Emerging Risks in the European Seafood Chain: Molecular Identification of Toxic Lagocephalus spp. and Evaluation of the Public DNA Libraries Reliability in Supporting Species Identification

Pufferfish may cause human intoxications due to Tetrodotoxin accumulation and their sale is banned in EU. In the last decades, some pufferfish (e. g. Lagocpehalus spp.) spread in the Mediterranean Sea through Lessepsian migration, representing an emerging risk within the seafood chain. Their illegal presence in mislabelled products is also reported. The identification of Lagocephalus spp. is hindered by morphological similarities among co-generic species and DNA amplification, sequencing and comparison to public DNA libraries is often required. This process can be affected by the presence of wrongly deposited sequences in the libraries. This study first aimed at finding a suitable molecular target for identifying all Lagocephalus spp. in commercial products. Moreover, the reliability of two the public libraries in supporting the discrimination of L. spadiceus and L. guentheri was assessed. All cytochrome oxidase subunit I (COI) and cytochrome b (cytb) genes sequences of Lagocephalus spp. were retrieved from official libraries and used to create phylogenetic trees depicting inter-species relationships. Given its higher inter-species variability, cytb was selected as target and 17 cytb sequences from 6 Lagocephalus spp. reference samples were produced. Then, a primer pair amplifying a 130bp cytb polymorphic fragment from all the Lagocephalus spp. was designed for identifying 16 mislabelled commercial products with degraded DNA. Also, 20 Mediterranean specimens of L. guentheri were collected; both COI and cytb sequences were ex-novo produced and phylogenetically analysed with the sequences from L. spadiceus and L. guentheri previously retrieved from official libraries. First, the presence of L. spadiceus in the commercial products was confirmed. Moreover, a scarce DNA libraries accuracy was observed since most sequences deposited as L. spadiceus of Mediterranean origin were instead misidentified L. guentheri, causing a significant underestimation of this species in the Mediterranean Sea. Overall, this study may provide a reliable tool to protect European consumers from the emerging risk associated to toxic pufferfish

Specie tossiche invasive (famiglia Tetraodontidae) lungo le coste italiane: un rischio emergente per la salute pubblica

Numerose specie appartenenti alla famiglia Tetraodontidae(“pescepalla”) sono attualmente presenti nel Mar Mediterraneo. Tra questeLagocephalus sceleratus, originario del Mar Rosso ed arrivato nelMar Mediterraneo attraverso il canale di Suez nel 2003, rappresenta una delle specie più tossiche ed altamente diffusive, ed è pertanto annoverato tra le specie aliene invasive. Dal 2013, anno delprimo ritrovamento ufficiale di L. sceleratus in acque italiane, è stato segnalato un numero crescente di esemplari. Oltre a questa specie, anche Lagocephalus lagocephaluse Sphoeroides pachygaster, a minor tossicità ed invasività, sono segnalate da tempo lungo le coste italiane. Questo studio rappresenta uno dei primi tentativi di descrivere sistematicamente la presenza nel Mediterraneo, e in particolare lungo le coste italiane, delle tre suddette specie, al fine di caratterizzare un rischio emergente per la salute pubblica

Accidental discovery of a Tetraodontidae (<i>Sphoeroides marmoratus</i>) within a cuttlefish (<i>Sepia officinalis</i>) bought in a fish shop in Italy: risk assessment associated with the presence of Tetrodotoxin

The discovery of a pufferfish specimen (Tetraodontidae) inside a frozen cuttlefish, purchased by a fishmonger, and caught in the central-east Atlantic (FAO 34) is reported. The consumer, who reported this case to FishLab (Department of Veterinary Sciences, University of Pisa) for investigation, was a student of Veterinary Medicine at the University of Pisa. He recognized the Tetraodontidae because he attended practical lessons on fish morphological identification during the course of food inspection and was aware of the risks to human health linked to the Tetrodotoxin (TTX). In this study, the pufferfish was identified morphologically, using the FAO morphological keys, and molecularly, analyzing two markers, the cytochrome oxidase I (COI) and the cytochrome b genes, by DNA barcoding. The pufferfish was identified morphologically as Sphoeroides spp., and molecularly as Sphoeroides marmoratus using the COI gene (99-100% identity values). Literature reports that S. marmoratus from the eastern Atlantic contains high concentrations of TTX in the gonads and the digestive tract. However, the possible passage of TTX from fish to other organisms linked to contact or ingestion has never been reported. This represents the first case of a potentially toxic pufferfish entering the market inside another organism. The fact that a student observed this occurrence highlights the key role of citizen science in the management of emerging risks

Safety and commercial issues in fresh mushrooms and mushroom-based products sold at retail in Tuscany region.

