Paralytic Shellfish Toxin Content Research Articles

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.

Reactive oxygen species are linked to the toxicity of the dinoflagellate Alexandrium spp. to protists

Short-term experiments were conducted to examine the response of the ciliate Tia- rina fusus and the heterotrophic dinoflagellate Polykrikos kofoidii to 3 strains in the Alexandrium tamarense species complex, each with a different paralytic shellfish toxin (PST) content. Both pro- tist species fed on all 3 Alexandrium strains, but significant mortality occurred within 24 h of initial exposure to high densities of each dinoflagellate isolate. Protist mortality was not related, how- ever, to the PST content of the Alexandrium strains, indicating a different mechanism of toxicity. Exposure of T. fusus to cell-free culture filtrates or cell extracts did not cause significant ciliate mortality. In contrast, significant mortality occurred when ciliates were separated physically from a live Alexandrium sp. culture by a 10 µm nylon mesh, suggesting that the toxicity is dependent upon the viability of the Alexandrium spp. cells but does not require physical contact or ingestion. Addition of antioxidant compounds significantly increased the survival of both protist species when exposed to Alexandrium, suggesting that reactive oxygen species and/or the secondary compounds produced by ROS-induced lipid peroxidation are involved in the toxicity of Alexan- drium spp. to ciliates and heterotrophic dinoflagellates. This mechanism of toxicity is previously unknown for Alexandrium spp. and may play an important role in bloom dynamics and toxin transfer within the food web.

Comparative Analysis of Paralytic Shellfish Toxin Content and Profile Produced by Dinoflagellate Gymnodinium Catenatum Isolated from Inokushi Bay, Japan

1 . The principal toxin were C1+2 (79.8 mole%) and GTX 5+6 (18.0 mole%), and minor components were characterized as GTX 2+3, dcGTX2+3, and neoSTX+STX. Interestingly, 7 strains were clearly distin- guished from those of other strains by the complete presence of GTX1+4. In the July strains, which were 33 strains isolated from bloom water, the average growth rate and toxin content was 0.34 divisions day -1 and 160 f mole cell -1 , respectively. C1+2 (83.3 mole%) and GTX 5+6 (10.6 mole%) were the principal toxins, as in the March strains. However, carbamoyl toxins in the July strains were increasing with decreasing N-sulfocarbamoyl toxins in the Mach strains. We also observed major changes in toxin profile, evident by the increase of C1+2 and the decrease of GTX 5+6 in response to increasing temperature, using a single March strain. Minor components of July strains were characterized by GTX2+3, neoSTX, STX and dcGTX2+3. Thus, if we were to consider the toxin profile percentage based on temperature variation, G. catenatum outbreaks in March and July may have originated from one and the same population.

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.

Trophic transfer of paralytic shellfish toxins from clams ( Ruditapes philippinarum) to gastropods ( Nassarius festivus)

A local strain of the dinoflagellate Alexandrium tamarense (ATCI01), which predominantly produces C2 toxin, was fed to the clams ( Ruditapes philippinarum) under laboratory conditions. Concentrations of paralytic shellfish toxins (PSTs) in the dosed clams were determined by High Performance Liquid Chromatographic (HPLC) analyses, and the clams were homogenized and then fed to the gastropods ( Nassarius festivus). In the toxin accumulation phase, which lasted for 42 days, concentrations of PSTs increased in the snails gradually, reaching a maximum of 1.10 nmole g −1 at the end of the exposure period. The toxin content of the homogenized clams (food) was 13.18 nmole g −1, which was about 12-fold higher than the PST content in the snails. Between day 43 and day 82, the snails were fed with non-toxic clams, and this period represented the depuration phase. Accumulation and depuration rates of PSTs in the snails, N. festivus, were determined by fitting the experimental data to user-defined parameters program using a one-compartment model. Two different modeling approaches were used to derive the accumulation and depuration rates. The first approach is to derive both values from the data for the toxin uptake. The second approach is to derive depuration rate from the depuration data and then to derive uptake rate, allowing for toxin depuration, from the data for toxin uptake. The first approach yielded more consistent results for the toxin concentration at the end of the uptake period, when compared with the experimental data. The toxin uptake and depuration rates were 1.64 (pmole of toxin into snail per day) per (nmole g −1 of toxin in food) and 0.06 ± 0.02 day −1 (mean ± SE), respectively. The toxin profiles of snails were similar to the clams, but different from the algae. Besides C toxins (C1 and C2), dcGTX2 and dcGTX3 were also detected in both clams and snails. The β:α epimer ratio gradually decreased during trophic transfer and approached a ratio of 1:3 (26.4 mol%:73.6 mol% at day 42) in the snails, near the end of the accumulation period.

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.

