Immediate and delayed effects of a heatwave and Prorocentrum lima ((Ehrenberg) Stein 1878) bloom on the toxin accumulation, physiology, and survival of the oyster Magallana gigas (Thunberg, 1793).

Warming could facilitate the intensification of toxic algal blooms, two important stressors for marine organisms that are predicted to co-occur more frequently in the future. We investigated the immediate and delayed effects of a heatwave and a simulated bloom (3 × 106 cells L-1) of the diarrhetic shellfish toxin (DST)-producing benthic dinoflagellate Prorocentrum lima on the survival, physiology (oxygen consumption rate, condition index, immune parameters), and toxin accumulation in the Pacific rock oyster Magallana (Crassostrea) gigas. Oysters exposed to both stressors contained higher mean DST concentrations (mean ± 1 SE: 173.3 ± 19.78 µg kg-1 soft tissue) than those exposed to P. lima bloom alone (120.4 ± 20.90 µg kg-1) and exceeded the maximum permitted levels for human consumption. Exposure to individual stressors and their combination modified the physiology of M. gigas. Oysters exposed to heatwave alone had significantly higher oxygen consumption rates (0.7 ± 0.06 mg O2 h-1 g-1) than the control (0.3 ± 0.06 mg O2 h-1 g-1). However, this was not observed in oysters exposed to both heatwave and P. lima (0.5 ± 0.06 mg O2 h-1 g-1). This alteration of the metabolic response to warming in the presence of P. lima may affect the ability of rock oysters to adapt to environmental stressors (i.e., a heatwave) to ensure survival. Immunomodulation, through changes in total hemocyte count, was observed in oysters exposed to P. lima alone and in combination with warming. Individual stressors and their combination did not influence the condition index, but one mortality was recorded in oysters exposed to both stressors. The findings of this study highlight the vulnerability of rock oysters to the predicted increased frequency of heatwaves and toxic algal blooms, and the increased likelihood of shellfish containing higher than regulatory levels of DST in warming coasts.

New Trends in the Occurrence of Yessotoxins in the Northwestern Adriatic Sea.

Yessotoxins (YTXs) are polycyclic toxic ether compounds produced by phytoplanktonic dinoflagellates which accumulate in filter-feeding organisms. We know that the water temperature in our areas Northwestern Adriatic Sea is optimal for the growth of potentially toxic algae (around 20 °C). In recent years, these temperatures have remained at these levels for longer and longer periods, probably due to global warming, which has led to an excessive increase in toxin levels. The interruption of mussel harvesting caused by algae negatively affects farmers' revenues and the availability of local fish, causing a major economic loss in Italy's main shellfish sector. METHODS: In the nine years considered, 3359 samples were examined: 1715 marine waters, 73 common clams; 732 mussels; 66 oysters; and 773 veracious clams. Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters. The method adopted for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. The enumeration of phytoplankton cells was performed according to the Utermhöl method. RESULTS: Between 2012 and 2020, 706 molluscs were tested for YTXs. In total, 246 samples tested positive, i.e., 34.84%. Of the positive samples, 30 exceeded the legal limit. CONCLUSION: In this regard, it is essential to develop and activate, as soon as possible, an "early warning" system that allows a better control of the production areas of live bivalve molluscs, thus allowing an optimal management of the plants in these critical situations.

The occurrence of lipophilic toxins in shellfish from the Middle Adriatic Sea.

The first survey of the phycotoxin profile in mussels (Mytilus galloprovincialis) from the coastal waters of Bosnia and Herzegovina (The Bay of Neum, Middle Adriatic Sea) in correlation to the Makarska City Bay (Croatia, Middle Adriatic Sea) was conducted in 2017. Throughout the monitoring period, occasions of gymnodimine (GYM) and azaspiracid (AZA2) shellfish toxicity were recorded in concentrations that do not endanger human health. The occurrence of yessotoxins (YTXs), the most common toxins found in the Adriatic Sea, was correlated to the presence of the Gonyaulax species, a potential source of YTX. The DSP group of toxins is represented by the ester-OA. Phytoplankton analysis confirmed the presence of dinoflagellates from the Prorocentrum genus, a species associated with DSP toxicity. Occurrence frequency and variability of toxin composition were investigated in conjunction to physico-chemical parameters in the surrounding sea water. In the central Adriatic Sea, the infestation period ranges in general from June to August. However, the depuration phase extended beyond September in the Bay of Neum, increasing the length of the decontamination period.

