Diarrhetic Shellfish Poisoning Toxins Research Articles

Small-Scale Biophysical Interactions and Dinophysis Blooms: Case Study in a Strongly Stratified Chilean Fjord

Diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins (PTXs) produced by endemic species of Dinophysis, mainly D. acuta and D. acuminata, threaten public health and negatively impact the shellfish industry worldwide. Despite their socioeconomic impact, research on the environmental drivers of DSP outbreaks in the Chilean fjords is scanty. From 22 to 24 March 2017, high spatial–temporal resolution measurements taken in Puyuhuapi Fjord (Northern Patagonia) illustrated the short-term (hours, days) response of the main phytoplankton functional groups (diatoms and dinoflagellates including toxic Dinophysis species) to changes in water column structure. Results presented here highlight the almost instantaneous coupling between time–depth variation in density gradients, vertical shifts of the subsurface chlorophyll maximum, and its evolution to a buoyancy-driven thin layer (TL) of diatoms just below the pycnocline the first day. A second shallower TL of dinoflagellates, including Dinophysis acuta, was formed on the second day in a low-turbulence lens in the upper part of the pycnocline, co-occurring with the TL of diatoms. Estimates of in situ division rates of Dinophysis showed a moderate growth maximum, which did not coincide with the cell density max. This suggests that increased cell numbers resulted from cell entrainment of off-fjord populations combined with in situ growth. Toxin profiles of the net tow analyses mirrored the dominance of D. acuminata/D. acuta at the beginning/end of the sampling period. This paper provides information about biophysical interactions of phytoplankton, with a focus on Dinophysis species in a strongly stratified Patagonian fjord. Understanding these interactions is crucial to improv predictive models and early warning systems for toxic HABs in stratified systems.

Physiological and genetic responses of the benthic dinoflagellate Prorocentrum lima to polystyrene microplastics

Microplastics are well known as contaminants in marine environments. With the development of biofilms, most microplastics will eventually sink and deposit in benthic environment. However, little research has been done on benthic toxic dinoflagellates, and the effects of microplastics on benthic dinoflagellates are unknown. Prorocentrum lima is a cosmopolitan toxic benthic dinoflagellate, which can produce a range of polyether metabolites, such as diarrhetic shellfish poisoning (DSP) toxins. In order to explore the impact of microplastics on marine benthic dinoflagellates, in this paper, we studied the effects of polystyrene (PS) on the growth and toxin production of P. lima. The molecular response of P. lima to microplastic stress was analyzed by transcriptomics. We selected 100 nm, 10 μm and 100 μm PS, and set three concentrations of 1 mg L−1, 10 mg L−1 and 100 mg L−1. The results showed that PS exposure had limited effects on cell growth, but increased the OA and extracellular polysaccharide content at high concentrations. After exposure to PS MPs, genes associated with DSP toxins synthesis, carbohydrate synthesis and energy metabolism, such as glycolysis, TCA cycle and pyruvate metabolism, were significantly up-regulated. We speculated that after exposure to microplastics, P. lima may increase the synthesis of DSP toxins and extracellular polysaccharides, improve the level of energy metabolism and gene expression of ABC transporter, thereby protecting algal cells from damage. Our findings provide new insights into the effects of microplastics on toxic benthic dinoflagellates.

Okadaic Acid Is at Least as Toxic as Dinophysistoxin-1 after Repeated Administration to Mice by Gavage.

Okadaic acid (OA) and its analogues cause diarrhetic shellfish poisoning (DSP) in humans, and risk assessments of these toxins require toxicity equivalency factors (TEFs), which represent the relative toxicities of analogues. However, no human death by DSP toxin has been reported, and its current TEF value is based on acute lethality. To properly reflect the symptoms of DSP, such as diarrhea without death, the chronic toxicity of DSP toxins at sublethal doses should be considered. In this study, we obtained acute oral LD50 values for OA and dinophysistoxin-1 (DTX-1) (1069 and 897 μg/kg, respectively) to set sublethal doses. Mice were treated with sublethal doses of OA and DTX-1 for 7 days. The mice lost body weight, and the disease activity index and intestinal crypt depths increased. Furthermore, these changes were more severe in OA-treated mice than in the DTX-1-treated mice. Strikingly, ascites was observed, and its severity was greater in mice treated with OA. Our findings suggest that OA is at least as toxic as DTX-1 after repeated oral administration at a low dose. This is the first study to compare repeated oral dosing of DSP toxins. Further sub-chronic and chronic studies are warranted to determine appropriate TEF values for DSP toxins.