Modelling changes in paralytic shellfish toxin content of dinoflagellates in response to nitrogen and phosphorus supply

A dynamic mathematical model is presented for the growth and Paralytic Shellfish Poison (PSP) content of Alexandrium fundyense. The model includes cellular nitrogen-cycling to enable the synthesis of toxins in the absence of an external nitrogen-source. PSP synthesis is pro- moted by phosphorus stress but depressed by nitrogen stress, with the model containing sigmoidal functions relating nitrogen and phosphorus status to toxin synthesis. The model was tuned simulta- neously to 4 sets of experimental data for ammonium- and nitrate-grown cultures of A. fundyense that were P-replete or P-stressed. The good fit of the model, with a single set of control constants, to almost all data (cellular N:C and P:C, cell size, chlorophyll a:carbon, toxin content, external nutrients) demonstrates the capabilities of mechanistic models under dynamic situations. The potential conse- quences of recycling toxin-N, or of not making toxins at all, were considered using the model. Loss of toxin from cells could be by turnover or by leakage/excretion; model output and experimental data sets suggest that turnover may be the fate in P-stressed nitrate-grown cells but not in ammonium- grown cells. From simulations, there appears to be no significant disadvantage in expending nitrogen on toxin synthesis, thus there need not be a specific evolutionary advantage in the process. If PSP synthesis were selection-neutral this could explain the significant diversity in PSP synthesis cap- abilities within the genus Alexandrium.

Cytological study and immunohistochemical location of PSP toxins in foot skin of the ormer, Haliotis tuberculata , from the Galician coast (NW Spain)

The ormer, Haliotis tuberculata Linnaeus, 1758, is able to accumulate paralytic shellfish toxins (PST) in the foot side skin. Epidermal and secretory cells are described separately for the pedal side and sole epithelia, as notable differences have been observed. Melanin and photosynthetic pigment granules in the epidermal cells of the foot side epithelium give this skin its appearance and characteristic color. This epidermis also has abundant secretory cells containing at least three kinds of secretory granules. Conversely, the epidermal cells of foot sole epithelium do not have pigment granules, and the secretory cells are mostly subepithelial. Immunohistochemical reaction against PST (saxitoxin and its derivatives) showed that the toxins are located in specific cells of the foot side epithelium, which are distinct from the epidermal cells. Immunofluorescence also reveals toxins throughout many areas of the mucilage covering the epithelium, suggesting the secretory nature of the cells containing the toxins. These results do not show any apparent relationship between melanin or photosynthetic pigment granules and PST content. PST were not detected in the foot sole epithelium either immunohistochemically or by HPLC. Considering the toxic characteristics of ormers described in previous studies and the results presented here, the authors discuss the role of toxins retained or produced by the foot side epithelium

Growth dynamics and toxicity of Alexandrium fundyense (Dinophyceae): the effect of changing N∶P supply ratios on internal toxin and nutrient levels

The effect of N- and P-limitation on the growth and paralytic shellfish toxin content of Alexandrium fundyense was studied in a series of batch cultures in which the N:P supply mass ratio was varied from less than 1 up to 160 (cf. Redfield ≡ 7.2). N- and P-limitation of growth was observed at the lower and higher ratios respectively, with dual limitation between. Cellular parameters were similar during the exponential phase of growth, regardless of external N:P ratio. Upon nutrient exhaustion cell yields and internal pools were affected, although these also varied according to the N-source (nitrate vs. ammonium). Generally N-limitation alone led to a higher C:N ratio, lower C:P and N:P ratios and a decreased toxin content in stationary-phase cells. P-limitation led to increased cell size and cell quotas, lower C:N ratios, and higher C:P and N:P ratios (to maxima of 350 and 35 respectively). Complex internal P-fractionation schemes were found unnecessary to determine the P-status of cells and extraction us...

Growth dynamics and toxicity of Alexandrium fundyense (Dinophyceae): the effect of changing N:P supply ratios on internal toxin and nutrient levels

The effect of N- and P-limitation on the growth and paralytic shellfish toxin content of Alexandrium fundyense was studied in a series of batch cultures in which the N:P supply mass ratio was varied from less than 1 up to 160 (cf. Redfield ≡ 7.2). N- and P-limitation of growth was observed at the lower and higher ratios respectively, with dual limitation between. Cellular parameters were similar during the exponential phase of growth, regardless of external N:P ratio. Upon nutrient exhaustion cell yields and internal pools were affected, although these also varied according to the N-source (nitrate vs. ammonium). Generally N-limitation alone led to a higher C:N ratio, lower C:P and N:P ratios and a decreased toxin content in stationary-phase cells. P-limitation led to increased cell size and cell quotas, lower C:N ratios, and higher C:P and N:P ratios (to maxima of 350 and 35 respectively). Complex internal P-fractionation schemes were found unnecessary to determine the P-status of cells and extraction using potassium persulphate, cold water and cold HCl alone were adequate. Severe P-limitation led to increased toxin content but only when N was also limiting, suggesting a synergistic effect of N and P availability on toxin synthesis and turnover. A positive relationship was found between toxin content and the intracellular concentration of arginine, but this varied with the nutrient status (in particular P-status) of the cells.