Kazuhiko Ichimi

Variety of PSP toxin profiles in various culture strains of Alexandrium tamarense and change of toxin profile in natural A. tamarense population

Paralytic shellfish poisoning (PSP) toxin profiles were compared between clonal and axenic culture strains of Alexandrium tamarense prepared from cysts. The cysts were collected from two stations in northern Japan. The major toxin components of A. tamarense were C2 and GTX4, however, the proportions of C2 and GTX4 varied largely 0.7–78.8 mol% and 79.4–8.5 mol%, respectively. Some culture strains contained significantly higher proportion of neoSTX than other strains. These results indicate that strains with various toxin profiles exist in the same region, and suggest that the comparison of the toxin profiles between strains at different localities is considerably difficult. A drastic change of the toxin profile was observed in natural planktonic populations containing A. tamarense. This may be explained by the presence of a lot of planktonic populations with various toxin profiles growing around the sea area.

The growth and cyst formation of a toxic dinoflagellate, Alexandrium tamarense, at low water temperatures in northeastern Japan.

A field survey was carried out in early spring to investigate the growth physiology and efficiency of cyst formation of Alexandrium tamarense in low water temperatures. A bloom of A. tamarense occurred in a stratified water column, formed by river inflow. The in situ growth rate estimated from daily cell abundance was high, 0.33 divisions day(-1), at 7.5-9 degrees C. New cysts began to be observed during the late growth phase. Maximum cyst flux (600 cysts cm(-2) day(-1)) was observed just after maximum cell abundance occurred. PO(4)-P Chl a(-1) gradually decreased and reached extremely low levels beyond the mid-growth phase of A. tamarense. As sinking cysts were also recognized at that time, it suggests cyst formation may have been induced by depletion of phosphorus source. The incidence of cyst formation (C.I) was 30%. The number of C.I was the same as reported previously for batch cultures under conditions suitable for vegetative growth. These results indicate that A. tamarense grows with considerably higher growth rate and transforms to cysts in high numbers, in low water temperatures in the field.

Kinetics of diarrhetic shellfish poisoning toxins, okadaic acid, dinophysistoxin‐1, pectenotoxin‐6 and yessotoxin in scallops Patinopecten yessoensis

Four toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-6 (PTX6), and yessotoxin (YTX), all associated with diarrhetic shellfish poisoning (DSP), were administered via syringe to Scallops Patinopecten yessoensis and their distribution in the hepatopancreas, adductor muscle, and combined other tissues (mantle, gill, gonad) was analyzed by liquid chromatography-mass spectrometry. Toxins exclusively remained in the hepatopancreas irrespective of the injection site, adductor muscle or hepatopancreas. When injected into hepatopancreas, OA, DTX1, and YTX were metabolized to 7-O-palmitoylOA, 7-O-palmitoylDTX1 and 45-hydroxyyessotoxin (45OH-YTX), respectively. Such metabolic changes were insignificant when toxins were injected into the adductor muscle. The residual ratio for each toxin in the hepatopancreas was less than 20%. Mortalities of scallops treated with PTX6 were lower than those treated with other toxins.

Paralytic shellfish poisoning (PSP) toxin profiles and short-term detoxification kinetics in mussels Mytilus galloprovincialis fed with the toxic dinoflagellate Alexandrium tamarense

Abstract Mussels ( Mytilus galloprovincialis ) were experimentally contaminated with paralytic shellfish poisoning (PSP) toxins by being fed with the toxic dinoflagellate Alexandrium tamarense , and changes in toxin content and specific composition during the decontamination period were analyzed by high-performance liquid chromatography (HPLC). Toxins excreted by the mussels into the seawater were also recovered using an activated charcoal column and analyzed by HPLC. The predominant toxins in A. tamarense , mussels, and seawater were the N -sulfocarbamoyl-11-hydrosulfate toxins (C1,2) and carbamate gonyautoxins-1,4 (GTX1,4). There were no remarkable differences in the relative proportions of the predominant toxins within A. tamarense , mussels and seawater. Because the relative proportion of the various toxin analogues excreted by the mussels was similar to that within their tissues during detoxification, it appeared that the selective release of particular toxins by the mussels was unlikely. The total amount of toxin lost from mussels was nearly equal to that which was found dissolved in the seawater, suggesting that, at least the early stages of mussel detoxification, most losses can be accounted for by excretion.

Effects of temperature and irradiance on growth of strains belonging to seven Skeletonema species isolated from Dokai Bay, southern Japan

