RESUMEN
The effects of yessotoxins (YTXs) produced by the dinoflagellate Protoceratium reticulatum in the early stages of bivalves have not been studied in detail. The present study evaluates the effects of P. reticulatum and YTXs on the survival and feed ingestion of veliger larvae of Argopecten purpuratus. Larvae were 96 h-exposed to 500, 1000 and 2000 P. reticulatum cells mL-1, and their equivalent YTX extract was prepared in methanol. Results show a survival mean of 82 % at the highest density of dinoflagellate, and 38 % for larvae with the highest amount of YTX extract. Feed ingestion is reduced in the dinoflagellate exposure treatments as a function of cell density. Therefore, the effect of YTXs on A. purpuratus represents a new and important area of study for investigations into the deleterious effects of these toxins in the early stages of the life cycle of this and, potentially, other bivalves.
Asunto(s)
Bivalvos , Dinoflagelados , Venenos de Moluscos , Oxocinas , Pectinidae , Animales , Toxinas Marinas/metabolismo , Larva , Dinoflagelados/metabolismo , Ingestión de AlimentosRESUMEN
The bloom-forming toxic dinoflagellate Alexandrium catenella was first detected in southern Chile (39.5-55° S) 50 years ago and is responsible for most of the area's cases of paralytic shellfish poisoning (PSP). Given the complex life history of A. catenella, which includes benthic sexual cysts, in this study, we examined the potential link between latitude, toxicity, and sexual compatibility. Nine clones isolated from Chilean Patagonia were used in self- and out-crosses in all possible combinations (n = 45). The effect of latitude on toxicity, reproductive success indexes, and cyst production was also determined. Using the toxin profiles for all strains, consisting of C1, C2, GTX4, GTX1, GTX3, and NeoSTX, a latitudinal gradient was determined for their proportions (%) and content per cell (pg cell-1), with the more toxic strains occurring in the north (-40.6° S). Reproductive success also showed a latitudinal tendency and was lower in the north. None of the self-crosses yielded resting cysts. Rather, the production of resting cysts was highest in pairings of clones separated by distances of 1000-1650 km. Our results contribute to a better understanding of PSP outbreaks in the region and demonstrate the importance of resting cysts in fueling new toxic events. They also provide additional evidence that the introduction of strains from neighboring regions is a cause for concern.
Asunto(s)
Dinoflagelados/genética , Dinoflagelados/metabolismo , Toxinas Marinas/metabolismo , Toxinas Marinas/toxicidad , Chile , ADN Espaciador Ribosómico/genética , Eutrofización , Toxinas Marinas/genética , ReproducciónRESUMEN
The Peruvian sea is one of the most productive ecosystems in the world. Phytoplankton production provides food for fish, mammals, mollusks and birds. This trophic network is affected by the presence of toxic phytoplankton species. In July 2017, samples of phytoplankton were obtained from Paracas Bay, an important zone for scallop (Argopecten purpuratus) aquaculture in Peru. Morphological analysis revealed the presence of the genus Pseudo-nitzschia, which was isolated and cultivated in laboratory conditions. Subsequently, the monoclonal cultures were observed by scanning electron microscopy (SEM), and identified as P. multistriata, based on both the morphological characteristics, and internal transcribed spacers region (ITS2) sequence phylogenetic analysis. Toxin analysis using liquid chromatography (LC) with high-resolution mass spectrometry (HRMS) revealed the presence of domoic acid (DA) with an estimated amount of 0.004 to 0.010 pg cell-1. This is the first report of DA from the coastal waters of Peru and its detection in P. multistriata indicates that it is a potential risk. Based on our results, routine monitoring of this genus should be considered in order to ensure public health.
