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Protoceratium reticulatum is the main yessotoxin-producer along the Chilean coast. Thus far, the yessotoxin levels recorded in this region have not posed a serious threat to human health. However, a bloom of P. reticulatum during the austral summer of 2022 caused the first ban of shellfish collection, due to the high toxin levels. A bloom of P. reticulatum during the austral summer of 2020 allowed an evaluation of the fine-scale distribution of the dinoflagellate during a tidal cycle. High-resolution measurements of biophysical properties were carried out in mid-summer (February 18-19) at a fixed sampling station in Puyuhuapi Fjord, Chilean Patagonia, as part of an intensive 24-h biophysical experiment to monitor the circadian distributions of P. reticulatum vegetative cells and yessotoxins. High P. reticulatum cell densities (>20 × 103 cells L-1) were found in association with a warmer (14.5-15 °C) and estuarine (23.5-24.5 g kg-1) sub-surface water layer (6-8 m). P. reticulatum cell numbers and yessotoxins followed a synchronic distribution pattern consistent with the excursions of the pycnocline. Nevertheless, the surface aggregation of the cells was modulated by the light cycle, suggesting daily vertical migration. The yessotoxin content per P. reticulatum cell ranged from 9.4 to 52.2 pg. This study demonstrates both the value of fine-scale resolution measurements of biophysical properties in a highly stratified system and the potential ecosystem impact of P. reticulatum strains producing high levels of yessotoxins.

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The effects of yessotoxins (YTXs) produced by the dinoflagellate Protoceratium reticulatum in the early stages of bivalves have not been studied in detail. The present study evaluates the effects of P. reticulatum and YTXs on the survival and feed ingestion of veliger larvae of Argopecten purpuratus. Larvae were 96 h-exposed to 500, 1000 and 2000 P. reticulatum cells mL-1, and their equivalent YTX extract was prepared in methanol. Results show a survival mean of 82 % at the highest density of dinoflagellate, and 38 % for larvae with the highest amount of YTX extract. Feed ingestion is reduced in the dinoflagellate exposure treatments as a function of cell density. Therefore, the effect of YTXs on A. purpuratus represents a new and important area of study for investigations into the deleterious effects of these toxins in the early stages of the life cycle of this and, potentially, other bivalves.

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Most of the shellfish fisheries of Mexico occur in the Gulf of California. In this region, known for its high primary productivity, blooms of diatoms and dinoflagellates are common, occurring mainly during upwelling events. Dinoflagellates that produce lipophilic toxins are present, where some outbreaks related to okadaic acid and dinophisystoxins have been recorded. From January 2015 to November 2017 samples of three species of wild bivalve mollusks were collected monthly in five sites in the southern region of Bahía de La Paz. Pooled tissue extracts were analyzed using LC-MS/MS to detect lipophilic toxins. Eighteen analogs of seven toxin groups, including cyclic imines were identified, fortunately individual toxins did not exceed regulatory levels and also the total toxin concentration for each bivalve species was lower than the maximum permitted level for human consumption. Interspecific differences in toxin number and concentration were observed in three species of bivalves even when the samples were collected at the same site. Okadaic acid was detected in low concentrations, while yessotoxins and gymnodimines had the highest concentrations in bivalve tissues. Although in low quantities, the presence of cyclic imines and other lipophilic toxins in bivalves from the southern Gulf of California was constant.

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Maitotoxins (MTXs) are among the most potent toxins known. These toxins are produced by epi-benthic dinoflagellates of the genera Gambierdiscus and Fukuyoa and may play a role in causing the symptoms associated with Ciguatera Fish Poisoning. A recent survey revealed that, of the species tested, the newly described species from the Canary Islands, G. excentricus, is one of the most maitotoxic. The goal of the present study was to characterize MTX-related compounds produced by this species. Initially, lysates of cells from two Canary Island G. excentricus strains VGO791 and VGO792 were partially purified by (i) liquid-liquid partitioning between dichloromethane and aqueous methanol followed by (ii) size-exclusion chromatography. Fractions from chromatographic separation were screened for MTX toxicity using both the neuroblastoma neuro-2a (N2a) cytotoxicity and Ca2+ flux functional assays. Fractions containing MTX activity were analyzed using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS) to pinpoint potential MTX analogs. Subsequent non-targeted HRMS analysis permitted the identification of a novel MTX analog, maitotoxin-4 (MTX4, accurate mono-isotopic mass of 3292.4860 Da, as free acid form) in the most toxic fractions. HRMS/MS spectra of MTX4 as well as of MTX are presented. In addition, crude methanolic extracts of five other strains of G. excentricus and 37 other strains representing one Fukuyoa species and ten species, one ribotype and one undetermined strain/species of Gambierdiscus were screened for the presence of MTXs using low resolution tandem mass spectrometry (LRMS/MS). This targeted analysis indicated the original maitotoxin (MTX) was only present in one strain (G. australes S080911_1). Putative maitotoxin-2 (p-MTX2) and maitotoxin-3 (p-MTX3) were identified in several other species, but confirmation was not possible because of the lack of reference material. Maitotoxin-4 was detected in all seven strains of G. excentricus examined, independently of their origin (Brazil, Canary Islands and Caribbean), and not detected in any other species. MTX4 may therefore serve as a biomarker for the highly toxic G. excentricus in the Atlantic area.

