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Ciguatera poisoning (CP), arising from ciguatoxins produced by toxic dinoflagellate Gambierdiscus, is one of the most common food-borne diseases in the South Pacific. Climate change as well as its related events have been hypothesized to a higher abundance and wider presence of toxic dinoflagellates, hence a higher risk of the disease. Yet existing studies assessing the relationship between climate factors and CP are limited or based on old data. In this study, we used prewhitened cross-correlation analysis and auto-regressive integrated moving-average (ARIMA) modeling to develop predictive models of monthly CP incidence in Cook Islands and French Polynesia, two ciguatera-endemic regions in the South Pacific, utilizing the latest epidemiological data. Results reveal the significant time-lagged associations between the monthly CP incidence rate and several indicators relating to sea surface temperature (SST). In particular, SST anomaly is proven to be a strong positive predictor of an increased ciguatera incidence for both countries. If these time-lags can be supported by more investigations, it will allow health authorities to take appropriate actions, to limit or avoid an epidemic risk, especially on high-risk climate scenarios.

An increase in cases of ciguatera poisoning (CP) and expansion of the causative species in the South Pacific region highlight the need for baseline data on toxic microalgal species to help identify new areas of risk and manage known hot spots. Gambierdiscus honu is a toxin producing and potential CP causing dinoflagellate species, first described in 2017. Currently no high-resolution geographical distribution, intraspecific genetic variation or toxin production diversity data is available for G. honu. This research aimed to further characterize G. honu by investigating its distribution using species-specific real-time polymerase chain reaction assays at 25 sites in an area spanning ∼8000 km of the Coral Sea/Pacific Ocean, and assessing intraspecific genetic variation, toxicity and toxin production of isolated strains. Assessment of genetic variation of the partial rRNA operon of isolates demonstrated no significant intraspecific population structure, in addition to a lack of adherence to isolation by distance (IBD) model of evolution. The detected distribution of G. honu in the Pacific region was within the expected tropical to temperate latitudinal ranges of 10° to -30° and extended from Australia to French Polynesia. In the lipophilic fractions, the neuroblastoma cell-based assay (CBA-N2a) showed no ciguatoxin (CTX)-like activity for nine of the 10 isolates, and an atypical pattern for CAWD233 isolate which showed cytotoxic activity in OV- and OV+ conditions. In the same way, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis confirmed no Pacific-CTXs (CTX-3B, CTX-3C, CTX-4A, CTX-4B) were produced by the ten strains. The CBA-N2a assessment of the hydrophilic fractions showed moderate to high cytotoxicity in both OV- and OV+ condition for all the strains showing a cytotoxic profile similar to that of gambierone. Indeed, this study is the first to show the cytotoxic activity of gambierone on mouse neuroblastoma cells while no cytotoxicity was observed when 44-MG was analysed at the same concentrations using the CBA-N2a. Analysis of the hydrophilic via LC-MS/MS confirmed production of gambierone in all isolates, ranging from 2.1 to 38.1 pg/cell, with 44-methylgambierone (44-MG) also produced by eight of the isolates, ranging from 0.3 to 42.9 pg/cell. No maitotoxin-1 was detected in any of the isolates. Classification of the G. honu strains according to the quantities of gambierone produced aligned with the classification of their cytotoxicity using the CBA-N2a. Finally, no maitotoxin-1 (MTX) was detected in any of the isolates. This study shows G. honu is widely distributed within the Pacific region with no significant intraspecific population structure present. This aligns with the view of microalgal populations as global metapopulations, however more in-depth assessment with other genetic markers could detect further structure. Toxicity diversity across 10 isolates assessed did not display any geographical patterns.

Ciguatera is one of the most widespread food poisonings caused by the ingestion of fish contaminated by ciguatoxins (CTXs). Snapper and grouper with high palatable and economic value are the primary food source and fish species for exportation in the Republic of Kiribati, but they are highly suspected CTX-contaminated species due to their top predatory characteristics. In this study, 60 fish specimens from 17 species of snappers and groupers collected from the Kiritimati Island and Marakei Island of the Republic of Kiribati were analyzed using mouse neuroblastoma (N2a) assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine Pacific CTX-1, -2 and -3 (P-CTX-1, -2 and -3). The LC-MS/MS results show that CTXs were detected in 74.5% of specimens from Marakei Island and 61.5% of specimens from Kiritimati Island. The most toxic fish Epinephelus coeruleopunctatus from Marakei Island and Cephalopholis miniata from Kiritimati Island were detected as 53-fold and 28-fold P-CTX-1 equivalents higher than the safety level of 10 pg/g P-CTX-1 equivalents, respectively. CTX levels and composition profiles varied with species and location. The N2a results suggested that fish specimens also contain high levels of other CTX-like toxins or sodium channel activators. The distribution patterns for ciguatoxic fish of the two islands were similar, with fish sampled from the northwest being more toxic than the southwest. This study shows that groupers and snappers are high-risk species for ciguatera in the Republic of Kiribati, and these species can further be used as indicator species in ciguatera endemic areas for risk assessment.