Asunto(s)
Diatomeas/metabolismo , Ácido Kaínico/análogos & derivados , Toxinas Marinas/metabolismo , Neurotoxinas/metabolismo , Fitoplancton/metabolismo , Bahías , Diatomeas/genética , Monitoreo del Ambiente , Ácido Kaínico/metabolismo , Perú , Filogenia , Fitoplancton/genéticaRESUMEN
Domoic acid (DA), the main toxin responsible for Amnesic Shellfish Poisoning, frequently affects the marine resources of Chile and other countries across the South Pacific, thus becoming a risk for human health. One of the affected resources is the scallop Argopecten purpuratus. Even though this species has a high commercial importance in Northern Chile and Peru, the characteristics of its DA depuration are not known. In this work, the DA depuration was studied by means of two experiments: one in controlled (laboratory) and another in natural conditions. All organs of A. purpuratus depurated the toxin very quickly in both experiments. In some organs, an increase or a very small decrease of toxin was detected in the early depuration steps. Several models were used to describe this kinetics. The one that included toxin transfer between organs and independent depuration from each organ was the model that best fit the data. It seems, therefore, that the DA in this species is quickly transferred from the digestive gland to all other organs, which release it into the environment. Physiological differences in the two experiments have been shown to have some effect on the depuration from each organ but the actual reasons are still unknown.
Asunto(s)
Sistema Digestivo/metabolismo , Ácido Kaínico/análogos & derivados , Toxinas Marinas/metabolismo , Pectinidae/metabolismo , Alimentos Marinos , Intoxicación por Mariscos , Animales , Carga Corporal (Radioterapia) , Ácido Kaínico/metabolismo , Ácido Kaínico/toxicidad , Cinética , Toxinas Marinas/toxicidad , Alimentos Marinos/efectos adversos , Distribución Tisular , ToxicocinéticaRESUMEN
Of all cyanobacteria, Microcystis aeruginosa is the most commonly found species in bloom episodes all over the world. This species is known to produce cyanopeptides with hepatotoxic effects, namely microcystins (MCs). In this regard, Advanced Oxidation Processes (AOPs) have been widely studied for cyanotoxin degradation, but very few studies focused on cyanobacteria inactivation combined with toxin removal. To our knowledge, this is the first report of the photo-Fenton process application focusing on M. aeruginosa inactivation and microcystin-LR (MC-LR) degradation. This research work aimed to evaluate the photo-Fenton process under three different conditions with regard to Fe2+/H2O2 ratios (0.6/10, 5/50, and 20/100 mg L-1) at the initial near-neutral pH. Process efficiency was measured by immediate cell density reduction, growth inhibition, effect on MC-LR concentrations, and scanning electron microscopy (SEM) to analyze any alterations in cell morphology. Growth inhibition test (GIT) results pointed to cell inactivation under all conditions tested, and MC-LR concentrations were reduced below WHO's maximum limit at medium and higher concentrations of reagents. The possible mechanisms of cell inactivation by oxidative species are discussed.
Asunto(s)
Toxinas Marinas/metabolismo , Microcistinas/metabolismo , Microcystis/metabolismo , Compuestos Ferrosos/análisis , Compuestos Ferrosos/farmacología , Peróxido de Hidrógeno/análisis , Peróxido de Hidrógeno/farmacología , Concentración de Iones de Hidrógeno , Microcystis/citología , Microcystis/efectos de los fármacos , Oxidación-ReducciónRESUMEN
Phytoplankton anti-grazer traits control zooplankton grazing and are associated with harmful blooms. Yet, how morphological versus chemical phytoplankton defenses regulate zooplankton grazing is poorly understood. We compared zooplankton grazing and prey selection by contrasting morphological (filament length: short vs. long) and chemical (saxitoxin: STX- vs. STX+) traits of a bloom-forming cyanobacterium (Raphidiopsis) offered at different concentrations in mixed diets with an edible phytoplankton to a copepod grazer. The copepod selectively grazed on the edible prey (avoidance of cyanobacteria) even when the cyanobacterium was dominant. Avoidance of the cyanobacterium was weakest for the "short STX-" filaments and strongest for the other three strains. Hence, filament size had an effect on cyanobacterial avoidance only in the STX- treatments, while toxin production significantly increased cyanobacterial avoidance regardless of filament size. Moreover, cyanobacterial dominance reduced grazing on the edible prey by almost 50%. Results emphasize that the dominance of filamentous cyanobacteria such as Raphidiopsis can interfere with copepod grazing in a trait specific manner. For cyanobacteria, toxin production may be more effective than filament size as an anti-grazer defense against selectively grazing zooplankton such as copepods. Our results highlight how multiple phytoplankton defensive traits interact to regulate the producer-consumer link in plankton ecosystems.
