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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 × 10 6 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 O 2 h -1  g -1 ) than the control (0.3 ± 0.06 mg O 2 h -1  g -1 ). However, this was not observed in oysters exposed to both heatwave and P. lima (0.5 ± 0.06 mg O 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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 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., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

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.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

"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., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier B.V. All rights reserved.)

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.

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., ((c) 2010 Elsevier Ltd. All rights reserved.)

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.

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.

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.

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.

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.

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.

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.

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.

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.

Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3',5'-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein-dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.

Blue mussels (Mytilus edulis) collected from Flødevigen Bay, Norway, in 2001 and 2002 were analysed for yessotoxins (YTXs) by ELISA and yessotoxin (YTX), 45-hydroxyYTX, and carboxyYTX by LC-MS. Results from the two methods were compared to evaluate the ELISA. The response in the ELISA was 3-13 times higher than LC-MS, probably due to the antibodies binding to other YTX analogues not included in the LC-MS analysis. Nevertheless, the correlation between ELISA and LC-MS was good, with r2 values> or =0.8. The results indicate that the ELISA is a reliable method for estimating the total level of YTXs in mussels, and are consistent with extensive metabolism of algal YTXs in mussels. YTX was a minor component in the blue mussels at all times compared to 45-hydroxyYTX and especially carboxyYTX, except when the P. reticulatum bloom occurred. The results also indicate the presence of significant amounts of YTX analogues in addition to those measured by LC-MS. All samples below 4 mg/kg by ELISA were below the current EU regulatory limit of 1 mg/kg by LC-MS. Therefore, we propose using ELISA as a screening tool with a cut-off limit at 4 mg/kg for negative samples, whereas samples above this limit would be reanalyzed by LC-MS.

It appears to be a general belief that stable isotopically labeled (SIL) internal standards yield better assay performance results for quantitative bioanalytical liquid chromatography/mass spectrometry (LC/MS) assays than does any other internal standard. In this article we describe our experiences with structural analogues and SIL internal standards and their merits and demerits. SIL internal standards are the first choice, but deuterium-labeled compounds may demonstrate unexpected behavior, such as different retention times or recoveries, than the analyte. In addition, a SIL internal standard with identical chemical properties as the analyte may cover up assay problems with stability, recovery, and ion suppression. Since SIL internal standards are not always available or are very expensive, structural analogues can be used, however, with consideration of several issues, which are usually displayed during method validation., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.

A red tide event occurred in El Salvador from August 2001 to January 2002. National health authorities usually measured toxin levels in Ostrea iridescens, however other species were analyzed during this microalgae bloom: Anadara similis, Anadara tuberculosa and Modiolus sp. El Salvador authorities consider 400 mouse units/100 g the highest value that is safe for human health. During this period toxin levels in 0. iridescens and Modiolus sp. increased from values under 400 to 3977 and 15,468 mouse units/100 g, respectively. Persistent and higher levels were recorded in oyster and mussel banks on the west part of the country. The Ministry of Health and Social Assistance treated 41 slight to moderate intoxications associated to bivalve mollusks consumption.

The study shown here provides the first indisputable evidence that shellfish can be contaminated with Paralytic Shellfish Poisoning (PSP) and Diarrheic Shellfish Poisoning (DSP) toxins during the summer season in the Southern Chilean fjords. Quantitative analysis of the simultaneous presence of PSP and DSP toxins in Mytilus chilensis samples collected in the Chiloe Island are shown. The High Performance Liquid Chromatography (HPLC) analysis with pre-column derivatization method for DSP toxins and the post-column derivatization methods for PSP toxins, both with fluorescent on-line detections, showed that both type of toxins were concentrated by the filter bivalve Mytilus chilensis in amounts above the international safe limits. The phytoplankton analysis showed the presence of both Alexandrium catenella and Dinophysis acuta in the water column. The data shows stratification of the toxic dinoflagellates in the water column, since the lowest amount of both DSP and PSP toxins were measured in the superficial and deeper levels of the water column. Moreover, the highest toxicities of both types of toxins were shown by the shellfish samples collected at a depth of 6 meters with 190 nanograms of DTX-1 / gram of digestive gland and 709.8 microg of PSP toxins / 100 grams of mussel meat.

