Paralytic Shellfish Toxin Production Research Articles

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Scientific questionParalytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in dinoflagellates is extremely limited owing to their unique features. The origin and evolution of PST biosynthesis in these two kingdoms are still controversial. Technical significanceHigh-throughput omics technologies, such as genomics, transcriptomics and proteomics provide powerful tools for the study of PST biosynthesis in cyanobacteria and dinoflagellates, and have shown their powerful potential with regard to revealing genes and proteins involved in PST biosynthesis in two kingdoms. Scientific significanceThis review summarizes the recent progress in PST biosynthesis in cyanobacteria and dinoflagellates with focusing on the novel insights from omics technologies, and discusses the evolutionary relationship of toxin biosynthesis genes between these two kingdoms.

Predator lipids induce paralytic shellfish toxins in bloom-forming algae

Interactions among microscopic planktonic organisms underpin the functioning of open ocean ecosystems. With few exceptions, these organisms lack advanced eyes and thus rely largely on chemical sensing to perceive their surroundings. However, few of the signaling molecules involved in interactions among marine plankton have been identified. We report a group of eight small molecules released by copepods, the most abundant zooplankton in the sea, which play a central role in food webs and biogeochemical cycles. The compounds, named copepodamides, are polar lipids connecting taurine via an amide to isoprenoid fatty acid conjugate of varying composition. The bloom-forming dinoflagellate Alexandrium minutum responds to pico- to nanomolar concentrations of copepodamides with up to a 20-fold increase in production of paralytic shellfish toxins. Different copepod species exude distinct copepodamide blends that contribute to the species-specific defensive responses observed in phytoplankton. The signaling system described here has far reaching implications for marine ecosystems by redirecting grazing pressure and facilitating the formation of large scale harmful algal blooms.

A multi-phylum study of grazer-induced paralytic shellfish toxin production in the dinoflagellate Alexandrium fundyense: A new perspective on control of algal toxicity

The present study surveyed grazer-induced stimulation of paralytic shellfish toxin (PST) production by the marine dinoflagellate Alexandrium fundyense. The survey included species, known to graze upon A. fundyense, from five phyla: the protists, Polykrikos kofoidii (Dinoflagellata) and Tiarina fusus (Ciliophora), the bivalve molluscs Mytilus edulis and Mya arenaria (Mollusca), the ascidians, Molgula manhattensis and Botrylloides violaceus (Chordata), and the copepod, Eurytemora herdmani (Arthropoda). Direct (grazers in contact with cells of A. fundyense) and indirect (grazers not in contact with cells of A. fundyense) induction assays were carried out with protists and copepods. Only indirect assays were carried out with molluscs and ascidians. Indirect assays also tested whether induction of PST production occurred via kairomones or feeding-related cues. All metazoan grazers induced PST production. By contrast, neither of the two species of protistan grazer induced PST production. Direct and indirect inductions of PST production were evident for the copepod, with direct induction being significantly higher than indirect induction. Effects upon PST production by phylum, species (nested within phylum), and interactions of phylum by diet, and species by diet, were evident. When induction of PST production occurred, a kairomone effect was apparent, except for M. edulis. Similarly, feeding-related cues were evident, except for E. herdmani. An asymptotic relationship between the magnitude of indirect induction of PST production and total cell ingestion by the grazers suggests a saturation response of grazer-induced PST production.

Bacterial community affects toxin production by Gymnodinium catenatum.

