Paralytic Shellfish Toxins Research Articles

Determination of Toxicity Equivalent Factors for Paralytic Shellfish Toxins by Electrophysiological Measurements in Cultured Neurons

The establishment of toxicity equivalent factors to develop alternative methods to animal bioassays for marine-toxin detection is an urgent need in the field of phycotoxin research. Paralytic shellfish poisoning (PSP) is one of the most severe forms of food poisoning. The toxins responsible for this type of poisoning are highly toxic natural compounds produced by dinoflagellates, which bind to voltage-gated Na(+) channels causing the blockade of action potential propagation. In spite of the fact that several standards of PSP toxins are currently commercially available, there is scarcity of data on the biological activity of these toxins, a fact that limits the calculation of their toxicity equivalent factors. We have evaluated the potency of the commercial PSP toxin standards for their ability to inhibit voltage-dependent sodium currents in cultured neuronal cells by electrophysiological measurements. The in vitro potencies of the PSP toxin standards as indicated by their IC(50) values were in the order Neosaxitoxin (NeoSTX) > decarbamoylsaxitoxin (dcSTX) > saxitoxin (STX) > gonyautoxin 1,4 (GTX1,4) > decarbamoylneosaxitoxin (dcNeoSTX) > gonyautoxin 2,3 (GTX2,3) > decarbamoylgonyautoxin 2,3 (dcGTX2,3) > gonyautoxin 5 (GTX5) > N-sulfocarbamoyl-gonyautoxin-2 and -3 (C1,2). The data obtained in this in vitro analysis correlated well with their previously reported toxicity values.

Improved phylogenetic resolution of toxic and non-toxic Alexandrium strains using a concatenated rDNA approach

Dinoflagellates of the genus Alexandrium are known producers of paralytic shellfish toxins. Species within the genus have similar phenotypes making morphological identification problematical. The use of Alexandrium rDNA sequence data is therefore increasing, resulting in the improved resolution of evolutionary relationships by phylogenetic inferences. However, the true branching pattern within Alexandrium remains unresolved, with minimal support shown for the main phylogentic branch. The aim of this study is to improve phylogenetic resolution via a concatenated rDNA approach with a broad sample of taxa, allowing inference of the evolutionary pattern between species and toxins. 27 Alexandrium strains from 10 species were tested with HPLC for PSP toxin presence and additionally sequenced for 18S, ITS1, 5.8S, ITS2 and 28S rDNA before being phylogenetically inferred together with all available orthologous sequences from NCBI. The resulting alignment is the largest to date for the genus, in terms of both inferred characters and taxa, thus allowing for the improved phylogenetic resolution of evolutionary patterns there in. No phylogenetic pattern between PSP producing and non-producing strains could be established, however the terminal tamarense complex was shown to produce more PSP analogues than basal clades. Additionally, we distinguish a high number of polymorphic regions between the two copies of A. fundyense rDNA, thus allowing us to demonstrate the presence of chimeric sequences within GenBank, as well as a possible over estimation of diversification within the tamarense complex.

Uptake, distribution and depuration of paralytic shellfish toxins from Alexandrium minutum in Australian greenlip abalone, Haliotis laevigata

Farmed greenlip abalone Haliotis laevigata were fed commercial seaweed-based food pellets or feed pellets supplemented with 8 × 105Alexandrium minutum dinoflagellate cells g−1 (containing 12 ± 3.0 μg STX-equivalent 100 g−1, which was mainly GTX-1,4) every second day for 50 days. Exposure of abalone to PST supplemented feed for 50 days did not affect behaviour or survival but saw accumulation of up to 1.6 μg STX-equivalent 100 g−1 in the abalone foot tissue (muscle, mouth without oesophagus and epipodial fringe), which is ∼50 times lower than the maximum permissible limit (80 μg 100 g−1 tissue) for PSTs in molluscan shellfish. The PST levels in the foot were reduced to 0.48 μg STX-equivalent 100 g−1 after scrubbing and removal of the pigment surrounding the epithelium of the epipodial fringe (confirmed by both HPLC and LC-MS/MS). Thus, scrubbing the epipodial fringe, a common procedure during commercial abalone canning, reduced PST levels by ∼70%. Only trace levels of PSTs were detected in the viscera (stomach, gut, heart, gonad, gills and mantle) of the abalone. A toxin reduction of approximately 73% was observed in STX-contaminated abalone held in clean water and fed uncontaminated food over 50 days. The low level of PST uptake when abalone were exposed to high numbers of A. minutum cells over a prolonged period may indicate a low risk of PSP poisoning to humans from the consumption of H. laevigata that has been exposed to a bloom of potentially toxic A. minutum in Australia. Further research is required to establish if non-dietary accumulation can result in significant levels of PSTs in abalone.

