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A double-DNA approach was developed to discriminate the three Trachurus species that abide in European waters: T. trachurus, T. mediterraneus, and T. picturatus. The analysis aimed at both mitochondrial and nuclear loci. Polymerase Chain Reaction (PCR) amplification of the cytochrome b gene of mtDNA was followed by restriction analysis with three species-specific enzymes: NlaIII, NciI, and BsmAI. Digestion with these endonucleases yielded species-specific electrophoretic profiles. The universality of the results was verified by screening a large number of individuals from 12 geographical regions covering most of the distribution of the species. Additionally, the nuclear multicopy 5S rRNA gene was selected as an alternative candidate for the discrimination of the three Trachurusspecies. A simple agarose gel electrophoretic analysis of the amplicons proved to be capable of leading to unambiguous identification of the three Trachurus species. Thus, the double-DNA methodology presented here allows the accurate discrimination of Trachurus fish species and the detection of commercial fraud.

DNA-based methodologies are increasingly used successfully to elucidate cases of misidentification of adult individuals of fish species with morphological similarities. However, the problem of misidentification can arise even earlier than adulthood, between congeneric and even distantly related species with similar morphology in the early developmental stages. Therefore, a double DNA approach was developed for Macrorhamphosus scolopax, to identify and discriminate it from European species of the genus Trachurus. These species overlap geographically and temporally and are characterized by morphologically similar eggs. The approach looks at both mitochondrial and nuclear loci. Polymerase Chain Reaction (PCR) amplification of the 16S rRNA mtDNA gene was followed by restriction analysis with two species-specific enzymes: EcoRV and PmlI. Digestion with these endonucleases yielded species-specific electrophoretic profiles. Additionally, the nuclear multi-copy 5S rRNA gene was selected as an alternative candidate for identifying M. scolopax. The universality of the results was verified by screening a large number of fish from five geographical regions, covering most of the overlapping distribution of the species. The output is a double DNA methodology that can be used for egg identification and which could be of value in the egg production method of biomass assessment. [Copyright &y& Elsevier]