The paralytic shellfish toxin (PST)-producing dinoflagellate Gymnodinium catenatum grows in association with a complex marine bacterial community that is both essential for growth and can alter culture growth dynamics. Using a bacterial community replacement approach, we examined the intracellular PST content, production rate, and profile of G. catenatum cultures grown with bacterial communities of differing complexity and composition. Clonal offspring were established from surface-sterilized resting cysts (produced by sexual crosses of strain GCDE06 and strain GCLV01) and grown with: 1) complex bacterial communities derived from each of the two parent cultures; 2) simplified bacterial communities composed of the G. catenatum-associated bacteria Marinobacter sp. strain DG879 or Alcanivorax sp. strain DG881; 3) a complex bacterial community associated with an untreated, unsterilized sexual cross of the parents. Toxin content (STX-equivalent per cell) of clonal offspring (134–197 fmol STX cell−1) was similar to the parent cultures (169–206 fmol STX cell−1), however cultures grown with single bacterial types contained less toxin (134–146 fmol STX cell−1) than offspring or parent cultures grown with more complex mixed bacterial communities (152–176 fmol STX cell−1). Specific toxin production rate (fmol STX day−1) was strongly correlated with culture growth rate. Net toxin production rate (fmol STX cell−1 day−1) did not differ among treatments, however, mean net toxin production rate of offspring was 8-fold lower than the parent cultures, suggesting that completion of the sexual lifecycle in laboratory cultures leads to reduced toxin production. The PST profiles of offspring cultures were most similar to parent GCDE06 with the exception of cultures grown with Marinobacter sp. DG879 which produced higher proportions of dcGTX2+3 and GC1+2, and lower proportions of C1+2 and C3+4. Our data demonstrate that the bacterial community can alter intracellular STX production of dinoflagellates. In G. catenatum the mechanism appears likely to be due to bacterial effects on dinoflagellate physiology rather than bacterial biotransformation of PST toxins.

Paralytic shellfish toxins or spirolides? The role of environmental and genetic factors in toxin production of the Alexandrium ostenfeldii complex

Dinoflagellates of the Alexandrium ostenfeldii complex (A. ostenfeldii, A. peruvianum) are capable of producing different types of neurotoxins: paralytic shellfish toxins (PSTs), spirolides and gymnodimines, depending on the strain and its geographic origin. While Atlantic and Mediterranean strains have been reported to produce spirolides, strains originating from the brackish Baltic Sea produce PSTs. Some North Sea, USA and New Zealand strains contain both toxins. Causes for such intraspecific variability in toxin production are unknown. We investigated whether salinity affects toxin production and growth rate of 5 A. ostenfeldii/peruvianum strains with brackish water (Baltic Sea) or oceanic (NE Atlantic) origin. The strains were grown until stationary phase at 7 salinities (6–35), and their growth and toxin production was monitored. Presence of saxitoxin (STX) genes (sxtA1 and sxtA4 motifs) in each strain was also analyzed. Salinity significantly affected both growth rate and toxicity of the individual strains but did not change their major toxin profile. The two Baltic Sea strains exhibited growth at salinities 6–25 and consistently produced gonyautoxin (GTX) 2, GTX3 and STX. The two North Sea strains grew at salinities 20–35 and produced mainly 20-methyl spirolide G (20mG), whereas the strain originating from the northern coast of Ireland was able to grow at salinities 15–35, only producing 13-desmethyl spirolide C (13dmC). The effects of salinity on total cellular toxin concentration and distribution of toxin analogs were strain-specific. Both saxitoxin gene motifs were present in the Baltic Sea strains, whereas the 2 North Sea strains lacked sxtA4, and the Irish strain lacked both motifs. Thus sxtA4 only seems to be specific for PST producing strains. The results show that toxin profiles of A. ostenfeldii/peruvianum strains are predetermined and the production of either spirolides or PSTs cannot be induced by salinity changes. However, changes in salinity may lead to changed growth rates, total cellular toxin concentrations as well as relative distribution of the different PST and spirolide analogs, thus affecting the actual toxicity of A. ostenfeldii/peruvianum populations.

An Overview on the Marine Neurotoxin, Saxitoxin: Genetics, Molecular Targets, Methods of Detection and Ecological Functions

Marine neurotoxins are natural products produced by phytoplankton and select species of invertebrates and fish. These compounds interact with voltage-gated sodium, potassium and calcium channels and modulate the flux of these ions into various cell types. This review provides a summary of marine neurotoxins, including their structures, molecular targets and pharmacologies. Saxitoxin and its derivatives, collectively referred to as paralytic shellfish toxins (PSTs), are unique among neurotoxins in that they are found in both marine and freshwater environments by organisms inhabiting two kingdoms of life. Prokaryotic cyanobacteria are responsible for PST production in freshwater systems, while eukaryotic dinoflagellates are the main producers in marine waters. Bioaccumulation by filter-feeding bivalves and fish and subsequent transfer through the food web results in the potentially fatal human illnesses, paralytic shellfish poisoning and saxitoxin pufferfish poisoning. These illnesses are a result of saxitoxin’s ability to bind to the voltage-gated sodium channel, blocking the passage of nerve impulses and leading to death via respiratory paralysis. Recent advances in saxitoxin research are discussed, including the molecular biology of toxin synthesis, new protein targets, association with metal-binding motifs and methods of detection. The eco-evolutionary role(s) PSTs may serve for phytoplankton species that produce them are also discussed.