Identification of Perna viridis contaminated with diarrhetic shellfish poisoning toxins in vitro using NIRS and a discriminative non-negative representation-based classifier.

Diarrhetic shellfish poisoning (DSP) toxins are one of the most widespread marine biotoxins that affect aquaculture and human health, and their detection has become crucial. In this study, near-infrared reflectance spectroscopy (NIRS) with non-destructive characteristics was used to identify DSP toxins in Perna viridis. The spectral data of the DSP toxin-contaminated and non-contaminated Perna viridis samples were acquired in the 950-1700nm range. To solve the discrimination of spectra with crossover and overlapping, a discriminative non-negative representation-based classifier (DNRC) has been proposed. Compared with collaborative and non-negative representation-based classifiers, the DNRC model exhibited better performance in detecting DSP toxins, with a classification accuracy of 99.44%. For a relatively small-scale sample dataset in practical applications, the performance of the DNRC model was compared with those of classical models. The DNRC model achieved the best results for both identification accuracy and F-measure, and its detection performance did not significantly decrease with decreasing sample size. The experimental results validated that a combination of NIRS and the DNRC model can facilitate rapid, convenient, and non-destructive detection of DSP toxins in Perna viridis.

Transcriptomic analysis of polyketide synthesis in dinoflagellate, Prorocentrum lima

The benthic dinoflagellate Prorocentrum lima is among the most common toxic morphospecies with a cosmopolitan distribution. P. lima can produce polyketide compounds, such as okadaic acid (OA), dinophysistoxin (DTX) and their analogues, which are responsible for diarrhetic shellfish poisoning (DSP). Studying the molecular mechanism of DSP toxin biosynthesis is crucial for understanding the environmental driver influencing toxin biosynthesis as well as for better monitoring of marine ecosystems. Commonly, polyketides are produced by polyketide synthases (PKS). However, no gene has been confirmatively assigned to DSP toxin production. Here, we assembled a transcriptome from 94,730,858 Illumina RNAseq reads using Trinity, resulting in 147,527 unigenes with average sequence length of 1035 nt. Using bioinformatics analysis methods, we found 210 unigenes encoding single-domain PKS with sequence similarity to type I PKSs, as reported in other dinoflagellates. In addition, 15 transcripts encoding multi-domain PKS (forming typical type I PKSs modules) and 5 transcripts encoding hybrid nonribosomal peptide synthetase (NRPS)/PKS were found. Using comparative transcriptome and differential expression analysis, a total of 16 PKS genes were identified to be up-regulated in phosphorus-limited cultures, which was related to the up regulation of toxin expression. In concert with other recent transcriptome analyses, this study contributes to the building consensus that dinoflagellates may utilize a combination of Type I multi-domain and single-domain PKS proteins, in an as yet undefined manner, to synthesize polyketides. Our study provides valuable genomic resource for future research in order to understand the complex mechanism of toxin production in this dinoflagellate.

Upregulation of Peridinin-Chlorophyll A-Binding Protein in a Toxic Strain of Prorocentrum hoffmannianum under Normal and Phosphate-Depleted Conditions.

Some strains of the dinoflagellate species Prorocentrum hoffmannianum show contrasting ability to produce diarrhetic shellfish poisoning (DSP) toxins. We previously compared the okadaic acid (OA) production level between a highly toxic strain (CCMP2804) and a non-toxic strain (CCMP683) of P. hoffmannianum and revealed that the cellular concentration of OA in CCMP2804 would increase significantly under the depletion of phosphate. To understand the molecular mechanisms, here, we compared and analyzed the proteome changes of both strains growing under normal condition and at phosphate depletion using two-dimensional gel electrophoresis (2-DE). There were 41 and 33 differential protein spots observed under normal condition and phosphate depletion, respectively, of which most were upregulated in CCMP2804 and 22 were common to both conditions. Due to the lack of matched peptide mass fingerprints in the database, de novo peptide sequencing was applied to identify the differentially expressed proteins. Of those upregulated spots in CCMP2804, nearly 60% were identified as peridinin-chlorophyll a-binding protein (PCP), an important light-harvesting protein for photosynthesis in dinoflagellates. We postulated that the high expression of PCP encourages the production of DSP toxins by enhancing the yields of raw materials such as acetate, glycolate and glycine. Other possible mechanisms of toxicity related to PCP might be through triggering the transcription of non-ribosomal peptide synthetase/polyketide synthase genes and the transportation of dinophysistoxin-4 from chloroplast to vacuoles.

