Optimization of a rapid QuEChERS sample treatment method for HILIC-MS2 analysis of paralytic shellfish poisoning (PSP) toxins in mussels

A rapid and simple QuEChERS sample treatment was proposed for the development of a selective hydrophilic interaction liquid chromatography-ESI-MS2-based method for the determination of saxitoxins (STXs) in mussel samples. Among different sorbents, ABS Elut-NEXUS phase, composed of polystyrene cross-linked with 50 % divinyl benzene and poly(methyl methacrylate), provided the best results. The effects of experimental parameters, including sorbent amount, vortexing time and centrifugation time were investigated and optimized by experimental design. In particular, regression models and desirability functions were applied to find the experimental conditions providing the highest global extraction response. The method was validated under the optimized conditions; detection and quantification limits in the 3-159 μg/kg and 7-436 μg/kg ranges respectively were obtained, except for C2 for which highest values were calculated due to its low ESI ionization efficiency. Finally, the analysis of twenty-eight mussel samples permitted to detect and quantify some of the investigated STXs, proving the applicability of the devised method

RNA sequencing and de novo assembly of the digestive gland transcriptome in Mytilus galloprovincialis fed with toxinogenic and non-toxic strains of Alexandrium minutum

Background The Mediterranean mussel Mytilus galloprovincialis is marine bivalve with a relevant commercial importance as well as a key sentinel organism for the biomonitoring of environmental pollution. Here we report the RNA sequencing of the mussel digestive gland, performed with the aim: a) to produce a high quality de novo transcriptome assembly, thus improving the genetic and molecular knowledge of this organism b) to provide an initial assessment of the response to paralytic shellfish poisoning (PSP) on a molecular level, in order to identify possible molecular markers of toxin accumulation. Results The comprehensive de novo assembly and annotation of the transcriptome yielded a collection of 12,079 non-redundant consensus sequences with an average length of 958 bp, with a high percentage of full-length transcripts. The whole-transcriptome gene expression study indicated that the accumulation of paralytic toxins produced by the dinoflagellate Alexandrium minutum over a time span of 5 days scarcely affected gene expression, but the results need further validation with a greater number of biological samples and naturally contaminated specimens. Conclusion The digestive gland reference transcriptome we produced significantly improves the data collected from previous sequencing efforts and provides a basic resource for expanding functional genomics investigations in M. galloprovincialis. Although not conclusive, the results of the RNA-seq gene expression analysis support the classification of mussels as bivalves refractory to paralytic shellfish poisoning and point out that the identification molecular biomarkers of PSP in the digestive gland of this organism is problematic

Marine phycotoxin levels in shellfish-14 years of data gathered along the Italian coast

Along the Italian coasts, toxins of algal origin in wild and cultivated shellfish have been reported since the 1970s. In this study, we used data gathered by the Veterinary Public Health Institutes (IZS) and the Italian Environmental Health Protection Agencies (ARPA) from 2006 to 2019 to investigate toxicity events along the Italian coasts and relate them to the distribution of potentially toxic species. Among the detected toxins (OA and analogs, YTXs, PTXs, STXs, DAs, AZAs), OA and YTX were those most frequently reported. Levels exceeding regulatory limits in the case of OA (≤2,448 μg equivalent kg-1) were associated with high abundances of Dinophysis spp., and in the case of YTXs (≤22 mg equivalent kg-1) with blooms of Gonyaulax spinifera, Lingulodinium polyedra, and Protoceratium reticulatum. Seasonal blooms of Pseudo-nitzschia spp. occur all along the Italian coast, but DA has only occasionally been detected in shellfish at concentrations always below the regulatory limit (≤18 mg kg-1). Alexandrium spp. were recorded in several areas, although STXs (≤13,782 μg equivalent kg-1) rarely and only in few sites exceeded the regulatory limit in shellfish. Azadinium spp. have been sporadically recorded, and AZAs have been sometimes detected but always in low concentrations (≤7 μg equivalent kg-1). Among the emerging toxins, PLTX-like toxins (≤971 μg kg-1 OVTX-a) have often been detected mainly in wild mussels and sea urchins from rocky shores due to the presence of Ostreopsis cf. ovata. Overall, Italian coastal waters harbour a high number of potentially toxic species, with a few HAB hotspots mainly related to DSP toxins. Nevertheless, rare cases of intoxications have occurred so far, reflecting the whole Mediterranean Sea conditions

