Marine algal toxins and their vectors in southern California cetaceans.

Published baseline data on biotoxin exposure in cetaceans is sparse but critical for interpreting mortality events as harmful algal blooms increase in frequency and duration. We present the first synthesis of domoic acid (DA), saxitoxin (STX), okadaic acid (OA), and microcystin detections in the feces and urine of stranded and bycaught southern California cetaceans, over an 18 year period (2001-2018), along with corresponding stomach content data. DA was detected in 13 out of 19 cetacean species, most often in harbor porpoise (Phocoena phocoena) (81.8%, n = 22) and long-beaked common dolphins (Delphinus delphis bairdii) (74%, n = 231). Maximum DA concentrations of 324,000 ng/g in feces and 271, 967 ng/ml in urine were observed in D. d. bairdii. DA was detected more frequently and at higher concentrations in male vs. female D. d. bairdii. Higher fecal DA concentrations in D. d. bairdii were associated with a greater proportion of northern anchovy (Engraulis mordax) in the diet, indicating it may be a primary vector of DA. Fecal DA concentrations for D. d. bairdii off Point Conception were greater than those from animals sampled off Los Angeles and San Diego counties, reflecting greater primary productivity and higher Pseudo-nitzschia spp. abundance in that region and a greater abundance of E. mordax in the diet. STX was detected at low levels (fecal max = 7.5 ng/g, urine max = 17 ng/ml) in 3.6% (n = 165) of individuals from 3 out of 11 species. The occurrence of E. mordax in 100% of the 3 examined stomachs suggests this species could be a primary vector of the detected STX. OA was detected in 2.4% of tested individuals (n = 85) at a maximum fecal concentration of 422.8 ng/g. Microcystin was detected in 14.3% (n = 7) of tested individuals with a maximum liver concentration of 96.8 ppb.

An assessment of temporal, spatial and taxonomic trends in harmful algal toxin exposure in stranded marine mammals from the U.S. New England coast.

Despite a long-documented history of severe harmful algal blooms (HABs) in New England coastal waters, corresponding HAB-associated marine mammal mortality events in this region are far less frequent or severe relative to other regions where HABs are common. This long-term survey of the HAB toxins saxitoxin (STX) and domoic acid (DA) demonstrates significant and widespread exposure of these toxins in New England marine mammals, across multiple geographic, temporal and taxonomic groups. Overall, 19% of the 458 animals tested positive for one or more toxins, with 15% and 7% testing positive for STX and DA, respectively. 74% of the 23 different species analyzed demonstrated evidence of toxin exposure. STX was most prevalent in Maine coastal waters, most frequently detected in common dolphins (Delphinus delphis), and most often detected during July and October. DA was most prevalent in animals sampled in offshore locations and in bycaught animals, and most frequently detected in mysticetes, with humpback whales (Megaptera novaeangliae) testing positive at the highest rates. Feces and urine appeared to be the sample matrices most useful for determining the presence of toxins in an exposed animal, with feces samples having the highest concentrations of STX or DA. No relationship was found between the bloom season of toxin-producing phytoplankton and toxin detection rates, however STX was more likely to be present in July and October. No relationship between marine mammal dietary preference and frequency of toxin detection was observed. These findings are an important part of a framework for assessing future marine mammal morbidity and mortality events, as well as monitoring ecosystem health using marine mammals as sentinel organisms for predicting coastal ocean changes.

Association between red tide exposure and detection of corresponding neurotoxins in bottlenose dolphins from Texas waters during 2007-2017.