Is photosynthesis a requirement for paralytic shellfish toxin production in the dinoflagellate Alexandrium minutum algal–bacterial consortium?

While there is increasing evidence that marine bacteria are involved in the production of paralytic shellfish toxins in algal blooms, the exact roles of the bacteria and microalgae have proved elusive. A novel experimental approach to this problem involved incubating parallel cultures of toxin producing Alexandrium minutum Anokoha A in the dark and in a natural daylight cycle. High- performance liquid chromatography was used to measure paralytic shellfish toxins while bacterial growth was monitored by culture on high- and low-nutrient agar media. After a 22-day incubation period in the dark, A. minutum Anokoha A failed to produce saxitoxin while parallel light- grown cultures produced 1.17 μg per 10,000 algal cells. However, both dark- and light-grown cultures showed comparable gonyautoxin production. Copiotrophic and oligotrophic algal-associated bacteria showed similar growth patterns over the incubation period except that a dip in bacterial numbers corresponded to a peak in algal numbers in the light-grown cultures. It appears that inhibition of photosynthesis resulted in changes in the toxin profile of A. minutum Anokoha A. When used with other methods, this approach may help to elucidate the algal-bacterial-toxin connection.

Effects of nitrate and phosphate on grazer‐induced toxin production in Alexandrium minutum

Two strains of Alexandrium minutum were exposed to waterborne cues from copepod grazers in factorial combinations of nitrate and phosphate concentrations to evaluate the importance of grazer‐induced paralytic shellfish toxin (PST) production under different nutrient regimes. In nitrate‐rich treatments, the presence of waterborne cues from grazers resulted in significantly increased cell‐specific PST content. In low nitrate treatments, however, waterborne cues from grazers did not result in any detectable increase in cell‐specific PST content. The grazer‐induced increase in cell‐specific PST content in nitrate‐rich treatments was comparable in size to the effects of nitrate in the absence of grazers. Effects of phosphate limitation were less consistent, resulting in increased PST content in nitrate‐rich treatments in one of the A. minutum strains, but had no significant effect in the other. The ability of dinoflagellates to sense and respond to the presence of grazers by increased PST production may be one of the most important factors affecting cell‐specific PST content under nitrogen replete conditions.

Induction of toxin production in dinoflagellates: the grazer makes a difference

The dinoflagellate Alexandrium minutum has previously been shown to produce paralytic shellfish toxins (PST) in response to waterborne cues from the copepod Acartia tonsa. In order to investigate if grazer-induced toxin production is a general or grazer-specific response of A. minutum to calanoid copepods, we exposed two strains of A. minutum to waterborne cues from three other species of calanoid copepods, Acartia clausi, Centropages typicus and Pseudocalanus sp. Both A. minutum strains responded to waterborne cues from Centropages and Acartia with significantly increased cell-specific toxicity. Waterborne cues from Centropages caused the strongest response in the A. minutum cells, with 5 to >20 times higher toxin concentrations compared to controls. In contrast, neither of the A. minutum strains responded with significantly increased toxicity to waterborne cues from Pseudocalanus. The absolute increase in PST content was proportional to the intrinsic toxicity of the different A. minutum strains that were used. The results show that grazer-induced PST production is a grazer-specific response in A. minutum, and its potential ecological importance will thus depend on the composition of the zooplankton community, as well as the intrinsic toxin-producing properties of the A. minutum population.

