Coupling of saxitoxin biosynthesis to the G1 phase of the cell cycle in the dinoflagellate Alexandrin fundyense: temperature and nutrient effects.

The correlation between changes in length of the different cell cycle stages and the toxicity of Alexandrium fundyense Balech was studied in semi-continuous cultures. Growth rates ranging from 0.031 d(-1) to 0.36 d(-1) were established at different temperatures or levels of phosphate limitation. In all treatments, G1 was the phase with the longest duration. Decrease in growth rate was associated with an increase in duration of the different cell cycle stages. Toxin content was always directly correlated to the duration of the G1 phase. In both the temperature treatments and the phosphate limitation experiments, toxin production rates remained constant for the respective range of conditions, implying that the variations in toxin content observed were a result of increasing periods of biosynthetic activity. Toxin accumulation was directly correlated to protein biosynthesis in all temperature treatments. In contrast, toxin content showed little correlation with protein content as phosphate limitation increased. Significant differences in toxin composition were observed between the temperature and phosphate treatments. Total concentrations of GTX II and III and C I and II were significantly higher in the phosphate-limited cultures, while the levels of STX, NEO and gonyautoxins I and IV remained virtually unchanged. We conclude that toxin biosynthesis in A. fundyense is coupled to the G1 phase of the cell cycle, that toxin synthesis is not down-regulated by phosphate deprivation and that interconversions among saxitoxin derivatives are influenced by the availability of phosphate.

Toxin production of Alexandrium minutum (Dinophyceae) from the Bay of Plenty, New Zealand.

Paralytic shellfish toxins of two clonal cultures of Alexandrium minutum isolated during the 1993 toxic shellfish events in the Bay of Plenty, New Zealand, were analyzed using high-performance liquid chromatography. Toxin composition profiles of both cultures showed neosaxitoxin (> 65 mole%) as the principal toxin, with saxitoxin and gonyautoxins (GTX1-4) as minor components. Neither C-toxins (C1-4) nor GTX5-6 were detectable in the two isolates. Bay of Plenty isolates of A. minutum have a unique toxin profile not found in any other isolates of this species that have been characterized. This weakens the hypothesis that A. minutum was recently introduced to New Zealand waters by ballast water or other long-distance transport mechanisms, and argues instead that the species was endemic to the area, but not noticed in the past. The average toxicity of the cultures was 8.8 and 11.0 pg saxitoxin equiv. cell-1 with acetic acid or HCl extraction, respectively. These are at the high end of the range of toxicity reported for A. minutum strains from around the world, and on a cell volume basis are comparable to the most toxic strains of the Alexandrium tamarense group. The toxin profile of A. minutum most closely matches that of mussels and to a lesser degree tuatua harvested from the Bay during the 1993 outbreak, but is quite different from the profile measured in scallops and pipi. Plausible mechanisms for bioconversion of the ingested algal toxins within the latter two shellfish species can be proposed, but it seems more likely that either other strains of A. minutum or other saxitoxin-producing dinoflagellates were ingested by those shellfish. This study established that A. minutum from the Bay of Plenty contains saxitoxins, has a unique toxin composition compared to all other isolates of this species, and was responsible for at least part of the PSP toxicity measured in shellfish during the 1993 outbreak.

Paralytic shellfish poisoning in southern China.