We reported that the common octopus, Octopus vulgaris, in common with vertebrates, possesses two members of the oxytocin/vasopressin superfamily: octopressin (OP) and cephalotocin (CT). This was the first observation of its kind in invertebrates. As OP and CT have different biological activities, the presence of specific receptors has been proposed. We cloned the cDNA of an orphan receptor from Octopus brain and found it to encode a polypeptide of 397 amino acids that displays sequences characteristic of G-protein coupled receptors. The orphan receptor showed high homology to receptors of the oxytocin/vasopressin superfamily and seemed to conserve the agonist-binding pocket common to the oxytocin and vasopressin receptors. Xenopus oocytes that express the orphan receptor responded to the application of CT by an induction of membrane Cl(-) currents coupled to the inositol phosphate/Ca(2+) pathway. OP and the other members of the oxytocin/vasopressin superfamily did not activate this receptor. HPLC fractionation of the Octopus brain extract combined with an oocyte assay yielded a single substance that was identical to CT. On the basis of these results, we conclude that the cloned receptor is the CT receptor (CTR). Expression of CTR mRNA in Octopus was detected in the central and the peripheral nervous systems, the pancreas, the oviduct and the ovary. This receptor may mediate physiological functions of CT in Octopus such as neurotransmission, reproduction and metabolism.

While the occurrence of yessotoxin (YTX) has been reported worldwide from Protoceratium reticulatum, the biogenetic origin of some YTX analogues is still unknown, thus raising an issue whether they are metabolites of YTX formed in mussels or true products of different dinoflagellate species. Findings reported herein suggest that P. reticulatum from the north-western Adriatic sea is responsible for production, together with YTX (1), of homoYTX (2), 45-OHYTX (3), carboxyYTX (5), and noroxoYTX (7). YTX and its analogues have been determined by high performance liquid chromatography coupled with electrospray ion trap mass spectrometry (HPLC-MS and HPLC-MSn experiments). The result is the first to confirm production of these YTX analogues from this species and indicate it as a producing organism of homoYTX, 45-OHYTX, carboxyYTX, and noroxoYTX found in shellfish.

The red whelk Neptunea antiqua (family Buccinidae) is a sublittoral species of marine prosobranch mollusc that occurs in the cold temperate waters of the Eastern Atlantic Boreal Region. A clearly defined seasonal cycle was revealed in the concentration of the whelk's salivary gland neurotoxin, tetramine, ranging from undetectable levels to 6530 microg/g over the course of an annual sampling period in the central western Irish Sea. Concentrations of this amine were indirectly related to feeding activity through the use of digestive gland indices. Additionally, laboratory observations confirmed the suspected ability of these whelks to actively predate on bivalves, supporting the use of this toxin in food procurement.

Enzyme-linked immunosorbent assays (ELISAs) were developed for amnesic, neurotoxic, and diarrhetic shellfish poisoning (ASP, NSP, and DSP) toxins and for yessotoxin. These assays, along with a commercially available paralytic shellfish poisoning (PSP) ELISA, were used to test the feasibility of an ELISA-based screening system. It was concluded that such a system to identify suspect shellfish samples, for subsequent analysis by methods approved by international regulatory authorities, is feasible. The assays had sufficient sensitivity and can be used on simple shellfish extracts. Alcohol extraction gave good recovery of all toxin groups. The ease of ELISAs permits the ready expansion of the system to screen for other toxins, as new ELISAs become available.

Repeatability and validity of a fluorimetric HPLC method in quantification of yessotoxin (YTX) in mussels related to the mouse bioassay was studied. Blue mussels (Mytilus edulis) from the Sognefjord, Norway were sampled from March to November, 1997, and October to December, 1998. A total of 75 samples were analysed for YTX by HPLC using 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3, 4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3, 5-dione (DMEQ-TAD) as a fluorimetric derivatization agent. Among these, 28 of the samples were analysed by HPLC in duplicate. All samples were analysed by the mouse bioassay using both chloroform and ether in the final step of extraction. The duplicate measurements using HPLC was found equal and the method repeatable (p<0.05). The absolute difference between the two measurements was found to increase with increasing level of measurements. This significant positive correlation (p<0.05) was mainly due to concentrations of YTX higher than 200 microg/100g mussel meat. However the precision of the results obtained was not found to be less in the upper level than in the lower level. Based on the internal correlation analysis including the mouse bioassay and the HPLC method a cut-off value of < or =10 microg YTX/5 g digestive gland was found preferable. The mouse bioassay of ether extracts often failed to detect high levels of YTX, and as demonstrated by the low kappa-values, the agreement between the mouse bioassay of ether extracts and the HPLC method was very weak. The HPLC method was found to give repeatable results and thereby found to be reliable. Consequently, the HPLC method seems to the method of choice for detection and quantification of YTX in mussels when compared with the mouse bioassay.

