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.

Structural Assignment of the Product Ion Generated from a Natural Ciguatoxin-3C Congener, 51-Hydroxyciguatoxin-3C, and Discovery of Distinguishable Signals in Congeners Bearing the 51-Hydroxy Group.

Ciguatoxins (CTXs) stand as the primary toxins causing ciguatera fish poisoning (CFP) and are essential compounds distinguished by their characteristic polycyclic ether structure. In a previous report, we identified the structures of product ions generated via homolytic fragmentation by assuming three charge sites in the mass spectrometry (MS)/MS spectrum of ciguatoxin-3C (CTX3C) using LC-MS. This study aims to elucidate the homolytic fragmentation of a ciguatoxin-3C congener. We assigned detailed structures of the product ions in the MS/MS spectrum of a naturally occurring ciguatoxin-3C congener, 51-hydroxyciguatoxin-3C (51-hydoxyCTX3C), employing liquid chromatography/quadrupole time-of-flight mass spectrometry with an atmospheric pressure chemical ionization (APCI) source. The introduction of a hydroxy substituent on C51 induced different fragmentation pathways, including a novel cleavage mechanism of the M ring involving the elimination of 51-OH and the formation of enol ether. Consequently, new cleavage patterns generated product ions at m/z 979 (C55H79O15), 439 (C24H39O7), 149 (C10H13O), 135 (C9H11O), and 115 (C6H11O2). Additionally, characteristic product ions were observed at m/z 509 (C28H45O8), 491 (C28H43O7), 481 (C26H41O8), 463 (C26H39O7), 439 (C24H39O7), 421 (C24H37O6), 171 (C9H15O3), 153 (C9H13O2), 141 (C8H13O2), and 123 (C8H11O).

An Unusual Presentation of Severe Ciguatera Poisoning: Case Report.

BACKGROUND Ciguatera poisoning is presently estimated to afflict approximately 16 000 people in the United States each year. Ciguatera toxicity is due to the ingestion of warm-water reef fish that are known to consume dinoflagellates that contain ciguatoxins (CTXs). Historically, the diagnosis of ciguatera poisoning is confirmed in the emergency department when this ingestion is followed by manifestations of neurologic and gastrointestinal organ symptoms. Some individuals also manifest cardiac symptoms. These symptoms can vary within each organ system in type and duration. CASE REPORT In this report, 2 patients (husband and wife) are presented that consumed the same barracuda, resulting in severe ciguatera poisoning diagnosed in the emergency department. One had the complete triad of symptoms of gastrointestinal (GI), neurologic, and cardiac involvement. The other patient lacked neurological symptoms but did experience severe gastrointestinal and cardiac symptoms. It was assumed by the inpatient team, the consulted infectious disease specialist, and, initially, the in-house toxicologist that the lack of neurologic symptoms excluded the diagnosis of ciguatera. CONCLUSIONS If these patients had presented separately, only the former would have been considered to have ciguatera poisoning. These 2 cases demonstrate the misunderstanding among healthcare practitioners of the literature on the diagnostic criteria. Therefore, this article aims to analyze ciguatera diagnosis in the emergency department, which will ultimately guide prognostics and management, particularly for severe cardiac symptoms.

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.

Gambierdiscus species diversity and community structure in St. Thomas, USVI and the Florida Keys, USA.

