Crassostrea virginica grazing on toxic and non-toxic diatoms.

Despite high abundances of toxic Pseudo-nitzschia spp. over Louisiana oyster beds (Crassostrea virginica; eastern oyster) there have been no documented cases of amnesic shellfish poisoning (ASP) in the state. Two possible explanations are that oysters do not readily feed on long pointed chains of Pseudo-nitzschia cells or they discriminate against toxic cells while grazing. To test these hypotheses, short-term grazing experiments were conducted with several diatoms, including the domoic acid (DA)-producing Pseudo-nitzschia multiseries (1.31+/-0.057 pg DA cell(-1)) and the non-toxic Pseudo-nitzschia delicatissima, Thalassiosira weissflogii, and Ditylum brightwellii. Grazing rates on the small centric species T. weissflogii were significantly higher than on the larger and pointier D. brightwellii and either Pseudo-nitzschia species. Grazing on toxic P. multiseries and non-toxic P. delicatissima was not significantly different. Pseudofeces production was higher and feces production was occasionally lower in oysters fed Pseudo-nitzschia spp. than in oysters fed the other two diatoms. Our data demonstrate lower filtration rates of C. virginica on Pseudo-nitzschia spp. relative to the other diatoms tested and comparable filtration on toxic and non-toxic Pseudo-nitzschia spp. These findings suggest that eastern oysters do not discriminate amongst food types due to DA content.

Isodomoic acids A and C exhibit low KA receptor affinity and reduced in vitro potency relative to domoic acid in region CA1 of rat hippocampus.

Several natural isomers of the seizurogenic neurotoxin domoic acid (DA) have been found to occur at up to mg/kg levels in shellfish. The aim of the current study was to assess the neurotoxic potency of isodomoic acids A and C (Iso-A and Iso-C), recently isolated from commercial shellfish. Hippocampal slices were obtained from young adult rats and maintained in a tissue recording chamber. Synaptically evoked population spikes were recorded in region CA1 before and after exposure to DA or its isomers. Both Iso-A and Iso-C produced transient neuronal hyperexcitability followed by a dose-dependent suppression of population spikes, but were, respectively, 4- and 20-fold less potent than DA (spike area: EC50 DA=237 nM; Iso-A=939 nM; Iso-C=4.6 microM). In the hippocampus, DA preconditioning induces tolerance to subsequent DA toxicity. However, in the present study neither Iso-A nor Iso-C were effective as preconditioning agents. Competitive binding studies using homomeric GluR6 kainate (kainic acid, KA) receptors showed the affinity of Iso-A to be 40-fold lower than DA (Ki DA=3.35 nM; Iso-A=130 nM). Together with earlier work showing Iso-C affinity at GluR6 receptors to be 240-fold lower than DA, our results suggest that neuroexcitatory effects of Iso-A in CA1 may involve both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and KA receptors, while Iso-C likely involves the activation of AMPA receptors alone.

Emerita analoga (Stimpson)--possible new indicator species for the phycotoxin domoic acid in California coastal waters.

Blooms of domoic acid (DA) synthesizing diatoms (Pseudo-nitzschia spp.) have been associated with the death and injury of hundreds of marine shorebirds and mammals, exposed humans to potentially serious health risks, and threatened to significantly impact coastal fisheries and commerce dependent on marine resources. While indicator organisms are widely utilized to monitor for marine biotoxins like paralytic shellfish poisoning toxins, a reliable intertidal indicator species to monitor DA remains to be identified. Here we evaluate and confirm the utility of the common sand crab (Emerita analoga) as an indicator for DA in comparison with sea mussels (Mytilus californianus). Mussels and sand crabs, collected from natural populations in Santa Cruz, California (April 1999-February 2000), were tested for DA using the HPLC-UV method. Toxin loads in sand crabs ranged from below detectable limits to 13.4 micro g DA g(-1) and coincided with the abundance of DA producing Pseudo-nitzschia species nearshore. Toxin levels in mussels collected during the study period were below HPLC-UV detectable limits. The rise and fall of DA in sand crabs in synchrony with Pseudo-nitzschia abundance, combined with this common intertidal species' accessibility and ease of DA extraction, clearly indicate the utility of sand crabs as a reliable, cost-effective monitoring tool for DA in the nearshore coastal environment.

Evaluation of 11-[3H]-tetrodotoxin use in a heterologous receptor binding assay for PSP toxins.

This report describes the preparative scale production of 11-[3H]-tetrodotoxin (TTX) and its evaluation as a substitute for [3H]-saxitoxin (STX) as the radioligand in a receptor binding assay for paralytic shellfish poisoning (PSP) toxins. Restrictions on the world-wide distribution of [3H]-STX imposed by the international Chemical Weapons Convention served as the primary impetus for this study. We have incorporated on a preparative scale, a nonexchangeable tritium label into the TTX molecule at a specific activity of 12.90 Ci/mmol and recovered material of high radiochemical purity (98%). The resulting 11-[3H]-TTX was found to exhibit site-specific binding characteristics in the receptor assay (dissociation constant(K(d))=4.77+/-1.54nM; maximum binding(B(max))=1. 62+/-0.24pmol/mg of synaptosomal protein). The inhibition constant (K(i)) for the assay was 1.46+/-0.28 nM STX equiv. (n=6), with an estimated detection limit of ca. 2-4 ng STX equiv./ml in a sample extract. Moreover, quantitative comparisons indicated that 11-[3H]-TTX could be used interchangeably with [3H]-STX in the receptor assay for determination of PSP toxicity in shellfish and algal extracts without compromising assay performance. We conclude that the 11-[3H]-TTX produced and evaluated herein exhibits physical, chemical and biological characteristics suitable not only for use in the PSP receptor binding assay, but likely for other applications employing [3H]-STX as the radioligand.

