Diarrheic Shellfish Poisoning Toxins Research Articles

Transcriptomic Analysis of the Response of the Toxic Dinoflagellate Prorocentrum lima to Phosphorous Limitation.

Some dinoflagellates cause harmful algal blooms, releasing toxic secondary metabolites, to the detriment of marine ecosystems and human health. Phosphorus (P) is a limiting macronutrient for dinoflagellate growth in the ocean. Previous studies have been focused on the physiological response of dinoflagellates to ambient P changes. However, the whole-genome's molecular mechanisms are poorly understood. In this study, RNA-Seq was utilized to compare the global gene expression patterns of a marine diarrheic shellfish poisoning (DSP) toxin-producing dinoflagellate, Prorocentrum lima, grown in inorganic P-replete and P-deficient conditions. A total of 148 unigenes were significantly up-regulated, and 30 unigenes were down-regulated under 1/4 P-limited conditions, while 2708 unigenes were significantly up-regulated, and 284 unigenes were down-regulated under 1/16 P-limited conditions. KEGG enrichment analysis of the differentially expressed genes shows that genes related to ribosomal proteins, glycolysis, fatty acid biosynthesis, phagosome formation, and ubiquitin-mediated proteolysis are found to be up-regulated, while most of the genes related to photosynthesis are down-regulated. Further analysis shows that genes encoding P transporters, organic P utilization, and endocytosis are significantly up-regulated in the P-limited cells, indicating a strong ability of P. lima to utilize dissolved inorganic P as well as intracellular organic P. These transcriptomic data are further corroborated by biochemical and physiological analyses, which reveals that under P deficiency, cellular contents of starch, lipid, and toxin increase, while photosynthetic efficiency declines. Our results indicate that has P. lima evolved diverse strategies to acclimatize to low P environments. The accumulation of carbon sources and DSP toxins could provide protection for P. lima to cope with adverse environmental conditions.

Responses of ABCB and ABCC transporters to the toxic dinoflagellate Prorocentrum lima in the mussel Perna viridis

Bivalve mollusks can accumulate diarrheic shellfish poisoning (DSP) toxins through filter-feeding, but they exhibit some resistance to the toxins. Previous studies have suggested that the ABC transporters may have an important role in the resistance to DSP toxins, but comprehensive studies are lacking. In this study, we comprehensively analyzed the distribution of ABC transporters in the mussel Perna viridis, and observed responses of ABCB and ABCC transporters to the DSP toxins-producing dinoflagellate Prorocentrum lima. Total 39 members of ABC transporters were identified in P. viridis, including 3 full PvABCBs, 3 half PvABCBs, and 7 PvABCCs transporters. We found that PvABCBs and PvABCCs subfamilies were expressed in hemocytes, gills and digestive gland with some difference, especially in hemocytes. After exposure to P. lima, PvABCBs and PvABCCs displayed different expression changes in different tissues. The short-term (3 h) exposure to P. lima induced the transcription of PvABCB1_like1, PvABCB6, PvABCC1, PvABCC1_like and PvABCC1/3, and the longer-term (96 h) exposure increased the transcription of PvABCB1, PvABCB1_like, PvABCB10, PvABCC1 and PvABCC1_like1 in gills and PvABCC10 in digestive gland. These results suggest that different types of PvABCBs and PvABCCs in P. viridis may contribute to the detoxification of DSP toxins in different tissues at different time after exposure to DSP toxins. Our finding provides new evidence for further understanding the role of ABC transporters in the tolerance of mussel to DSP toxins.

Responses of JNK signaling pathway to the toxic dinoflagellate Prorocentrum lima in the mussel Perna viridis

