Deng Fwu Hwang

Influence of environmental and nutritional factors on growth, toxicity, and toxin profile of dinoflagellate Alexandrium minutum

The toxic dinoflagellate Alexandrium minutum T1 was isolated from southern Taiwan in February 1996, grown under various environmental and nutritional conditions, and then examined for growth, toxicity, and toxin profile. It was found that the optimal environmental conditions for cell growth and toxin production of A. minutum T1 was as follows: temperature 25 degrees C, pH 7.5, light strength 120 microEm(-2) s(-1), and salinity 15 ppt. The optimal level of nutrients supplemented in the 50% natural seawater medium was as follows: phosphate 0.002%, nitrate 0.01%, cupric ion 5.0 ppb, ferric ion 270 ppb and humic acid free. Both cell toxicity and total toxicity reached the maximum level at the post-stationary growth phase and decreased quickly. The toxic components of A. minutum T1 were assayed by high performance liquid chromatography, and found to be gonyautoxin (GTX) 1-4 only. Among these four toxin components, toxins GTX 1 and 4 were the predominant components throughout the growth curve when the cells were grown in the optimal environmental and nutritional conditions. But toxins GTX 2 and 3 increased when the cells were cultured in high salinity medium.

Occurrence of tetrodotoxin in the gastropod mollusk Natica lineata (lined moon shell)

Paralytic toxicity was detected in 28 of 38 specimens of the gastropod mollusk Natica lineata (lined moon shell). The highest toxicity, expressed as tetrodotoxin (TTX), was 720 mouse units per gram (MU/g) muscle, followed by other parts which included salivary gland, brain and mouth organs (28 MU/g) and digestive gland (12 MU/g). The toxin was partially purified by ultrafiltration and Bio-Gel P-2 column chromatography. The toxin showed a specific toxicity (as TTX) of 620 MU/mg. Results of analyses by thin layer chromatography, cellulose acetate membrane electrophoresis and high performance liquid chromatography showed that the toxin was composed of TTX and anhydrotetrodotoxin. This is the first time that TTX and its related substance have been found in this species of gastropod.

Tetrodotoxin and derivatives in several species of the gastropod Naticidae.

The organs of 195 specimens of 4 species of the gastropod Naticidae, collected from fish markets in Taiwan, were assayed for toxicity. The calf moon shell Natica vitellus contained weak toxicity (10-99 MU/g) in the muscle and digestive gland. The bladder moon shell Polinices didyma contained moderate (100-999 MU/g) and weak toxicity in the muscle and digestive gland, respectively. The digestive gland in 1 out of 20 specimens of the pear-shaped moon shell P. tumidus was toxic (4 MU/g). All tissues of the butterfly moon shell N. alapapilionis were non-toxic. The toxins were partially purified from the toxic specimens of the calf moon shell and the bladder moon shell, and identified to be tetrodotoxin and anhydrotetrodotoxin.

Gonyautoxin-3 as a minor toxin in the gastropod Niotha clathrata in Taiwan

Paralytic toxicity was detected in the gastropod mollusc Niotha clathrata collected from South Taiwan in April and November 1993. Each seasonal toxin was partially purified from toxic specimens of N. clathrata by ultrafiltration using a membrane (Diaflo YM-2), followed by chromatography on a column (Bio-Gel P-2). Two toxin fractions (I and II) were then obtained for each seasonal shell toxin. The ratio of fraction I to fraction II for each seasonal shell toxin was about 4:1 according to tetrodotoxin bioassay. Based on analyses by TLC, electrophoresis, and HPLC, fraction I toxin contained tetrodotoxin and its derivative anhydrotetrodotoxin, and fraction II toxin contained gonyautoxin-3 for each seasonal shell toxin.

Occurrence of a new toxin and tetrodotoxin in two species of the gastropod mollusk Nassariidae.

The lethalities of 102 specimens of three species of the gastropod mollusk Nassariidae, collected from fish markets in Taiwan, were examined. The frequency of toxicity in Zeuxis scalaris and Z. castus-like specimens was 94 and 41%, respectively. The range of lethal potency in toxic specimens of Zeuxis scalaris and Z. castus-like was 2-140 and 2-13 mouse units, respectively, while all tissues of Z. castus were non-toxic. The toxins were partially purified from the toxic specimens of Z. scalaris and Z. castus-like. Two toxin fractions were obtained from the extract of each species of Nassariidae by using Bio-Gel P-2 column chromatography. Analyses by thin layer chromatography, electrophoresis, high performance liquid chromatography and ultraviolet spectroscopy, showed that toxin fraction I contained tetrodotoxin while toxin fraction II contained a new neurotoxin.

Comparison of paralytic toxins in aquaculture of purple clam in Taiwan

Food poisoning incidents due to ingesting the cultured purple clam Soletellina diphos occurred in western Taiwan in February 1991. Clam specimens, sediment and the dinoflagellate Alexandrium tamarensis were collected and assayed for lethality as paralytic shellfish poison (PSP). The lethality of purple clam, wet sediment and phytoplankton was 1700 mouse units per specimen (MU/specimen), 0.05 MU/g and 3.6 x 10(-5) MU/cell, respectively. The toxins obtained from clam, sediment and phytoplankton consisted of gonyautoxins 1-4, along with trace amounts of neosaxitoxin.

Tetrodotoxin secretion from the lined moon shell Natica Lineata in response to external stimulation

The lined moon shell Natica lineata secretes tetrodotoxin (TTX) in response to an external stimulation such as removal from the seawater. The toxin released from the shellfish contained 14-361 mouse units of TTX per specimen. The shellfish did not secrete any further toxin when seawater removal was repeated over four times at about 1 hr intervals. All specimens recovered TTX secreting ability when they were kept in an aquarium for 5 days.

Variation and secretion of toxins in gastropod mollusc Niotha clathrata

Nearly 300 specimens of the gastropod mollusc Niotha clathrata were collected in South Taiwan. All specimens were assayed for toxicity by the official method for tetrodotoxin (TTX). The frequency value of toxic specimens in N. clathrata was 30.0%, 68.0% and 80.4% at Anping, Chiating and Tungkang, respectively. The highest lethal potency of a gastropod specimen was 1900 mouse units (MU). The specimens collected from Tungkang showed the highest frequency of toxic specimens and toxicity, followed by those from Chiating and Anping. The specimens collected in autumn and spring showed higher toxicity than those collected in other seasons. Moreover, another 17 specimens of N. clathrata were collected for testing the toxin secretion by electric shock treatment. It is found that the gastropod did not secrete any additional toxin when electric shock treatment was performed twice at approximately 1-hr intervals. The toxicity of secreted toxin was 206 MU.