Harmful algal blooms produced by the phytoplankton species Karenia brevis and its associated neurotoxin, brevetoxin (PbTx), occur throughout the Gulf of Mexico and have had devastating impacts on co-occurring populations of bottlenose dolphins (Tursiops truncatus), an important marine sentinel species. The majority of documented impacts, however, are from the eastern Gulf of Mexico, with a critical lack of information on the degree and frequency of PbTx exposure in bottlenose dolphins from Texas coastal waters. This study documents PbTx exposure in Texas bottlenose dolphins between 2007 and 2017 and their association with co-occurring K. brevis blooms. PbTx was detected in 60% (n = 112) of the animals tested. Liver tissue samples had the highest frequency of detection (62%), followed by feces (41.4%) and gastric contents (30.4%). PbTx was not detected in urine or intestinal tissue. The concentration ranges of PbTx detected in feces (1.2-216, mean 38.4 ng/g), gastric contents (3.3-1016, mean 158 ng/g) and liver (0.6-52.4, mean 8.5 ng/g) samples were an order of magnitude less than values reported for Florida dolphins for the same sample types. The proportion of dolphins recovered within 4 weeks of a bloom that tested positive for PbTx ('Bloom' group; 75%) was significantly higher compared to those that were recovered 5-8 weeks after termination of a bloom ('Post-Bloom' group; 36%; p = 0.004). The proportion of PbTx-positive animals with no observed bloom association ('Baseline' group; 60%) was also significantly greater than the Post-Bloom group (p = 0.012). No significant difference in proportion of PbTx-positive animals was detected between Bloom and Baseline groups (p = 0.242). No significant differences in liver PbTx concentrations were observed between any pairwise combinations of the 3 exposure groups (p = 0.261). Overall, these findings suggest persistent PbTx exposure for many individuals in these populations, although the health impacts of such exposure are not known.

The Southeast Alaska Tribal Ocean Research (SEATOR) Partnership: Addressing Data Gaps in Harmful Algal Bloom Monitoring and Shellfish Safety in Southeast Alaska.

Many communities in Southeast Alaska harvest shellfish such as mussels and clams as an important part of a subsistence or traditional diet. Harmful algal blooms (HABs) of phytoplankton such as Alexandrium spp. produce toxins that can accumulate in shellfish tissues to concentrations that can pose a hazard for human health. Since 2013, several tribal governments and communities have pooled resources to form the Southeast Alaska Tribal Ocean Research (SEATOR) network, with the goal of minimizing risks to seafood harvest and enhancing food security. SEATOR monitors toxin concentrations in shellfish and collects and consolidates data on environmental variables that may be important predictors of toxin levels such as sea surface temperature and salinity. Data from SEATOR are publicly available and are encouraged to be used for the development and testing of predictive algorithms that could improve seafood risk assessment in Southeast Alaska. To date, more than 1700 shellfish samples have been analyzed for paralytic shellfish toxins (PSTs) in more than 20 locations, with potentially lethal concentrations observed in blue mussels (Mytilus trossulus) and butter clams (Saxidomus gigantea). Concentrations of PSTs exhibit seasonality in some species, and observations of Alexandrium are correlated to sea surface temperature and salinity; however, concentrations above the threshold of concern have been found in all months, and substantial variation in concentrations of PSTs remain unexplained.

Tissue distribution of amino acid- and lipid-brevetoxins after intravenous administration to C57BL/6 mice.

Brevetoxins produced during algal blooms of the dinoflagellate Karenia are metabolized by shellfish into reduction, oxidation, and conjugation products. Brevetoxin metabolites comprising amino acid- and lipid conjugates account for a large proportion of the toxicity associated with the consumption of toxic shellfish. However, the disposition of these brevetoxin metabolites has not been established. Using intravenous exposure to C57BL/6 mice, we investigated the disposition in the body of three radiolabeled brevetoxin metabolites. Amino acid-brevetoxin conjugates represented by S-desoxy-BTX-B2 (cysteine-BTX-B) and lipid-brevetoxin conjugates represented by N-palmitoyl-S-desoxy-BTX-B2 were compared to dihydro-BTX-B. Tissue concentration profiles were unique to each of the brevetoxin metabolites tested, with dihydro-BTX-B being widely distributed to all tissues, S-desoxy-BTX-B2 concentrated in kidney, and N-palmitoyl-S-desoxy-BTX-B2 having the highest concentrations in spleen, liver, and lung. Elimination patterns were also unique: dihydro-BTX-B had a greater fecal versus urinary elimination, whereas urine was a more important elimination route for S-desoxy-BTX-B2, and N-palmitoyl-S-desoxy-BTX-B2 persisted in tissues and was eliminated equally in both urine and feces. The structures particular to each brevetoxin metabolite resulting from the reduction, amino acid conjugation, or fatty acid addition of BTX-B were likely responsible for these tissue-specific distributions and unique elimination patterns. These observed differences provide further insight into the contribution each brevetoxin metabolite class has to the observed potencies.

Semisynthesis of radiolabeled amino acid and lipid brevetoxin metabolites and their blood elimination kinetics in C57BL/6 mice.