Diarrheic shellfish poisoning (DSP) toxins are widely distributed over the world, causing diarrhea, vomiting, and even tumor in human. However, bivalves, the main carrier of the DSP toxins, have some tolerant mechanisms to DSP toxins, though it remains unclear. In this study, we scrutinized the role of Jun N-terminal kinases (JNK) in tolerance of DSP toxins and the relationship between JNK, apoptosis and nuclear factor E2-related factor/antioxidant response element (Nrf2/ARE) pathways. We found that the phosphorylated level of JNK protein was significantly increased both in hemocytes (6 h) and gills (3 h) of the mussel Perna viridis after short-term exposure to DSP toxins-producing dinoflagellate Prorocentrum lima. Exposure of P. lima induced oxidative stress in mussels. Hemocytes and gills displayed different sensitivities to the cytotoxicity of DSP toxins. Exposure of P. lima activated caspase-3 and induced apoptosis in gills but did not induce caspase-3 and apoptosis in hemocytes. The short-term exposure of P. lima could activate Nrf2/ARE signaling pathway in hemocytes (6 h), while longer-term exposure could induce glutathione reductase (GR) expression in hemocytes (96 h) and glutathione-S-transferases (GST) in gills (96 h). Based on the phylogenetic tree of Nrf2, Nrf2 in P. viridis was closely related to that in other mussels, especially Mytilus coruscus, but far from that in Mus musculus. The most likely phosphorylated site of Nrf2 in the mussels P. viridis is threonine 504 for JNK, which is different from that in M. musculus. Taken all together, the tolerant mechanism of P. viridis to DSP toxins might be involved in JNK and Nrf2/ARE signaling pathways, and JNK play a key role in the mechanism. Our findings provide a new clue to further understand tolerant mechanisms of bivalves to DSP toxins.

DSP Toxin Distribution across Organs in Mice after Acute Oral Administration.

Okadaic acid (OA) and its main structural analogs dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine lipophilic phycotoxins distributed worldwide that can be accumulated by edible shellfish and can cause diarrheic shellfish poisoning (DSP). In order to study their toxicokinetics, mice were treated with different doses of OA, DTX1, or DTX2 and signs of toxicity were recorded up to 24 h. Toxin distribution in the main organs from the gastrointestinal tract was assessed by liquid chromatography-mass spectrometry (LC/MS/MS) analysis. Our results indicate a dose-dependency in gastrointestinal absorption of these toxins. Twenty-four hours post-administration, the highest concentration of toxin was detected in the stomach and, in descending order, in the large intestine, small intestine, and liver. There was also a different toxicokinetic pathway between OA, DTX1, and DTX2. When the same toxin doses are compared, more OA than DTX1 is detected in the small intestine. OA and DTX1 showed similar concentrations in the stomach, liver, and large intestine tissues, but the amount of DTX2 is much lower in all these organs, providing information on DSP toxicokinetics for human safety assessment.

Associated Bacteria and Their Effects on Growth and Toxigenicity of the Dinoflagellate Prorocentrum lima Species Complex From Epibenthic Substrates Along Mexican Coasts

The dinoflagellate genus Prorocentrum is globally represented by a wide variety of species found upon benthic and/or epiphytic substrates. Many epibenthic Prorocentrum species produce lipophilic polyether toxins, some of which act as potent protein phosphatase inhibitors and tumor-promoters associated with Diarrheic Shellfish Poisoning (DSP). Most members of the P. lima species complex (PLSC) commonly found in the tropics and sub-tropics are toxigenic. Epiphytic and planktonic bacteria co-occur with toxigenic Prorocentrum but reciprocal allelochemical interactions are under-investigated. The aim of the present study was to identify the culturable bacteria collected together with isolates of the PLSC from seagrass (Thalassia testudinum) and macroalgae (Laurencia sp. and Sargassum sp.) along tropical Mexican coasts, and to explore potential species interactions with selected isolates. Twenty-one bacterial genera belonging to Proteobacteria, Actinobacteria and Bacteroidetes were identified by amplification of the 16S rRNA gene marker from nine clonal Prorocentrum cultures, with γ-proteobacteria comprising the dominant class. A positive correlation was found between the bacterial genera associated with two Prorocentrum clones and the esterified toxin analog DTX1a-D8, but there was no apparent correlation between the other PLSC clones and their associated bacteria with the other five DSP toxins detected. No bacteriostatic or allelochemical response was found for cell- and culture medium extracts of five Prorocentrum isolates assayed for bioactivity against Staphylococcus sp. DMBS2 and Vibrio sp. HEL66. Bulk cell-washing of Prorocentrum PA1, followed by growth with antibiotics, was only effective in reducing bacterial load in the initial growth stages, but did not yield axenic cultures or lower bacterial cell densities throughout the culture cycle. Antibiotic treatment did not impair growth or survival of the dinoflagellate, or apparently affect DSP toxin production. There was no significant correlation between Prorocentrum cell volume, growth rate, bacterial cell counts or cellular toxin concentration over the entire time-series culture cycle. Benthic Prorocentrum and associated bacterial communities comprise highly diverse and characteristic microbiomes upon substrates, and among compartments in culture, but this study provides little evidence that allelochemical interactions among Prorocentrum cells and associated bacteria originating from epibenthic substrates play a definable role in growth and toxigenicity.

