Morphological, Toxicological, and Biochemical Characterization of Two Species of Gambierdiscus from Bahía de La Paz, Gulf of California.

We describe five new isolates of two Gambierdiscus species from Bahía de La Paz in the southern Gulf of California. Batch cultures of Gambierdiscus were established for morphological characterization using light microscopy (LM) and scanning electron microscopy (SEM). Pigment and amino acid profiles were also analyzed using high-performance liquid chromatography (HPLC-UV and HPLC-DAD). Finally, toxicity (CTX-like and MTX-like activity) was evaluated using the Artemia salina assay (ARTOX), mouse assay (MBA), marine fish assay (MFA), and fluorescent receptor binding assay (fRBA). These strains were identified as Gambierdiscus cf. caribaeus and Gambierdiscus cf. carpenteri. Toxicity for CTX-like and MTX-like activity was confirmed in all evaluated clones. Seven pigments were detected, with chlorophyll a, pyridine, Chl2, and diadinoxanthin being particularly noteworthy. For the first time, a screening of the amino acid profile of Gambierdiscus from the Pacific Ocean was conducted, which showed 14 amino acids for all strains except histidine, which was only present in G. cf. caribeaus. We report the presence of Gambierdiscus and Fukuyoa species in the Mexican Pacific, where ciguatera fish poisoning (CFP) cases have occurred.

Changes at small intestine induced by food-fish contaminated with ciguatoxins.

Ciguateric syndrome is a food poisoning associated with the consumption of some species of fish that have accumulated ciguatoxins (CTXs) in their tissues. The effects of the syndrome occur with nervous imbalances which have been described for quite some time, and mentioned in sailing literature for centuries. In the last decade, research has been focused on the implementation of analytical methods for toxin identification and the study of action modes of CTXs to design effective treatments. However, an important aspect is to determine the damage that CTXs caused in the organs of affected individuals. In this work, the damages observed in tissues of mice, mainly in the small intestine, were analyzed. The animals were fed with CTX-contaminated fish muscle at concentrations 10-times below the median lethal dose (LD50) for 10 weeks. The analysis of tissues derived from the oral treatment resulted in an increased occurrence of Paneth cells, presence of lymphoid tissue infiltrating the mucosa and fibrous lesions in the mucosal layer of the small intestine. A decreasing weight in animals fed with toxic muscle was observed.

Mouse N2a Neuroblastoma Assay: Uncertainties and Comparison with Alternative Cell-Based Assays for Ciguatoxin Detection.

The growing concern about ciguatera fish poisoning (CF) due to the expansion of the microorganisms producing ciguatoxins (CTXs) increased the need to develop a reliable and fast method for ciguatoxin detection to guarantee food safety. Cytotoxicity assay on the N2a cells sensitized with ouabain (O) and veratridine (V) is routinely used in ciguatoxin detection; however, this method has not been standardized yet. This study demonstrated the low availability of sodium channels in the N2a cells, the great O/V damage to the cells and the cell detachment when the cell viability is evaluated by the classical cytotoxicity assay and confirmed the absence of toxic effects caused by CTXs alone when using the methods that do not require medium removal such as lactate dehydrogenase (LDH) and Alamar blue assays. Different cell lines were evaluated as alternatives, such as human neuroblastoma, which was not suitable for the CTX detection due to the greater sensitivity to O/V and low availability of sodium channels. However, the HEK293 Nav cell line expressing the α1.6 subunit of sodium channels was sensitive to the ciguatoxin without the sensitization with O/V due to its expression of sodium channels. In the case of sensitizing the cells with O/V, it was possible to detect the presence of the ciguatoxin by the classical cytotoxicity MTT method at concentrations as low as 0.0001 nM CTX3C, providing an alternative cell line for the detection of compounds that act on the sodium channels.

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.

Chronic Ciguatera Poisoning: A Case Report.

