[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.

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.

Isolation of Caribbean Ciguatoxin-5 (C-CTX5) and confirmation of its structure by NMR spectroscopy.

Ciguatera poisoning occurs throughout subtropical and tropical regions globally. The Virgin Islands in the Caribbean Sea is a known hyperendemic region for ciguatera and has been associated with Caribbean ciguatoxin (C-CTX) contamination in fish. An algal C-CTX (C-CTX5) was identified in Gambierdiscus silvae and G. caribeaus isolated from benthic algal samples collected in waters south St. Thomas, US Virgin Islands. The highest CTX-producing isolate, G. silvae 1602 SH-6, was grown at large-scale to isolate sufficient C-CTX5 for structural confirmation by NMR spectroscopy. A series of orthogonal extraction and fractionation procedures resulted in purification of approximately 40 µg of C-CTX5, as estimated by quantitative NMR. A suite of 1D and 2D NMR experiments were acquired that verified the structure originally proposed for C-CTX5. The structural confirmation and successful isolation of C-CTX5 opens the way for work on the stability, toxicology and biotransformation of C-CTXs, as well as for the production of quantitative reference materials for analytical method development and validation. The strategies developed for purification of C-CTX5 may also apply to isolation and purification of CTXs from the Pacific Ocean and other regions.

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]+.

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.

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.

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.

Synthesis of the A-F Fragment of the Pacific Ciguatoxin CTX3C by Iterative Ring-Closing Metathesis and Tsuji-Trost Allylation.

The fully functionalized A-F fragment of the Pacific ciguatoxin CTX3C has been synthesized from a derivative of D-glucal, which serves as the B-ring. Rings A and C were introduced to either side of ring B by ring-closing diene and enyne metathesis (RCM). The seven-membered D-ring and eight-membered E-ring were assembled by iterative use of a six-step reaction sequence in which RCM was used for ring construction and Tsuji-Trost allylation was employed for subsequent stereoselective functionalization. The nine-membered F-ring was formed by use of an RCM reaction and bears the functionality required for attachment of the I-M fragment and subsequent closure of rings G and H.

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.

Algal ciguatoxin identified as source of ciguatera poisoning in the Caribbean.

Ciguatera poisoning (CP) is a severe seafood-borne disease, caused by the consumption of reef fish contaminated with Caribbean ciguatoxins (C-CTXs) in the Caribbean and tropical Atlantic. However, C-CTXs have not been identified from their presumed algal source, so the relationship to the CTXs in fish causing illness remains unknown. This has hindered the development of detection methods, diagnostics, monitoring programs, and limited fundamental knowledge on the environmental factors that regulate C-CTX production. In this study, in vitro and chemical techniques were applied to unambiguously identify a novel C-CTX analogue, C-CTX5, from Gambierdiscus silvae and Gambierdiscus caribaeus strains from the Caribbean. Metabolism in vitro by fish liver microsomes converted algal C-CTX5 into C-CTX1/2, the dominant CTX in ciguatoxic fish from the Caribbean. Furthermore, C-CTX5 from G. silvae was confirmed to have voltage-gated sodium-channel-specific activity. This finding is crucial for risk assessment, understanding the fate of C-CTXs in food webs, and is a prerequisite for development of effective analytical methods and monitoring programs. The identification of an algal precursor produced by two Gambierdiscus species is a major breakthrough for ciguatera research that will foster major advances in this important seafood safety issue.

Model of the Origin of a Ciguatoxic Grouper (Plectropomus leopardus).