Effects of the DSP-toxic dinoflagellate Dinophysis acuta on clearance and respiration rate of the blue mussel, Mytilus edulis.

Diarrheic Shellfish Poisoning toxins (DST) are a severe health risk to shellfish consumers and can be a major problem for the shellfish industry. Bivalve molluscs can accumulate DST via ingestion of toxic dinoflagellates like Dinophysis spp., which are the most prominent producers of DST. The effects of DST-containing dinoflagellate Dinophysis acuta on bivalve clearance and respiration rate were investigated in the blue mussel (Mytilus edulis) exposed to different algal densities in a controlled laboratory study. Results showed that M. edulis exposed to D. acuta displayed a reduced clearance rate compared to M. edulis exposed to equivalent bio-volumes of the non-toxic cryptophyte Rhodomonas salina. Furthermore, M. edulis ceased to feed on D. acuta after 1 to 4 h, depending on D. acuta densities. The quickest response was observed at the highest densities of D. acuta. The estimated total amount of DST accumulated in the M. edulis exceeded the regulatory limit for human consumption and furthermore, intoxication of the M. edulis seemed to occur faster at high cell toxicity rather than at high cell density. However, respiration rates were, similar, irrespective of whether M. edulis were fed single diets of R. salina, D. acuta or a mixed diet of both algal species. In conclusion, the DST-containing D. acuta had a severe negative effect on the clearance of M. edulis, which can affect the conditions of the M. edulis negatively. Hence, DST may cause low quality M. edulis, due to reduced feeding when exposed to DST-containing D. acuta.

De novo transcriptome analysis of the mussel Perna viridis after exposure to the toxic dinoflagellate Prorocentrum lima.

Diarrheic shellfish poisoning (DSP) toxins are produced by harmful microalgae and accumulate in bivalve mollusks, causing various toxicity. These toxic effects appear to abate with increasing DSP concentration and longer exposure time, however, the underlying mechanisms remain unclear. To explore the underlying molecular mechanisms, de novo transcriptome analysis of the digestive gland of Perna viridis was performed after Prorocentrum lima exposure. RNA-seq analysis showed that 1886 and 237 genes were up- and down-regulated, respectively after 6h exposure to P. lima, while 265 genes were up-regulated and 217 genes were down-regulated after 96h compared to the control. These differentially expressed genes mainly involved in Nrf2 signing pathways, immune stress, apoptosis and cytoskeleton, etc. Combined with qPCR results, we speculated that the mussel P. viridis might mainly rely on glutathione S-transferase (GST) and ABC transporters to counteract DSP toxins during short-term exposure. However, longer exposure of P. lima could activate the Nrf2 signaling pathway and inhibitors of apoptosis protein (IAP), which in turn reduced the damage of DSP toxins to the mussel. DSP toxins could induce cytoskeleton destabilization and had some negative impact on the immune system of bivalves. Collectively, our findings uncovered the crucial molecular mechanisms and the regulatory metabolic nodes that underpin the defense mechanism of bivalves against DSP toxins and also advanced our current understanding of bivalve defense mechanisms.

Systems-level analysis of metabolic mechanism following nitrogen limitation in benthic dinoflagellate Prorocentrum lima

