Docking Simulation of the Binding Interactions of Saxitoxin Analogs Produced by the Marine Dinoflagellate Gymnodinium catenatum to the Voltage-Gated Sodium Channel Nav1.4

Saxitoxin (STX) and its analogs are paralytic alkaloid neurotoxins that block the voltage-gated sodium channel pore (Nav), impeding passage of Na+ ions into the intracellular space, and thereby preventing the action potential in the peripheral nervous system and skeletal muscle. The marine dinoflagellate Gymnodinium catenatum produces an array of such toxins, including the recently discovered benzoyl analogs, for which the mammalian toxicities are essentially unknown. We subjected STX and its analogs to a theoretical docking simulation based upon two alternative tri-dimensional models of the Nav1.4 to find a relationship between the binding properties and the known mammalian toxicity of selected STX analogs. We inferred hypothetical toxicities for the benzoyl analogs from the modeled values. We demonstrate that these toxins exhibit different binding modes with similar free binding energies and that these alternative binding modes are equally probable. We propose that the principal binding that governs ligand recognition is mediated by electrostatic interactions. Our simulation constitutes the first in silico modeling study on benzoyl-type paralytic toxins and provides an approach towards a better understanding of the mode of action of STX and its analogs

Presencia de toxinas tipo benzoato en una cepa de Gymnodinium catenatum (Dinophyceae) aislada de Manzanillo, Colima, México

The extract of a Gymnodinium catenatum strain, isolated from Manzanillo, Colima, in the Mexican Pacific coast, was analized by nuclear magnetic resonance (1 H-NMR) to determine the presence of benzoate-type analogs. The extract was fractioned using solid-phase extraction (SPE) partitioning on a C-18 cartridge, using a stepwise gradient from 0 to 100% (v/v). Fraction 2 (10% methanol) showed two aromatic doublet signals at 7.91 and 7.53 ppm, consistent with an AABB system, corresponding to a para substituted bencenic ring, attributed to the hydroxybenzoate moiety on C-17 of the toxin basic structure. These findings indicate the presence of benzoate type analogs in this Mexican strain. These analogs have been poorly studied, and the report on their existence in our country is of great importance because they could represent a public health risk, but in the other hand, their existence generates the opportunity to continue the research to determine their toxicity in mammals and, eventually, their pharmacological potential.Se analizó, por espectroscopía de resonancia magnética nuclear de protón (1 H-RMN), el extracto de una cepa de Gymnodinium catenatum Graham, aislada de las costas de Manzanillo, Colima, para determinar la existencia de toxinas tipo benzoato. El extracto fue fraccionado con un gradiente ascendente de metanol de 0 a 100% (v/v) por extracción en fase sólida con cartuchos LC-18 en pasos de 10%. La fracción 2 (10% metanol) exhibió dos señales dobles centradas en 7.91 y 7.53 ppm, correspondientes a un sistema AABB, que confirmó la presencia de un anillo bencénico para sustituido, atribuido al p-hidroxibenzoato en el carbono 17 del esqueleto base de la toxina. Con ello se confirmó la presencia de análogos tipo benzoato en la cepa de Gymnodinium catenatum proveniente de esta zona del Pacífico Mexicano, los cuales han sido poco estudiados, y el hecho de detectarlos en cepas de México es importante por el peligro potencial que representan para la salud pública. Asímismo, se generó la oportunidad de continuar estudiando estos compuestos, tanto para determinar su toxicidad en mamíferos como para, eventualmente, conocer su potencial farmacológico

LC-MS/MS Method Development for the Discovery and Identification of Amphidinols Produced by Amphidinium

Amphidinols are polyketides produced by dinoflagellates suspected of causing fish kills. Here, we demonstrate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the identification and quantification of amphidinols (AM). Novel AM were detected by neutral loss (NL) scan and then quantified together with known AM by selection reaction monitoring (SRM). With the new method, AM were detected in four of eight analyzed strains with a maximum of 3680 fg toxin content per cell. In total, sixteen novel AM were detected by NL scan and characterized via their fragmentation patterns. Of these, two substances are glycosylated forms. This is the first detection of glycosylated AM

Durinskia yucatanensis sp. nov. (Peridiniales: Kryptoperidiniaceae), una nueva especie de dinoflagelado planctónico, y su hábitat en aguas costeras de Yucatán, Golfo de México

