Kristina Kappel

Identifying fishes through DNA barcodes and microarrays.

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 hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products.

DNA Microarrays for Identifying Fishes

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 commercially 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.

Low mislabeling rates indicate marked improvements in European seafood market operations

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.

Species identification in mixed tuna samples with next-generation sequencing targeting two short cytochrome b gene fragments

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.

Tuna labels matter in Europe: Mislabelling rates in different tuna products

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.