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Paralytic shellfish toxins (PSTs) comprise a suite of potent neurotoxins that act by blocking sodium channels in nerve axons (3). These toxins may cause severe human poisoning upon consumption of contaminated shellfish. The production of PSTs by dinoflagellates has been thoroughly documented and confirmed worldwide. Strains of the dinoflagellates Alexandrium lusitanicum Balech and Gymnodinium catenatum Graham isolated from Portuguese waters have been shown by different detection methods to produce PSTs (1,6,7,16).The potential involvement of dinoflagellate-associated bacteria in PST production, specifically the autonomous synthesis of these toxins, has been addressed using bacterial strains isolated from laboratory dinoflagellate cultures (5, 10, 15) in conjunction with high-performance liquid chromatography (HPLC) (5, 15) and in vitro assay-based toxin detection (10). Two bacterial strains isolated previously by our group from A. lusitanicum and G. catenatum and identified as Pseudomonas stutzeri and Pseudomonas diminuta, respectively, were reported to produce PSTs (5, 7). Nevertheless, the autonomous production of PSTs by bacteria remains a controversial subject, and there have been several recent reports of the incorrect identification of bacterial metabolites as PSTs by HPLC analysis (2,17,18,20,21).In the context of such findings and the continuing uncertainty regarding bacterial involvement in PST toxigenesis, the present investigation was undertaken to reevaluate our previous results on the toxin content and profile in P. stutzeri and P. diminuta. Both biological in vitro (mouse neuroblastoma [MNB] assay) and chemical (HPLC) methods were employed to account for sodium channel-blocking (SCB) activity and/or compounds with the same chromatographic behavior as PSTs produced by these two bacterial isolates. Additionally, since nutritional status has been reported to influence bacterial toxicity (4), analyses for intra-and extracellular toxins under both nutrient-replete and phosphorus-limited growth conditions were conducted.For this study, P. stutzeri and P. diminuta (5, 7) were individually inoculated into 500-ml volumes of two growth media: phosphorus-limiting artificial seawater experimental (ASWE) medium (11) containing 0.5 M Na 2 HPO 4 as well as 37.5 mM sodium succinate as the sole carbon and energy source, and a complex organic seawater complete medium (SWC) (4, 11) rich in phosphate (5 g of Bacto Tryptone, 3 g of yeast extract, and 6 ml of 50% glycerol per liter of seawater). Cultures were incubated at 19 to 21°C (150 oscillations min Ϫ1 ) under 22-E m Ϫ2 s Ϫ1 irradiance until late log phase. At that time, a cell count (in CFU per milliliter) was obtained and cells were separated from growth medium by centrifugation (10,000 ϫ g, 20 min, 8°C).For toxin extraction, cell pellets were lyophilized after centrifugation and extracted with 1 ml of 0.5 N acetic acid per 100 mg of lyophilized weight. Bacterial cells were mechanically disrupted, and the suspension was centrifuged (2,000 ϫ g, 10 min, room temper...

Laboratory grazing experiments compared ingestion of two subclones of the dinoflagellate Alexandrium lusitanicum by gastropod veliger larvae (Nassarius sp.). While the two prey subclones originated from the same monoclonal isolate of A. lusitanicum, one possessed the ability to produce paralytic-shellfish-poisoning toxins (PSTs), while the other did not. Ingestion rates on the two Alexandrium subclones were not significantly different over a range of prey concentrations (approximately 100 – 660 cells ml-1), indicating that PSTs did not serve as a grazing deterrent for these larvae. However, ingestion rates on both subclones were low at the higher prey concentrations tested. Mortality of the predators also increased linearly with concentration of either subclone. These observations indicated that both A. lusitanicum subclones produced an unknown substance that inhibited and killed the grazers. Veliger mortality was not induced by culture filtrates or lysates, suggesting either that the substance was either highly labile or that contact with intact cells was required. Because toxic algae can produce multiple bioactive substances, experimental demonstrations of alleopathic effects of toxic species should not be assigned to known toxins without supporting evidence. In addition, the results show that the effectiveness of algal grazing deterrents can increase with cell concentration, which may have implications for bloom dynamics.

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