Temperate fish are particularly sensitive to low temperatures, especially in the northern Mediterranean area, where the cold season decreases fish-farm production and affects fish health. Recent studies have suggested that the skin mucus participates in overall fish defense and welfare, and therefore propose it as a target for non-invasive studies of fish status. Here, we determine the mucus interactome of differentially expressed proteins in a temperate fish model, gilthead sea bream (Sparus aurata), after chronic exposure to low temperatures (7 weeks at 14°C). The differentially expressed proteins were obtained by 2D-PAGE of mucus soluble proteins and further assessed by STRING analyses of the functional interactome based on protein-protein interactions. Complementarily, we determined mucus metabolites, glucose, and protein, as well as enzymes involved in innate defense mechanisms, such as total protease and esterase. The cold mucus interactome revealed the presence of several subsets of proteins corresponding to Gene Ontology groups. “Response to stress” formed the central core of the cold interactome, with up-regulation of proteins, such as heat shock proteins (HSPs) and transferrin; and down-regulation of proteins with metabolic activity. In accordance with the low temperatures, all proteins clustered in the “Single-organism metabolic process” group were down-regulated in response to cold, evidencing depressed skin metabolism. An interactome subset of “Interspecies interaction between species” grouped together several up-regulated mucus proteins that participate in bacterial adhesion, colonization, and entry, such as HSP70, lectin-2, ribosomal proteins, and cytokeratin-8, septin, and plakins. Furthermore, cold mucus showed lower levels of soluble glucose and no adaptation response in total protease or esterase activity. Using zymography, we detected the up-regulation of metalloprotease-like activity, together with a number of fragments or cleaved keratin forms which may present antimicrobial activity. All these results evidence a partial loss of mucus functionality under chronic exposure to low temperatures which would affect fish welfare during the natural cold season under farm conditions.

Aquaculture is a growing industry that is increasingly providing a sizable proportion of fishery products for human consumption. Dietary energy and temperature fluctuations affect fish health and may even trigger mortality, causing great losses in fish production during winter. To better understand this unproductive winter period in aquaculture, the redox status in a cultured marine species, the gilthead sea bream, was analysed for the first time by inducing controlled temperature fluctuations and reducing dietary lipid content. Two groups of fish (by triplicate), differing in their dietary lipid content (18% vs 14%), were subjected to 30 days at 22ºC (Pre-Cold), 50 days at 14ºC (Cold) and then 35 days at 22ºC (Recovery). Plasma and liver redox metabolites (oxidised lipid, oxidised protein and thiol groups), liver glutathione forms (total, oxidised and reduced) and liver antioxidant enzyme activities were measured. Reducing dietary lipid content did not affect gilthead sea bream growth, glutathione levels or enzyme activities, but did reduce the amount of oxidised lipids. A sustained low temperature of 14ºC showed a lack of adaptation of antioxidant enzyme activities, mainly catalase and glutathione reductase, which subsequently affected the glutathione redox cycle and caused an acute reduction in total hepatic glutathione levels, irrespective of diet. Antioxidant enzyme activities were gradually restored to their pre-cold levels, but the glutathione redox cycle was not restored to its pre-cold values during the recovery period used. Moreover, the lower lipid diet was associated with transiently increased liver oxidised protein levels. Thus, we propose that fish should be fed a low lipid diet during pre-cold and cold periods, which would reduce oxidised lipid levels without affecting fish growth, and a higher energy diet during the recovery period. Moreover, diets supplemented with antioxidants should be considered, especially during temperature recovery.