Ciguatera Poisoning (CP) is a widespread and complex poisoning syndrome caused by the consumption of fish or invertebrates contaminated with a suite of potent neurotoxins collectively known as ciguatoxins (CTXs), which are produced by certain benthic dinoflagellates species in the genera Gambierdiscus and Fukuyoa. Due to the complex nature of this HAB problem, along with a poor understanding of toxin production and entry in the coral reef food web, the development of monitoring, management, and forecasting approaches for CP has lagged behind those available for other HAB syndromes. Over the past two decades, renewed research on the taxonomy, physiology, and toxicology of CP-causing dinoflagellates has advanced our understanding of the species diversity that exists within these genera, including identification of highly toxic species (so called "superbugs") that likely contribute disproportionately to ciguatoxins entering coral reef food webs. The recent development of approaches for molecular analysis of field samples now provide the means to investigate in situ community composition, enabling characterization of spatio-temporal species dynamics, linkages between toxic species abundance and toxin flux, and the risk of ciguatoxin prevalence in fish. In this study we used species-specific fluorescent in situ hybridization (FISH) probes to investigate Gambierdiscus species composition and dynamics in St. Thomas (USVI) and the Florida Keys (USA) over multiple years (2018-2020). Within each location, samples were collected seasonally from several sites comprising varying depths, habitats, and algal substrates to characterize community structure over small spatial scales and across different host macrophytes. This approach enabled the quantitative determination of communities over spatiotemporal gradients, as well as the selective enumeration of species known to exhibit high toxicity, such as Gambierdiscus silvae. The investigation found differing community structure between St. Thomas and Florida Keys sites, driven in part by differences in the distribution of toxin-producing species G. silvae and G. belizeanus, which were present throughout sampling sites in St. Thomas but scarce or absent in the Florida Keys. This finding is significant given the high toxicity of G. silvae, and may help explain differences in fish toxicity and CP incidence between St. Thomas and Florida. Intrasite comparisons along a depth gradient found higher concentrations of Gambierdiscus spp. at deeper locations. Among the macrophytes sampled, Dictyota may be a likely vector for toxin transfer based on their widespread distribution, apparent colonization by G. silvae, and palatability to at least some herbivore grazers. Given its ubiquity throughout both study regions and sites, this taxa may also serve as a refuge, accumulating high concentrations of Gambierdiscus and other benthic dinoflagellates, which in turn can serve as source populations for highly palatable and ephemeral habitats nearby, such as turf algae. These studies further demonstrate the successful application of FISH probes in examining biogeographic structuring of Gambierdiscus communities, targeting individual toxin-producing species, and characterizing species-level dynamics that are needed to describe and model ecological drivers of species abundance and toxicity.

CiguaMOD I: A conceptual model of ciguatoxin loading in the Greater Caribbean Region.

Ciguatera poisoning (CP) is the most common form of phycotoxin-borne seafood poisoning globally, affecting thousands of people on an annual basis. It most commonly occurs in residential fish of coral reefs, which consume toxin-laden algae, detritus, and reef animals. The class of toxins that cause CP, ciguatoxins (CTXs), originate in benthic, epiphytic dinoflagellates of the genera, Gambierdiscus and Fukuyoa, which are consumed by herbivores and detritivores that facilitate food web transfer. A number of factors have hindered adequate environmental monitoring and seafood surveillance for ciguatera including the low concentrations in which the toxins are found in seafood causing illness (sub-ppb), a lack of knowledge on the toxicity equivalence of other CTXs and contribution of other benthic algal toxins to the disease, and the limited availability of quantified toxin standards and reference materials. While progress has been made on the identification of the dinoflagellate taxa and toxins responsible for CP, more effort is needed to better understand the dynamics of toxin transfer into reef food webs in order to implement a practical monitoring program for CP. Here, we present a conceptual model that utilizes empirical field data (temperature, Gambierdiscus cell densities, macrophyte cover) in concert with other published studies (grazing rates and preference) to produce modeling outputs that suggest approaches that may be beneficial to developing monitoring programs: 1) targeting specific macrophytes for Gambierdiscus and toxin measurements to monitor toxin levels at the base of the food web (i.e., toxin loading); and 2) adjusting these targets across sites and over seasons. Coupling this approach with other methodologies being incorporated into monitoring programs (artificial substrates; FISH probes; toxin screening) may provide an "early warning" system to develop strategic responses to potential CP flare ups in the future.

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.