Mortality of sea lions along the central California coast linked to a toxic diatom bloom.

Over 400 California sea lions (Zalophus californianus) died and many others displayed signs of neurological dysfunction along the central California coast during May and June 1998. A bloom of Pseudo-nitzschia australis (diatom) was observed in the Monterey Bay region during the same period. This bloom was associated with production of domoic acid (DA), a neurotoxin that was also detected in planktivorous fish, including the northern anchovy (Engraulis mordax), and in sea lion body fluids. These and other concurrent observations demonstrate the trophic transfer of DA resulting in marine mammal mortality. In contrast to fish, blue mussels (Mytilus edulus) collected during the DA outbreak contained no DA or only trace amounts. Such findings reveal that monitoring of mussel toxicity alone does not necessarily provide adequate warning of DA entering the food web at levels sufficient to harm marine wildlife and perhaps humans.

16S rRNA Targeted Probes for the Identification of Bacterial Strains Isolated from Cultures of the Toxic Dinoflagellate Alexandrium tamarense.

A BSTRACTBacteria have been implicated in the production of paralytic shellfish poison (PSP) toxins, which are normally associated with bloom-forming algal species, specifically toxic dinoflagellate algae. To clarify the role that these bacteria may play in the production of PSP toxins, it is desirable to identify and localize the bacteria associated with the dinoflagellates. 16S rRNA-targeted probes offer the possibility for both, and thus, probes have been made to putatively toxigenic bacteria isolated from the PSP-related dinoflagellate Alexandrium tamarense and tested for their specificity in dot blot and in situ hybridization experiments.

A receptor binding assay for paralytic shellfish poisoning toxins: recent advances and applications.

We recently described a high throughput receptor binding assay for paralytic shellfish poisoning (PSP) toxins, the use of the assay for detecting toxic activity in shellfish and algal extracts, and the validation of 11-[3H]-tetrodotoxin as an alternative radioligand to the [3H]-saxitoxin conventionally employed in the assay. Here, we report a dramatic increase in assay efficiency through application of microplate scintillation technology, resulting in an assay turn around time of 4 h. Efforts are now focused on demonstrating the range of applications for which this receptor assay can provide data comparable to the more time consuming, technically demanding HPLC analysis of PSP toxins, currently the method of choice for researchers. To date, we have compared the results of both methods for a variety of sample types, including different genera of PSP toxin producing dinoflagellates (e.g. Alexandrium lusitanicum, r2 = 0.9834, n = 12), size-fractioned field samples of Alexandrium spp. (20-64 microm; r2 = 0.9997, n = 10) as well as its associated zooplankton grazer community (200-500 microm: r2 = 0.6169, n = 10; >500 microm: r2 = 0.5063, n = 10), and contaminated human fluids (r2 = 0.9661, n = 7) from a PSP outbreak. Receptor-based STX equivalent values for all but the zooplankton samples were highly correlated and exhibited close quantitative agreement with those produced by HPLC. While the PSP receptor binding assay does not provide information on toxin composition obtainable by HPLC, it does represent a robust and reliable means of rapidly assessing PSP-like toxicity in laboratory and field samples. Moreover, this assay should be effective as a screening tool for use by public health officials in responding to suspected cases of PSP intoxication.

Detection of domoic acid in northern anchovies and California sea lions associated with an unusual mortality event.

The occurrence of an unusual mortality event involving California sea lions (Zalophus californianus) along the central California coast in May 1998 was recently reported. The potent neurotoxin domoic acid (DA), produced naturally by the diatom Pseudo-nitzschia australis and transmitted to the sea lions via planktivorous northern anchovies (Engraulis mordax), was identified as the probable causative agent. Details of DA analyses for anchovy tissues and sea lion feces are described. Domoic acid levels were estimated in anchovy samples by HPLC-UV, and in sea lion feces using the same method as well as a microplate receptor binding assay, with absolute confirmation by tandem mass spectrometry. The highest DA concentrations in anchovies occurred in the viscera (223 +/- 5 microg DA g(-1)), exceeding values in the body tissues by seven-fold and suggesting minimal bioaccumulation of DA in anchovy tissue. HPLC values for DA in sea lion fecal material (ranging from 152 to 136.5 microg DA g(-1)) required correction for interference from an unidentified compound. Inter-laboratory comparisons of HPLC data showed close quantitative agreement. Fecal DA activity determined using the receptor binding assay corresponded with HPLC values to within a factor of two. Finally, our detection of P. australis frustules, via scanning electron microscopy, in both anchovy viscera and fecal material from sea lions exhibiting seizures provides corroborating evidence that this toxic algal species was involved in this unusual sea lion mortality event.

