Survival and behavior patterns associated with hypoxia at different life stages of the pen shell Atrina cf. japonica.

Pen shell (Atrina cf. japonica) resources have been devastated in Ariake Bay, Japan, and to facilitate the recovery of this species, there is an urgent need to fully understand the factors contributing to its high levels of mortality. Pen shells living in natural waters grow through successive life stages, and environmental factors may affect these bivalves differently at different stages. Accordingly, to elucidate the causes of mortality in natural waters, it is necessary to gain an understanding of the quantitative effects of environmental factors on bivalves at each life stage. In this study, we sought to determine the differential effects of hypoxic conditions on 1-year-old (average shell length: 93.9 mm) and 2-year-old (146.5 mm) pen shells bred under artificial conditions. We exposed shells of each age group to six different dissolved oxygen (DO) concentrations for 96 h and monitored their behavior and survival rate. Based on the survival status, we estimated the lethal DO concentrations that induced 50%, 95%, and 5% mortality (LC50, LC95, and LC5, respectively) at each age. We found that for 1-year-old shells, the LC50 values at 48, 72, and 96 h were 0.51, 0.74, and 0.84 mg/L, respectively, whereas the corresponding values for 2-year-old shells were 0.74, 1.27, and 1.80 mg/L. Furthermore, we found that for 1- and 2-year-old shells, the estimated ranges from LC95 to LC5 at 48, 72, and 96 h were 0.39-0.68, 0.62-0.88, and 0.64-1.12 mg/L, and 0.31-1.75, 0.77-2.09, and 1.29-2.53 mg/L, respectively. Under low DO concentrations (0.47 to 1.93 mg/L and 0.49 to 3.30 mg/L for 1- and 2-year-old shells, respectively), we observed pen shells with more than half of their shell length protruding above the substrate. In addition to age and body size, the 1- and 2-year-old pen shells used in the present study also differed with respect to reproductive status, with 7.6% of 1-year-old and 96.7% of 2-year-old shells considered to be fully ripe. Collectively, our observations indicate that 2-year-old pen shells are less tolerant to hypoxic conditions than are 1-year-old pen shells, and we suspect that the differences in hypoxic tolerance could be attributable to differences in the physiological status of the pen shells during gonadal development. We believe the findings of this study will make an important contribution to enhancing our understanding of the effects of hypoxia on the viability of A. cf. japonica in natural waters.

Effects of Chattonella antiqua on the swimming behavior and brain monoamine metabolism of juvenile yellowtail (Seriola quinqueradiata).

Being the precursor of serotonin and melatonin, dietary supplementation with tryptophan (TRP) may modulates behavior, stress responses, and antioxidant capacity in fish. In this study, effects of Chattonella exposure on the swimming behavior and brain monoamine metabolism of yellowtail fed a commercial diet (control diet) or that enriched by 1.5% L-TRP (TRP + diet) were investigated. A 7-day dietary TRP supplementation elevated spontaneous swimming speed of yellowtail and mitigated their behavioral response to Chattonella (250 cells/mL) exposure. A 30-day dietary TRP supplementation elevated growth of juvenile yellowtail. Lethal exposure to Chattonella (1000 cells/mL) significantly elevated the turnover rates of serotonin, dopamine, and norepinephrine metabolism in fish fed control diet, but did not alter the serotonin turnover rate in fish fed TRP + diet. Our results suggested that dietary supplementation with TRP had potential to mitigate the stress response in yellowtail to Chattonella, partly via mediating their brain monoamine metabolism.

Toxin Profiles of Okadaic Acid Analogues and Other Lipophilic Toxins in Dinophysis from Japanese Coastal Waters.

The identification and quantification of okadaic acid (OA)/dinophysistoxin (DTX) analogues and pectenotoxins (PTXs) in Dinophysis samples collected from coastal locations around Japan were evaluated by liquid chromatography mass spectrometry. The species identified and analyzed included Dinophysis fortii, D. acuminata, D. mitra (Phalacroma mitra), D. norvegica, D. infundibulus, D. tripos, D. caudata, D. rotundata (Phalacroma rotundatum), and D. rudgei. The dominant toxin found in D. acuminata was PTX2 although some samples contained DTX1 as a minor toxin. D. acuminata specimens isolated from the southwestern regions (Takada and Hiroshima) showed characteristic toxin profiles, with only OA detected in samples collected from Takada. In contrast, both OA and DTX1, in addition to a larger proportion of PTX2, were detected in D. acuminata from Hiroshima. D. fortii showed a toxin profile dominated by PTX2 although this species had higher levels of DTX1 than D. acuminata. OA was detected as a minor toxin in some D. fortii samples collected from Yakumo, Noheji, and Hakata. PTX2 was also the dominant toxin found among other Dinophysis species analyzed, such as D. norvegica, D. tripos, and D. caudata, although some pooled picked cells of these species contained trace levels of OA or DTX1. The results obtained in this study re-confirm that cellular toxin content and profiles are different even among strains of the same species.