Background: In the coastal waters of the northern Yucatan Peninsula, in the southeastern Gulf of Mexico, numerous pelagic algal blooms have been recorded in the 21st century. In August 2010, an unknown small-sized Peridiniales species caused an intense bloom in the Sisal marina. In subsequent years, it was occasionally found at other sites along the Yucatan coast. Goals: The main objective of the present study was to name this dinoflagellate as a new species and determine its ecological preferences. Methods: Phytoplankton blooms were monitored from August 2011 to September 2014. Fixed cells of the studied species were examined in a JEOL JSM-7600F scanning electron microscope. Its ecological preferences were evaluated using multivariate permutational analysis and generalized additive models (GAM). Results: The name Durinskia yucatanensis (Dinophyceae: Peridiniales) with the thecal plate formula Po X 4’ 2a 6” 5c 4s(?) 5”’ 2”” is proposed for a previously recorded Kryptoperidiniaceae species from the northern Yucatan coastal waters. Dissolved inorganic nitrogen positively correlated with cell abundances for both the exposed coast and marinas, especially in July-August, characterized by high water temperature (31-32 °C). Chlorophyll-a was the only parameter that presented significant spatio-temporal variability among years, months, and sampling sites. The GAM showed that temperature and salinity can predict changes in abundance in different study zones (exposed coast and marinas). The highest values were observed in the Progreso-Chicxulub area along the exposed coast in 2011 and only at Dzilam in marinas during all studied years. Conclusions: The species appears to prefer eutrophic conditions typical for marinas along the northern coast of Yucatan.Antecedentes: En las aguas costeras del norte de la península de Yucatán, en el sureste del Golfo de México, numerosos florecimientos pelágicos de microalgas se han registrado en el siglo 21. En agosto de 2010, una especie pequeña y desconocida de Peridiniales causó un florecimiento intenso en el puerto de abrigo de Sisal. En los años siguientes, se encontró ocasionalmente en otros sitios a lo largo de la costa de Yucatán. Objetivo: Nombrar a este dinoflagelado como una especie nueva para la ciencia y determinar sus preferencias ecológicas. Métodos: Los florecimientos de fitoplancton fueron monitoreados desde agosto de 2011 hasta septiembre de 2014. Las células se examinaron en un microscopio electrónico de barrido JEOL JSM-7600F. Sus preferencias ecológicas se evaluaron mediante análisis permutacional multivariante y modelos aditivos generalizados (GAM). Resultados: El nombre Durinskia yucatanensis (Dinophyceae: Peridiniales), cuya fórmula de placa tecal Po X 4’ 2a 6” 5c 4s(?) 5”’ 2””, se propone para una especie de Kryptoperidiniaceae previamente registrada de las aguas costeras del norte de Yucatán. El nitrógeno inorgánico disuelto se correlacionó positivamente con la abundancia de células, tanto para la costa expuesta como para los puertos deportivos, especialmente en julio-agosto, caracterizados por una alta temperatura del agua (31-32 °C). La clorofila-a fue el único parámetro que presentó variabilidad espacio-temporal significativa entre años, meses y sitios de muestreo. Los GAM demostraron que la temperatura y la salinidad pueden predecir cambios en la abundancia en diferentes zonas de estudio (costa expuesta y puertos deportivos). Los valores más altos se observaron en el área de Progreso-Chicxulub a lo largo de la costa expuesta en 2011 y solo en Dzilam en marinas durante todos los años estudiados. Conclusiones: La especie parece preferir las condiciones eutróficas típicas de los puertos deportivos a lo largo de la costa norte de Yucatán

Paralytic Toxin Producing Dinoflagellates in Latin America: Ecology and Physiology

In this review we summarize the current state of knowledge regarding taxonomy, bloom dynamics, toxicity, autoecology, and trophic interactions, of saxitoxin producing dinoflagellates in this region. The dinoflagellates Gymnodinium catenatum, Pyrodinium bahamense and several species of Alexandrium are saxitoxin producers, and have been responsible of paralytic shellfish poisoning in different regions of Latin America, causing intoxications and important fisheries losses. The species distribution differ; most harmful algal blooms of G. catenatum are from the northern region, however this species has also been reported in central and southern regions. Blooms of P. bahamense are mostly reported in North and Central America, while blooms of Alexandrium species are more common in South America, however this genus is widely spread in Latin America. Species and regional differences are contrasted, with the aim to contribute to future guidelines for an international scientific approach for research and monitoring activities that are needed to increase our understanding of paralytic toxin producing dinoflagellates in this region

First Identification of Amphidinols from Mexican Strains and New Analogs

The genus Amphidinium has been the subject of recent attention due to the production of polyketide metabolites. Some of these compounds have shown significant bioactivities and could be related to species interactions in the natural benthic microenvironment. Among these compounds, amphidinols (AMs) are suspected to be related to fish kills and probably implicated in ciguatera symptoms associated with the occurrence of benthic harmful algal blooms (bHABs). Here, we present the first report of a variety of AMs produced by cultured strains from several species from the Mexican Pacific, the Gulf of California, and the Gulf of Mexico. Through ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS), ten previously known AMs (AM02, -04, -05, -06, -07, -09, -11, -14, -15, and -17), four recently reported AMs (N7, N8/N9, N12, and N13), and three new variants (U1, U2, and U3) were identified. Of the twelve analyzed Amphidinium cultures, five were not AM producers, and the cell quotas of the remaining seven strains ranged from close to nondetectable to a maximum of 1694 fg cell-1, with many intermediate levels in between. The cultures from the Mexican North Pacific coast produced AMs in a higher quantity and variety than those from worldwide locations. This is the first study of AMs from Mexican Amphidinium strains, and our results confirm the relevance of continuing the investigation of the genus bioactive metabolites