Brevetoxin B (BTX-B), produced by dinoflagellates of the species Karenia, is a highly reactive molecule, due in part to an α,ß-unsaturated aldehyde group at the terminal side chain, leading to the production of metabolites in shellfish by reduction, oxidation, and conjugation. We have investigated in mice the blood elimination of three common bioactive brevetoxin metabolites found in shellfish, which have been semisynthesized from BTX-B in radioactive forms. BTX-B was reduced at C42 to yield [(3)H] dihydro-BTX-B. [(3)H] S-desoxy-BTX-B2 (cysteine brevetoxin B) was semisynthesized from BTX-B by the conjugation of cysteine at the C50 olefinic group then [(3)H] radiolabeled by C42 aldehyde reduction. [(14)C] N-Palmitoyl-S-desoxy-BTX-B2 was prepared using S-desoxy-BTX-B2 as the starting material with addition of the [(14)C] radiolabeled fatty acid via cysteine-amide linkage. The elimination of intravenously administered [(3)H] S-desoxy-BTX-B2, [(14)C] N-palmitoyl-S-desoxy-BTX-B2, or [(3)H] dihydro-BTX-B was measured in blood collected from C57BL/6 mice over a 48 h period. Each brevetoxin metabolite tested exhibited biexponential elimination kinetics and fit a two-compartment model of elimination that was applied to generate toxicokinetic parameters. The rate of transfer between the central compartment (i.e., blood) and the peripheral compartment (e.g., tissue) for each brevetoxin differed substantially, with dihydro-BTX-B exchanging rapidly with the peripheral compartment, S-desoxy-BTX-B2 eliminating rapidly from the central compartment, and N-palmitoyl-S-desoxy-BTX-B2 eliminating slowly from the central compartment. Toxicokinetic parameters were analyzed in the context of the unique structure of each brevetoxin metabolite resulting from a reduction, amino acid conjugation, or fatty acid addition to BTX-B.

Comparative analysis of three brevetoxin-associated bottlenose dolphin (Tursiops truncatus) mortality events in the Florida Panhandle region (USA).

In the Florida Panhandle region, bottlenose dolphins (Tursiops truncatus) have been highly susceptible to large-scale unusual mortality events (UMEs) that may have been the result of exposure to blooms of the dinoflagellate Karenia brevis and its neurotoxin, brevetoxin (PbTx). Between 1999 and 2006, three bottlenose dolphin UMEs occurred in the Florida Panhandle region. The primary objective of this study was to determine if these mortality events were due to brevetoxicosis. Analysis of over 850 samples from 105 bottlenose dolphins and associated prey items were analyzed for algal toxins and have provided details on tissue distribution, pathways of trophic transfer, and spatial-temporal trends for each mortality event. In 1999/2000, 152 dolphins died following extensive K. brevis blooms and brevetoxin was detected in 52% of animals tested at concentrations up to 500 ng/g. In 2004, 105 bottlenose dolphins died in the absence of an identifiable K. brevis bloom; however, 100% of the tested animals were positive for brevetoxin at concentrations up to 29,126 ng/mL. Dolphin stomach contents frequently consisted of brevetoxin-contaminated menhaden. In addition, another potentially toxigenic algal species, Pseudo-nitzschia, was present and low levels of the neurotoxin domoic acid (DA) were detected in nearly all tested animals (89%). In 2005/2006, 90 bottlenose dolphins died that were initially coincident with high densities of K. brevis. Most (93%) of the tested animals were positive for brevetoxin at concentrations up to 2,724 ng/mL. No DA was detected in these animals despite the presence of an intense DA-producing Pseudo-nitzschia bloom. In contrast to the absence or very low levels of brevetoxins measured in live dolphins, and those stranding in the absence of a K. brevis bloom, these data, taken together with the absence of any other obvious pathology, provide strong evidence that brevetoxin was the causative agent involved in these bottlenose dolphin mortality events.

Determination of paralytic shellfish toxins in shellfish by receptor binding assay: collaborative study.