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

Ethnopharmacological relevance In French Polynesia, many pathologies common or endemic to the territory cause diarrhea. This is the case for rotavirus gastroenteritis, salmonella food poisoning, ingestion of water contaminated by bacteria, and ciguatera. To treat these ailments, the population may employ traditional medicine for cultural reasons, geographical isolation, and poor health coverage. Polynesian remedies are often used without medical consultation and there is no data on their benefit-risk balance. A few ethnobotanical studies have been carried out in order to identify the traditional remedies used for various ailments, but few studies have focused on gastrointestinal pathologies. In this context, an ethnobotanical survey was carried out to identify treatments used for diarrhea and ciguatera, inventory the plants used, better understand the local representation of these remedies, and provide efficacy and safety data on these uses. Materials and methods From February to April 2021, a semi-structured survey was conducted on six islands in French Polynesia, including one island in the Windward Islands archipelago (Tahiti), three islands in the Marquesas archipelago (Hiva Oa, Nuku Hiva, Tahuata), and two islands in the Leeward Islands archipelago (Raiatea, Tahaa). A total of 133 people was interviewed including 34 specialists (of which 29 experts in herbalism). Results These people mentioned the use of 27 plants for the treatment of diarrhea, and 24 for the treatment of ciguatera. Citrus aurantiifolia, Psidium guajava and Cordyline fruticosa were the three most cited plant species used for treating diarrhea, while Cocos nucifera, Punica granatum and Barringtonia asiatica were the most cited for ciguatera. A large majority of plants are widespread and introduced plants, which is congruent with the history of Polynesian people. While some plants are well known for similar uses (e.g. Psidium guajava for diarrhea, Heliotropium arboreum for ciguatera), others are less well known and may present toxicity risks (e.g. Barringtonia asiatica for ciguatera). Conclusion Traditional Polynesian medicine is an integral part of the local culture so important to be preserved and valued. However, more pharmacological and toxicological studies are still needed to determine the benefit-risk balance of some of these remedies and to allow their official integration into the Polynesian health system.

Ciguatoxins are marine compounds that share a ladder-shaped polyether structure produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa, and include maitotoxins (MTX1 and MTX3), ciguatoxins (CTX3C) and analogues (gambierone), components of one of the most frequent human foodborne illness diseases known as ciguatera fish poisoning. This disease was previously found primarily in tropical and subtropical areas but nowadays, the dinoflagellates producers of ciguatoxins had spread to European coasts. One decade ago, the European Food Safety Authority has raised the need to complete the toxicological available data for the ciguatoxin group of compounds. Thus, in this work, the in vivo effects of ciguatoxin-related compounds have been investigated using internationally adopted guidelines for the testing of chemicals. Intraperitoneal acute toxicity was tested for maitotoxin 1 at doses between 200 and 3200 ng/kg and the acute oral toxicity of Pacific Ciguatoxin CTX3C at 330 and 1050 ng/kg and maitotoxin 1 at 800 ng/kg were also evaluated showing not effects on mice survival after a 96 h observation period. Therefore, for the following experiments the oral subchronic doses were between 172 and 1760 ng/kg for gambierone, 10 and 102 ng/kg for Pacific Ciguatoxin CTX3C, 550 and 1760 ng/kg for maitotoxin 3 and 800, 2560 and 5000 ng/kg for maitotoxin 1. The results presented here raise the need to reevaluate the in vivo activity of these agents. Although the intraperitoneal lethal dose of maitotoxin 1 is assumed to be 50 ng/kg, without chemical purity identifications and description of the bioassay procedures, in this work, an intraperitoneal lethal dose of 1107 ng/kg was obtained. Therefore, the data presented here highlight the need to use a common procedure and certified reference material to clearly establish the levels of these environmental contaminants in food Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The research leading to these results has received funding from the following FEDER cofunded-grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01). From Ministerio de Ciencia e Innovación PID 2020-11262RB-C21. From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX SI

Tetrodotoxin (TTX) is a crystalline, weakly basic, colorless organic substance and is one of the most potent marine toxins known. Although TTX was first isolated from pufferfish, it has been found in numerous other marine organisms and a few terrestrial species. Moreover, tetrodotoxication is still an important health problem today, as TTX has no known antidote. TTX poisonings were most commonly reported from Japan, Thailand, and China, but today the risk of TTX poisoning is spreading around the world. Recent studies have shown that TTX-containing fish are being found in other regions of the Pacific and in the Indian Ocean, as well as the Mediterranean Sea. This review aims to summarize pertinent information available to date on the structure, origin, distribution, mechanism of action of TTX and analytical methods used for the detection of TTX, as well as on TTX-containing organisms, symptoms of TTX poisoning, and incidence worldwide. info:eu-repo/semantics/publishedVersion