Asunto(s)
Toxinas Bacterianas/metabolismo , Copépodos/fisiología , Cianobacterias/fisiología , Cadena Alimentaria , Floraciones de Algas Nocivas , Toxinas Marinas/metabolismo , Animales , Cianobacterias/crecimiento & desarrollo , Cianobacterias/metabolismoRESUMEN
In fjord systems, Harmful Algal Blooms (HABs) not only constitute a serious problem when affecting the wildlife and ecosystems, but also human health and economic activities related to the marine environment. This is mostly due to a broad spectrum of toxic compounds produced by several members of the phytoplankton. Nevertheless, a deep coverage of the taxonomic diversity and composition of phytoplankton species and phycotoxin profiles in HAB prone areas are still lacking and little is known about the relationship between these fundamental elements for fjord ecosystems. In this study, a detailed molecular and microscopic characterization of plankton communities was performed, together with an analysis of the occurrence and spatial patterns of lipophilic toxins in a HAB prone area, located in the Southeastern Pacific fjord region. Microscopy and molecular analyses based on the 18S rRNA gene fragment indicated high diversity and taxonomic homogeneity among stations. Four toxigenic genera were identified: Pseudo-nitzschia, Dinophysis, Prorocentrum, and Alexandrium. In agreement with the detected species, liquid chromatography coupled with mass spectrometry revealed the presence of domoic acid (DA), pectenotoxin-2 (PTX-2), dinophysistoxin-2 (DTX-2), and 13-desmethyl spirolide C (SPX-1). Furthermore, a patchy distribution among DA in different net haul size fractions was found. Our results displayed a complex phytoplankton-phycotoxin pattern and for the first time contribute to the characterization of high-resolution phytoplankton community composition and phycotoxin distribution in fjords of the Southeastern Pacific region.
Asunto(s)
Biodiversidad , Estuarios , Toxinas Marinas/química , Toxinas Marinas/metabolismo , Fitoplancton/genética , Fitoplancton/metabolismo , Chile , Secuenciación de Nucleótidos de Alto RendimientoRESUMEN
This study evaluated an 8-year dataset (2007 to 2015, except 2008) in the attempt to identify the most susceptible periods for the occurrence of diarrheic shellfish poisoning (DSP) episodes associated with the presence of toxigenic dinoflagellates, Dinophysis spp., in the mussel farming area of Babitonga Bay (southern Brazil). Dinophysis acuminata complex was the most frequent (present in 66% of the samples) and abundant (max. 4100 cells L-1) taxon, followed by D. caudata (14%; max. 640 cells L-1) and D. tripos (0.9%; max. 50 cells L-1). There was a marked onset of the annual rise in Dinophysis spp. abundance during weeks 21-25 (early winter) of each year, followed by a second peak on week 35 (spring). Mussel (Perna perna) samples usually started testing positive in DSP mouse bioassays (MBA) in late winter. Positive results were more frequent in 2007 and 2011 when the mean D. acuminata complex abundance was ~ 500 cells L-1. Although positive DSP-MBA results were observed in only 11% of the samples during the studied period, the toxin okadaic acid (OA) was present in 90% of the analyzed mussels (max. 264 µg kg-1). MBA results were positive when D. acuminata complex cell densities exceed 1200 ± 300 cells L-1, while trace toxin amounts could be detected at cell densities as low as 150 ± 50 cells L-1 (free OA) to 200 ± 100 cells L-1 (conjugated OA). Low salinity and the meteorological conditions triggered by La Niña events were the main factors associated with both Dinophysis abundance and OA accumulation in mussels.