Toxin Levels and Profiles in Microalgae from the North-Western Adriatic Sea—15 Years of Studies on Cultured Species

The Northern Adriatic Sea is the area of the Mediterranean Sea where eutrophication and episodes related to harmful algae have occurred most frequently since the 1970s. In this area, which is highly exploited for mollusk farming, the first occurrence of human intoxication due to shellfish consumption occurred in 1989, nearly 10 years later than other countries in Europe and worldwide that had faced similar problems. Until 1997, Adriatic mollusks had been found to be contaminated mostly by diarrhetic shellfish poisoning toxins (i.e., okadaic acid and dinophysistoxins) that, along with paralytic shellfish poisoning toxins (i.e., saxitoxins), constitute the most common marine biotoxins. Only once, in 1994, a toxic outbreak was related to the occurrence of paralytic shellfish poisoning toxins in the Adriatic coastal waters. Moreover, in the past 15 years, the Adriatic Sea has been characterized by the presence of toxic or potentially toxic algae, not highly widespread outside Europe, such as species producing yessotoxins (i.e., Protoceratium reticulatum, Gonyaulax spinifera and Lingulodinium polyedrum), recurrent blooms of the potentially ichthyotoxic species Fibrocapsa japonica and, recently, by blooms of palytoxin-like producing species of the Ostreopsis genus. This review is aimed at integrating monitoring data on toxin spectra and levels in mussels farmed along the coast of the Emilia-Romagna region with laboratory studies performed on the species involved in the production of those toxins; toxicity studies on toxic or potentially toxic species that have recently appeared in this area are also reviewed. Overall, reviewed data are related to: (i) the yessotoxins producing species P. reticulatum, G. spinifera and L. polyedrum, highlighting genetic and toxic characteristics; (ii) Adriatic strains of Alexandrium minutum, Alexandrium ostenfeldii and Prorocentrum lima whose toxic profiles are compared with those of strains of different geographic origins; (iii) F. japonica and Ostreopsis cf. ovata toxicity. Moreover, new data concerning domoic acid production by a Pseudo-nitzschia multistriata strain, toxicity investigations on a Prorocentrum cf. levis, and on presumably ichthyotoxic species, Heterosigma akashiwo and Chattonella cf. subsalsa, are also reported

A new therapy for transthyretin amyloidosis, no longer an orphan condition

Amyloid cardiomyopathy is a condition characterized by intra-myocardial deposit of protein-like material, in fibrillar shape (amyloid), which presence determine a progressive thickening and stiffening of the cardiac walls leading to a cardiac dysfunction. The proteins most often involved with cardiac amyloid are the light chains of the immunoglobulin, typical of amyloidosis AL, and transthyretin, responsible for transthyretin amyloidosis, in both its forms, hereditary and wild type. An accurate estimate of the incidence of cardiac amyloidosis is still difficult due to the variety and complexity of the clinical presentation of the condition. Nonetheless, the condition has stimulated the interest of the scientific community, so that a specific diagnostic path has been developed, beginning from the clinical suspicion and first-line testing, such as electrocardiogram, echocardiogram, and blood work, to progress to the diagnostic confirmation using more sophisticated testing such as magnetic resonance, scintiscan, and eventually cardiac biopsy. To understand and recognize this condition is very important, stemming from the availability of 'aetiology oriented therapies' (designed to prevent, control and possibly regress amyloid deposition), which should be added to the 'supportive therapies', used for the treatment of the complication of the condition, namely heart failure

Development of a data dependent acquisition-based approach for the identification of unknown fast-acting toxins and their ester metabolites