Ciguatoxins (CTX) cause ciguatera poisoning, which is the most common reported human food poisoning related to natural marine toxins. Pacific ciguatoxins are the most abundant and studied CTX analogues; however, the growing distribution of Caribbean analogues and the limited data available on their biological effects make necessary to re-evaluate their relative potency. For decades, the guidelines established by regulatory agencies have assumed that the potency of the Caribbean CTXs were tenfold lower than the Pacific CTXs. We present here an integrated study involving Neuro-2a cells (the method used worldwide to test ciguatoxins), electrophysiological assays, and in silico simulations that evidence the similar cytotoxicity of Caribbean and Pacific ciguatoxins and their asymmetry binding within sodium channels. The binding mode of the toxins was first explored by molecular docking using the GOLD program and the resulting binary complexes were further studied by Molecular Dynamics simulation studies using the molecular mechanics force field AMBER. The simulation studies explain their distinct impact on the activation potential of the channel as experimentally observed and provide a detailed picture of the effects caused by these toxins on an atomic scale Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The research leading to these results has received funding from the following FEDER-co-funded grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01, ED431C 2021/29, and the Centro singular de investigación de Galicia accreditation 2019–2022 ED431G 2019/03). From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX, and the EUROCIGUA project: “Risk Characterization of Ciguatera Fish Poisoning in Europe” GP/EFSA/AFSCO/2015/03, co-funded by the European Food Safety Authority (EFSA). From Ministerio de Ciencia e Innovación PID2020-115010RB-I00/AEI/10.13039/501100011033. David Castro (D.C.) financial support for the PhD studies was obtained through EUROCIGUA project: Risk characterization of Ciguatera Fish Poisoning in Europe, framework partnership agreement GP/EFSA/AFSCO/2015/03, co-funded by the EFSA. Pablo Estevez (P.E.) acknowledges financial support from the Xunta de Galicia (Regional Government, Spain) under grant ED481A-2018/207 SI

8 pages, Harmful algal blooms (HABs) are a signifi­cant global environmental management challenge, especially with respect to micro­algae that produce dangerous natural toxins. Examples of HAB toxin diseases with major global health impact include: ciguatera poi­soning, paralytic shellfish poisoning (PSP), amnesic shellfish poisoning (ASP), diarrhetic shellfish poisoning (DSP), and neurotoxic (brevetoxin) shellfish poisoning (NSP). Such diseases affect communities globally and contribute to health inequalities within the United States and beyond. Sharing data and lessons learned about the factors deter­mining bloom occurrence and associated exposure to contaminated seafood across locations can reduce public health risks. Knowledge sharing is particularly important as ongoing global environmental changes seem to alter the intensity, location, and timing of toxic HAB events, reducing the reliability of conventional guidance where toxin risks have been endemic and leading to emerging challenges in new settings. Political changes that disrupt membership in knowledge-sharing networks may impede efforts to share scientific expertise and best practices. In this commentary, we stress the importance of community and expert knowledge sharing for reducing HAB risks, both for vulnerable communities in the United States and globally. Considering the impacts of political changes, we note the indirect engagement sometimes required for continued participation in international coordination programs. As an example, we highlight how lessons learned from a Native-led toxin monitoring and testing program (the Southeast Alaska Tribal Ocean Research partnership) can inform programs in other settings. We also describe how in­ternational knowledge is mutually valuable for this program in Southeast Alaska, Funding comes from the National Institutes of Health grant number 7R01ES029165, Prevention of Paralytic Shellfish Poisoning in Subsistence Shellfish Harvest Communities of Southeast Alaska, and the T32 Cancer Prevention and Control Training Program at the University of Alabama at Birmingham, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Ciguatoxins (CTXs) which are produced by dinoflagellates of the genus Gambierdiscus and Fukuyoa and share a ladder-shaped polyether structure, are causative compounds of one of the most frequent foodborne illness disease known as ciguatera fish poisoning (CFP). CFP was initially found in tropical and subtropical areas but nowadays the dinoflagellates producers of ciguatoxins had spread to European coasts. Therefore, this raises the need of establishing toxicity equivalency factors for the different compounds that can contribute to ciguatera fish poisoning, since biological methods have been replaced by analytical techniques. Thus, in this work, the effects of six compounds causative of ciguatera, on their main target, the human voltage-gated sodium channels have been analyzed for the first time. The results presented here led to the conclusion that the order of potency was CTX1B, CTX3B, CTX4A, gambierol, gambierone and MTX3. Furthermore, the data indicate that the activation voltage of sodium channels is more sensitive to detect ciguatoxins than their effect on the peak sodium current amplitude The research leading to these results has received funding from the following FEDER cofunded-grants. From Conselleria de Cultura, Educacion e Ordenación Universitaria, Xunta de Galicia, GRC (ED431C 2021/01). From Ministerio de Ciencia e Innovación IISCIII/PI19/001248, PID 2020-11262RB-C21. From European Union Interreg AlertoxNet EAPA-317-2016, Interreg Agritox EAPA-998-2018, and H2020 778069-EMERTOX SI