Effects of toxic Alexandrium tamarense on behavior, hemocyte responses and development of brown ring disease in Manila clams

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 430:35-48 (2011) - DOI: https://doi.org/10.3354/meps09111 Effects of toxic Alexandrium tamarense on behavior, hemocyte responses and development of brown ring disease in Manila clams V. M. Bricelj1,*, Susan E. Ford2, Christophe Lambert3, Annaick Barbou3, Christine Paillard3 1Institute of Marine and Coastal Sciences, Rutgers University, New Brunswick, New Jersey 08901, USA 2Haskin Shellfish Research Laboratory, Rutgers University, Port Norris, New Jersey 08349, USA 3Institut Universitaire Européen de la Mer, LEMAR, Technopôle Brest Iroise, 29280 Plouzané, France *Email: mbricelj@marine.rutgers.edu ABSTRACT: In this study of short-term exposure of Ruditapes philippinarum from Brittany, France, to an Alexandrium tamarense isolate that produces high concentrations of paralytic shellfish toxins (PSTs), the effects of the isolate on clam fitness, toxin uptake, and the response of hemocytes (responsible for internal defense in bivalves) were examined. Adults exhibited individual variability in ­resistance to the effects of PSTs, measured by their burrowing capacity. If genetically based, this may (1) indicate that resistance is under natural selection and (2) provide a new model to advance our understanding of the molecular basis for PST resistance in bivalves, so far determined only in Mya arenaria. Juvenile clams were more vulnerable to PSTs than adults, experiencing 100% burrowing incapacitation within 1 d of exposure to A. tamarense. They also experienced growth suppression and high mortalities following 4 d of toxification. Juveniles challenged with Vibrio tapetis, the cause of brown ring disease (BRD), and exposed to A. tamarense, developed significantly fewer BRD symptoms relative to controls fed non-toxic algae, but suffered higher mortality. Adult clams exposed to A. tamarense showed a significant increase in hemocyte concentrations and a small, but significant, decrease in phagocytic activity, and no effect on hemocyte viability or other functional parameters. We speculate that the inhibitory effects on BRD progression may be attributable to toxicity of PSTs to V. tapetis, inability of juveniles to activate the shell conchiolin-deposition response, and/or an overall increase in phagocytic cells induced by A. tamarense. Harmful algae and pathogens may thus ­interact and modulate the effects of disease in bivalve populations. KEY WORDS: Ruditapes philippinarum · Paralytic shellfish toxins · Vibrio · Burrowing · Hemocytes Full text in pdf format PreviousNextCite this article as: Bricelj VM, Ford SE, Lambert C, Barbou A, Paillard C (2011) Effects of toxic Alexandrium tamarense on behavior, hemocyte responses and development of brown ring disease in Manila clams. Mar Ecol Prog Ser 430:35-48. https://doi.org/10.3354/meps09111 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 430. Online publication date: May 26, 2011 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2011 Inter-Research.