Phycotoxins in the marine food-web represent a serious threat to human health. Consumption of contaminated shellfish and/or finfish poses risk to consumer safety: several cases of toxins-related seafood poisoning have been recorded so far worldwide. Cyclic imines are emerging lipophilic toxins, which have been detected in shellfish from different European countries. Currently, they are not regulated due to the lack of toxicological comprehensive data and hence the European Food Safety Authority has required more scientific efforts before establishing a maximum permitted level in seafood. In this work, a novel data dependent liquid chromatography – high resolution mass spectrometry (LC-HRMS) approach has been successfully applied and combined with targeted studies for an in-depth investigation of the metabolic profile of shellfish samples. The proposed analytical methodology has allowed: i) to discover a plethora of unknown fatty acid esters of gymnodimines and ii) to conceive a brand new MS-based strategy, termed as backward analysis, for discovery and identification of new analogues. In particular, the implemented analytical workflow has broadened the structural diversity of cyclic imine family through the inclusion of five new congeners, namely gymnodimine –F, -G, –H, –I and -J. In addition, gymnodimine A (376.5 μg/kg), 13-desmethyl spirolide C (11.0–29.0 μg/kg) and pinnatoxin G (3.1–7.7 μg/kg) have been detected in shellfish from different sites of the Mediterranean basin (Tunisia and Italy) and the Atlantic coast of Spain, with the confirmation of the first finding of pinnatoxin G in mussels harvested in Sardinia (Tyrrhenian Sea, Italy)

Marine Biotoxins: Occurrence, Toxicity, Regulatory Limits and Reference Methods

Harmful algal blooms are natural phenomena caused by the massive growth of phytoplankton that may contain highly toxic chemicals, the so-called marine biotoxins causing illness and even death to both aquatic organisms and humans. Their occurrence has been increased in frequency and severity, suggesting a worldwide public health risk. Marine biotoxins can accumulate in bivalve molluscs and regulatory limits have been set for some classes according to European Union legislation. These compounds can be distinguished in water- and fat-soluble molecules. The first group involves those of Paralytic Shellfish Poisoning and Amnesic Shellfish Poisoning, whereas the toxins soluble in fat can cause Diarrheic Shellfish Poisoning and Neurotoxic Shellfish Poisoning. Due to the lack of long-term toxicity studies, establishing tolerable daily intakes for any of these marine biotoxins was not possible, but an acute reference dose can be considered more appropriate, because these molecules show an acute toxicity. Dietary exposure assessment is linked both to the levels of marine biotoxins present in bivalve molluscs and the portion that could be eaten by consumers. Symptoms may vary from a severe gastrointestinal intoxication with diarrhea, nausea, vomiting, and abdominal cramps to neurological disorders such as ataxia, dizziness, partial paralysis, and respiratory distress. The official method for the detection of marine biotoxins is the mouse bioassay (MBA) showing some limits due to ethical restrictions and insufficient specificity. For this reason, the liquid chromatography-mass spectrometry method has replaced MBA as the reference technique. However, the monitoring of algal blooms producing marine biotoxins should be regularly assessed in order to obtain more reliable, accurate estimates of bloom toxicity and their potential impacts

Tetrodotoxin an Emerging Threat to Humans in the Mediterranean Area: First Detection in Italian Mussels

