Paralytic Shellfish Toxin Content Research Articles

Mechanistic insights into the effects of diuron exposure on Alexandrium pacificum

Diuron (N-(3,4-dichlorophenyl)-N,N‑dimethylurea, DCMU), a ureic herbicide, is extensively used in agriculture to boost crop productivity; however, its extensive application culminates in notable environmental pollution, especially in aquatic habitats. Therefore, the present study investigated the effect of diuron on the dinoflagellate Alexandrium pacificum, which is known to induce harmful algal blooms (HAB), and its potential to biodegrade DCMU. Following a four-day DCMU exposure, our results revealed that A. pacificum proficiently assimilated DCMU at concentrations of 0.05 mg/L and 0.1 mg/L in seawater, attaining a complete reduction (100 % efficiency) after 96 h for both concentrations. Moreover, evaluations of paralytic shellfish toxins content indicated that cells subjected to higher DCMU concentrations (0.1 mg/L) exhibited reductions of 73.4 %, 86.7 %, and 75 % in GTX1, GTX4, and NEO, respectively. Exposure to DCMU led to a notable decrease in A. pacificum's photosynthetic efficacy, accompanied by increased levels of reactive oxygen species (ROS) and suppressed cell growth, with a growth inhibition rate of 41.1 % at 72 h. Proteomic investigations pinpointed the diminished expression levels of specific proteins like SxtV and SxtW, linked to paralytic shellfish toxins (PSTs) synthesis, as well as key proteins associated with Photosystem II, namely PsbA, PsbD, PsbO, and PsbU. Conversely, proteins central to the cysteine biosynthesis pathways exhibited enhanced expression. In summary, our results preliminarily resolved the molecular mechanisms underlying the response of A. pacificum to DCMU and revealed that DCMU affected the synthesis of PSTs. Meanwhile, our data suggested that A. pacificum has great potential in scavenging DCMU.

Novel insights into toxin changes associated with the growth of Alexandrium pacificum: Revealing active toxin-secretion ability and toxin cell quota variation

Paralytic shellfish toxins (PSTs) are widely distributed globally and are produced by Alexandrium pacificum in marine system. However, the characteristics of toxins producing and secreting associated with growth phases are still unclear, especially whether A. pacificum has the ability to actively secrete PSTs is controversial. In this study, variation characteristics of intracellular and extracellular PSTs contents associated with A. pacificum growth phases were investigated thoroughly. The results showed that intracellular and extracellular PSTs contents increased sharply during the exponential phase. But during the stationary phase, the intracellular PSTs content increased by only 26 %, and the extracellular PSTs content did not increase significantly. Since the increase in extracellular PSTs content mainly occurred at the exponential phase, when most cells were living, we speculated that active PSTs secretion of living cells might be an important production pathway of extracellular toxins besides leakage from dead cells. Furthermore, toxin cell quota variation associated with the growth phase was analysed. In the exponential phase, the toxin cell quota first increased and then decreased, with a maximum of 19.02 ± 1.80 fmol/cell at 6 d. However, after entering the stationary phase, this value slowly increased again, suggesting that vigilance should be raised for the plateau of Alexandrium blooms. In addition, cells in the exponential phase mainly produced O-sulfated components such as GTX1&4, cells in the stationary phase mainly produced O-sulfate-free components such as GTX5. In this study, the toxigenic rules of A. pacificum were comprehensively uncovered, which provided theoretical guidance for the prevention and mitigation of A. pacificum blooms.

