José J. Bustillos-Guzmán

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.

Variations of PSP toxin profiles during different growth phases in Gymnodinium catenatum (Dinophyceae) strains isolated from three locations in the Gulf of California, Mexico

In vitro experiments were performed with Gymnodinium catenatum Graham strains isolated from three locations in the Gulf of California to determine the variability in toxicity and toxin profiles. Strains were cultivated in GSe at 20°C±1°C, 150 μmol photons·m−2·s−1 (12:12 light:dark cycle), and harvested during different growth phases. Growth rates were higher than in previous studies, varying between 0.70 and 0.82 day−1. The highest cell yields were reached at 16 and 19 days, with maximum densities between 1090 and 3393 cells·mL−1. Bahía de La Paz (BAPAZ) and Bahía de Mazatlán (BAMAZ) were the most toxic (101 pg STXeq·cell−1), whereas strains from Bahía Concepción (BACO) were the least toxic (13 pg STXeq·cell−1). A strain isolated from cyst germination was one of the least toxic strains. No significant changes in toxin content with culture age were observed (0.2 and 0.6 pmol paralytic shellfish poisoning·cell−1). All strains contained neosaxitoxin (NEOSTX), decarbamoyl‐saxitoxin (dcSTX), decarbamoyl‐gonyautoxin‐2,‐3, (dcGTX2‐3), N‐sulfo‐carbamoylsaxitoxin (B1), N‐sulfo‐carbamoylneosaxitoxin (B2), and N‐sulfo‐carbamoylgonyautoxin‐2,‐3 (C1‐2). Bahía Concepción strains had the highest content of C1; BAPAZ and BAMAZ strains had a higher percentage of NEOSTX. Differences in toxin composition with culture age were observed only in BAMAZ and BAPAZ strains. Cultures with a higher percentage of long chains had more NEOSTX, while those with a higher proportion of short chains had a lower content of NEOSTX. Gulf of California strains are characterized by a high proportion of NEOSTX, and seem to have evolved particular physiological responses to their environment that are reflected in the toxin profile, suggesting different populations.

Presence of benzoate type toxins in Gymnodinium catenatum Graham isolated from the Mexican Pacific

Benzoate type toxins have been described as an important component of Gymnodinium catenatum cells. In this paper we study these toxins in a G. catenatum strain isolated from the Mexican coast. A partition of the toxins was done by solid-phase extraction on a COOH cartridge and detected by HPLC coupled to fluorescence after pre-column periodate oxidation. Two groups of the hydrophobic analogues of saxitoxin were identified: those containing a sulphate group in the benzoate moiety instead of a hydroxyl group like GC1/2 or GC3 and the hydroxy-benzoate analogues, with a sulphate group at the eleventh position of the STX core present or absent (GCs-GTX and GCs-STX analogues, respectively). These toxins are more abundant, in a relative basis, when comparing with a G. catenatum toxin content isolated from Portugal. This is the first report of the presence of these toxins in a Mexican strain.

Growth potential of wild juvenile Penaeus stylirostris in earthen ponds receiving chemical and organic fertilizers, and pelleted feed

Abstract The growth potential of wild juvenile Penaeus stylirostris was analyzed in experimental trials using chemical and organic fertilizers and pelleted feed in 0·25 ha earthen ponds. Shrimp were stocked at a low density (1·6 ind/m2). After 103 days, final weight ranged from 9·5 to 26·0 g. The significance of differences in growth response was determined using a reparameterization of the von Bertalanffy growth curve. Maximum growth rates observed in the trials ranged from 0·06 to 0·33 g/day, averaging 0·22 g/day. Maximum rates were related to weight by the equation: ( dw dt ) max =0·034w 0·69 r 2 =0·9,P Pelleted feed produced best growth, chemical fertilizers were acceptable, and organic fertilizers gave poor results. The high growth potential of the species stimulates future research in order to adapt a culture technology to the semi-arid conditions of northwest Mexico, where the species is indigenous.

Allelopathic effect of Chattonella marina var. marina (Raphidophyceae) on Gymnodinium catenatum (Dinophycea)

The allelopathic effect of the raphidophyte Chattonella marina var. marina on the dinoflagellate Gymnodinium catenatum was determined. Both species are harmful algal bloom forming algae, produce toxic metabolites, and can co-exist in the environment. In general, raphidophytes tend to dominate over dinoflagellates, which may indicate an allelopathic effect of the former algae. Strains of C. marina var. marina and G. catenatum isolated from Bahía de La Paz were cultured in bi-algal cultures with and without cell contact. Additionally, cultures of G. catenatum were exposed to cell-free culture filtrates of the raphidophyte to test whether soluble allelopathic molecules are active. During late stationary phase, both species were cultivated in mixed cultures for 72h using the following cell abundance proportions: 20×103cellsL-1: 20×103cellsL-1 (1:1; G. catenatum: C. marina); 10×103cellsL-1: 20×103cellsL-1 (1:2), and 20×103cellsL-1: 10×103cellsL-1 (2:1). Cells of G. catenatum were also exposed to different volumes of cell filtrates of C. marina (10, 20, and 50mL) using the same cell abundance proportions for 24h. Samples were taken daily for cell counts and microscopic observations. Growth inhibition was higher when there was cell contact between both species, however mortality of G. catenatum was also observed without direct cell contact, indicating that toxic metabolites are liberated to the culture medium. Changes in cell morphology of G. catenatum occurred in the presence of cells and filtrates of C. marina, such as loss of flagella and motility, swelling, loss of girdle and sulci, prominent nucleus, rupture of cell membrane, and cell lysis. Induction of temporary cysts was also observed. These results suggest that toxic metabolites are liberated to the medium by C. marina, affecting G. catenatum by inhibiting its growth and causing changes in its life history, providing new insights of interactions between raphidophytes and dinoflagellates that could happen in the natural environment when both species are present.