A collaborative study was conducted on a microplate format receptor binding assay (RBA) for paralytic e shellfish toxins (PST). The assay quantifies the composite PST toxicity in shellfish samples based on the ability of sample extracts to compete with (3)H saxitoxin (STX) diHCl for binding to voltage-gated sodium channels in a rat brain membrane preparation. Quantification of binding can be carried out using either a microplate or traditional scintillation counter; both end points were included in this study. Nine laboratories from six countries completed the study. One laboratory analyzed the samples using the precolumn oxidation HPLC method (AOAC Method 2005.06) to determine the STX congener composition. Three laboratories performed the mouse bioassay (AOAC Method 959.08). The study focused on the ability of the assay to measure the PST toxicity of samples below, near, or slightly above the regulatory limit of 800 (microg STX diHCl equiv./kg). A total of 21 shellfish homogenates were extracted in 0.1 M HCl, and the extracts were analyzed by RBA in three assays on separate days. Samples included naturally contaminated shellfish samples of different species collected from several geographic regions, which contained varying STX congener profiles due to their exposure to different PST-producing dinoflagellate species or differences in toxin metabolism: blue mussel (Mytilus edulis) from the U.S. east and west coasts, California mussel (Mytilus californianus) from the U.S. west coast, chorito mussel (Mytilus chiliensis) from Chile, green mussel (Perna canaliculus) from New Zealand, Atlantic surf clam (Spisula solidissima) from the U.S. east coast, butter clam (Saxidomus gigantea) from the west coast of the United States, almeja clam (Venus antiqua) from Chile, and Atlantic sea scallop (Plactopecten magellanicus) from the U.S. east coast. All samples were provided as whole animal homogenates, except Atlantic sea scallop and green mussel, from which only the hepatopancreas was homogenized. Among the naturally contaminated samples, five were blind duplicates used for calculation of RSDr. The interlaboratory RSDR of the assay for 21 samples tested in nine laboratories was 33.1%, yielding a HorRat value of 2.0. Removal of results for one laboratory that reported systematically low values resulted in an average RSDR of 28.7% and average HorRat value of 1.8. Intralaboratory RSDr based on five blind duplicate samples tested in separate assays, was 25.1%. RSDr obtained by individual laboratories ranged from 11.8 to 34.9%. Laboratories that are routine users of the assay performed better than nonroutine users, with an average RSDr of 17.1%. Recovery of STX from spiked shellfish homogenates was 88.1-93.3%. Correlation with the mouse bioassay yielded a slope of 1.64 and correlation coefficient (r(2)) of 0.84, while correlation with the precolumn oxidation HPLC method yielded a slope of 1.20 and an r(2) of 0.92. When samples were sorted according to increasing toxin concentration (microg STX diHCl equiv./kg) as assessed by the mouse bioassay, the RBA returned no false negatives relative to the 800 microg STX diHCl equiv./kg regulatory limit for shellfish. Currently, no validated methods other than the mouse bioassay directly measure a composite toxic potency for PST in shellfish. The results of this interlaboratory study demonstrate that the RBA is suitable for the routine determination of PST in shellfish in appropriately equipped laboratories.

Brevetoxin B is a clastogen in rats, but lacks mutagenic potential in the SP-98/100 Ames test.

Brevetoxins are polyether toxins produced by the dinoflagellate Karenia brevis that are released into the air and are known to cause respiratory hemorrhage in manatees and irritation in humans. Brevetoxin has been previously reported to cause DNA breakage and chromosomal aberrations in in vitro cell assays. The toxin is subject to epoxidation reaction and the formation of nucleic acid adducts in cultured lung fibroblasts and in lung tissue after intratracheal administration to rats. We have exposed rats intratracheally to brevetoxin B (45 microg/kg) and analyzed liver cells for DNA fragmentation using a comet assay. Brevetoxin B (PbTx2) treated rats showed a two to three-fold increase in the amount of DNA in the comet tails, indicating that brevetoxin has in vivo clastogenic activity. We next tested brevetoxin B for mutagenic activity using the Ames 98/100 mutagenesis assay. Brevetoxin B at concentrations from 0.064 to 200 microg/mL failed to cause histidine revertants. Oxidative metabolism of brevetoxin B resulting from Aroclor 1259-induced rat liver microsomes also failed to cause histidine revertants. Finally, direct application of the brevetoxin B epoxide (PbTx6) in the Ames 98/100 assay at concentrations from 0.064 to 200 microg/mL failed to induce histidine revertants. These studies indicate that brevetoxin B retains clastogenic activity after intratracheal administration to the rat. Although brevetoxin B has been shown to form nucleic acid adducts in the lung, neither brevetoxin B nor its epoxide metabolite has mutagenic potential as assessed by the Ames 98/100 test.

