Novel foods, old issues: Metabarcoding revealed mislabeling in insect-based products sold by e-commerce on the EU market.

Insects intended for human consumption are considered Novel Foods according to EU legislation. marketed in form of powders, bars, snacks are increasingly available on the EU market, especially on e-commerce. The commercial form and the way of distribution make IBPs particularly prone to mislabeling. Literature concerning the mislabeling occurrence in IBPs is extremely scarce. In this study, 46 processed IBPs were collected on nine EU e-commerce platforms (e-CO) to be authenticated by metabarcoding. A 200 bp region from 16S rRNA gene was used as molecular target. Sequencing data were processed using DADA2 R package, and sequences were taxonomically assigned through BLAST analysis against GenBank. Procedural blanks and positive controls were included in the analysis, and threshold values were established to filter the final data. The mislabeling rate (i. e. the mismatch between the species declared on the IBP label and the species identified by metabarcoding) was calculated. Overall, a high mislabeling rate (33.3 %) was observed, although this percentage is influenced by the e-CO platform and the insect species, with A. domesticus particularly involved. The use of species not listed in authorized Novel Food (e. g. Gryllus locorojo), and/or the partial replacement of high value species with lower value species was highlighted for the first time in processed IBPs. The presence of insect pests was also detected. Metabarcoding was confirmed as an effective tool for IBPs authentication. Also, outcomes from this study can provide useful data on the main issues involving the EU IBPs' market, that can represent an incentive to reinforce both official controls and FBO's self-controls on these poorly investigated products.

Is the metabarcoding ripe enough to be applied to the authentication of foodstuff of animal origin? A systematic review.

Food authentication using molecular techniques is of great importance to fight food fraud. Metabarcoding, based on the next-generation sequencing (NGS) technologies, allowing large-scale taxonomic identification of complex samples via massive parallel sequencing of fragments (called DNA barcodes) simultaneously, has become increasingly popular in many scientific fields. A systematic review to answer the question "Is the metabarcoding ripe enough to be applied to the authentication of foodstuff of animal origin?" is presented. The inclusion criteria were focused on the selection of scientific papers (SPs) only applying metabarcoding to foodstuff of animal origin collected on the market. The 23 included SPs were first analyzed with respect to the metabarcoding phases: library preparation (target genes, primer pairs, and fragment length), sequencing (NGS platforms), and final data analysis (bioinformatic pipelines). Given the importance of primer selection, the taxonomic coverage of the used primers was also evaluated. In addition, the SPs were scored based on the use of quality control measures (procedural blanks, positive controls, replicates, curated databases, and thresholds to filter the data). A lack of standardized protocols, especially with respect to the target barcode/s and the universal primer/s, and the infrequent application of the quality control measures, leads to answer that metabarcoding is not ripe enough for authenticating foodstuff of animal origin. However, the observed trend of the SP quality improvement over the years is encouraging. Concluding, a proper protocol standardization would allow a wider use of metabarcoding by both official and private laboratories, enabling this method to become the primary for the authentication of foodstuffs of animal origin.

First Toxicological Analysis of the Pufferfish Sphoeroides pachygaster Collected in Italian Waters (Strait of Sicily): Role of Citizens Science in Monitoring Toxic Marine Species.

Pufferfish (Tetraodontidae) inhabiting the Mediterranean Sea may represent an emerging public health risk due to the possible accumulation of marine neurotoxins such as tetrodotoxin (TTXs) and saxitoxin (STXs) in their tissues. In this study, the presence of pufferfish species in the Strait of Sicily (Lampedusa Island, Italy) was investigated using a citizen science (CS) approach, involving local fishermen. Samples (liver, intestine, gonads, muscle, skin) from 20 specimens were sent to the National Reference Laboratory on Marine Biotoxins for TTXs detection using a validated HILIC-MS/MS method on fish tissue. The presence of STXs was also screened in part of the specimens. Overall, 56 specimens identified as Sphoeroides pachygaster (Müller &Troschel, 1848) were collected. Data on their total length, body weight, fishing method and catch area (with relative depth temperature and salinity) were analyzed and compared with the S. pachygaster records reported in literature which were updated to 2022. All the analysed tissues were found to be negative for both TTXs and STXs. CS played an essential role in monitoring potentially toxic marine species in this investigation. Outcomes from this study, which is the first investigating S. pachygaster toxicity in Italian waters, may provide useful data for the proper assessment of this emerging risk.

Accidental discovery of a Tetraodontidae (Sphoeroides marmoratus) within a cuttlefish (Sepia officinalis) 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 Eastern Central 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.

