Establishing a Receptor Binding Assay for Ciguatoxins: Challenges, Assay Performance and Application.

Ciguatera, a global issue, lacks adequate capacity for ciguatoxin analysis in most affected countries. The Caribbean region, known for its endemic ciguatera and being home to a majority of the global small island developing states, particularly needs established methods for ciguatoxin detection in seafood and the environment. The radioligand receptor binding assay (r-RBA) is among the in vitro bioassays currently used for ciguatoxin analysis; however, similarly to the other chemical-based or bioassays that have been developed, it faces challenges due to limited standards and interlaboratory comparisons. This work presents a single laboratory validation of an r-RBA developed in a Cuban laboratory while characterizing the performance of the liquid scintillation counter instrument as a key external parameter. The results obtained show the assay is precise, accurate and robust, confirming its potential as a routine screening method for the detection and quantification of ciguatoxins. The new method will aid in identifying high-risk ciguatoxic fish in Cuba and the Caribbean region, supporting monitoring and scientific management of ciguatera and the development of early warning systems to enhance food safety and food security, and promote fair trade fisheries.

Ciguatoxin Occurrence in Food-Web Components of a Cuban Coral Reef Ecosystem: Risk-Assessment Implications.

In Cuba, ciguatera poisoning associated with fish consumption is the most commonly occurring non-bacterial seafood-borne illness. Risk management through fish market regulation has existed in Cuba for decades and consists of bans on selected species above a certain weight; however, the actual occurrence of ciguatoxins (CTXs) in seafood has never been verified. From this food safety risk management perspective, a study site locally known to be at risk for ciguatera was selected. Analysis of the epiphytic dinoflagellate community identified the microalga Gambierdiscus. Gambierdiscus species included six of the seven species known to be present in Cuba (G. caribaeus, G. belizeanus, G. carpenteri, G. carolinianus, G. silvae, and F. ruetzleri). CTX-like activity in invertebrates, herbivorous and carnivorous fishes were analyzed with a radioligand receptor-binding assay and, for selected samples, with the N2A cell cytotoxicity assay. CTX activity was found in 80% of the organisms sampled, with toxin values ranging from 2 to 8 ng CTX3C equivalents g-1 tissue. Data analysis further confirmed CTXs trophic magnification. This study constitutes the first finding of CTX-like activity in marine organisms in Cuba and in herbivorous fish in the Caribbean. Elucidating the structure-activity relationship and toxicology of CTX from the Caribbean is needed before conclusions may be drawn about risk exposure in Cuba and the wider Caribbean.

Morphology, toxicity and molecular characterization of Gambierdiscus spp. towards risk assessment of ciguatera in south central Cuba.

Ciguatera poisoning is caused by the consumption of reef fish or shellfish that have accumulated ciguatoxins, neurotoxins produced by benthic dinoflagellates of the genera Gambierdiscus or Fukuyoa. Although ciguatera constitutes the primary cause of seafood intoxication in Cuba, very little information is available on the occurrence of ciguatoxins in the marine food web and the causative benthic dinoflagellate species. This study conducted on the south-central coast of Cuba reports the occurrence of Gambierdiscus and Fukuyoa genera and the associated benthic genera Ostreopsis and Prorocentrum. Gambierdiscus/Fukuyoa cells were present at low to moderate abundances depending on the site and month of sampling. This genus was notably higher on Dictyotaceae than on other macrophytes. PCR analysis of field-collected samples revealed the presence of six different Gambierdiscus and one Fukuyoa species, including G. caribaeus, G. carolinianus, G. carpenteri, G. belizeanus, F. ruetzleri, G. silvae, and Gambierdiscus sp. ribotype 2. Only Gambierdiscus excentricus was absent from the eight Gambierdiscus/Fukuyoa species known in the wider Caribbean region. Eleven clonal cultures were established and confirmed by PCR and SEM as being either G. carolinianus or G. caribaeus. Toxin production in each isolate was assessed by a radioligand receptor binding assay and found to be below the assay quantification limit. These novel findings augment the knowledge of the ciguatoxin-source dinoflagellates that are present in Cuba, however further studies are needed to better understand the correlation between their abundance, species-specific toxin production in the environment, and the risk for fish contamination, in order to develop better informed ciguatera risk management strategies.

A radioligand receptor binding assay for ciguatoxin monitoring in environmental samples: Method development and determination of quality control criteria.