Sam, Sahh and Map gene expression during cell division and paralytic shellfish toxin production of Alexandrium catenella (Dinophyceae)

L.D. Harlow, A. Negri, G.M. Hallegraeff and A. Koutoulis. 2007. Sam, Sahh and Map gene expression during cell division and paralytic shellfish toxin production of Alexandrium catenella (Dinophyceae). Phycologia 46: 666–674. DOI: 10.2216/07-25.1The expression of genes potentially involved in paralytic shellfish toxin (PST) biosynthesis was quantified during cell division of the dinoflagellate Alexandrium catenella. Basic precursors of the neurotoxic PST molecule(s), such as S-adenosylmethionine (SAM), have been hypothesised, but no genes coding for PST production have been definitively identified in dinoflagellates. Sam encodes the enzyme SAM synthetase and two other candidate PST genes S-adenosylhomocysteine hydrolase (Sahh) and methionine aminopeptidase (Map) have been previously identified in the closely related PST producing dinoflagellate Alexandrium fundyense. PST production and expression of Sam, Sahh and Map were examined concurrently during cell division in A. catenella. The PST profile of A. catenella was dominated by C2 (30–37 mol%), GTX5 (12–19 mol%) and GTX6 (27–33 mol%) and was constant over the cell cycle. However, the net cellular PST production rate varied over the cell cycle and was highest (127 fmol cell−1 h−1) during the G2+M phase of the cell cycle (56% cells in G2+M), slowing considerably (−14 fmol cell−1 h−1) as cells entered the G1 phase (66% cells in G1). Quantitative real time PCR revealed that Sam, Sahh and Map were up-regulated (doubling in each case) during PST production and the G2+M phase of the cell division cycle before irradiance. Alexandrium catenella actively produced PSTs during this dark period, suggesting that photon irradiance was not a direct trigger for PST production. Interactions between cell division and PST production appear complex and the present study has demonstrated correlations between PST production and expression of Sam, Sahh and Map, which warrants further investigation.

Copepods induce paralytic shellfish toxin production in marine dinoflagellates

Among the thousands of unicellular phytoplankton species described in the sea, some frequently occurring and bloom-forming marine dinoflagellates are known to produce the potent neurotoxins causing paralytic shellfish poisoning. The natural function of these toxins is not clear, although they have been hypothesized to act as a chemical defence towards grazers. Here, we show that waterborne cues from the copepod Acartia tonsa induce paralytic shellfish toxin (PST) production in the harmful algal bloom-forming dinoflagellate Alexandrium minutum. Induced A. minutum contained up to 2.5 times more toxins than controls and was more resistant to further copepod grazing. Ingestion of non-toxic alternative prey was not affected by the presence of induced A. minutum. The ability of A. minutum to sense and respond to the presence of grazers by increased PST production and increased resistance to grazing may facilitate the formation of harmful algal blooms in the sea.

Taxonomy and production of paralytic shellfish toxins by the freshwater cyanobacterium Aphanizomenon gracile LMECYA40

Detection of Paralytic Shellfish Toxins (PST) in an algal bloom in a Portuguese drinking water reservoir (Lake Crato) was followed by isolation of a strain of Aphanizomenon gracile. The strain, coded as LMECYA40, was cultured and identified by combining a morphological study with 16S rRNA gene sequencing. The toxin profile of this isolate, as revealed by HPLC-FLD analysis, was similar to that of other Aphanizomenon strains (e.g. NH-5) isolated from North America, consisting of two PST analogues: neoSTX (0.27 fmol cell−1) and STX (0.05 fmol cell−1). Based on these toxin levels, we estimated that a culture with a cell density of 107 cells ml−1 should contain approximately 910 μg of STX equivalents per litre. Small volumes of water containing PST concentrations similar to those estimated from the cultured A. gracile would therefore contain PST amounts comparable to those used as limit enforcement for harvesting and consumption of shellfish in marine environments (80 μg of STX equivalents per 100 g of shellfish).

Accumulation and depuration of cyanobacterial paralytic shellfish toxins by the freshwater mussel Anodonta cygnea