Asunto(s)
Acuicultura , Monitoreo del Ambiente , Estuarios , Toxinas Marinas/metabolismo , Perna/fisiología , Contaminantes Químicos del Agua/metabolismo , Animales , Bivalvos , Brasil , Dinoflagelados , Toxinas Marinas/análisis , Ratones , Alimentos Marinos , Estaciones del Año , Mariscos/análisis , Intoxicación por Mariscos , Contaminantes Químicos del Agua/análisisRESUMEN
In order to assess the effects of Fe-enrichment on the growth and domoic acid (DA) production of the toxigenic diatom Pseudo-nitzschia multiseries, static cultures that received the addition of different iron (Fe) concentrations were maintained for 30 days. Intra- and extracellular DA concentrations were evaluated over time, and growth and chain-formation were compared to those of non-toxic diatoms, Bacillaria sp. Growth rates of P. multiseries (µ = 0.45-0.73 d-1) were similar among cultures containing different Fe concentrations. Likewise, the similar incidence and length of P. multiseries stepped cell chains (usually 2-4; up to 8-cell long) among the treatments reinforces that the cultures were not growth-inhibited under any condition tested, suggesting an efficient Fe acquisition mechanism. Moreover, DA concentrations were significantly higher under the highest Fe concentration, indicating that Fe is required for toxin synthesis. Bacillaria sp. reached comparable growth rates under the same Fe concentrations, except when the dissolved cell contents from a P. multiseries culture was added. The 50-70% reduction in cell density and 70-90% decrease in total chlorophyll-a content of Bacillaria sp. at early stationary growth phase indicates, for the first time, an allelopathic effect of undetermined compounds released by Pseudo-nitzschia to another diatom species.
Asunto(s)
Alelopatía/efectos de los fármacos , Diatomeas/fisiología , Hierro/farmacología , Toxinas Marinas/metabolismo , Neurotoxinas/metabolismo , Alimentos Marinos/toxicidad , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Clorofila/metabolismo , Clorofila A , Diatomeas/efectos de los fármacos , Humanos , Ácido Kaínico/análogos & derivados , Ácido Kaínico/metabolismo , Ácido Kaínico/toxicidad , Toxinas Marinas/toxicidad , Neurotoxinas/toxicidadRESUMEN
The toxin profile and hemolytic activity of a strain of Ostreopsis cf. ovata (UFBA013) isolated from Todos os Santos Bay (northeastern Brazil) were evaluated under different levels of N and P. Phylogenetic analyses based on ITS rDNA region (ITS1-5.8S-ITS2) placed UFBA013 within the Atlantic/Mediterranean/Pacific clade of O. cf. ovata. Growth experiments were conducted in f/2 medium modified by adding N and P (P: 0-36 µM; N: 0-882 µM). The growth kinetics was adequately described by logistic equations. The best growth (highest Gm) was recorded under levels of N/P = 0/18, 129/5 and 441/36, while one of the lowest Gm was obtained under P-depletion. The maximum and specific maximum growth rates (as vm; cells mL-1 d-1 and µm; d-1) were achieved with N limitation (N/P = 441/36) and P-limitation/depletion (753/5.3 and 441/0) and are the highest values reported in the literature, most similar to isolates from Pacific and Mediterranean areas. The control experiment (N/P = 441/18) also yielded similar values to those from some Mediterranean isolates, but higher than formerly reported for Brazilian isolates. In all conditions assayed, no palytoxin (PLTX) was detected. The ovatoxins (OVTXs) a, b, c, d and e did not show significant differences in cell quota between exponential and stationary phases. A significant relationship was detected between OVTXs concentration and hemolytic activity.
Asunto(s)
Dinoflagelados/crecimiento & desarrollo , Dinoflagelados/metabolismo , Toxinas Marinas/metabolismo , Animales , Brasil , ADN Ribosómico , Dinoflagelados/clasificación , Floraciones de Algas Nocivas , Hemólisis , Nitrógeno/metabolismo , Fósforo/metabolismo , Filogenia , Agua de Mar/química , Análisis de Secuencia de ADN , OvinosRESUMEN
Dinophysis is a cosmopolitan genus of marine dinoflagellates, considered as the major proximal source of diarrheic shellfish toxins and the only producer of pectenotoxins (PTX). From three oceanographic expeditions carried out during autumn, spring and late summer along the Argentine Sea (â¼38-56°S), lipophilic phycotoxins were determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in size-fractionated plankton samples. Lipophilic toxin profiles were associated with species composition by microscopic analyses of toxigenic phytoplankton. Pectenotoxin-2 and PTX-11 were frequently found together with the presence of Dinophysis acuminata and Dinophysis tripos. By contrast, okadaic acid was rarely detected and only in trace concentrations, and dinophysistoxins were not found. The clear predominance of PTX over other lipophilic toxins in Dinophysis species from the Argentine Sea is in accordance with previous results obtained from north Patagonian Gulfs of the Argentine Sea, and from coastal waters of New Zealand, Chile, Denmark and United States. Dinophysis caudata was rarely found and it was confined to the north of the sampling area. Because of low cell densities, neither D. caudata nor Dinophysis norvegica could be biogeographically related to lipophilic toxins in this study. Nevertheless, the current identification of D. norvegica in the southern Argentine Sea is the first record for the southwestern Atlantic Ocean. Given the typical toxigenicity of this species on a global scale, this represents an important finding for future surveillance of plankton-toxin associations.