Degradation of paralytic shellfish toxins during flocculation of Alexandrium pacificum by an oxidized modified clay: A laboratory experiment

Paralytic shellfish toxins (PSTs), produced by Alexandrium pacificum in the marine environment, are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells. During the toxigenic A. pacificum blooms outbreaks, PSTs can be accumulated through the food chain and finally enter the human body, posing a significant threat to human health and safety. This study experimented with a novel type of oxidized modified clay, potassium peroxymonosulfate modified clay (PMPS-MC), which could remove A. pacificum cells as well as reduce intracellular and extracellular PSTs toxicity rapidly. For the extracellular PSTs, its content decreased to below the detection limit rapidly through oxidative degradation within 15 min of 10 mg/L PMPS-MC treatment. Whereafter, although the residual cells in water column and some viable cells in flocculated sediment continued to secrete toxins, the extracellular PSTs content and toxicity in the PMPS-MC treatment groups remained significantly lower than those in the control group. For the intracellular PSTs, PMPS-MC might induce the transformation of more toxic GTX1&4 to less toxic GTX2&3 and C1&2, resulting in intracellular PSTs toxicity reduced within 15 min. In addition, intracellular PSTs content and toxicity in the PMPS-MC treatment groups were consistently lower than the control group within 48 h, possibly by inhibiting the A. pacificum cells growth. These results will provide a scientific basis for the field application of modified clay to control A. pacificum blooms.

Variations in the toxicity and condition index of five bivalve species throughout a red tide event caused by Alexandrium catenella: A field study

Harmful red tides in China have caused paralytic shellfish toxins (PSTs) pollution and led to severe socioeconomic effects in shellfish aquaculture. Although shellfish can survive harmful algal blooms, the effects on their Condition Index (CI) have been underestimated. This study sought to evaluate the effects of the profiles and levels of paralytic shellfish toxins on variations in the CI in bivalves under natural blooming conditions. We observed clear soft tissue lesions to varying degrees except in Mytilus galloprovincialis after toxin exposure. Among the five species of shellfish exposed in situ, only M. galloprovincialis accumulated PSTs content above the maximum permitted level (800 μg STX di-HCl eq./kg). The highest toxin content in all sample tissues was observed in Patinopecten yessoensis. Significant interspecies differences in PSTs accumulation among the five bivalve species were observed in the hepatopancreas. A total of nine PSTs components and four new C-11 hydroxyl metabolites (so-called M-toxins) toxins were detected, and detoxification diversity was observed among bivalves. We observed a higher proportion of M-toxin in early stages, and the proportions changed only slightly over time in M. galloprovincialis and Magallana gigas, thus accounting for the significantly higher metabolism rate. Notably, the CI in M. gigas and Argopecten irradians was positively correlated with lowest toxin accumulation of PSTs content, but significantly inhibited. In conclusion, our results revealed a significant inhibitory effect on the CI in shellfish, in a species specific manner, with distinct levels of inhibition correlated with different toxin metabolites. Our study revealed the toxin content of different bivalves exposed to a natural red tide environment and the consequent effects on growth, thus building a foundation for research on the mechanisms underlying the effects of PSTs on growth. These data establish the ecological and economic significance of the effects of harmful algal blooms on bivalves.

Transcriptome and Network Analyses Reveal the Gene Set Involved in PST Accumulation and Responses to Toxic Alexandrium minutum Exposure in the Gills of Chlamys farreri.

Bivalve molluscs are filter-feeding organisms that can accumulate paralytic shellfish toxins (PST) through ingesting toxic marine dinoflagellates. While the effects of PST accumulation upon the physiology of bivalves have been documented, the underlying molecular mechanism remains poorly understood. In this study, transcriptomic analysis was performed in the gills of Zhikong scallop (Chlamys farreri) after 1, 3, 5, 10, and 15 day(s) exposure of PST-producing dinoflagellate Alexandrium minutum. Higher numbers of differentially expressed genes (DEGs) were detected at day 1 (1538) and day 15 (989) than that at day 3 (77), day 5 (82), and day 10 (80) after exposure, and most of the DEGs were only regulated at day 1 or day 15, highlighting different response mechanisms of scallop to PST-producing dinoflagellate at different stages of exposure. Functional enrichment results suggested that PST exposure induced the alterations of nervous system development processes and the activation of xenobiotic metabolism and substance transport processes at the acute and chronic stages of exposure, respectively, while the immune functions were inhibited by PST and might ultimately cause the activation of apoptosis. Furthermore, a weighted gene co-expression network was constructed, and ten responsive modules for toxic algae exposure were identified, among which the yellow module was found to be significantly correlated with PST content. Most of the hub genes in the yellow module were annotated as solute carriers (SLCs) with eight being OCTN1s, implying their dominant roles in regulating PST accumulation in scallop gills. Overall, our results reveal the gene set responding to and involved in PST accumulation in scallop gills, which will deepen our understanding of the molecular mechanism of bivalve resistance to PST.