The rapidly expanding mariculture and commercial region along the southern coast of China has experienced sporadic outbreaks of paralytic shellfish poisoning for nearly 30 years, yet virtually nothing is known of the nature of that toxicity or of the causative organisms. This study presents the first direct comparisons of the high performance liquid chromatography toxin composition profiles of shellfish implicated in paralytic shellfish poisoning outbreaks in Daya Bay with Alexandrium tamarense cultures established from those waters. The three cultures that were analyzed produced an unusually high proportion of the low potency N-sulfocarbamoyl toxins C1 and C2 (nearly 90% of the total), and only trace quantities of the other saxitoxin derivatives. Total toxicity was thus very low with mild acid extraction, ranging between 7.2 and 12.7 fmole cell-1, or 0.7-0.9 pg saxitoxin equiv. cell-1. Following acid hydrolysis using the standard AOAC extraction method, the dominant toxins in the cultures were gonyautoxins 2 and 3 and decarbamoyl gonyautoxins 2 and 3. Total potency increased fourfold to 2.6-3.4 pg saxitoxin equiv. cell-1 following acid hydrolysis. These cultures are thus at the low end of the range of toxicities recorded for members of the A. tamarense species complex. Two scallop samples and one mussel sample collected from Daya Bay during paralytic shellfish poisoning episodes in 1990 and 1991 were also analyzed following the AOAC extraction procedure. The toxin profiles were similar for the three shellfish samples, in that the same suite of toxins were present in each, but the relative proportion of those toxins varied. The dominant toxins were gonyautoxins 2 and 3 and toxins C1-C4. Total toxicity was 336 and 654 micrograms saxitoxin per 100 g meat for the scallop samples, and 723 for the mussels. Toxins C3,4 were present in the shellfish at up to 22 mole%, but were not detected in cultures, even when mild acid was used for extraction. Despite the otherwise similar nature of the culture versus the shellfish toxin signatures, the presence of C3,4 indicates that another strain or species of Alexandrium, or possibly a paralytic shellfish poisoning-producing species of another genus was responsible for the 1990 and 1991 paralytic shellfish poisoning outbreaks in Daya Bay. Since the cultures analyzed were of low intrinsic toxicity, A. tamarense may be more widespread along the south coast of China than is suggested by the sporadic pattern of past paralytic shellfish poisoning outbreaks. Blooms with high cell density are required to generate sufficient toxin to be dangerous. The alarming increase in algal blooms in Chinese waters due to persistent and growing pollution may make these low toxicity populations more problematic in the future.

Growth and toxin production of the toxic dinoflagellate Pyrodinium bahamense var. compressum in laboratory cultures.

Toxin production of a Malaysian isolate of the toxic red tide dinoflagellate Pyrodinium bahamense var. compressum was investigated at various stages of the batch culture growth cycle and under growth conditions affected by temperature, salinity, and light intensity variations. In all the experiments conducted, only 5 toxins were ever detected. Neosaxitoxin (NEO) and gonyautoxin V (GTX5) made up 80 mole percent or more of the cellular toxin content and saxitoxin (STX), GTX6 and decarbamoylsaxitoxin (dcSTX) made up the remainder. No gonyautoxins I-IV or C toxins were ever detected. In nutrient-replete batch cultures, toxin content rapidly peaked during early exponential phase and just as rapidly declined prior to the onset of plateau phase. Temperature had a marked effect on toxin content, which increased 3-fold as the temperature decreased from the optimum of 28 degrees C to 22 degrees C. Toxin content was constant at salinities of 24% or higher, but increased 3-fold at 20%. Toxin content decreased 2-fold and chlorophyll content increased 3-fold when light intensity was reduced from 90 to 15 microE m-2 s-1. This accompanied a 30% decrease in growth rate. Toxin composition (mole % individual toxin cell-1) remained constant throughout the course of the nutrient-replete culture and during growth at various salinities, but varied significantly with temperature and light intensity changes. At 22 degrees C, GTX5 was 25 mole % and NEO was 65 mole %, while at 34 degrees C, GTX5 increased to 55 mole % and NEO decreased proportionally to 40 mole %. When light intensity was reduced from 90 to 15 microE m-2 s-1, NEO decreased from 55 to 38 mole %, while GTX5 increased from 40 to 58 mole %. These data suggest that low light and high temperature both somehow enhance sulfo-transferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxin composition variations in one isolate of the dinoflagellate Alexandrium fundyense.

A commonly accepted paradigm in the study of saxitoxin-producing dinoflagellates is that the total concentration of all toxins (toxin content) in one isolate can vary with growth conditions, but that the relative abundance of each toxin (toxin composition) does not change. We demonstrate here that dramatic changes in toxin composition do occur in one isolate of Alexandrium fundyense. In nitrogen- and phosphorus-limited semi-continuous cultures, toxin composition varied systematically with growth rate. When cells grew slowly under severe nutrient limitation, toxin composition was dominated by one or at most two toxin epimer pairs; as nutrient stresses eased at higher growth rates, the toxin profiles became more heterogeneous. Steady-state, sustained nitrogen limitation favored the production of toxins C 1,2 and GTX I,IV, whereas phosphorus limitation produced cells with high relative abundance of GTX II,III. STX reached its highest relative abundance when growth was most rapid. The lack of observed compositional changes in most past studies is probably not due to inherent differences in toxin biosynthetic pathways between the strains of Alexandrium examined, but rather to differences in the physiology of cells grown under different culturing modes (batch vs semi-continuous), methods of toxin analysis, and dominant toxins in the particular isolates examined.