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.

Three different immunoglobulin isotypes can be found in teleost fish, IgM, IgD and the teleost specific IgT. IgM is considered to have a systemic activity and IgT is attributed a mucosal role, similar to mammalian IgA. In this study the complete sequence of gilthead sea bream IgM and IgT in their membrane (m) and soluble (s) forms are described for the first time in a perciform fish. Their constitutive gene expression is analyzed in different tissues, and their regulation upon viral, bacterial, parasitic, mucosal vaccination and dietary challenges are studied. Gilthead sea bream IgM and IgT have the prototypical structure when compared to other fish immunoglobulins. The constitutive expression of sIgM was the highest overall in all tissues, whereas mIgT expression was highest in mucosal tissues, such as gills and intestine. IgM and IgT were differentially regulated upon infection. IgT was highly up-regulated locally upon infection with the intestinal parasite Enteromyxum leei, or systemically after Nodavirus infection. Long term intestinal parasitic infections increased the serum titer of both isotypes. Mucosal vaccination against Photobacterium damselae subsp. piscicida finely regulated the immunoglobulin response inducing a systemic increase of IgM titers in serum and a local IgT response in skin mucus when animals were exposed to the pathogen by bath challenge. Interestingly, plant-based diets inhibit IgT up-regulation upon intestinal parasitic challenge, which was related to a worse disease outcome. All these results corroborate the mucosal role of IgT and emphasize the importance of a finely tuned regulation of immunoglobulin isotypes upon infection, which could be of special interest in vaccination studies.

In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8⁺ memory T cells while downregulating CD4⁺ memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM⁺ B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM⁺ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10.

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.

Background: The key effects of intestinal microbiota in animal health have led to an increasing interest in manipulating these bacterial populations to improve animal welfare. The aquaculture sector is no exception and in the last years, many studies have described these populations in different fish species. However, this is not an easy task, as intestinal microbiota is composed of very dynamic populations that are influenced by different factors, such as diet, environment, host age, and genetics. In the current study, we aimed to determine whether the genetic background of gilthead sea bream (Sparus aurata) influences the intestinal microbial composition, how these bacterial populations are modulated by dietary changes, and the effect of selection by growth on intestinal disease resistance. To that aim, three different groups of five families of gilthead sea bream that were selected during two generations for fast, intermediate, or slow growth (F3 generation) were kept together in the same open-flow tanks and fed a control or a well-balanced plant-based diet during 9 months. Six animals per family and dietary treatment were sacrificed and the adherent bacteria from the anterior intestinal portion were sequenced. In parallel, fish of the fast- and slow-growth groups were infected with the intestinal parasite Enteromyxum leei and the disease signs, prevalence, intensity, and parasite abundance were evaluated. Results: No differences were detected in alpha diversity indexes among families, and the core bacterial architecture was the prototypical composition of gilthead sea bream intestinal microbiota, indicating no dysbiosis in any of the groups. The plant-based diet significantly changed the microbiota in the intermediate- and slow-growth families, with a much lower effect on the fast-growth group. Interestingly, the smaller changes detected in the fast-growth families potentially accounted for more changes at the metabolic level when compared with the other families. Upon parasitic infection, the fast-growth group showed significantly lower disease signs and parasite intensity and abundance than the slow-growth animals. Conclusions: These results show a clear genome-metagenome interaction indicating that the fast-growth families harbor a microbiota that is more flexible upon dietary changes. These animals also showed a better ability to cope with intestinal infections.

Both endotherms and ectotherms (e.g., fish) increase their body temperature to limit pathogen infection. Ectotherms do so by moving to warmer places, hence the term "behavioral fever." We studied the manifestation of behavioral fever in the common carp infected by cyprinid herpesvirus 3, a native carp pathogen. Carp maintained at 24°C died from the infection, whereas those housed in multi-chamber tanks encompassing a 24°C-32°C gradient migrated transiently to the warmest compartment and survived as a consequence. Behavioral fever manifested only at advanced stages of infection. Consistent with this, expression of CyHV-3 ORF12, encoding a soluble decoy receptor for TNF-α, delayed the manifestation of behavioral fever and promoted CyHV-3 replication in the context of a temperature gradient. Injection of anti-TNF-α neutralizing antibodies suppressed behavioral fever, and decreased fish survival in response to infection. This study provides a unique example of how viruses have evolved to alter host behavior to increase fitness.