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PubMed2001

Inhibition of egg hatching success and larvae survival of the scallop, Chlamys farreri, associated with exposure to cells and cell fragments of the dinoflagellate Alexandrium tamarense.

We report an apparently novel toxic effect of the dinoflagellate Alexandrium tamarense, manifested by inhibition of the egg hatching success of the scallop, Chlamys farreri. The hatching rate of C. farreri approached only 30% of controls when its fertilised eggs were exposed for 36h to A. tamarense cells or cellular fragments at a concentration of 100 cells/ml, and the hatching rate was just 5% after exposure to A. tamarense of 500 cells/ml. Similar exposures of the fertilised scallop eggs to two other algal species, the diatom Phaeodactylum tricornutum and the raphidophyte Heterosigma carterae, resulted in no such toxicity or inhibitory effects. Likewise, exposure of eggs to standard STX toxin, as well as to A. tamarense cell contents (supernant of re-suspended algal cells following ultrasonication and centrifugation), did not elicit this inhibitory response. However, exposure of the scallop eggs to cell cultures, intact algal cells, or cell fragments of A. tamarense produced marked toxicity. The alga also influenced larvae at early D-shape stage of scallop. The survival rates began to decrease significantly after exposed for 6 days at concentration of 3000 cells/ml and above; no larvae could survive after 14-day exposure to A. tamarense at 10,000 cells/ml or 20-day at 5000 cells/ml. The results indicated the production of novel substances from A. tamarense which can cause adverse effects on egg hatching and survival of the scallop larvae. The experiment also found that the developmental stages before blastula was the developmental period most sensitive to the A. tamarense toxin(s) and the alga at early exponential stage had the strongest effect on egg hatching comparing with other growth phases. The adverse effect of A. tamarense on early development of scallops may cause decline of shellfish population and may have further impact on marine ecosystem.

Animals↗

PubMed2001

The validity of two HPLC methods and a colorimetric PP2A assay related to the mouse bioassay in quantification of diarrhetic toxins in blue mussels (Mytilus edulis).

Validity of two HPLC methods and a PP2A assay in relation to the mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins was evaluated. The mouse bioassay for DSP toxins was performed on a total of 177 mussel samples from the Sognefjord, Norway, using diethyl ether in the final step of extraction. For fluorimetric HPLC analyses, either 4-bromomethyl-7-methoxycoumarin (BrMMC) or 9-anthryl diazomethane (ADAM) were used for analysis of 48 and 118 of the samples, respectively. The colorimetric PP2A inhibition assay was performed on all 177 samples that were analysed with the mouse bioassay. When comparing the HPLC-BrMMC, the HPLC-ADAM and the PP2A assays with the mouse bioassay, cut off values of < or =4, 5 and 6 microg okadaic acid (OA) equivalents (eq.)/5 g digestive gland (DG) was used. With reference to the results from the mouse bioassay, the total number of failure and correct classification by HPLC-ADAM and the PP2A method was compared for the three cut off values. No significant differences between the methods were detected. However, all differences were found in favour of HPLC-ADAM. All three methods could replace the mouse bioassay in detecting levels of diarrhetic toxins approved internationally for safe consumption of mussels. However, HPLC-ADAM seems to be the method of choice.

Animals↗

PubMed2001

Dynamics of C2 toxin and chlorophyll-a formation in the dinoflagellate Alexandrium tamarense during large scale cultivation.

The production of paralytic shellfish toxins (PSTs) by the dinoflagellate Alexandrium tamarense ATCI01, a toxigenic strain isolated from South China coastal waters, was studied in batch cultures in relatively large volumes (20l). Under nutrient-replete conditions, this strain produced C2 toxin (C2T) as a predominant PST. In a 15-day production culture, phosphate was depleted by day 4, the stationary phase began at day 6, and the toxin productivity peaked at day 10, in which the cell content of C2T reached 76 fmol per cell. Much of the toxin was produced after the depletion of phosphate in the medium suggesting that C2T is a secondary metabolite. Aeration with small bubbles was useful in increasing cell mass and toxin yield. Chlorophyll-a (Chl-a) was formed in algal cells until the culture entered the stationary phase, after which Chl-a began to disappear rapidly from the culture while the C2T content continued to rise. These results suggest a metabolic relationship between Chl-a and C2T.

