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Later on P. The symptoms were considered to be characteristic of paralytic shellfish poisoning PSP 6.

The definitive link between P. These dinoflagellates produced a water-soluble neurotoxic compound, different from PSP, which was able to rapidly kill mice after neurotoxic symptoms 7 , 8. This unknown compound was able to block sodium and calcium channels and was accumulated by shellfish 7. However, since some of the toxic episodes related to P.

This coexistence does not eliminate the potential risk of P. In this sense, although most P. Tetrodotoxin TTX is one of the most important neurotoxins known to block sodium channels and thus it inhibits the propagation of action potentials in muscle and nerve cells. This compound blocks site 1 of voltage dependent sodium channels 10 and it has interesting pharmacological applications in the study of excitable membranes as well as therapeutic uses in treating migraines, addictions, or as an anaesthetic agent for pain TTX has been described in different aquatic animals such as fish, arthropods, echinoderms, molluscs, worms, newts, frogs or toads as well as several bacteria species and dinoflagellates 11 , However, the origin of TTX is still unclear, although the presence of this toxin in such a diverse group of animals suggests that bacteria might be as the primary source In this sense, TTX can be produced without bacteria, but the presence of microorganisms could, in some way, simplify TTX biosynthesis To date, few appearances of TTX in bivalve molluscs have been reported 14 , In addition in , in Greece, during routine controls of shellfish, an unexplained toxicity associated with neurological symptoms was observed in a series of mouse bioassays MBA.

This atypical toxicity coincided with the absence of known dinoflagellates and other toxins, while only P. Therefore a potential TTX presence-dinoflagellate link was hypothesized. In this context the aim of this work was to further study the relationship between TTX and P. To further study the occurrence and origin of TTXs in mussels collected in the unusual toxic episode in Greece, it was evaluated the presence of bacteria as TTX producers as well as the effect on Na v channels. Levels of A rarefaction plot of observed bacterial species in control white non-contaminated tissue and TTX-mussel samples showed higher species diversity in the uncontaminated material Fig.

Although high species diversity was achieved, uncontaminated sample was under represented when compared to TTX-mussel samples. Jackknifed hierarchical clustering by UPGMA unweighted pair group method with arithmetic mean showed a high support for the depurated samples clustering separate from the non-depurated one, while a lower support was observed for contaminated samples Fig. However these genera were not represented in the Proteobacteria of non-contaminated sample. The distribution of these bacteria showed that in both samples Vibrio parahaemolyticus and Vibrio alginolyticus were the dominant species, while Pseudomonas appeared at lower levels as well as other Vibrio species.

B Bootstrapped tree for hierarchical samples clustering.

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A Primer on LC/NMR/MS

Next, to further support the existence of TTX in mussel extracts, the effect over Na v channels, the main cellular target of TTX, was checked by patch clamp measurements in stable transfected hNa v 1. As Fig. At the highest concentration tested, an inhibition of Automated patch clamp experiments were carried out in hNav1. I Na magnitude is expressed as percentage of basal current. Although the source of TTXs in contaminated mussels seemed to be related to bacteria, the dinoflagellate P. Three strains of P.


After 15 days of growth, cells were harvested and the extracts obtained dissolved in acetic acid 0. Then, the effect of dinoflagellate extracts on I Na was checked. This effect was not dependent on light intensity. Therefore, these results indicate the presence of some compound produced by P.

Next, we proceeded to the identification of compounds produced by P. The five compounds initially observed in P. However, since P. This technology allows us to predict the formula using ad oc predictor software based on the accurate mass data recorded. TTX standard was employed as control to predict the molecular formula of precursor and ion products. These data are summarized in Table 1 and shown in Figs 5 , 6 and 7. The errors between these measured ion products and the theoretical values range from 0. Since these compounds are present in low concentration in P. The errors between these measured product-ions and the theoretical values calculated are shown in Table 1.

In this case the higher range observed was probably related to the low concentration. In addition the predicted structure proposed for each compound based on the high resolution masses of fragment ions are shown in Figs 5 , 6 and 7. Both compounds were present in all P.

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While the amount of compound did not show a direct relation with the effect on I Na , the extracts with higher amount of compound produced higher I Na inhibition. Finally, the presence of TTX-producing bacteria was checked in P. Firstly, we checked for the presence of bacteria described in contaminated mussels, Vibrio and Pseudomonas spp. After PCR amplification, V. When the same assay was performed using specific primers for Pseudomonas, no positive amplification product was detected results not shown. In addition, P.