Occurrence of lipophilic marine toxins in shellfish from Galicia (NW of Spain) and synergies among them.

Lipophilic marine toxins pose a serious threat for consumers and an enormous economic problem for shellfish producers. Synergistic interaction among toxins may play an important role in the toxicity of shellfish and consequently in human intoxications. In order to study the toxic profile of molluscs, sampled during toxic episodes occurring in different locations in Galicia in 2014, shellfish were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS), the official method for the detection of lipophilic toxins. The performance of this procedure was demonstrated to be fit for purpose and was validated in house following European guidelines. The vast majority of toxins present in shellfish belonged to the okadaic acid (OA) group and some samples from a particular area contained yessotoxin (YTX). Since these toxins occur very often with other lipophilic toxins, we evaluated the potential interactions among them. A human neuroblastoma cell line was used to study the possible synergies of OA with other lipophilic toxins. Results show that combination of OA with dinophysistoxin 2 (DTX2) or YTX enhances the toxicity triggered by OA, decreasing cell viability and cell proliferation, depending on the toxin concentration and incubation time. The effects of other lipophilic toxins as 13-desmethyl Spirolide C were also evaluated in vitro.

Transient isotachophoresis-capillary zone electrophoresis with contactless conductivity and ultraviolet detection for the analysis of paralytic shellfish toxins in mussel samples.

The accumulation of paralytic shellfish toxins (PSTs) in contaminated shellfish is a serious health risk making early detection important to improve shellfish safety and biotoxin management. Capillary electrophoresis (CE) has been proven as a high resolution separation technique compatible with miniaturization, making it an attractive choice in the development of portable instrumentation for early, on-site detection of PSTs. In this work, capillary zone electrophoresis (CZE) with capacitively coupled contactless conductivity detector (C(4)D) and UV detection were examined with counter-flow transient isotachophoresis (tITP) to improve the sensitivity and deal with the high conductivity sample matrix. The high sodium concentration in the sample was used as the leading ion while l-alanine was used as the terminating electrolyte (TE) and background electrolyte (BGE) in which the toxins were separated. Careful optimization of the injected sample volume and duration of the counter-flow resulted in limit of detections (LODs) ranging from 74.2 to 1020 ng/mL for tITP-CZE-C(4)D and 141 to 461 ng/mL for tITP-CZE-UV, an 8-97 fold reduction compared to conventional CZE. The LODs were adequate for the analysis of PSTs in shellfish samples close to the regulatory limit. Intra-day and inter-day repeatability values (percentage relative standard deviation, n=3) of tITP-CZE-C(4)D and tITP-CZE-UV methods for both migration time and peak height were in the range of 0.82-11% and 0.76-10%, respectively. The developed method was applied to the analysis of a contaminated mussel sample and validated against an Association of Official Analytical Chemists (AOAC)-approved method for PSTs analysis by high performance liquid chromatography (HPLC) with fluorescence detection (FLD) after pre-column oxidation of the sample. The method presented has potential for incorporation in to field-deployable devices for the early detection of PSTs on-site.

Tracing the origin of paralytic shellfish toxins in scallop Patinopecten yessoensis in the northern Yellow Sea.

Some dinoflagellate species within the genera Alexandrium, Gymnodinium and Pyrodinium are well-known producers of paralytic shellfish toxins (PST), which led to many poisoning incidents around the world. In the northern Yellow Sea, an important mariculture zone for scallop Patinopecten yessoensis, PST have been frequently detected from scallops. However, there is little knowledge concerning PST-producing microalgae in this region so far. In cruises carried out in 2011 and 2012, scallop and phytoplankton samples were collected from the northern Yellow Sea. PST were detected from scallops by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Toxin content and profile were remarkably different among the four tissues, i.e. viscera, adductor muscle, mantle and gonad, suggesting apparent toxin transfer and transformation in scallops. Viscera always had the highest content of PST dominated by low-potency N-sulfocarbamoyl toxins C1 and C2, which closely resembled the toxin profiles of net-concentrated phytoplankton samples in spring. Based on the morphological features, cells of Alexandrium spp. in net-concentrated phytoplankton samples were picked out and a partial sequence of the large subunit ribosomal RNA gene (LSU rDNA) was amplified using a single-cell polymerase chain reaction (PCR) method. Cells of both toxic A. tamarense species complex and non-toxic A. affine were identified from the phytoplankton samples based on the partial LSU rDNA sequence information. According to these findings, it is implied that A. tamarense species complex is the major toxic species related to PST contamination in scallops of the northern Yellow Sea. The presence of both toxic and non-toxic Alexandrium spp. in this region requires for a species-specific method to monitor the distribution and dynamics of A. tamarense species complex.

