The sxt Gene and Paralytic Shellfish Poisoning Toxins as Markers for the Monitoring of Toxic Alexandrium Species Blooms

Paralytic shellfish poisoning (PSP) is a serious human illness caused by the ingestion of seafood contaminated with saxitoxin and its derivatives (STXs). These toxins are produced by some species of marine dinoflagellates within the genus Alexandrium. In the Mediterranean Sea, toxic Alexandrium spp. blooms, especially of A. minutum, are frequent and intense with negative impact to coastal ecosystem, aquaculture practices and other economic activities. We conducted a large scale study on the sxt gene and toxin distribution and content in toxic dinoflagellate A. minutum of the Mediterranean Sea using both quantitative PCR (qPCR) and HILIC-HRMS techniques. We developed a new qPCR assay for the estimation of the sxtA1 gene copy number in seawater samples during a bloom event in Syracuse Bay (Mediterranean Sea) with an analytical sensitivity of 2.0 × 10° sxtA1 gene copy number per reaction. The linear correlation between sxtA1 gene copy number and microalgal abundance and between the sxtA1 gene and STX content allowed us to rapidly determine the STX-producing cell concentrations of two Alexandrium species in environmental samples. In these samples, the amount of sxtA1 gene was in the range of 1.38 × 105 − 2.55 × 108 copies/L and the STX concentrations ranged from 41−201 nmol/L. This study described a potential PSP scenario in the Mediterranean Sea.Versión del editor5,228

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

Palytoxins: Chemistry and Detection

Palytoxin is one of the most potent non protein marine toxins known and it is suspected to cause severe, and sometimes even lethal intoxications in humans. Its origin has long been controversial and different potential production sources have been proposed. Nowadays, dinoflagellates belonging to the genus Ostreopsis are the most accredited producers of palytoxins. Since the isolation of palytoxin in 1971, a number of palytoxin congeners have been isolated so far from different biological sources. In this chapter the main aspects of chemistry of palytoxins are reported

Palytoxins from marine coastal environments and home aquaria. What role do they play in human inhalatory poisonings?

Since the late 90s’, hundreds of cases of respiratory illness and/or dermatitis have been repeatedly recorded in people concomitantly with massive proliferations of the benthic dinoflagellate Ostreopsis cf. ovata in the Mediterranean area. Thanks to development of a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, we have characterized O. cf. ovata as a producer of congeners of palytoxin, a highly potent toxin whose inhalation hazard is however unknown. On the basis of the concomitance of Ostreopsis blooms, respiratory illness in humans, and detection of palytoxin congeners in algal samples, a cause and effect relationship between the cases of malaise and the algal toxins has been postulated but never substantiated. Further cases of respiratory illness tentatively attributed to palytoxins have been reported for aquarium hobbyists from incidental inhalation of steams generated during cleaning operations of home aquaria containing soft corals belonging to Palythoa genus. The only common feature between Palythoa spp. and Ostreopsis spp. is that both, although phylogenetically distinct, may produce palytoxins. The study reported herein serves the double purpose of demonstrating the presence of palytoxins in home marine aquaria involved in several inhalatory poisonings and of correlating the symptoms shown by patients while handling Palythoa spp. in home aquaria with those reported for Ostreopsis-related poisonings. From the chemical and symptomatological data, it is reasonable to hold palytoxins responsible for respiratory disorders following inhalation. Although the exact mechanism through which palytoxin congeners from Palythoa spp. and Ostreopsis spp. exert toxicity by inhalation is still unknown, this represents a step toward inhalatory risk assessment of palytoxin congeners in domestic and open-air environments

LC-HRMSn versus LC-MS/MS of Paralytic Shellfish Poisoning toxins and tetrodotoxin in microalgae and seafood

Paralytic shellfish poisoning toxins (PST) are a group of neurotoxins produced by dinoflagellate belonging to genera Alexandrium and Gymnodinium. The accumulation of these toxins in seafood represent a major concern to humans since following ingestion of contaminated seafood human poisonings with neurological symptomatology may occur. Consequently, official control monitoring of these toxins is carried out in Europe. Tetrodotoxin (TTX) is a neurotoxin firstly detected in fish parrotfish and triggerfish; it has been recently found in European seafood (Spain, Portugal, England, Greece, etc). Due to its chemical similarity with PST, TTX may be co-extracted with any of the extraction procedures currently used for PST. Thus, a method that allows the combined analysis of PST and TTX is highly desirable. The aim of this work was to set up a method based on hydrophilic interaction liquid chromatography coupled to high resolution multiple stage mass spectrometry (HILIC-HRMSn) for the combined determination of PST and TTX in different matrices. The method has been successfully applied to the analysis of real samples, including marine phytoplankton, algal cultures of Alexandrium minutum, A. pacificum and mussel extracts collected in the frame of a monitoring study of these toxins in the Mediterranean Sea carried out from 2012 to nowadays. Different HILIC columns were tested and results obtained by HILIC-HRMSn on an LTQ Orbitrap XL were compared with those obtained by HILIC-MS/MS on a triple quadrupole MS

