Characterization of Benzoyl Saxitoxin Analogs from the Toxigenic Marine Dinoflagellate Gymnodinium catenatum by Hydrophilic Interaction Liquid Ion-Chromatography-Tandem Mass Spectrometry

The chain-forming marine dinoflagellate Gymnodinium catenatum Graham has a remarkable capacity to produce a wide array of neurotoxic alkaloids associated with Paralytic Shellfish Poisoning (PSP). More than a decade ago, a completely new group of benzoyl saxitoxin analogs produced exclusively by this species was discovered, but the exact structural assignments and diversity among global population has remained elusive and nconfirmed in most cases. In the current study, fifteen among eighteen hypothetical benzoyl analogs were partially purified and identified from cultured isolates of G. catenatum from the Pacific coast of Mexico. Combined serial application of flash chromatography, preparative liquid chromatography and tandem mass spectrometry (LC-MS/MS) in multiple steps yielded a richness of benzoyl analogs that has not been reported nor confirmed before. Two sub-fractions were analyzed by 1H-NMR; results from one fraction showed a probable AMX pattern for three protons, consistent with the presence of a 3,4-dihydroxylated benzoyl ring. These findings could be interpreted to correct the 2,4-dihydroxylated structure previously proposed for the GCa benzoyl analog series. The revised and enhanced structural information on proposed benzoyl derivatives is necessary to provide further insights into biogeographical diversity of these potentially potent toxins produced by marine dinoflagellates and their role in seafood safety

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

Variabilidad del estado trófico y la biomasa del fitoplancton de Bahía Concepción, Golfo de California (1997-1999)

To determine the effect of the phytoplankton biomass and the trophic status in Bahía Concepción in the centralwest part of the Gulf of California an hydro-biologic study was conducted from 1997 to 1999. Temperature, dissolved oxygen, inorganic nutrients (nitrate, nitrite, phosphate and silicate) and phytoplankton biomass (chlorophyll a) concentrations were measured. Hydrographic conditions shown a mixing period (October-April), two transition periods (April-May and September-October) and a stratification period (June-September). Warmest temperatures were observed during 1998 and coldest in 1997. Dissolved oxygen values shown an hypoxic layer (<2.7±0.78 ml l-1), during the stratified period below 20 m depth. During the transition period two peaks of phytoplankton biomass were observed, on spring and summer, which matched with those reported for coastal lagoons at similar latitudes, however the highest biomasses were observed in the stratification period, particularly below 15 m depth. The scale of nutrients showed an eutrophy for phosphate and silicate all the year. The trophic index revealed an oligotrophy state for phosphate, nitrate and nitrite had a dominant status with the exception for the transition periods, when the mesotrophic conditions were presents. These results suggest that phytoplankton biomass is associated with the natural inputs of nutrients and that trophic conditions (mesotrophy or eutrophy) were determined for the hydrographic of the area.Con el propósito de determinar la variación de la biomasa del fitoplancton (clorofila a) y el estado trófico de Bahía Concepción ubicada en la parte central oeste del Golfo de California, se realizó un estudio hidro-biológico durante el período de 1997 a 1999. Se determinó la temperatura, la concentración del oxígeno disuelto y de nutrientes inorgánicos (nitrato, nitrito, fosfato y silicato), y la biomasa fitoplanctónica (clorofila a). Las condiciones hidrográficas muestran un período de mezcla (octubre-abril), dos de transición (abril-mayo, septiembre-octubre) y uno de estratificación (junio-septiembre). Las temperaturas más cálidas durante el periodo de estudio fueron observadas durante 1998 y las más frías en 1997 para todos los períodos hidrográficos. Los valores de oxígeno disuelto muestran una zona de hipoxia durante el período estratificado (< 2.7±0.78 ml l-1), por abajo de los 20 metros de profundidad. Durante los períodos de transición se observó un incremento de la biomasa fitoplanctónica similar al que se observa como máximos de primavera y verano de algunas lagunas costeras en latitudes similares, sin embargo la mayor biomasa se detectó en el período de estratificación, particularmente por abajo de los 15 m. La escala de nutrientes mostró un estado de eutrofía para fosfato y silicato todo el año. El índice trófico reveló que la oligotrofía para fosfatos, nitratos y nitritos es el estado dominante, con excepción de los períodos de transición, que es cuando la mesotrofía se presentó. Los resultados sugieren que los incrementos fitoplanctónicos en Bahía Concepción se deben al suministro de nutrientes de origen natural y que las condiciones tróficas (mesotrofía o eutrofía), son determinadas por las condiciones hidrográficas del área