Asunto(s)
Dinoflagelados/química , Toxinas Marinas/análisis , Plancton/química , Océano Atlántico , Dinoflagelados/metabolismo , Toxinas Marinas/metabolismo , Plancton/metabolismoRESUMEN
High resolution mass spectrometry investigation of an extract of the toxic cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 led to the discovery of four new spumigin congeners. The structures for these peptides were postulated on the basis of accurate mass data and isotopic pattern information of both full scan and product ion spectra. This is the first reported evidence of spumigins on Sphaerospermopsis species.
Asunto(s)
Cianobacterias/metabolismo , Toxinas Marinas/química , Oligopéptidos/química , Fraccionamiento Químico , Cromatografía Liquida , Cianobacterias/química , Toxinas Marinas/aislamiento & purificación , Toxinas Marinas/metabolismo , Espectrometría de Masas , Oligopéptidos/aislamiento & purificación , Oligopéptidos/toxicidadRESUMEN
In northern Chile, domoic acid (DA) has been detected in several bivalve species. In Mesodesma donacium, one of the most important commercial species for local fishermen, no information is available on depuration, or on the anatomical distribution of this toxin and its potential use as a palliative measure to minimize the consequences of ASP outbreaks. Deputation of DA is very fast in M. donacium, and can be adequately described by means of a two-compartment model. The estimated rates for the first and second compartments were 1.27 d(-1) and 0.24 d(-1), respectively, with a transfer rate between compartments of 0.75. Having high depuration rates protects this species from being affected by Pseudo-nitzschia blooms for an extended period of time. Taking this into account, the time in which the bivalves are unsafe for consumers is very short, and therefore the economic losses that could result by the DA outbreaks in local fisheries should be moderate. In relation to anatomical distribution, at least during the uptake phase, the toxin was evenly distributed within the soft tissues, with a total toxin burden corresponding to 27%, 32% and 41% for Digestive Gland (DG), Foot (FT) and Other Body Fractions (OBF), respectively. Since the contribution of each organ to the toxin concentration is a function of both weight contribution and toxin burden, the pattern of toxin distribution showed the following trend: "all other body fractions" (OBF) > Foot (FT) > Digestive Gland (DG). Thus, the highest concentration of DA, with a contribution close to 72%, corresponds to the edible tissues (OBF + FT), while the DG (non-edible tissue) only contributes the remaining 28%. Consequently, in view of the anatomical distribution of domoic acid in M. donacium, the elimination of the digestive gland does not substantially reduce the toxicity of the final product and therefore selective evisceration would not improve their quality for human consumption.
Asunto(s)
Bivalvos/química , Diatomeas/química , Ácido Kaínico/análogos & derivados , Toxinas Marinas/análisis , Toxinas Marinas/metabolismo , Animales , Transporte Biológico , Bivalvos/anatomía & histología , Chile , Ácido Kaínico/metabolismo , Distribución TisularRESUMEN
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.
