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.

Humanization and characterization of an anti-ciguatoxin CTX3C monoclonal antibody.

Ciguatera poisoning (CP), caused by ciguatoxins (CTXs), is one of the most common food-borne diseases, affecting more than 50,000 people each year. In most cases, CP are managed with symptomatic and supportive remedies, and no specific treatment has been devised. In this study, toward the development of therapeutic antibodies for CP, we examined to humanize mouse anti-CTX3C antibody 10C9 (m10C9), which exhibited neutralizing activity against ciguatoxin in vitro and in vivo. The complementarity determining regions were grafted onto a human germline sequence with high sequence identity to m10C9, and the backmutations were examined to maintain the binding affinity. The optimized humanized antibody, Opt.h10C9Fab, showed a strong binding affinity to CTX3C with a high affinity (KD = 19.0 nM), and only two backmutations of ArgL46 and CysH94 in the framework regions were involved in determining the antigen binding affinity.

[Universal Method for Determination of Ciguatoxins by LC-MS/MS].

Ciguatera poisoning (CP) is one of the most frequent seafood poisonings across the globe. CP results from the consumption of fish flesh that has accumulated principal toxins known as ciguatoxins (CTXs), and it mainly occurs in tropical and subtropical regions. In Japan, incidents of CP have been reported primarily from Okinawa and Amami Islands in the subtropical area. Meanwhile, there have also been reports from Mainland sporadically. Since the amount of CTXs contained in fish flesh is extremely low, a highly sensitive detection method by LC-MS/MS is required. But the currently reported detection method is applicable only to specific equipment, and many laboratories have difficulty to respond CP. In this study, to prepare for the risk of nationwide CP, we researched a universal analytical method for CTXs based on LC-MS/MS. Using a water/acetonitrile mobile phase supplemented with lithium hydroxide and formic acid gave rise to prominent peaks of the stable [M+Li]+ions. As the [M+Li]+ions did not produce valid product ions even with high collision energy, the [M+Li]+ions of each analog were set for both precursor and product ions ([M+Li]+>[M+Li]+) and monitored under the multiple reaction monitoring (MRM) mode. With the method described above, analyses of nine CTX congeners were carried out. The limit of detection (LOD, S/N>5) and quantitation (LOQ, S/N>10) were estimated as 0.005-0.030 ng/mL and 0.010-0.061 ng/mL, respectively. When the 1 mL of extract solution is prepared from 5 g of the fish tissue, the LOD and LOQ will be at 0.001-0.006 µg/kg and 0.002-0.012 µg/kg, respectively. This result indicates that we could detect the required level of 0.175 µg/kg CTX1B equivalent in fish flesh which is recommended for safe consumption in Japan. This method is considered to be a universal analytical method without depending on the specific equipment. Thus it could contribute to improving the CP investigations in nationwide laboratories.

The Prevalence of Marine Lipophilic Phycotoxins Causes Potential Risks in a Tropical Small Island Developing State.

Tropical small island developing states (SIDS), with their geographical isolation and limited resources, heavily rely on the fisheries industry for food and revenue. The presence of marine lipophilic phycotoxins (MLPs) poses risks to their economy and human health. To understand the contamination status and potential risks, the Republic of Kiribati was selected as the representative tropical SIDS and 55 species of 256 coral reef fish encompassing multiple trophic levels and feeding strategies were collected to analyze 17 typical MLPs. Our results showed that the potential risks of ciguatoxins were the highest and approximately 62% of fish species may pose risks for consumers. Biomagnification of ciguatoxins was observed in the food web with a trophic magnification factor of 2.90. Brevetoxin-3, okadaic acid, and dinophysistoxin-1 and -2 were first reported, but the risks posed by okadaic acid and dinophysistoxins were found to be negligible. The correlation analysis revealed that fish body size and trophic position are unreliable metrics to indicate the associated risks and prevent the consumption of contaminated fish. The potential risks of MLPs in Kiribati are of concern, and our findings can serve as valuable inputs for developing food safety policies and fisheries management strategies specific to tropical SIDS contexts.

Detection of Extremely Low Level Ciguatoxins through Monitoring of Lithium Adduct Ions by Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry.