The compliance to European and National safety and labelling requirements relating to the sale of spontaneous and cultivated mushrooms and mushroom-based products in Tuscany was assessed. The evidence was collected by the Mycological Inspectorate of North-West Tuscany Local Health Authority during 90 inspections (from 2016 to 2020) at large-scale distribution stores, wholesalers, and restaurants in 10 cities belonging to 3 provinces, and on the labelling analysis of 98 commercial products collected at retail in 2021. Despite a substantial compliance of the inspected activities and products with the regulatory requirements, critical issues were highlighted: 1) EU legislative gap in the definition of specific measures for the safe sale of spontaneous mushrooms; 2) improper shelf storage temperatures of fresh-cut products; 3) incorrect condition of use on the labels of pre-packaged products; 4) lack of countryof- origin declaration in pre-packaged products. Furthermore, the labelling analysis highlighted that 18.4% and 15.3% of the products presented issues in the validity and correctness of the scientific names respect to national requirements in. A revision of the current EU legislation is needed to guarantee consumers safety, also considering the relevant number of poisoning cases related to false mycetisms (ingestion of edible mushrooms unproperly stored or used). Also, a specific revision and harmonization of the EU labelling of mushrooms would be desirable to protect consumers.

Building of an Internal Transcribed Spacer (ITS) Gene Dataset to Support the Italian Health Service in Mushroom Identification.

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.

Molecular Characterization of Clistobothrium sp. Viable Plerocercoids in Fresh Longfin Inshore Squid (Doryteuthis pealeii) and Implications for Cephalopod Inspection.

Cephalopods, an appreciated seafood product, are common hosts of marine cestodes. The aim of this work is to report visible alive plerocercoids in longfin inshore squid (Doryteuthis pealeii), a cephalopod species commercialized as fresh and whole in Italy. Seventy D. pealeii from the Northwest Atlantic (FAO area 21) were collected and visually inspected. In total, 18 plerocercoid larvae were found in the viscera of 10 host specimens (P: 14.3% 95% CI 7.1-24.7; MI: 1.8, MA: 0.26; range 1-4) and molecularly analyzed targeting the variable D2 region of the large subunit (LSU) rRNA gene and the cytochrome c oxidase subunit I (COI) gene. The molecular characterization allowed to identify all the plerocercoids as Clistobothrium sp., a cestode of the Phyllobothriidae family with Lamnidae sharks as definitive hosts, and cephalopods as second intermediate hosts. These findings represent the first molecular record of Clistobothrium sp. in D. pealeii, thus contributing to elucidate its poorly known life cycle. Even if not affecting consumer's health, these visible parasites may represent a reason for disgust for consumers. Therefore, the results suggest that Food Business Operators should also check for the presence of these visible parasites during inspection and underline the importance of a correct consumers' education.

Analysis of the sanitary survey 2015-2017 conducted in the gulf of La Spezia (Italy): Reclassification of the areas of production of live bivalve molluscs.

The sanitary survey is aimed at classifying and monitoring the production areas of live bivalve molluscs (LBM) and it is performed using standards that are provided by the Centre for Environment, Fisheries and Aquaculture Science's Guide to Good Practice. In this study, data from the sanitary survey carried out by the Asl5 Spezzino on the production areas of the gulf of La Spezia during the period 2015-2017 were analysed. The number and type of the analysis performed both on the total sampling and on the individual target species, as well as the number and type of found non-compliance (assessed on both mandatory parameters and on parameters fixed by the local monitoring plan) were considered. Data were also compared with those from the sanitary survey 2012-2014. Appropriate statistic tests were used to evaluate data from E. coli and Norovirus monitoring. Overall, 4306 analysis were performed, especially on the species M. galloprovincialis (89%) and they were mostly focused on to the search of biological agents. 160 NC were detected. Most of the NC concerns the Norovirus's positivity (93.75%) in M. galloprovincialis and C. gigas. A correlation between the levels of E. coli and rainfall/ seasonality (higher levels in the colder months) was proved, especially in the sampling points located in the inner part of the dam and in the Portovenere Bay. Class B was reconfirmed for M. galloprovincialis, the production areas of C. gigas were reclassified as A and those of V. verrucosa were definitively closed. The sanitary survey was therefore confirmed as a useful tool for reclassification and for monitoring LBM production areas.

DNA barcoding for the verification of supplier's compliance in the seafood chain: How the lab can support companies in ensuring traceability.