Development of a Method for Detecting Alexandrium pacificum Based on the Quantification of sxtA4 by Chip-Based Digital PCR.

Alexandrium pacificum, which produces the paralytic shellfish toxin (PST) saxitoxin (STX), is one of the causative species of paralytic shellfish poisoning outbreaks in coastal areas of Korea. In this study, we developed a chip-based digital PCR (dPCR) method for A. pacificum detection and tested it for monitoring in Jinhae-Masan Bay. Using the sequence of an A. pacificum strain isolated in 2017, species-specific primers targeting sxtA4 (a STX biosynthesis-related gene) were designed and used in a dPCR, detecting 2.0 ± 0.24 gene copies per cell of A. pacificum. Cell abundance in field samples, estimated by a chip-based dPCR, was compared with the PST content, and measured using a mouse bioassay. A comparison with shellfish PST concentrations indicated that cell concentrations above 500 cells L−1, as measured using the dPCR assay, may cause shellfish PST concentrations to exceed the allowed limits for PSTs. Concordance rates between dPCR and PST results were 62.5% overall in 2018–2021, reaching a maximum of 91.7% in 2018–2019. The sensitivity of the dPCR assay was higher than that of microscopy and sxtA4-based qPCRs. Absolute quantification by chip-based dPCRs targeting sxtA4 in A. pacificum exhibits potential as a complementary approach to mouse bioassay PST monitoring for the prevention of toxic blooms.

Relationship between paralytic shellfish toxin content and sxtA gene copy number in different growth phases of Gymnodinium catenatum (Dinophyceae)

Paralytic shellfish toxin (PST) content in the dinoflagellate Gymnodinium catenatum changes with culture age, with a higher toxin concentration in the logarithmic phase that decreases when the culture ages. The gene copy number (GCN) of domains sxtA1 and sxtA4 was higher in the lag and stationary phase, and lag phase, respectively. No relationship was found between the GCN of the domains sxtA4 and sxtA1 with the PST content in G. catenatum.

Uptake of Paralytic Shellfish Toxins by Blacklip Abalone (Haliotis rubra rubra Leach) from direct exposure to Alexandrium catenella microalgal cells and toxic aquaculture feed

The Tasmanian abalone fishery represents the largest wild abalone resource in the world, supplying close to 25% of the annual wild-caught global harvest. Prompted by the need to manage Paralytic Shellfish Toxin (PST) contamination of Blacklip Abalone (Haliotis rubra rubra) from east coast Tasmania, the uptake of toxins by this species is investigated in a land-based, controlled aquaculture setting. Abalone were exposed to either live Alexandrium catenella microalgal cultures or PST contaminated feed pellets during a 28 day exposure period and toxins quantified in viscera, foot muscle and epipodium tissues. PST profiles of abalone foot tissues were dominated by saxitoxin and neosaxitoxin, whilst viscera more closely resembled those of the toxin source (A. catenella cells rich in gonyautoxin 1&4 and 2&3 or feed pellets containing A. catenella extracts rich in these analogues). This indicates direct uptake of PST in the viscera via browsing/grazing on the pellet and /or sedimented microalgal cells. After exposure to A. catenella cell culture, PST concentrations in the foot (muscle + epipodium) were on average 8 times higher than in the viscera. Higher toxicity of foot tissue was caused by higher PST content of the epipodium (up to 1,085 µg STX.2HCl equiv. kg−1), which despite its small contribution to total animal weight significantly added to the overall toxin burden. Higher PST levels in the abalone foot suggest that toxin monitoring programmes may not need to routinely analyse both foot and viscera, potentially allowing for a 50% reduction of analytical costs. This option is being further investigated with continuing field studies.

