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The German National Reference Centre for Authentic Food (NRZ-Authent) and the competent German food control authorities of the federal states cooperated within the framework of the 10th joint Europol INTERPOL operation OPSON (OPSON X) in the detection of adulterated meat products. A total of 63 meat product samples were collected and analysed by the authorities using standard analytical procedures and subjected to a recently published 16S rDNA metabarcoding analysis. The sequence reads were analysed using 3 bioinformatics data processing strategies. The study aimed to gain additional data on the test samples regarding the authenticity of the declared species and to validate the 16S rDNA metabarcoding method with representative samples. The method was tested not only on 63 test samples, but also on 5 commercial samples from 2 interlaboratory comparison studies and 9 mock mixtures in parallel. The 16S rDNA metabarcoding method was able to detect species that were not target species of the used standard analytical methods, but failed, as shown previously, to detect fallow deer. Otherwise, the qualitative results of the 16S rDNA metabarcoding method were very similar to those of the methods currently in use by the German food control laboratories. Thus, the method has great potential to be used as a screening method for the authentication of mammal and poultry species in meat products.

Graphical abstract This proof-of-principle study describes the development of a rapid and easy-to-use DNA microarray assay for the authentication of giant tiger prawns and whiteleg shrimp. Following DNA extraction and conventional end-point PCR of a 16S rDNA segment, the PCR products are hybridised to species-specific oligonucleotide probes on DNA microarrays located at the bottom of centrifuge tubes (ArrayTubes) and the resulting signal patterns are compared to those of reference specimens. A total of 21 species-specific probes were designed and signal patterns were recorded for 47 crustacean specimens belonging to 16 species of seven families. A hierarchical clustering of the signal patterns demonstrated the specificity of the DNA microarray for the two target species. The DNA microarray can easily be expanded to other important crustaceans. As the complete assay can be performed within half a day and does not require taxonomic expertise, it represents a rapid and simple alternative to tedious DNA barcoding and could be used by crustacean trading companies as well as food control authorities for authentication of crustacean commodities. Supplementary Information The online version contains supplementary material available at 10.1007/s00216-021-03440-2.

Fishery products are traded worldwide, have long and branched supply chains and are among the most counterfeit foods. Food control authorities are responsible of controlling food labelling in order to ensure the safety and quality of products. This is particularly important for tropical and subtropical fish species, such as snappers (Lutjanidae), because certain species are known to be frequently contaminated with ciguatoxins that cause harmful Ciguatera Fish Poisoning outbreaks. The analytical method of choice for the identification of a sample at species level is ‘DNA barcoding’, the sequencing of specific genetic markers and the comparison of the sequences with international nucleotide databases, such as GenBank and BOLD. However, the results of these analyses can be severely impaired by an insufficient data basis and especially by database entries with incorrect species annotations. In this work, the available nucleotide sequences for common genetic markers (COI, cytb, 16S rDNA, 12S rDNA, rag1 and ITS) of snapper species in GenBank were subjected to careful examination with regard to their unambiguousness and clarity. Phylogenetic neighbour joining trees were prepared and checked for ambiguous and contradictory placement of species' sequences with special emphasis on the Malabar blood snapper (Lutjanus malabaricus), the two-spot red snapper (L. bohar) and the crimson snapper (L. erythropterus). The results indicate that ambiguous and contradictory database nucleotide sequences impede the DNA barcoding-based authentication of snapper products at species level. A species assignment for the Malabar snapper and the crimson snapper based solely on database queries seems questionable.

Seafood is particularly susceptible to the substitution of species. In order to guarantee authentic seafood products, seafood processors and traders must perform self-checks on the authenticity of imported and purchased goods. However, the conventional Sanger sequencing of PCR products for the authentication of seafood species is time-consuming and requires advanced infrastructure. DNA microarrays (DNA chips) with species-specific oligonucleotide probes represent a rapid alternative to sequencing-based species authentication. So far, though, only DNA microarrays for the authentication of land vertebrate species have achieved market success. In this work, a user-friendly DNA microarray assay was developed for the authentication of ten important food fish species that can be performed in four to five hours from start to end. The assay was tested with authenticated specimens from 67 different fish species, and by comparing the probe signal patterns all target species and even closely related non-target species could be distinguished.

