Temporal Prediction of Paralytic Shellfish Toxins in the Mussel Mytilus galloprovincialis Using a LSTM Neural Network Model from Environmental Data

Paralytic shellfish toxins (PSTs) are produced mainly by Alexandrium catenella (formerly A. tamarense). Since 2000, the National Institute of Fisheries Science (NIFS) has been providing information on PST outbreaks in Korean coastal waters at one- or two-week intervals. However, a daily forecast is essential for immediate responses to PST outbreaks. This study aimed to predict the outbreak timing of PSTs in the mussel Mytilus galloprovincialis in Jinhae Bay and along the Geoje coast in the southern coast of the Korea Peninsula. We used a long-short-term memory (LSTM) neural network model for temporal prediction of PST outbreaks from environmental data, such as water temperature (WT), tidal height, and salinity, measured at the Geojedo, Gadeokdo, and Masan tidal stations from 2006 to 2020. We found that PST outbreaks is gradually accelerated during the three years from 2018 to 2020. Because the in-situ environmental measurements had many missing data throughout the time span, we applied LSTM for gap-filling of the environmental measurements. We trained and tested the LSTM models with different combinations of environmental factors and the ground truth timing data of PST outbreaks for 5479 days as input and output. The LSTM model trained from only WT had the highest accuracy (0.9) and lowest false-alarm rate. The LSTM-based temporal prediction model may be useful as a monitoring system of PSP outbreaks in the coastal waters of southern Korean.

Proteome Response of Meretrix Bivalves Hepatopancreas Exposed to Paralytic Shellfish Toxins Producing Dinoflagellate Gymnodinium catenatum

Paralytic shellfish toxins (PSTs) contamination of seafood has become a growing global problem. However, the molecular response of bivalves, some of the most popular seafoods, to PSP toxins has seldom been reported and the underlying molecular mechanisms of the interactions between Meretrix meretrix bivalves and PSTs-producing dinoflagellates are scarcely known. This study compared the protein expression profiles between PSP toxin-contaminated and non-PSP toxin contaminated M. meretrix, determined proteome responses and identified potential biomarkers based on feeding experiments. Results showed that the content of total PSP toxins in contaminated bivalves was 40.63 ± 4.08 μg saxitoxin (STX) equivalents per gram, with 95.3% in hepatopancreas, followed by gill (1.82%) and foot (1.79%). According to two-dimensional gel electrophoresis (2-DE), 15 differentially expressed proteins (at least 2-fold difference) between the hepatopancreas of bivalves with and without PSP toxins were detected. Eight of them were successfully identified by MALDI-TOF MS. These were catalase, protein ultraspiracle homolog, G2 and S phase-expression protein, paramyosin, Mn-superoxide dismutase, response regulator receiver domain-containing protein, sarcoplasmic calcium-binding protein and major facilitator superfamily transporters. The differences in the expression levels of the last three proteins involving in cell signaling, structure and membrane transport were 4.2, 5.3 and 4.9-fold, respectively. These proteins could be further developed as potential biomarkers. The other two up-regulated proteins, Mn-superoxide dismutase and catalase, were involved in cell defence mechanisms against oxidative stress, suggesting PSP toxin acts as xenobiotics and poses oxidative stress in bivalves. This study gives insights into the response of bivalves to PSP toxin-producing dinoflagellate at the proteomic level and the potential of using 2-DE to develop specific protein markers in bivalves.

Sub-Acute Feeding Study of Saxitoxin to Mice Confirms the Effectiveness of Current Regulatory Limits for Paralytic Shellfish Toxins

Regulatory limits for shellfish toxins are required to protect human health. Often these limits are set using only acute toxicity data, which is significant, as in some communities, shellfish makes up a large proportion of their daily diet and can be contaminated with paralytic shellfish toxins (PSTs) for several months. In the current study, feeding protocols were developed to mimic human feeding behaviour and diets containing three dose rates of saxitoxin dihydrochloride (STX.2HCl) were fed to mice for 21 days. This yielded STX.2HCl dose rates of up to 730 µg/kg bw/day with no effects on food consumption, growth, blood pressure, heart rate, motor coordination, grip strength, blood chemistry, haematology, organ weights or tissue histology. Using the 100-fold safety factor to extrapolate from animals to humans yields a dose rate of 7.3 µg/kg bw/day, which is well above the current acute reference dose (ARfD) of 0.5 µg STX.2HCl eq/kg bw proposed by the European Food Safety Authority. Furthermore, to reach the dose rate of 7.3 µg/kg bw, a 60 or 70 kg human would have to consume 540 or 630 g of shellfish contaminated with PSTs at the current regulatory limit (800 µg/kg shellfish flesh), respectively. The current regulatory limit for PSTs therefore seems appropriate.

Inhibition of Diarrheal Shellfish Toxins Accumulation in the Mussel Perna viridis by Curcumin and Underlying Mechanisms

Diarrheal shellfish toxins (DSTs) are among the most widely distributed phytotoxins, and are associated with diarrheal shellfish poisoning (DSP) events in human beings all over the world. Therefore, it is urgent and necessary to identify an effective method for toxin removal in bivalves. In this paper, we found that curcumin (CUR), a phytopolylphenol pigment, can inhibit the accumulation of DSTs (okadaic acid-eq) in the digestive gland of Perna viridis after Prorocentrum lima exposure. qPCR results demonstrated that CUR inhibited the induction of DSTs on the aryl hydrocarbon receptor (AhR), hormone receptor 96 (HR96) and CYP3A4 mRNA, indicating that the CUR-induced reduction in DSTs may be correlated with the inhibition of transcriptional induction of AhR, HR96 and CYP3A4. The histological examination showed that P. lima cells caused severe damage to the digestive gland of P. viridis, and the addition of curcumin effectively alleviated the damage induced by P. lima. In conclusion, our findings provide a potential method for the effective removal of toxins from DST-contaminated shellfish.

A Comparative Analysis of Methods (LC-MS/MS, LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters, Mussels and Pipis

Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish–Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chromatography—Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0–150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60–262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25–100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r2 = 0.86) and the PP2A kit (r2 = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.

In Vitro Effects of Paralytic Shellfish Toxins and Lytic Extracellular Compounds Produced by Alexandrium Strains on Hemocyte Integrity and Function in Mytilus edulis

Harmful effects caused by the exposure to paralytic shellfish toxins (PSTs) and bioactive extracellular compounds (BECs) on bivalves are frequently difficult to attribute to one or the other compound group. We evaluate and compare the distinct effects of PSTs extracted from Alexandrium catenella (Alex5) cells and extracellular lytic compounds (LCs) produced by A. tamarense (NX-57-08) on Mytilus edulis hemocytes. We used a 4 h dose–response in vitro approach and analyzed how these effects correlate with those observed in a previous in vivo feeding assay. Both bioactive compounds caused moderated cell death (10–15%), being dose-dependent for PST-exposed hemocytes. PSTs stimulated phagocytic activity at low doses, with a moderate incidence in lysosomal damage (30–50%) at all tested doses. LCs caused a dose-dependent impairment of phagocytic activity (up to 80%) and damage to lysosomal membranes (up to 90%). PSTs and LCs suppressed cellular ROS production and scavenged H2O2 in in vitro assays. Neither PSTs nor LCs affected the mitochondrial membrane potential in hemocytes. In vitro effects of PST extracts on M. edulis hemocytes were consistent with our previous study on in vivo exposure to PST-producing algae, while for LCs, in vivo and in vitro results were not as consistent.