Geographical distribution and seasonal variation in paralytic shellfish toxins in the coastal water of the South China Sea

Paralytic shellfish toxins (PSTs) are a group of the most harmful neurotoxins distributed worldwide. Marine organism samples, including mollusks, crustaceans and fish, collected from 11 sites around the coastal water of the South China Sea, were analyzed in terms of the PST and toxicity via high–performance liquid chromatography. The PST geographical distribution, detectable rate for PST and toxin content all increased slightly from 2006 to 2008 to 2015. High PST content with more than 2 nmol g−1 appeared in Shantou (ST), Shanwei (SW), Zhanjiang (ZJ), Beihai (BH), Xuwen (XW), Haikou (HK), Dongfang (DF), Wanning (WN) and Sanya (SY). Low PST content with less than 2 nmol g−1 appeared in Shenzhen (SZ) and Yangjiang (YJ). High PST toxicity over the safety limit 800 μg STXeq/kg appeared in ST, SW, ZJ, BH, DF, SY and XW. PST has significant seasonal characteristics and was mostly detected in spring, early summer (March–June) and autumn (September–November) in the coastal water of the South China Sea. PST was usually detected in some specific species of scallop, mussel, bloody clam, hard clam, razor clam, oyster, crab, shrimp and fish. Toxin profile variation of marine organism samples was influenced greatly by species, sampling time and site.

The Cost of Toxicity in Microalgae: Direct Evidence From the Dinoflagellate Alexandrium.

Empirical evidence of the cost of producing toxic compounds in harmful microalgae is completely lacking. Yet costs are often assumed to be high, implying substantial ecological benefits with adaptive significance exist. To study potential fitness costs of toxin production, 16 strains including three species of the former Alexandrium tamarense species complex were grown under both carbon limitation and unlimited conditions. Growth rates, levels of intracellular paralytic shellfish toxins (PSTs), and effects of lytic compounds were measured to provide trade-off curves of toxicity for both PST and lytic toxicity under high light (300 μmol photons m−2 s−1) and under low light (i.e., carbon limited; 20 μmol photons m−2 s−1). Fitness costs in terms of reduced growth rates with increasing PST content were only evident under unlimited conditions, but not under carbon limitation, in which case PST production was positively correlated with growth. The cost of production of lytic compounds was detected both under carbon limitation and unlimited conditions, but only in strains producing PST. The results may direct future research in understanding the evolutionary role and ecological function of algal toxins. The intrinsic growth rate costs should be accounted for in relation to quantifying benefits such as grazer avoidance or toxin-mediated prey capture in natural food web settings.

Modelling paralytic shellfish toxins (PST) accumulation in Crassostrea gigas by using Dynamic Energy Budgets (DEB)

