Christine J. Band-Schmidt

Raphidophyceans on the coasts of Mexico

The presence of ichthyotoxic phytoflagellates Chattonella marina, Fibrocapsa japonica, and Heterosigma akashiwo of the algal class Raphidophyceae are reported for first time in the Gulf of Mexico and the Pacific coast of Mexico. Phytoplankton were sampled to isolate and identify species, and to develop growth experiments in different media. We observed living material of the three species, but were unable to recognize specimens in preserved samples. Cultures were established by isolating single cells and diluting phytoplankton samples in modified f/2 and Erd–Schreiber media. Features, including morphological variations and pigment composition, of living cells under various nutrient conditions are described. These ichthyotoxic species produce harmful blooms in several parts of the world, although no documented cases have been reported in Mexico.

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, 150u2003μmol 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 0.82u2003day−1. The highest cell yields were reached at 16 and 19 days, with maximum densities between 1090 and 3393u2003cells·mL−1. Bahía de La Paz (BAPAZ) and Bahía de Mazatlán (BAMAZ) were the most toxic (101u2003pg STXeq·cell−1), whereas strains from Bahía Concepción (BACO) were the least toxic (13u2003pg 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 0.6u2003pmol 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.

Morphology, biochemistry, and growth of raphidophyte strains from the Gulf of California

Strains of raphidophytes from different regions of the Gulf of California were established for identification and biochemical description. Raphidophyte in our coasts have been traditionally identified based on microscopic observations, a biochemical analyses of strains present in our coast is needed for a more detailed characterization and species confirmation. Strains were identified by morphological observations and sequencing of the 5.8S and COI regions. Cells were cultivated in modified f/2 media at 20°C with a light–dark cycle (12xa0h:12xa0h) and 150xa0μmolxa0m−2xa0s−1 light intensity. Pigments were identified by HPLC, brevetoxins by LC–MS/MS, fatty acids by gas chromatography, superoxide radicals by spectrophotometry, and lipid peroxidation by the determination of thiobarbituric acid reactive substances. Strains were identified as Chattonella subsalsa, C. marina, and Fibrocapsa japonica. In all strains, the main pigment was chlorophyll a, followed by fucoxanthin, chlorophyll c1 and c2, violaxanthin, β-carotene, and diadinoxanthin. Strains were tested for PbTx-1, PbTx-2, PbTx-3, PbTx-6, PbTx-9, PbTx-carboxylic acid, brevenal, and brevisin; none were detected. All strains presented superoxide radical production and lipid peroxidation. The main fatty acids were 18:4 (n-3) and 20:5 (n-3). Strains had typical fatty acid composition for raphidophytes and produced brevetoxin-like compounds, had superoxide radical production, and lipid peroxidation. With this contribution, we confirm the presence of C. subsalsa, C. marina and F. japonica in the Gulf of California.

Bloom of Scrippsiella trochoidea (Gonyaulacaceae) in a shrimp pond in the southwestern Gulf of California, Mexico

a Laboratorio de Fitoplancton, Departamento de Plancton y Ecología Marina, CICIMAR-IPN, Apdo. Postal 592, La Paz, B.C.S. 23000, Mexico b Centro de Investigaciones Biológicas del Noroeste (CIBNOR), Mar Bermejo 195, Col. Playa Palo de Santa Rita, La Paz, B.C.S. 23090, Mexico c Centro de Estudios Tecnológicos del Mar No. 4, Avenida Instituto Politécnico Nacional y Calle Cetmar s/n, 23096 La Paz, B.C.S. 23000, Mexico

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.

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.

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.

Mortality in the initial ontogeny of Paralabrax maculatofasciatus (Actinopterygii, Perciformes, Serranidae) caused by Chattonella spp. (Raphidophyceae)

Fish are particularly sensitive to metabolites produced by Raphidophyte species and these have caused intensive fish kills in several countries. However, the effects on embryos of marine fish are unknown but could probably provoke an important impact on new stock recruitment and hence on fisheries. We evaluated the toxic effects of Chattonella spp. strains from the Gulf of California on three development stages of spotted sand bass (Paralabraxmaculatofasciatus): embryo in segmentation stage (ES), embryo (EM), and eleutheroembryo (EL). Embryos (ES) were exposed to different cell concentrations of Chattonella subsalsa, Chattonella marina, Prorocentrum micans, and f/2 medium as control. Also, one set of embryos was tested with cell-free media for C. subsalsa cultures. Incubation lasted until embryos reached apterolarva phase. The ES was the most sensitive stage reaching 98% mortality with C. subsalsa, followed by cell-free media of C. subsalsa cultures, with mortalities close to 90%, whereas EM and EL phases presented mortalities below 60%. This work demonstrates that larval stages of P. maculatofasciatus are highly sensitive to short time exposure to all Chattonella spp. strains tested, that direct physical contact with cells is not required to cause mortality, and that the toxic effect is more pronounced when embryos hatch.

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 na 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.