Ciguatera poisoning (CP) is the most common type of marine biotoxin food poisoning worldwide, and it is caused by ciguatoxins (CTXs), thermostable polyether toxins produced by dinoflagellate Gambierdiscus and Fukuyoa spp. It is typically caused by the consumption of large fish high on the food chain that have accumulated CTXs in their flesh. CTXs in trace amounts are found in natural samples, and they mainly induce neurotoxic effects in consumers at concentrations as low as 0.2 µg/kg. The U.S. Food and Drug Administration has established CTX maximum permitted levels of 0.01 µg/kg for CTX1B and 0.1 µg/kg for C-CTX1 based on toxicological data. More than 20 variants of the CTX1B and CTX3C series have been identified, and the simultaneous detection of trace amounts of CTX analogs has recently been required. Previously published works using LC-MS/MS achieved the safety levels by monitoring the sodium adduct ions of CTXs ([M+Na]+ > [M+Na]+). In this study, we optimized a highly sensitive method for the detection of CTXs using the sodium or lithium adducts, [M+Na]+ or [M+Li]+, by adding alkali metals such as Na+ or Li+ to the mobile phase. This work demonstrates that CTXs can be successfully detected at the low concentrations recommended by the FDA with good chromatographic separation using LC-MS/MS. It also reports on the method's new analytical conditions and accuracy using [M+Li]+.

First Detection of Algal Caribbean Ciguatoxin in Amberjack Causing Ciguatera Poisoning in the Canary Islands (Spain).

Ciguatera Poisoning (CP) is an illness associated with the consumption of fish contaminated with potent natural toxins found in the marine environment, commonly known as ciguatoxins (CTXs). The risk characterization of CP has become a worldwide concern due to the widespread expansion of these natural toxins. The identification of CTXs is hindered by the lack of commercially available reference materials. This limitation impedes progress in developing analytical tools and conducting toxicological studies essential for establishing regulatory levels for control. This study focuses on characterizing the CTX profile of an amberjack responsible for a recent CP case in the Canary Islands (Spain), located on the east Atlantic coast. The exceptional sensitivity offered by Capillary Liquid Chromatography coupled with High-Resolution Mass Spectrometry (cLC-HRMS) enabled the detection, for the first time in fish contaminated in the Canary Islands, of traces of an algal ciguatoxin recently identified in G. silvae and G. caribeaus from the Caribbean Sea. This algal toxin was structurally characterized by cLC-HRMS being initially identified as C-CTX5. The total toxin concentration of CTXs was eight times higher than the guidance level proposed by the Food and Drug Administration (0.1 ng C-CTX1/g fish tissue), with C-CTX1 and 17-hydroxy-C-CTX1 as major CTXs.

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.

Targeted Metabolite Fingerprints of Thirteen Gambierdiscus, Five Coolia and Two Fukuyoa Species.

The genus Gambierdiscus produces an array of bioactive hydrophilic and lipophilic secondary metabolites that range in mode of action and toxicity. In this study, the metabolite fingerprint was mapped for thirteen Gambierdiscus, five Coolia and two Fukuyoa species (34 isolates) by assessing the production of 56 characterised secondary metabolites. Gambierdiscus polynesiensis was the only species to produce Pacific-ciguatoxin-3B (P-CTX3B), P-CTX3C, iso-P-CTX3B/C, P-CTX4A, P-CTX4B and iso-P-CTX4A/B. G. australes produced maitotoxin-1 (MTX-1) and MTX-5, G. cheloniae produced MTX-6 and G. honu produced MTX-7. Ubiquitous production of 44-methylgambierone was observed amongst all the Gambierdiscus isolates, with nine species also producing gambierone. Additional gambierone analogues, including anhydrogambierone (tentatively described herein), were also detected in all Gambierdiscus species, two Coolia and two Fukuyoa species. Gambieroxide was detected in G. lewisii and G. pacificus and gambieric acid A was detected in ten Gambierdiscus species, with G. australes (CAWD381) being the only isolate to produce gambieric acids A-D. This study has demonstrated that the isolates tested to date produce the known CTXs or MTXs, but not both, and highlighted several species that produced 'unknown' compounds displaying characteristics of cyclic polyethers, which will be the focus of future compound discovery efforts.

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).

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.

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.

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.

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.

Synergistic Effect of Brevetoxin BTX-3 and Ciguatoxin CTX3C in Human Voltage-Gated Nav1.6 Sodium Channels.

Emerging marine biotoxins such as ciguatoxins and brevetoxins have been widely and independently studied as food pollutants. Their maximum levels in food components were set without considering their possible synergistic effects as consequence of their coexistence in seafood and their action at the same cellular target. The absolute lack of data and regulations of the possible combined effects that both marine biotoxins may have raised the need to analyze their direct in vitro effects using electrophysiology techniques. The results presented in this study indicate that ciguatoxins and brevetoxins had a synergistic effect on human Nav1.6 voltage-gated sodium channels by hyperpolarizing their activation and inactivation states. The results presented here indicate that brevetoxin 3 (BTX-3) acts as partial agonist of human sodium channels, while ciguatoxin 3C (CTX3C) was a full agonist, explaining the differences in the effect of each toxin in the channel. Therefore, this work sets the cellular basis to further apply this type of studies to other food toxicants that may act synergistically and thus implement the corresponding regulatory limits considering their coexistence and the risks to human and animal health derived from it.

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.

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.