The increasing frequency by which the production of paralytic shellfish toxins (PST) by freshwater bloom-forming cyanobacteria is being noticed world-wide raises the possibility of PST bioaccumulation by freshwater mussels. This study evaluates PST accumulation and depuration by the freshwater mussel Anodonta cygnea exposed over a 14-day period to high densities (mean=1.4×10 9 cells l −1, S.D.=0.29×10 9 cells l −1) of the toxic cyanobacterium Aphanizomenon issatschenkoi (corresponding to a mean toxin concentration of 25.5 nmol PST l −1, S.D.=9.9 nmol PST l −1). Mussels were subsequently detoxified either by starvation or by feeding on the non-toxic green-algae Ankistodesmus falcatus. Filter feeding activity and toxin uptake by the mussels were followed by cell counting and toxin analysis in water samples taken before and after each daily water renewal. The accumulation and depuration of PST as well as the anatomical distribution of toxins were monitored throughout the experiment by HPLC analysis of mussel extracts. Mussels fed the toxic cyanobacterium removed on average 65.3% of cells and 40.36% of total PST daily provided. Daily rates of cell clearance (% of initial) were negatively correlated with the amounts of PST daily provided (but not with the amount of cells). This suggests a negative effect of toxins on the feeding behaviour of mussels. Small amounts of toxins could be detected in the mussels after the second day of exposure, reaching a maximum of 26 μg PST 100 g −1 by day 7. The viscera contained the greatest proportion of toxins (78%) at the start of the toxification. However, increasing amounts of PST were found in the remaining tissues (gills, mantle and foot) over time. Toxins detected in the mussel extracts were the same provided in the dietary A. issatschenkoi. Nevertheless, mussels showed a higher proportion of saxitoxin and decarbomoylsaxitoxin and a lower proportion of gonyautoxin-5 than the fed cyanobacterium. Similar depuration efficiencies were observed among starved individuals (6.9% day −1) and those fed with A. falcatus (8.2% day −1) indicating that both treatments had comparable effects on toxin metabolism. Mussels showed a typical S shaped depuration kinetics curve consisting of a first short period of slow toxin decay followed by a rapid loss and a subsequent slower release of toxins. Trace to undetectable levels of PST were found in mussels after the 14-day depurating period. Although freshwater mussels are not widely consumed by humans, their capacity to accumulate PST points to the risk of PST propagation through the food chain of freshwater ecosystems via filter-feeding mussels.

Accumulation and depuration of cyanobacterial paralytic shellfish toxins by the freshwater mussel Anodonta cygnea

The increasing frequency by which the production of paralytic shellfish toxins (PST) by freshwater bloom-forming cyanobacteria is being noticed world-wide raises the possibility of PST bioaccumulation by freshwater mussels. This study evaluates PST accumulation and depuration by the freshwater mussel Anodonta cygnea exposed over a 14-day period to high densities (mean=1.4×10 9 cells l −1 , S.D.=0.29×10 9 cells l −1 ) of the toxic cyanobacterium Aphanizomenon issatschenkoi (corresponding to a mean toxin concentration of 25.5 nmol PST l −1 , S.D.=9.9 nmol PST l −1 ). Mussels were subsequently detoxified either by starvation or by feeding on the non-toxic green-algae Ankistodesmus falcatus . Filter feeding activity and toxin uptake by the mussels were followed by cell counting and toxin analysis in water samples taken before and after each daily water renewal. The accumulation and depuration of PST as well as the anatomical distribution of toxins were monitored throughout the experiment by HPLC analysis of mussel extracts. Mussels fed the toxic cyanobacterium removed on average 65.3% of cells and 40.36% of total PST daily provided. Daily rates of cell clearance (% of initial) were negatively correlated with the amounts of PST daily provided (but not with the amount of cells). This suggests a negative effect of toxins on the feeding behaviour of mussels. Small amounts of toxins could be detected in the mussels after the second day of exposure, reaching a maximum of 26 μg PST 100 g −1 by day 7. The viscera contained the greatest proportion of toxins (78%) at the start of the toxification. However, increasing amounts of PST were found in the remaining tissues (gills, mantle and foot) over time. Toxins detected in the mussel extracts were the same provided in the dietary A. issatschenkoi . Nevertheless, mussels showed a higher proportion of saxitoxin and decarbomoylsaxitoxin and a lower proportion of gonyautoxin-5 than the fed cyanobacterium. Similar depuration efficiencies were observed among starved individuals (6.9% day −1 ) and those fed with A. falcatus (8.2% day −1 ) indicating that both treatments had comparable effects on toxin metabolism. Mussels showed a typical S shaped depuration kinetics curve consisting of a first short period of slow toxin decay followed by a rapid loss and a subsequent slower release of toxins. Trace to undetectable levels of PST were found in mussels after the 14-day depurating period. Although freshwater mussels are not widely consumed by humans, their capacity to accumulate PST points to the risk of PST propagation through the food chain of freshwater ecosystems via filter-feeding mussels.