Prorocentrum lima is a typical diarrheic shellfish poisoning (DSP) toxin-producing benthic dinoflagellate usually found attached to or associated with macrophytes, floating detritus, debris, or other substrates. In this paper, to gain a deeper understanding of the molecular and cellular responses to N limitation in P. lima, transcriptome profiling was performed. We found that most genes related to photosynthesis, porphyrin and chlorophyll metabolism were down-regulated following N limitation, while some genes concerning fatty acid biosynthesis, starch synthesis and ubiquitin pathway were up-regulated. Meanwhile, ABC transports ABCB1 and ABCG2 and glutathione S-transferase were up-regulated under N-limited conditions. These transcriptomic data were further corroborated by the biochemical analyses which revealed that starch, lipid content was increased, while photosynthetic efficiency was decreased. In addition, TEM analysis demonstrated that chloroplasts appeared smaller and were less abundant, while a major fraction of the cell volume was occupied by lipid bodies and starch granules, and thylakoid was twisted in the cells. Taking together, we proposed that N limitation could induce the accumulation of neutral lipid and starch in P. lima cells for carbon fixation trough recycling chloroplast membranes by autophagy. ABCB and ABCG transporters might be involved in the transport of DSP toxins. Our findings might provide global information for the response or adaptation of P. lima to N limitation.

Lipophilic marine biotoxins SERS sensing in solutions and in mussel tissue

To detect and recognise three structurally related marine biotoxins responsible for the diarrheic shellfish poisoning (DSP) symptom, namely okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) respectively, as well as the structurally different yessotoxin (YTX), we developed a novel surface-enhanced micro-Raman scattering (micro-SERS) approach to investigate for the first time their micro-SERS signalling in solution and jointly analysed them in conjunction with the normal and toxic mussel tissue. YTX provided the main SERS feature surprisingly similar to DTX-1 and DTX-2, suggesting similar molecular adsorption mechanism with respect to the AgNPs. A fingerprint SERS band at 1017 cm−1 characteristic for the C-CH3 stretching in DTX-1 and DTX-2 and absent in OA SERS signal, allowed direct SERS discrimination of DTX-1,2 from OA. In acid form or as dissolved potassium salt, OA showed reproducible SERS feature for 0.81 μM to 84.6 nM concentrations respectively, while its ammonium salt slightly changed the overall SERS signature. The inherently strong fluorescence of the shellfish tissue, which hampers Raman spectroscopy analysis, further increases when toxins are present in tissue. Through SERS, tissue fluorescence is partially quenched. Artificially intoxicated mussel tissue with DSP toxins and incubated with AgNPs allowed direct SERS evidence of the toxin presence, opening a novel avenue for the in situ shellfish tracking and warning via micro-SERS. Natural toxic tissue containing 57.91 μg kg−1 YTX (LC-MS confirmed) was micro-SERS assessed to validate the new algorithm for toxins detection. We showed that a portable Raman system was able to reproduce the lab-based SERS results, being suitable for in situ raw seafood screening. The new approach provides an attractive, faster, effective and low-cost alternative for seafood screening, with economic, touristic and sustainable impact in aquaculture, fisheries, seafood industry and consumer trust.

Toxic Action Reevaluation of Okadaic Acid, Dinophysistoxin-1 and Dinophysistoxin-2: Toxicity Equivalency Factors Based on the Oral Toxicity Study

Background/Aims: Okadaic acid (OA) and the structurally related compounds dinophysistoxin-1 (DTX1) and dinophysistoxin-2 (DTX2) are marine phycotoxins that cause diarrheic shellfish poisoning (DSP) in humans due to ingestion of contaminated shellfish. In order to guarantee consumer protection, the regulatory authorities have defined the maximum level of DSP toxins as 160 µg OA equivalent kg^-1 shellfish meat. For risk assessment and overall toxicity determination, knowledge of the relative toxicities of each analogue is required. In absence of enough information from human intoxications, oral toxicity in mice is the most reliable data for establishing Toxicity Equivalence Factors (TEFs). Methods: Toxins were administered to mice by gavage, after that the symptomatology and mice mortality was registered over a period of 24 h. Organ damage data were collected at necropsy and transmission electron microscopy (TEM) was used for ultrastructural studies. Toxins in urine, feces and blood were analyzed by HPLC-MS/MS. The evaluation of in vitro potencies of OA, DTX1 and DTX2 was performed by the protein phosphatase 2A (PP2A) inhibition assay. Results: Mice that received DSP toxins by gavage showed diarrhea as the main symptom. Those toxins caused similar gastrointestinal alterations as well as intestine ultrastructural changes. However, DSP toxins did not modify tight junctions to trigger diarrhea. They had different toxicokinetics and toxic potency. The lethal dose 50 (LD₅₀) was 487 µg kg^-1 bw for DTX1, 760 µg kg^-1 bw for OA and 2262 µg kg^-1 bw for DTX2. Therefore, the oral TEF values are: OA = 1, DTX1 = 1.5 and DTX2 = 0.3. Conclusion: This is the first comparative study of DSP toxins performed with accurate well-characterized standards and based on acute toxicity data. Results confirmed that DTX1 is more toxic than OA by oral route while DTX2 is less toxic. Hence, the current TEFs based on intraperitoneal toxicity should be modified. Also, the generally accepted toxic mode of action of this group of toxins needs to be reevaluated.

