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Resumen del trabajo presentado en el Mucosal Health in Aquaculture, celebrado en Madrid (España) del 03 al 06 de octubre de 2022., Secreted mucins are highly O-glycosylated glycoproteins of high molecular mass, produced by goblet cells in mucosal epithelia. These complex biomolecules have a long peptide backbone, the apomucin of 5,000-13,000 amino acids, coated by hundreds of extremely diverse carbohydrate sidechains accounting for 80% of the mucin’s molecular mass. Mucin molecules multimerize in a mesh, constituting the viscous gel layer overlying the epithelia, which renders physical protection against erosion and dehydration and maintains homeostasis, offers the substrate for the resident and transient microbial communities, and is involved in pathogen recognition, adhesion and expulsion. The monogenean Sparicotyle chrysophrii is responsible for the widespread sparicotylosis in gilthead sea bream (GSB), invoking severe anaemia and compromising the fish’s immune condition. This ectoparasite attaches to the secondary lamellae of the gills with numerous haptor clamps and feeds on fish blood. This work aimed to identify changes in the mucus secretion of the gills of GSB suffering sparicotylosis, particularly changes related to mucus composition and goblet cell abundance and distribution. Understanding the underlying molecular mechanisms of goblet cell and mucin regulation will be addressed at a transcriptional level. Control unexposed (C, n = 23) and recipient (R, n = 28) GSB experimentally infected with S. chrysophrii were sampled at seven consecutive times: 0, 11, 20, 32, 41, 61 and 81 days post-exposure (dpe). Gills were sampled for paraffin histology (Bouin-fixed) and PCR array (RNAlater-preserved). Paraffin sections were stained with periodic acid Schiff (PAS) to demonstrate neutral mucins or with alcian blue (pH 2.5) to identify acidic mucins, and distinct terminal glycoconjugates were detected by lectin labelling (Concanavalina ensiformis, ConA; Ulex europaeus, UEA; Triticum vulgaris, WGA; Glycine max, SBA; Griffonia simplicifolia, BSL I; Sambucus nigra, SNA). Goblet cell abundance was estimated semiquantitatively using the different stainings at four different gill locations (filament tip, interlamellar pockets, lamellar epithelium and raker epithelium), and the intensity of each lectin labelling in these goblet cells was semiquantitatively registered. In addition, the presence and staining of discharged extracellular mucus were registered. Neutral goblet cells were ubiquitous in all gill locations. Their amount significantly increased at filament tips, interlamellar pockets and lamellar epithelia of R fish from 32 dpe on. Mucins did not acidify during the course of infection, but their glycosylation pattern varied depending on the specific gill site. The increase of SBA-label and decrease of SNA- and UEAlabel were signs of immature mucin secretion and a reduced capacity for microbial trapping. Histological observations demonstrated the onset of goblet cell hyperplasia after the first month of parasite exposure, matching a host response for adult worm expelling. The immature mucin features of the increased mucus secretion might limit the host’s capacity to regulate mucosal protection. The transcriptomic study of goblet cell and mucin regulation will shed some light on the underlying mechanisms.