As other filter-feeders, Crassostrea gigas can concentrate paralytic shellfish toxins (PST) by consuming dinoflagellate phytoplankton species like Alexandrium minutum. Intake of PST in oyster tissues mainly results from feeding processes, i.e. clearance rate, pre-ingestive sorting and ingestion that are directly influenced by environmental conditions (trophic sources, temperature). This study aimed to develop a mechanistic model coupling the kinetics of PST accumulation and bioenergetics in C. gigas based on Dynamic Energy Budget (DEB) theory. For the first time, the Synthesizing Units (SU) concept was applied to formalize the feeding preference of oysters between non-toxic and toxic microalgae. Toxin intake and accumulation were both dependent on the physiological status of oysters. The accumulation was modelled through the dynamics of two toxin compartments: (1) a compartment of ingested but non-assimilated toxins, with labile toxins within the digestive gland eliminated via faeces production; (2) a compartment of assimilated toxins with a rapid detoxification rate (within a few days). Firstly, the DEB-PST model was calibrated using data from two laboratory experiments where oysters have been exposed to A. minutum. Secondly, it was validated using data from another laboratory experiment and from three field surveys carried out in the Bay of Brest (France) from 2012 to 2014. To account for the variability in PST content of A. minutum cells, the saxitoxin (STX) amount per energy units in a toxic algae (ρPST) was adjusted for each dataset. Additionally, the effects of PST on the oyster bioenergetics were calibrated during the first laboratory experiment. However, these effects were shown to depend on the strain of A. minutum. Results of this study could be of great importance for monitoring agencies and decision makers to identify risky conditions (e.g. production areas, seawater temperature), to properly assess detoxification step (e.g. duration, modalities) before any commercialization or to improve predictions regarding closing of shellfish areas.

Predator cues reduce intraspecific trait variability in a marine dinoflagellate

BackgroundPhenotypic plasticity is commonplace and enables an organism to respond to variations in the environment. Plastic responses often modify a suite of traits and can be triggered by both abiotic and biotic changes. Here we analysed the plastic response towards a grazer of two genotypes of the marine dinoflagellate Alexandrium fundyense, evaluated the similarity of this response and discuss potential strain-specific trade-offs. We compared the expression of the known inducible defensive traits paralytic shellfish toxin content, and chain length. The effectiveness of the induced defense was assessed by monitoring grazing rates in both strains.ResultsOur results show that the grazer cues diminish phenotypic variability in a population by driving the phenotype towards a common defended morphotype. We further showed that the expression of the sxtA gene that initiates the paralytic shellfish toxin biosynthesis pathway does not correlate with an observed increase in the paralytic shellfish toxin analogue saxitoxin, and that toxin induction differs in its physiological characteristics in both strains.ConclusionInduced defense response in Alexandrium thus can directly affect further species interactions by reducing phenotypic variation and can result in genotype-dependent ecological trade-offs.

Paralytic shellfish toxins in phytoplankton and shellfish samples collected from the Bohai Sea, China

Phytoplankton and shellfish samples collected periodically from 5 representative mariculture zones around the Bohai Sea, Laishan (LS), Laizhou (LZ), Hangu (HG), Qinhuangdao (QHD) and Huludao (HLD), were analysed for paralytic shellfish toxins (PSTs) using an high-performance liquid chromatography (HPLC) method. Toxins were detected in 13 out of 20 phytoplankton samples, and N-sulfocarbamoyl toxins (C1/2) were predominant components of PSTs in phytoplankton samples with relatively low toxin content. However, two phytoplankton samples with high PST content collected from QHD and LS had unique toxin profiles characterized by high-potency carbamoyl toxins (GTX1/4) and decarbamoyl toxins (dcGTX2/3 and dcSTX), respectively. PSTs were commonly found in shellfish samples, and toxin content ranged from 0 to 27.6nmol/g. High level of PSTs were often found in scallops and clams. Shellfish from QHD in spring, and LZ and LS in autumn exhibited high risks of PST contamination.

Paralytic shellfish toxin content is related to genomic sxtA4 copy number in Alexandrium minutum strains.

Dinoflagellates are microscopic aquatic eukaryotes with huge genomes and an unusual cell regulation. For example, most genes are present in numerous copies and all copies seem to be obligatorily transcribed. The consequence of the gene copy number (CPN) for final protein synthesis is, however, not clear. One such gene is sxtA, the starting gene of paralytic shellfish toxin (PST) synthesis. PSTs are small neurotoxic compounds that can accumulate in the food chain and cause serious poisoning incidences when ingested. They are produced by dinoflagellates of the genera Alexandrium, Gymnodium, and Pyrodinium. Here we investigated if the genomic CPN of sxtA4 is related to PST content in Alexandrium minutum cells. SxtA4 is the 4th domain of the sxtA gene and its presence is essential for PST synthesis in dinoflagellates. We used PST and genome size measurements as well as quantitative PCR to analyze sxtA4 CPN and toxin content in 15 A. minutum strains. Our results show a strong positive correlation between the sxtA4 CPN and the total amount of PST produced in actively growing A. minutum cells. This correlation was independent of the toxin profile produced, as long as the strain contained the genomic domains sxtA1 and sxtA4.