Comparison of biosensor platforms for surface plasmon resonance based detection of paralytic shellfish toxins

Paralytic shellfish poisoning (PSP) toxins are produced by certain marine dinoflagellates and may accumulate in bivalve molluscs through filter feeding. The Mouse Bioassay (MBA) is the internationally recognised reference method of analysis, but it is prone to technical difficulties and regarded with increasing disapproval due to ethical reasons. As such, alternative methods are required. A rapid surface plasmon resonance (SPR) biosensor inhibition assay was developed to detect PSP toxins in shellfish by employing a saxitoxin polyclonal antibody (R895). Using an assay developed for and validated on the Biacore Q biosensor system, this project focused on transferring the assay to a high-throughput, Biacore T100 biosensor in another laboratory. This was achieved using a prototype PSP toxin kit and recommended assay parameters based on the Biacore Q method. A monoclonal antibody (GT13A) was also assessed. Even though these two instruments are based on SPR principles, they vary widely in their mode of operation including differences in the integrated μ-fluidic cartridges, autosampler system, and sensor chip compatibilities. Shellfish samples (n=60), extracted using a simple, rapid procedure, were analysed using each platform, and results were compared to AOAC high performance liquid chromatography (HPLC) and MBA methods. The overall agreement, based on statistical 2×2 comparison tables, between each method ranged from 85% to 94.4% using R895 and 77.8% to 100% using GT13A. The results demonstrated that the antibody based assays with high sensitivity and broad specificity to PSP toxins can be applied to different biosensor platforms.

Past trends and future scenarios for environmental conditions favoring the accumulation of paralytic shellfish toxins in Puget Sound shellfish

The risk of harmful algal blooms (HABs) of the dinoflagellate Alexandrium catenella in Puget Sound, Washington State, can be assessed by identifying and predicting climate and environmental conditions that are favorable for bloom development and the accumulation of paralytic shellfish toxins (PSTs) in shellfish. When these favorable conditions occur in combination, a harmful algal bloom window of opportunity (HAB-WOO) exists for A. catenella. The original HAB-WOO was identified by Moore et al. (2009) for the time period 1993–2007. In general, it showed that warm air and water temperatures, low streamflow, low winds, and small tidal variability precede PST events. Here, we use the HAB-WOO model to examine (i) changes in the annual HAB-WOO duration over the period from 1967 to 2006, and (ii) the potential effect of future climate change on HAB risk through the late 21st century. The annual HAB-WOO duration increased between 1978 and 2006, as did the frequency and geographic extent of PST events. Two step-like changes occurred in 1978 and 1992 with higher annual values attained by the HAB-WOO compared to previous years. The 1978 step change may be related to the 1977 reversal of the Pacific Decadal Oscillation from cool to warm phase. Climate change projections from global climate models and regionally downscaled climate models for the Pacific Northwest are used to evaluate scenarios for the future HAB-WOO. Under a moderate greenhouse gas emissions scenario (i.e., A1B), the annual HAB-WOO for A. catenella in Puget Sound is projected to increase by an average of 13 days by the end of the 21st century. Furthermore, the annual HAB-WOO may begin up to 2 months earlier in the year and persist for up to 1 month later in the year compared to the present day. This research provides managers, health authorities, and shellfish growers in Washington State with critical information for anticipating climate impacts on toxic HABs in the Pacific Northwest now and in a future warmer climate.

Alexandrium catenella and Alexandrium tamarense in the North Lake of Tunis: bloom characteristics and the occurrence of paralytic shellfish toxin

The dinoflagellate Alexandrium is thought to be the main species responsible for paralytic shellfish poisoning (PSP) throughout the world. The genus Alexandrium is present in unusually high diversity in the North Lake of Tunis, a productive Mediterranean fishing and shellfish collecting area. High abundances of Alexandrium tamarense (7 × 103 cells l–1) were recorded here in August 2007, and of A. catenella (5.5 × 04 cells l–1) in January 2008 for the first time. High densities of A. tamarense were associated with high surface temperature (29.3 °C) and high salinity (40.6), while A. catenella blooms occurred at lower temperature (15.9 °C) and salinity (36.7). Alexandrium tamarense cell densities correlated positively with NH4 + (47.10 μmol l–1), but negatively with NO3 − (0.55 μmol l–1). In contrast, A. catenella proliferated when NO3 − was high (2.15 μmol l–1) and NH4 + was relatively low (8.38 μmol l–1). To study the toxicity of A. catenella and A. tamarense, extracts from clams Ruditapes decussatus were analysed by mouse bioassay and Jellett rapid PSP test from July 2007 to April 2008. The December 2007 and January 2008 samples were positive, corresponding to blooms of Alexandrium catenella, but the PSP concentrations were below 80 μg STX equivalent 100 g–1 of shellfish meat.