The role of domoic acid in abortion and premature parturition of California sea lions (Zalophus californianus) on San Miguel Island, California.

Domoic acid is a glutaminergic neurotoxin produced by marine algae such as Pseudo-nitzschia australis. California sea lions (Zalophus californianus) ingest the toxin when foraging on planktivorous fish. Adult females comprise 60% of stranded animals admitted for rehabilitation due to acute domoic acid toxicosis and commonly suffer from reproductive failure, including abortions and premature live births. Domoic acid has been shown to cross the placenta exposing the fetus to the toxin. To determine whether domoic acid was playing a role in reproductive failure in sea lion rookeries, 67 aborted and live-born premature pups were sampled on San Miguel Island in 2005 and 2006 to investigate the causes for reproductive failure. Analyses included domoic acid, contaminant and infectious disease testing, and histologic examination. Pseudo-nitzschia spp. were present both in the environment and in sea lion feces, and domoic acid was detected in the sea lion feces and in 17% of pup samples tested. Histopathologic findings included systemic and localized inflammation and bacterial infections of amniotic origin, placental abruption, and brain edema. The primary lesion in five animals with measurable domoic acid concentrations was brain edema, a common finding and, in some cases, the only lesion observed in aborted premature pups born to domoic acid-intoxicated females in rehabilitation. Blubber organochlorine concentrations were lower than those measured previously in premature sea lion pups collected in the 1970s. While the etiology of abortion and premature parturition was varied in this study, these results suggest that domoic acid contributes to reproductive failure on California sea lion rookeries.

An extract of Hydrilla verticillata and associated epiphytes induces avian vacuolar myelinopathy in laboratory mallards.

Avian vacuolar myelinopathy (AVM) is a neurological disease affecting bald eagles (Haliaeetus leucocephalus), American coots (Fulica americana), waterfowl, and other birds in the southeastern United States. The cause of the disease is unknown, but is thought to be a naturally produced toxin. AVM is associated with aquatic macrophytes, most frequently hydrilla (Hydrilla verticillata), and researchers have linked the disease to an epiphytic cyanobacterial species associated with the macrophytes. The goal of this study was to develop an extraction protocol for separating the putative toxin from a hydrilla-cyanobacterial matrix. Hydrilla samples were collected from an AVM-affected reservoir (J. Strom Thurmond Lake, SC) and confirmed to contain the etiologic agent by mallard (Anas platyrhynchos) bioassay. These samples were then extracted using a solvent series of increasing polarity: hexanes, acetone, and methanol. Control hydrilla samples from a reference reservoir with no history of AVM (Lake Marion, SC) were extracted in parallel. Resulting extracts were administered to mallards by oral gavage. Our findings indicate that the methanol extracts of hydrilla collected from the AVM-affected site induced the disease in laboratory mallards. This study provides the first data documenting for an "extractable" AVM-inducing agent.

Single-laboratory validation of the microplate receptor binding assay for paralytic shellfish toxins in shellfish.

A single-laboratory validation (SLV) study was conducted for the microplate receptor binding assay (RBA) for paralytic shellfish poisoning (PSP) toxins in shellfish. The basis of the assay is the competition between [3H]saxitoxin (STX) and STX in a standard or sample for binding to the voltage dependent sodium channel. A calibration curve is generated by the addition of 0.01-1000 nM STX, which results in the concentration dependent decrease in [3H]STX-receptor complexes formed and serves to quantify STX in unknown samples. This study established the LOQ, linearity, recovery, accuracy, and precision of the assay for determining PSP toxicity in shellfish extracts, as performed by a single analyst on multiple days. The standard curve obtained on 5 independent days resulted in a half-maximal inhibition (IC50) of 2.3 nM STX +/- 0.3 (RSD = 10.8%) with a slope of 0.96 +/- 0.06 (RSD = 6.3%) and a dynamic range of 1.2-10.0 nM. The LOQ was 5.3 microg STX equivalents/100 g shellfish. Linearity, established by quantification of three levels of purified STX (1.5, 3, and 6 nM), yielded an r2 of 0.97. Recovery from mussels spiked with three levels (40, 80, and 120 microg STX/100 g) averaged 121%. Repeatability (RSD(r)), determined on six naturally contaminated shellfish samples on 5 independent days, was 17.7%. A method comparison with the AOAC mouse bioassay yielded r2 = 0.98 (slope = 1.29) in the SLV study. The effects of the extraction method on RBA-based toxicity values were assessed on shellfish extracted for PSP toxins using the AOAC mouse bioassay method (0.1 M HCI) compared to that for the precolumn oxidation HPLC method (0.1% acetic acid). The two extraction methods showed linear correlation (r2 = 0.99), with the HCl extraction method yielding slightly higher toxicity values (slope = 1.23). A similar relationship was observed between HPLC quantification of the HCI- and acetic acid-extracted samples (r2 = 0.98, slope 1.19). The RBA also had excellent linear correlation with HPLC analyses (r2 = 0.98 for HCl, r2 = 0.99 for acetic acid), but gave somewhat higher values than HPLC using either extraction method (slope = 1.39 for HCl extracts, slope = 1.32 for acetic acid). Overall, the excellent linear correlations with the both mouse bioassay and HPLC method and sufficient interassay repeatability suggest that the RBA can be effective as a high throughput screen for estimating PSP toxicity in shellfish.