Identification of the Gene sxtA (Domains sxtA1 and sxtA4) in Mexican Strains of Gymnodinium catenatum (Dinophyceae) and Their Evolution

Saxitoxin (STX) and its analogs are a broad group of natural neurotoxic alkaloids, commonly known as paralytic shellfish toxins. SxtA is the initial gene in the biosynthesis of saxitoxin. It has been proposed that the genes for STX biosynthesis had a bacterial origin and were acquired in the dinoflagellates by a horizontal gene transfer (HGT). In Gymnodinium catenatum, the origin of the STX genes is not well established. In this paper, we sequenced sxtA gene (domains sxtA1 and sxtA4) and determined the gene copy number in the genome in four Mexican strains of G. catenatum. We compare them with sequences of G. catenatum, Pyrodinium bahamense, and Alexandrium spp. from other geographic regions, and non-toxic producing dinoflagellates. Amplifications were performed for domains sxtA1 and sxtA4 from strains of G. catenatum and the phylogenetic analyses was done by maximum likelihood and Bayesian inference. The copy number determination was carried out using qPCR. The phylogenetic tree of domain sxtA4 showed the formation of two clades where G. catenatum sequences separated from the Alexandrium/Pyrodinium clade. The domain sxtA1 formed a higher number of clades than sxtA4. Sequences of G. catenatum were grouped together with sequences of Alexandrium. Dinoflagellates sequences that do not produce saxitoxin formed a separate clade. The gene copy number was 64 ± 30 and 110 ± 50 copies of sxtA1 and sxtA4 respectively. The identification of the gene sxtA of G. catenatum shows that the sequences are similar to those of Alexandrium species with low variations between species. These results may indicate that the acquisition of the gene sxtA was an early HGT event in the evolution of dinoflagellates. The possible loss of the ability to produce STX in some species suggests that the HGT from Alexandrium species toward G. catenatum is not possible

El estado actual del estudio de florecimientos algales nocivos en México

This review presents an analysis of the state of knowledge of harmful algae studies in the marine environment in Mexico, in order to evaluate how we can optimize the available resources to study these events, identify unattended issues, and propose improvements in monitoring and/or research programs that could help to further understand the complexity of HAB in our coasts. An exhaustive bibliographic revision, from 1940 to 2011, was done consulting specialized data analyses, conference meetings, and specialized books and manuals. The number of researchers and institutions involved in HAB (harmful algal blooms) studies has increased significantly during the last decades, which is reflected in the number and quality of publications. However most of the existing HAB reports are based on sporadic sampling and short-term research programs. Monitoring programs and research activities have been focused in only a few HAB species; many toxic and harmful microalgal species have not been considered. To date, there is no laboratory with the capacity and techniques to analyze all the phycotoxins detected in Mexican coasts. Also, many research areas have not been attended adequately in Mexico such as cyst studies, allelophaty, exotic species, effect of climate change, use of molecular tools and teledetection, socioeconomic analyses, and mitigation strategies among others. This review shows that there has been a significantly improvement in the study of HAB in Mexico. However the studies are still addressed under an individual and disperse way and lack coordination. Greater achievements would be obtained if we concentrate the efforts of researchers and institutions were coordinated in order to optimize the available resources for HAB studies.Esta revisión da a conocer el estado de conocimiento de los estudios de Florecimientos Algales Nocivos (FAN) en el ámbito marino en México, con el objeto de evaluar la manera de optimizar los recursos destinados para el estudio de FAN en nuestro país, identificando líneas no abordadas y proponiendo mejoras en las actividades de monitoreo y de investigación que puedan aumentar nuestro conocimiento sobre la complejidad de FAN en nuestras costas. Se realizó una búsqueda bibliográfica de 1940 a 2011 consultando bases de datos, reuniones académicas y libros especializados. El número de investigadores e instituciones involucrados con el estudio de FAN se ha incrementado significativamente en las últimas décadas, lo cual se ve reflejado en el número y calidad de las publicaciones. Sin embargo, la mayoría de los programas y actividades de investigación están basados en muestreos puntuales y programas de investigación a corto plazo, enfocándose a pocas especies de FAN. Actualmente, varias líneas de investigación relacionadas con los FAN se han abordado pobremente o no se han considerado como son los estudios sobre quistes, alelopatía, transportación de especies por aguas de lastre, efecto del cambio climático, utilización de imágenes de satélite, uso de herramientas moleculares, análisis socioeconómicos y estrategias de mitigación, entre otros. Este análisis muestra que ha habido un avance significativo en el estudio de FAN en México, si bien la manera de abordar su estudio ha sido individualizada y dispersa. Se obtendría un mayor avance si se coordinaran los esfuerzos entre los diversos investigadores e instituciones para optimizar los recursos disponibles para el estudio de los FAN

