Yukihiko Matsuyama

MICROSATELLITE MARKERS REVEAL POPULATION GENETIC STRUCTURE OF THE TOXIC DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN JAPANESE COASTAL WATERS1

This is the first report to explore the fine‐scale diversity, population genetic structure, and biogeography of a typical planktonic microbe in Japanese and Korean coastal waters and also to try to detect the impact of natural and human‐assisted dispersals on the genetic structure and gene flow in a toxic dinoflagellate species. Here we present the genetic analysis of Alexandrium tamarense (Lebour) Balech populations from 10 sites along the Japanese and Korean coasts. We used nine microsatellite loci, which varied widely in number of alleles and gene diversity across populations. The analysis revealed that Neis genetic distance correlated significantly with geographic distance in pair‐wise comparisons, and that there was genetic differentiation in about half of 45 pair‐wise populations. These results clearly indicate genetic isolation among populations according to geographic distance and restricted gene flow via natural dispersal through tidal currents among the populations. On the other hand, high P‐values in Fishers combined test were detected in five pair‐wise populations, suggesting similar genetic structure and a close genetic relationship between the populations. These findings suggest that the genetic structure of Japanese A. tamarense populations has been disturbed, possibly by human‐assisted dispersal, which has resulted in gene flow between geographically separated populations.

Interactions between the red tide dinoflagellates Heterocapsa circularisquama and Gymnodinium mikimotoi in laboratory culture

Abstract Interactions between Heterocapsa circularisquama and Gymnodinium mikimotoi, causative red tide dinoflagellates, were investigated using bialgal cultures. G. mikimotoi was killed by H. circularisquama when the initial cell density of each species was set at 200 cells ml−1. However, cells of H. circularisquama transformed to temporary cysts when the initial cell density of G. mikimotoi was increased to 2000 cells ml−1. Thus the interaction between H. circularisquama and G. mikimotoi was found to be dependent upon the initial cell densities of the two species. Culture filtrate of H. circularisquama induced no inhibitory effect on the growth of G. mikimotoi. Similarly when separated by a membrane filter, G. mikimotoi grew well when cultured with H. circularisquama. G. mikimotoi appear to be killed by cell contact with H. circularisquama. In growth experiments using a culture filtrate of G. mikimotoi and cultures separated by a membrane filter, G. mikimotoi was shown to secrete a substance that inhibited the growth of H. circularisquama. However, the inhibitory effect of the medium was found at higher cell densities of G. mikimotoi than in the bialgal cultures at which the growth of H. circularisquama was suppressed and formed temporary cysts. It is likely that the inhibitory effect of G. mikimotoi on H. circularisquama in the bialgal cultures occurred mainly by direct cell contact. The growth of H. circularisquama and G. mikimotoi in the bialgal cultures was simulated using a mathematical model to quantify the interaction. The degree that G. mikimotoi was inhibited by H. circularisquama was found to be three times larger than the inhibitory effect of G. mikimotoi on H. circularisquama.

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.

Toxicity and LD50 levels of the red tide dinoflagellate Heterocapsa circularisquama on juvenile pearl oysters

Abstract Laboratory experiments were conducted to clarify the mechanism of the mass mortality of pearl oysters ( Pinctada fucata ) associated with the red tide dinoflagellate Heterocapsa circularisquama such as occurred in Ago Bay, Japan, in 1992. Two-month-old pearl oysters were treated with various densities of H. circularisquama, Heterosigma akashiwo and a sterile control. Immediately after the exposure to H. circularisquama cells, juvenile pearl oysters rapidly contracted their mantles and closed their shell valves. Then, they contracted their gills and experienced irregular heartbeat patterns until the heart stopped permanently. The mortality of the juveniles was closely correlated with the cell density of H. circularisquama . The LD 50 was approximately 20 000 cells ml −1 after 24 h and 10000 cells ml −1 after 48 h from the start of the experiments. However, in the presence of either H. akashiwo at 100 000 cells ml −1 or supernatant of H. circularisquama culture, juvenile pearl oysters did not die within 72 h. During the red tide in Ago Bay, 1992, the maximum cell density of H. circularisquama reached over 80 000 cells ml −1 . Therefore, we consider that the mass mortality of cultured pearl oysters during this red tide was caused by the direct action of H. circularisquama cells.

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.