SxtA and sxtG gene expression and toxin production in the Mediterranean Alexandrium minutum (Dinophyceae).

The dinoflagellate Alexandrium minutum is known for the production of potent neurotoxins affecting the health of human seafood consumers via paralytic shellfish poisoning (PSP). The aim of this study was to investigate the relationship between the toxin content and the expression level of the genes involved in paralytic shellfish toxin (PST) production. The algal cultures were grown both in standard f/2 medium and in phosphorus/nitrogen limitation. In our study, LC-HRMS analyses of PST profile and content in different Mediterranean A. minutum strains confirmed that this species was able to synthesize mainly the saxitoxin analogues Gonyautoxin-1 (GTX1) and Gonyautoxin-4 (GTX4). The average cellular toxin content varied among different strains, and between growth phases, highlighting a decreasing trend from exponential to stationary phase in all culture conditions tested. The absolute quantities of intracellular sxtA1 and sxtG mRNA were not correlated with the amount of intracellular toxins in the analysed A. minutum suggesting that the production of toxins may be regulated by post-transcriptional mechanisms and/or by the concerted actions of alternative genes belonging to the PST biosynthesis gene cluster. Therefore, it is likely that the sxtA1 and sxtG gene expression could not reflect the PST accumulation in the Mediterranean A. minutum populations under the examined standard and nutrient limiting conditions.

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.

Capillary electrophoresis for the analysis of paralytic shellfish poisoning toxins in shellfish: Comparison of detection methods

Paralytic shellfish toxins (PSTs) are produced by marine and freshwater microalgae and accumulate in shellfish including mussels, oysters, and scallops, causing possible fatalities when inadvertently consumed. Monitoring of PST content of shellfish is therefore important for food safety, with currently approved methods based on HPLC, using pre- or postcolumn oxidation for fluorescence detection (HPLC-FLD). CE is an attractive alternative for screening and detection of PSTs as it is compatible with miniaturization and could be implemented in portable instrumentation for on-site monitoring. In this study, CE methods were developed for C(4) D, FLD, UV absorption detection, and MS-making this first report of C(4) D and FLD for PSTs detection. Because most oxidized toxins are neutral, MEKC was used in combination with FLD. The developed CZE-UV and CZE-C(4) D methods provide better resolution, selectivity, and separation efficiency compared to CZE-MS and MEKC-FLD. The sensitivity of the CZE-C(4) D and MEKC-FLD methods was superior to UV and MS, with LOD values ranging from 140 to 715 ng/mL for CZE-C(4) D and 60.9 to 104 ng/mL for MEKC-FLD. With the regulatory limit for shellfish samples of 800 ng/mL, the CZE-C(4) D and MEKC-FLD methods were evaluated for the screening and detection of PSTs in shellfish samples. While the CZE-C(4) D method suffered from significant interferences from the shellfish matrix, MEKC-FLD was successfully used for PST screening of a periodate-oxidized mussel sample, with results confirmed by HPLC-FLD. This confirms the potential of MEKC-FLD for screening of PSTs in shellfish samples.