Forecasting biotoxin contamination in mussels across production areas of the Portuguese coast with Artificial Neural Networks

Harmful algal blooms (HABs) and the consequent contamination of shellfish are complex processes depending on several biotic and abiotic variables, turning prediction of shellfish contamination into a challenging task. Not only the information of interest is dispersed among multiple sources, but also the complex temporal relationships between the time-series variables require advanced machine methods to model such relationships. In this study, multiple time-series variables measured in Portuguese shellfish production areas were used to forecast shellfish contamination by diarrhetic shellfish poisoning (DSP) toxins one to four weeks in advance. These time series included DSP concentration in mussels (Mytilus galloprovincialis), toxic phytoplankton cell counts, meteorological, and remotely sensed oceanographic variables. Several data pre-processing and feature engineering methods were tested, as well as multiple autoregressive and artificial neural network (ANN) models. The best results regarding the mean absolute error of prediction were obtained for a bivariate long short-term memory (LSTM) neural network based on biotoxin and toxic phytoplankton measurements, with higher accuracy for short-term forecasting horizons. When evaluating all ANNs model ability to predict the contamination state (below or above the regulatory limit for contamination) and changes to this state, multilayer perceptrons (MLP) and convolutional neural networks (CNN) yielded improved predictive performance on a case-by-case basis. These results show the possibility of extracting relevant information from time-series data from multiple sources which are predictive of DSP contamination in mussels, therefore placing ANNs as good candidate models to assist the production sector in anticipating harvesting interdictions and mitigating economic losses.

Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for assessing okadaic acid and its derivatives in shellfish

ABSTRACT Okadaic acid (OA) and the analogs of dinophysistoxin (DTX) are important members of diarrhetic shellfish poisoning (DSP) toxins. In this study, five OA-specific mAbs (monoclonal antibodies) were developed from five stable cells of hybridoma. The anti-OA mAb-2D7 showed high sensitivity to OA, the IC50 of the antibody was 0.24 ng/mL, and its cross-reactivity was 91.6% with DTX-1 and 110.5% with DTX-2., In dcELISA, the IC50 was set at 0.182 ng/mL, and the detection limit was set at 0.023 ng/mL by using the anti-OA mAb-2D7. The level of OA recovered from spiked mussel samples was 2–50 ng/g, ranging from 97.6 ± 7.2% to 106.4 ± 9.8%. In contrast, the immunostrip assay with a limit of 5 ng/mL, conducted for detecting OA, was completed in 10 min. The mAb-based dcELISA and immunostrip assay developed were precise and sensitive enough to quickly assess OA, DTX-1, and DTX-2 in shellfish specimens.

Toxic Responses of Different Shellfish Species after Exposure to Prorocentrum lima, a DSP Toxins Producing Dinoflagellate

Prorocentrum lima is a global benthic dinoflagellate that produces diarrhetic shellfish poisoning (DSP) toxins, which can be ingested by filter-feeding bivalves, and eventually pose a great threat to human health through food chain. After being exposed to P. lima, different bivalves may accumulate various levels of DSP toxins and display different toxic responses. However, the underlying mechanism remains unclear. Here, we found that the content of okadaic acid-equivalents (OA-eq) varied in the digestive glands of the three bivalves including Crassostrea gigas, Mytilus coruscus and Tegillarca granosa after P. lima exposure. The degree of esterification of OA-eq in the three bivalves were opposite to the accumulation of OA-eq. The digestive gland tissues of the three bivalve species were damaged to different degrees. The transcriptional induction of Nrf2 targeted genes such as ABCB1 and GPx indicates the functionality of Nrf2 pathway against DSP toxins in bivalves. The oyster could protect against DSP toxins mainly through ABC transporters and esterification, while the mussel and clam reduce the damage induced by DSP toxins mainly by regulating the expression of antioxidant genes. Our findings may provide some explanations for the difference in toxic response to DSP toxins in different shellfish.

