Application of NMR-based metabolomics techniques to biological systems: a case study on bivalves

In the past years, metabolomics has progressed greatly, providing a reliable and high-throughput approach particularly feasible for the study of complex biological systems. Indeed, thanks to the development of powerful analytical methods capable of screening a large number of chemical compounds in a sample, nowadays metabolomics plays an important role in gaining biological insights toward the influence of internal (genetic and developmental) and external (environmental) factors on phenotypes. The work presented in this Ph.D. thesis shows examples of applications of NMR-based metabolomics to the study of bivalves, facing challenges of interest in both food and environmental sciences. The outcome of such studies yield insights, at molecular level, into several aspects concerning the impact of different storage conditions on shellfish quality and the effect of natural and anthropogenic environmental stressors on bivalves’ metabolic profiles. Firstly, the effect of different cold storage conditions on the hydrosoluble chemical components of Mytilus galloprovincialis (Lamarck, 1819) was investigated for the first time by NMR-based metabolomics. The data revealed substantial time-related changes in the metabolic profiles of mussels stored at 0 °C and 4 °C. The observed biochemical modifications were in good agreement with the microbiological quality of samples, reflecting changes in their microbial loads. These results confirmed the potential use of metabolomics as a reliable method to assess seafood freshness. Secondly, a metabolomic approach was also applied to study the effect of short-term exposure to heavy metals on two different clams’ species: Ruditapes decussatus (Linnaeus, 1758) and Ruditapes philippinarum (Adams & Reeve, 1850). Heavy metals are considered to be among the most harmful pollutants that can contaminate marine environments. The toxicity of trace metals gives rise from their persistent nature which lead to environmental accumulation. Given the high accumulation rates associated with their filter feeding attitude, bivalves mollusks are considered to be feasible monitoring organisms and are widely used in biomonitoring programs. R. decussatus and R. philippinarum, two bivalves’ species widely distributed along the Italian coasts, were selected in this Ph.D. project for assessing lead and zinc effects on their metabolic profiles. The results evidenced a sensible short-time metabolic response upon metal exposure, pointing out a main variability in the content of amino acids and organic osmolytes in relation to both metal nature and bivalve species. These findings show that NMR-based metabolomics has the required sensitivity and specificity to gain insights into the biochemical consequences arising upon heavy metals exposure, providing thus a useful tool for the identification of putative biomarkers as fast and sensitive indicators of contaminant-induced stress. Finally, 1H NMR-based metabolomics was applied with the aim to assess the effects of seasonal change on Ruditapes decussatus metabolic profile. The results of the present study demonstrated that the combined use of advanced multivariate statistical techniques with NMR spectroscopy is a feasible approach to discriminate specimens of R. decussatus according to the sampling season. Moreover, the sensitivity of this analytical tool allowed the individuation of those metabolites whose relative amount significantly varied according to seasonal change (alanine and glycine), paving the way for further investigations that would contribute to achieve additional insights on bivalves’ bio-ecological framework.