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Maitotoxin (MTX) is the most potent marine toxin known to date. It is responsible for a particular human intoxication syndrome called ciguatera fish poisoning (CFP). Several reports indicate that MTX is an activator of non-selective cation channels (NSCC) in different cell types. The molecular identity of these channels is still an unresolved topic, and it has been proposed that the transient receptor potential (TRP) channels are involved in this effect. In Xenopus laevis oocytes, MTX at picomolar (pM) concentrations induces the activation of NSCC with functional and pharmacological properties that resemble the activity of TRP channels. The purpose of this study was to characterize the molecular identity of the TRP channel involved in the MTX response, using the small interference RNA (siRNA) approach and the two-electrode voltage-clamp technique (TEVC). The injection of a specifically designed siRNA to silence the transient receptor potential canonical type 1 (TRPC1) protein expression abolished the MTX response. MTX had no effect on oocytes, even at doses 20-fold higher compared to cells without injection. Total mRNA and protein levels of TRPC1 were notably diminished. The TRPC4 siRNA did not change the MTX effect, even though it was important to note that the protein level was reduced by the silencing of TRPC4. Our results suggest that MTX could be a selective activator of TRPC1 channels in X. laevis oocytes and a useful pharmacological tool for further studies on these TRP channels.

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ResumenEl aumento de la resistencia bacteriana a los antibióticos ha causado preocupación a nivel mundial, por lo que se ha promovido la búsqueda de nuevos compuestos. Debido a su abundancia y diversidad, el fitoplancton marino constituye una importante fuente potencial de tales compuestos. La investigación sobre dinoflagelados ha llevado al descubrimiento de inhibidores de crecimiento bacteriano. El dinoflagelado marino Lingulodinium polyedrum causa proliferaciones algales en diferentes regiones del mundo, incluyendo México, y también se sabe que regula el crecimiento de otras especies en las aguas costeras. En este trabajo, se investiga la taxonomía de este dinoflagelado y se caracteriza la capacidad de sus extractos para inhibir el crecimiento de dos bacterias de importancia médica (Vibrio vulnificus y Staphylococcus aureus) en placas de cultivo de agar. La caracterización taxonómica se realizó por PCR y amplificación del gen de ITS, y se confirmó que la especie aislada en la costa del Pacífico de México fue L. polyedrum. Para demostrar el efecto inhibidor de los extractos de L. polyedrum, los cultivos se cosecharon por centrifugación. Los pellets de tres abundancias celulares se extrajeron con agua, metanol, hexano y cloroformo. Los experimentos en V. vulnificus mostraron una inhibición alta del crecimiento para los cuatro extractos, variando entre 77 y 98 %. Sorprendentemente, la inhibición del crecimiento fue menor cuando los extractos se originaron a partir de una mayor abundancia de células L. polyedrum, varía de 0 a 34 %. Para S. aureus, la inhibición del crecimiento también fue alta, pero no estadísticamente diferente para todos los extractos y abundancias de células, con un rango de 62 hasta 99 %. Esto resultados son prometedores para futuras aplicaciones farmacológicas. La cepa mexicana de L. polyedrum no produjo yesotoxinas detectables.

AbstractThe increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins. Rev. Biol. Trop. 64 (2): 805-816. Epub 2016 June 01.

