Tissue Distribution and Metabolization of Ciguatoxins in an Herbivorous Fish following Experimental Dietary Exposure to Gambierdiscus polynesiensis.

Ciguatoxins (CTXs), potent neurotoxins produced by dinoflagellates of the genera Gambierdiscus and Fukuyoa, accumulate in commonly consumed fish species, causing human ciguatera poisoning. Field collections of Pacific reef fish reveal that consumed CTXs undergo oxidative biotransformations, resulting in numerous, often toxified analogs. Following our study showing rapid CTX accumulation in flesh of an herbivorous fish, we used the same laboratory model to examine the tissue distribution and metabolization of Pacific CTXs following long-term dietary exposure. Naso brevirostris consumed cells of Gambierdiscus polynesiensis in a gel food matrix over 16 weeks at a constant dose rate of 0.36 ng CTX3C equiv g-1 fish d-1. CTX toxicity determination of fish tissues showed CTX activity in all tissues of exposed fish (eight tissues plus the carcass), with the highest concentrations in the spleen. Muscle tissue retained the largest proportion of CTXs, with 44% of the total tissue burden. Moreover, relative to our previous study, we found that larger fish with slower growth rates assimilated a higher proportion of ingested toxin in their flesh (13% vs. 2%). Analysis of muscle extracts revealed the presence of CTX3C and CTX3B as well as a biotransformed product showing the m/z transitions of 2,3-dihydroxyCTX3C. This is the first experimental evidence of oxidative transformation of an algal CTX in a model consumer and known vector of CTX into the fish food web. These findings that the flesh intended for human consumption carries the majority of the toxin load, and that growth rates can influence the relationship between exposure and accumulation, have significant implications in risk assessment and the development of regulatory measures aimed at ensuring seafood safety.

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.

A study of the influence of brevetoxin exposure on trace element bioaccumulation in the blue mussel Mytilus edulis.

Marine organisms are exposed to and affected by a multitude of chemicals present in seawater and can accumulate in their tissues a wide range of contaminants as well as natural biotoxins associated with harmful algal blooms (HABs). Trace elements and biotoxins may modify physiological functions in exposed organisms, and studies have been conducted to better understand their respective kinetics and effects in marine species. Despite the increasing concern of concurrent toxic HABs and pollution events due to anthropogenic pressures and global change, very little information is available on their combined effects. Chemical interactions between biotoxins and trace elements have been reported, and exposure to certain biotoxins is known to modify ion transport pathways, suggesting that biotoxins have the potential to alter trace element uptake. Using specific and sensitive radiotracer techniques (radioligand receptor binding assay and γ-spectrometry), this laboratory study examined the influence of pre-exposure to the brevetoxins (PbTxs)-producing microalgae Karenia brevis on the bioaccumulation of selected non-essential (Cd) and essential (Co, Mn and Zn) trace elements in the blue mussel Mytilus edulis. PbTxs are a group of neurotoxins known to accumulate in bivalves but also to have lethal effects on a number of marine organisms including fish and mammals. We found that, over 23 days exposure to the radiotracers, the bioaccumulation of the dissolved essential trace elements Co, Mn and Zn in M. edulis was not significantly affected by pre-exposure to toxic K. brevis. In contrast, the uptake rate constant ku of Cd was significantly higher in the pre-exposed group (p < 0.05), likely caused by a decrease in mussel clearance rates after K. brevis exposure. These results suggest that the effects of algal toxin exposure on bioaccumulation of trace elements in mussels may be trace element-dependent.

Experimental evidence of dietary ciguatoxin accumulation in an herbivorous coral reef fish.

Ciguatoxins (CTXs) are potent algal toxins that cause widespread ciguatera poisoning and are found ubiquitously in coral reef food webs. Here we developed an environmentally-relevant, experimental model of CTX trophic transfer involving dietary exposure of herbivorous fish to the CTX-producing microalgae Gambierdiscus polynesiensis. Juvenile Naso brevirostris were fed a gel-food embedded with microalgae for 16 weeks (89 cells g-1 fish daily, 0.4 µg CTX3C equiv kg-1 fish). CTXs in muscle tissue were detectable after 2 weeks at levels above the threshold for human intoxication (1.2 ±â€¯0.2 µg CTX3C equiv kg-1). Although tissue CTX concentrations stabilized after 8 weeks (∼3 ±â€¯0.5 µg CTX3C equiv kg-1), muscle toxin burden (total µg CTX in muscle tissue) continued to increase linearly through the end of the experiment (16 weeks). Toxin accumulation was therefore continuous, yet masked by somatic growth dilution. The observed CTX concentrations, accumulation rates, and general absence of behavioural signs of intoxication are consistent with field observations and indicate that this method of dietary exposure may be used to develop predictive models of tissue-specific CTX uptake, metabolism and depuration. Results also imply that slow-growing fish may accumulate higher CTX flesh concentrations than fast-growing fish, which has important implications for global seafood safety.

The role of marine biotoxins on the trophic transfer of Mn and Zn in fish.

Essential nutrients are critical for physiological processes of organisms. In fish, they are obtained primarily from the diet, and their transfer and accumulation are known to be impacted by environmental variables such as water temperature, pH and salinity, as well as by diet composition and matrices. Yet, prey items consumed by fish may also contain toxic compounds such as marine toxins associated with harmful algae. These biotoxins have the potential to affect essential trace element assimilation in fish through chemical interactions such as the formation of trace element-toxin complexes or by affecting general fish physiology as in the modification of ion-specific transport pathways. We assessed the influence of dietary exposure to brevetoxins (PbTxs), ichthyotoxic neurotoxins produced by the dinoflagellate Karenia brevis, on trophic transfer of two essential trace elements, Mn and Zn, in a fish model. Using ecologically relevant concentrations of PbTxs and trace elements in controlled laboratory conditions, juvenile turbots Scophthalmus maximus were given food containing PbTxs before or at the same time as a feeding with radiotracers of the chosen essential elements (54Mn and 65Zn). Treatments included simultaneous exposure (PbTxs + 54Mn + 65Zn) in a single-feeding, 3-week daily pre-exposure to dietary PbTx followed by a single feeding with 54Mn and 65Zn, and a control (54Mn and 65Zn only). After a 21-day depuration period, turbot tissue brevetoxin levels were quantified and assimilation efficiencies of 54Mn and 65Zn were assessed. PbTxs were found in turbot tissues in each exposure treatment, demonstrating dietary trophic transfer of these toxins; yet, no differences in assimilation efficiencies of Mn or Zn were found between treatments or the control (p > 0.05). These results indicate that, in our experimental conditions, PbTx exposure does not significantly affect the trophic transfer of Mn and Zn in fish.

Empirical data demonstrates risk-tradeoffs between landscapes for herbivorous fish may promote reef resilience.

Herbivores balance resource requirements with predation risk, which can differ among landscapes; hence, landscape can shape these trade-offs, influencing herbivore distribution and behavior. While this paradigm has been well established on coral-dominated reefs, tropical reefs worldwide are shifting to algal dominance. If herbivores avoid algae due to higher risk and forage in coral, these algal states may be stabilized. However, if herbivores forage more in resource-rich algal states, this may promote coral recovery. We assessed the distribution and behavior of herbivorous fishes in Moorea, French Polynesia in coral and algal turf-dominated fringing reef sites. Acanthuridae were more abundant in coral states and Labridae, tribe Scarinae, in algal turf states, though total fish abundances were equivalent in the two states. Fish in both families spent more time feeding in algal states and hiding/swimming in coral states. Thus, behavior reflects the trade-off between resource acquisition and refuge in these two landscapes and may promote recovery to coral.