Application of LDH-release assay to cellular-level evaluation of the toxic potential of harmful algal species.

Lactate dehydrogenase (LDH)-release assay was applied to estimate the toxic potential of harmful algal species at the cellular level. African green monkey kidney (Vero), yellowtail fin epithelia (MJF), and rainbow trout gill (RTgill-W1) cells were used as target cells. A live cell suspension of Karenia mikimotoi (SUO-1) induced the release of LDH from these cell lines, while the activity of another strain, FUK, was much lower. The cell-free culture supernatants and ruptured cell suspensions of both strains of K. mikimotoi were less effective on LDH-release assay. Exposure experiments against abalone and shrimp revealed that SUO-1 showed much stronger lethal effects on these organisms than FUK. Among six phytoplankton species, three species known to be harmful algal species induced the release of LDH to different extents depending on the cell line, whereas the other three species, known to be non-toxic, showed no effects on any cell lines. These results suggest that LDH-release assay is a useful micro-plate assay for estimation of the toxic potential of harmful phytoplankton.

Comparative study on the toxic effects of red tide flagellates Heterocapsa circularisquama and Chattonella marina on the short-necked clam (Ruditapes philippinarum).

Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.

Evaluation of the potential biological toxicities of aqueous extracts from red tide phytoplankton cultures in in vitro and in vivo systems.

The biological toxic potentials of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined in both in vitro and in vivo systems. Interestingly, the extract from A. affine was the only one that showed potent cytotoxicities towards HeLa, Vero, and Neuro-2a cells in a concentration-dependent manner. Mice given intraperitoneal injections of the extracts revealed that none of the extracts exhibited serious toxicities in mice. However, temporal body weight loss was observed in the mice injected with the extract from A. affine during the early stage, and the dramatic enlargement of spleens was also observed in the mice on the 7th day after injection. Since A. affine extract showed potent hemolytic activity in vitro towards mouse erythrocytes, hemolytic anemia may be a possible mechanism responsible for the splenomegaly in the mice injected with A. affine extract. Similar marginal effects were observed in the mice injected with the extract from C. ovata; however, no significant toxic or detrimental effects were detected in the mice injected with the extract from G. impudicum. These results suggest that the extract from G. impudicum may not be contaminated with detectable levels of biologically hazardous compounds and may be relatively safe compared with the other two extracts.

The possibility of reactive oxygen species (ROS)-independent toxic effects of Cochlodinium polykrikoides on damselfish (Chromis caerulea).

To elucidate the ichthyotoxic mechanism of the harmful dinoflagellate Cochlodinium polykrikoides, a bioassay using damselfish was conducted. After exposure to a live-cell suspension of C. polykrikoides, all the fish were died within 90 min. In the presence of catalase and superoxide dismutase (SOD), no significant reduction in the toxicity of C. polykrikoides on the fish was observed. Furthermore, no significant levels of reactive oxygen species (ROS) were detected in five strains of C. polykrikoides isolated in Japan. Our results support the idea that certain toxic substances, rather than ROS, are mainly responsible for the fish-killing activity of C. polykrikoides.

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.

A discrepancy in superoxide scavenging activity between the ESR-spin trapping method and the luminol chemiluminescence method.

In a previous study of ours, the superoxide scavenging activity of aqueous extracts from dinophycean red tide flagellates was detected by an electron spin resonance (ESR)-spin trapping method, but not by an L-012 (luminol analog)-dependent chemiluminescence (CL) method. To investigate the discrepancy between the two methods, the effect of ferric-protein complexes on superoxide scavenging activity was examined. The reduced signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OOH due to superoxide dismutase (SOD) did not change with the addition of horseradish peroxidase (HRP), while the reduced CL response due to SOD was restored by the addition of different concentrations of HRP. Since HRP is a ferric-protein complex, the effects of other ferric-protein complexes, catalase and hemoglobin, on the reduced CL response due to SOD were examined, and similar results were obtained. As is the case with SOD, the reduced CL response activity due to an aqueous extract from a raphidophycean red tide flagellate, Chattonella ovata, was also enhanced by HRP, catalase, and hemoglobin. ESR spectra analyzed at 77 K indicated that aqueous extracts of Gymnodinium impudicum and Alexandrium affine, both of which are dinophycean red tide flagellates, contained a ferric-protein complex, and that an extract of C. ovata did not. These results suggest that the presence of such a ferric-protein complex is a causative factor in the discrepancy between the ESR and luminol CL methods when determining superoxide scavenging activity.

Antioxidant properties of aqueous extracts from red tide plankton cultures.