Published data were used to model the transfer of ciguatoxins (CTX) across three trophic levels of a marine food chain on the Great Barrier Reef (GBR), Australia, to produce a mildly toxic common coral trout (Plectropomus leopardus), one of the most targeted food fishes on the GBR. Our model generated a 1.6 kg grouper with a flesh concentration of 0.1 µg/kg of Pacific-ciguatoxin-1 (P-CTX-1 = CTX1B) from 1.1 to 4.3 µg of P-CTX-1 equivalents (eq.) entering the food chain from 0.7 to 2.7 million benthic dinoflagellates (Gambierdiscus sp.) producing 1.6 pg/cell of the P-CTX-1 precursor, P-CTX-4B (CTX4B). We simulated the food chain transfer of ciguatoxins via surgeonfishes by modelling Ctenochaetus striatus feeding on turf algae. A C. striatus feeding on ≥1000 Gambierdiscus/cm2 of turf algae accumulates sufficient toxin in <2 days that when preyed on, produces a 1.6 kg common coral trout with a flesh concentration of 0.1 µg/kg P-CTX-1. Our model shows that even transient blooms of highly ciguatoxic Gambierdiscus can generate ciguateric fishes. In contrast, sparse cell densities of ≤10 Gambierdiscus/cm2 are unlikely to pose a significant risk, at least in areas where the P-CTX-1 family of ciguatoxins predominate. The ciguatera risk from intermediate Gambierdiscus densities (~100 cells/cm2) is more difficult to assess, as it requires feeding times for surgeonfish (~4-14 days) that overlap with turnover rates of turf algae that are grazed by herbivorous fishes, at least in regions such as the GBR, where stocks of herbivorous fishes are not impacted by fishing. We use our model to explore how the duration of ciguatoxic Gambierdiscus blooms, the type of ciguatoxins they produce, and fish feeding behaviours can produce differences in relative toxicities between trophic levels. Our simple model indicates thresholds for the design of risk and mitigation strategies for ciguatera and the variables that can be manipulated to explore alternate scenarios for the accumulation and transfer of P-CTX-1 analogues through marine food chains and, potentially, for other ciguatoxins in other regions, as more data become available.

Dereplication of Gambierdiscusbalechii extract by LC-HRMS and in vitro assay: First description of a putative ciguatoxin and confirmation of 44-methylgambierone.

Marine toxins have a significant impact on seafood resources and human health. Up to date, mainly based on bioassays results, two genera of toxic microalgae, Gambierdiscus and Fukuyoa have been hypothesized to produce a suite of biologically active compounds, including maitotoxins (MTXs) and ciguatoxins (CTXs) with the latter causing ciguatera poisoning (CP) in humans. The global ubiquity of these microalgae and their ability to produce (un-)known bioactive compounds, necessitates strategies for screening, identifying, and reducing the number of target algal species and compounds selected for structural elucidation. To accomplish this task, a dereplication process is necessary to screen and profile algal extracts, identify target compounds, and support the discovery of novel bioactive chemotypes. Herein, a dereplication strategy was applied to a crude extract of a G. balechii culture to investigate for bioactive compounds with relevance to CP using liquid chromatography-high resolution mass spectrometry, in vitro cell-based bioassay, and a combination thereof via a bioassay-guided micro-fractionation. Three biologically active fractions exhibiting CTX-like and MTX-like toxicity were identified. A naturally incurred fish extract (Sphyraena barracuda) was used for confirmation where standards were unavailable. Using this approach, a putative I/C-CTX congener in G. balechii was identified for the first time, 44-methylgambierone was confirmed at 8.6 pg cell-1, and MTX-like compounds were purported. This investigative approach can be applied towards other harmful algal species of interest. The identification of a microalgal species herein, G. balechii (VGO920) which was found capable of producing a putative I/C-CTX in culture is an impactful advancement for global CP research. The large-scale culturing of G. balechii could be used as a source of I/C-CTX reference material not yet commercially available, thus, fulfilling an analytical gap that currently hampers the routine determination of CTXs in various environmental and human health-relevant matrices.

Cyclodextrin polymer clean-up method for the detection of ciguatoxins in fish with cell-based assays.

