A global retrospective study on Hhuman cases of tetrodotoxin (TTX) 3 poisoning intoxication due to the ingestion ofafter seafood products 4 containing tetrodotoxin: a global retrospective study consumption 5 6

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Pufferfish may be responsible for human intoxications due to the accumulation of a potentially lethal neurotoxin, called tetrodotoxin (TTX). While traditionally some species of Pufferfish are consumed in Japan, their marketing is banned in the EU. However, their illegal presence in mislabelled products has been reported. Moreover, some species of the genus Lagocephalus spread in the Mediterranean Sea during the last decades due to the Lessepsian migration phenomenon and they may represent a significant emerging risk within the European seafood chain. This study aimed at finding a suitable molecular marker for quickly identifying Lagocephalus species in fresh and processed products. All the available sequences of COI and cytb mitochondrial genes were used to create different length datasets (long and short fragments) to be used to produce NJ trees depicting genetic relationships for Lagocephalus spp. The cytb was selected as molecular target and 17 new complete sequences of 6 Lagocephalus species, deriving from reference samples, were produced and included in the datasets. Then, a primer pair for amplifying a ⁓130bp cytb polymorphic fragment from all the Lagocephalus spp. was projected for identifying sixteen mislabelled commercial products with degraded DNA containing pufferfish. Cytb dataset's phylogenetic analysis supported the most recent species classification of the Lagocephalus genus and highlighted the presence of the toxic L. spadiceus in the products. The analysis of the proposed short fragment could represent a reliable tool to protect European consumers from emerging risk associated to toxic Lagocephalus spp.

This study aims at building an ITS gene dataset to support the Italian Health Service in mushroom identification. The target species were selected among those mostly involved in regional (Tuscany) poisoning cases. For each target species, all the ITS sequences already deposited in GenBank and BOLD databases were retrieved and accurately assessed for quality and reliability by a systematic filtering process. Wild specimens of target species were also collected to produce reference ITS sequences. These were used partly to set up and partly to validate the dataset by BLAST analysis. Overall, 7270 sequences were found in the two databases. After filtering, 1293 sequences (17.8%) were discarded, with a final retrieval of 5977 sequences. Ninety-seven ITS reference sequences were obtained from 76 collected mushroom specimens: 15 of them, obtained from 10 species with no sequences available after the filtering, were used to build the dataset, with a final taxonomic coverage of 96.7%. The other 82 sequences (66 species) were used for the dataset validation. In most of the cases (n = 71; 86.6%) they matched with identity values ≥ 97–100% with the corresponding species. The dataset was able to identify the species involved in regional poisoning incidents. As some of these species are also involved in poisonings at the national level, the dataset may be used for supporting the National Health Service throughout the Italian territory. Moreover, it can support the official control activities aimed at detecting frauds in commercial mushroom-based products and safeguarding consumers.