Ciguatera poisoning (CP) is one of the most abundant seafood poisonings in the world. CP frequently occurred in the tropical and subtropical Indo-Pacific Ocean and the Caribbean Sea. In Japan, CP cases have been reported annually, from the subtropical regions, including Okinawa Prefecture and Amami Islands, Kagoshima Prefecture. The principal toxins, named ciguatoxins (CTXs), are bio-synthesized by benthic dinoflagellate of genera Gambierdiscus and Fukuyoa. They are bio-transferred herbivorous animals to carnivorous fishes via the food chain.The Ogasawara Islands comprise more than 30 islands, Mukojima Islands, Chichijima (Bonin) Islands, Hahajima Islands, Iwo Islands, Nishinoshima, Minamitorishima, and Okinotorishima, which locate in the tropical to subtropical regions. The Mukojima Islands, Chichijima Islands, and Hahajima Islands locate approximately the same latitude as Okinawa. The distance from Tokyo is approximately 1,000 km for Chichijima, 1,700 km for Okinotorishima (the southernmost tip of Japan), and 1,900 km for Minamitorishima (the easternmost tip of Japan). These islands exist in a wide range of waters, latitudes from 20°25' to 27°44' North and longitudes from 136°04' to 153° 59' East. We collected 65 specimens of a grouper, Variola louti, the most frequent species implicated in CP in Japan, from the waters around the Chichijima, Mukojima, and Hahajima islands. The fish flesh specimens were analyzed CTXs using the liquid chromatography-tandem mass spectrometer (LC-MS/MS). While the peak whose retention time is almost identical to that of CTX1B was detected in all specimens on our routine protocol, no 52-epi-54-deoxyCTX1B nor 54-deoxyCTX1B was detected. The peak retention time was quite different from that of CTX1B when re-analyzing by changing the analytical column. Thus, the CTXs in the specimens in the waters of these islands seemed to be undetectable levels.

Modeling ciguatoxin (CTX) trophic transfer in marine food webs has significant implications for the management of ciguatera poisoning, a circumtropical disease caused by human consumption of CTX-contaminated seafood. Current models associated with CP risk rely on modeling abundance/presence of CTX-producing epi-benthic dinoflagellates, e.g., Gambierdiscus spp., and are based on studies showing that toxin production is site specific and occurs in pulses driven by environmental factors. However, food web models are not yet developed and require parameterizing the CTX exposure cascade in fish which has been traditionally approached through top-down assessment of CTX loads in wild-caught fish. The primary goal of this study was to provide critical knowledge on the kinetics of C-CTX-1 bioaccumulation and depuration in the marine omnivore Lagodon rhomboides. We performed a two-phase, 17 week CTX feeding trial in L. rhomboides where fish were given either a formulated C-CTX-1 (n = 40) or control feed (n = 37) for 20 days, and then switched to a non-toxic diet for up to 14 weeks. Fish were randomly sampled through time with whole muscle, liver, and other pooled viscera dissected for toxin analysis by a sodium channel-dependent MTT-based mouse neuroblastoma (N2a) assay. The CTX levels measured in all tissues increased with time during the exposure period (days 1 to 20), but a decrease in CTX-specific toxicity with depuration time only occurred in viscera extracts. By the end of the depuration, muscle, liver, and viscera samples had mean toxin concentrations of 189%, 128%, and 42%, respectively, compared to fish sampled at the start of the depuration phase. However, a one-compartment model analysis of combined tissues showed total concentration declined to 56%, resulting in an approximate half-life of 97 d (R2 = 0.43). Further, applying growth dilution correction models to the overall concentration found that growth was a major factor reducing C-CTX concentrations, and that the body burden was largely unchanged, causing pseudo-elimination and a half-life of 143–148 days (R2 = 0.36). These data have important implications for food web CTX models and management of ciguatera poisoning in endemic regions where the frequency of environmental algal toxin pulses may be greater than the growth-corrected half-life of C-CTX in intermediate-trophic-level fish with high site fidelity.

Ciguatera poisoning (CP) cases linked to the consumption of deep-water fish occurred in 2003 in the Gambier Islands (French Polynesia). In 2004, on the request of two local fishermen, the presence of ciguatoxins (CTXs) was examined in part of their fish catches, i.e., 22 specimens representing five deep-water fish species. Using the radioactive receptor binding assay (rRBA) and mouse bioassay (MBA), significant CTX levels were detected in seven deep-water specimens in Lutjanidae, Serranidae, and Bramidae families. Following additional purification steps on the remaining liposoluble fractions for 13 of these samples (kept at −20 °C), these latter were reanalyzed in 2018 with improved protocols of the neuroblastoma cell-based assay (CBA-N2a) and liquid chromatography tandem mass spectrometry (LC–MS/MS). Using the CBA-N2a, the highest CTX-like content found in a specimen of Eumegistus illustris (Bramidae) was 2.94 ± 0.27 µg CTX1B eq. kg−1. Its toxin profile consisted of 52-epi-54-deoxyCTX1B, CTX1B, and 54-deoxyCTX1B, as assessed by LC–MS/MS. This is the first study demonstrating that deep-water fish are potential ciguatera vectors and highlighting the importance of a systematic monitoring of CTXs in all exploited fish species, especially in ciguatera hotspots, including deep-water fish, which constitute a significant portion of the commercial deep-sea fisheries in many Asian–Pacific countries.

Ciguatera poisoning is caused by the ingestion of fish or shellfish contaminated with ciguatoxins produced by dinoflagellate species belonging to the genera Gambierdiscus and Fukuyoa. Unlike in the Pacific region, the species producing ciguatoxins in the Atlantic Ocean have yet to be definitely identified, though some ciguatoxins responsible for ciguatera have been reported from fish. Previous studies investigating the ciguatoxin-like toxicity of Atlantic Gambierdiscus species using Neuro2a cell-based assay identified G. excentricus as a potential toxin producer. To more rigorously characterize the toxin profile produced by this species, a purified extract from 124 million cells was prepared and partial characterization by high-resolution mass spectrometry was performed. The analysis revealed two new analogs of the polyether gambierone: sulfo-gambierone and dihydro-sulfo-gambierone. Algal ciguatoxins were not identified. The very low ciguatoxin-like toxicity of the two new analogs obtained by the Neuro2a cell-based assay suggests they are not responsible for the relatively high toxicity previously observed when using fractionated G. excentricus extracts, and are unlikely the cause of ciguatera in the region. These compounds, however, can be useful as biomarkers of the presence of G. excentricus due to their sensitive detection by mass spectrometry.