Phylogenetic and functional diversity of the cultivable bacterial community associated with the paralytic shellfish poisoning dinoflagellate Gymnodinium catenatum

Gymnodinium catenatum is one of several dinoflagellates that produce a suite of neurotoxins called the paralytic shellfish toxins (PST), responsible for outbreaks of paralytic shellfish poisoning in temperate and tropical waters. Previous research suggested that the bacteria associated with the surface of the sexual resting stages (cyst) were important to the production of PST by G. catenatum. This study sought to characterise the cultivable bacterial diversity of seven different strains of G. catenatum that produce both high and abnormally low amounts of PST, with the long-term aim of understanding the role the bacterial flora has in bloom development and toxicity of this alga. Sixty-one bacterial isolates were cultured and phylogenetically identified as belonging to the Proteobacteria (70%), Bacteroidetes (26%) or Actinobacteria (3%). The Alphaproteobacteria were the most numerous both in terms of the number of isolates cultured (49%) and were also the most abundant type of bacteria in each G. catenatum culture. Two phenotypic (functional) traits inferred from the phylogenetic data were shown to be a common feature of the bacteria present in each G. catenatum culture: firstly, Alphaproteobacteria capable of aerobic anoxygenic photosynthesis, and secondly, Gammaproteobacteria capable of hydrocarbon utilisation and oligotrophic growth. In relation to reports of autonomous production of PST by dinoflagellate-associated bacteria, PST production by bacterial isolates was investigated, but none were shown to produce any PST-like toxins. Overall, this study has identified a number of emergent trends in the bacterial community of G. catenatum which are mirrored in the bacterial flora of other dinoflagellates, and that are likely to be of especial relevance to the population dynamics of natural and harmful algal blooms.

Effects of salinity and two coastal waters on the growth and toxin content of the dinoflagellate Alexandrium minutum

The dinoflagellate Alexandrium minutum strain AM89BM was studied to investigate its capacity to adapt to different salinities and the influence of salinity on cellular content of paralytic shellfish toxins (PST), in batch culture in enriched offshore seawater media. The strain shows optimal growth (average rate � 0.5 div day � 1 ) at salinities between 20 and 37 p.s.u., peaking at 25 p.s.u. (0.63 � 0.07 div day � 1 ). Under a high NO3 ‐ :PO4 3‐ mole ratio (76), cell PST content increased during growth phase, peaking toward the end of growth phase or in the early stationary phase. The PST content was low (� 10 fmol PST cell � 1 ) from 30 to 37 p.s.u., but it increased at lower salinities, with the maximum value (� 50 fmol PST cell � 1 ) recorded at 15 p.s.u. We then compared growth rate and toxin content of A. minutum cells grown in 3- and 0.2-m filtered estuarine water (27 p.s.u.), and a similarly filtered seawater collected from an oyster farming area (36 p.s.u.), with a N- and P-enriched offshore seawater (37 p.s.u.). In both estuarine water treatments, growth was fast (� 0.8 div day � 1 ) and the cell PST content increased only in the early growth phase, peaking in mid-growth phase at 9.56 � 1.06 and 7.63 � 0.44 fmol PST cell � 1 in the 3- and 0.2-m filtered waters, respectively. In the 3-m filtered oyster farm water, although inorganic nutrient concentrations were very low, A. minutum grew well (0.68 � 0.06 div day � 1 ), suggesting mixotrophic nutrition; as PST production was nearly zero, the cell toxin content decreasing to 1.11 fmol PST cell � 1 , we hypothesize that toxin biosynthesis was greatly weakened due to the lack of amino acid precursors in prey material. In the offshore seawater, A. minutum grew slowly (0.18 � 0.04 div day � 1 ) and cells lost toxins down to 1.08 fmol PST cell � 1 , suggesting that growth was limited and PST production stopped due to the lack of some vitamins, and/or trace metals.

Reevaluation of production of paralytic shellfish toxin by bacteria associated with dinoflagellates of the Portuguese coast.