Unusual mortality of krill (Crustacea: Euphausiacea) in Bahía de La Paz, Gulf of California

ABSTRACT Surface aggregations and beach strandings of a species of krill, Nematoscelis difficilis Hansen, were observed in June 2003 at locations along the shore of Bahía de La Paz in the Gulf of California. For 10 days before the krill die-off, a steady wind blew from the south at speeds between 4 and 5 m/sec. For that period, satellite images showed water temperatures between 18 and 22 °C along this coast, which is low compared with typical seasonal water temperatures of 26 to 28 °C for June. Phytoplankton biomass, determined by pigment concentration and cell counts, was the highest in the area in June. The diatom Chaetoceros debilis represented more than 96% of the phytoplankton community. Nutrients were in relatively higher concentrations. These data suggest that upwelling conditions occurred and the diatom bloom was in its final phase. Based on this limited data set, we present a hypothetical scenario describing the sea-surface aggregations and beach strandings of N. difficilis.

Effect of temperature on growth and paralytic toxin profiles in isolates of Gymnodinium catenatum (Dinophyceae) from the Pacific coast of Mexico

The effects of temperature on growth, cell toxicity, toxin content, and profile of paralytic shellfish toxins was determined in eight isolates of Gymnodinium catenatum from several localities along the Pacific Coast of Mexico. The isolates were cultivated in modified f/2 media with Se (10(-8) M), and a reduced concentration of Cu (10(-8) M), under a 12 h:12 h day-night cycle with an irradiance of 150 μE m(-2) s(-1). Isolates were progressively adapted for three generations to each of the temperatures (16, 19, 22, 24, 27, 30, and 33 °C). The cultures were grown in 125 mL Erlenmeyer flasks with 60 mL of media and harvested by filtration in late exponential growth. Toxins were analyzed by HPLC with a post-column oxidation and fluorescent detection (FLD). G. catenatum isolates tolerate temperatures between 16 and 33 °C, with maximum growth rates of 0.32 and 0.39 div day(-1) at 21 °C and 24 °C, respectively; maximum cell densities of 4700 and 5500 cells mL(-1) were obtained at 27 and 21 °C, respectively. No effect of toxicity per cell with temperature was observed, varying between 10.10 and 28.19 pgSXTeq cell(-1). Ten saxitoxin analogues were detected in all isolates, observing changes in the toxin profile with temperature. C1/2 toxins decreased from 80% mol at 16 °C to 20% mol at 33 °C, B1/2 toxins increased from 19% mol at 16 °C to 42% mol at 33 °C, and decarbamoyl toxins were more abundant at 21 °C. These results show that G. catenatum isolates from different regions of the Pacific coast of Mexico have a similar response to temperature and that this parameter can modify growth rate, cell density, and toxin profile of the species, particularly the decarbamoyl and sulfocarbamoyl toxins.

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.

Condiciones ambientales durante un florecimiento de Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) en la Ensenada de La Paz, Golfo de California

Abstract .- Environmental conditions are described during a bloom of Cochlodinium polykrikoides that occurred betweenSeptember 12 th and November 9 th 2012, in Ensenada de La Paz, Gulf of California. At the beginning of the bloom, the numberof cells, chlorophyll a , and peridinin was 6.2 x10 2 cells mL -1 , 10.1 mg m -3 , and 3.5 mg m -3 , respectively; 8.6 x 10 3 cells mL -1 ,121.2 mg m -3 and 40.2 mg m -3 in their maximum intensity; and 5 cells mL -1 , 1.02 mg m -3 of chlorophyll a , and 0.03 mg m -3 ofperidinin, at the end of the bloom period. Thermal interval fluctuated between 30.3 and 31.3 °C; salinity varied from 35.2to 36.8; dissolved O 2 ranged from 4.8 to 10.8 mL L -1 ; saturation percentage was > 200. The values of nitrate, phosphate, andthe N:P ratio at the initiation of the bloom were 1.8, 0.9 (μM) and 2.8, during the maximum intensity they were 8.5, 3.4 (μM),and 2.9. At the end of the bloom they were 5.3, 0.6 (μM) and 9.0. Prevailing winds were from the NNE (0.4-5.0 m s

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.