Fast Detection of Diarrhetic Shellfish Poisoning Toxins in Mussels Using NIR Spectroscopy and Improved Twin Support Vector Machines

Diarrhetic shellfish poisoning (DSP) toxins are potent marine biotoxins. It can cause a severe gastrointestinal illness by the consumption of mussels contaminated by DSP toxins. New methods for effectively and rapidly detecting DSP toxins-contaminated mussels are required. In this study, we used near-infrared (NIR) reflection spectroscopy combined with pattern recognition methods to detect DSP toxins. In the range of 950-1700 nm, the spectral data of healthy mussels and DSP toxins-contaminated mussels were acquired. To select optimal waveband subsets, a waveband selection algorithm with a Gaussian membership function based on fuzzy rough set theory was applied. Considering that detecting DSP toxins-contaminated mussels from healthy mussels was an imbalanced classification problem, an improved approach of twin support vector machines (TWSVM) was explored, which is based on a centered kernel alignment. The influences of parameters of the waveband selection algorithm and regularization hyperparameters of the improved TWSVM (ITWSVM) on the performance of models were analyzed. Compared to conventional SVM, TWSVM, and other state-of-the-art algorithms (such as multi-layer perceptron, extreme gradient boosting and adaptive boosting), our proposed model exhibited better performance in detecting DSP toxins and was little affected by the imbalance ratio. For the proposed model, the F-measure reached 0.9886, and detection accuracy reached 98.83%. We explored the physical basis for the detection model by analyzing the relationship between the occurrence of overtone and combination bands and selected wavebands. This study supports NIR spectroscopy as an innovative, rapid, and convenient analytical method to detect DSP toxins in mussels.

Bayesian Networks modeling of diarrhetic shellfish poisoning in Mytilus edulis harvested in Bantry Bay, Ireland

Diarrhetic Shellfish Poisoning (DSP) results from the human consumption of contaminated shellfish with marine biotoxins, which are produced by some species of marine dinoflagellates, mainly belonging to the genus Dinophysis. Shellfish contamination with marine biotoxins not only pose a threat to human health, but also lead to financial loss to aquaculture operations from the temporary closure of production areas when toxin concentrations exceed regulatory levels. In this study, we developed a Bayesian Network (BN) model for forecasting the short-term variations of DSP toxins in blue mussels (Mytilus edulis) from Bantry Bay, Southwest Ireland. Data inputs to a BN model from 10 production sites in Bantry Bay included plankton cell densities in sea water, DSP toxin concentration in mussels and sea surface temperature. The model was trained with data from 2014 to 2018, and validated with data of 2019. Validation consisted of predicting the DSP toxin concentration at one production site using the model parameters from the other locations as input values. Model validation showed that the prediction accuracy was higher than 86%. Sensitivity analysis indicated that in general, DSP toxin concentration was more relevant than plankton abundance. This initial work has demonstrated the usefulness of BN modeling as an approach to short term forecasting. Further work is ongoing to use the model for scenario testing and to increase the number of environmental parameters used as inputs to the model.

The diarrhetic shellfish-poisoning toxin, okadaic acid, provokes gastropathy, dysbiosis and susceptibility to bacterial infection in a non-rodent bioassay, Galleria mellonella

Diarrhetic shellfish-poisoning (DSP) toxins such as okadaic acid and dinophysistoxins harm the human gastrointestinal tract, and therefore, their levels are regulated to an upper limit of 160 μg per kg tissue to protect consumers. Rodents are used routinely for risk assessment and studies concerning mechanisms of toxicity, but there is a general move toward reducing and replacing vertebrates for these bioassays. We have adopted insect larvae of the wax moth Galleria mellonella as a surrogate toxicology model. We treated larvae with environmentally relevant doses of okadaic acid (80–400 μg/kg) via intrahaemocoelic injection or gavage to determine marine toxin-related health decline: (1) whether pre-exposure to a sub-lethal dose of toxin (80 μg/kg) enhances susceptibility to bacterial infection, or (2) alters tissue pathology and bacterial community (microbiome) composition of the midgut. A sub-lethal dose of okadaic acid (80 μg/kg) followed 24 h later by bacterial inoculation (2 × 105Escherichia coli) reduced larval survival levels to 47%, when compared to toxin (90%) or microbial challenge (73%) alone. Histological analysis of the midgut depicted varying levels of tissue disruption, including nuclear aberrations associated with cell death (karyorrhexis, pyknosis), loss of organ architecture, and gross epithelial displacement into the lumen. Moreover, okadaic acid presence in the midgut coincided with a shift in the resident bacterial population over time in that substantial reductions in diversity (Shannon) and richness (Chao-1) indices were observed at 240 μg toxin per kg. Okadaic acid-induced deterioration of the insect alimentary canal resembles those changes reported for rodent bioassays.