Dinoflagellates of the genera Gambierdiscus and Fukuyoa are able to produce potent neurotoxins like ciguatoxins (CTXs), which, after biooxidation in fish, are responsible for ciguatera intoxication. An isolate of G. australes from the Canary Islands, that revealed the presence of CTX-like compounds by immunosensing tools, was studied by immunocytochemistry to localize intracellular CTX-like compounds, using 8H4 monoclonal antibody that specifically recognizes the right wing of CTX1B and CTX3C analogues. Confocal microscopy observations of immunostained whole cells revealed a strong positive reaction on cell surface and all along the cell outline, while no reaction was detected inside the cells, probably because the antibody was not able to pass through thecal plates. Cell sections showed a positive antibody staining not only on thecal plates, but also inside cytoplasm, with numerous small dots and larger tubule-like reticulate structures. Small fluorescent dots were detected also on the nuclear surface. These observations indicate that CTX-like compounds are present in G. australes cytoplasm, and then are, at least in part, released to cover the cell surface. info:eu-repo/semantics/acceptedVersion

The proliferation of harmful microalgae endangers aquatic ecosystems and can have serious economic implications on a global level. Harmful microalgae and their associated toxins also pose a threat to human health since they can cause seafood-borne diseases such as ciguatera. Implementation of DNA-based molecular methods together with appropriate detection strategies in monitoring programs can support the efforts for effective prevention of potential outbreaks. A PCR-lateral flow assay (PCR-LFA) in dipstick format was developed in this work for the detection of two Gambierdiscus species, G. australes and G. excentricus, which are known to produce highly potent neurotoxins known as ciguatoxins and have been associated with ciguatera outbreaks. Duplex PCR amplification of genomic DNA from strains of these species utilizing species-specific ssDNA tailed primers and a common primer containing the binding sequence of scCro DNA binding protein resulted in the generation of hybrid ssDNA-dsDNA amplicons. These were captured on the dipsticks via hybridization with complementary probes and detected with a scCro/carbon nanoparticle (scCro/CNPs) conjugate. The two different test zones on the dipsticks allowed the discrimination of the two species and the assay exhibited high sensitivity, 6.3 pg/μL of genomic DNA from both G. australes and G. excentricus. The specificity of the approach was also demonstrated using genomic DNA from non-target Gambierdiscus species and other microalgae genera which did not produce any signals. The possibility to use cells directly for amplification instead of purified genomic DNA suggested the compatibility of the approach with field sample testing. Future work is required to further explore the potential use of the strategy for on-site analysis and its applicability to other toxic species.

In the coastal countries of Southeast Asia, fish is a staple diet and certain fish species are food delicacies to local populations or commercially important to individual communities. Although there have been several suspected cases of ciguatera fish poisoning (CFP) in Southeast Asian countries, few have been confirmed by ciguatoxins identification, resulting in limited information for the correct diagnosis of this food-borne disease. In the present study, ciguatoxin-1B (CTX-1B) in red snapper (Lutjanus bohar) implicated in a CFP case in Sabah, Malaysia, in December 2017 was determined by single-quadrupole selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS). Continuous consumption of the toxic fish likely resulted in CFP, even when the toxin concentration in the fish consumed was low. The identification of the fish species was performed using the molecular characterization of the mitochondrial cytochrome c oxidase subunit I gene marker, with a phylogenetic analysis of the genus Lutjanus. This is the first report identifying the causative toxin in fish-implicated CFP in Malaysia.

Ciguatera poisoning is mainly caused by the consumption of reef fish that have accumulated ciguatoxins (CTXs) produced by the benthic dinoflagellates Gambierdiscus and Fukuyoa. China has a long history of problems with ciguatera, but research on ciguatera causative organisms is very limited, especially in the Beibu Gulf, where coral reefs have been degraded significantly and CTXs in reef fish have exceeded food safety guidelines. Here, five strains of Gambierdiscus spp. were collected from Weizhou Island, a ciguatera hotspot in the Beibu Gulf, and identified by light and scanning electron microscopy and phylogenetic analyses based on large and small subunit rDNA sequences. Strains showed typical morphological characteristics of Gambierdiscus caribaeus, exhibiting a smooth thecal surface, rectangular-shaped 2′, almost symmetric 4″, and a large and broad posterior intercalary plate. They clustered in the phylogenetic tree with G. caribaeus from other locations. Therefore, these five strains belonged to G. caribaeus, a globally distributed Gambierdiscus species. Toxicity was determined through the mouse neuroblastoma assay and ranged from 0 to 5.40 fg CTX3C eq cell−1. The low level of toxicity of G. caribaeus in Weizhou Island, with CTX-contaminated fish above the regulatory level in the previous study, suggests that the long-term presence of low toxicity G. caribaeus might lead to the bioaccumulation of CTXs in fish, which can reach dangerous CTX levels. Alternatively, other highly-toxic, non-sampled strains could be present in these waters. This is the first report on toxic Gambierdiscus from the Beibu Gulf and Chinese waters and will provide a basis for further research determining effective strategies for ciguatera management in the area.