Repeatability in the quantification of diarrhetic shellfish toxins was investigated for two fluorometric HPLC methods and a colorimetric PP2A assay, using agreement analysis. Blue mussels (Mytilus edulis) from the Sognefjord on the southwest coast of Norway were sampled during the periods of July to October, 1996, March to November, 1997, and October to December, 1998. The results from the first and the duplicate measurement were found to be very similar for both HPLC methods using two fluorogenic reagents, 4-bromomethyl,7-methoxycoumarin and 9-anthryl diazomethane, and the colorimetric PP2A inhibition assay in detection of diarrhetic shellfish toxins. The levels of agreement between the measurements were satisfactory for all three methods. Significant correlations were found between the level of observation and the absolute difference between the two measurements and were mainly due to concentrations of OA/DTX-1 higher than 100 microg/100 g mussel meat. However, the precision of quantifying DSP toxins in the upper level was not found to be less than in the lower level. Both the HPLC methods and the PP2A assay were found to give repeatable results and thereby found to be reliable.

Automated pre-column oxidation (the method of Lawrence) was implemented on a routine basis since the end of 1996 to study paralytic shellfish poisoning (PSP) toxins in Portuguese shellfish. Liquid chromatography confirmed the presence of PSP toxins when the known toxic algae were present: Gymnodinium catenatum and/or Alexandrium cf. lusitanicum. On the other side, it has eliminated PSP toxins as a possible recurrent contaminant in oysters from Sado estuary. These oysters were already known to contain high levels of some metals (mainly zinc, copper and cadmium) due to their location in a contaminated area and their particular physiology prone to accumulate metals. The presence of PSP toxins in Scrobicularia plana from Mondego estuary and Tellina crassa from the northern coast, during the absence of the above toxic microalgae in the water column, was confirmed. Unlike other shellfish, these two genera have the feeding habit of aspirating more sediment than organisms in suspension, and probably ingest from the sediment resting cysts of PSP producing microalgae. This is another route of contamination that may help to explain why after a bloom certain shellfish species maintain toxicity for long periods. The method revealed to have a fast implementation on a daily basis, short analysis time (around 20 min between samples), high sensitivity and robustness, and therefore, it is one of the best HPLC methods for screening a large number of shellfish samples for monitoring purposes.

Three piscivorous Conus species, C. ermineus, C. consor and C. catus were acclimatized in aquaria. The study of their strategy to capture the prey and details of their radula's morphology revealed that all of them used a 'hook and line' strategy which consists of immobilizing the prey rapidly before engulfing it. The venoms from these piscivorous species clearly elicit, when injected into fish, an excitotoxic shock characterized by a sudden tetanus of the prey. In mammals, the venoms induce both flaccid paralysis via i.p. injection and seizures via i.c.v. injection. Intracellular recordings from frog nerve-muscle preparations revealed that the venoms from these Conus species first caused spontaneous synaptic potentials which in turn triggered muscle action potentials. Such spontaneous activity is due to an increased nerve terminal excitability. In addition, the venoms suppressed neuromuscular transmission probably by blocking postsynaptic nicotinic acetylcholine receptors. No direct effect of these Conus venoms was observed on the membrane of skeletal muscle fibres. In conclusion, C. ermineus, C. consor and C. catus, which have not securely tethered their prey used a mixture of toxins which target both pre-and postsynaptic elements of the neuromuscular junction and which produce rapid immobilization of their prey.

