DIETARY EFFECTS OF METHIONINE IN ATLANTIC SEABASS ANALYSED BY SHOTGUN PROTEOMICS

Outline: Optimizing fish growth and welfare are challenging goals for the aquaculture industry and new strategies are needed to keep pace with the increasing and high demands of fish consumption worldwide. Supplementation of diets with essential amino acids can help to improve both fish nutritional status and the health/immune response. Diet supplementation with methionine (Met) in particular, may help to improve the fish immune system, as it plays an essential role in the inflammatory and oxidative stress response. A high-throughput shotgun proteomic analysis was employed to evaluate liver changes after feeding fish at different requirements of Met to identify key proteins either involved in growth or immune response/health with a potential role in fish welfare.

Methodology: Juvenile Atlantic seabass with an initial body weight of 10 g were fed during 85 days with experimental diets (ED) at 0.65%, 0.85%, 1.25% and 1.5% Methionine (w/w) of the estimated requirements for this species. Fish were fed twice a day, ad libitum and maintained in a flow-through system. Dissolved oxygen in seawater was maintained above 5 mg L^-1 with a mean temperature of 22.4 ᵒC ± 1.9 ᵒC. Liver samples were obtained after 18 and 85 days of growth, being immediately collected after lethal anesthesia and kept at -80 ᵒC until further use. Proteins were extracted with 7M Urea, 2M Thiourea, 4% CHAPS and 30mM Tris (pH 8.8) plus protease inhibitors and cleaned-up with the Bio-Rad ReadyPrep kit. Samples were prepared for high resolution MS (LTQ Orbitrap Velos Pro-ETD) according to Kulak et al. (2014) by resuspending protein pellets in 1% sodium deoxycholate, 10mM TCEP, 40mM CAA and 100mM Tris (pH 8.5). MS data processed on Proteome discoverer (ThermoFisher Scientific) was loaded into ScaffoldQ+ (Proteome Software) for label-free quantitative comparisons of protein relative abundance.

Results and Conclusions: A dose-dependent effect was observed on growth performance in fish fed with ED at different concentrations of Met. About 300 liver proteins with significant changes in protein abundance (FDR <0.1%) were identified after pairwise comparisons across all diets. Proteome changes were observed already after 18 days, though more pronounced after 85 days. GO enrichment and network analyses evidenced the influence of amino acid supplementation in the liver proteome of Atlantic seabass, with most proteins with significant changes being related with anti-oxidant activity, response to stimulus and growth (Fig.1), suggesting that supplementation at 1.5 % Met may have a beneficial effect in global fish welfare.