[Results]: All fish grew efficiently, with feed conversion ratios between 1.09 and 1.19 (no statistical differences). Specific growth rates did not vary significantly between CTRL and 50LSAqua fish, though a slight decrease of growth rate (5%) was found with the total FM replacement (100LSAqua). Organosomatic weight indexes of liver, mesenteric fat and intestine were not altered by dietary treatment, but intestine length was shortened by the strategy of FM replacement. In addition, the concentration of lactic acid, was increased in stripped faeces of fish fed LSAqua diets. When fish of fish fed LS-Aqua diets were put together (50/100LSAqua), the bacterial richness was similar in both fish groups, but a remarkably lower diversity was found in CTRL fish. At the phylum level, Proteobacteria was the most abundant phylum, constituting ≥ 55% of the total resident bacteria in the anterior intestine. Firmicutes was the second most abundant phylum in the CTRL group (~20%) with a significant decrease (10.3%) in the 50/100LSAqua group. Conversely, Actinobacteria raised from 11.9% in CTRL fish to 23.2% in 50/100LSAqua fish, whereas the abundance of Bacteroidetes remained almost constant (~4.5%) in both fish groups. PLS-DA analyses showed significant differences in the microbial composition among dietary groups. For these discriminant bacteria, a first type of response was mediated by 27 OTUs overrepresented in fish fed 50/100LSAqua diets. In this group, it was remarkable the presence of Verrucomicrobia and Chlamydiae phyla, the class Betaproteobacteria and the genera Paracoccus, Omithinimicrobium, Tetrasphaera, Rubellimicrobium and Butivibrio. A second type of response grouped 20 OTUs with a significantly higher proportion in CTRL fish. This group included Bacteroidetes and Cyanobacteria phyla, Chromatiales, Bacillales and Methylococcales orders and Aggregatibacter, Clostridium sensu stricto, Acinetobacter, Rhodolyulum, Albimonas, Propioniclava and Psychrobacter genera. Inferred metagenome analysis showed that 27 pathways could be significantly changing in the fish fed 50/100LSAqua diets compared to CTRL ones. Pathways related with IL-17 signalling pathway, Th17 cell differentiation, and antigen processing and presentation were underrepresented in 50/100LSAqua fish, whereas quorum sensing, flavone and flavonoid biosynthesis, isoflavonoid biosynthesis, Fc gamma R-mediated phagocytosis, and antimicrobial production were markedly overrepresented (Fig. 1).

[Methods]: The feeding trial (8 weeks, May-July) with control (CTRL) and experimental diets (50LSAqua, 1000LSAqua) was conducted in triplicate 500 L tanks under natural photoperiod and temperature conditions in a flow through system with well aerated sea water (O2 concentration > 5.5 ppm). Fish with an initial body weight of 23 g were fed near to visual satiety one time per day, six days per week. Nine fish per group were sacrificed, and the adherent bacteria from the anterior intestinal portion were collected and immediately used for DNA extraction. The V3-V4 region of the 16S rRNA of each individual sample was amplified and sequenced by Illumina MiSeq. After quality filtering, taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq, and beta diversity using PERMANOVA and PLS-DA models. Metagenome prediction and pathway analysis were performed using Piphillin.

[Conclusions]: These results point to a reduced activity of IL-17 pathways in fish fed LSAqua diets, which, among their functions, is to limit proliferation of resident bacteria (Douzanez-Mobarrez et al., 2019). This anti-inflammatory response would be reinforced by the over-representation in the gut microbiota of OTUs involved in flavones and flavonoids biosynthetic processes. At the same time, host defence and inflammation processes would be counter-regulated by the overrepresentation of mucosal microbes involved in the natural production of antimicrobials, which would contribute to control proliferation of specific bacteria in our model of carnivorous fish fed with FM-free diets.

[Introduction]: The sustainability and physiological effects of high inclusion levels of new feed ingredients is often questioned, and their potential application requires the use of conventional methodologies, but also cutting-edge tools, for unravelling the close talk between diets, host metabolism and gut microbiota (Fontinha et al., 2020). In this challenging scenario, the aim of the present study was to evaluate, in a high valuable farmed fish model (gilthead sea bream), the effects on growth performance and gut microbiota of partial (50%) and total (100%) replacement of fish meal (FM) by a combination of processed animal proteins and single cell proteins.

This work was funded by the TNA programme (project ID AE170009) within H2020 AQUAEXCEL2020 project (652831) to PSJ and DVM for accessing to IATS-CSIC facilities.

Trabajo presentado en Aquaculture Europe 2020, celebrado en modalidad virtual del 12 al 15 de abril de 2021.