Development and preliminary validation of a microtiter plate-based receptor binding assay for paralytic shellfish poisoning toxins.

More than 20 countries have either established or proposed regulatory limits for one or more of the paralytic shellfish poisoning (PSP) toxins as they occur in seafood products. PSP toxin levels are generally estimated using the standard AOAC mouse bioassay, yet because of various limitations of this method [e.g. high variability (+/-20%), low sensitivity, limited sample throughput and use of live animals], there remains a need for alternative testing protocols. A sensitive and selective, high capacity assay was developed for the PSP toxins which exploits the highly specific interaction of these toxins with their biological receptor (i.e. voltage-dependent sodium channel) and is thus based on functional activity. This receptor binding assay provides a radioactive endpoint, and is performed in a microtiter filter plate format with results determined by standard liquid scintillation counting within 24 hr. The Ki for the assay is 3.66 +/- 0.86 nM saxitoxin, with a limit of detection of c. 5 ng saxitoxin/ml in a sample extract. Good quantitative agreement of the assay with both mouse bioassay and high-performance liquid chromatographic analysis of crude extracts of contaminated shellfish, as well as PSP toxin-producing algae, was observed. Our findings indicate that the receptor binding assay has a strong predictive value for toxicity determined by mouse bioassay, and that this approach warrants consideration as a rapid, reliable and cost-effective alternative to live animal testing for detection and estimation of PSP-related toxicity in seafood and toxic algae.

Hypertension and identification of toxin in human urine and serum following a cluster of mussel-associated paralytic shellfish poisoning outbreaks.

Following four outbreaks of paralytic shellfish poisoning on Kodiak Island, Alaska, during 1994, medical records of ill persons were reviewed and interviews were conducted. Urine and serum specimens were analyzed at three independent laboratories using four different saxitoxin binding assays. High-performance liquid chromatography was used to determine the presence of specific toxin congeners. Among 11 ill persons, three required mechanical ventilation and one died. Mean peak systolic and diastolic blood pressure measurements were 172 (range 128-247) and 102 (range 78-165) mmHg, respectively, and blood pressure measurements corresponded with ingested toxin dose. All four different laboratory methodologies detected toxin in serum at 2.8-47 nM during acute illness and toxin in urine at 65-372 nM after acute symptom resolution. The composition of specific paralytic shellfish poisons differed between mussels and human biological specimens, suggesting that human metabolism of toxins had occurred. The results of this study indicate that saxitoxin analogues may cause severe hypertension. In addition, we demonstrate that saxitoxins can be detected in human biological specimens, that nanomolar serum toxin levels may cause serious illness and that human metabolism of toxin may occur. Clearance of paralytic shellfish poisons from serum was evident within 24 hr and urine was identified as a major route of toxin excretion in humans.

Interactions between bacteria and harmful algae: a review.

The ways in which bacteria interact with eukaryotic, unicellular algae are extremely diverse. Such relationships vary widely according to a number of criteria, including spatial and temporal scales, the degree of specificity, and if the relationship can be characterized as beneficial or detrimental to any of the organisms involved. These criteria can be applied to our assessment of how microbes interact with those species involved in the formation of harmful algal blooms (HABs). The aim of this paper is to assess the current state of our knowledge of bacterial/HAB interactions as they pertain to the influence of bacteria on HAB population dynamics, the role of bacteria in the production of toxins normally attributed to the algae, and the suggestion that HABs may act as vectors for pathogenic bacteria. Given that viruses are now considered to play a potentially important role in structuring phytoplankton communities, the possible effects of viruses on the population dynamics of harmful algal species are also addressed.

Enhancement of domoic acid production by reintroducing bacteria to axenic cultures of the diatom Pseudo-nitzschia multiseries.

Axenic cultures of Pseudo-nitzschia multiseries (formerly Pseudonitzschia pungens f. multiseries) produce less domoic acid (DA) than the original bacteria-containing cultures. Bacterial strains isolated from two nonaxenic P. multiseries clones were reintroduced individually into cultures of three axenic P. multiseries strains. The bacteria did not substantially affect division rates or cell yields. However, they did cause a 2- to 95-fold enhancement of DA production (per cell basis) relative to the axenic culture, depending on the P. multiseries and bacterial strain used. Bacteria isolated from a nontoxic Chaetoceros sp. culture also enhanced DA per cell (by 115-fold), showing that it is not necessary for the bacteria to be isolated from a toxic culture in order to enhance toxin production. There was no evidence of intracellular bacteria in disrupted P. multiseries cells obtained from axenic cultures. Our results demonstrate an important, but nonessential, role of extracellular bacterial in DA production. Characterization of the bacterial strains using morphology, substrate utilization, and restriction fragment length polymorphism (RFLP) analyses clearly showed that we had isolated different species of bacteria from the various nonaxenic cultures. We conclude that not one but several bacterial species enhance DA production by P. multiseries.

Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins.

The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.