Ciguatera food poisoning affects consumer health and fisheries’ economies worldwide in tropical zones, and specifically in the Pacific area. The wide variety of ciguatoxins bio-accumulated in fish or shellfish responsible for this neurological illness are produced by marine dinoflagellates of the genus Gambierdiscus and bio-transformed through the food web. The evaluation of the contents of ciguatoxins in strains of Gambierdiscus relies on the availability of standards and on the development of sensitive and specific tools to detect them. There is a need for sensitive methods for the analysis of pacific ciguatoxins with high resolution mass spectrometry to ensure unequivocal identification of all congeners. We have applied a fractional factorial design of experiment 2^8-3 for the screening of the significance of eight parameters potentially influencing ionization and ion transmission and their interactions to evaluate the behavior of sodium adducts, protonated molecules and first water losses of CTX4A/B, CTX3B/C, 2-OH-CTX3C and 44-methylgambierone on a Q-TOF equipment. The four parameters that allowed to significantly increase the peak areas of ciguatoxins and gambierones (up to a factor ten) were the capillary voltage, the sheath gas temperature, the ion funnel low pressure voltage and the ion funnel exit voltage. The optimized method was applied to revisit the toxin profile of G. polynesiensis (strain TB92) with a confirmation of the presence of M-seco-CTX4A only putatively reported so far and the detection of an isomer of CTX4A. The improvement in toxin detection also allowed to obtain informative high resolution targeted MS/MS spectra revealing high similarity in fragmentation patterns between putative isomer (4) of CTX3C, 2-OH-CTX3C and CTX3B on one side and between CTX4A, M-seco-CTX4A and the putative isomer on the other side, suggesting a relation of constitutional isomerism between them for both isomers.

Currently, digital technologies influence information dissemination in all business sectors, with great emphasis put on exploitation strategies. Public administrations often use information systems and establish open data repositories, primarily supporting their operation but also serving as data providers, facilitating decision-making. As such, risk analysis in the public health sector, including food safety authorities, often relies on digital technologies and open data sources. Global food safety challenges include marine biotoxins (MBs), being contaminants whose mitigation largely depends on risk analysis. Ciguatera Fish Poisoning (CFP), in particular, is a MB-related seafood intoxication attributed to the consumption of fish species that are prone to accumulate ciguatoxins. Historically, CFP occurred endemically in tropical/subtropical areas, but has gradually emerged in temperate regions, including European waters, necessitating official policy adoption to manage the potential risks. Researchers and policy-makers highlight scientific data inadequacy, under-reporting of outbreaks and information source fragmentation as major obstacles in developing CFP mitigation strategies. Although digital technologies and open data sources provide exploitable scientific information for MB risk analysis, their utilization in counteracting CFP-related hazards has not been addressed to date. This work thus attempts to answer the question, “What is the current extent of digital technologies’ and open data sources’ utilization within risk analysis tasks in the MBs field, particularly on CFP?”, by conducting a systematic literature review of the available scientific and grey literature. Results indicate that the use of digital technologies and open data sources in CFP is not negligible. However, certain gaps are identified regarding discrepancies in terminology, source fragmentation and a redundancy and downplay of social media utilization, in turn constituting a future research agenda for this under-researched topic.

Ciguatera poisoning is a globally occurring seafood disease caused by the ingestion of marine products contaminated with dinoflagellate produced neurotoxins. Persistent forms of ciguatera, which prove to be highly debilitating, are poorly studied and represent a significant medical issue. The present study aims to better understand chronic ciguatera manifestations and identify potential predictive factors for their duration. Medical files of 49 patients were analyzed, and the post-hospitalization evolution of the disease assessed through a follow-up questionnaire. A rigorous logistic lasso regression model was applied to select significant predictors from a list of 37 patient characteristics potentially predictive of having chronic symptoms. Missing data were handled by complete case analysis, and a survival analysis was implemented. All models used standardized variables, and multiple comparisons in the survival analyses were handled by Bonferroni correction. Among all studied variables, five significant predictors of having symptoms lasting ≥3 months were identified: age, tobacco consumption, acute bradycardia, laboratory measures of urea, and neutrophils. This exploratory, hypothesis-generating study contributes to the development of ciguatera epidemiology by narrowing the list from 37 possible predictors to a list of five predictors that seem worth further investigation as candidate risk factors in more targeted studies of ciguatera symptom duration.