Asunto(s)
Dinoflagelados/crecimiento & desarrollo , Dinoflagelados/metabolismo , Toxinas Marinas/metabolismo , Agua de Mar , TemperaturaRESUMEN
The aim of this study was to analyse and determine the composition of paralytic shellfish poisoning (PSP) toxins and lipophilic toxins in the Region of Aysén, Chile, in wild endemic mussels (Mytilus chilensis, Venus antiqua, Aulacomya ater, Choromytilus chorus, Tagelus dombeii and Gari solida) and in two endemic carnivorous molluscs species (Concholepas concholepas and Argobuccinum ranelliforme). PSP-toxin contents were determined by using HPLC with fluorescence detection, while lipophilic toxins were determined by using LC-MS/MS. Mean concentrations for the total of PSP toxins were in the range 55-2505 µg saxitoxin-equivalent/100 g. The two most contaminated samples for PSP toxicity were bivalve Gari solida and carnivorous Argobuccinum ranelliforme with 2505 ± 101 and 1850 ± 137 µg saxitoxin-equivalent/100 g, respectively (p < 0.05). The lipophilic toxins identified were okadaic acid, dinophysistoxin-1 (DTX-1), azaspiracid-1 (AZA-1), pectenotoxin-2 (PTX-2) and yessotoxins (YTX). All analysed molluscs contained lipophilic toxins at levels ranging from 56 ± 4.8 to 156.1 ± 8.2 µg of okadaic acid-equivalent/kg shellfish together with YTX at levels ranging from 1.0 ± 0.1 to 18 ± 0.9 µg of YTX-equivalent/kg shellfish and AZA at levels ranging from 3.6 ± 0.2 to 31 ± 2.1 µg of AZA-equivalent/kg shellfish. Furthermore, different bivalves and gastropods differ in their capacity of retention of lipophilic toxins, as shown by the determination of their respective lipophilic toxins levels. In all the evaluated species, the presence of lipophilic toxins associated with biotransformation in molluscs and carnivorous gastropods was not identified, in contrast to the identification of PSP toxins, where the profiles identified in the different species are directly related to biotransformation processes. Thus, this study provides evidence that the concentration of toxins in the food intake of the evaluated species (Bivalvia and Gastropoda class) determines the degree of bioaccumulation and biotransformation they will thereafter exhibit.
Asunto(s)
Bivalvos/química , Dinoflagelados/metabolismo , Contaminación de Alimentos , Inspección de Alimentos , Gastrópodos/química , Toxinas Marinas/metabolismo , Mariscos/análisis , Animales , Biotransformación , Bivalvos/crecimiento & desarrollo , Bivalvos/metabolismo , Chile , Cromatografía Líquida de Alta Presión , Dinoflagelados/crecimiento & desarrollo , Dinoflagelados/aislamiento & purificación , Gastrópodos/crecimiento & desarrollo , Gastrópodos/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Toxinas Marinas/química , Toxinas Marinas/toxicidad , Estructura Molecular , Islas del Pacífico , Océano Pacífico , Agua de Mar/parasitología , Mariscos/efectos adversos , Intoxicación por Mariscos/etiología , Espectrometría de Fluorescencia , Espectrometría de Masas en Tándem , Distribución TisularRESUMEN
Sea anemones represent one of the emerging groups of interest concerning venomous animals in toxinology and the goal of the present work was the prospection, and the structural and functional characterization of the compounds present in the secretion of the sea anemone Stichodactyla duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low molecular weight fractions were further submitted to RP-HPLC resulting in 35 new subfractions that were subsequently analyzed by offline MALDI-TOF mass spectrometry. MALDI peptide mass fingerprinting yielded up to 134 different molecular masses, ranging from m/z 901 to 10,833. Among these subfractions, a new peptide of 3431Da, named U-SHTX-Sdd1, was purified and completely sequenced by automated Edman's degradation and tandem mass spectrometry. An analysis of U-SHTX-Sdd1 revealed a modified O-HexNAc-Threonine at position 1, which, at the best of our knowledge, constitutes the first sea anemone toxin reported with such post-translational modification. Because of its sequence similarity with other sea anemone toxins, the pharmacological activity of U-SHTX-Sdd1 was assessed by electrophysiological measurements using the two electrode voltage-clamp technique on cloned voltage-gated potassium channel subtypes, expressed in Xenopus laevis oocytes. However, U-SHTX-Sdd1 did not show activity on these channels. A large-scale proteomic approach was also employed to shed lights on the sea anemone compounds, and a total 67 proteins and peptides were identified. BIOLOGICAL SIGNIFICANCE: In this manuscript, we report an extensive characterization of S. duerdeni secretion by means of peptide mass fingerprinting and high-throughput proteome analyses. Also, we report the structure of a new glycopeptide by a combination of biochemical techniques. Despite the previous studies that described proteinaceous compounds present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy of analysis is perfectly suitable to other sea anemone secretions and animal venoms. Moreover, new peptide structures can arise contributing to the knowledge of the diversity of these animal peptides.