Two different strains of the dinoflagellate Protoceratium reticulatum collected at Harima Nada and Yamada Bay in Japan were cultured and analyzed by fluorometric HPLC for yessotoxin production. Only the Yamada Bay strain produced yessotoxin. The toxin together with its analog, 45,46,47-trinoryessotoxin, were isolated from larger scale culture and unambiguously confirmed by (1)H NMR and MS measurements. This is the first confirmation of the biogenetic origin of yessotoxin in Japan, where the toxin was first reported. The results also indicate that the production of yessotoxins by P. reticulatum differs from strain to strain, in a similar way to that observed in many other toxigenic dinoflagellates such as Dinophysis spp. and Alexandrium spp., (Copyright 1999 John Wiley & Sons, Ltd.)

A rapid HPLC method with fluorescence detection of yessotoxin (YTX) and its two analogs (45-OHYTX and norYTX) in mussels and scallops is presented. A dienophile reagent, DMEQ-TAD, was used for fluorescence labeling. YTX was measured in the range 1-100 ng. The method confirmed the occurrence of YTX and 45-OHYTX for the first time in mussels from Chile and New Zealand.

The bioactive components in the venoms of cone snails are largely small peptides which target to a diverse set of ion channels and neuronal receptors. We report here the presence of substantial levels of serotonin in the venom of the imperial cone snail, Conus imperialis. This venom also contains many small bioactive peptides. The identification of serotonin has been confirmed by mass spectroscopy, NMR, HPLC mobility and UV spectroscopy. Although serotonin has been reported in arthropod venoms, it was previously reported to be absent in Conus venoms. We examined a total of fourteen different Conus species, and found detectable serotonin only in the venom of C. imperialis. Conus imperialis specializes in feeding upon the amphinomid polychaete Eurythoe complanata. We speculate that serotonin may play a role in the capture of this prey.

Butter clams obtained from a variety of locations along the northern British Columbia coast were assayed for the presence of individual paralytic shellfish poisoning toxins (PSPT) by HPLC and total PSPT toxicity using the mouse bioassay. Specific organs, namely the siphon, adductor muscle, foot and mantle were examined for soluble antigens that crossreacted with crab Saxitoxin-Induced Protein (SIP) using immunochemical (Western blotting) techniques. Butter clams containing high concentrations of PSPT also had several proteins that crossreacted with crab anti-SIP serum. In particular, soluble proteins with distinctly different molecular weights were found in the siphon and foot, respectively, in toxic shellfish. These proteins were absent in nontoxic butter clams. The concept of using PSPT-induced proteins in the butter clam as a screen for identifying toxic shellfish is introduced.

The lethalities of 102 specimens of three species of the gastropod mollusk Nassariidae, collected from fish markets in Taiwan, were examined. The frequency of toxicity in Zeuxis scalaris and Z. castus-like specimens was 94 and 41%, respectively. The range of lethal potency in toxic specimens of Zeuxis scalaris and Z. castus-like was 2-140 and 2-13 mouse units, respectively, while all tissues of Z. castus were non-toxic. The toxins were partially purified from the toxic specimens of Z. scalaris and Z. castus-like. Two toxin fractions were obtained from the extract of each species of Nassariidae by using Bio-Gel P-2 column chromatography. Analyses by thin layer chromatography, electrophoresis, high performance liquid chromatography and ultraviolet spectroscopy, showed that toxin fraction I contained tetrodotoxin while toxin fraction II contained a new neurotoxin.

Mouse bioassay results of rock scallops occasionally monitored by official agencies of British Columbia, Washington and California before and/or after algal blooms from 1980-1990 revealed that 24%, 38% and 87% of adductor muscles, whole body and viscera, respectively, were unsafe (greater than or equal to 80 micrograms saxitoxin equivalents/100 g tissue) for human consumption. The safety of adductor muscles could not be predicted from corresponding toxicity levels of the viscera but the absence of detectable toxin could.

Monoclonal antibodies have been prepared against omega-conotoxin GVI A, a peptide isolated from marine snails of the genus Conus (Conus geographus and Conus magus). This toxin is a blocker of select presynaptic Ca2+ channels in the central nervous system. Antigenic omega-conotoxin GVI A was synthesized as a covalent conjugate with bovine serum albumin and injected s.c. An ELISA assay combined with a competitive inhibition assay was used to select and characterize monoclonal antibodies able to recognize and bind the free toxin. Several of the antibodies were found to block omega-conotoxin GVI A inhibition of 45Ca transport into rat brain synaptosomes and to block omega-conotoxin GVI A binding to membranes from the same preparation. The antibodies recognize native, synthetic toxin, and are useful for analysis of toxin in biological fluids.