Preparation of Calibration Standards of N1-H Paralytic Shellfish Toxin Analogues by Large-Scale Culture of Cyanobacterium Anabaena circinalis (TA04)

Mouse bioassay is the official testing method to quantify paralytic shellfish toxins (PSTs) in bivalves. A number of alternative analytical methods have been reported. Some methods have been evaluated by a single laboratory validation. Among the different types of methods, chemical analyses are capable of identifying and quantifying the toxins, however a shortage of the necessary calibration standards hampers implementation of the chemical analyses in routine monitoring of PSTs in bivalves. In our present study, we studied preparation of major PST analogues as calibrants by large-scale cultivation of toxic freshwater cyanobacteria Anabaena circinalis TA04. The cells were steadily grown in 10 L bottle for 28 days. The primary N1-H toxins, C1/C2, were produced at a concentration of 1.3 ±0.1 μmol/L. The intracellular and extracellular toxins occupied 80% and 20%, respectively. Over 220 μmol of the toxins was obtained from approximately 200 L of the culture over six months, demonstrating that it is sufficient to prepare saxitoxin analogues. The toxins were chemically converted to six N1-H analogues. Preparation of the analogues was carried out at relatively high yields (50–90%). The results indicate that our preparation method is useful to produce N1-H toxins. In our present study, detailed conditions for preparation of one of the rare N1-H analogues, gonyautoxin-5, were investigated.

First detection of cyanobacterial PSP (paralytic shellfish poisoning) toxins in Spanish freshwaters

Presence of the paralytic shellfish toxins (PSTs) saxitoxin, neosaxitoxin, decarbamoyl saxitoxin and gonyautoxin-5 was analyzed by mass spectrometry in 41 Spanish freshwaters and 13 strains of potential PST-producing planktonic cyanobacteria. Toxins were detected in five waterbodies, but were absent from the isolated strains. PST containing samples belonged to the same geographical region (South Western Spain) and were most likely related to the presence of the genus Aphanizomenon.

An improved immunoassay for detection of saxitoxin by surface plasmon resonance biosensors

Saxitoxin and its analogs, the causative agents of paralytic shellfish poisoning (PSP), are a worldwide threat to seafood safety. Effective monitoring of potentially contaminated fishing areas as well as screening of seafood samples is necessary to adequately protect the public. While many analytical methods exist for detecting paralytic shellfish toxins (PSTs), each technique has challenges associated with routine use. One recently developed method [1] that overcomes ethical or performance-related issues of other techniques is the surface plasmon resonance (SPR) bioassay. Notwithstanding the advantages of this method, much research remains in optimizing the sensor substrate and assay conditions to create a robust technique for rapid and sensitive measurement of PSTs. This manuscript describes a more rigorous and stable SPR inhibition immunoassay through optimization of the surface chemistry as well as determination of optimum mixture ratios and mixing times. The final system provides rapid substrate formation (18 h saxitoxin conjugation with low reagent consumption), contains a reference channel for each assay, and is capable of triplicate measurements in a single run with detection limits well below the regulatory action level.