Air↗

PubMed2002

Evaluation of marine biotoxin's accumulation by Acanthocardia tuberculatum from Algarve, Portugal.

Acanthocardia tuberculatum is a bivalve mollusc that presents recurrent problems of paralytic shellfish poisoning (PSP) contamination in the Mediterranean coasts of Spain and Morocco. Although not commercially exploited from the Portuguese south coast, it represents an alternative for reducing the harvest pressure on species presently exploited. Evaluation of accumulation of marine biotoxins was carried out by HPLC in this species, harvested during a campaign carried out in April 2001 aimed at evaluating bivalve's resources that covered the entire Portuguese south coast.PSP toxins were studied by automated pre-column oxidation. Toxins were found in specimens from all stations, but always under the regulatory limit of 80 microg STX eq./100g. Saxitoxin and decarbamoyl-saxitoxin were the only PSP toxins unambiguously identified. In commercially exploited species, saxitoxin and analogues were not detected. The amnesic toxin domoic acid was found, but in levels similar to those found in species commercially exploited, and always under the regulatory limit of 20 microg/g. A fast elimination, and not a prolonged retention, of domoic acid seems to occur in A. tuberculatum in view of the levels close to or higher than 20 microg/g found in commercial bivalves harvested the two preceding months. The diarrhoeic toxin okadaic acid was found at trace levels much under the allowable level, similar to what was happening with other species harvested during the same period.

Animals↗

PubMed2002

A tetrodotoxin-binding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties.

The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein.

Amino Acids↗

PubMed2002

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.

Animals↗

PubMed2003

GTX(4) imposters: characterization of fluorescent compounds synthesized by Pseudomonas stutzeri SF/PS and Pseudomonas/Alteromonas PTB-1, symbionts of saxitoxin-producing Alexandrium spp.

Saxitoxins, the etiological agent of paralytic shellfish poisoning, are synthesized by dinoflagellates and cyanobacteria. Several reports indicate that bacteria are capable of saxitoxin synthesis. Two bacterial strains were isolated from saxitoxin-producing dinoflagellates, Alexandrium tamarense and A. lusitanicum (=Alexandrium minutum), and grown under a variety of culture conditions including those previously reported to induce saxitoxin synthesis in bacteria. Five fluorescent compounds were accumulated by the bacteria that had HPLC-FLD retention times similar to a reference standard of GTX(4), one of the saxitoxin congeners. However, we were unable to detect GTX(1), the epimeric partner of GTX(4), in the bacterial samples. The GTX(4) standard was hydrolyzed by NaOH/heat treatment but four of the bacterial compounds were stable. Unlike GTX(4), none of the five bacterial compounds were detectable by HPLC-FLD following electrochemical oxidation. The fluorescence emission spectrum of each of the five bacterial compounds was unique and readily discernable from the spectrum of GTX(4). None of the samples containing the putative GTX(4) toxin yielded positive results when analyzed by a 3H-saxitoxin receptor-binding assay for saxitoxin-like activity. We cannot rule out the possibility that these bacteria produce saxitoxins, however, our data clearly demonstrate that they accumulate at least five different fluorescent compounds that could be easily mistaken for GTX(4). We conclude that these five fluorescent compounds are GTX(4) imposters and that fluorescence scanning and chemical/heat stability should, at a minimum, be incorporated into HPLC-FLD protocols for identification of saxitoxins.

Animals↗

PubMed2003

Paralytic toxins in three new gastropod (Olividae) species implicated in food poisoning in southern Taiwan.

The toxins in the new gastropods Oliva miniacea, O. mustelina and O. nirasei implicated in a food paralytic poisoning incident in South Taiwan in February 2002 were studied. It was found that the three species of gastropods contained moderate amounts of toxin in edible portion only, and the highest toxicity score was 18 MU/g for O. miniacea, 10 MU/g for O. mustelina, and 27 MU/g for O. nirasei. The toxin was partially purified from the toxic specimens of each species by ultrafiltration using a YM-1 membrane, followed by chromatography on Bio-Gel P-2 column. Analyses by HPLC, GC-MS and LC-MS showed that the toxin from O. miniacea, O. nirasei and O. mustelina contained TTX, and related compounds 4-epi TTX and anhydro-TTX. The paralytic shellfish poisons were not found.

Animals↗

PubMed2003

Complex yessotoxins profile in Protoceratium reticulatum from north-western Adriatic sea revealed by LC-MS analysis.