Ciguatoxins (CTXs) are marine toxins produced by microalgae of the genera Gambierdiscus and Fukuyoa, which are transferred through the food webs, reaching humans and causing a poisoning known as ciguatera. The cell-based assay (CBA) is commonly used for their detection because of its high sensitivity and the provided toxicological information. However, matrix effects may interfere in the CBA. In this work, γ-cyclodextrin-hexamethylene diisocyanate (γ-CD-HDI), γ-cyclodextrin-epichlorohydrin (γ-CD-EPI) and γ-CD-EPI conjugated to magnetic beads (γ-CD-EPI-MB) have been evaluated as clean-up materials for fish flesh extracts containing CTXs. The best results were achieved with γ-CD-HDI in column format, which showed a CTX1B recovery of 42% and 32% for Variola louti and Seriola dumerili, respectively, and allowed exposing cells to at least 400 mg/mL of fish flesh. This clean-up strategy provides at least 4.6 and 3.0-fold higher sensitivities to the assay for V.louti and S.dumerili, respectively, improving the reliability of CTX quantification.

[Analysis of Ciguatoxins in the Spotted Knifejaw, Oplegnathus punctatus from the Waters of Japan].

Ciguatera fish poisoning (CFP) is recognized as the most frequent seafood poisoning due to the consumption of fish containing the principal toxins, ciguatoxins (CTXs). In Japan, CFP events have been reported annually from Okinawa and Amami Islands, locating subtropical regions. In addition, there have been reported several outbreaks due to consumption of the fish caught from the Pacific coast of the Mainland and they were often caused by the matured spotted knifejaw, Oplegnathus punctatus. As part of our research on CFP in Japan, we investigated CTXs analysis by LC-MS/MS on 176 individuals of O. punctatus (weight: 100-6,350 g, standard length: 13-60 cm) from the coast of the Mainland (Honshu, Shikoku, and Kyushu), Amami, Okinawa, and Ogasawara (Bonin) Islands. CTXs were detected from only two specimens collected from Okinawa. Total CTXs levels of the two specimens were at 0.014 and 0.040 µg/kg, respectively, exceeding FDA guidance level at 0.01 µg CTX1B equivalent/kg. However, they might be little risk of CFP because consuming over 1.5 kg of flesh is needed to develop intoxication. The toxins consisted of CTX1B analogs including CTX1B, 52-epi-54-deoxyCTX1B, CTX4A, and CTX4B, and no CTX3C analogs, supporting the finding that ciguatoxic fishes in Okinawan Waters containing only CTX1B analogs.

Bimodal Cell Size and Fusing Cells Observed in a Clonal Culture of the Ciguatoxin-Producing Benthic Dinoflagellate Gambierdiscus (WC1/1).

Cells in a clonal culture of the WC1/1 strain of Gambierdiscus that produced ciguatoxin and maitotoxin-3 were observed to spontaneously fuse during the light phase of culture growth. Cells in the process of fusion were indistinguishable from other cells under the light microscope, except that at least one (often both) of the fusing cells displayed an extendible, finger-like protrusion (presumed peduncle) arising from near the sulcul region. Fusion started with one of the cells turning 90° to place the planes of the girdles approximately at right angles to each other, and movement of the transverse flagella ceased in both cells, or in the cell seen in girdle (lateral) view. The cell in girdle view appeared to fuse into the theca of the other cell. The cell that had turned 90° often rounded up and become egg shaped (obovoid) during early fusion. Fusion can be quick (<10 min) or can take more than an hour. We saw no evidence of the theca being shed during fusion. Measurement of the dorsoventral and transdiameters revealed a wide range for cell sizes that were distributed as a bimodal population in the clonal culture. This bimodal cell population structure was maintained in clonal cultures reisolated from a small or large cell from the original WC1/1 culture. Cellular production of ciguatoxins by the WC1/1 clone increased during the first two years in culture with a corresponding decrease in production of maitotoxin-3, but this inverse relationship was not maintained over the following ~1.5 years.

Selective and Efficient Capture and Release of vic-Diol-Containing Pacific and Caribbean Ciguatoxins from Fish Extracts with a Boronate Affinity Polymer.