The effect of temperature on the growth of Skeletonema ardens, S. costatum sensu stricto, the S. marinoi–dohrnii complex, S. japonicum, S. menzelii, S. pseudocostatum and S. tropicum isolated from Dokai Bay in southern Japan were examined under five to seven different temperatures and an irradiance of 150 µmol m−2 s−1. The effect of irradiance on the growth of the seven Skeletonema species was also examined under a wide range of irradiances ranging from 7–700 µmol m−2 s−1 at 20°C. All Skeletonema species were able to grow at temperatures ranging from 15 to 25°C. Intra-species differences in specific growth rates of four strains for S. menzelii, and five strains for S. ardens, the S. marinoi–dohrnii complex, S. japonicum and S. tropicum were not significant (Kruskal–Wallis test, P > 0.05). Significant inter-species differences in specific growth rates were observed at 10, 15, 25 and 30°C (Kruskal–Wallis test, P < 0.01; Steel–Dwass test, P < 0.01). The S. marinoi–dohrnii complex and S. japonicum grew faster than other species at the lower temperatures of 10 and 15°C, and S. ardens and S. menzelii grew at the highest temperature of 35°C. The maximum specific growth rates (μ max) from growth–irradiance curves ranged from 1.50 to 3.44 d−1. Threshold values of irradiance (I 0) and saturation irradiance (S) for growth ranged from 3.9 to 7.6 µmol m−2 s−1, and from 250 to 740 µmol m−2 s−1, respectively. In Dokai Bay, our results suggested that the occurrence of Skeletonema species should be affected mainly by temperature and less by irradiance. In particular, only two species, the S. marinoi–dohrnii complex and S. japonicum could maintain their populations in the surface water during the cold season, whereas during other periods, all seven species could maintain their population under the strong influence of estuarine circulation, which rapidly flushed the surface water out of Dokai Bay. Temperature and irradiance dependent growth values were in good agreement with their geographical distributions. The S. marinoi–dohrnii complex and S. japonicum were capable of growing in cold regions, but our results suggested that S. ardens and S. menzelii will tend to prefer tropical regions.

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.

EXTREMELY HIGH GROWTH RATE OF THE SMALL DIATOM CHAETOCEROS SALSUGINEUM ISOLATED FROM AN ESTUARY IN THE EASTERN SETO INLAND SEA, JAPAN1

Small single‐celled Chaetoceros sp. are often widely distributed, but frequently overlooked. An estuarine diatom with an extremely high growth potential under optimal conditions was isolated from the Shinkawa‐Kasugagawa estuary in the eastern part of the Seto Inland Sea, western Japan. It was identified as Chaetoceros salsugineum based on morphological observations. This strain had a specific growth rate of 0.54 h−1 at 30°C under 700 μmol · m−2 · s−1 (about 30% of natural maximal summer light) with a 14:10 L:D cycle; there was little growth in the dark. However, under continuous light it grew at only 0.35 h−1 or a daily specific growth rate of 8.4 d−1. In addition, cell density, chlorophyll a, and particulate organic carbon concentrations increased by about 1000 times in 24 h at 30°C under 700 μmol · m−2 · s−1 with a 14:10 L:D cycle, showing a growth rate of close to 7 d−1. This very rapid growth rate may be the result of adaptation to this estuarine environment with high light and temperature. Thus, C. salsugineum can be an important primary producer in this estuary in summer and also an important organism for further physiological and genetic research.

Chemical composition of Coscinodiscus wailesii and the implication for nutrient ratios in a coastal water, Seto Inland Sea, Japan

Chemical compositions of Coscinodiscus wailesii were determined for four samples of natural cells. Results revealed that the cellular Si:N ratio of C. wailesii cell was 2.4:1.0-5.2:1.0. The impacts of C. wailesii on surrounding coastal water were evaluated from hydrographic observations, in which C. wailesii cell density, nutrients concentrations and temperature were monitored from November 2001 to February 2005 in Harima-Nada, the Seto Inland Sea, Japan. In low temperature periods, from October to December, two peaks of C. wailesii cell density were observed and nutrient concentrations were depleted. The draw-down ratio of Si(OH)(4) and DIN (Si(OH)(4):DIN ratio) in the water column were similar to the cellular Si:N ratio of C. wailesii cells, which have high Si contents. In addition the effects of different Si(OH)(4):DIN ratio were determined for in situ bottle incubation experiments. In the culture experiments, picoplankton (0.2-2.0 microm) which consisted of small flagellates became dominant under low Si(OH)(4):DIN ratios. These results suggested that the sizes distribution shifted to small size and the phytoplankton community was changed to small flagellates after the C. wailesii bloom. These changes would influence predators at higher trophic levels in its coastal ecosystem.

Negligible effect of the benthic fauna on measuring the nutrient upward fluxes from coastal sediments

Although the influence of benthic fauna on estimating the nutrient upward fluxes from the coastal sediments are a concern, there are few comprehensive studies. We conducted monthly observations in a coastal embayment to examine the effect of benthic fauna on measuring the nutrient flux. Nutrient fluxes were measured by the core incubation method and the densities of organisms in benthic communities were also measured. Numerical composition of benthic communities, according to taxonomic group, was dominated by Polychaeta composed of a few Capitellida and Spionidae species. A negligible effect of the benthic communities on measuring nutrient fluxes was demonstrated.

Research on the Discoloration of Nori (Porphyra) and Characteristics of Water Quality Using the Numerical Model

In and around the Seto Inland Sea Japan, there are many aquaculture facilities for Nori (Porphyra). Recently, the production of Nori (Porphyra) severely decreased at Kagawa prefectural coast, eastern Bisan Seto. We have conducted the field research and calculated the trajectories of nutrient particles under the condition in which western wind velocity changes using the numerical model. From the results of field research and numerical simulation, we suggested that Nori bad harvest was caused due to a little rain in the autumn season. It was also suggested that at Nori bad harvest area, the nutrient supply by river discharge was so important for aquaculture of Nori.