Ciguatera is a common marine, toxin-borne illness caused by the consumption of fish that contain toxins that activate voltage-sensitive sodium channels. The clinical manifestations of ciguatera are typically self-limited, but chronic symptoms may occur in a minority of patients. This report describes a case of ciguatera poisoning with chronic symptoms, including pruritus and paresthesias. A 40-y-old man was diagnosed with ciguatera poisoning after consuming amberjack while vacationing in the US Virgin Islands. His initial symptoms, including diarrhea, cold allodynia, and extremity paresthesias, evolved into chronic, fluctuating paresthesias and pruritus that became worse after the consumption of alcohol, fish, nuts, and chocolate. After a comprehensive neurologic evaluation failed to reveal another cause for his symptoms, he was diagnosed with chronic ciguatera poisoning. His neuropathic symptoms were treated with duloxetine and pregabalin, and he was counseled to avoid foods that triggered his symptoms. Chronic ciguatera is a clinical diagnosis. Signs and symptoms of chronic ciguatera can include fatigue, myalgias, headache, and pruritus. The pathophysiology of chronic ciguatera is incompletely understood but may involve genetic factors or immune dysregulation. Treatment involves supportive care and avoidance of foods and environmental conditions that may exacerbate symptoms.

Evaluating Age and Growth Relationship to Ciguatoxicity in Five Coral Reef Fish Species from French Polynesia.

Ciguatera poisoning (CP) results from the consumption of coral reef fish or marine invertebrates contaminated with potent marine polyether compounds, namely ciguatoxins. In French Polynesia, 220 fish specimens belonging to parrotfish (Chlorurus microrhinos, Scarus forsteni, and Scarus ghobban), surgeonfish (Naso lituratus), and groupers (Epinephelus polyphekadion) were collected from two sites with contrasted risk of CP, i.e., Kaukura Atoll versus Mangareva Island. Fish age and growth were assessed from otoliths' yearly increments and their ciguatoxic status (negative, suspect, or positive) was evaluated by neuroblastoma cell-based assay. Using permutational multivariate analyses of variance, no significant differences in size and weight were found between negative and suspect specimens while positive specimens showed significantly greater size and weight particularly for E. polyphekadion and S. ghobban. However, eating small or low-weight specimens remains risky due to the high variability in size and weight of positive fish. Overall, no relationship could be evidenced between fish ciguatoxicity and age and growth characteristics. In conclusion, size, weight, age, and growth are not reliable determinants of fish ciguatoxicity which appears to be rather species and/or site-specific, although larger fish pose an increased risk of poisoning. Such findings have important implications in current CP risk management programs.

Comparative Study on the Performance of Three Detection Methods for the Quantification of Pacific Ciguatoxins in French Polynesian Strains of Gambierdiscus polynesiensis.

Gambierdiscus and Fukuyoa dinoflagellates produce a suite of secondary metabolites, including ciguatoxins (CTXs), which bioaccumulate and are further biotransformed in fish and marine invertebrates, causing ciguatera poisoning when consumed by humans. This study is the first to compare the performance of the fluorescent receptor binding assay (fRBA), neuroblastoma cell-based assay (CBA-N2a), and liquid chromatography tandem mass spectrometry (LC-MS/MS) for the quantitative estimation of CTX contents in 30 samples, obtained from four French Polynesian strains of Gambierdiscus polynesiensis. fRBA was applied to Gambierdiscus matrix for the first time, and several parameters of the fRBA protocol were refined. Following liquid/liquid partitioning to separate CTXs from other algal compounds, the variability of CTX contents was estimated using these three methods in three independent experiments. All three assays were significantly correlated with each other, with the highest correlation coefficient (r2 = 0.841) found between fRBA and LC-MS/MS. The CBA-N2a was more sensitive than LC-MS/MS and fRBA, with all assays showing good repeatability. The combined use of fRBA and/or CBA-N2a for screening purposes and LC-MS/MS for confirmation purposes allows for efficient CTX evaluation in Gambierdiscus. These findings, which support future collaborative studies for the inter-laboratory validation of CTX detection methods, will help improve ciguatera risk assessment and management.