Influence of Environmental Factors on the Paralytic Shellfish Toxin Content and Profile of Alexandrium catenella (Dinophyceae) Isolated from the Mediterranean Sea

Laboratory experiments were designed to study the toxin content and profile of the Alexandrium catenella strain ACT03 (isolated from Thau Lagoon, French Mediterranean) in response to abiotic environmental factors under nutrient-replete conditions. This dinoflagellate can produce various paralytic shellfish toxins with concentrations ranging from 2.9 to 50.3 fmol/cell. The toxin profile was characterized by carbamate toxins (GTX3, GTX4 and GTX5) and N-sulfocarbamoyl toxins (C1, C2, C3 and C4). C2 dominated at 12–18 °C, but only for salinities ranging from 10 to 25 psu, whereas GTX5 became dominant at temperatures ranging from 21 to 30 °C at almost all salinities. There was no significant variation in the cellular toxin amount from 18 °C to 27 °C for salinities ranging between 30 and 40 psu. At salinities of 10 to 25 psu, the toxin concentrations always remained below 20 fmol/cell. Toxin content was stable for irradiance ranging from 10 to 70 μmol photons/m2/s then slightly increased. Overall, the toxin profile was more stable than the toxin content (fmol/cell), except for temperature and/or salinity values different from those recorded during Alexandrium blooms in Thau Lagoon.

Single-cell analysis of paralytic shellfish toxins in Alexandrium tamarense by HPLC with post-column fluorescent derivatization

We developed a methodology for analyzing the C-toxin (C2) content in single Alexandrium tamarense cells; this method was based on high performance liquid chromatography (HPLC). C2 is the main paralytic shellfish toxin (PST) detected in a clonal culture of A. tamarense, which is a common causative organism in cases of paralytic shellfish poisoning in Japan. This HPLC method employs post-column fluorescent derivatization (FL). Mobile phase, column size, flow rate, reagent concentrations, and lamp type for the fluorescent detector were all optimized for the detection of C2. With this improved methodology, we could measure 1 fmol of C2 with a signal to noise ratio (S/N)=2. Clonal heterogeneity within the toxic strain, which was maintained for 13 years after re-isolation from the original clonal culture, ranged from <1fmol to 700fmolcell−1. This report is the first to demonstrate definitively that PST content varies on a cell-by-cell basis in a clonal culture of a dinoflagellate that causes paralytic shellfish poisoning.

Investigation of extraction and analysis techniques for Lyngbya wollei derived Paralytic Shellfish Toxins

Paralytic Shellfish Toxins (PSTs) are highly toxic metabolic by-products of cyanobacteria and dinoflagellates. The filamentous cyanobacterium Lyngbya wollei produces a unique set of PSTs, including L. wollei toxins (LWT) 1–6. The accurate identification and quantification of PSTs from Lyngbya filaments is challenging, but critical for understanding toxin production and associated risk, as well as for providing baseline information regarding the potential for trophic transfer. This study evaluated several approaches for the extraction and analysis of PSTs from field-collected L. wollei dominated algal mats. Extraction of PSTs from lyophilized Lyngbya biomass was assessed utilizing hydrochloric acid and acetic acid at concentrations of 0.001–0.1 M. Toxin profiles were then compared utilizing two analysis techniques: pre-column oxidation (peroxide and periodate) High Performance Liquid Chromatography (HPLC) with Fluorescence (FL) detection and LC coupled with Mass Spectrometry (MS). While both acid approaches efficiently extracted PSTs, hydrochloric acid was found to convert the less toxic LWT into the more toxic decarbamoylgonyautoxins 2&3 (dcGTX2&3) and decarbamoylsaxitoxin (dcSTX). In comparison, extraction with 0.1 M acetic acid preserved the original toxin profile and limited the presence of interfering co-extractants. Although pre-chromatographic oxidation with HPLC/FL was relatively easy to setup and utilize, the method did not resolve the individual constituents of the L. wollei derived PST profile. The LC/MS method allowed characterization of the PSTs derived from L. wollei, but without commercially available LWT 1–6 standards, quantitation was not possible for the LWT. In future work, evaluation of the risk associated with L. wollei derived PSTs will require commercially available standards of LWT 1–6 for accurate determinations of total PST content and potency.