Diversity and distribution of benthic dinoflagellates in Tonga include the potentially harmful genera Gambierdiscus and Fukuyoa.

Benthic dinoflagellates that can cause illness, such as ciguatera poisoning (CP), are prevalent around the Pacific but are poorly described in many locations. This study represents the first ecological assessment of benthic harmful algae species in the Kingdom of Tonga, a country where CP occurs regularly. Surveys were conducted in June 2016 in the Tongatapu island group, and in June 2017 across three island groups: Ha'apai, Vava'u, and Tongatapu. Shallow subtidal coastal habitats were investigated by measuring water quality parameters and conducting quadrat surveys. Microalgae samples were collected using either macrophyte collection or the artificial substrate method. Benthic dinoflagellates (Gambierdiscus and/or Fukuyoa, Ostreopsis, and Prorocentrum) were counted using light microscopy, followed by molecular analyses (real-time PCR in 2016 and high throughput sequencing (metabarcoding) in 2017) to identify Gambierdiscus and Fukuyoa to species level. Six species were detected from the Tongatapu island group in 2016 (G. australes, G. carpenteri, G. honu, G. pacificus, F. paulensis, and F. ruetzleri) using real-time PCR. Using the metabarcoding approach in 2017, a total of eight species (G. australes, G. carpenteri, G. honu, G. pacificus, G. cheloniae, G. lewisii, G. polynesiensis, and F. yasumotoi) were detected. Species were detected in mixed assemblages of up to six species, with G. pacificus and G. carpenteri being the most frequently observed. Ha'apai had the highest diversity with eight species detected, which identifies this area as a Gambierdiscus diversity 'hotspot'. Vava'u and Tongatapu had three and six species found respectively. Gambierdiscus polynesiensis, a described ciguatoxin producer and proposed causative agent of CP was found only in Ha'apai and Vava'u in 2017, but not in Tongatapu in either year. Ostreopsis spp. and Prorocentrum spp. were also frequently observed, with Prorocentrum most abundant at the majority of sites. In 2016, the highest number of Gambierdiscus and/or Fukuyoa cells were observed on seagrass (Halodule uninervis) from Sopu, Tongatapu. In 2017, the highest numbers of Gambierdiscus and/or Fukuyoa from artificial substrate samples were recorded in the Halimeda dominant habitat at Neiafu Tahi, Vava'u, a low energy site. This raised the question of the effect of wave motion or currents on abundance measurements from artificial substrates. Differences in detection were noticed between macrophytes and artificial substrates, with higher numbers of species found on artificial substrates. This study provides a baseline of benthic dinoflagellate distributions and diversity for Tonga that may be used for future studies and the development of monitoring programmes.

Outbreak of ciguatera poisoning in a commercial vessel: lessons learnt and prospects of early diagnosis, management, and prophylaxis.

An outbreak of food poisoning of unknown origin was notified to Central Queensland Public Health Unit on 9 December 2021. The bulk carrier sailing from Higashiharima, Japan to Gladstone, Australia reported an incident of sudden illness, with 19 out of 20 sailors on board reporting a combination of gastrointestinal and neurological symptoms. Central Queensland Public Health Unit started the outbreak investigation as per Queensland Health public health management guidelines. All 20 of the sailors consumed a self-caught barracuda and squid, prepared by the ship's cook, the day before. Unconsumed samples of the fish and squid were sent for testing. The affected sailors were triaged on arrival and were provided with medical care as required. The barracuda sample contained ciguatoxins (CTXs; P-CTX-1, P-CTX-2, P-CTX-3) with a total count of 3.40 ug/kg confirming the diagnosis. We propose the usage of the combination of gastrointestinal symptoms and paraesthesia in the light of a recent intoxication event for early detection of ciguatera poisoning (CP) in the eastern seaboard of Australia.

Ciguatera poisoning in French Polynesia: A review of the distribution and toxicity of Gambierdiscus spp., and related impacts on food web components and human health.