Co-ocurrencia de Chattonella marina y Gymnodinium catenatum en laBahía de LaPaz, Golfo de California (primavera2009)

During the period of 24 March to 14 May 2009, water and net samples were obtained for phytoplankton analyses; one aliquot was fixed in Lugol´s solution for species quantification and identification. Net samples were used for the identification of live phytoplankton species. Additionally, temperature in situ, chlorophyll a and inorganic nutrients were measured weekly in three stations. Two species were identified: Chattonella marina (Subrahmanyan) Y. Hara et Chihara and Gymnodinium catenatum Graham. Chattonella marina was the most abundant species with a density at subsurface level of 14 × 103 to 36 × 103 cell L-1 . Gymnodinium catenatum had densities were of 1 × 103 to 19 × 103 cell L-1 . Water column temperature was quasi homogeneous from surface to 20 m in depth, with an interval of 19.5 to 22.0 °C and 20.0 to 21.5 °C from March to April respectively. In the middle of May stratification of the water column initiated with a slight temperature difference of 5.0 °C recorded from surface to 20 m in depth. Nitrates varied from 0.5 to 11.0 µM and orthophosphates from 0.4 to 2.6 µM. Chlorophyll a showed a value at surface level of 7.5 mg m-3 and 3.5 mg m-3 at 20 m in March; in May chlorophyll a values ranged between 2.0 to 7.0 mg m-3 . We conclude that the presence of these two species during this study period was associated to the relaxation of the mixing processes in the zoneDurante el período del 24 de marzo al 14 de mayo de 2009 en la Bahía de La Paz, se obtuvieron muestras de agua y de red para análisis de fitoplancton. Una alícuota se fijó con una solución de Lugol para la identificación y cuantificación, la de red se utilizó para la identificación in vivo. Semanalmente se midió la temperatura in situ, se determinaron nutrientes inorgánicos y clorofila a en tres estaciones. Se identificaron dos especies: Chattonella marina (Subrahmanyan) Y. Hara et Chihara y Gymnodinium catenatum Graham. Chattonella marina fue la especie más abundante con densidades de 14 × 103 a 36 × 103 cél. L-1 a nivel sub-superficial. Gymnodinium catenatum se registró con densidades de 1 × 103 a 19 × 103 cél. L-1 . La temperatura del agua fue homogénea desde la superficie hasta los 20 m de profundidad, con un intervalo de 19.5 a 22.0 °C y de 20.0 a 21.5 °C a finales de marzo y abril respectivamente. En la primera mitad de mayo se inició la estratificación, con una diferencia térmica de 5.0 °C entre la superficie y los 20 m. Los nitratos variaron de 0.5 a 11.0 µM y los ortofosfatos de 0.4 a 2.6 µM. La clorofila a en marzo presentó un valor en superficie de 7.5 mg m-3 y a 20 m de 3.5 mg m-3 , en mayo varió de 2.0 a 7.0 mg m-3 . Se concluye que la aparición de las dos especies observadas en este estudio se asocia a la disminución de los procesos de mezcla en esta bahía

Mass fish die-off during a diatom bloom in the Bahía de La Paz, Gulf of California