Ciguatoxins are algal toxins responsible for tens of thousands of human intoxications yearly, both in tropical and subtropical endemic regions as well as worldwide through fish exportation. Previously developed methods for biotoxin surveillance in the environment and seafood include analytical methods and in vivo and in vitro bioassays. The radioligand receptor binding assay (r-RBA) is among the in vitro methodologies currently used for the detection and quantification of marine biotoxins. For the ciguatoxin group, the r-RBA has been widely used as a means to characterize the mode of action and as detection method in various biological matrices. Yet, screening methods have not been standardized, and the details of the ciguatoxin-specific r-RBA are not well-documented, which limit interlaboratory comparison and progress toward method validation. This work presents the development of an optimized r-RBA for ciguatoxins and provides guidance on its use and quality control checks for analysis of environmental samples. We focus on the analysis of critical parameters involved in determining assay acceptability. Calculation of toxin concentrations in fish samples is illustrated with four examples. Thus, this paper provides the detailed information required for a full validation of the r-RBA, a necessary step toward the development and implementation of a regulatory monitoring programme for ciguatoxins in seafood products using the r-RBA.

Two-year temporal response of benthic macrofauna and sediments to hypoxia in a tropical semi-enclosed bay (Cienfuegos, Cuba) / Respuesta temporal durante dos años de la macrofauna bentónica y sedimentos a la hipoxia en una bahía semi-cerrada tropical (Cienfuegos, Cuba)

AbstractHypoxia is the depletion of dissolved oxygen below 2 mg O2/L. Relatively few studies on hypoxia and its effects on benthic macrofauna have been done in tropical marine ecosystems. This study describes the temporal response of the water column, sediments and macrofauna to seasonal hypoxia in a semi-enclosed bay (Cienfuegos, Caribbean Sea). The Calisito site was sampled monthly from June 2010 until February 2012, yielding 21 sampling times. At each sampling event water and sediment samples were collected for measuring the abiotic variables (temperature, salinity, dissolved oxygen, nutrients, redox potential discontinuity, silt/clay and organic matter content) and macrofauna (abundance and species richness). Temperature and surface salinity followed a typical temporal pattern during the summer/rainy and the winter/dry periods. Salinity stratification occurred in the rainy period, lasting three months in 2010 and six months in 2011. The bottom water dissolved oxygen indicated hypoxic and anoxic events during the wet periods of 2010 and 2011 associated with salinity stratification, low hydrodynamics and oxidation of the accumulated organic matter. Over the study period, 817 individuals were collected and identified. Polychaetes were the dominant group in terms of abundance (57 % of total) followed by mollusks (41%). Hypoxia (and occasionally anoxia) caused strong deleterious effects on the abundance and species richness of macrofaunal communities in the study site. The most abundant polychaetes were opportunistic species with high tolerance to hypoxic conditions: Prionospio steenstrupi, Polydora sp.and Paraprionospio pinnata. Most of them colonized relatively fast once hypoxia ended. Persistent species such as Caecum pulchellum and Parvanachis obesa were present during hypoxia with fluctuating densities and apparently recover to higher abundances when normoxic conditions are re-established. Macoma tenta and Tellina consobrina colonized approximately 1-2 months later than the first polychaete peak during normoxia. Probably, the deleterious effects of hypoxia on the macrofauna were intensified by negative interspecific relationships such as competition by suitable space and predation. The recolonization of macrofauna depended possibly on local transport by currents within the bay because the connection with the Caribbean Sea is relatively limited. In summary, seasonal hypoxia in Cienfuegos Bay influences the water and sediment geochemistry and reduces both the abundance and diversity of macrofauna.