Conformational analysis of conotoxin GI, one of the neurotoxic peptides produced by a marine snail, genus Conus, was performed by a combination of nuclear magnetic resonance spectroscopy (NMR) and distance geometry calculations. The resulting conformers on minimization of the target function were classified into two groups. The difference in the structures of the conformers is mainly due to the difference in the orientation of the side chain of the tyrosyl residue. The results show that the solution structure of conotoxin GI satisfies the conformational requirements for the biological activity of an antagonist toward nicotinic cholinergic receptors elucidated in a series of studies on alkaloids. The structure is discussed on the basis of the results of comparison of the atomic arrangements of the active sites of snake venom peptides and molecular models based on the results of secondary structure prediction.

The heat-stable enterotoxin ST Ib produced by enterotoxigenic E. coli strains shares a sequence homology with the sea snail neurotoxin, conotoxin GI. Rabbit antisera were raised against synthetic analogs of these toxins and to a six-residue peptide representing the region common to both toxins. Results from enzyme-linked immunosorbent assays indicate that the homologous region of both toxins represents part of their antigenic site. The lack of cross-reactivity exhibited by the six-residue common domain with serum directed against either toxin suggests that this region probably retains a similar conformation in the intact toxins but not in the isolated fragment.

Recent progress in separation techniques has enabled us to work with water-soluble components which, more often than not, are responsible for biological activities found in marine organisms. Isolation techniques and chemical and biological aspects of water-soluble marine natural products will be discussed with some representative examples.

Onchidal has been identified as the major lipid-soluble component of the defensive secretion of the mollusc Onchidella binneyi, and it has been proposed as the compound responsible for the chemical protection of Onchidella [Bioorg. Chem. 7:125-131 (1978)]. In support of this hypothesis, we now report that onchidal can be found in several different species of Onchidella and that it is toxic to fish. Because onchidal is an acetate ester similar to acetylcholine, its ability to interact with nicotinic acetylcholine receptors and acetylcholinesterase was investigated. Although onchidal did not prevent the binding of 125I-alpha-bungarotoxin to nicotinic acetylcholine receptors, it inhibited acetylcholinesterase in a progressive, apparently irreversible, manner. The apparent affinity of onchidal for the initial reversible binding to acetylcholinesterase (Kd) was approximately 300 microM, and the apparent rate constant for the subsequent irreversible inhibition of enzyme activity (kintact) was approximately 0.1 min-1. Onchidal was a substrate for acetylcholinesterase, and approximately 3250 mol of onchidal were hydrolyzed/mol of enzyme irreversibly inhibited. The calculated kcat for onchidal was 325 min-1. Irreversible inhibition resulted from either onchidal itself or a reactive intermediate in the enzyme-catalyzed hydrolysis of onchidal, rather than from the hydrolysis products of onchidal. Irreversible inhibition of enzyme activity was prevented by coincubation with reversible agents that either sterically block (edrophonium and decamethonium) or allosterically modify (propidium) the acetylcholine binding site. Enzyme activity was not regenerated by incubation with oxime reactivators; therefore, the mechanism of irreversible inhibition does not appear to involve acylation of the active site serine. Because onchidal contains a potentially reactive alpha,beta-unsaturated aldehyde, irreversible inhibition of acetylcholinesterase may result from formation of a novel covalent bond between the toxin and the enzyme. Thus, this novel toxin could potentially be exploited in the design of a new class of anticholinesterase insecticides and in the identification of amino acids that contribute to the binding and hydrolysis of acetylcholine.

Putative N-type voltage-sensitive calcium channels were localized autoradiographically in thaw-mounted rat brain slices using [125I]omega-conotoxin GVIA as a ligand. Density of the toxin binding sites were highly heterogeneous throughout the brain. The highest density of the binding sites was observed in the glomerular layer of the olfactory bulb, cerebral cortex, molecular layer of the hippocampus, amygdaloid complex, reticular part of the substantia nigra, molecular layer of the cerebellar cortex, and nucleus of the solitary tract. White matter tract regions such as the internal capsule, corpus callosum, fimbria of the hippocampus, fornix, and fasciculus retroflexus showed an extremely low density.