Food Business Operators (FBOs) rely on laboratory analysis to ensure seafood traceability. DNA barcoding and Forensically Informative Nucleotide Sequencing may represent a support within self-checking programs finalized to suppliers' qualification and products identity certification. The present study aimed at verifying the usefulness of a decisional procedure (decision tree) set up at the FishLab (Department of Veterinary Sciences, University of Pisa, Italy) for seafood species identification by DNA analysis, to cope with FBOs' needs. The decision tree was applied to the analysis of 182 seafood (fish and molluscs) products, conferred to the FishLab by different FBOs between 2014 and 2015 as result of their self-checking activities. The analysis relied on a standard COI gene fragment eventually integrated by the analysis of alternative or supportive molecular targets (cytb and 16S rRNA). It also included a mini-DNA barcoding approach for processed products. Overall, 96.2% of the samples were unambiguously identified at species level using the elective target alone (92.4%) or a multi-target approach (3.8%). The lack of species identification (3.8%) was attributable to the absence of reference sequences or to the low resolution of the molecular targets. Nonetheless, all the molecular results were deemed adequate to evaluate the sample's compliance to the label information. Non-compliances were highlighted in 18.1% of the products. The protocol was proven as an effective supportive tool for the seafood identity verification within the supply chain self-checking activities. In addition, a considerable fraud rate was confirmed and the species most frequently involved in substitution were pointed out.

Advances in the analysis of complex food matrices: Species identification in surimi-based products using Next Generation Sequencing technologies.

The Next Generation Sequencing (NGS) technologies represent a turning point in the food inspection field, particularly for species identification in matrices composed of a blend of two or more species. In this study NGS technologies were applied by testing the usefulness of the Ion Torrent Personal Genome Machine (PGM) in seafood traceability. Sixteen commercial surimi samples produced both in EU and non-EU countries were analysed. Libraries were prepared using a universal primer pair able to amplify a short 16SrRNA fragment from a wide range of fish and cephalopod species. The mislabelling rate of the samples was also evaluated. Overall, DNA from 13 families, 19 genera and 16 species of fish, and from 3 families, 3 genera and 3 species of cephalopods was found with the analysis. Samples produced in non-EU countries exhibited a higher variability in their composition. 37.5% of the surimi products were found to be mislabelled. Among them, 25% voluntary declared a species different from those identified and 25% (all produced in non-EU countries) did not report the presence of molluscs on the label, posing a potential health threat for allergic consumers. The use of vulnerable species was also proved. Although the protocol should be further optimized, PGM platform proved to be a useful tool for the analysis of complex, highly processed products.

Seafood Identification in Multispecies Products: Assessment of 16SrRNA, cytb, and COI Universal Primers' Efficiency as a Preliminary Analytical Step for Setting up Metabarcoding Next-Generation Sequencing Techniques.

Few studies applying NGS have been conducted in the food inspection field, particularly on multispecies seafood products. A preliminary study screening the performance and the potential application in NGS analysis of 14 "universal primers" amplifying 16SrRNA, cytb, and COI genes in fish and cephalopods was performed. Species used in surimi preparation were chosen as target. An in silico analysis was conducted to test primers' coverage capacity by assessing mismatches (number and position) with the target sequences. The 9 pairs showing the best coverage capacity were tested in PCR on DNA samples of 53 collected species to assess their amplification performance (amplification rate and amplicon concentration). The results confirm that primers designed for the 16SrRNA gene amplification are the most suitable for NGS analysis also for identification of multispecies seafood products. In particular, the primer pair of Chapela et al. (2002) is the best candidate.

A Conventional Multiplex PCR Assay for the Detection of Toxic Gemfish Species (Ruvettus pretiosus and Lepidocybium flavobrunneum): A Simple Method To Combat Health Frauds.

The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries have banned the sale of these species, mislabeling cases have been reported in sushi catering. This work developed a simple conventional multiplex PCR, which discriminates the two toxic gemfishes from other potentially replaced species, such as tunas, cod, and sablefish. A common degenerate forward primer and three species-specific reverse primers were designed to amplify cytochrome oxidase subunit I (COI) gene regions of different lengths (479, 403, and 291 bp) of gemfishes, tunas, and sablefish, respectively. A primer pair was designed to amplify a fragment (193 bp) of the cytb gene of cod species. Furthermore, a primer pair targeting the 16S rRNA gene was intended as common positive control (115 bp). The method developed in this study, by producing the expected amplicon for all of the DNA samples tested (reference and commercial), provides a rapid and reliable response in identifying the two toxic species to combat health frauds.

Pentaplex PCR as screening assay for jellyfish species identification in food products.

Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market.