Ciguatera Poisoning (CP) is a seafood poisoning highly prevalent in French Polynesia. This illness results from the consumption of seafood contaminated with ciguatoxins (CTXs) produced by Gambierdiscus, a benthic dinoflagellate. Ciguatera significantly degrades the health and economic well-being of local communities largely dependent on reef fisheries for their subsistence. French Polynesia has been the site of rich and active CP research since the 1960's. The environmental, toxicological, and epidemiological data obtained in the frame of large-scale field surveys and a country-wide CP case reporting program conducted over the past three decades in the five island groups of French Polynesia are reviewed. Results show toxin production in Gambierdiscus in the natural environment may vary considerably at a temporal and spatial scale, and that several locales clearly represent Gambierdiscus spp. "biodiversity hotspots". Current data also suggest the "hot" species G. polynesiensis could be the primary source of CTXs in local ciguateric biotopes, pending formal confirmation. The prevalence of ciguatoxic fish and the CTX levels observed in several locales were remarkably high, with herbivores and omnivores often as toxic as carnivores. Results also confirm the strong local influence of Gambierdiscus spp. on the CTX toxin profiles characterized across multiple food web components including in CP-prone marine invertebrates. The statistics, obtained in the frame of a long-term epidemiological surveillance program established in 2007, point towards an apparent decline in the number of CP cases in French Polynesia as a whole; however, incidence rates remain dangerously high in some islands. Several of the challenges and opportunities, most notably those linked to the strong cultural ramifications of CP among local communities, that need to be considered to define effective risk management strategies are addressed.

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.

A 15-Year Retrospective Review of Ciguatera in the Madeira Islands (North-East Atlantic, Portugal).

The first ciguatera fish poisoning (CFP) in Portugal dates from 2008 when 11 people reported CFP symptoms after consuming a 30 kg amberjack caught around the Selvagens Islands (Madeira Archipelago). Since then, 49 human poisonings have been reported. The emergence of a new threat challenged scientists and regulators, as methods for toxic microalgae analyses and ciguatoxin (CTX) detection were not implemented. To minimise the risk of ciguatera, the Madeira Archipelago authorities interdicted fisheries in Selvagens Islands and banned the capture of amberjacks weighing more than 10 kg in the entire region of Madeira Archipelago. The accurate identification and quantification of the benthic toxin-producing algae species spreading to new areas require efforts in terms of both microscopy and molecular techniques. Two ciguatera-causing dinoflagellates, Gambierdiscus excentricus and Gambierdiscus australes, were identified in the Madeira Island and Selvagens sub-archipelago, respectively. Regarding the CTX analysis (N2a cell-based assay and LC-MS) in fish, the results indicate that the Selvagens Islands are a ciguatera risk area and that fish vectoring CTX are not limited to top predator species. Nevertheless, advances and improvements in screening methods for the fast detection of toxicity in seafood along with certified reference material and sensitive and selective targeted analytical methods for the determination of CTX content are still pending. This study aims to revise the occurrence of ciguatera cases in the Madeira Archipelago since its first detection in 2008, to discuss the risk management strategy that was implemented, and to provide a summary of the available data on the bioaccumulation of CTX in marine fish throughout the marine food web, taking into consideration their ecological significance, ecosystem dynamics, and fisheries relevance.

Characterizing the Influence of a Heterotrophic Bicosoecid Flagellate Pseudobodo sp. on the Dinoflagellate Gambierdiscus balechii.