Epidemiology and Toxicology of Ciguatera Poisoning in the Colombian Caribbean.

Ciguatera is a food intoxication caused by the consumption of primarily coral fish; these species exist in large numbers in the seas that surround the Colombian territory. The underreported diagnosis of this clinical entity has been widely highlighted due to multiple factors, such as, among others, ignorance by the primary care practitioner consulted for this condition as well as clinical similarity to secondary gastroenteric symptoms and common food poisonings of bacterial, parasitic or viral etiology. Eventually, it was found that people affected by ciguatoxins had trips to coastal areas hours before the onset of symptoms. Thanks to multiple studies over the years, it has been possible to identify the relation between toxigenic dinoflagellates and seagrasses, as well as its incorporation into the food chain, starting by fish primarily inhabiting reef ecosystems and culminating in the intake of these by humans. Identifying the epidemiological link, its cardinal symptoms and affected systems, such as gastrointestinal, the peripheral nervous system and, fortunately with a low frequency, the cardiovascular system, leads to a purely clinical diagnostic impression without necessitating further complementary studies; in addition, what would also help fight ciguatera poisoning is performing an adequate treatment of the symptoms right from the start, without underestimating or overlooking any associated complications.

Rapid detection of ciguatoxins in Gambierdiscus and Fukuyoa with immunosensing tools.

Consumption of seafood contaminated with ciguatoxins (CTXs) leads to a foodborne disease known as ciguatera. Primary producers of CTXs are epibenthic dinoflagellates of the genera Gambierdiscus and Fukuyoa. In this study, thirteen Gambierdiscus and Fukuyoa strains were cultured, harvested at exponential phase, and CTXs were extracted with an implemented rapid protocol. Microalgal extracts were obtained from pellets with a low cell abundance (20,000 cell/mL) and were then analyzed with magnetic bead (MB)-based immunosensing tools (colorimetric immunoassay and electrochemical immunosensor). It is the first time that these approaches are used to screen Gambierdiscus and Fukuyoa strains, providing not only a global indication of the presence of CTXs, but also the ability to discriminate between two series of congeners (CTX1B and CTX3C). Analysis of the microalgal extracts revealed the presence of CTXs in 11 out of 13 strains and provided new information about Gambierdiscus and Fukuyoa toxin profiles. The use of immunosensing tools in the analysis of microalgal extracts facilitates the elucidation of further knowledge regarding these dinoflagellate genera and can contribute to improved ciguatera risk assessment and management.

Ciguatoxins activate the Calcineurin signalling pathway in Yeasts: Potential for development of an alternative detection tool?

Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1µM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products.

Ciguatera in Mexico (1984⁻2013).

Historical records of ciguatera in Mexico date back to 1862. This review, including references and epidemiological reports, documents 464 cases during 25 events from 1984 to 2013: 240 (51.72%) in Baja California Sur, 163 (35.12%) in Quintana Roo, 45 (9.69%) in Yucatan, and 16 (3.44%) cases of Mexican tourists intoxicated in Cuba. Carnivorous fish, such as snapper (Lutjanus) and grouper (Epinephelus and Mycteroperca) in the Pacific Ocean, and great barracuda (Sphyraena barracuda) and snapper (Lutjanus) in the Atlantic (Gulf of Mexico and Caribbean Sea), were involved in all cases. In the Mexican Caribbean, a sub-record of ciguatera cases that occurred before 1984 exists. However, the number of intoxications has increased in recent years, and this food poisoning is poorly studied in the region. Current records suggest that ciguatera fish poisoning in humans is the second most prevalent form of seafood poisoning in Mexico, only exceeded by paralytic shellfish poisoning (505 cases, 21 fatalities in the same 34-year period). In this study, the status of ciguatera in Mexico (epidemiological and treatment), and the fish vectors are reviewed. Dinoflagellate species Gambierdiscus, Ostreopsis, and Prorocentrum are related with the reported outbreaks, marine toxins, ecological risk, and the potential toxicological impact.