Screening for multiple classes of marine biotoxins by liquid chromatography–high-resolution mass spectrometry

Marine biotoxins pose a significant food safety risk when bioaccumulated in shellfish, and adequate testing for biotoxins in shellfish is required to ensure public safety and long-term viability of commercial shellfish markets. This report describes the use of a benchtop Orbitrap system for liquid chromatography-mass spectrometry (LC-MS) screening of multiple classes of biotoxins commonly found in shellfish. Lipophilic toxins such as dinophysistoxins, pectenotoxins, and azaspiracids were separated by reversed phase LC in less than 7min prior to MS data acquisition at 2Hz with alternating positive and negative scans. This approach resulted in mass accuracy for analytes detected in positive mode (gymnodimine, 13-desmethyl spirolide C, pectenotoxin-2, and azaspiracid-1, -2, and -3) of less than 1ppm, while those analytes detected in negative mode (yessotoxin, okadaic acid, and dinophysistoxin-1 and -2) exhibited mass errors between 2 and 4ppm. Hydrophilic toxins such as domoic acid, saxitoxin, and gonyautoxins were separated by hydrophilic interaction LC (HILIC) in less than 4min, and MS data was collected at 1Hz in positive mode, yielding mass accuracy of less than 1ppm error at a resolving power of 100,000 for the analytes studied (m/z 300-500). Data were processed by extracting 5ppm mass windows centered around the calculated masses of the analytes. Limits of detection (LOD) for the lipophilic toxins ranged from 0.041 to 0.10μg/L (parts per billion) for the positive ions, 1.6-5.1μg/L for those detected in negative mode, while the domoic acid and paralytic shellfish toxins yielded LODs ranging from 3.4 to 14μg/L. Toxins were detected in mussel tissue extracts free of interference in all cases.

A putative gene cluster from a Lyngbya wollei bloom that encodes paralytic shellfish toxin biosynthesis.

Saxitoxin and its analogs cause the paralytic shellfish-poisoning syndrome, adversely affecting human health and coastal shellfish industries worldwide. Here we report the isolation, sequencing, annotation, and predicted pathway of the saxitoxin biosynthetic gene cluster in the cyanobacterium Lyngbya wollei. The gene cluster spans 36 kb and encodes enzymes for the biosynthesis and export of the toxins. The Lyngbya wollei saxitoxin gene cluster differs from previously identified saxitoxin clusters as it contains genes that are unique to this cluster, whereby the carbamoyltransferase is truncated and replaced by an acyltransferase, explaining the unique toxin profile presented by Lyngbya wollei. These findings will enable the creation of toxin probes, for water monitoring purposes, as well as proof-of-concept for the combinatorial biosynthesis of these natural occurring alkaloids for the production of novel, biologically active compounds.

Pre- versus post-column oxidation liquid chromatography fluorescence detection of paralytic shellfish toxins

Both pre- and post-column oxidation liquid chromatography methods with fluorescence detection are available for detecting paralytic shellfish toxins. Each method has been evaluated in multiple laboratories and validated as a potential alternative to the mouse bioassay. This communication compares the advantages and limitations of both methods. For a given laboratory, the selection of either method may be based primarily on practicality and less on any deficiencies in scientific merit.

Matrix effect on paralytic shellfish toxins quantification and toxicity estimation in mussels exposed to Gymnodinium catenatum

Paralytic shellfish toxins were quantified in whole tissues of the mussel Mytilus galloprovincialis exposed to blooms of the dinoflagellate Gymnodinium catenatum in Portuguese coastal waters. A validated liquid chromatography method with fluorescence detection, involving pre-chromatographic oxidation was used to quantify carbamoyl, N-sulfocarbamoyl and decarbamoyl toxins. In order to test for any matrix effect in the quantification of those toxins, concentrations obtained from solvent and matrix matched calibration curves were compared. A suppression of the fluorescence signal was observed in mussel extract or fraction in comparison to solvent for the compounds dcGTX2 + 3, GTX2 + 3 and GTX1 + 4, while an enhancement was found for C1 + 2, dcSTX, STX, B1, dcNEO and NEO. These results showed that a matrix effect varies among compounds. The difference of concentrations between solvent and matrix matched calibration curves for C1 + 2 (median = 421 ng g−1) exceeded largely the values for the other quantified compounds (0.09–58 ng g−1). Those differences were converted into toxicity differences, using Oshima toxicity equivalence factors. The compounds C1 + 2 and dcNEO were the major contributors to the differences of total toxicity in the mussel samples. The differences of total toxicity were calculated in ten mussel samples collected during a 10-week blooming period in Portuguese coastal lagoon. Values varied between 53 and 218 µg STX equivalents kg−1. The positive differences mean that the estimated toxicity using solvent calibration curves exceed the values taking into account the matrix. For the toxicity interval 200–800 µg STX equivalents kg−1 an increase was found between 44 and 28%.