While the occurrence of yessotoxin (YTX) has been reported worldwide from Protoceratium reticulatum, the biogenetic origin of some YTX analogues is still unknown, thus raising an issue whether they are metabolites of YTX formed in mussels or true products of different dinoflagellate species. Findings reported herein suggest that P. reticulatum from the north-western Adriatic sea is responsible for production, together with YTX (1), of homoYTX (2), 45-OHYTX (3), carboxyYTX (5), and noroxoYTX (7). YTX and its analogues have been determined by high performance liquid chromatography coupled with electrospray ion trap mass spectrometry (HPLC-MS and HPLC-MSn experiments). The result is the first to confirm production of these YTX analogues from this species and indicate it as a producing organism of homoYTX, 45-OHYTX, carboxyYTX, and noroxoYTX found in shellfish.

Animals↗

PubMed2003

Accumulation of gliotoxin, a cytotoxic mycotoxin from Aspergillus fumigatus, in blue mussel (Mytilus edulis).

A strain of Aspergillus fumigatus has been isolated from sediments of a mussel bed. When cultured in hyper saline conditions (with sea-water), it produces a cytotoxic and immunosuppressive toxin, gliotoxin, which is excreted in an exudate. In order to know if this toxin could represent a risk for shellfish consumers, an experiment of bioaccumulation of gliotoxin in mussel has been carried out. After 6 days of contamination, toxin was accumulated in the meat of the mussels, at a level up to 2.9 microg/mg of extract weight, with a mode of contamination different to the classical digestive process described for a majority of marine toxins, but similar to the contamination mode of domoic acid.

Animals↗

PubMed2003

Cylindrospermopsin occurrence in two German lakes and preliminary assessment of toxicity and toxin production of Cylindrospermopsis raciborskii (Cyanobacteria) isolates.

Cylindrospermopsis raciborskii, a freshwater cyanobacterium of tropical origin, is not only increasingly found in (sub) tropical water bodies, but also in temperate regions. Since this species may produce potent toxins such as cylindrospermopsin (CYN) and paralytic shellfish poisons, its massive occurrence in water bodies used as drinking water sources or for recreation is of major concern. The proliferation of C. raciborskii in German water bodies has been documented for the past decade. We investigated the occurrence of CYN in field populations and isolates of C. raciborskii from two lakes, and assessed the toxicity of culture isolates using the mouse bioassay, primary rat hepatocytes and human derived cell lines. We show for the first time the occurrence of CYN in German water bodies. None of seven isolates of C. raciborskii contained CYN, however, all isolates were toxic to primary rat hepatocytes, human hepatoblastoma (HEP-G2) and human colon adenocarcinoma (CACO-2) cells. Methanolic extracts were more toxic than aqueous extracts. Three isolates tested in the mouse bioassay were toxic at a concentration of 800 mg kg(-1) showing liver and spleen damage and inflammation of the intestine. These results give strong evidence that the German isolates of C. raciborskii contain currently not identified or unknown toxins.

Alkaloids↗

PubMed1997

Determination of okadaic acid by micellar electrokinetic chromatography with ultraviolet detection.

Micellar electrokinetic chromatography (MEKC) with ultraviolet (UV) detection was applied for the determination of non-derivatized phycotoxins associated with diarrhoetic shellfish poisoning. A detection limit for 40 pg of okadaic acid (OA) was achieved. The UV intensities of this toxin measured at 200 nm showed good linearity in the range 40-640 pg. OA was detected in mussels spiked with 10 ng/g whole tissue. The presence of OA and dinophysistoxin-2 was observed in the crude extract of the dinoflagellate Prorocentrum lima.

Animals↗

PubMed1997

A rapid tissue culture assay for the detection of okadaic acid and related compounds in mussels.

Okadaic acid, the main toxin of the diarrhoetic shellfish poisoning (DSP) group, shows high cytotoxicity to Buffalo green monkey (BGM) kidney cell cultures after different times according to concentration. A rapid method of detecting the presence of okadaic acid and related compounds in contaminated mussels by means of a cytotoxicity test is presented. The method is based on direct microscopic observation of toxin-induced morphological changes in BGM cell cultures. A high correlation (r = 0.950; P < 0.001) was found between the official mouse bioassay and the cytotoxicity test conducted on naturally contaminated samples of Mytilus galloprovincialis.