Toxicological Investigations on the Sea Urchin Tripneustes gratilla (Toxopneustidae, Echinoid) from Anaho Bay (Nuku Hiva, French Polynesia): Evidence for the Presence of Pacific Ciguatoxins.

The sea urchin Tripneustes gratilla (Toxopneustidae, Echinoids) is a source of protein for many islanders in the Indo-West Pacific. It was previously reported to occasionally cause ciguatera-like poisoning; however, the exact nature of the causative agent was not confirmed. In April and July 2015, ciguatera poisonings were reported following the consumption of T.gratilla in Anaho Bay (Nuku Hiva Island, Marquesas archipelago, French Polynesia). Patient symptomatology was recorded and sea urchin samples were collected from Anaho Bay in July 2015 and November 2016. Toxicity analysis using the neuroblastoma cell-based assay (CBA-N2a) detected the presence of ciguatoxins (CTXs) in T.gratilla samples. Gambierdiscus species were predominant in the benthic assemblages of Anaho Bay, and G.polynesiensis was highly prevalent in in vitro cultures according to qPCR results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed that P-CTX-3B was the major ciguatoxin congener in toxic sea urchin samples, followed by 51-OH-P-CTX-3C, P-CTX-3C, P-CTX-4A, and P-CTX-4B. Between July 2015 and November 2016, the toxin content in T.gratilla decreased, but was consistently above the safety limit allowed for human consumption. This study provides evidence of CTX bioaccumulation in T.gratilla as a cause of ciguatera-like poisoning associated with a documented symptomatology.

Ciguatoxins and Maitotoxins in Extracts of Sixteen Gambierdiscus Isolates and One Fukuyoa Isolate from the South Pacific and Their Toxicity to Mice by Intraperitoneal and Oral Administration.

Ciguatoxins (CTXs), and possibly maitotoxins (MTXs), are responsible for Ciguatera Fish Poisoning, an important health problem for consumers of reef fish (such as inhabitants of islands in the South Pacific Ocean). The habitational range of the Gambierdiscus species is expanding, and new species are being discovered. In order to provide information on the potential health risk of the Gambierdiscus species, and one Fukuyoa species (found in the Cook Islands, the Kermadec Islands, mainland New Zealand, and New South Wales, Australia), 17 microalgae isolates were collected from these areas. Unialgal cultures were grown and extracts of the culture isolates were analysed for CTXs and MTXs by liquid chromatography tandem mass spectrometry (LC-MS/MS), and their toxicity to mice was determined by intraperitoneal and oral administration. An isolate of G. carpenteri contained neither CTXs nor MTXs, while 15 other isolates (including G. australes, G. cheloniae, G. pacificus, G.honu, and F. paulensis) contained only MTX-1 and/or MTX-3. An isolate of G. polynesiensis contained both CTXs and MTX-3. All the extracts were toxic to mice by intraperitoneal injection, but those containing only MTX-1 and/or -3 were much less toxic by oral administration. The extract of G. polynesiensis was highly toxic by both routes of administration.

The Epiphytic Genus Gambierdiscus (Dinophyceae) in the Kermadec Islands and Zealandia Regions of the Southwestern Pacific and the Associated Risk of Ciguatera Fish Poisoning.