Ciguatera poisoning can occur following the consumption of fish contaminated with trace levels of ciguatoxins (CTXs). These trace levels represent an analytical challenge for confirmation by LC-MS due to matrix interferences and the high instrument sensitivity required. Sample preparation procedures are laborious and require extensive cleanup procedures to address these issues. The application of a selective isolation technique employing boronate affinity polymers was therefore investigated for the capture of vic-diol-containing Caribbean and Pacific CTXs from fish extracts. A dispersive SPE procedure was developed where nearly complete binding of CTXs in fish extracts occurred with boric acid gel in less than 1 h. Release of the bound CTXs resulted in >95% recovery of C-CTX1/2, C-CTX3/4, CTX1B, 54-deoxyCTX1B, and 52-epi-54-deoxyCTX1B from the extracts. This selective extraction tool has the potential to greatly simplify both analytical sample preparation and preparative extraction and isolation of CTXs for structure elucidation and production of standards.

Immobilisation of Neuro-2a cells on electrodes and electrochemical detection of MTT formazan crystals to assess their viability.

Marine toxins are potent toxic compounds that may reach humans and poison them. Therefore, their detection in seafood is crucial to prevent intoxication cases. Colorimetric cell-based assays (CBAs) have been developed to analyse marine neurotoxins, such as ciguatoxins (CTXs) and tetrodotoxins (TTXs), and are based on the toxicological effect of these toxins on the cells. Cell viability can be quantified by measuring the mitochondrial activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). With the purpose of moving forward in the development of cell-based biosensors (CBBs) for neurotoxins, Neuro-2a cells were immobilised on electrodes of different materials (carbon, carbon/polyaniline, carbon/poly-l-lysine, carbon/poly(3,4-ethylenedioxythiophene) and gold) and their presence and viability were assessed by the detection of MTT formazan crystals with cyclic voltammetry (CV). Best results in terms of oxidation potential and current intensity were achieved with carbon and carbon/polyaniline electrodes. Light microscopy also proved the presence of immobilised and living cells on electrodes. Cell density, incubation time and MTT concentration were optimised. Appropriate electrochemical responses were obtained incubating 100,000 cells/electrode for 2 h and using 0.86 mg/mL MTT. The system was able to detect toxicity when exposed to CTX1B and TTX standard solutions as well as Seriola dumerili and Lagocephalus sceleratus fish extracts containing these toxins.

Origin of Ciguateric Fish: Quantitative Modelling of the Flow of Ciguatoxin through a Marine Food Chain.

To begin to understand the impact of food chain dynamics on ciguatera risk, we used published data to model the transfer of ciguatoxins across four trophic levels of a marine food chain in Platypus Bay, Australia. The data to support this first attempt to conceptualize the scale of each trophic transfer step was limited, resulting in broad estimates. The hypothetical scenario we explored generated a low-toxicity 10 kg Spanish mackerel (Scomberomorus commerson) with a flesh concentration of 0.1 µg/kg of Pacific-ciguatoxin-1 (P-CTX-1, also known as CTX1B) from 19.5-78.1 µg of P-CTX-1 equivalents (eq.) that enter the marine food chain from a population of 12-49 million benthic dinoflagellates (Gambierdiscus sp.) producing 1.6 × 10-12 g/cell of the P-CTX-1 precursor, P-CTX-4B. This number of Gambierdiscus could be epiphytic on 22-88 kg of the benthic macroalgae (Cladophora) that carpets the bottom of much of Platypus Bay, with the toxin transferred to an estimated 40,000-160,000 alpheid shrimps in the second trophic level. This large number of shrimps appears unrealistic, but toxic shrimps would likely be consumed by a school of small, blotched javelin fish (Pomadasys maculatus) at the third trophic level, reducing the number of shrimps consumed by each fish. The Spanish mackerel would accumulate a flesh concentration of 0.1 µg/kg P-CTX-1 eq. by preying upon the school of blotched javelin and consuming 3.6-14.4 µg of P-CTX-1 eq. However, published data indicate this burden of toxin could be accumulated by a 10 kg Spanish mackerel from as few as one to three blotched javelin fish, suggesting that much greater amounts of toxin than modelled here must at certain times be produced and transferred through Platypus Bay food chains. This modelling highlights the need for better quantitative estimates of ciguatoxin production, biotransformation, and depuration through marine food chains to improve our understanding and management of ciguatera risk.