Analysis of Manganese Bioaccumulated in Mediterranean Blue Mussel (Mytilus galloprovincialis) from the Bay of Mali Ston (Adriatic Sea, Croatia) during Diarrhetic Shellfish Poisoning Toxicity

Diarrhetic Shellfish Poisoning (DSP) toxicity was revealed in the Mediterranean blue mussel (Mytilus galloprovincialis) from the Bay of Mali Ston, in the south part of the Eastern Adriatic Sea, through the Croatian National Monitoring Programme in the period from January until June of 2011. A survey of DSP toxicity within the frame of regular controls carried out through the mouse bioassay (MBA, at the time the official method for DSP toxins) demonstrated that in some incidents, positive MBA, which manifested by the atypical symptomatology of the animals, dominated. Additional studies were done to explain the atypical results of the conducted biological tests at the time. In the current study, the bioaccumulated manganese concentration in the soft tissues of mussels was measured to investigate its influence on the MBA results. In both DSP negative and DSP positive samples, which were prepared for the analysis according to the modified US EPA 3052 method, the concentration of the bioaccumulated manganese was performed on the atomic absorption spectrometer using flame atomic absorption spectroscopy technique. The analysis revealed higher concentration of manganese in 87% of DSP positive samples and the expressed per wet weight ranged from 0.15 to 5.38 mg kg−1. The mean concentration of manganese for all DSP positive samples was 1.78 mg kg−1, while for DSP negative samples, it was 48% lower (0.93 mg kg−1). The highest concentration of manganese in DSP positive samples was measured in February 2011. Since the low concentrations of lipophilic biotoxins gymnodimines (GYMs) and spirolides (SPXs) were also detected in the analysed DSP positive samples in the parallel studies, the results obtained in this study suggest future investigations of the connection between the concentration of manganese and lipophilic biotoxins.

Effects of the Marine Biotoxins Okadaic Acid and Dinophysistoxins on Fish

Natural high proliferations of toxin-producing microorganisms in marine and freshwater environments result in dreadful consequences at the socioeconomically and environmental level due to water and seafood contamination. Monitoring programs and scientific evidence point to harmful algal blooms (HABs) increasing in frequency and intensity as a result of global climate alterations. Among marine toxins, the okadaic acid (OA) and the related dinophysistoxins (DTX) are the most frequently reported in EU waters, mainly in shellfish species. These toxins are responsible for human syndrome diarrhetic shellfish poisoning (DSP). Fish, like other marine species, are also exposed to HABs and their toxins. However, reduced attention has been given to exposure, accumulation, and effects on fish of DSP toxins, such as OA. The present review intends to summarize the current knowledge of the impact of DSP toxins and to identify the main issues needing further research. From data reviewed in this work, it is clear that exposure of fish to DSP toxins causes a range of negative effects, from behavioral and morphological alterations to death. However, there is still much to be investigated about the ecological and food safety risks related to contamination of fish with DSP toxins.

Molecular networking as a novel approach to unravel toxin diversity of four strains of the dominant Dinophysis species from French coastal waters

Some species of the genus Dinophysis contain Diarrhetic shellfish Poisoning (DSP) toxins and are the main threat to shellfish farming in Europe including France. Dinophysis species are known to produce two families of bioactive lipophilic toxins: (i) okadaic acid (OA) and their analogues dinophysistoxins (DTXs) and (ii) pectenotoxins (PTXs). Only six toxins (OA, DTX1, DTX2, DTX3, PTX1 and PTX2) regulated by the European Union Legislation (EC No. 15/2011; 3) are routinely monitored using targeted chemical analysis by liquid chromatography coupled to mass spectrometry (LC-MS/MS) while toxic species of Dinophysis produce many other analogues. To tentatively identify unknown toxin analogues, a recent approach (Molecular Networking, MN) was used based on fragmentation data obtained by untargeted high resolution mass spectrometry (HRMS). An optimization of the data-dependent LC-HRMS/MS acquisition conditions was conducted to obtain more informative networks. The MN was applied to provide an overview of the chemical diversity of four strains belonging to three major Dinophysis species isolated from French coastal waters (D. acuta, D. caudata and the “D. acuminata complex” species D. acuminata and D. sacculus). This approach highlighted species-specific chemical patterns and also that Dinophysis chemical diversity is largely unexplored. Using MN allowed to identify directly known toxins and their relationship between species of Dinophysis, leading to the discovery of five new putative PTX analogues.