Species in the genus Gambierdiscus produce ciguatoxins (CTXs) and/or maitotoxins (MTXs), which may cause ciguatera fish poisoning (CFP) in humans if contaminated fish are consumed. Species of Gambierdiscus have previously been isolated from macroalgae at Rangitahua (Raoul Island and North Meyer Islands, northern Kermadec Islands), and the opportunity was taken to sample for Gambierdiscus at the more southerly Macauley Island during an expedition in 2016. Gambierdiscus cells were isolated, cultured, and DNA extracted and sequenced to determine the species present. Bulk cultures were tested for CTXs and MTXs by liquid chromatography-mass spectrometry (LC-MS/MS). The species isolated were G. australes, which produced MTX-1 (ranging from 3 to 36 pg/cell), and G. polynesiensis, which produced neither MTX-1 nor, unusually, any known CTXs. Isolates of both species produced putative MTX-3. The risk of fish, particularly herbivorous fish, causing CFP in the Zealandia and Kermadec Islands region is real, although in mainland New Zealand the risk is currently low. Both Gambierdiscus and Fukuyoa have been recorded in the sub-tropical northern region of New Zealand, and so the risk may increase with warming seas and shift in the distribution of Gambierdiscus species.

Recent Trends in Marine Phycotoxins from Australian Coastal Waters.

Phycotoxins, which are produced by harmful microalgae and bioaccumulate in the  marine food web, are of growing concern for Australia. These harmful algae pose a threat to  ecosystem and human health, as well as constraining the progress of aquaculture, one of the fastest  growing food sectors in the world. With better monitoring, advanced analytical skills and an  increase in microalgal expertise, many phycotoxins have been identified in Australian coastal  waters in recent years. The most concerning of these toxins are ciguatoxin, paralytic shellfish  toxins, okadaic acid and domoic acid, with palytoxin and karlotoxin increasing in significance. The  potential for tetrodotoxin, maitotoxin and palytoxin to contaminate seafood is also of concern,  warranting future investigation. The largest and most significant toxic bloom in Tasmania in 2012  resulted in an estimated total economic loss of~AUD$23M, indicating that there is an imperative to  improve  toxin  and  organism  detection  methods,  clarify  the  toxin  profiles  of  species  of  phytoplankton and carry out both intra- and inter-species toxicity comparisons. Future work also  includes the application of rapid, real-time molecular assays for the detection of harmful species  and toxin genes. This information, in conjunction with a better understanding of the life histories  and  ecology  of  harmful  bloom  species,  may  lead  to  more  appropriate  management  of  environmental, health and economic resources.

Contribution to the risk characterization of ciguatoxins: LOAEL estimated from eight ciguatera fish poisoning events in Guadeloupe (French West Indies).

From 2010 to 2012, 35 ciguatera fish poisoning (CFP) events involving 87 individuals who consumed locally-caught fish were reported in Guadeloupe (French West Indies). For 12 of these events, the presence of ciguatoxins (CTXs) was indicated in meal remnants and in uncooked fish by the mouse bioassay (MBA). Caribbean ciguatoxins (C-CTXs) were confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Using a cell-based assay (CBA), and the only available standard Pacific ciguatoxin-1 (P-CTX-1), the lowest toxins level detected in fish samples causing CFP was 0.022 µg P-CTX-1 equivalent (eq.)·kg(-1) fish. Epidemiological and consumption data were compiled for most of the individuals afflicted, and complete data for establishing the lowest observable adverse effects level (LOAEL) were obtained from 8 CFP events involving 21 individuals. Based on toxin intakes, the LOAEL was estimated at 4.2 ng P-CTX-1 eq./individual corresponding to 48. 4 pg P-CTX-1 eq.kg(-1) body weight (bw). Although based on limited data, these results are consistent with the conclusions of the European Food Safety Authority (EFSA) opinion which indicates that a level of 0.01 µg P-CTX-1 eq.kg(-1) fish, regardless of source, should not exert effects in sensitive individuals when consuming a single meal. The calculated LOAEL is also consistent with the U.S. Food and Drug Administration guidance levels for CTXs (0.1 µg C-CTX-1 eq.kg(-1) and 0.01 µg P-CTX-1 eq.kg(-1) fish).

Ciguatera fish poisoning in East Asia and southeast Asia.

