce (< 1 cell mL^?1), and C-killers occurred at a low level (≤ 3.4 mL^?1). Conversely, red tides of H. akashiwo occurred there in June of both years. The dynamics of H-killers revealed a close relationship with that of H. akashiwo populations. H-killers followed the increase of H. akashiwo cells, reached a maximum level after the beginning of decline of H. akashiwo, maintained a high level for at least 1 week after the crash of bloom, and then decreased. C-killers consistently remained at low densities during the period of H. akashiwo red tides in both 1992 and 1993. Hence, algicidal micro-organisms specifically associated with the occurrence and crash of H. akashiwo red tides, and presumably contributed to the rapid termination of the red tides in the coastal seas such as northern Hiroshima Bay.     

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.     

Haemolytic activity and reactive oxygen species production of four harmful algal bloom species

Based on haemolytic activity and reactive oxygen species (ROS) production of Chattonella marina, Chattonella antiqua, Heterocapsa circularisquama, Alexandrium tamiyavanichii and Karenia mikimotoi, the species were categorized into four types. (1) H. circularisquama: haemolytic activity was detected in both cell suspension and cell-free culture supernatant, but with greater activity in cell suspension than in the supernatant suggesting the presence of both cell surface and secreted haemolytic agents. (2) A. tamiyavanichii: equal haemolytic activities were detected in both the cell suspension and cell-free culture supernatant suggesting the presence of only secreted haemolytic agents. (3) K. mikimotoi: haemolytic activity was detected only in the cell suspension, indicating haemolytic agents occur only on the cell surface. (4) C. marina and C. antiqua: no significant haemolytic activity was detected in either cell suspension or cell-free culture supernatant, but high ROS were detected in the cell suspensions. Heterocapsa circularisquama and K. mikimotoi showed lethal effects on rotifers (Brachionus plicatilis), whereas A. tamiyavanichii, C. marina and C. antiqua had no effect. Our results suggest that H. circularisquama, K. mikimotoi and A. tamiyavanichii produce haemolytic agents with distinct characteristics, whereas C. marina and C. antiqua have an extremely potent ability to produce ROS.     

Isolation and characterization of a marine algicidal bacterium against the harmful raphidophyceae <Emphasis Type="Italic">Chattonella marina</Emphasis>

A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of ULjin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal activity by different culture filtrate volumes, the 10% (100 μl/ml) concentration showed the biggest change in algicidal activity; there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5. Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina.     

Dynamics of seagrass bed microbial communities in artificial Chattonella blooms: A laboratory microcosm study

The influence of algicidal and growth-inhibiting bacteria in a seagrass (Zostera marina) bed, and their capability of controlling blooms of the fish-killing raphidophyte flagellate, Chattonella antiqua, were examined in laboratory microcosm experiments. Bacterial communities in seawater collected from the seagrass bed and Z. marina biofilm suppressed artificial Chattonella blooms in the presence of their natural competitors and predators. Phylogenetic analysis suggest that considerable numbers of bacteria that suppress Chattonella, including algicidal or growth-inhibiting bacteria isolated from seagrass biofilm and seawater from the seagrass bed, are members of Proteobacteria that can decompose lignocellulosic compounds. A direct comparison of partial 16S rRNA gene sequences (500 bp) revealed that the growth-limiting bacterium (strain ZM101) isolated from Z. marina biofilm belonged to the genus Phaeobacter (Alphaproteobacteria) showed 100% similarity with strains of growth-limiting bacteria isolated from seawater of both the seagrass bed and nearshore region, suggesting that the origin of these growth-limiting bacteria are the seagrass biofilm or seawater surrounding the seagrass bed. This study demonstrates that Chattonella growth-limiting bacteria living on seagrass biofilm and in the adjacent seawater can suppress Chattonella blooms, suggesting the possibility of Chattonella bloom prevention through restoration, protection, or introduction of seagrass in coastal areas.     

Anti-Angiogenesis Activities of Novel Peptide Complexes: Mitochondria-Disruptive 9mer Peptides Conjugated with the Integrin alpha V beta 3-Homing Cyclic RGD Motif

Chattonella is one of the most toxic red tide phytoplankton and causes severe damage to fish farming. Recent studies demonstrated that Chattonella sp. generates superoxide and hydroxyl radicals, which may be responsible for the toxicity of this plankton. However, little is known about the mechanism of the production of oxygen radicals by Chattonella, and the role of oxygen radicals in Chattonella themselves is also unclear. In this study, we found that superoxide dismutase (SOD) and catalase inhibited the growth of Chattonella marina concomitant with their morphological changes. In the presence of these enzymes, the shape of vegetative C. marina cells changed from spindle to round. Furthermore, the generation of oxygen radicals by C. marina depended on the growth phase; the rate of superoxide and hydrogen peroxide generation was the highest during exponentially growing phase and subsequently decreased to one-fifth of the maximal level in the stationary growth phase. These results suggest that oxygen radicals generated by C. marina play an essential role in their own survival, especially in cell division.