Detection of yessotoxin by three different methods in Mytilus galloprovincialis of Adriatic Sea, Italy.

Samples of Mytilus galloprovincialis collected from shellfish aquaculture plans located in the Adriatic Sea were analysed for yessotoxin (YTX) by three methods, in vivo (Mouse Bioassay, MBA), in vitro (functional assay) and chemical test (Liquid Chromatography-Mass Spectrometry, LC-MS/MS). As YTX coexists with other phycotoxins in shellfish, namely the diarrhoetic shellfish poisoning, okadaic acid, dinophysistoxins, pectenotoxins, and azaspirazids, the MBA is not completely satisfactory because it is difficult to identify which toxin causes the death of the mice. So, the two other techniques were proposed to detect and quantify YTX and its analogues in order to avoid this problem. The global results showed no difference among the three methods and the correlation between the functional assay and LC-MS/MS was positive (Spearman r=0.72). Both analytical methods demonstrated advantages; the functional assay is specific, very sensitive and correlates well with real toxicity, whereas LC-MS/MS is convenient because it allows the detection of YTX and some analogues which are currently included in the EU regulation. For this reason LC-MS/MS will become the official method starting 1st January 2015 (Regulation 15/2011/EU). Only four samples exceeded the current EU regulation limit of 1mg of YTX equivalent kg(-1). However, all samples belonged to a monitoring program and they were not suitable for consumers.

Analysis of a cone snail's killer cocktail--the milked venom of Conus geographus.

"Snails can kill" is a statement that receives much disbelief. Yet the venom from Conus geographus, as delivered by a disposable hypodermic-like needle, has indeed killed many unsuspecting human victims. Our understanding of their milked venom the essence of these fatalities, is in itself non-existent. Here, we present the molecular mass analysis of the milked venom of C. geographus, providing the first insight into the composition of its deadly cocktail.

Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach.

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.

A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus.

Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.

[Rapid biochemical method for the detection of paralytical shellfish poisoning toxins in shellfish from seafood market].

A rapid biochemical method was discussed in order to detect low concentration of paralytic shellfish poisoning (PSP) in sea food. The mice were injected with PSP extract of bivalves (1 and 0.2 microg/kg respectively, as STX equivalents) purchased from seafood market. ACh, AChE, NO and NOS in blood were studied at 15, 60, 120 min respectively. The results showed that at low dose (0.2 microg/kg) and 15 min, only the contents of ACh changed significantly compared with control group (p < 0.05), which was (141.2 +/- 14.8) microg/mg, while the contents of NO and the activities of NOS changed until 120 min, compared to control group (p < 0.05) ,which were (68.7 +/- 3.8) micromol/g and (40.1 +/- 4.9) U/mg respectively. At high dose the contents of ACh changed at all three time point. It can be suggested that the contents of ACh is the only one of four indexes which can response to PSP at low dose in an early stage (15 min) and may be selected as a biochemical index for rapid detection of PSP.

Venom on ice: first insights into Antarctic octopus venoms.

The venom of Antarctic octopus remains completely unstudied. Here, a preliminary investigation was conducted into the properties of posterior salivary gland (PSG) extracts from four Antarctica eledonine (Incirrata; Octopodidae) species (Adelieledone polymorpha, Megaleledone setebos, Pareledone aequipapillae, and Pareledone turqueti) collected from the coast off George V's Land, Antarctica. Specimens were assayed for alkaline phosphatase (ALP), acetylcholinesterase (AChE), proteolytic, phospholipase A(2) (PLA(2)), and haemolytic activities. For comparison, stomach tissue from Cirroctopus sp. (Cirrata; Cirroctopodidae) was also assayed for ALP, AChE, proteolytic and haemolytic activities. Dietary and morphological data were collected from the literature to explore the ecological importance of venom, taking an adaptive evolutionary approach. Of the incirrate species, three showed activities in all assays, while P. turqueti did not exhibit any haemolytic activity. There was evidence for cold-adaptation of ALP in all incirrates, while proteolytic activity in all except P. turqueti. Cirroctopus sp. stomach tissue extract showed ALP, AChE and some proteolytic activity. It was concluded that the AChE activity seen in the PSG extracts was possibly due to a release of household proteins, and not one of the secreted salivary toxins. Although venom undoubtedly plays an important part in prey capture and processing by Antarctica eledonines, no obvious adaptations to differences in diet or morphology were apparent from the enzymatic and haemolytic assays. However, several morphological features including enlarged PSG, small buccal mass, and small beak suggest such adaptations are present. Future studies should be conducted on several levels: Venomic, providing more detailed information on the venom compositions as well as the venom components themselves; ecological, for example application of serological or genetic methods in identifying stomach contents; and behavioural, including observations on capture of different types of prey.

