Masaaki Kodama

Effect of water temperature and light intensity on growth rate and toxicity change in Protogonyaulax tamarensis

The effect of water temperature and light intensity on the growth rate and the toxicity of Protogonyaulax tamarensis was examined using a monoclonal culture isolated from Ofunato Bay, Japan in March, 1984. The growth rate decreased with the decrease of either light intensity or temperature. The amount of toxin produced increased concomitantly with the decrease of the growth rate. However, the increase of the toxicity under low growth rate was less remarkable when the growth rate was lowered by the decrease of light intensity. This indicates that photosynthesis plays an important role in the production of toxin in P. tamarensis.

DOMOIC ACID PRODUCTION IN NITZSCHIA SP. (BACILLARIOPHYCEAE) ISOLATED FROM A SHRIMP-CULTURE POND IN DO SON, VIETNAM

Domoic acid (DA), a neuroexcitatory amino acid, was detected in batch culture of the newly recognized species Nitzschia navis‐varingica Lundholm et Moestrup. The production of DA by this diatom was confirmed by electrospray ionization mass spectrometry. The diatom was collected from a shrimp‐culture pond in Do Son, Vietnam. The production of DA (1.7 pg·cell−1) is within the levels reported for Pseudo‐nitzschia multiseries (Hasle) Hasle. The DA production started during the late exponential growth phase and reached a maximum during the early stationary growth phase. Maximum DA levels in the axenic culture decreased to about half that of the nonaxenic culture (0.9 pg·cell−1 vs. 1.7 pg·cell−1), suggesting that DA production by the new species is influenced by bacteria.

Production of paralytic shellfish toxins by a bacterium Moraxella Sp. isolated from Protogonyaulax tamarensis

A bacterium Moraxella sp. isolated from Protogonyaulax tamarensis was cultured in various conditions. Changes of toxicity and toxin components of the cells during culture were analyzed by bioassay and HPLC-fluorometric analysis. Toxin productivity of Moraxella sp. increased when it was cultured in nutrition-deficient environments. The main toxins produced by Moraxella sp. in these conditions were gonyautoxins (GTXs), mainly GTX 1 and 4 which are major toxins of P. tamarensis.

Relationships Between Bacteria and Harmful Algae

The interactions of harmful algal (HA) species with their physico-chemical and biological environments ultimately determine their abundance and distribution. While an algal taxon’s physiological tolerance limits (e.g., temperature, salinity, light) and intrinsic phenotypic traits (e.g., growth rate, nutrient uptake, vertical migration) largely define its ecological niche, relationships with the biological components of an ecosystem (e.g., grazers, microbes, pathogens, competing algal species) play a critical role in its ability to achieve concentrations that lead to the many negative impacts characterizing harmful species. A biological factor of potentially great significance in regulating the population and even toxin dynamics of HA that has received increasing yet comparatively little attention to date is their relationship with the ubiquitous and diverse bacterial community. These microbes, which exist as free-living forms as well as securely attached to algal cells, occasionally with a high degree of taxonomic specificity, have now been demonstrated to modulate (either positively or negatively) algal growth rates and transitions between life history stages, influence toxin production, and even induce the rapid lysis of algal cells. Previous reviews of this topic (e.g., Doucette 1995; Doucette et al. 1998) described an exciting, emerging field in which molecular-based approaches were just beginning to supplement traditional bacteriological techniques, yielding new insights into the nature and ecological implications of bacterialalgal interactions. During the intervening time, continued application of molecular techniques, novel experimental methods, and targeted studies of natural bacterial communities have contributed valuable details of the relationships between bacteria and HA that will be the focus of this chapter.

Toxin production in the dinoflagellate Protogonyaulax tamarensis

Many clones of Protogonyaulax tamarensis were collected from Ofunato Bay in the same season and cultured under the same conditions. The toxicity of the cultured cells of these clones was remarkably different from each other (maximum of 100-fold). Significant differences (maximum of 20-fold) in toxicity of cultured cells were also observed among subclones isolated from a single clonal culture. It is unlikely that this variation in toxicity among subclones is due to mutation during culture. From these observations we suggest that toxin production in P. tamarensis is not a hereditary characteristic.

External secretion of tetrodotoxin from puffer fishes stimulated by electric shock

Four toxic species of puffers (Takifugu pardalis, T. poecilonotus, T. vermicularis and T. niphobles) secreted large amounts of tetrodotoxin into the surrounding water immediately after being stimulated by electric shock. The total amount of toxin secreted ranged from 2 000 to 50 000 MU depending upon the specimen. The amount of toxin secreted seemed to be related to the toxicity of individuals. Non-toxic specimens of T. rubripes did not secrete any toxin. The stimulation always caused inflation of puffers, and this is generally accepted as a repelling behavior of these species. The secreted tetrodotoxin may act as a repellent to predators.

Confirmation of domoic acid production of Pseudo-nitzschia multiseries isolated from Ofunato Bay, Japan.

Production of domoic acid (DA), the responsible toxin for amnesic shellfish poisoning, was examined for 44 strains of Pseudo-nitzschia spp. isolated from Ofunato Bay, Japan. Only one strain which was identified as Pseudo-nitzschia multiseries produced DA in a level comparable to Canadian strains. No significant DA was detected in the rest of the strains, indicating that toxic P. multiseries does not bloom in a high density in the bay.

Occurrence of saxitoxin and other toxins in the liver of the pufferfish Takifugu pardalis

Highly toxic livers of the pufferfish Takifugu pardalis were extracted with acidic ethanol. The toxins extracted were partially purified by chromatography on Bio-Gel P-2 and then Bio-Rex 70, resulting in separation into three fractions I, II and III. ratios of total mouse units per fraction were approximately 0.1:100:0.01, respectively, with tetrodotoxin (TTX) as standard. By TLC, electrophoresis and a TTX analyzer, Fr. II was identified as TTX and, unexpectedly, Fr. III as saxitoxin, while Fr. I remains unidentified.

Frequent occurrence of paralytic shellfish poisoning toxins as dominant toxins in marine puffer from tropical water

Considerably high toxicity was detected in marine puffers collected from Masinloc Bay, Philippines. The toxicity was detected in the liver, intestine, muscle and skin. Noteworthy, the specimens, the muscle of which showed high toxicity, appeared in high frequency, indicating that puffers from this area is not safe for human consumption. These puffer specimens contained paralytic shellfish poisoning (PSP) toxins, often as major toxin components, the profile of which was similar to that of freshwater puffers reported from tropical areas. These results indicate that PSP toxins are common in tropical puffers both from marine and freshwater.

Tetrodotoxin secreting glands in the skin of puffer fishes.

Unique exocrine glands or gland-like structures were found in the skin of several species of puffer fishes of the genus Takifugu. The glands of T. pardalis and T. vermiculare porphyreum consisted only of secretory cells with a large vacuole. These cells were completely enclosed by epithelial cells with developed microfilaments, except at their opening to the lumen. The contents of the large vacuole in the peculiar secretory cell were forced out when the puffer was stimulated. Exocrine glands or gland-like structures with peculiar secretory cells were also found in the skin of T. poecilonotus, T. niphobles and T. vermiculare radiatum. A high concentration of TTX was detected in the gland contents collected directly from live specimens of T. pardalis. We therefore conclude that these glands are TTX secreting glands.