Tetrodotoxin (TTX) is one of the most potent neurotoxins, originally found in ovary and liver of pufferfish (Tetraodontidae) [1]. Successively, TTX was isolated from many other marine and terrestrial animals, as xanthid crab, trumpet shellfish, blue-ringed octopus, chaetognatha, gastropods, starfish, and frogs. The wide distribution of TTX in genetically unrelated organisms has made TTX origin for long time controversial, with different kind of bacteria being identified as TTX-producing organisms [2]. Even Alexandrium tamarense – one of the paralytic shellfish poisoning toxins (PST) producing organisms – was proposed as potential biogenetic source of TTX [3]. Although fatal human poisonings following consumption of TTX-contaminated seafood have been reported so far only in Japan, the accumulation of TTX in fish, oysters and mussels collected in Europe (Spain, Portugal, England, Greece) has been recently reported. So, in the frame of a collaborative study on evaluation of PST-related risk in the Mediterranean area, mussels collected in the Siracuse bay (Sicily, Italy) over a three year period (2015-2017), were analyzed by hydrophilic interaction liquid chromatography coupled with both high resolution and tandem mass spectrometry (HILIC-HRMS and HILIC-MS/MS). Both techniques highligted the presence of high PST contamination levels, with samples collected in 2016 containing up to 10851 g STX eq/kg. Unexpectedly, together with PST, tetrodotoxin was detected in Sicilian mussels. Although this was the first report of TTX in Italy, contamination levels found in mussels (0.8-6.4 µg/kg) were well below the regulatory limit of 2 mg TTX eq/kg established for TTX in Japan. Interestingly, much higher contamination levels of TTX (413 g/kg) have been detected in mussels collected in 2017 in the northeastern Adriatic coasts of Italy (Lagoon of Marano), in the frame of the monitoring programme for marine biotoxins regulated in the EU. [1] Wu Z, Xie L, Xia G, Zhang J, Nie Y, Hu J, Wang S, Zhang R. 2005. A new tetrodotoxin-producing actinomycete, Norcardiopsis dassonvillei, isolated from the ovaries of puffer fish Fugu rubripes. Toxicon 45:851-859. [2] Yasumoto T, Yasumura D, Yotsu M, Michishita T, Endo A, Kotak Y. 1986. Bacterial production of tetrodotoxin and anhydrotetrodotoxin. Agric. Biol. Chem. 50:793–795. [3] Kodama M, Sato S, Sakamoto S, Ogata T. 1996. Occurrence of tetrodotoxin in Alexandrium tamarense, a causative dinoflagellate of paralytic shellfish poisoning. Toxicon. 34:1101-1105

First Detection of Tetrodotoxin in Italian Mussels. Is it an Emerging Threat to Humans in the Mediterranean Area?

Tetrodotoxin (TTX) is one of the most potent neurotoxins, originally found in ovary and liver of pufferfish (Tetraodontidae). Successively, TTX was isolated from many other marine and terrestrial animals, as xanthid crab, trumpet shellfish, blue-ringed octopus, chaetognatha, gastropods, starfish, and frogs. The wide distribution of TTX in genetically unrelated organisms has made TTX origin for long time controversial, with different kind of bacteria being identified as TTX-producing organisms. Even Alexandrium tamarense – one of the paralytic shellfish poisoning toxins (PST) producing organisms – was proposed as potential biogenetic source of TTX . Although fatal human poisonings following consumption of TTX-contaminated seafood have been reported so far only in Japan, the accumulation of TTX in fish, oysters and mussels collected in Europe (Spain, Portugal, England, Greece) has been recently reported. So, in the frame of a collaborative study on evaluation of PST-related risk in the Mediterranean area, mussels collected in the Siracuse bay (Sicily, Italy) over a three year period (2015-2017), were analyzed by hydrophilic interaction liquid chromatography coupled with both high resolution and tandem mass spectrometry (HILIC-HRMS and HILIC-MS/MS). Both techniques highligted the presence of high PST contamination levels, with samples collected in 2016 containing up to 10851 ug STX eq/kg. Unexpectedly, together with PST, tetrodotoxin was detected in Sicilian mussels. Although this was the first report of TTX in Italy, contamination levels found in mussels (0.8-6.4 µg/kg) were well below the regulatory limit of 2 mg TTX eq/kg established for TTX in Japan. Interestingly, much higher contamination levels of TTX (413 ug/kg) have been detected in mussels collected in 2017 in the northeastern Adriatic coasts of Italy (Lagoon of Marano), in the frame of the monitoring programme for marine biotoxins regulated in the EU

Monitoraggio di aree costiere interessate da fioriture di fitoplancton tossico: approcci molecolari e nuove proposte di controllo = Monitoring of coastal areas affected by HAB phytoplankton blooms: molecular approaches and new proposal for management

Molecular technologies of PCR were applied on seawater fixed samples and molluscs to reveal the presence of some potentially toxic phytoplankton taxa in the Mediterranean Sea. PCR assays on field samples permitted a rapid identification of different genera and species of HAB microalgae. The molecular techniques are able to discriminate HAB target cells at lower detection limit for the traditional microscopy methods. Species-specific PCR assay for HABs applied on molluscs permitted to reveal the presence of toxic cells also in absence of toxicity determined by chemical and mouse bioassay analyses