Resumen del trabajo presentado en Mucosal Health in Aquaculture (MHA 2022), celebrado en Madrid (España) del 03 al 06 de octubre de 2022., In parallel with the growth of the aquaculture industry, an increasing interest in fish microbiota research has grown to improve fish welfare and nutrition, as it plays a critical role in protection against pathogens, nutrient digestion and absorption, excretion of nitrogenous wastes, and osmotic regulation. Thus, the characterization of microbial communities is pivotal for detecting potential alterations that lead to fish homeostatic imbalance. During intensive fish farming, high rearing densities can be achieved, and this can dramatically reduce water oxygen availability, especially with the summer increase of temperature. This study aimed to assess how mild hypoxia and relatively high stocking densities affect gilthead sea bream skin microbiota, and how these results could correlate with different operational welfare indicators (OWIs), in order to improve fish health and aquaculture productivity. Hereby, two-year-old gilthead sea bream (450-500 g) were tagged and distributed in 3,000 L tanks to achieve three different initial rearing densities (low, LD: 6 kg/m3; medium, MD: 12 kg/m3, high, HD: 22 kg/m3). Fish were fed close to satiety with a commercial diet from May to June (8 weeks) under natural photoperiod and temperature conditions at our latitude (40°5’N;0°10’E), varying the concentration of dissolved oxygen from 6-5 ppm in LD fish to 5-4 ppm in MD fish, and 4-3 ppm in HD fish. At the end of the trial, growth rates of HD fish were 40% lower in comparison to MD/LD fish. At this stage, randomly selected fish (12 fish per group) were evaluated for external tissue OWIs, using a scoring system from 1 to 5, which detected different levels of fin damage and skin epidermal lesions in HD fish and to a lower extent in MD fish. The same fish were used to obtain skin mucus samples for microbiota analysis, the 16S rRNA v1-v9 regions were sequenced with the Oxford Nanopore Technology (ONT) MinION device, and then processed with an in-house bioinformatics pipeline to analyze skin microbial composition. Discriminant analysis showed that the skin microbiome of the three different groups differed in bacterial abundance. However, in all the groups the dominant phyla were Proteobacteria (73-94%), Firmicutes (2-15 %), and Bacteroidetes (1-7%), and so as the family Alteromonadaceae (11-45%). Changes in bacterial composition, together with OWIs of external tissue damage can provide new insights into how crowding and hypoxia conditions have an effect on fish welfare and microbiota.

Resumen del trabajo presentado en Aquaculture Europe, celebrado en Rimini (Italia) del 27 al 30 de septiembre de 2022., [Introduction]: The AquaIMPACT H2020 EU project aims to integrate selective breeding and nutrition for producing more sustainable marine fish with a higher phenotypic plasticity to deal with environmental challenges. At this interplay between diet and genetics, gut microbiota emerge as a reliable criterion to assess the selection practices. However, the influence of the host genome on the composition and activity of gut microbiota is still scarcely investigated in gilthead sea bream (Piazzon et al., 2020). Thus, the aim of this study was to unravel the main effects of alternative diet formulations along the production cycle of gilthead sea bream selected for high growth (HG) compared to reference (REF) fish, establishing correlations between intestinal gene expression and gut microbiota abundance., [Methods]: HG and REF gilthead sea bream were fed CTRL (15% FM; 6-8% FO) and FUTURE (7.5% FM; 12% poultry sub-products and 3% DHA-rich algae oil) diets during 12 months at the experimental facilities of ECOAQUA, at ULPGC, under natural photoperiod and temperature conditions. Initial (t0; Nov-19; 10 g body weight; 18 fish), intermediate (Jul-20; t1; 200 g; 36 fish) and final (Nov-20; t2; 350 g; 40 fish) sampling points were established to assess the dynamics of anterior intestine (AI) mucosal adherent microbiota composition across the production cycle. As the most prominent differences in gut microbiota among groups were found in t1, fish of this sampling point were selected for RNA-sequencing. V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq, and taxonomy was assigned using the RDP database. RNA-seq libraries were sequenced by Illumina NovaSeq6000. Good-quality reads were mapped against CSIC gilthead sea bream genome and differential expressed transcripts (DET) were retrieved using DESeq2 (FDR < 0.05). Overrepresentation analyses of GO and KEGG terms were implemented in goseq and protein-protein association networks were retrieved using STRING (FDR < 0.05). DET expression and OTUs abundances of the equivalent fish of t1 were correlated using Spearman correlation tests (P < 0.001)., [Results]: Illumina sequencing and taxonomic assignment of the 94 samples allowed the identification of 1,897 OTUs at 97% identity threshold along the gilthead sea bream production cycle. Taking into account the entire population, Chao1 