Microbial interactions including competition, mutualism, commensalism, parasitism, and predation, which can be triggered by nutrient acquisition and chemical communication, are universal phenomena in the marine ecosystem. The interactions may influence the microbial population density, metabolism, and even their environmental functions. Herein, we investigated the interaction between a heterotrophic bicosoecid flagellate, Pseudobodo sp. (Bicoecea), and a dinoflagellate, Gambierdiscus balechii (Dinophyceae), which is a well-known ciguatera food poisoning (CFP) culprit. The presence of Pseudobodo sp. inhibited the algal proliferation and decreased the cardiotoxicity of zebrafish in the algal extract exposure experiment. Moreover, a significant difference in microbiome abundance was observed in algal cultures with and without Pseudobodo sp. Chemical analysis targeting toxins was performed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with molecular networking (MN), showing a significant alteration in the cellular production of gambierone analogs and some super-carbon chain compounds. Taken together, our results demonstrated the impact of heterotrophic flagellate on the photosynthetic dinoflagellates, revealing the complex dynamics of algal toxin production and the ecological relationships related to dinoflagellates in the marine environment.

Toxic neuropathies.

PURPOSE OF REVIEW: Immunotherapy has had a significant impact on the treatment of an increasing number of cancers as well as in inflammatory, rheumatological and gastroenterological conditions.Recreational nitrous oxide use is now a global epidemic. Linezolid is now recommended for the treatment of drug-resistant tuberculosis (TB); neuropathy is a significant cause of morbidity.Global warming will result in increasing toxin exposure, such as ciguatera, in previously unaffected areas. RECENT FINDINGS: With increasing experience, the pathophysiology underlying the neuropathic complications of these drugs has become clear with guidelines now available, for the complications of immune check-point inhibitors and nitrous oxide toxicity. The optimum dose and duration of treatment for resistant TB with regimens, including linezolid, has been ascertained. SUMMARY: Although neuropathic complications with immunotherapy are relatively rare, it is essential that they are recognized and treated early. Nitrous oxide toxicity should be in the differential diagnosis for all patients, particularly those of younger age, presenting with a neuropathy or myleo-neuropathy. Ciguatera toxicity is under recognized and its geographical spread will increase due to global warming. Further research is necessary on the mechanisms and treatment of both acute and chronic effects, which at present, are only symptomatic.

Benthic harmful microalgae and their impacts in South America.

Public awareness about Benthic Harmful Algal Blooms (BHABs) and their negative impacts has increased substantially over the past few decades. Even so, reports of BHABs remain relatively scarce in South America (SA). This paper provides a comprehensive overview of the current state of knowledge on BHABs in the continent, by integrating data from published articles, books, and technical reports. We recorded ∼300 different occurrences of potentially toxic BHAB species over the Caribbean, Atlantic and Pacific coasts, mostly in marine (>95%) but also in estuarine areas located from 12°36' N to 54°53' S. Over 70% of the data was published/released within the past 10 years, and ∼85% were concentrated in Brazil, Venezuela, Ecuador and Colombia. Benthic species were mainly associated with macroalgae, seagrass and sediment. Incidental detection in the plankton was also relevant, mainly in places where studies targeting BHAB species are still rare, like Argentina, Uruguay, Chile and Peru. The study listed 31 infrageneric taxa of potentially toxic benthic dinoflagellates and eight of estuarine cyanobacteria occurring in SA, with the greatest species diversity recorded in the equatorial-tropical zone, mainly in northeastern Brazil (Atlantic), Venezuela and Colombia (Caribbean), and the Galapagos Islands, Ecuador (Pacific). Local strains of Amphidinium, Gambierdiscus, Coolia and Prorocentrum spp. produced toxic compounds of emerging concern. Prorocentrum lima species complex was the most common and widely distributed taxon, followed by Ostreopsis cf. ovata. In fact, these two dinoflagellates were associated with most BHAB events in SA. Whereas the former has caused the contamination of multiple marine organisms and cases of Diarrhetic Shellfish Poisoning in subtropical and temperate areas, the latter has been associated with faunal mortalities and is suspected of causing respiratory illness to beach users in tropical places. Ciguatera Poisoning has been reported in Colombia (∼240 cases; no deaths) and Venezuela (60 cases; two deaths), and may be also a risk in other places where Gambierdiscus spp. and Fukuyoa paulensis have been reported, such as the Galapagos Islands and the tropical Brazilian coast. Despite the recent advances, negative impacts from BHABs in SA are intensified by limited research/training funding, as well as the lack of official HAB monitoring and poor analytical capability for species identification and toxin detection in parts of the continent.