Characterization of the dinophysistoxin-2 acute oral toxicity in mice to define the Toxicity Equivalency Factor

Ingestion of shellfish with dinophysistoxin-2 (DTX2) can lead to diarrheic shellfish poisoning (DSP). The official control method of DSP toxins in seafood is the liquid chromatography-mass spectrometry analysis (LC-MS). However in order to calculate the total toxicity of shellfish, the concentration of each compound must be multiplied by individual Toxicity Equivalency Factor (TEF). Considering that TEFs caused some controversy and the scarce information about DTX2 toxicity, the aim of this study was to characterize the oral toxicity of DTX2 in mice.A 4-Level Up and Down Procedure allowed the characterization of DTX2 effects and the estimation of DTX2 oral TEF based on determination of the lethal dose 50 (LD50). DTX2 passed the gastrointestinal barrier and was detected in urine and feces. Acute toxicity symptoms include diarrhea and motionless, however anatomopathology study and ultrastructural images restricted the toxin effects to the gastrointestinal tract. Nevertheless enterocytes microvilli and tight junctions were not altered, disconnecting DTX2 diarrheic effects from paracellular epithelial permeability. This is the first report of DTX2 oral LD50 (2262 μg/kg BW) indicating that its TEF is about 0.4. This result suggests reevaluation of the present TEFs for the DSP toxins to better determine the actual risk to seafood consumers.

Occurrence of Harmful Algal Species and Shellfish Toxicity in Sardinia (Italy).

Sardinia (Italy, north-western Mediterranean) is a commercially important producer of edible bivalve molluscs. Since the early 2000s, it was subjected to recurring cases of mussel farm closures due to toxic algal poison. Here, we present the studies on toxin concentrations and the associated potentially toxic phytoplankton distribution and abundances carried out by a regular monitoring programme in Sardinian shellfish areas, from January to May 2015. Diarrheic shellfish poisoning (DSP) toxins were detected in several bivalve molluscs samples, while paralytic shellfish poisoning (PSP) and paralytic shellfish poisoning toxins were present just once, without exceeding the legal limits. Potentially toxic algal species have been constantly present. Pseudo-nitzschia species were present during the entire study often with high abundances, while Dinophysis species reached high densities sporadically. Among PSP phytoplankton, only Alexandrium minutum Halim was found. The data obtained in this study showed an increase in the DSP toxicity in mussels in Sardinia. No clear relation between the occurrence of toxins in shellfish and the presence of potentially toxic algal species was found, although a slight correlation between DSP toxins and Dinophysis species could be supported.

Okadaic Acid: More than a Diarrheic Toxin

Okadaic acid (OA) is one of the most frequent and worldwide distributed marine toxins. It is easily accumulated by shellfish, mainly bivalve mollusks and fish, and, subsequently, can be consumed by humans causing alimentary intoxications. OA is the main representative diarrheic shellfish poisoning (DSP) toxin and its ingestion induces gastrointestinal symptoms, although it is not considered lethal. At the molecular level, OA is a specific inhibitor of several types of serine/threonine protein phosphatases and a tumor promoter in animal carcinogenesis experiments. In the last few decades, the potential toxic effects of OA, beyond its role as a DSP toxin, have been investigated in a number of studies. Alterations in DNA and cellular components, as well as effects on immune and nervous system, and even on embryonic development, have been increasingly reported. In this manuscript, results from all these studies are compiled and reviewed to clarify the role of this toxin not only as a DSP inductor but also as cause of alterations at the cellular and molecular levels, and to highlight the relevance of biomonitoring its effects on human health. Despite further investigations are required to elucidate OA mechanisms of action, toxicokinetics, and harmful effects, there are enough evidences illustrating its toxicity, not related to DSP induction, and, consequently, supporting a revision of the current regulation on OA levels in food.

