Faster ciguatoxin extraction methods for toxicity screening.

Ciguatera poisoning (CP) is a severe global public health problem caused by the consumption of seafood products contaminated with ciguatoxins (CTXs). The growing demand for seafood products requires high-throughput testing for CTX-susceptible seafood, however complex extraction and slow cleanup methods inhibit this goal. Herein, several methods for extracting CTXs from fish tissue were established and compared; these methods are sensitive, specific, and valid while achieving higher sample extraction throughput than currently established protocols. The trial fish material was generated from multiple species, with different physical conditions (wet and freeze-dried tissue), and naturally contaminated with various CTXs (i.e., CTX-1B, CTX-3C, and C-CTX-1), thus ensuring these methods are robust and broadly applicable. The extraction methods used were based on mechanical maceration with acetone or methanol or enzymatic digestion followed by acetone and ethyl acetate extraction. Crude extracts were investigated for CTX-like toxicity using an in vitro mouse neuroblastoma (N2a) cell-based assay (CBA). Among the three methods, there was no significant difference in toxin estimates (p = 0.219, two-way ANOVA), indicating their interchangeability. For speed (> 16 samples/day), accuracy (100%), and CTX analog retention confirmation by liquid chromatography-tandem mass spectrometry (LC‒MS/MS), the preferred extraction methods were both methanol and enzyme-based. All extraction methods post hoc confirmation of CTX analogs successfully met international seafood market-based CTX contaminant guidance. These methods can drastically increase global CTX screening capabilities and subsequently relieve sample processing bottlenecks, inhibiting environmental and human health-based CTX analysis.

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

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

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

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

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.

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.

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.

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.