Paralytic Shellfish Poisoning (PSP) in Mussels from the Eastern Cantabrian Sea: Toxicity, Toxin Profile, and Co-Occurrence with Cyclic Imines

In the late autumn of 2018 and 2019, some samples taken by the official monitoring systems of Cantabria and the Basque Country were found to be paralytic shellfish poisoning (PSP)-positive using a mouse bioassay. To confirm the presence of PSP toxins and to obtain their profile, these samples were analyzed using an optimized version of the Official Method AOAC 2005.06 and using LC–MS/MS (HILIC). The presence of some PSP toxins (PSTs) in that geographical area (~600 km of coast) was confirmed for the first time. The estimated toxicities ranged from 170 to 983 µg STXdiHCl eq.·kg−1 for the AOAC 2005.06 method and from 150 to 1094 µg STXdiHCl eq.·kg−1 for the LC–MS/MS method, with a good correlation between both methods (r2 = 0.94). Most samples contained STX, GTX2,3, and GTX1,4, and some also had NEO and dcGTX2. All of the PSP-positive samples also contained gymnodimine A, with the concentrations of the two groups of toxins being significantly correlated. The PSP toxin profiles suggest that a species of the genus Alexandrium was likely the causative agent. The presence of gymnodimine A suggests that A. ostenfeldii could be involved, but the contribution of a mixture of Alexandrium species cannot be ruled out.

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.

Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America

With the move away from use of mouse bioassay (MBA) to test bivalve mollusc shellfish for paralytic shellfish poisoning (PSP) toxins, countries around the world are having to adopt non-animal-based alternatives that fulfil ethical and legal requirements. Various assays have been developed which have been subjected to single-laboratory and multi-laboratory validation studies, gaining acceptance as official methods of analysis and approval for use in some countries as official control testing methods. The majority of validation studies conducted to date do not, however, incorporate shellfish species sourced from Latin America. Consequently, this study sought to investigate the performance of five alternative PSP testing methods together with the MBA, comparing the PSP toxin data generated both qualitatively and quantitatively. The methods included a receptor binding assay (RBA), two liquid chromatography with fluorescence detection (LC-FLD) methods including both pre-column and post-column oxidation, liquid chromatography with tandem mass spectrometry (LC-MS/MS) and a commercial lateral flow assay (LFA) from Scotia. A total of three hundred and forty-nine shellfish samples from Argentina, Mexico, Chile and Uruguay were assessed. For the majority of samples, qualitative results compared well between methods. Good statistical correlations were demonstrated between the majority of quantitative results, with a notably excellent correlation between the current EU reference method using pre-column oxidation LC-FLD and LC-MS/MS. The LFA showed great potential for qualitative determination of PSP toxins, although the findings of high numbers of false-positive results and two false negatives highlighted that some caution is still needed when interpreting results. This study demonstrated that effective replacement methods are available for countries that no longer wish to use the MBA, but highlighted the importance of comparing toxin data from the replacement method using local shellfish species of concern before implementing new methods in official control testing programs.

Paralytic Shellfish Toxins in Surf Clams Mesodesma donacium during a Large Bloom of Alexandrium catenella Dinoflagellates Associated to an Intense Shellfish Mass Mortality

In late February 2016, a harmful algal bloom (HAB) of Alexandrium catenella was detected in southern Chiloé, leading to the banning of shellfish harvesting in an extended geographical area (~500 km). On April 24, 2016, this bloom produced a massive beaching (an accumulation on the beach surface of dead or impaired organisms which were drifted ashore) of surf clams Mesodesma donacium in Cucao Bay, Chiloé. To determine the effect of paralytic shellfish poisoning (PSP) toxins in M. donacium, samples were taken from Cucao during the third massive beaching detected on May 3, 2016. Whole tissue toxicity evidence a high interindividual variability with values which ranged from 1008 to 8763 μg STX eq 100 g−1 and with a toxin profile dominated by GTX3, GTX1, GTX2, GTX4, and neoSTX. Individuals were dissected into digestive gland (DG), foot (FT), adductor muscle (MU), and other body fractions (OBF), and histopathological and toxin analyses were carried out on the obtained fractions. Some pathological conditions were observed in gill and digestive gland of 40–50% of the individuals that correspond to hemocyte aggregation and haemocytic infiltration, respectively. The most toxic tissue was DG (2221 μg STX eq 100 g−1), followed by OBF (710 μg STX eq 100 g−1), FT (297 μg STX eq 100 g−1), and MU (314 μg STX eq 100 g−1). The observed surf clam mortality seems to have been mainly due to the desiccation caused by the incapability of the clams to burrow. Considering the available information of the monitoring program and taking into account that this episode was the first detected along the open coast of the Pacific Ocean in southern Chiloé, it is very likely that the M. donacium population from Cucao Bay has not had a recurrent exposition to A. catenella and, consequently, that it has not been subjected to high selective pressure for PSP resistance. However, more research is needed to determine the effects of PSP toxins on behavioral and physiological responses, nerve sensitivity, and genetic/molecular basis for the resistance or sensitivity of M. donacium.

Multi-Toxin Occurrences in Ten French Water Resource Reservoirs

Cyanobacteria are known to produce a wide array of metabolites, including various classes of toxins. Among these, hepatotoxins (Microcystins), neurotoxins (Anatoxin-A and PSP toxins) or cytotoxins (Cylindrospermopsins) have been subjected to numerous, individual studies during the past twenty years. Reports of toxins co-occurrences, however, remain scarce in the literature. The present work is an inventory of cyanobacteria with a particular focus on Nostocales and their associated toxin classes from 2007 to 2010 in ten lakes used for drinking water production in France. The results show that potential multiple toxin producing species are commonly encountered in cyanobacteria populations. Individual toxin classes were detected in 75% of all samples. Toxin co-occurrences appeared in 40% of samples as two- or three-toxin combinations (with 35% for the microcystins–anatoxin combination), whereas four-toxin class combinations only appeared in 1% of samples. Toxin co-occurrences could be partially correlated to species composition and water temperature. Peak concentrations however could never be observed simultaneously and followed distinct, asymmetrical distribution patterns. As observations are the key for preventive management and risk assessment, these results indicate that water monitoring should search for all four toxin classes simultaneously instead of focusing on the most frequent toxins, i.e., microcystins.

Allelopathic Effects of Alexandium spp. on Growth of Prorocentrum donghaiense Lu: Is It Toxin Dependent?

The allelopathic properties of three Alexandrium species: A tamarense (ADHK), A. minimus (AM-1) and A. catenella (ACDH), on one species of dinoflagellate Prorocentrum donghaiense were determined using bioassays of allelopathic test. Their culture filtrates were prepared at different growth phases of exponential (EP) and stationary one (SP), and were applied for the cultivation of P. donghaiense in the co-culture. Results revealed that all the culture filtrates presented growth inhibition on the co-cultured P. donghaiense, among which ADHK presented the most severe inhibition. Culture filtrates at SP showed stronger inhibition as compard to that in EP, and the dilution of the culture filtrates could alleviate their negative effects. Analysis on hemolytic activity presented that all of the Alexandrum species had hemolytic activity, which was consistent with their reported PSP toxins. It was thus speculated that the toxin-independent allelochemical(s) were involved in the noxious effects of Alexandrum spp. on P. donghaiense.