In the coastal countries of East Asia and Southeast Asia, ciguatera should be common because of the extensive tropical and subtropical coral reefs along the coasts and in the neighboring seas with ciguatoxic fishes. An extensive search of journal databases, the Internet and the government websites was performed to identify all reports of ciguatera from the regions. Based on the official data and large published case series, the incidence of ciguatera was higher in the coastal cities (Hong Kong, Foshan, Zhongshan) of southern China than in Japan (Okinawa Prefecture). In Singapore, ciguatera appeared to be almost unknown. In other countries, only isolated cases or small case series were reported, but under-reporting was assumed to be common. Ciguatera may cause severe acute illness and prolonged neurological symptoms. Ciguatera represents an important public health issue for endemic regions, with significant socio-economic impact. Coordinated strategies to improve risk assessment, risk management and risk communication are required. The systematic collection of accurate data on the incidence and epidemiology of ciguatera should enable better assessment and management of its risk. Much more work needs to be done to define the size threshold for important coral reef fish species from different regions, above which the risk of ciguatera significantly increases.

3-Epimers of Caribbean ciguatoxins in fish and algae.

Ciguatera poisoning (CP) is endemic to several subtropical and tropical regions and is caused by the consumption of fish contaminated with ciguatoxins (CTXs). The recent discovery of Caribbean CTXs (C-CTXs) in Gambierdiscus spp. isolated from the Caribbean resulted in the identification of a precursor analogue, C-CTX5, that is reduced into C-CTX1. C-CTX5 has two reducible sites, a ketone at C-3 and hemiketal at C-56. Chemical reductions of C-CTX5 into C-CTX3/4 resulted in two peaks in the LC-HRMS chromatograms with a ratio that differed markedly from that observed in fish extracts and the reduction of C-CTX1 isolated from fish. Reduction of C-CTX5 should have produced four diastereoisomers of C-CTX3/4, prompting a more detailed study of the reduction products. LC-HRMS with a slow gradient was used to separate and detect the four stereoisomers of C-CTX3/4, and to determine the distribution of these analogues in naturally contaminated fish tissues and following chemical reduction of isolated analogues. The results showed that in naturally contaminated fish tissues C-CTX1/2 is a mixture of two diastereoisomers at C-3 and that C-CTX3/4 is a mixture of two pairs of diastereoisomers at C-3 and C-56. The data suggests that there is variability in the enzymatic reduction at C-3 and C-56 of C-CTXs in reef fish, leading to variations in the ratios of the four stereoisomers. Based on these findings, a naming convention for C-CTXs is proposed which aligns with that used for Pacific CTX congeners and will aid in the identification of the structure and stereochemistry of the different CTX analogues.

Effects on Biochemical Parameters and Animal Welfare of Dusky Grouper (Epinephelus marginatus, Lowe 1834) by Feeding CTX Toxic Flesh.

Ciguatera is a foodborne disease caused by ciguatoxins (CTXs), produced by dinoflagellates (genera Gambierdiscus and Fukuyoa), which bioaccumulate in fish through the food web, causing poisoning in humans. Currently, the physiological mechanisms of the species with the highest amount of toxins in their adult stage of life that are capable of causing these poisonings are poorly understood. Dusky grouper (Epinephelus marginatus) is a relevant fishing species and is part of the CTX food chain in the Canary Islands. This study developed an experimental model of dietary exposure featuring adult dusky groupers with two diets of tissue naturally contaminated with CTXs (amberjack and moray eel flesh) with two different potential toxicities; both groups were studied at different stages of exposure (4, 6, 10, 12, and 18 weeks). The results showed that this species did not show changes in its behavior due to the provided feeding, but the changes were recorded in biochemical parameters (mainly lipid and hepatic metabolism) that may respond to liver damage and alterations in the homeostasis of the fish; more research is needed to understand histopathological and cytotoxic changes.

Sensitive Detection of Ciguatoxins Using a Neuroblastoma Cell-Based Assay with Voltage-Gated Potassium Channel Inhibitors.

Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 µg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.