Dinoflagellate species of the genera Gambierdiscus and Fukuyoa are known to produce ciguatera poisoning-associated toxic compounds, such as ciguatoxins, or other toxins, such as maitotoxins. However, many species and strains remain poorly characterized in areas where they were recently identified, such as the western Mediterranean Sea. In previous studies carried out by our research group, a G. australes strain from the Balearic Islands (Mediterranean Sea) presenting MTX-like activity was characterized by LC-MS/MS and LC-HRMS detecting 44-methyl gambierone and gambieric acids C and D. However, MTX1, which is typically found in some G. australes strains from the Pacific Ocean, was not detected. Therefore, this study focuses on the identification of the compound responsible for the MTX-like toxicity in this strain. The G. australes strain was characterized not only using LC-MS instruments but also N2a-guided HPLC fractionation. Following this approach, several toxic compounds were identified in three fractions by LC-MS/MS and HRMS. A novel MTX analogue, named MTX5, was identified in the most toxic fraction, and 44-methyl gambierone and gambieric acids C and D contributed to the toxicity observed in other fractions of this strain. Thus, G. australes from the Mediterranean Sea produces MTX5 instead of MTX1 in contrast to some strains of the same species from the Pacific Ocean. No CTX precursors were detected, reinforcing the complexity of the identification of CTXs precursors in these regions. European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03 Xunta de Galicia | Ref. ED481A-2018/207 Generalitat de Catalunya | Ref. CERCA

Ciguatera poisoning is a food intoxication associated with the consumption of fish or shellfish contaminated, through trophic transfer, with ciguatoxins (CTXs). In this study, we developed an experimental model to assess the trophic transfer of CTXs from herbivorous parrotfish, Chlorurus microrhinos, to carnivorous lionfish, Pterois volitans. During a 6-week period, juvenile lionfish were fed naturally contaminated parrotfish fillets at a daily dose of 0.11 or 0.035 ng CTX3C equiv. g−1, as measured by the radioligand-receptor binding assay (r-RBA) or neuroblastoma cell-based assay (CBA-N2a), respectively. During an additional 6-week depuration period, the remaining fish were fed a CTX-free diet. Using r-RBA, no CTXs were detectable in muscular tissues, whereas CTXs were measured in the livers of two out of nine fish sampled during exposure, and in four out of eight fish sampled during depuration. Timepoint pooled liver samples, as analyzed by CBA-N2a, confirmed the accumulation of CTXs in liver tissues, reaching 0.89 ng CTX3C equiv. g−1 after 41 days of exposure, followed by slow toxin elimination, with 0.37 ng CTX3C equiv. g−1 measured after the 6-week depuration. These preliminary results, which need to be pursued in adult lionfish, strengthen our knowledge on CTX transfer and kinetics along the food web.

Ciguatoxins (CTXs) are polyether marine biotoxins that can cause ciguatera poisoning (CP) after the consumption of fish or invertebrates containing sub ppb levels, concentrations that present a challenge for current extraction and analysis methods. Here, a newly developed and (partly) validated single-day extraction protocol is presented. First, the fish sample is broken-down by enzymatic digestion, followed by extraction and extract clean-up by defatting and two solid-phase extractions. Final extracts were investigated using two different CTX-analysis methods, an in vitro cytotoxicity assay (N2a-assay) and by LC-MS/MS. Validation was performed for both fillet and freeze-dried samples of snapper, parrotfish, and grouper spiked with CTX1B, 52-epi-54-deoxyCTX1B, 54-deoxyCTX1B, and CTX3C. Based on recovery rates (35–88%) and matrix effects (66–116%) determined by LC-MS/MS, the enzyme protocol is applicable to various matrices. The protocol was applied to naturally contaminated fish tissue (Lutjanus bohar) obtained during a CP incident in Germany. Several potential CTX congeners were identified by a two-tier LC-MS/MS approach (screening of sodium adducts, high-resolution or low-resolution confirmation via ammonium adducts). Inclusion of &gt, 30 known CTX congeners into the LC-MS/MS methods and single-day sample preparation make the method suitable for analysis of ciguatera suspect samples at sub ppb levels also with undisclosed CTX profiles.

Invasive species can precede far-reaching environmental and economic consequences. In the Hawai'ian Archipelago Cephalopholis argus (family Serranidae) is an established invasive species, now recognized as the dominant local reef predator, negatively impacting the native ecosystem and local fishery. In this region, no official C. argus fishery exists, due to its association with Ciguatera seafood poisoning (CP); a severe intoxication in humans occurring after eating (primarily) fish contaminated with ciguatoxins (CTXs). Pre-harvest prediction of CP is currently not possible; partly due to the ubiquitous nature of the microalgae producing CTXs and the diverse bioaccumulation pathways of the toxins. This study investigated the perceived risk of CP in two geographically discrete regions (Leeward and Windward) around the main island of Hawai'i, guided by local fishers. C. argus was collected and investigated for CTXs using the U.S. Food and Drug Administration (FDA) CTX testing protocol (in vitro neuroblastoma N2a-assay and LC-MS/MS). Overall, 76% of fish (87/113) exceeded the FDA guidance value for CTX1B (0.01 ng g-1 tissue equivalents); determined by the N2a-assay. Maximum CTX levels were ≅2× higher at the Leeward vs Windward location and, respectively, 95% (64/67) and 54% (25/46) of fish were positive for CTX-like activity. Fisher persons and environmental understandings, regarding the existence of a geographic predictor (Leeward vs Windward) for harvest, were found to be (mostly) accurate as CTXs were detected in both locations and the local designation of C. argus as a risk for CP was confirmed. This study provides additional evidence that supports the previous conclusions that this species is a severe CP risk in the coastal food web of Hawai'i, and that ocean exposure (wave power) may be a prominent factor influencing the CTX content in fish within a hyperendemic region for CP.

