Molecular biomarkers for fish welfare and species authentication.

Aquaculture is currently contributing almost half of fish consumed by the human population and it keeps growing more rapidly than other animal food production sectors. The introduction of molecular techniques, such as various genome projects, gene expression analysis and functional genomics, and the monitoring of stress levels through very early indicators such as molecular biomarkers, can bring considerable benefits to the quantity and quality of production and improve welfare of reared animals. Moreover in recent years, some teleost species have become model organisms, such as zebrafish and pufferfish, and the knowledge about them could be transferred to further improve reared animals and husbandry. Therefore, species of interest in aquaculture could, in turn, become new animal models. In this context, we looked for a new molecular biomarker for stress in Dicentrarchus labrax. Stress could involve alterations of brain functioning that may precipitate to mood disorders. The neurotrophin Brain Derived Neurotrophic Factor (BDNF) has recently been involved in stress-induced adaptation. BDNF is a key regulator of neuronal plasticity and adaptive processes. Regulation of BDNF is complex and may reflect not only stressspecific mechanisms, but also hormonal and emotional responses. For this reason, we used, as an animal model of stress, a fish, D. labrax, whose brain organization is very similar to that of higher vertebrates, but is generally considered free of emotional reactions. We provide, for the first time in a species of great interest in aquaculture, a comprehensive characterization of BDNF gene and its transcriptional, translational and post-translational regulation following acute stress. While total BDNF mRNA levels are unchanged, BDNF splicing variants 1c and 1d resulted down regulated after acute stress. Acute stress induces also a significant increase in proBDNF levels and reduction in mature BDNF suggesting altered regulation of proBDNF proteolytic processing. Notably, we provide here the first evidence that fishes possess a simplified proteolytic regulation of BDNF since the pro28kDa form, generated by the SKI-1 protease in mammals, is absent in fishes. The cleavage site, in fact, has first emerged in reptilians. Finally, we show that the proBDNF/totBDNF ratio is a highly predictive novel quantitative biomarker to detect stress in fishes with sensitivity = 100%, specificity = 87%, and Negative Predictive Value = 100%. The high predictivity of proBDNF/totBDNF ratio for stress in lower vertebrates indicates that processing of BDNF is a central mechanism in adaptation to stress and predicts that a similar regulation of pro/mature BDNF has likely been conserved throughout evolution of vertebrates from fish to man. The second part of this thesis is focused on the problem of seafood and fish species authentication. This is an important issue within the seafood industry to protect consumers from fraudulent practices, like species substitution, resulting from the increasingly wide diversification of species and globalization of fish trade. DNA-based methods for species identification are by far the best: they are generally based on PCR amplification of a target sequence, followed by a post-PCR analysis of amplified products, which could consist in sequencing or obtaining species-specific patterns of restriction fragments. The gene coding for the 5S ribosomal RNA is a suitable target for fish species identification because, for its particular sequence features, it does not require any further treatment after PCR. This gene consists of a small coding conserved region and a variable region of noncoding DNA, which is termed not transcribed spacer (NTS). Both regions are tandem repeated in the genome. The NTS, which is species-specific for length and sequence, has been used, here, to discriminate species subjected to substitution in the Italian fish market. Although preliminary, our results have demonstrated the value of this approach.