Nel presente studio sono stati analizzati gli aspetti igienico-sanitari e commerciali relativi alla vendita al consumo presso la grande distribuzione organizzata (GDO) di funghi freschi, spontanei e coltivati, eprodotti a base di funghi, attraverso: 1) l’analisi dei risultati dei con-trolli ufficiali; 2) la valutazione dell’etichettatura. Per quanto riguardail punto 1, i dati si riferiscono ai risultati delle attività di controllo ufficiale svolte dall’azienda ASL Toscana Nord Ovest -Dipartimento della prevenzione gruppo Micologico, nel periodo 2016-2020. Durante icontrolli sono stati verificati gli item aziendali e le disposizioni del LR.n. 16/99 (articoli 20 e 21) in merito all’autorizzazione al commercioe alla somministrazione dei funghi epigei spontanei, nonché lo stato di conservazione e la detenzione da parte dell’OSA di attestati per lavendita di funghi sfusi. Per l’analisi di cui al punto 2 è stata valutatala conformità delle etichette di 98 prodotti acquistati presso la GDOalla normativa europea in materia di etichettatura dei prodotti ali-mentari e alla normativa nazionale concernente la commercializzazione dei funghi epigei freschi e conservati. In particolare, attraverso lavalutazione della presenza delle informazioni di cui all’articolo 37 del Reg. UE n. 1169/2011 integrate con le informazioni specifiche per questa tipologia di prodotti di cui all’articolo 10 del DPR 14 luglio1995, n. 376. I risultati dei sopralluoghi delle attività di controllo uffi-ciale hanno evidenziato non conformità relative alle modalità di conservazione di prodotti di IV gamma (per lo più funghi coltivati, sottoposti a trattamenti di minima entità e confezionati). In particolare, le non conformità erano riferibili ad una non corretta conservazione che, ai sensi Decreto del Ministero dello sviluppo Economico n. 3746 del 20/06/2014 (articolo 6), dovrebbe avvenire ad una temperatura uniforme inferiore a 8°C, dal confezionamento all’acquisto. Durante i sopralluoghi sono state inoltre riportate osservazioni in merito alla conservazione di funghi spontanei a temperatura ambiente con decadimento organolettico del prodotto stesso. L’analisi delle etichette deiprodotti acquistati ha evidenziato una sostanziale conformità ai requisiti imposti dalle normative di riferimento. Ciò nonostante, sono state osservate criticità relative all’indicazione delle condizioni d’uso e relativi tempi di cottura suggeriti dagli OSA che sembrerebbero non garantire l’inattivazione di tossine termolabili presenti anche in funghi dichiarati edibili. Infatti, se per alcune specie come Agaricus bisporus si consiglia un trattamento a 70°C per almeno 10 minuti, per altre specie potrebbe essere necessario raggiungere i 100°C. Questo infunzione della differenti tossine presenti nei funghi. Alla luce delle criticità emerse si rende necessario redigere una proposta di integrazione e revisione della normativa vigente in materia di gestione dellacatena del freddo e informazioni al consumatore per la conservazione e utilizzo dei funghi e prodotti a base di funghi. Questa revisione è necessaria anche in virtù dei dati di intossicazione da funghi a livello nazionale. Infatti, oltre ai casi dovuti al consumo di specie fungine tossiche, sono documentati episodi occasionali descritti come falsi micetismi, dovuti all’ingestione di funghi edibili conservati e/o utilizzati non correttamente

Molecular authentication of mushroom products: First survey on the Italian market