Ciguatera Fish Poisoning in the Caribbean Sea and Atlantic Ocean: Reconciling the Multiplicity of Ciguatoxins and Analytical Chemistry Approach for Public Health Safety.

Ciguatera is a major circumtropical poisoning caused by the consumption of marine fish and invertebrates contaminated with ciguatoxins (CTXs): neurotoxins produced by endemic and benthic dinoflagellates which are biotransformed in the fish food-web. We provide a history of ciguatera research conducted over the past 70 years on ciguatoxins from the Pacific Ocean (P-CTXs) and Caribbean Sea (C-CTXs) and describe their main chemical, biochemical, and toxicological properties. Currently, there is no official method for the extraction and quantification of ciguatoxins, regardless their origin, mainly due to limited CTX-certified reference materials. In this review, the extraction and purification procedures of C-CTXs are investigated, considering specific objectives such as isolating reference materials, analysing fish toxin profiles, or ensuring food safety control. Certain in vitro assays may provide sufficient sensitivity to detect C-CTXs at sub-ppb levels in fish, but they do not allow for individual identification of CTXs. Recent advances in analysis using liquid chromatography coupled with low- or high-resolution mass spectrometry provide new opportunities to identify known C-CTXs, to gain structural insights into new analogues, and to quantify C-CTXs. Together, these methods reveal that ciguatera arises from a multiplicity of CTXs, although one major form (C-CTX-1) seems to dominate. However, questions arise regarding the abundance and instability of certain C-CTXs, which are further complicated by the wide array of CTX-producing dinoflagellates and fish vectors. Further research is needed to assess the toxic potential of the new C-CTX and their role in ciguatera fish poisoning. With the identification of C-CTXs in the coastal USA and Eastern Atlantic Ocean, the investigation of ciguatera fish poisoning is now a truly global effort.

Evaluation of relative potency of calibrated ciguatoxin congeners by near-infrared fluorescent receptor binding and neuroblastoma cell-based assays.

Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.

Gambierone and Sodium Channel Specific Bioactivity Are Associated with the Extracellular Metabolite Pool of the Marine Dinoflagellate Coolia palmyrensis.

Tropical epibenthic dinoflagellate communities produce a plethora of bioactive secondary metabolites, including the toxins ciguatoxins (CTXs) and potentially gambierones, that can contaminate fishes, leading to ciguatera poisoning (CP) when consumed by humans. Many studies have assessed the cellular toxicity of causative dinoflagellate species to better understand the dynamics of CP outbreaks. However, few studies have explored extracellular toxin pools which may also enter the food web, including through alternative and unanticipated routes of exposure. Additionally, the extracellular exhibition of toxins would suggest an ecological function and may prove important to the ecology of the CP-associated dinoflagellate species. In this study, semi-purified extracts obtained from the media of a Coolia palmyrensis strain (DISL57) isolated from the U.S. Virgin Islands were assessed for bioactivity via a sodium channel specific mouse neuroblastoma cell viability assay and associated metabolites evaluated by targeted and non-targeted liquid chromatography tandem and high-resolution mass spectrometry. We found that extracts of C. palmyrensis media exhibit both veratrine enhancing bioactivity and non-specific bioactivity. LC-HR-MS analysis of the same extract fractions identified gambierone and multiple undescribed peaks with mass spectral characteristics suggestive of structural similarities to polyether compounds. These findings implicate C. palmyrensis as a potential contributor to CP and highlight extracellular toxin pools as a potentially significant source of toxins that may enter the food web through multiple exposure pathways.