56 pages, 5 figures, Seafood authenticity is a key parameter for seafood quality, particularly in Europe where regulations provide a strict framework for seafood labeling. A wide variety of methods are commonly used in control laboratories (private or public) to identify seafood species, but emergent approaches for the development of new and fast DNA- and protein-based methods for species differentiation are also considered. To address the challenges in controlling further labeling requirements in the latest European legislation on seafood product traceability and labeling (Regulation (EU) 1379/2013), a review of the development of methods to identify fishing areas and to distinguish between wild and farmed fish, as well as an overview of the advanced methods that could be used for differentiation of fresh and frozen-thawed fish, is given. These methods will become increasingly important in the near future as the risk-based control of food authenticity is prescribed by the new EU control regulation (Regulation (EU) 2017/625)

High-priced fish and seafood prepared as restaurant dish is especially prone to fraudulent substitution due to the lack of morphologic characters and less stringent labelling requirements. Common sole (Solea solea) is considered one of the most valuable and tasty fish species on the European markets and fraudulent substitutions of common sole in restaurant stores have been reported by official German food authorities in the past (from 2005 to 2011). The aim of the present study was to assess the substitution of common sole prepared as dishes in restaurants and to identify species used as substitutes for common sole. Furthermore it was investigated whether substitution takes place at the level of the restaurants or earlier within the food supply chain. Altogether, 47 common sole dishes were ordered incognito in 24 restaurants and 98 whole fish specimens or fish fillets were purchased from 35 different shops (including wholesale dealers and speciality markets). Species identities were determined by cytochrome b gene sequencing and isoelectric focusing of sarcoplasmic proteins, which are official German methods for fish species identification afforded by §64 of the German Food and Feed Code (LFGB). 50 % of the restaurant samples were shown to be substituted by species of lower commercial values, such as catfish species (Pangasidae), Senegalese tonguesole (Cynoglossus senegalensis) or Portuguese sole (Synaptura lusitanica). As almost all samples from the retail were shown to be authentic – only one sample was identified as lemon sole (Microstomus kitt) – we presume that fraudulent substitution is performed by restaurant owners and staff rather than by persons responsible for fishing, manufacturing or retail.

Due to the huge variety of fish species on the European market and the loss of morphologic characters upon fish processing, molecular methods are needed for reliable species identification of fishery products. There are 3 official methods for fish species authentication in Germany and a 4th method is currently considered to be included into the official collection of methods. Within the framework of the EU project ‘Labelfish’ (the Atlantic network on genetic control of fish and seafood labelling and traceability) species authenticity of fish samples from the German market and from German restaurants was analysed with 2 official methods in the years 2013 and 2014. Substitution of fish species was remarkably high among common sole restaurant samples (50 %), followed by substitution of tinned tuna species (17.4 %) and fresh and frozen tuna products (13.2 %). However there was no adulteration of market cod samples at all. Substitution of fish used in restaurants presumably is taking place directly within the restaurants rather than earlier in the retail sector, because species used as substitutes in restaurants were not be found in the respective fresh and frozen products from the retail.