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In summer 2009, during a survey in Bahía Mejillones, a dense bloom of a dinoflagellate from the genus Gonyaulax was detected, as well as the presence of yessotoxin. Phytoplankton samples were analyzed in detail by light and scanning electron microscopy (SEM), revealing the presence of Gonyaulax taylorii. Morphological examination showed that the cells in the bloom fit in Gonyaulax jollifei Murray et Whitting sensu Dodge, subsequently classified as Gonyaulax taylorii by Carbonell-Moore. In this context, some inconsistencies have been found in regard to the holotype; the plate 1"' appears as two plates, 1‴ and 2‴, showing a suture that does not exist in Dodge's figure of G. jollifei, from where the holotype was drawn, nor within the samples collected. Therefore, this plate has been originally described erroneously as two plates named 1"' and 2"' instead of only one named 1‴. After this correction, this species has five instead of six postcingular plates. For this reason, the description of this species must be emended. Phytoplankton net samples were found to contain yessotoxin and homoyessotoxin, with concentrations below 1pgcell-1. The present study identifies, therefore, the dinoflagellate G. taylorii as a new source of yessotoxins.

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The increased bacterial resistance to antibiotics has caused global concern, prompting the search for new compounds. Because of their abundance and diversity, marine phytoplankton are an important potential source of such compounds. Research on dinoflagellates has led to the discovery of inhibitors of bacterial growth. The marine dinoflagellate Lingulodinium polyedrum blooms in different regions of the world, including Mexico, and is also known to regulate the growth of other species in coastal waters. Here, we investigated the taxonomy of this dinoflagellate and characterized the ability of its extracts to inhibit the growth of two bacteria of medical importance (Vibrio vulnificus and Staphylococcus aureus). Taxonomic characterization was performed by PCR and gene amplification of ITS, and confirmed that the species isolated off the Pacific coast of Mexico was L. polyedrum. To prove the inhibitory effect of L. polyedrum extracts, cultures were harvested by centrifugation. Pellets from three cellular abundances were extracted with water, methanol, hexane and chloroform. The experiments on V. vulnificus showed a high growth inhibition for the four extracts, ranging from 77 to 98 %. Surprisingly, the growth inhibition was lower when the extracts originated from a higher L. polyedrum cell abundance, ranging from 0 to 34 %. For S. aureus, the growth inhibition was also high, but not statistically different for all extracts and cell abundances, ranging from 62 to 99 %. This study obtained promising results for future pharmacological applications. Our Mexican strain of L. polyedrum did not produce any detectable yessotoxins.

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A single cell of the dinoflagellate genus Fukuyoa was isolated from the island of Formentera (Balearic Islands, west Mediterranean Sea), cultured, and characterized by morphological and molecular methods and toxin analyses. This is the first report of the Gambierdiscus lineage (genera Fukuyoa and Gambierdiscus) from the western Mediterranean Sea, which is cooler than its eastern basin. Molecular analyses revealed that the Mediterranean strain belongs to F. paulensis and that it bears LSU rDNA sequences identical to New Zealand, Australian, and Brazilian strains. It also shared an identical sequence of the more variable ITS-rDNA with the Brazilian strain. Toxin analyses showed the presence of maitotoxin, 54-deoxyCTX1B, and gambieric acid A. This is the first observation of the two latter compounds in a Fukuyoa strain. Therefore, both Gambierdiscus and Fukuyoa should be considered when as contributing to ciguatera fish poisoning. Different strains of Fukuyoa form a complex of morphologically cryptic lineages where F. paulensis stands as the most distantly related nominal species. The comparison of the ITS2 secondary structures revealed the absence of CBCs among strains. The study of the morphological and molecular traits depicted an unresolved taxonomic scenario impacted by the low strains sampling.

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The presence of phytoplankton responsible for the production of lipophilic marine biotoxins is well recognised throughout parts of South America. To date, the quantitation of lipophilic toxins in Argentinean shellfish has been limited to select and highly focussed geographical studies. This work reports the analysis for lipophilic marine biotoxins in shellfish harvested across five regions of Argentina between 1992 and 2012. LC-MS/MS analysis was used for the quantitation of all regulated lipophilic toxins. High concentrations of okadaic acid group toxins were quantified, with a clear dominance of the parent okadaic acid and more than 90% of the toxin present as esters. Results showed DSP toxins in shellfish from the Buenos Aires Province during 2006 and 2007, earlier than previously described. There was also strong evidence linking the presence of okadaic acid to human intoxications. Other lipophilic toxins detected were yessotoxin, pectenotoxin-2 and 13-desMeC spirolide. With evidence published recently for the presence of azaspiracid producers, this work reports the detection of low concentrations of azaspiracid-2 in shellfish. As such the data provides the first published evidence for yessotoxins and azaspiracids in Argentinean shellfish and further evidence for the continuing presence of lipophilic marine toxins in Argentinean waters.

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