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain dinoflagellate and cyanobacterial species. The autonomous production of PSTs by bacteria remains controversial. In this study, PST production by two bacterial strains, isolated previously from toxic dinoflagellates, was evaluated using biological and analytical methods. Analyses were performed under conditions determined previously to be optimal for toxin production and detection. Our data are inconsistent with autonomous bacterial PST production under these conditions, thereby challenging previous findings for the same strains.

PARALYTIC SHELLFISH TOXINS ARE RESTRICTED TO FEW SPECIES AMONG AUSTRALIA'S TAXONOMIC DIVERSITY OF CULTURED MICROALGAE1

There are at least 40,000 species of microalgae in the aquatic environment. Fifteen species of marine dinoflagellates and freshwater cyanobacteria are known to produce paralytic shellfish toxins (PSTs) and represent a threat to human and/or livestock health. Although known toxic species are regularly monitored, the wider cross‐section of microalgae has not been systematically tested for PSTs. Advances in rapid screening techniques have resulted in the development of highly sensitive and specific methods to detect PSTs, including the sodium channel and saxiphilin binding assays. These assays were used in this study in 96‐well formats to screen 234 highly diverse isolates of Australian freshwater and marine microalgae for PSTs. The screening assays detected five toxic species, representing one freshwater cyanobacterium (Anabaena circinalis Rabenhorst) and four species of marine dinoflagellates (Alexandrium minutum Halim, A. catenella Balech, A. tamarense Balech, and Gymnodinium catenatum Graham). Liquid chromatography‐fluorescence detection was used to identify 14 saxitoxin analogues across the five species, and each species exhibited distinct toxin profiles. These results indicate that PST production is restricted to a narrow range of microalgal species found in Australian waters.

Particle size fractionation of paralytic shellfish toxins (PSTs): seasonal distribution and bacterial production in the St Lawrence estuary, Canada

We determined the seasonal distribution of paralytic shellfish toxins (PSTs) and PST producing bacteria in >15, 5–15, and 0.22–5 μm size fractions in the St Lawrence. We also measured PSTs in a local population of Mytilus edulis. PST concentrations were determined in each size fraction and in laboratory incubations of sub-samples by high performance liquid chromatography (HPLC), including the rigorous elimination of suspected toxin ‘imposter’ peaks. Mussel toxin levels were determined by mouse bioassay and HPLC. PSTs were detected in all size fractions during the summer sampling season, with 47% of the water column toxin levels associated with particles smaller than Alexandrium tamarense (<15 μm). Even in the >15 μm size fraction, we estimated that as much as 92% of PSTs could be associated with particles other than A. tamarense. Our results stress the importance of taking into account the potential presence of PSTs in size fractions other than that containing the known algal producer when attempting to model shellfish intoxication, especially during years of low cell abundance. Finally, our HPLC results confirmed the presence of bacteria capable of autonomous PST production in the St Lawrence as well as demonstrating their regular presence and apparent diversity in the plankton.

Seasonal occurrence at a Scottish PSP monitoring site of purportedly toxic bacteria originally isolated from the toxic dinoflagellate genus Alexandrium

There is increasing evidence that bacterial–algal interactions play a role in Harmful Algal Bloom (HAB) ecology. Bacteria that are associated with bloom-forming algal species, specifically toxic dinoflagellate algae, have been implicated in the production and biotransformation of paralytic shellfish toxins (PSTs). To clarify the role that these bacteria may play in the production of PSTs, it is desirable to identify and localize the bacteria associated with the dinoflagellates and enumerate them during the course of the algal blooms that the toxic dinoflagellates produce. Because 16S rRNA-targeted probes offer the possibility of both, we previously made and tested probes for some putatively toxigenic bacteria isolated from cultures of the PSP-related dinoflagellates Alexandrium tamarense, A. affine and A. lusitanicum. The bacteria isolated from the dinoflagellates belong primarily to the alpha-proteobacterial group of Roseobacter and the gamma-proteobacterial group of Alteromonas. Here, we report the successful application of these probes to Lugol's-fixed seawater samples. We detected these bacteria in high numbers in the water column when Alexandrium spp. were both present and absent, and during periods when mussels contained PSTs.