Inhibition Equivalency Factors for Dinophysistoxin-1 and Dinophysistoxin-2 in Protein Phosphatase Assays: Applicability to the Analysis of Shellfish Samples and Comparison with LC-MS/MS

The protein phosphatase inhibition assay (PPIA) is a well-known strategy for the determination of diarrheic shellfish poisoning (DSP) lipophilic toxins, which deserves better characterization and understanding to be used as a routine screening tool in monitoring programs. In this work, the applicability of two PPIAs to the determination of okadaic acid (OA), dinophysistoxin-1 (DTX-1), dinophysistoxin-2 (DTX-2), and their acyl ester derivatives in shellfish has been investigated. The inhibitory potencies of the DSP toxins on a recombinant and a wild PP2A have been determined, allowing the establishment of inhibition equivalency factors (IEFs) (1.1 and 0.9 for DTX-1, and 0.4 and 0.6 for DTX-2, for recombinant and wild PP2A, respectively). The PPIAs have been applied to the determination of OA equivalent contents in spiked and naturally contaminated shellfish samples. Results have been compared to those obtained by LC-MS/MS analysis, after application of the IEFs, showing good agreements.

Production of monoclonal antibody and application in indirect competitive ELISA for detecting okadaic acid and dinophytoxin-1 in seafood

Okadaic acid (OA) and analogues of dinophysistoxin (DTX) are key diarrheic shellfish poisoning (DSP) toxins, which possibly arouse DSP symptoms by consuming the contaminated shellfish. Because of the stable toxicity in high temperature and the long-term carcinogenicity, the outbreaks of DSP related to consumption of bivalve mollusks contaminated by DSP toxins pose a hazard to public health. Therefore, it is worth developing a fast and reliable analytical method for the detection of OA and analogues in shellfish. In this paper, an indirect competitive enzyme-linked immunosorbent assay (ELISA) (icELISA) for detecting OA and DTX-1 in seafood was developed based on monoclonal antibody (McAb). The OA was conjugated to human immunoglobulin G (IgG) and bovine serum albumin (BSA) by the active ester method as the immune antigen and the detective antigen. The spleen cells from BALB/c mice immunized with OA-IgG were fused with SP2/0 myeloma cells. A hybridoma cell line, which secreted McAb against OA, was selected by "limiting dilution" cloning. An icELISA was developed based on immobilized conjugate (OA-BSA) competing the McAb with the free OA in seafood sample. A hybridoma cell line, which secreted IgG1 subclass monoclonal antibody (McAb) against OA, was selected. The IC(50) of the McAb for OA and dinophytoxin-1 (DTX-1) were 4.40 and 3.89ng/mL, respectively. Based on the McAb, an indirect competitive ELISA for detection of OA and DTX-1 in seafood was developed. The regression equation was y = 54.713x - 25.879 with a coefficient correlation of R (2) = 0.9729. The linear range and the limit of detection were 0.4-12.5 and 0.45ng/mL, respectively. The average recovery of OA and DTX-1 spiked shellfish was 82.29% with the coefficient of variation of 7.67%. The developed icELISA is a fast, sensitive, and convenient assay for detecting of total amount of OA and DTX-1 in seafood.

Love wave biosensor for real-time detection of okadaic acid as DSP phycotoxin

This paper reports the detection of okadaic acid (OA) as a Diarrheic Shellfish Poisoning (DSP) toxin with an acoustic wave platform in real-time. According to the FDA, the threshold for safe consumption of shellfish is 20μg of OA for 100g of shellfish tissue. The high gravimetric sensitivity of Love wave acoustic devices allow, in liquid media, immuno-detection thanks to immobilized specific antibodies. The biosensor is composed of two lines, one for the test and one used as a reference. Sensitive films were deposited through a PolyDiMethylSiloxane (PDMS) microfluidic chip. On both lines, anti-okadaic acid antibodies (OA-Ab) and saturating agents were successively injected under continuous flow with controlled flow rate. On the test line, okadaic acid (OA) was injected while an unspecific peptide (6×Histidine, 6×His) was used on the control line. On both lines, polyclonal OA-Ab were re-injected a second time to reveal previously fixed OA on the test line. Measured frequency shifts were three times higher on test lines than on control lines. In these conditions, for only 2μg of OA used for detection, the acoustic wave platform could detect DSP toxins with only 10g of shellfish tissue.