HEMOLYTIC ACTIVITY OF HETEROCAPSA CIRCULARISQUAMA (DINOPHYCEAE) AND ITS POSSIBLE INVOLVEMENT IN SHELLFISH TOXICITY

Heterocapsa circularisquama Horiguchi is lethal to shellfish, particularly bivalves such as pearl oysters (Pinctada fucata Gould). No detrimental effects of this flagellate on fish have been observed thus far. In this study, we found that H. circularisquama causes mammalian erythrocytes to lyse. Among the erythrocytes tested, rabbit erythrocytes showed the highest susceptibility, whereas erythrocytes from cattle, sheep, and human were relatively insensitive. Heterocapsa triquetra Stein, which is morphologically similar to H. circularisquama but not toxic to bivalves, showed no hemolytic activity toward rabbit erythrocytes. Culture supernatant or ultrasonic‐ruptured cells of H. circularisquama showed only weak hemolytic activity. Hemolytic activity was found in the ethanol extract of H. circularisquama cells, suggesting that the hemolytic agents may be more stable in ethanol than in aqueous solution. Both an intact flagellate cell suspension and the ethanol extract caused morphological changes and eventual collapse of unfertilized eggs of Pacific oyster. Furthermore, the ethanol extract was lethal to the microzooplankton rotifer Brachionus plicatilis Müller, which is highly sensitive to H. circularisquama. Our results suggest that a hemolytic toxin produced by H. circularisquama may be one of the causative agents responsible for the shellfish toxicity.

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.

The life cycle of Gyrodinium instriatum (Dinophyceae) in culture

The life cycle events of an unarmored dinoflagellate Gyrodinium instriatum Freudenthal et Lee has been investigated using clonal cultures. After the inoculation of vegetative cells into fresh medium, clumping of gametes was observed after a period of 10 days. In the clumps, a number of gametes were observed to be swimming in close contact with each other, pairing successively and then forming a plano‐zygote. This clumping behavior is considered to be useful in the performance of sexual reproduction, particularly in the event of low cell density, because it increases the chance of fusion. Gametes of this species were most often isogarnous, although apparent amsogamous fusions were occasionally observed. When planozygotes were isolated and placed in fresh medium, they enlarged their size and finally divided into two cells. The daughter cells continued to multiply by binary fission and produced vegetative cells. Thus, G. instriatum has an alternative cycle between vegetative cells and zygotes without a hypnozygote stage. However, cysts of this species were transformed from large motile cells (pre‐cyst cells) which are oblong and dorso‐ventrally flattened in shape. These mottle pre‐cyst cells have two longitudinal flagella, which may indicate that cysts of this species are of zygote origin. On the basis of these results, the relationship between zygotes and pre‐cysts of G. instriatum is discussed. Excystment was enhanced by dark and cold treatments prior to the incubation for germination experiments with a germination success rate of 26–64%. Encystment was greatly inhibited by the lack of dark and cold treatments.

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 °C and 200 °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.

Effects of irradiance of various wavelengths from light-emitting diodes on the growth of the harmful dinoflagellate Heterocapsa circularisquama and the diatom Skeletonema costatum

We investigated the effects of specific light wavelengths from light-emitting diodes (LEDs) on the growth of the dinoflagellate Heterocapsa circularisquama, which kills bivalves, and the diatom Skeletonema costatum, which is an important food source for bivalves. Growth of H. circularisquama was obviously inhibited at 590 nm and a photon flux density less than 75 μmol quanta/m2/s. However, growth of S. costatum was not suppressed by irradiance from any LEDs tested from near-ultraviolet to near-infrared wavelengths at 75 μmol quanta/m2/s. The growth rate of H. circularisquama in an experimental treatment group with irradiance provided by both cool-white fluorescent lamps (12∶12 h L:D cycle) and a 590-nm LED (continuous irradiance) was 0.43/day. In the control group with irradiance provided only by cool-white fluorescent lamps (12∶12 h L:D cycle), the growth rate was 0.63/day, indicating that growth of H. circularisquama was suppressed by 590 nm (less than 75 μmol quanta/m2/s) irradiance from the LED and the continuous irradiance. The use of 590-nm LEDs in bivalve culture at irradiance levels less than 75 μmol quanta/m2/s might encourage the growth of the useful diatom S. costatum without stimulating growth of the harmful dinoflagellate H. circularisquama.