Asunto(s)
Glicopéptidos , Activación del Canal Iónico/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Proteómica , Anémonas de Mar , Animales , Glicopéptidos/química , Glicopéptidos/genética , Glicopéptidos/metabolismo , Glicopéptidos/farmacología , Activación del Canal Iónico/genética , Toxinas Marinas/química , Toxinas Marinas/genética , Toxinas Marinas/metabolismo , Toxinas Marinas/farmacología , Oocitos , Canales de Potasio con Entrada de Voltaje/biosíntesis , Canales de Potasio con Entrada de Voltaje/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anémonas de Mar/química , Anémonas de Mar/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Xenopus laevisRESUMEN
Paralytic shellfish poisoning toxins (PSTs) are a family of more than 30 natural alkaloids synthesized by dinoflagellates and cyanobacteria whose toxicity in animals is mediated by voltage-gated Na(+) channel blocking. The export of PST analogues may be through SxtF and SxtM, two putative MATE (multidrug and toxic compound extrusion) family transporters encoded in PSTs biosynthetic gene cluster (sxt). sxtM is present in every sxt cluster analyzed; however, sxtF is only present in the Cylindrospermopsis-Raphidiopsis clade. These transporters are energetically coupled with an electrochemical gradient of proton (H(+)) or sodium (Na(+)) ions across membranes. Because the functional role of PSTs remains unknown and methods for genetic manipulation in PST-producing organisms have not yet been developed, protein structure analyses will allow us to understand their function. By analyzing the sxt cluster of eight PST-producing cyanobacteria, we found no correlation between the presence of sxtF or sxtM and a specific PSTs profile. Phylogenetic analyses of SxtF/M showed a high conservation of SxtF in the Cylindrospermopsis-Raphidiopsis clade, suggesting conserved substrate affinity. Two domains involved in Na(+) and drug recognition from NorM proteins (MATE family) of Vibrio parahaemolyticus and V. cholerae are present in SxtF/M. The Na(+) recognition domain was conserved in both SxtF/M, indicating that Na(+) can maintain the role as a cation anti-transporter. Consensus motifs for toxin binding differed between SxtF and SxtM implying differential substrate binding. Through protein modeling and docking analysis, we found that there is no marked affinity between the recognition domain and a specific PST analogue. This agrees with our previous results of PST export in R. brookii D9, where we observed that the response to Na(+) incubation was similar to different analogues. These results reassert the hypothesis regarding the involvement of Na(+) in toxin export, as well as the motifs L(398)XGLQD(403) (SxtM) and L(390)VGLRD(395) (SxtF) in toxin recognition.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cylindrospermopsis/metabolismo , Toxinas Marinas/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte Biológico Activo , Simulación por Computador , Cylindrospermopsis/química , Cylindrospermopsis/genética , Toxinas Marinas/química , Toxinas Marinas/genética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Modelos Moleculares , Familia de Multigenes , Filogenia , Conformación Proteica , Saxitoxina/análogos & derivados , Saxitoxina/genética , Saxitoxina/metabolismoRESUMEN
The dinoflagellate Gymnodinium catenatum produces paralyzing shellfish poisons that are consumed and accumulated by bivalves. We performed short-term feeding experiments to examine ingestion, accumulation, biotransformation, histopathology, and paralysis in the juvenile Pacific calico scallop Argopecten ventricosus that consume this dinoflagellate. Depletion of algal cells was measured in closed systems. Histopathological preparations were microscopically analyzed. Paralysis was observed and the time of recovery recorded. Accumulation and possible biotransformation of toxins were measured by HPLC analysis. Feeding activity in treated scallops showed that scallops produced pseudofeces, ingestion rates decreased at 8 h; approximately 60% of the scallops were paralyzed and melanin production and hemocyte aggregation were observed in several tissues at 15 h. HPLC analysis showed that the only toxins present in the dinoflagellates and scallops were the N-sulfo-carbamoyl toxins (C1, C2); after hydrolysis, the carbamate toxins (epimers GTX2/3) were present. C1 and C2 toxins were most common in the mantle, followed by the digestive gland and stomach-complex, adductor muscle, kidney and rectum group, and finally, gills. Toxin profiles in scallop tissue were similar to the dinoflagellate; biotransformations were not present in the scallops in this short-term feeding experiment.