For the first time, mushroom products sold on the Italian market were authenticated using DNA barcoding. The analysis was performed on 71 market products (MPs) of different types (canned, dried, frozen, ready-to-cook-dishes) collected in supermarket chains. The ITS complete region was amplified and sequenced. The obtained sequences were then submitted to a BLAST analysis against both an internal dataset developed in a previous study and GenBank for species identification. Issues during amplification and sequencing were especially highlighted for canned products, plausibly due to the processing. The internal dataset was able to support the identification at species level. However, the need to improve it with sequences of commercially relevant species it is essential to increase its identification capability. Cases of suspected mislabeling were observed, with Agaricus bisporus and Bjerkandera adusta respectively found in two products labeled as Boletus edulis and its group. The presence of B. adusta, a not edible plant pathogen, also highlighted a scarce attention in the application of good hygiene and manufacturing practices. Since only 1 or 2 sequence/s were produced from each MP, we cannot exclude that mislabeling rate (2.8%) may be under-estimated. Specific guidelines on sampling strategy should be therefore fixed for mislabeling assessment purposes. Also, the use of alternative DNA based methods, such as metabarcoding, able to identify all the species present in a sample, should be considered

Toxic invasive pufferfish (Tetraodontidae family) along Italian coasts: Assessment of an emerging public health risk

Several pufferfish species belonging to the Tetraodontidae family are currently present in the Mediterranean Sea. Since 2013 Lagocephalus sceleratus is increasingly reported also along Italian coasts, where other two species of less concern, Lagocephalus lagocephalus and Sphoeroides pachygaster, have also occured since a long time. This study represents one of the first attempts to sistematically describe the occurrence in the Mediterranean, and in particular along Italian coasts, of the three aforesaid species, in order to characterize an emerging public health hazard. Reports were searched in scientific articles and dedicated online databases. The following data were collected: number of specimens and type of record, geographical location and date of report, fish size, depth and type of seabed. Overall, at least 111079 individuals of the three species were found in the Mediterranean Sea, including 110237 specimens of L. sceleratus (since 2003), 126 of L. lagocephalus (1878–2017) and 716 of S. pachygaster (1979–2017). The evident differences confirm the invasive character of the toxic Lessepsian immigrant L. sceleratus, the species of main public health concern. Despite this species was recorded along Italian coasts in low numbers (0.08% of the total individuals of L. sceleratus) and its current distribution is limited to southern regions, the picture could change rapidly. Moreover, the collected data show that most L. sceleratus specimens have a large size and are mainly caught by commercial fishing gears. These factors may increase the risk of this species entering the seafood chain with serious consequences for consumers’ health. Results suggest that the presence of L. sceleratus should be strictly monitored. Institutional measures should be implemented to inform people as regards a new hazard that has the potential to affect the Italian seafood chain

Emerging risks in the European seafood chain: Molecular identification of toxic Lagocephalus spp. in fresh and processed products

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 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. On the basis of its higher variability, cytb gene was selected as the 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 of the Lagocephalus spp. was designed for identifying sixteen mislabelled commercial products containing pufferfish with degraded DNA. Cytb dataset's phylogenetic analysis supported the most recent species classification of the Lagocephalus genus and highlighted the presence of toxic L. spadiceus in the products. The analysis of the proposed short fragment could represent a reliable tool to protect European consumers from the emerging risk associated to toxic Lagocephalus spp

Molecular Identification of Mushroom Species in Italy: An Ongoing Project Aimed at Reinforcing the Control Measures of an Increasingly Appreciated Sustainable Food

Proper investment in mushroom production (farming and wild mushroom picking activities) may represent a winning strategy for many countries, including Italy, to better face the problems of food security and environmental impact, and to break away from imports, enhancing the local products. However, the risk related to the consumption of poisoning species requires governments to implement or reinforce effective control measures to protect consumers. Mushroom identification by phenotype observation is hardly applicable if morphologically-similar species, non-whole specimens, or clinical samples are involved. Genotypic analysis is a valid alternative. An ongoing research project involving the Experimental Zooprophylactic Institute of Lazio and Tuscany, the regional Mycological Inspectorate, the Tuscany Mycological Groups Association, and the Department of Veterinary Sciences of the University of Pisa aims to reinforce the collaboration among institutions for the management of mushroom poisoning. The core&rsquo;s project aims to develop an internal genetic database to support the identification of wild and cultivated mushroom species in the Italian territory. The database will include Internal Transcribed Spacer (ITS) sequences retrieved from official databases (the NCBI GenBank and the BOLD system) which are considered to be reliable, after a proper selection process, and sequences from specimens collected directly and identified by expert mycologists. Once it is validated, the database will be available and further implementable by the official network of national laboratories