Identification of yessotoxin in mussels from the Caucasian Black Sea Coast of the Russian Federation.

The toxin load of shellfish hepatopancreas harvested from the Caucasian Black Sea Coast of the Russian Federation was investigated. The majority of the toxin load was shown to be yessotoxin (YTX), 45-hydroxy-yessotoxin (45-OH-YTX), and homoyessotoxin (homoYTX). Concurrent with the mussel intoxication, the dinoflagellates Lingulodinium polyedrum and Gonyaulax spinifera were found in high concentrations.

Saxitoxin puffer fish poisoning in the United States, with the first report of Pyrodinium bahamense as the putative toxin source.

BACKGROUND: From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. METHODS: We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. RESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 microg STX equivalents (eq)/100 g tissue (action level, 80 microg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. CONCLUSIONS: Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.

Brevetoxicosis: red tides and marine mammal mortalities.

Potent marine neurotoxins known as brevetoxins are produced by the 'red tide' dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels.

Multi-Laboratory Study of Five Methods for the Determination of Brevetoxins in Shellfish Tissue Extracts.

A thirteen-laboratory comparative study tested the performance of four methods as alternatives to mouse bioassay for the determination of brevetoxins in shellfish. The methods were N2a neuroblastoma cell assay, two variations of the sodium channel receptor binding assay, competitive ELISA, and LC/MS. Three to five laboratories independently performed each method using centrally prepared spiked and naturally incurred test samples. Competitive ELISA and receptor binding (96-well format) compared most favorably with mouse bioassay. Between-laboratory relative standard deviations (RSDR) ranged from 10 to 20% for ELISA and 14 to 31% for receptor binding. Within-laboratory (RSDr) ranged from 6 to 15% for ELISA, and 5 to 31% for receptor binding. Cell assay was extremely sensitive but data variation rendered it unsuitable for statistical treatment. LC/MS performed as well as ELISA on spiked test samples but was inordinately affected by lack of toxin-metabolite standards, uniform instrumental parameters, or both, on incurred test samples. The ELISA and receptor binding assay are good alternatives to mouse bioassay for the determination of brevetoxins in shellfish.

Permanent expression of a cyclin B homologue in the cell cycle of the dinoflagellate Karenia brevis.

The eukaryotic cell cycle is driven by a set of cyclin-dependent kinases associated with their regulatory partners, the cyclins, which confer activity, substrate specificities and proper localization of the kinase activity. We describe the cell cycle of Karenia brevis and provide evidence for the presence of a cyclin B homologue in this dinoflagellate using two antibodies with different specificities. This cyclin B homologue has an unusual behavior, since its expression is permanent and it has a cytoplasmic location throughout the cell cycle. There is no evidence for translocation to the nucleus during mitosis. However, it appears also to be specifically bound to the nucleolus throughout the cell cycle. The permanent expression and the cytoplasmic localization during mitosis of this cyclin B homologue is similar to p56, a cyclin B homologue previously described in a different species of dinoflagellate, Crypthecodinium cohnii. Here we discuss this unusual behavior of the cyclin B homologue in dinoflagellates, its relationship to the unusual characteristics of dinomitosis, and its potential implications regarding the evolution of cell cycle regulation among eukaryotes.

Evidence for cAMP-dependent protein kinase in the dinoflagellate, Amphidinium operculatum.

A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.