Multiscale physical background to an exceptional harmful algal bloom of Dinophysis acuta in a fjord system

Dinophysis acuta produces diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins (PTX). It blooms in thermally-stratified shelf waters in late summer in temperate to cold temperate latitudes. Despite its major contribution to shellfish harvesting bans, little effort has been devoted to study its population dynamics in Chilean Patagonia. In 2017–2018, mesoscale distribution of harmful algal species (75 monitoring stations) revealed the initiation (late spring) and seasonal growth of a dense D. acuta population in the Aysén region, with maximal values at Puyuhuapi Fjord (PF). Vertical phytoplankton distribution and fine-resolution measurements of physical parameters along a 25-km transect in February 16th identified a 15-km (horizontal extension) subsurface thin layer of D. acuta from 4 to 8 m depth. This layer, disrupted at the confluence of PF with the Magdalena Sound, peaked at the top of the pycnocline (6 m, 15.9 °C, 23.4 psu) where static stability was maximal. By February 22nd, it deepened (8 m, 15.5 °C; 23.62 psu) following the excursions of the pycnocline and reached the highest density ever recorded (664 × 103 cells L−1) for this species. Dinophysis acuta was the dominant Dinophysis species in all microplankton net-tows/bottle samples; they all contained DSP toxins (OA, DTX-1) and PTX-2. Modeled flushing rates showed that Puyuhuapi, the only fjord in the area with 2 connections with the open sea, had the highest water residence time. Long term climate variability in the Southern hemisphere showed the effects of a Southern Annular Mode (SAM) in positive mode (+1.1 hPa) overwhelming a moderate La Niña. These effects included positive spring precipitation anomalies with enhanced salinity gradients and summer drought with positive anomalies in air (+1 °C) and sea surface (+2 °C) temperature. Locally, persistent thermal stratification in PF seemed to provide an optimal physical habitat for initiation and bloom development of D. acuta. Thus, in summer 2018, a favourable combination of meteorological and hydrographic processes of multiple scales created conditions that promoted the development of a widespread bloom of D. acuta with its epicentre at the head of Puyuhuapi fjord.

Characterization of toxin-producing strains of Dinophysis spp. (Dinophyceae) isolated from French coastal waters, with a particular focus on the D. acuminata-complex

Dinoflagellates of the genus Dinophysis are the most prominent producers of Diarrhetic Shellfish Poisoning (DSP) toxins which have an impact on public health and on marine aquaculture worldwide. In particular, Dinophysis acuminata has been reported as the major DSP agent in Western Europe. Still, its contribution to DSP events in the regions of the English Channel and the Atlantic coast of France, and the role of the others species of the Dinophysis community in these areas are not as clear. In addition, species identification within the D. acuminata complex has proven difficult due to their highly similar morphological features. In the present study, 30 clonal strains of the dominant Dinophysis species have been isolated from French coasts including the English Channel (3 sites), the Atlantic Ocean (11 sites) and the Mediterranean Sea (6 sites). Morphologically, strains were identified as three species: D. acuta, D. caudata, D. tripos, as well as the D. acuminata-complex. Sequences of the ITS and LSU rDNA regions confirmed these identifications and revealed no genetic difference within the D. acuminata-complex. Using the mitochondrial gene cox1, two groups of strains differing by only one substitution were found in the D. acuminata-complex, but SEM analysis of various strains showed a large range of morphological variations. Based on geographical origin and morphology, strains of the subclade A were ascribed to ‘D. acuminata’ while those of the subclade B were ascribed to ‘D. sacculus’. Nevertheless, the distinction into two separate species remains questionable and was not supported by our genetic data. The considerable variations observed in cultured strains suggest that physiological factors might influence cell contour and bias identification. Analyses of Dinophysis cultures from French coastal waters using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) revealed species-conserved toxin profiles for D. acuta (dinophysistoxin 2 (DTX2), okadaic acid (OA), pectenotoxin 2 (PTX2)), D. caudata (PTX2) and D. tripos (PTX2), irrespective of geographical origin (Atlantic Ocean or Mediterranean Sea). Within the D. acuminata-complex, two different toxin profiles were observed: the strains of ‘D. acuminata’ (subclade A) from the English Channel and the Atlantic Ocean contained only OA while strains of ‘D. sacculus’ (subclade B) from Mediterranean Sea/Atlantic Ocean contained PTX2 as the dominant toxin, with OA and C9-esters also being present, albeit in lower proportions. The same difference in toxin profiles between ‘D. sacculus’ and ‘D. acuminata’ was reported in several studies from Galicia (NW- Spain). This difference in toxin profiles has consequences in terms of public health, and consequently for monitoring programs. While toxin profile could appear as a reliable feature separating ‘D. acuminata’ from ‘D. sacculus’ on both French and Spanish coasts, this does not seem consistent with observations on a broader geographical scale for the D. acuminata complex, possibly due to the frequent lack of genetic characterization.