TxXIIIA, an atypical homodimeric conotoxin found in the Conus textile venom.

Venoms of predatory Conus snails are composed of several hundreds of peptide toxins. Many of these peptides display a high selectivity for particular membrane receptors such as ionic channels or G-protein coupled receptors. This property makes them very promising tools for the study of receptors and potential new drugs. Conus snails synthesize toxins under various folds, each fold related to particular pharmacological activities. Aiming the discovery of new conotoxins, we looked for toxins with original fold in the Conus textile venom by offline LC-MALDI-TOF/TOF mass spectrometry. Venom fractions were analysed by MALDI-TOF (in 2,5-dihydroxybenzoic acid) before and after the "in-solution" reduction of the disulfide bridges. Comparison of the spectra allows the classification of a large number of conotoxins according to the number of disulfide bridges. We focussed on a component at m/z 2785.7 (non-reduced)/ 1398.4 (reduced), which might represent a novel type of homodimeric toxin. The sequence TSDCCFYHNCCC was determined by De novo sequencing on the reduced species and represent a new fold. This sequence has already been described as the C-terminus part of a conotoxin scaffold IX precursor (expasy: Q9BPH1) but the power of our study resides in the fact that mass spectrometry highlights the right length of the toxin as well as its homodimeric form which could not be determined by the previous cDNA study. TxXIIIA is also the first homodimeric conotoxin with five disulfide bonds and composed of two monomers containing an odd number of cysteins.

Purification and characterization of a novel excitatory peptide from Conus distans venom that defines a novel gene superfamily of conotoxins.

An excitatory peptide, di16a, with 49 amino acids and 10 cysteine residues was purified and characterized from the venom of Conus distans. Five AA residues were modified: one gamma-carboxyglutamate (Gla), and four hydroxyproline (Hyp) residues. A cDNA clone encoding the precursor for the peptide was characterized; the peptide has a novel cysteine framework and a distinctive signal sequence that differs from any other conotoxin superfamily. The peptide was chemically synthesized and folded, and synthetic and native materials were shown to co-elute. Injection of the synthetic peptide causes a hyperexcitable phenotype in mice greater than 3 weeks of age at lower doses, and lethargy at higher doses. The peptide defines both a previously uncharacterized gene superfamily of conopeptides, and a new Cys pattern with three vicinal Cys residues.

[Phycotoxins of ASP, PSP and DSP groups in aquaris organisms].

In this article the results of analysis of ASP, PSP and DSP phycotoxins content in aquatic organisms are presented. Methods of determination of toxins were ELISA and HPLS.

Advanced studies for the application of high-performance capillary electrophoresis for the analysis of yessotoxin and 45-hydroxyyessotoxin.

Yessotoxins (YTXs) are a group of polyether toxins which have been previously reported as responsible for seafood contamination in several places worldwide. Despite their toxicity, which is not yet fully discussed, YTXs have been reported as an interference in the success of mouse bioassay for the determination of diarrhetic shellfish poisoning (DSP) toxins, and therefore, efficient and reliable analytical methodologies are required to evaluate their presence, avoiding false positives for DSP. High-performance capillary electrophoresis (HPCE) is presented in this work as an alternative to HPLC technique widely used for the analysis of YTXs. Improvements in the applicability of HPCE have been carried out through the development of different CE modes as well as different detection modes. With this aim, micellar electrokinetic chromatography (MEKC) has been considered for an increased selectivity while an increased sensitivity was achieved by using sample stacking. Moreover, the coupling of CE with mass spectrometry allowed the confirmation of YTXs present in the contaminated samples evaluated in this work. The results obtained showed the potential of CE as an alternative to HPLC for the analysis of YTXs present in naturally contaminated samples.