Asunto(s)
Dinoflagelados/metabolismo , Toxinas Marinas/toxicidad , Parálisis/inducido químicamente , Pectinidae/efectos de los fármacos , Pectinidae/metabolismo , Animales , Biotransformación , Cromatografía Líquida de Alta Presión/métodos , Métodos de Alimentación , Branquias/efectos de los fármacos , Branquias/metabolismo , Branquias/patología , Hemocitos/metabolismo , Hemocitos/patología , Hidrólisis , Cinética , Toxinas Marinas/metabolismo , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Parálisis/metabolismo , MariscosRESUMEN
Lipophilic toxins associated with diarrhoeic toxins were found in Mytilus chilensis (Blue mussels) and Aulacomya ater (Ribbed mussels). These shellfish samples were collected from Chiloe Island, Southern Chile. The samples were tested by liquid chromatography-tandem mass spectrometry (LC-MS/MS). After the analysis, four toxins were found: DTX-1, DTX-3, YTX and PTX. All toxins were identified by comparing their HPLC retention times with those of analytical standards and confirmed by LC-MS/MS. Dinophysistoxin-1 (DTX-1) and dinophysistoxin-3 (DTX-3) toxins were the major components within the mussel extracts. Nevertheless, the percentages of these toxins differed depending on the area they were collected from and/or the sampling date. The levels detected in Butacheuques Island for okadaic acid (OA) was 267 ± 3.5 µg OA eq kg(-1) (p < 0.05) and for DTX-3 was 183.4 ± 7.5 µg kg(-1) in ribbed mussels. Pectenotoxin (PTX) and yessotoxin (YTX) were the toxins detected in minor proportions in the toxic profile of the bivalves. The maximum concentration of YTX detected in ribbed mussels was 85.2 ± 2.8 µg kg(-1) in Mechuque Island, whereas the PTX-2 level in ribbed mussels was 82.0 ± 2.4 µg kg(-1) in Cailin Island. Analogues of YTX and PTX-2 were not detected in any of the analysed mussels, which did not support the supposed presence of isomers of toxins as a result of the enzymatic metabolism of bivalves. This study found evidence proving co-occurrence of lipophilic toxins - like PTX and YTX - with diarrhoeic toxin in samples collected in Southern Chile, which is, to date, the more complex mix of lipophilic toxins ever found in mussels samples from Southern Chile.
Asunto(s)
Acuicultura , Bivalvos/metabolismo , Toxinas Marinas/metabolismo , Animales , Chile , Cromatografía Liquida , Espectrometría de Masas en TándemRESUMEN
Sea anemones are known to contain a wide diversity of biologically active peptides, mostly unexplored according to recent peptidomic and transcriptomic studies. In the present work, the neurotoxic fractions from the exudates of Stichodactyla helianthus and Bunodosoma granulifera were analyzed by reversed-phase chromatography and mass spectrometry. The first peptide fingerprints of these sea anemones were assessed, revealing the largest number of peptide components (156) so far found in sea anemone species, as well as the richer peptide diversity of B. granulifera in relation to S. helianthus. The transcriptomic analysis of B. granulifera, performed by massive cDNA sequencing with 454 pyrosequencing approach allowed the discovery of five new APETx-like peptides (U-AITX-Bg1a-e - including the full sequences of their precursors for four of them), which together with type 1 sea anemone sodium channel toxins constitute a very distinguishable feature of studied sea anemone species belonging to genus Bunodosoma. The molecular modeling of these new APETx-like peptides showed a distribution of positively charged and aromatic residues in putative contact surfaces as observed in other animal toxins. On the other hand, they also showed variable electrostatic potentials, thus suggesting a docking onto their targeted channels in different spatial orientations. Moreover several crab paralyzing toxins (other than U-AITX-Bg1a-e), which induce a variety of symptoms in crabs, were isolated. Some of them presumably belong to new classes of crab-paralyzing peptide toxins, especially those with molecular masses below 2kDa, which represent the smallest peptide toxins found in sea anemones.