An unusual fish die-off occurred during a bloom of diatoms from June 10th July 3rd 2006 in Bahía de La Paz in the Gulf of California. The prevalent organisms were Pseudo-nitzschia spp. (2.4 × 106 cells L1), Thalassiosira eccentrica (2.3 × 106 cells L1) and Chaetoceros spp. (9.65 x 105 cells L1). Three toxic species were identified: P. pseudodelicatissima-complex, P. fraudulenta, and P. pungens. Fucoxanthin was the dominant pigment during the bloom, peaking at 9.3 ?g L1. Sea surface temperature dramatically increased from 19.0 °C to 27.0 °C during the bloom, with inorganic nitrogen (1.0 ± 0.6 ?M) and Si(OH)4 (15.5 ± 8.0 ?M). Low content of domoic acid measured by HPLC-UV from net samples ranged from 24.0 to 52.0 ng per filter and tissue of the chocolate clam Megapitaria squalida (0.55 ?g g1) and the white clam Dosinia ponderosa (0.06 ?g g1). Domoic acid analysis of dead fish tissues was negative. Fish necropsy indicated that death by asphyxia was probably caused by Thalassiosira eccentrica and Chaetoceros spp. Alternate mixing processes and instability of the water column, as well as sudden changes of both temperature and nutrients created conditions for proliferation of the diatoms

Toxinas paralizantes en moluscos bivalvos durante una proliferación de Gymnodinium catenatum Graham en la Bahía de La Paz, México

From February to March 2007 a harmful algae bloom of Gymnodinium catenatum Graham ocurred in Bahía de La Paz, Mexico, with cell densities from 6 x 105 to 2,39 x 106 cells L-1. During this event the toxin concentration and toxin profile of paralytic shellfish toxins in mollusks (Pinna rugosa, Modiolus capax, Megapitaria squalida, Periglypta multicostata, Dosinia ponderosa, and Megapitaria aurantiaca), and in phytoplankton samples were determined. Some physicochemical data were obtained. The average values of the surface temperature and dissolved oxygen were 20.9 ± 0.7 °C and 6.9 ± 0.3 mg L-1. The average concentrations of nitrites, nitrates, ammonium, phosphates, and silicates were 0.22 ± 0.05, 1.04 ± 0.33, 0.89 ± 0.88, 0.81 ± 0.76, and 8.85 ± 1.60 µM, respectively. The concentration of paralytic shellfish toxins in the net phytoplankton samples varied from 4.32 to 79.60 ng saxitoxin equivalents filter-1, identifying 8 toxins, the most abundant being C1 and C2. The highest toxin concentration found in mollusks were 31.14, 37.74, and 25.89 µg STXeq 100 g-1 in M. capax, P. rugosa, and M. aurantiaca, respectively. The variations in the paralytic toxin profile in the different mollusks species are given.De febrero a marzo de 2007, se presentó una proliferación de Gymnodinium catenatum Graham en la Bahía de La Paz (México), con densidades de 6 x 105 a 2,39 x 106 cél. L-1. Durante este evento se determinó la concentración y el perfil de toxinas paralizantes en moluscos bivalvos (Pinna rugosa, Modiolus capax, Megapitaria squalida, Periglypta multicostata, Dosinia ponderosa y Megapitaria aurantiaca) y en muestras de fitoplancton. También se obtuvieron algunos parámetros físicos y químicos. Los valores promedios de la temperatura superficial y del oxígeno disuelto fueron de 20.9 ± 0.7 °C y 6.9 ± 0.3 mg L-1, respectivamente. Las concentraciones promedios de nitritos, nitratos, amonio, fosfatos y silicatos fueron de 0.22 ± 0.05, 1.04 ± 0.33, 0.89 ± 0.88, 0.81 ± 0.76 y 8.85 ± 1.60 µM, respectivamente. El contenido de toxinas paralizantes en las muestras de arrastres de red variaron de 4.32 a 79.60 ng saxitoxina equivalente filtro-1, identificándose 8 toxinas, siendo la C1 y C2 las dominantes. Las máximas concentraciones de toxinas paralizantes en los moluscos fueron de 31.14, 37.74 y 25.89 µg STXeq. 100 g-1 en M. capax, P. rugosa y M. aurantiaca, respectivamente. Se dan a conocer las variaciones en el perfil de toxinas paralizantes en las diversas especies de moluscos