The antioxidant properties of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined. An electron spin resonance (ESR)-spin trapping method coupled with steady state kinetic analysis showed that all of the extracts directly scavenge superoxide, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. As for hydroxyl radical scavenging, the Fenton reaction and the method of ultraviolet radiation to hydrogen peroxide were used as hydroxyl radical generation systems. All of extracts reduced the level of hydroxyl radicals in both of the systems, indicating that the extracts also directly scavenge hydroxyl radicals. Since the levels of phenolic compounds did not correlate with the antioxidant activities of the extracts, substances other than phenolic compounds also appeared to be attributable to the activities. It is of our interest that the scavenging activities of extract from G. impudicum against superoxide and hydroxyl radicals were increased by heat exposure at 100 degrees C and 200 degrees C respectively. Although the reason for the increased activities of the aqueous extract from G. impudicum is not clear, the heat-resistance of the extract from G. impudicum might make it a desirable antioxidant.

Some dinophycean red tide plankton species generate a superoxide scavenging substance.

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.

Producing mechanism of an algicidal compound against red tide phytoplankton in a marine bacterium gamma-proteobacterium.

Strain MS-02-063, gamma-proteobacterium, isolated from a coast area of Nagasaki, Japan, produced a red pigment which belongs to prodigiosin members. This pigment, PG-L-1, showed potent algicidal activity against various red tide phytoplanktons in a concentration-dependent manner. An understanding of a mechanism of PG-L-1 production by this marine bacterium may yield important new insights and strategies for preventing blooms of harmful flagellate algae in natural marine environments. Therefore, we analyzed the mechanisms of PG-L-1 production. In our previous study, the pigment production by this marine bacterium was completely inhibited at 1.56 microg/ml of erythromycin or 3.13 microg/ml of chloramphenicol, while minimal inhibitory concentrations for cell growth of erythromycin and chloramphenicol against this bacterium were >100 and 25 microg/ml, respectively. It is interesting to note that the ability of the pigment production in erythromycin-treated bacterium recovered by an addition of homoserine lactone. In fact, the pigment production was inhibited by beta-cyclodextrin that inhibits autoinducer activities by a complex with N-acyl homoserine lactones. N-acyl homoserine lactones with autoinducer activities are ubiquitous bacterial signaling molecules that regulate gene expression in a cell density dependent process known as quorum sensing. Therefore, it was suggested that PG-L-1 produced by strain MS-02-063 is controlled by the homoserine lactone quorum sensing. It is speculated that this quorum sensing is involved in the production of algicidal agents of other marine bacteria. This bacterium and other algicidal bacteria might be concerned in regulating the blooms of harmful flagellate algae through the quorum sensing system.

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 O2- specific probe, a slightly lower but significant level of O2- 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, H2O2 was also detected in the K. mikimotoi cell suspension, but the level of H2O2 was approximately 10% of that in the C. marina suspension based on the cell number. No significant increase in O2- 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 O2- in response to these stimuli. Furthermore, O2- production in C. marina, but not in K. mikimotoi, was inhibited by treatment with proteinase K.

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(2)O(2)), and the ROS-mediated ichthyotoxicity has been proposed. In this study, we found that the levels of O(2)(-) and H(2)O(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.

Comparison of hemolytic activities among strains of Heterocapsa circularisquama isolated in various localities in Japan.

Heterocapsa circularisquama (Dinophyceae), a red tide dinoflagellate, is toxic to bivalve molluscs such as the pearl oyster (Pinctada fucata), but no harmful effects of this alga on fish have been observed so far. We found that 7 strains of H. circularisquama showed hemolytic activities toward rabbit erythrocytes in a cell-density dependent manner, but to quite different extents. The strains which are known to be highly toxic to bivalves tend to show stronger hemolytic activities and vice versa, suggesting that the hemolytic activity is parallel with the shellfish toxicity of H. circularisquama. Since the hemolytic assay is simple, semiquantitative, and reproducible, this assay is useful not only to search for certain toxins responsible for the shellfish toxicity of H. circularisquama but also to estimate the potential toxicity of a newly appeared strain of H. circularisquama.

Photosensitizing hemolytic toxin in Heterocapsa circularisquama, a newly identified harmful red tide dinoflagellate.

Red tides of Heterocapsa circularisquama (H. circularisquama), recently identified as a novel species of dinoflagellate, have frequently caused mass mortality of several species of bivalves in Japan, while no harmful effects of this flagellate on fish have been reported so far. We found that the cell-free ethanol extract prepared from H. circularisquama caused hemolysis of rabbit erythrocytes and demonstrated cytotoxic effects in HeLa cells and on the microzooplankton rotifer (B. plicatilis) in a dose- and time-dependent manner. Interestingly, the hemolytic activity and cytotoxic effects of the extract were completely dependent on the presence of light. When the experiments were conducted in the dark, no hemolysis was observed even at very high concentration of the extract. These results suggest that H. circularisquama has photosensitizing hemolytic toxin which can be easily extracted into ethanol. This may be the first report documenting the occurrence of photosensitizing hemolytic toxin in marine phytoplankton species.