The Selvagens Islands, which are a marine protected area located at the southernmost point of the Portuguese maritime zone, have been associated with fish harboring ciguatoxins (CTX) and linked to ciguatera fish poisonings. This study reports the results of a field sampling campaign carried out in September 2018 in these remote and rarely surveyed islands. Fifty-six fish specimens from different trophic levels were caught for CTX-like toxicity determination by cell-based assay (CBA) and toxin content analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Notably, high toxicity levels were found in fish with an intermediate position in the food web, such as zebra seabream (Diplodus cervinus) and barred hogfish (Bodianus scrofa), reaching levels up to 0.75 µg CTX1B equivalent kg−1. The LC-MS/MS analysis confirmed that C-CTX1 was the main toxin, but discrepancies between CBA and LC-MS/MS in D. cervinus and top predator species, such as the yellowmouth barracuda (Sphyraena viridis) and amberjacks (Seriola spp.), suggest the presence of fish metabolic products, which need to be further elucidated. This study confirms that fish from coastal food webs of the Selvagens Islands represent a high risk of ciguatera, raising important issues for fisheries and environmental management of the Selvagens Islands. European Food Safety Authority | Ref. GP/EFSA/AFSCO/2015/03 Fundação para a Ciência e a Tecnologia | Ref. PTDC/CTA-AMB/30557/2017 Fundação para a Ciência e a Tecnologia | Ref. UID/Multi/04326/2020 Xunta de Galicia | Ref. ED481A-2018/207 Generalitat de Catalunya | Ref. CERCA

Ciguatera poisoning is mainly caused by the consumption of reef fish that have accumulated ciguatoxins (CTXs) produced by the benthic dinoflagellates Gambierdiscus and Fukuyoa. China has a long history of problems with ciguatera, but research on ciguatera causative organisms is very limited, especially in the Beibu Gulf, where coral reefs have been degraded significantly and CTXs in reef fish have exceeded food safety guidelines. Here, five strains of Gambierdiscus spp. were collected from Weizhou Island, a ciguatera hotspot in the Beibu Gulf, and identified by light and scanning electron microscopy and phylogenetic analyses based on large and small subunit rDNA sequences. Strains showed typical morphological characteristics of Gambierdiscus caribaeus, exhibiting a smooth thecal surface, rectangular-shaped 2′, almost symmetric 4″, and a large and broad posterior intercalary plate. They clustered in the phylogenetic tree with G. caribaeus from other locations. Therefore, these five strains belonged to G. caribaeus, a globally distributed Gambierdiscus species. Toxicity was determined through the mouse neuroblastoma assay and ranged from 0 to 5.40 fg CTX3C eq cell−1. The low level of toxicity of G. caribaeus in Weizhou Island, with CTX-contaminated fish above the regulatory level in the previous study, suggests that the long-term presence of low toxicity G. caribaeus might lead to the bioaccumulation of CTXs in fish, which can reach dangerous CTX levels. Alternatively, other highly-toxic, non-sampled strains could be present in these waters. This is the first report on toxic Gambierdiscus from the Beibu Gulf and Chinese waters and will provide a basis for further research determining effective strategies for ciguatera management in the area.

The genus Gambierdiscus is a marine benthic/epiphytic dinoflagellate considered the causative agent of ciguatera poisoning (CP). Clarifying the geographical distribution of this genus to understand the potential risk of CP is important. Many studies have focused only on the species/phylotype composition of Gambierdiscus in shallow waters, but no study has investigated the species/phylotype composition of the genus in deep waters. In the present study, the distributions of Gambierdiscus species/phylotypes at two depths (2-8 and 30 m) and two sampling sites (temperate and subtropical) in Japan was investigated using high throughput sequencing (HTS) with a newly developed primer set that preferentially amplifies the 18S rDNA V8-V9 region of Alveolata. A phylogenetic analysis using 89 samples collected over three years revealed of ten Gambierdiscus species/phylotypes including not only two species that have not been reported in Japan (G. caribaeus and G. silvae) but also four novel phylotypes (Gambierdiscus spp. Clade II_1, Clade II_2, Clade II_3, and Clade VI_1). Uncorrected genetic distances also supported that these new phylotypes clearly diverged from other Gambierdiscus species. All four new phylotypes, G. caribaeus, and G. silvae were distributed in the subtropical region. Among them, Clade II_2, Clade VI_1, and G. silvae were also distributed in the temperate region. Four species/phylotypes previously reported from Japan showed a similar distribution as reported previously. Among the ten species/phylotypes, Gambierdiscus sp. type 3 and Clade VI_1 were found only in deep waters, whereas five species/phylotypes were observed only in shallow waters. The other three species/phylotypes were found in both deep and shallow waters. The results of the horizontal and vertical distribution suggest that the growth characteristics of each species/phylotypes found in Japan might adapt to the ambient environmental conditions. This study revealed an inclusive assemblage of Gambierdiscus species/phylotypes in Japan through metabarcoding using the Alveolata primer set. In the future, the abundance and toxicities/toxin productions of the newly reported species/phylotypes need to be clarified to understand the mechanism of CP outbreaks in Japan.