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The EuroCigua project main objective is to characterize the risk of Ciguatera Poisoning (CP) in Europe including several specific objectives: to determine the incidence of ciguatera in Europe and the epidemiological characteristics of cases; to assess the presence of ciguatoxin in food and the environment in Europe and to develop and validate methods for the detection, quantification and confirmation of the presence of ciguatoxin contaminated specimens. This report compiles the activities carried out during the EuroCigua project from the signing in April 2016 until December 2020. The present document corresponds to Deliverable No. 6: “Final Scientific Report” on Risk characterization of ciguatera food poisoning in Europe of the Specific Agreement no. 1 “MANAGEMENT AND SCIENTIFIC COORDINATION” within the Framework Partnership Agreement GP/EFSA/AFSCO/2015/03 “Risk characterization of ciguatera food poisoning in Europe”.

Over the last decade, knowledge has significantly increased on the taxonomic identity and distribution of dinoflagellates of the genera Gambierdiscus and Fukuyoa. Additionally, a number of hitherto unknown bioactive metabolites have been described, while the role of these compounds in ciguatera poisoning (CP) remains to be clarified. Ciguatoxins and maitotoxins are very toxic compounds produced by these dinoflagellates and have been described since the 1980s. Ciguatoxins are generally described as the main contributors to this food intoxication. Recent reports of CP in temperate waters of the Canary Islands (Spain) and the Madeira archipelago (Portugal) triggered the need for isolation and cultivation of dinoflagellates from these areas, and their taxonomic and toxicological characterization. Maitotoxins, and specifically maitotoxin-4, has been described as one of the most toxic compounds produced by these dinoflagellates (e.g., G. excentricus) in the Canary Islands. Thus, characterization of toxin profiles of Gambierdiscus species from adjacent regions appears critical. The combination of liquid chromatography coupled to either low- or high-resolution mass spectrometry allowed for characterization of several strains of Gambierdiscus and Fukuyoa from the Mediterranean Sea and the Canary Islands. Maitotoxin-3, two analogues tentatively identified as gambieric acid C and D, a putative gambierone analogue and a putative gambieroxide were detected in all G. australes strains from Menorca and Mallorca (Balearic Islands, Spain) while only maitotoxin-3 was present in an F. paulensis strain of the same region. An unidentified Gambierdiscus species (Gambierdiscus sp.2) from Crete (Greece) showed a different toxin profile, detecting both maitotoxin-3 and gambierone, while the availability of a G. excentricus strain from the Canary Islands (Spain) confirmed the presence of maitotoxin-4 in this species. Overall, this study shows that toxin profiles not only appear to be species

Two small Lagocephalus sceleratus juveniles were captured in picarel targeting catches from North Aegean Sea (Greece) in the autumn of 2017. An electrochemical immunosensing tool using magnetic beads as immobilisation support was developed and applied to the rapid screening of tetrodotoxins (TTXs), potent neurotoxins that constitute a food safety hazard when present in seafood. This tool revealed the presence of TTXs in both individuals. Results were compared with those provided by mELISA and LC-HRMS, the latter confirming the presence of TTX. Some of the tissues contained TTX contents close to or above 2 mg/kg. L. sceleratus juveniles had been considered as non-toxic and, to our knowledge, this is the first report of high TTX levels in small L. sceleratus individuals. Such specimens can be mistaken with other edible species, posing a threat to consumers. The availability of low-cost and user-friendly tools for TTXs detection will contribute to guarantee seafood safety. © 2019 Elsevier Ltd

The marine dinoflagellate genus Ostreopsis includes species producing potent toxic compounds, such as paliytoxin and palytoxin analogs, which might cause problematic blooms in the Mediterranean Sea and other tropical or temperate areas. Phylogenetic and phylogeographical studies based on sequences of ribosomal genes, revealed the existence of distinct species and, within them, clades in relation to geographical distribution potentially representing new or cryptic species. The morphological variability of Ostreopsis complicates the identification of species; thus, molecular analyses of isolates or field samples can be helpful. The aim of this study was to improve the characterization of Ostreopsis species and investigate the geographical distribution by using large dataset composed of both new and old sampled isolates. To determine if mitochondrial genes can be used to identify Ostreopsis species, we designed new primers sets then amplified and sequenced representative regions of the COI (cytochrome c oxydase 1) and cob (cytochrome b) genes. Phylogenetic analyses of the resulting mitochondrial DNA (mtDNA) and existing or new ribosomal DNA (rDNA) sequence data showed little divergence in the mtDNA sequences among Ostreopsis species indicating that neither the COI or cob genes are phylogenetically informative. In contrast, the ribosomal gene phylogeny indicated the existence of distinct Ostreopsis species. A network of haplotypes (based on ITS-5.8S rDNA) inferred from O. cf. ovata isolates collected worldwide revealed that Atlantic/Mediterranean and Indo/Pacific areas might host two separated large populations. In conclusion, it appears that rDNA gene sequences provide an effective molecular means of distinguishing the phylogenetic and phylogeographical relationships among Ostreopsis species.

This study reports some recent phylogeographical considerations on the genus Ostreopsis distribution worldwide, with particular attention to the Mediterranean Sea, and new recent advances on the quali-quantitiative detection of Ostreopsis species along coastal areas of the Mediterranean Sea based on the PCR and quantitative real time PCR (qrt-PCR) assays. It was found that O. cf. ovata is widely dispersed throughout tropical and warm temperate coastal areas. In the Atlantic/Mediterranean region it represents a panmictic population that is highly divergent from Indo-Pacific populations. Furthermore, we demonstrated that the developed qrt-PCR assay is specific, robust and high sample throughput for the quantification of the toxic O. cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional methods of microscopy and applied for the survey of benthic toxic microalgal species in marine ecosystems

Of the known benthic, toxigenic microalgae, species from the genera, Gambierdiscus and Ostreopsis, present the biggest threats to human and environmental health. Gambierdiscus spp. produce gambiertoxins which are precursors of ciguatoxins, the compounds that cause ciguatera, the most prevalent, phycotoxin-related form of seafood poisoning across the globe, affecting 25,000 to 500,000 people annually. Recent Ostreopsis spp. blooms in the Mediterranean Sea and coastal waters of New Zealand have led to concerns for seafood safety due to the palytoxin analogs produced by these dinoflagellates. Recent advances in molecular genetics have demonstrated that the taxonomy of both genera were in dire need of revision. Subsequently, Gambierdiscus was revised, and efforts to do the same are ongoing for Ostreopsis. The purpose of this review, therefore, is to update the state of knowledge of these genera, with particular emphasis on the validation or dismissal of earlier findings in light of these revisions. The review is divided into four sections (taxonomy, geography, ecophysiology, and toxicology) in an effort to cover all major aspects of study of these genera. A concluding section closes the review with a synthesis of the state of knowledge on these dinoflagellates and identifies gaps as suggested areas for future research efforts.