12 pages, 4 figures.-- Carmen G. Sotelo ... et al.-- This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited, Tuna fisheries and processing represent economic activities of paramount importance around the world. Most of these products are traded for human consumption and in general are highly demanded commodities. However, not all tuna products achieve the same market price, some consumers are willing to pay a huge amount of money for certain species (i.e. Japanese market for Bluefin tuna) while other species are rather affordable (i.e. Skipjack tuna), therefore mislabelling has been observed frequently. We collected and analysed 545 tuna samples in six European countries, including fresh, frozen and canned products, and we have investigated whether or not these products were correctly labelled under European and national legislations. We found an overall mislabelling rate of 6.79%; in particular, 6.70% of the fresh and frozen tuna products and 7.84% of canned tuna were mislabelled, and only in the case of fresh and frozen tuna samples significant differences among countries were found. Mislabelling rates for Atlantic Bluefin tuna labelled products were very high, ranging from 50 up to 100%. In general, mislabelling was higher when specific names were included in the labels. The “tuna” umbrella term is a very popular one with consumers, but also one that remains vulnerable to ambiguity, hampering efforts towards market transparency and with potential negative consequences to the adequate management of tuna species stocks, The work was funded by LABELFISH project, supported by the EU Atlantic Area Programme Project Number 2011-1/163 and the UK Department for Environment, Food & Rural Affairs grant FA0116

8 páginas, 3 figuras, 1 tabla, Conventional Sanger sequencing of PCR products is the gold standard for species authentication of seafood products. However, this method is inappropriate for the analysis of products that might contain mixtures of species, such as tinned tuna. The purpose of this study was to test whether next-generation sequencing (NGS) can be a solution for the authentication of mixed products. Nine tuna samples containing mixtures of up to four species were prepared and subjected to an NGS approach targeting two short cytochrome b gene (cytb) fragments on the Illumina MiSeq platform. Sequence recovery was precise and admixtures of as low as 1% could be identified, depending on the species composition of the mixtures. Duplicate samples as well as two individual NGS runs produced very similar results. A first test of three commercial tinned tuna samples indicated the presence of different species in the same tin, although this is forbidden by EU law, This work was funded by the European Union INTERREG Atlantic Area Program (“LabelFish”, project 2011-1/163)

In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commer- cially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products.

5 pages, 2 figures, 1 table, Over the span of a decade, genetic identification methods have progressively exposed the inadequacies of the seafood supply chain, revealing previously unrecognized levels of seafood fraud, raising awareness among the public, and serving as a warning to industry that malpractice will be detected. Here we present the outcome of the latest and largest multi-species, transnational survey of fish labeling accuracy to date, which demonstrates an apparent sudden reduction of seafood mislabeling in Europe. We argue that recent efforts in legislation, governance, and outreach have had a positive impact on industry regulation. Coordinated, technology-based, policy-oriented actions can play a pivotal role in shaping a transparent, sustainable global seafood market and in bolstering healthier oceans., This study is part of the LABELFISH project (www.labelfish.eu), supported by the EU Atlantic Area Programme (project number 2011-1/163) and the UK Department for Environment, Food & Rural Affairs (grant FA0116).

Background: International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings: This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of "DNA barcoding" and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the "position of label" effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridization experiments (, SCOPUS: ar.j, info:eu-repo/semantics/published

DNA microarrays are currently in use almost exclusively as research tools for gene expression analysis and their application for the identification of organisms is still in its infancy, only documented by a few studies on mammals, bacteria, and viruses. The "Fish & Chips" project aims to demonstrate that DNA chips can be a new innovative tool for the identification of marine animals and phytoplankton. To achieve this goal, fishes, invertebrates, and phytoplankton were sampled in European seas and taxonomically classified. Fragments of their mitochondrial 16S, cyt b, and COI genes and from the nuclear 18S genes were sequenced and served as molecular markers to enable probe design for the microarrays. An on-line data base containing the sequences and all relevant information of the samples has been implemented. A first prototype of a "Fish Chip",is based on more than 400 sequences from the 16S rRNA gene belonging to 46 species. A second microarray prototype serves to identify flatfishes from the North Sea based on COI-and 16S sequences from 70 individuals of 17 fish species. A "Phytoplankton Chip" is now available with probes for all microalgal classes and many toxic species, as well as a "Invertebrate Chip". The results show that this approach is feasible.