Clinical Characteristics of Ciguatera Poisoning in Martinique, French West Indies-A Case Series.

Ciguatera poisoning (CP) is one of the most common causes worldwide of marine poisoning associated with fish consumption from tropical areas. Its incidence is underreported. CP cases seem to increase with grouped cases reported during summer. Exposure to ciguatoxins, toxins responsible for CP with sodium-channel agonistic, voltage-gated potassium channel blocking, cholinergic, and adrenergic activities, may result in a large spectrum of manifestations. We aimed to describe the clinical characteristics, management, and outcome of CP in Martinique, French West Indies. We conducted an observational retrospective single-center study during six years (October 2012 to September 2018) including all CP patients managed by the prehospital medical services, admitted to the university hospital emergency department, or declared to the regional health agency. A total of 149 CP patients (81 females/63 males; median age, 46 years (interquartile range, 34-61)) were included. Acute features consisted in general (91%; mainly, myalgia pruritus, and asthenia), gastrointestinal (90%; mainly diarrhea, abdominal pain, and nausea), neurological (72%; mainly, paresthesia, dysgeusia, and impairment of hot/cold feeling), and cardiovascular manifestations (22%; bradycardia, hypotension, and heart conduction disorders). Management was supportive. No patient died but symptoms persisted in 40% of the 77 patients with follow-up at day 15. CP was mainly attributed to the ingestion of trevallies (59%), snappers (13%), and king mackerels (8%) with collective contaminations (71%). Unusual fish (tuna, salmon, and spider conchs) were suspected in rare cases. Ingestion of trevallies was associated with significantly higher persistent symptoms (odds ratio, 3.00; 95% confidence interval, (1.20-8.00); p = 0.03). CP incidence was 0.67 cases per 10,000 patient-years in Martinique over the study period. To conclude, CP represents an increasing public health issue in Martinique, as is the case in other Caribbean islands. Patients present usual but possibly life-threatening features. Outcome is excellent despite frequently prolonged manifestations.

Reductive Amination for LC-MS Signal Enhancement and Confirmation of the Presence of Caribbean Ciguatoxin-1 in Fish.

Ciguatera poisoning is a global health concern caused by the consumption of seafood containing ciguatoxins (CTXs). Detection of CTXs poses significant analytical challenges due to their low abundance even in highly toxic fish, the diverse and in-part unclarified structures of many CTX congeners, and the lack of reference standards. Selective detection of CTXs requires methods such as liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) or high-resolution MS (LC-HRMS). While HRMS data can provide greatly improved resolution, it is typically less sensitive than targeted LC-MS/MS and does not reliably comply with the FDA guidance level of 0.1 µg/kg CTXs in fish tissue that was established for Caribbean CTX-1 (C-CTX-1). In this study, we provide a new chemical derivatization approach employing a fast and simple one-pot derivatization with Girard's reagent T (GRT) that tags the C-56-ketone intermediate of the two equilibrating C-56 epimers of C-CTX-1 with a quaternary ammonium moiety. This derivatization improved the LC-MS/MS and LC-HRMS responses to C-CTX-1 by approximately 40- and 17-fold on average, respectively. These improvements in sensitivity to the GRT-derivative of C-CTX-1 are attributable to: the improved ionization efficiency caused by insertion of a quaternary ammonium ion; the absence of adduct-ions and water-loss peaks for the GRT derivative in the mass spectrometer, and; the prevention of on-column epimerization (at C-56 of C-CTX-1) by GRT derivatization, leading to much better chromatographic peak shapes. This C-CTX-1-GRT derivatization strategy mitigates many of the shortcomings of current LC-MS analyses for C-CTX-1 by improving instrument sensitivity, while at the same time adding selectivity due to the reactivity of GRT with ketones and aldehydes.