Esterification of okadaic acid in the mussel Mytilus galloprovincialis

Okadaic acid and other toxins of the diarrheic shellfish poisoning (DSP) group are transformed mainly to their acyl-derivatives in bivalves. Some recent studies suggest that bacteria present in the bivalve gut could contribute substantially to the acylation of the toxins. By feeding microcapsules containing okadaic acid to mussels we have shown unequivocally that the ingested okadaic acid is nearly completely transformed to its fatty acid esters (acyl-derivatives). Treating mussels with antibiotics did not have any significant effect on the acylation of the supplied okadaic acid, suggesting that bacteria do not play any significant role in this process. The microsomal and mitochondrial subcellular fractions of the cells of the digestive gland have been shown to have contain enzymes that are able to transfer a fatty acid molecule from Coenzyme A to okadaic acid (so, that have Acyl-CoA:OA acyltransferase activity). This activity was related to that of the enzyme Cytochrome C reductase (NADPH), a marker of endoplasmic reticulum, suggesting that this organelle is the main responsible for the acylation process. Acylation of DSP toxins seems to be a key step in the depuration of these toxins from mussels, as these compounds are found in feces as acyl-derivatives. This is probably true for most bivalves. The proportion of acyl-derivatives accumulated can point to the key process of the depuration: acylation or excretion of acylated derivatives. In the mussels Mytilus galloprovincialis, Mytilus edulis and in Donax trunculus, the first process seems to be the most important, but in most bivalve species it seems to be the second one. Other aspects of the relationship between depuration and acylation are also discussed.

Mediterranean Mussel Gene Expression Profile Induced by Okadaic Acid Exposure

Seasonal seawater temperature increases define optimal growth conditions for Dinoflagellate species which can reach high concentrations in water column and also in filter-feeding organisms like Mytilus galloprovincialis. Commonly produced by Dinophysis and Prorocentrum spp., okadaic acid (OA) and its analogues are responsible for the Diarrheic Shellfish Poisoning (DSP) syndrome in humans. Closure of shellfishing grounds is therefore recommended by the EU when DSP toxin levels in shellfish exceed 16 μg OA 100 g(-1) flesh. Despite not being responsible for casualties either in humans or mussels, DSP outbreaks are considered natural events causing health and economic issues due to the frequency of their occurrence. Since gene expression studies offer a wide range of different solutions, we used a mussel cDNA microarray to evaluate gene expression changes in the digestive gland of mussels fed for five weeks with OA-contaminated nutrient. Among the differentially expressed genes we observed a general up-regulation of transcripts coding for stress proteins, proteins involved in cellular synthesis, and a few not annotated proteins. Overall, at the first time point analyzed we identified 58 candidate transcripts for OA-induced stress in mussels, half of which have unknown function. In this paper we present the first gene expression analysis performed on Mediterranean mussels exposed to okadaic acid. The characterization of these transcripts could be useful for the identification of an early physiological response to OA exposure.

Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells

Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process.

A simple short range model for the prediction of harmful algal events in the bays of southwestern Ireland

A simple model is described which predicts harmful algal events in the bays of southwestern Ireland. Fundamental to the model is the physical forcing of circulation in these bays in summer. The predominant hydrographic feature at this time is a wind-driven two-layer oscillatory flow acting in a thermally stratified water column. This mechanism exchanges substantial proportions of the bays' volumes, and harmful algal events arise with the associated transport of harmful populations into them. The model is therefore based on the criterion that wind-driven water exchanges result in exchanges of phytoplankton, which, if the time of year is correct, result in toxic events. Utilising Bantry Bay as an example, hindcasting showed that the model has a high degree of success using a wind index based on the sequence of winds that results in water exchange. The model was implemented by estimating indices from the five-day weather forecast, and trialled in 2005. Results were published on the web in real time, during which a predicted water exchange event in mid-June was accompanied by an influx of Dinophysis acuminata into Bantry Bay with an associated contamination of shellfish with Diarrheic Shellfish Poisoning toxins.