Ingestion and clearance rates of the red Noctiluca scintillans fed on the toxic dinoflagellate Alexandrium minutum (Halim)

In this study, we evaluated the ingestion and clearance rates of the heterotrophic dinoflagellate Noctiluca scintillans (Macartney) fed on the toxic microalgae Alexandrium minutum Halim in laboratory cultures. Specimens of the red form of N. scintillans were collected in the Ría de Vigo and kept in culture. The results showed that N. scintillans actively fed on A. minutum, with maximum ingestion rates of 0.37 μg C ind −1 day −1, showing that there was non-satiated feeding by N. scintillans on A. minutum. The mean cellular toxin content of A. minutum in the stock culture ranged from 2.57 to 3.44 fmol cell −1. The HPLC analyses of the extracts from N. scintillans fed with 4 different abundances of A. minutum revealed no detectable amounts of toxin. This suggested that although N. scintillans was able to ingest a toxic dinoflagellate such as A. minutum into its vacuoles, the ingested toxin was bioconverted or excreted by Noctiluca. Our findings support the theory that Noctiluca may inflict grazing pressure on the growth of Paralytic Shellfish Toxin (PST) species in the field, and could therefore play an important role as a regulator against PST-producing phytoplankton. This is the first report about grazing rates of N. scintillans on Alexandrium cells.

Solid core column technology applied to HPLC-FD of paralytic shellfish toxins

Pre-column oxidation liquid chromatography with fluorescence detection is a chemical method for analyzing paralytic shellfish toxins. In order to improve the sample throughput and efficiency of AOAC Method 2005.06, solid core particle column technology was evaluated. We demonstrate that supplanting the original fully porous particle column with a solid core particle column reduces sample analysis time from 15 to 5min per sample and improves resolution.

Germination characteristics of Alexandrium catenella cysts from surface sediments in Quartermaster Harbor, Puget Sound, Washington, USA

The dinoflagellate Alexandrium catenella causes frequent outbreaks of paralytic shellfish toxins (PSTs) in Puget Sound, Washington; however, little is known about its basic biology and ecology. Most of what is known is inferred mainly from shellfish toxin records and recent work on cyst distribution and germination potential. We report on a year-long study of cyst dormancy and germination potential based on experiments using surface sediment collected from a shallow embayment, Quartermaster Harbor, in Puget Sound. Cyst abundance in Quartermaster Harbor was 1550–1750 cysts cm −3 when sediment was collected in mid-October 2006. Germination experiments set up monthly had germination occur in all months when cysts were provided with adequate growth supporting conditions. Germination rates were highest in May and June when 100% germination occurred within 2 days. Longer incubation periods observed for the first two months of the study (November and December) may indicate a mandatory dormancy period of up to 5 months if the majority of cysts were deposited in the sediments following a late summer bloom that apparently occurred in Quartermaster Harbor in 2006 as indicated by Washington State Department of Health's toxin records. The data provide no evidence of an endogenous clock restricting germination to a specific annual timeframe for these shallow water cysts. This contrasts with the presence of a circannual endogenous clock, for deep water cysts of A. fundyense in the Gulf of Maine. The significance of these results is that germination of the A. catenella Puget Sound population appears to be primarily regulated by physiological requirements. However, once physiological requirements are met cysts can germinate rapidly, often within 24 h, when provided with adequate environmental conditions.