ResumenLa hipoxia es la disminución del oxígeno disuelto por debajo de 2 mg O2/L. Relativamente pocos estudios sobre la hipoxia y sus efectos en la macrofauna bentónica han sido realizados en ecosistemas marinos tropicales. Este estudio describe la respuesta temporal de la columna de agua, los sedimentos y la macrofauna a la hipoxia estacional en una bahía semicerrada en Cienfuegos, Mar Caribe. La estación de Calisito fue muestreada mensualmente de Junio 2010 a Febrero 2012, resultando en 21 eventos de muestreo. En cada oportunidad se recolectaron muestras de agua y sedimento para la medición de las variables abióticas (temperatura, salinidad, oxígeno disuelto, discontinuidad del potencial redox, contenido de limo/arcilla y materia orgánica) y macrofauna (abundancia y riqueza de especies). La temperatura y la salinidad superficial presentaron un patrón temporal típico durante los periodos de verano/ lluvia e invierno/seca. Ocurrió estratificación por salinidad en el periodo lluvioso, que duró tres meses en 2010 y seis en 2011. El oxígeno disuelto de fondo indicó eventos de hipoxia y anoxia durante los periodos lluviosos de 2010 y 2011 asociados con la estratificación de la columna de agua, la hidrodinámica lenta y la oxidación de la materia orgánica acumulada. Esto causó efectos deletéreos en la abundancia y diversidad de la macrofauna. Los poliquetos más abundantes fueron especies oportunistas con alta tolerancia a condiciones hipóxicas: Prionospio steenstrupi, Polydora sp. y Paraprionospio pinnata. La mayoría de estos colonizaron relativamente rápido una vez que terminó la hipoxia. Especies persistentes como Caecum pulchellum y Parvanachis obesa estuvieron presentes durante la hipoxia con densidades fluctuantes y aparentemente recuperaron sus abundancias cuando las condiciones normóxicas fueron re-establecidas. Macoma tenta y Tellina consobrina colonizaron 1-2 meses después que el primer pico de poliquetos durante la normoxia.Probablemente, los efectos deletéreos de la hipoxia en la macrofauna fueron intensificados por relaciones interespecíficas negativas como la competencia por espacio y la depredación. La recolonización de la macrofauna dependió probablemente del transporte local por las corrientes dentro de la bahía pues la conexión con el mar Caribe es relativamente limitada. En resumen, la hipoxia estacional en la bahía de Cienfuegos influencia la geoquímica del agua y los sedimentos y reduce la abundancia y diversidad de la macrofauna.

Two-year temporal response of benthic macrofauna and sediments to hypoxia in a tropical semi-enclosed bay (Cienfuegos, Cuba).

Hypoxia is the depletion of dissolved oxygen below 2 mg O(2)/L. Relatively few studies on hypoxia and its effects on benthic macrofauna have been done in tropical marine ecosystems. This study describes the temporal response of the water column, sediments and macrofauna to seasonal hypoxia in a semi-enclosed bay (Cienfuegos, Caribbean Sea). The Calisito site was sampled monthly from June 2010 until February 2012, yielding 21 sampling times. At each sampling event water and sediment samples were collected for measuring the abiotic variables (temperature, salinity, dissolved oxygen, nutrients, redox potential discontinuity, silt/clay and organic matter content) and macrofauna (abundance and species richness). Temperature and surface salinity followed a typical temporal pattern during the summer/rainy and the winter/dry periods. Salinity stratification occurred in the rainy period, lasting three months in 2010 and six months in 2011. The bottom water dissolved oxygen indicated hypoxic and anoxic events during the wet periods of 2010 and 2011 associated with salinity stratification, low hydrodynamics and oxidation of the accumulated organic matter. Over the study period, 817 individuals were collected and identified. Polychaetes were the dominant group in terms of abundance (57 % of total) followed by mollusks (41%). Hypoxia (and occasionally anoxia) caused strong deleterious effects on the abundance and species richness of macrofaunal communities in the study site. The most abundant polychaetes were opportunistic species with high tolerance to hypoxic conditions: Prionospio steenstrupi, Polydora sp.and Paraprionospio pinnata. Most of them colonized relatively fast once hypoxia ended. Persistent species such as Caecum pulchellum and Parvanachis obesa were present during hypoxia with fluctuating densities and apparently recover to higher abundances when normoxic conditions are re-established. Macoma tenta and Tellina consobrina colonized approximately 1-2 months later than the first polychaete peak during normoxia. Probably, the deleterious effects of hypoxia on the macrofauna were intensified by negative interspecific relationships such as competition by suitable space and predation. The recolonization of macrofauna depended possibly on local transport by currents within the bay because the connection with the Caribbean Sea is relatively limited. In summary, seasonal hypoxia in Cienfuegos Bay influences the water and sediment geochemistry and reduces both the abundance and diversity of macrofauna.

Effects of organic enrichment on nematode assemblages in a microcosm experiment.

Marine nematodes from subtidal tropical sediments of Cienfuegos Bay were subjected to organic enrichment in a microcosm experiment for 32 days. Nematode abundance and diversity decreased, and the taxonomic and trophic structure was altered. The results suggested that the nematodes were not food limited in the microcosms or in their natural environment. Chemical stressors such as ammonia and hydrogen sulfide derived from reduced conditions in sediments may be important factors affecting the assemblages. Hypoxic conditions occurred in all experimental units, as well as in the field, suggesting a nematode assemblage adapted to naturally enriched sediments. However, tolerant species showed a grade of sensitivity to reduced conditions. In agreement with the model by Pearson and Rosenberg (1978), we predict that further organic enrichment in sediments from Cienfuegos Bay may cause a phase shift into a strongly depleted benthic fauna and reduced conditions in water and sediments.