Distribution of tetrodotoxin in the body of the blue-ringed octopus (Hapalochlaena maculosa).

Tetrodotoxin (TTX) was quantitatively assayed in six specimens of semi-adult blue-ringed octopus, Hapalochlaena maculosa, by a post-column fluorescent-HPLC system. TTX was found to be present in all body parts, e.g. in high concentrations in the arms followed by the abdomen and cephalothorax. The toxin is not associated exclusively with the posterior salivary gland.

Isolation and structure-activity of mu-conotoxin TIIIA, a potent inhibitor of tetrodotoxin-sensitive voltage-gated sodium channels.

Mu-conotoxins are three-loop peptides produced by cone snails to inhibit voltage-gated sodium channels during prey capture. Using polymerase chain reaction techniques, we identified a gene sequence from the venom duct of Conus tulipa encoding a new mu-conotoxin-TIIIA (TIIIA). A 125I-TIIIA binding assay was established to isolate native TIIIA from the crude venom of Conus striatus. The isolated peptide had three post-translational modifications, including two hydroxyproline residues and C-terminal amidation, and <35% homology to other mu-conotoxins. TIIIA potently displaced [3H]saxitoxin and 125I-TIIIA from rat brain (Nav1.2) and skeletal muscle (Nav1.4) membranes. Alanine and glutamine scans of TIIIA revealed several residues, including Arg14, that were critical for high-affinity binding to tetrodotoxin (TTX)-sensitive Na+ channels. We were surprised to find that [E15A]TIIIA had a 10-fold higher affinity than TIIIA for TTX-sensitive sodium channels (IC50, 15 vs. 148 pM at rat brain membrane). TIIIA was selective for Nav1.2 and -1.4 over Nav1.3, -1.5, -1.7, and -1.8 expressed in Xenopus laevis oocytes and had no effect on rat dorsal root ganglion neuron Na+ current. 1H NMR studies revealed that TIIIA adopted a single conformation in solution that was similar to the major conformation described previously for mu-conotoxin PIIIA. TIIIA and analogs provide new biochemical probes as well as insights into the structure-activity of mu-conotoxins.

[Lipophilic toxin profiles associated with diarrhetic shellfish poisoning in scallops, Patinopecten yessoensis, collected in Hokkaido and comparison of the quantitative results between LC/MS and mouse bioassay].

Lipophilic toxins associated with diarrhetic shellfish poisoning (DSP) in scallops, Patinopecten yessoensis, collected in Hokkaido, Japan were quantified by liquid chromatography-mass spectrometry (LC/MS). Pectenotoxin-6 (PTX6) and yessotoxin (YTX) were the dominant toxins in the scallops, although the percentages of these toxins were different depending on the production area or the sampling period. The quantitative results obtained for the scallops in LC/MS and in mouse bioassay (MBA) were compared. Fifty of the 55 samples found to be exceeding the local quarantine level (0.025 MU/g whole meat) in Hokkaido by LC/MS were quantified by MBA as being below the quarantine level. It is suggested that this discrepancy is due to poor detection of YTX by MBA. These results indicate that LC/MS is a better method than MBA in terms of sensitivity and accuracy to quantify known lipophilic toxins, including YTX.

Screening for post-translational modifications in conotoxins using liquid chromatography/mass spectrometry: an important component of conotoxin discovery.

Mass spectrometry has emerged as an important technique for conotoxin analysis due to its capacity for selective, sensitive, information-rich analyses. Using liquid chromatography/mass spectrometry, Conus venom can be fractionated and the peptides surveyed for specific post-translational modifications, indicating those toxin components likely to have an important biological function. With Conus striatus and Conus victoriae venom as models, bromination, carboxylation and glycosylation modifications are identified through characteristics such as isotopic distribution and labile losses observed during mass spectrometric analysis. This modification screening approach enables the identification of a C. victoriae bromo-carboxy-conotoxin, designated vc5c, as a candidate for detailed mass spectrometric analysis. Using a cDNA sequence coupled with liquid chromatography/mass spectrometry and nanoelectrospray ionization-ion trap-mass spectrometry, the sequence of vc5c is determined to be ICCYPNXWCCD, where W is 6-bromotryptophan, X is gamma-carboxy glutamate and C is disulfide-linked cysteine. This represents the ninth T-superfamily (-CC-CC- scaffold) toxin that has been isolated from venom and characterized.