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

Identification of Palytoxin–Ca²⁺ Complex by NMR and Molecular Modeling Techniques

More than 40 years after its isolation, the understanding of how palytoxin interacts with biological systems has yet to be fully determined. The Na⁺,K⁺-ATPase pump constitutes a molecular receptor for palytoxin that is able to convert the pump into an open channel, with consequent loss of cellular K⁺ and remarkable rise of cytosolic Na⁺ levels. In addition, a slight permeability to Ca²⁺ is detected when palytoxin binds to the pump. It has been demonstrated that the increase of cytosolic free Ca²⁺ concentration gives rise to downstream events ultimately leading to cell death. The widely accepted recognition of the dependence of important cellular events on calcium ion concentration propelled us to investigate the occurrence of palytoxin–Ca²⁺ complex in aqueous solution by NMR- and molecular modeling-based approach. We identified two specific regions of palytoxin where Ca²⁺ is preferentially coordinated. This study constitutes the first characterization of a calcium complex with palytoxin and, as such, is expected to support the investigation of the toxin molecular bioactivity

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

Paralytic Shellfish Poisoning toxins from Mediterranean Alexandrium minutum and A. catenella: toxin profile and sxt gene content

Paralytic shellfish poisoning (PSP) toxins are potent water-soluble neurotoxins including the parent compound saxitoxin (STX) and a number of its congeners. They are tetrahydropurine derivatives that can be subdivided into three main groups according to substitution of the side chain: carbamoyl-, N-sulfocarbamoyl-, and decarbamoyl-toxins. The carbamoyl derivatives (STX, NEO and GTX1-4) are reported to be the most potent. Due to their accumulation in filter feeding shellfish, PSP toxins can move through the food chain inducing a toxic syndrome in seafood consumers. Symptoms are neurological with rapid onset (30-60 min from ingestion) and include paraesthesia, vertigo, numbness, tingling of the face, tongue, and lip, ataxia, blocking of respiration and even death. Due to the high risk posed to human health by PSP toxins, a multidisciplinary integrated approach based on liquid chromatography high resolution mass spectrometry (LC-HRMS and MS2) and qPCR-based assay has been used to depict the PSP toxin scenario in the Mediterranean Sea. As the sxtA and the sxtG genes are known as the starting genes of PSP toxin synthesis in dinoflagellates, different populations of the Mediterranean A. minutum from NW Adriatic, Ionian, Tyrrhenian and Catalan Seas were grown in culture and analyzed by qPCR in order to obtain the quantification of these genes. In parallel, LC-HRMS2 analyses were performed on the A. minutum cultured strains and revealed for all of them a toxin profile consisting of only GTX1 and GTX4. Toxin production was in the fg/cell range. Concomitantly with a massive bloom of A. minutum and A. catenella that occurred in Spring 2014 along the Syracuse coasts (Sicily, Italy), four seawater samples were collected and analyzed by LC-HRMS and MS2. The analyzed extracts were found to contain a variety of PSP toxins, namely STX, NEO, the gonyautoxins GTX1-4, the N-sulfocarbamoyl derivatives C1/C2, B1 and B2 and the decarbamoyl ST

Untargeted and targeted LC-MS and data processing workflow for the comprehensive analysis of oligopeptides from cyanobacteria

Cyanobacteria produce a plethora of structurally diverse bioactive secondary metabolites, including cyanotoxins which pose a serious threat to humans and other living organisms worldwide. Currently, a wide variety of mass spectrometry-based methods for determination of microcystins (MCs), the most commonly occurring and studied class of cyanotoxins, have been developed and employed for research and monitoring purposes. The scarcity of commercially available reference materials, together with the ever-growing range of mass spectrometers and analytical approaches, make the accuracy of quantitative analyses a critical point to be carefully investigated in view of a reliable risk evaluation. This study reports, a comparative investigation of the qualitative and quantitative MCs profile obtained using targeted and untargeted liquid chromatography–mass spectrometry approaches for the analyses of cyanobacterial biomass from Lake Kastoria, Greece. Comparison of the total MCs content measured by the two approaches showed good correlation, with variations in the range of 3.8–13.2%. In addition, the implementation of an analytical workflow on a hybrid linear ion trap/orbitrap mass spectrometer is described, based on combining data-dependent acquisition and a powerful database of cyanobacterial metabolites (CyanoMetDB) for the annotation of known and discovery of new cyanopeptides. This untargeted strategy proved highly effective for the identification of MCs, microginins, anabaenopeptins, and micropeptins. The systematic interpretation of the acquired fragmentation patterns allowed the elucidation of two new MC structural variants, MC-PrhcysR and MC-Prhcys(O)R, and proposal of structures for two new microginins, isomeric cyanostatin B and MG 821A, and three isomeric micropeptins at m/z 846.4715, 846.4711 and 846.4723