Managing fish stress is crucial to ensure a sustainable aquaculture production. In a research context, high plasma levels of cortisol, glucose and/or lactate are universally used to assess fish welfare. However, the heterogeneity of responses demonstrate that these should be interpreted with caution in cases of long-term stressors. The standardization of stress biomarkers would be an important contribute to the existing species-specific stress management protocols. Proteomics was employed in this study as a tool to discover more robust fish stress biomarkers, since proteins are ubiquitously affected by abiotic and biotic stimuli in a slower timescale when compared with endocrine responses. The analysis of proteome changes in different tissues, e.g. liver and mucus may offer not only tissue-specific protein fingerprints, but can be also highly advantageous in the context of fish welfare, as skin mucus allows for sampling in a non-invasive way. Moreover, this combined proteomic analysis provides a more detailed insight into the molecular mechanisms, as a complementary picture of the animal´s physiological state under stress.
Sparus aurata was exposed to different suboptimal rearing conditions in three separated trials: overcrowding (OC30 and OC45), repetitive net handling (NET2 and NET4), and hypoxia (HYP30 and HYP15), using fish reared under optimal conditions for the species, as control. By the end of the trials, fish were sampled and protein extracts from liver and mucus samples were prepared for further analysis by reverse phase nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Proteins identified with high confidence (protein FDR <0.5%; peptide FDR <0.1%) within each trial were analyzed by One-way ANOVA followed by Tukey’s HSD test (p < 0.05). Proteins were screened for enriched KEGG and Reactome pathways. Pairwise comparisons between control and stressed fish samples were also established within each trial (T-test, q <0.05) and up- and down-regulated proteins were further classified according to Gene Ontology Enrichment (Fisher’s Exact test, FDR <0.05). Cortisol, glucose and lactate were measured from blood samples, and glycogen stores were assessed in the liver.
Results and conclusions
Label-free shotgun proteomics reproducibly identified a mean of 1300 proteins in at least 4 out of 6 fish per treatment, either in the liver or in the skin mucus of gilthead seabream. A total of 297 (liver) and 250 (skin mucus) differentially regulated proteins were identified between stressed and control fish across the three trials. A tissue-specific stress regulation was observed, although 40 common proteins, mostly implicated in translation and protein folding processes, were found to be differentially regulated in both tissues. Liver-specific proteins were involved in metabolism regulation, while mucus-unique proteins were implicated in signal transduction and immune response. Plasma cortisol levels and liver glycogen storages were significantly altered exclusively in net-handled fish. The overall results suggest that the net-handling was the most impactful stressor, and the fish physiological stress response was tuned according to the challenge’s severity. This integrated approach provides a starting point for the development of more reliable fish welfare assessment measures in comparison to cortisol levels solely, to further improve aquaculture sustainability.