Comparative dynamics of paralytic shellfish toxins (PST) in a tolerant and susceptible population of the copepod Acartia hudsonica

In common-environment experiments, we measured the uptake, accumulation, retention, transformation and depuration of paralytic shellfish toxins (PSTs) of the dinoflagellate Alexandrium fundyense in adult females of two geographically separated populations (Maine and New Jersey, USA) of the copepod Acartia hudsonica. These populations were previously shown to differ in their history of exposure to Alexandrium blooms, and in their degree of adaptation to this dinoflagellate. Toxin accumulation was measured at several times in incubations lasting up to 72 h. Toxin depuration was assessed after a 60 h incubation of females with toxic A. fundyense and subsequent 60 h incubation in a sole diet of the non-toxic green flagellate Tetraselmis sp. As previously observed, the Maine population had significantly higher toxin ingestion rates. By contrast, both toxin accumulation (up to ∼2.5 ng STX eq. female −1) and the depuration rate (∼0.73 d −1) were not significantly different between populations. Hence, faster depuration is ruled out as a tolerance mechanism in the Maine population. Some toxin transformation during both accumulation and depuration was evident in both populations. However, differential toxin transformation does not appear to be a tolerance mechanism either. In contrast to these results, toxin retention (ratio of accumulation/cumulative ingestion) was significantly lower in the Maine population. The retentions of gonyautoxin 3 (GTX3) and of two of the most potent toxins, neosaxitoxin (NEO) and saxitoxin (STX), were also significantly lower in that population. At steady state, toxin absorption efficiency was estimated to be 6% for the Maine population and 9% for the New Jersey population. These results suggest that lower toxin absorption is a possible tolerance mechanism for the Maine population. The results of the present study also suggest that copepod adaptation to toxic dinoflagellates does not necessarily lead to higher toxin transfer up the food web.

The relative influences of El Niño‐Southern Oscillation and Pacific Decadal Oscillation on paralytic shellfish toxin accumulation in northwest Pacific shellfish

Historical records of paralytic shellfish toxins (PSTs) in shellfish from a coastal embayment in the Pacific Northwest of the United States are used to examine the influence of large‐scale climate variations on aspects of Alexandrium catenella bloom dynamics on interannual and interdecadal timescales. An annual index of shellfish toxicity covaries with the number of days annually that sea surface temperature (SST) exceeds 13°C—a known temperature threshold for increased shellfish toxicity in this region—and with an index of the Pacific Decadal Oscillation (PDO). In contrast, no robust relationship exists between our shellfish toxicity index and an index of the El Niño‐Southern Oscillation (ENSO). We hypothesize that this is because anomalously warm water temperatures created during El Niño winters do not generally persist into the seasonal time period that shellfish in this region accumulate PSTs, which is typically in the summer and fall. In contrast, anomalously warm water temperatures created during warm‐phase PDO winters and springs typically persist into the summer and fall, thereby increasing the number of days annually that SST exceeds 13°C, and increasing the window of opportunity for Alexandrium blooms that ultimately lead to shellfish acquiring higher concentrations of PSTs.

Effects of mitomycin C and colchicine on toxin production and cell cycle regulation in the dinoflagellate Alexandrium tamarense

Paralytic shellfish toxin (PST) production and cell proliferation in the dinoflagellate Alexandrium tamarense were investigated under the influence of two metabolic inhibitors (mitomycin C and colchicine) that have different mechanisms of action. Intracellular PST levels in cells treated with 2 μM mitomycin C increased gradually, reaching a maximum of 176 fmol/cell (a 6-fold increase). High concentrations of colchicine prolonged G 1 phase in A. tamarense cells, even though colchicine arrests several other eukaryotic cell types in M phase. The cells in prolonged G 1 phase under the influence of colchicine were apparently unable to produce PST. Cell proliferation and toxin production recovered after removal of colchicine; in contrast, the effect of mitomycin C was irreversible. While the timing of toxin production within the cell cycle was not conclusively determined, A. tamarense cells in S phase were able to produce PST.