Diversity of Bacterioplankton and Bacteriobenthos from the Veracruz Reef System, Southwestern Gulf of Mexico

Bacterial diversity was explored among field samples and cultured isolates from coral reefs within the Veracruz Reef System. Bacterioplankton and bacteriobenthos were characterized by pyrosequencing 16S rRNA genes. Identified sequences belonged to the kingdom Bacteria and classified into 33 phyla. Proteobacteria (likely SAR11 clade) dominated in collective field samples, whereas Firmicutes were the most abundant taxa among cultured isolates. Bioinformatic sorting of sequences to family level revealed 223 bacterial families. Pseudomonadaceae, Exiguobacteraceae and Bacillaceae were dominant among cultured isolates. Vibrionaceae, Alteromonadaceae, and Flavobacteriaceae dominated in reef-associated sediments, whereas Rickettsiaceae and Synechoccaceae were more highly represented in the water column. Bacterial communities from sediments were more diverse than from the water column. This study reveals cryptic bacterial diversity among microenvironmental components of marine microbial reef communities subject to differential influence of anthropogenic stressors. Such investigations are critical for constructing scenarios of environmentally induced shifts in bacterial biodiversity and species composition

Phylogeography and Diversity Among Populations of the Toxigenic Benthic Dinoflagellate Prorocentrum From Coastal Reef Systems in Mexico

The marine dinoflagellate genus Prorocentrum Ehrenberg comprises many species occupying primarily benthic or epiphytic habitats, particularly in tropical and sub-tropical waters. Despite concerted efforts to establish phylogenetic associations, there remain unresolved issues in defining morphospecies and membership in species complexes. The study described herein addressed the inter- and infraspecific relationships of members of the Prorocentrum lima and Prorocentrum hoffmannianum species complexes (PLSC and PHSC, respectively) by applying multivariate approaches in morphotaxonomy, molecular phylogenetics and chemodiversity to establish affinities among multiple clonal isolates. Morphotaxonomic analysis showed consistency with classical morphospecies descriptors, and high variability in cell size and dimensions, but did not challenge current species complex concepts. Phylogenetic analysis of ITS/5.8S rDNA sequences from isolates from the Gulf of California, Caribbean Sea, and Gulf of Mexico coasts compared with archived global GenBank sequences served to define five consistent clades with separation of the PLSC and PHSC. Secondary structure modeling of ITS2 rRNA variation based on compensatory base changes (CBC) was effective in resolving details of the respective species complexes and even indicated putative incipient or cryptic speciation due to potential hybridization barriers. This study represents the largest (n = 67 isolates) chemodiversity analysis of polyketide-derived toxins associated with diarrheic shellfish poisoning (DSP) from a benthic dinoflagellate genus. Relative composition of some analogs (OA, OA-D8, DTX1, DTX1a, and DTX1a-D8), including two new undescribed isomers, distinguished P. lima from P. hoffmannianum sensu lato, but without clear associations with substrate type or geographical origin. Although all P. lima and most (one exception) P. hoffmannianum were toxigenic, the total cell toxin content could not be linked at the species level. This research demonstrates that clonal chemodiversity in toxin composition cannot yet be effectively applied to define ecological niches or species interactions within local assemblages. Phylogenetic analysis of the ITS/5.8 rDNA, particularly when combined with secondary structure modeling, rather than only a comparison of LSU rDNA sequences, is a more powerful approach to identify cryptic speciation and to resolve species complexes within benthic dinoflagellate groups

Paralytic toxin profile of the marine dinoflagellate Gymnodinium catenatum Graham from the Mexican Pacific as revealed by LC-MS/MS

The paralytic shellfish toxin (PST) profiles of Gymnodinium catenatum Graham have been reported for several strains from the Pacific coast of Mexico cultured under different laboratory conditions, as well as from natural populations. Up to 15 saxitoxin analogues occurred and the quantity of each toxin depended on the growth phase and culture conditions. Previous analysis of toxin profiles of G. catenatum isolated from Mexico have been based on post-column oxidation liquid chromatography with fluorescence detection (LC-FLD), a method prone to artefacts and non-specificity, leading to misinterpretation of toxin composition. We describe, for the first time, the complete toxin profile for several G. catenatum strains from diverse locations of the Pacific coast of Mexico. The new results confirmed previous reports on the dominance of the less potent sulfocarbamoyl toxins (C1/2); significant differences, however, in the composition (e.g., absence of saxitoxin, gonyautoxin 2/3 and neosaxitoxin) were revealed in our confirmatory analysis. The LC-MS/MS analyses also indicated at least seven putative benzoyl toxin analogues and provided support for their existence. This new toxin profile shows a high similarity (> 80%) to the profiles reported from several regions around the world, suggesting low genetic variability among global populations