Specification of the Okadaic Acid Equivalent for Okadaic Acid, Dinophysistoxin-1, and Dinophysistoxin-2 Based on Protein Phosphatase 2A Inhibition and Cytotoxicity Assays Using Neuro 2A Cell Line

Diarrhetic shellfish poisoning (DSP) is a globally occurring disease threatening public health and trade. The causative toxins, okadaic acid (OA), dinophysistoxin-1 (DTX1), and dinophysistoxin-2 (DTX2) are collectively called OAs, and are quantified using the LC-MS/MS method. The hazardous effect of total OAs is expressed as the sum of OA equivalents defined for respective OAs based on mouse lethality, produced by either intraperitoneal (OAip) or oral administration (OAor). OAs are potent inhibitors of protein phosphatase 2A (PP2A) and are cytotoxic, necessitating expansion of the concept of OA equivalents to all relevant bioactivities. In this study, we determined OA equivalents for respective OA members in PP2A inhibition and cytotoxicity assays. To secure result credibility, we used certified OAs, reference materials, and PP2A produced using genetic engineering. The relative ratio of the OA equivalents determined by PP2A inhibition assays for OA, DTX1, and DTX2 were 1.0:1.6:0.3, while the ratio determined using the cytotoxicity assays indicated 1.0:1.5:0.5. OA equivalents showed a similar tendency in the PP2A inhibition and cytotoxicity assays, and matched better with oral toxicity data than intraperitoneal toxicity in mice. The PP2A inhibition assay, which measures the core activity of the OAs, suggested a higher OA equivalent for DTX1 than that currently used.

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.

New evidence of pectenotoxins in farmed bivalve molluscs from Sardinia (Italy)

Several planktonic dinoflagellates can produce lipophilic phycotoxins that represent a significant threat to public health as well as to shellfish and fish farming. Poisoning related to some of these toxins is categorised as diarrhetic shellfish poisoning. We analysed 975 shellfish samples from Tortolì in the central-eastern region of Sardinia (Italy) from January 2016 to March 2020, to investigate the prevalence of different lipophilic marine biotoxins in mollusc bivalves. The results highlighted the predominant presence of toxins belonging to the okadaic acid group in all samples with toxin concentrations exceeding legal limits, and revealed the new occurrence of pectenotoxins in oysters and clams with a winter seasonality in recent years. The origin of shellfish toxicity was associated with the same Dinophysis species, mainly D. acuminata. Based on both these results and other precedents, monitoring and recording systems are strongly recommended.

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.

Morphology and Phylogenetics of Benthic Prorocentrum Species (Dinophyceae) from Tropical Northwestern Australia

Approximately 70 species of Prorocentrum are known, of which around 30 species are associated with benthic habitats. Some produce okadaic acid (OA), dinophysistoxin (DTX) and their derivatives, which are involved in diarrhetic shellfish poisoning. In this study, we isolated and characterized Prorocentrum concavum and P. malayense from Broome in north Western Australia using light and scanning electron microscopy as well as molecular sequences of large subunit regions of ribosomal DNA, marking the first record of these species from Australian waters. The morphology of the motile cells of P. malayense was similar to P. concavum in the light microscopy, but differed by the smooth thecal surface, the pore pattern and the production of mucous stalk-like structures and a hyaline sheath around the non-motile cells. P. malayense could also be differentiated from other closely related species, P. leve and P. foraminosum, despite the similarity in thecal surface and pore pattern, by its platelet formula and morphologies. We tested the production of OA and DTXs from both species, but found that they did not produce detectable levels of these toxins in the given culturing conditions. This study aids in establishing more effective monitoring of potential harmful algal taxa in Australian waters for aquaculture and recreational purposes.

Outbreak of diarrhetic shellfish poisoning associated with consumption of mussels, United Kingdom, May to June 2019

We report on six cases of diarrhetic shellfish poisoning following consumption of mussels harvested in the United Kingdom. Dinophysis spp. in the water column was found to have increased rapidly at the production site resulting in high levels of okadaic acid-group lipophilic toxins in the flesh of consumed mussels. Clinicians and public health professionals should remain aware of algal-derived toxins being a potential cause of illness following seafood consumption.

Morphology, molecular phylogeny and toxinology of Coolia and Prorocentrum strains isolated from the tropical South Western Atlantic Ocean

The morphology, molecular phylogeny and toxinology of two Coolia and one Prorocentrum dinoflagellate strains from Brazil were characterized. They matched with Coolia malayensis and Coolia tropicalis morphotypes, while the Prorocentrum strain fitted well with the morphology of Prorocentrum emarginatum. Complementary identification by molecular analyses was carried out based on LSU and ITS-5.8S rDNA. Phylogenetic analyses of Coolia strains (D1/D2 region, LSU rDNA), showed that C. malayensis (strain UFBA044) segregated together with sequences of this species from other parts of the world, but diverged earlier in a separate branch to sequences from São Paulo (Brazil) or Caribbean areas. Coolia tropicalis (strain UFBA055) grouped with other sequences of this species, in a subclade with an isolate from Belize, closer to a subgroup including isolates from Thailand, Australia and Hong Kong. Phylogenetic analyses (ITS-5.8S rDNA) of P. emarginatum (strain UFBA033) grouped together with another sequence of this species from China Sea. Diarrhetic shellfish poisoning toxins (OA, DTXs and PTX2) were not detected in P. emarginatum by mass spectrometry analyses. However, hemolytic assays in P. emarginatum and both Coolia strains in this study showed positive results.