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The presence of TTX in mussels is an important issue from both a toxicological and human-health risk standpoint. Although this compound is a regulated toxin in Europe and around the world, it is not included in regular monitoring programs because the consumption of species that usually accumulate the toxin is prohibited. Before only one highly toxic TTX-accumulation in scallops from Japan had been reported However, from to date three reports have identified the presence of TTX in shellfish and it was also detected in several edible gastropods 15 , 16 , 17 , 22 , In addition, the MBA, that could detect all toxins, is no longer used in Europe as an official assay.

In this paper the presence of TTX in contaminated samples collected in Greece was confirmed through the effect on I Na. In addition, V. Besides this, several species from Pseudomonas genus were also observed. This genus had previously been reported in TTX production, although the species were not specified 24 , From our data, both Vibrio and Pseudomonas sp.

Therefore, the connection between shellfish, bacteria and TTX in the toxic episode earlier described in Greece was clear, however, a P. Since the toxin production by this dinoflagellate has not to date been clarified, we studied its growth and toxicity in different conditions. In this way, depending on the salinity, temperature and light, P.

The inhibitory effect on transfected hNa v 1. In the Malaysia strain the effect was also dependent of light and salinity conditions. These results confirm previous data where a water-soluble component produced by P. The variations of P.

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Our results also confirm these hypotheses, since depending on the strain and on the growth conditions the effect on I Na is different, as well as the toxin production. From electrophysiological experiments with P. In this case a column for Hydrophilic Interaction Chromatography HILIC with polar analytes was used and 5 compounds with apparently the same product ions but different retention times to TTX and analogues were obtained 22 , 26 , However, these compounds did not follow the characteristic fragmentation pathway of TTXs.

These conditions are deemed necessary to affirm that the compounds are TTX analogues 27 , Therefore, P. Based on the predicted elemental composition and the structure of a parent compound, the structures of product ions can be obtained with a high degree of confidence 21 , 29 , 30 , 31 , Taking into account the structure for the TTX, the loss of water can occur in different carbons but it is considered that the easiest loss is in C4 This fragmentation was confirmed with a MS 3 spectrum. In the first case it is a C8 molecule while in the second it is a C9.

These structures and molecular formula were also proposed as fragment ions of 5,6,trideoxyTTX In addition, a similar molecule C 9 H 9 N 3 O 2 , molecular weight When the errors between theoretical and experimental data are compared some differences are observed. In the case of TTX, both precursor and product ions have a narrow mass difference 0. Therefore the increase in error should be related to the decrease in the amount of compound used. In addition the sample were not purified, and some matrix effect is also present.

These molecules could be TTX precursors in mussels.

However, the quantification of both molecules was done with TTX standard and can be not accurate. It is important to emphasize that the presence and above all the position of CH 2 OH group is important for TTX-Na v channels binding, and the compounds reported in this work have the same group.

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This structure is common to all TTX active analogues However, the affinity and toxicity of analogues should be different 22 , A symbiosis between bacteria and dinoflagellates has been often proposed, however little is known about this phenomenon. Sometimes the bacteria induce death of the algae, while in other situations it provides essential vitamins for microalgae. This is the case for Roseobacter sp and P minimum cultures, which the symbiotic association increases the production of B1 and B12 vitamins essential for the dinoflagellate 35 , Roseobacter sp have been associated with the production of TTX in the copepod Pseudocaligus fugu As it was reported before, TTX and analogues have been detected in many different animals and environments, in addition considerable differences were found in the analogues identified in pufferfish TTX derivatives and amphibians chiquiritoxin 21 , It has been suggested that this wide range of compounds identified is probably due to the different biosynthesis or metabolism of TTX between animals This symbiotic production may explain why the strain is not always toxic.

As far as we know, this is the first time that the toxic compounds produce by P. The structures proposed for the C9 base in these two compounds are shown in Figs 6C and 7C. To finally clarify this, the structural NMR determination and crystallographic analysis should be performed, although this would require a massive scaling of cultures to obtain enough pure compound.

A consequence of TTX being produced by bacteria in algae is that this would potentially be highly influenced by environmental conditions, hence opening a justification as to why climate change provide a link to the recent increase of TTX presence in previously unreported areas. Lugo, Spain.

Expanding the Limits of Structure Elucidation

Acetonitrile and methanol were obtained from Panreac Barcelona, Spain. Formic acid was purchased from Merck Darmstadt, Germany.