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

EVALUATION OF ANTI-INFLAMMATORY ACTIVITY OF MACROALGAE COLLECTED FROM BAJA CALIFORNIA SUR, MEXICO

Objective: This study aimed to establish the anti-inflammatory potential of macroalgae from Baja California Sur, Mexico. Methods: The anti-inflammatory activity of the extracts from 10 macroalgae species was evaluated in vitro and in vivo. Dichloromethane, ethanolic, and aqueous extracts were obtained from each species. The criteria used to determine anti-inflammatory activity included 1) the membrane protection of human red blood cells (HRBCs) against hemolysis induced by hypotonicity, 2) the inhibition of heat-induced protein denaturation (IDP), and 3) the inhibition of mouse ear edema and the myeloperoxidase (MPO) enzyme. Results: The dichloromethane extracts of Gracilaria vermiculophylla and Opuntiella californica were the most active, both in vitro and in vivo. These extracts resulted in values of HRBC membrane protection above 95%, which were similar to those obtained with diclofenac sodium. And reduce the inflammation with edema inhibition percentage up to 60 % while the control indomethacin was able to inhibit edema by 82.3 %. Conclusion: The bioprospection of several macroalgae species from Bahía de La Paz, Mexico, allowed the identification of two species with promising anti-inflammatory activity. Gracilaria vermiculophylla showed interesting topical anti-inflammatory effects and a profile of compounds (flavonoids and terpenes) that may contribute to the potential biological properties of this seaweed. This study represents the first report of the anti-inflammatory potential of O. californica

Changes in mortality rates during the larval stage of the Pacic white shrimp (Litopenaeus vannamei ) on the basis of algal (Chaetoceros calcitrans or Tetraselmis suecica) food density/Cambios en las tasas de mortalidad durante el estadio larval del camarón blanco del Pacíco (Litopenaeus vannamei ) en base a la densidad de alimento algal (Chaetoceros

In the shrimp culture, larval stages have high mortality rates, particularly in the zoea stage, this because to the star of phytoplankton feeding and an inadequate microalga species and cell density used in commercial hatcheries. Several microalgae species are used as food in shrimp larviculture, and the most common are Chaetoceros calcitrans and Tetraselmis suecica. Therefore, in this study, we quantied changes in the mortality rates of zoea larvae of L. vannamei fed either C. calcitrans or T. suecica at dierent cell densities. Results showed higher mortality rates when fed L. vannamei larvae with T. suecica than C. calcitrans. This study demonstrates that when zoea begin to feed on phytoplankton, they are highly sensitive to the microalga diet and cell densities supplied, which signicantly aect the survival of L. vannamei larvae

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

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

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

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

Ecological and Physiological Studies of Gymnodinium catenatum in the Mexican Pacific: A Review

This review presents a detailed analysis of the state of knowledge of studies done in Mexico related to the dinoflagellate Gymnodinium catenatum, a paralytic toxin producer. This species was first reported in the Gulf of California in 1939; since then most studies in Mexico have focused on local blooms and seasonal variations. G. catenatum is most abundant during March and April, usually associated with water temperatures between 18 and 25 ºC and an increase in nutrients. In vitro studies of G. catenatum strains from different bays along the Pacific coast of Mexico show that this species can grow in wide ranges of salinities, temperatures, and N:P ratios. Latitudinal differences are observed in the toxicity and toxin profile, but the presence of dcSTX, dcGTX2-3, C1, and C2 are usual components. A common characteristic of the toxin profile found in shellfish, when G. catenatum is present in the coastal environment, is the detection of dcGTX2-3, dcSTX, C1, and C2. Few bioassay studies have reported effects in mollusks and lethal effects in mice, and shrimp; however no adverse effects have been observed in the copepod Acartia clausi. Interestingly, genetic sequencing of D1-D2 LSU rDNA revealed that it differs only in one base pair, compared with strains from other regions