Animals↗

PubMed1997

Yessotoxin in mussels of the northern Adriatic Sea.

This study investigated the composition of diarrhoetic shellfish toxins in the hepatopancreas of mussels from the northern Adriatic Sea. The major toxins were shown to be yessotoxin, identified by its chromatographic properties and spectral data, and okadaic acid, detected both by fluorometric high-performance liquid chromatography and by comparison of its spectral properties with those of an authentic sample.

Animals↗

PubMed1997

Specificity of the test based on modification of cell morphology for detection of lipophilic inhibitors of protein phosphatases.

The test based on morphological changes in KB cells was assayed with different toxins. Only lipophilic inhibitors of protein phosphatases, such as okadaic acid or calyculin A, induced visible changes in cell morphology. The activity of contaminated mussel extracts on KB cells was evaluated comparatively by direct interpretation of morphological changes and by a colorimetric method estimating the number of viable cells after staining. The latter technique revealed interferences (not detected by the former) with mussel cytotoxins. These results show that the technique, based on determination of the minimal active concentration of toxic extracts inducing morphological changes, is more specific, faster and preferable to the determination of IC50 for the detection of protein phosphatase inhibitors in shellfish.

Animals↗

PubMed1997

Bioaccumulation and clearance of microcystins from salt water mussels, Mytilus edulis, and in vivo evidence for covalently bound microcystins in mussel tissues.

Over a period of 3 days saltwater mussels, Mytilus edulis, were fed a cyanobacteria, Microcystis aeruginosa, that contained a high concentration of microcystins. The mussels were killed on a periodic basis over the course of 2 months. Mussels were also collected at two sites were high levels of microcystins in tissues had been noted. A strategy based on the chemically unique nature of the C20 beta-amino acid, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid (Adda), portion of the microcystins was used in conjunction with a protein phosphatase (PPase) assay to analyse for both covalently bound microcystins and free microcystins in the mussel tissues. The mussel PPase assay results were compared with the Lemieux oxidation gas chromatography-mass spectrometry (GCMS) analysis. Less than 0.1% of the total microcystin burden in the mussel tissue was found to be extractable with MeOH. Thus, direct evidence was provided for the existence of covalently bound microcystins in mussel tissues in vivo. The mussels rapidly cleared the covalently bound microcystins when transferred to untreated seawater. Within 4 days the total microcystin burden dropped from a high of 336.9 (+/- 45.8) micrograms/g wet tissue to 11.3 (+/- 2.6) micrograms/g. After 4 days postexposure until completion of the experiment the total levels remained below the detection limits of the GCMS method. The levels of free microcystins, extracted with MeOH and detected by the PPase assay, fell from 204 ng/g wet tissue to a residual 14 ng/g over a 53 day postexposure period. Presumably the bound microcystin present in the mussel tissue exists as a covalent complex with the PP-1 and PP-2A enzymes. We conclude that in any shellfish monitoring program it is the total tissue microcystin burden that needs to be considered.

Animals↗

PubMed1998

Okadaic acid production from the marine dinoflagellate Prorocentrum belizeanum Faust isolated from the Belizean coral reef ecosystem.

A species of epiphytic Prorocentrum (Dinophyta, Prorocentrales, collected from the macroalga Dictyota dichotoma, was brought into culture. Based on morphological characteristics, this isolate was identified as Prorocentrum belizeanum. Analysis of the culture extract using high-performance liquid chromatography and liquid chromatography-mass spectrometry indicated the production of okadaic acid, a toxic polyether linked with the human diseases, diarrhetic shellfish poisoning and ciguatera fish poisoning.

Animals↗

PubMed1998

Combined use of analytical high-performance liquid chromatography and cell morphology transformation assay to detect new protein phosphatase inhibitors of okadaic acid type.

The search for new protein phosphatase inhibitors in shellfish contaminated by toxin-producing dinoflagellates generally relies on preliminary separation techniques followed by biological tests. To detect such substances without purifying them initially, we developed an approach based on a correlation of the results of two different analytical techniques applied to toxic extracts: high-performance liquid chromatography after derivation of the toxins and the cell morphology transformation assay on KB cells. Application of this protocol to stored frozen mussels showed a decrease in okadaic acid concentration during storage, with formation of degradation derivatives, some of which possessed notable protein phosphatase inhibition activity.

Animals↗