Drastic hypothermia after intraperitoneal injection of okadaic acid, a diarrhetic shellfish poisoning toxin, in mice.

The mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins had been used as the official method in Japan and also used in the world. In this study, hypothermia, one of the symptoms observed in mice after inoculation with DSP toxins, were characterized. Lethal and sublethal doses of okadaic acid (OA), a representative component of DSP toxins, were inoculated intraperitoneally into mice. Body-temperature changes over time were measured by an electronic thermometer or monitored by an infrared camera. Drastic hypothermia (<30°C in some mice) was observed in a few hours after administration of a lethal dose of OA. Dose-dependency was clearly seen between doses of OA inoculated and body-temperature decrease. Drastic hypothermia was also detected by using an infrared camera. These results suggest that hypothermia could be used as an index for the humane endpoint in experimental animal toxicological studies.

Shellfish contamination with marine biotoxins in Portugal and spring tides: a dangerous health coincidence.

Bivalve molluscs can acquire marine biotoxins by filter-feeding upon certain toxin-producing microalgae. The two most common syndromes observed in temperate coastal waters have been diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP). While DSP is a non-fatal gastrointestinal syndrome, PSP is a neurological syndrome which can lead to death by respiratory paralysis in high intoxication scenarios. In Portugal, the presence of both DSP and PSP toxins leads to recurrent seasonal bans of bivalve harvesting. On a few occasions, the bans were not placed in time, not properly disseminated to the public or were disregarded by recreational harvesters. Several cases of poisonings have been studied in collaboration between health authorities and the laboratory in charge of the biotoxin monitoring programme. Some of the outbreaks have even called the attention of the local media. In several of these recorded cases, a common trait has emerged throughout the years: bivalve harvest had often been done during very low tides attributed to either new or full moons. These tides expose intertidal bivalves more widely, increase harvesting time, and allow picking of larger-sized specimens. In some occasions, the consumers were extremely unfortunate because a noxious coincidence had occurred: larger-sized specimens were available but had attained the highest toxin content of the toxic season. This review alerts that despite costly monitoring programmes have been perfected through the years, human poisonings still take place due to the rapid increase in toxin levels and/or disrespect of harvest bans.

First detection of pectenotoxin-2 in shellfish associated with an intense spring bloom of Dinophysis acuminata on the central Chilean coast

Diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins (PTX) produced by endemic species of the genus Dinophysis, mainly D. acuta and D. acuminata, pose a big threat to public health, artisanal fisheries and the aquaculture industry in Southern Chile. This work reports the first detection of lipophilic toxins, including pectenotoxin-2 (PTX-2) and gymnodimine-A (GYM-A), in hard razor clam (Tagelus dombeii) associated with an unprecedented spring bloom −38.4 × 103 cells L−1 in integrated hose sampler (0–10 m) – of Dinophysis acuminata in coastal waters of central Chile. The socio-economic challenges to small-scale fisheries are discussed. The study points to the pressing need for sound policies to face unexpected HAB event, probably due to biogeographical expansions, with a focus on fisheries management, participation of stakeholders, and development of adaptive capacities.