The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested. The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome. This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.

Intestinal microbiota is key for many host functions, such as digestion, nutrient metabolism, disease resistance, and immune function. With the growth of the aquaculture industry, there has been a growing interest in the manipulation of fish gut microbiota to improve welfare and nutrition. Intestinal microbiota varies with many factors, including host species, genetics, developmental stage, diet, environment, and sex. The aim of this study was to compare the intestinal microbiota of adult gilthead sea bream (Sparus aurata) from three groups of age and sex (1-year-old males and 2- and 4-year-old females) maintained under the same conditions and fed exactly the same diet. Microbiota diversity and richness did not differ among groups. However, bacterial composition did, highlighting the presence of Photobacterium and Vibrio starting at 2 years of age (females) and a higher presence of Staphylococcus and Corynebacterium in 1-year-old males. The core microbiota was defined by 14 Operational Taxonomic Units (OTUs) and the groups that showed more OTUs in common were 2- and 4-year-old females. Discriminant analyses showed a clear separation by sex and age, with bacteria belonging to the phyla Firmicutes, Proteobacteria and Actinobacteria driving the separation. Pathway analysis performed with the inferred metagenome showed significant differences between 1-year-old males and 4-year-old females, with an increase in infection-related pathways, nitrotoluene degradation and sphingolipid metabolism, and a significant decrease in carbohydrate metabolism pathways with age. These results show, for the first time, how intestinal microbiota is modulated in adult gilthead sea bream and highlight the importance of reporting age and sex variables in these type of studies in fish.

The Gh/Prl/Sl family has evolved differentially through evolution, resulting in varying relationships between the somatotropic axis and growth rates within and across fish species. This is due to a wide range of endogenous and exogenous factors that make this association variable throughout season and life cycle, and the present minireview aims to better define the nutritional and environmental regulation of the endocrine growth cascade over precisely defined groups of fishes, focusing on Mediterranean farmed fishes. As a result, circulating Gh and Igf-i are revitalized as reliable growth markers, with a close association with growth rates of gilthead sea bream juveniles with deficiency signs in both macro- or micro-nutrients. This, together with other regulated responses, promotes the use of Gh and Igf-i as key performance indicators of growth, aerobic scope, and nutritional condition in gilthead sea bream. Moreover, the sirtuin-energy sensors might modulate the growth-promoting action of somatotropic axis. In this scenario, transcripts of igf-i and gh receptors mirror changes in plasma Gh and Igf-i levels, with the ghr-i/ghr-ii expression ratio mostly unaltered over season. However, this ratio is nutritionally regulated, and enriched plant-based diets or diets with specific nutrient deficiencies downregulate hepatic ghr-i, decreasing the ghr-i/ghr-ii ratio. The same trend, due to a ghr-ii increase, is found in skeletal muscle, whereas impaired growth during overwintering is related to increase in the ghr-i/ghr-ii and igf-ii/igf-i ratios in liver and skeletal muscle, respectively. Overall, expression of insulin receptors and igf receptors is less regulated, though the expression quotient is especially high in the liver and muscle of sea bream. Nutritional and environmental regulation of the full Igf binding protein 1–6 repertoire remains to be understood. However, tissue-specific expression profiling highlights an enhanced and nutritionally regulated expression of the igfbp-1/-2/-4 clade in liver, whereas the igfbp-3/-5/-6 clade is overexpressed and regulated in skeletal muscle. The somatotropic axis is, therefore, highly informative of a wide-range of growth-disturbing and stressful stimuli, and multivariate analysis supports its use as a reliable toolset for the assessment of growth potentiality and nutrient deficiencies and requirements, especially in combination with selected panels of other nutritionally regulated metabolic biomarkers.

The skin mucus of gilthead sea bream was mapped by one-dimensional gel electrophoresis followed by liquid chromatography coupled to high resolution mass spectrometry using a quadrupole time-of-flight mass analyzer. More than 2000 proteins were identified with a protein score filter of 30. The identified proteins were represented in 418 canonical pathways of the Ingenuity Pathway software. After filtering by canonical pathway overlapping, the retained proteins were clustered in three groups. The mitochondrial cluster contained 59 proteins related to oxidative phosphorylation and mitochondrial dysfunction. The second cluster contained 79 proteins related to antigen presentation and protein ubiquitination pathways. The third cluster contained 257 proteins where proteins related to protein synthesis, cellular assembly, and epithelial integrity were over-represented. The latter group also included acute phase response signaling. In parallel, two-dimensional gel electrophoresis methodology identified six proteins spots of different protein abundance when comparing unstressed fish with chronically stressed fish in an experimental model that mimicked daily farming activities. The major changes were associated with a higher abundance of cytokeratin 8 in the skin mucus proteome of stressed fish, which was confirmed by immunoblotting. Thus, the increased abundance of markers of skin epithelial turnover results in a promising indicator of chronic stress in fish.

Background: The increased demand for fish protein has led to the intensification of aquaculture practices which are hampered by nutritional and health factors affecting growth performance. To solve these problems, antibiotics have been used for many years in the prevention, control and treatment against disease as well as growth promoters to improve animal performance. Nowadays, the use of antibiotics in the European Union and other countries has been completely or partially banned as a result of the existence of antibiotic cross-resistance. Therefore, a number of alternatives, including enzymes, prebiotics, probiotics, phytonutrients and organic acids used alone or in combination have been proposed for the improvement of immunological state, growth performance and production in livestock animals. The aim of the present study was to evaluate two commercially available feed additives, one based on medium-chain fatty acids (MCFAs) from coconut oil and another with aBacillus-based probiotic, in gilthead sea bream (GSB,Sparus aurata), a marine farmed fish of high value in the Mediterranean aquaculture.Methods: The potential benefits of adding two commercial feed additives on fish growth performance and intestinal health were assessed in a 100-days feeding trial. The experimental diets (D2 and D3) were prepared by supplementing a basal diet (D1) with MCFAs in the form of a sodium salt of coconut fatty acid distillate (DICOSAN^®; Norel, Madrid, Spain), rich on C-12, added at 0.3% (D2) or with the probioticBacillus amyloliquefaciensCECT 5940, added at 0.1% (D3). The study integrated data on growth performance, blood biochemistry, histology and intestinal gene expression patterns of selected markers of intestinal function and architecture.Results: MCFAs in the form of a coconut oil increased feed intake, growth rates and the surface of nutrient absorption, promoting the anabolic action of the somatotropic axis. The probiotic (D3) induced anti-inflammatory and anti-oxidant effects with changes in circulating cortisol, immunoglobulin M, leukocyte respiratory burst, and mucosal expression levels of cytokines, lymphocyte markers and immunoglobulin T.Discussion: MCFA supplementation showed positive effects on GSB growth and intestinal architecture acting mainly in the anterior intestine, where absorption takes place. The probioticB. amyloliquefaciensCECT 5940 exhibited key effects in the regulation of the immune status inducing anti-inflammatory and anti-oxidant effects which can be potentially advantageous upon infection or exposure to other stressors. The potential effects of these feed additives in GSB are very promising to improve health and disease resistance in aquaculture.