richness index progressively decreased with age, whereas the α-diversity Simpson index was significantly higher in t1 and t2 than in t0. Proteobacteria was the most abundant phylum (64-78%) in all the sampling times, followed by Firmicutes (13-15%), Actinobacteria (6-12%), and Bacteroidetes (~2%). The core microbiota of gilthead sea bream along the production cycle was constituted by 25 OTUs (>1% in at least one of the three sampling times). A validated PLS-DA (Figure 1A) rendered the separation of the three experimental time points. The FUTURE diet induced a shift in the gut microbiota composition of REF fish, validated by PLS-DA, but not in HG fish. The influence of the genetic background was already evidenced at t0, when discriminant analyses separated HG from REF fish. The magnitude of this separation and the number of discriminant taxa persisted along time, but progressively decreased in t1 and t2 in fish were fed CTRL diet. By contrast, HG and REF fish microbiota was not distinguished when fed the FUTURE diet. In t1, RNA-seq mapped reads were associated to 41,582 AI transcripts, with 1,429 DET (1,218 unique descriptions). Group comparisons displayed 1,057 and 459 DET differentially regulated between HG-CTRL and REF-CTRL fish, and between HG-FUTURE and HG-CTRL, respectively. A total of 1,025 significant associations were established between the abundance of 96 OTUs and the expression of 476 DET. A total of 35 correlations between 20 abundant (>1%) OTUs and 25 DET were stablished (Figure 1B). The 60% of the correlated OTUs were associated to DET related with immune system, with a clear presence of Bacilli linked to anti-inflammatory responses. The microbiota-independent list of DET (953) showed the over-representation of transcripts involved in β-oxidative processes of very long chain fatty acids (up-regulated in HG-CTRL) and smooth muscle contraction (upregulated in HG-FUTURE)., [Conclusions]: These results highlight the plasticity of gilthead sea bream gut microbiota along its production cycle, showing the effects of size and season. As in previous studies (Naya-Català et al., 2022), the genetic background strongly influenced the gut microbial composition and the intestinal response of this species, as selected fish exhibited a more plastic microbiota and more changes at the transcriptomic level, when facing dietary changes. Genes related with cell membrane fluency were up-regulated in HG-CTRL fish, whereas FUTURE diet boosted genes involved in intestinal motility in both fish groups. Correlation studies denoted the close link between immune system genes and the intestinal microbiota. Altogether, these results show how diet and genetics can affect the microbiota and the digestive process, and highlight that certain taxa could be interacting with the gilthead sea bream transcriptome., AquaIMPACT (H2020; #818367). RYC2018-024049-I & ESF.

Trabajo presentado en Aquaculture Europe, celebrado en Rimini (Italia) del 27 al 30 de septiembre de 2022., [Introduction]: Studies in gilthead sea bream evidenced that high stocking densities exacerbate the negative impact of reduced oxygen availability, showing transcriptional analyses a different contribution of analyzed tissues (liver ≥ heart > muscle > blood) to the hypoxic- and crowding stress-mediated responses to cope with a changing environment (Martos-Sitcha et al., 2019). Moreover, blood metabolic and muscle transcriptomic landmarks indicate that mild hypoxia induces a hypometabolic state, increasing the contribution of lipid metabolism to the whole energy supply to preserve the aerobic energy production during exercise (Naya-Català et al., 2021a). The intestinal microbiota also has key effects on host health and welfare, and these complex populations tightly interact with the host affecting local and systemic physiological functions (Naya-Català et al., 2021b). Thus, the aim of the current study was to assess how mild hypoxia and high stocking rearing conditions affect the intestinal health of gilthead sea bream by applying a hologenomic approach to determine the host-microbiota interactions., [Methods]. Two-year-old gilthead sea bream (450-500 g) were tagged and distributed in 3,000 L tanks to achieve three different initial rearing densities (low, LD: 6 kg/m3; medium, MD: 12 kg/m3; high, HD: 22 kg/m3). Fish were fed close to satiety with a commercial diet from May to June (8 weeks) under natural photoperiod and temperature conditions at our latitude (40°5’N;0°10’E), varying the concentration of dissolved oxygen from 6-5 ppm in LD fish to 5-4 ppm in MD fish, and 4-3 ppm in HD fish. At the end of the trial, the growth rate of HD fish was 40% lower than that of MD/LD fish, and randomly selected fish from each group were taken to obtain tissue and mucus samples from the anterior intestine. RNA extracted from the anterior intestine from 10 fish/group was sequenced using NovaSeq PE150 (50 M reads/sample), and reads were assembled and mapped against the gilthead sea bream genome. DESeq2 was used to extract genes with P < 0.05 in all comparisons, that were used to perform discriminant and cluster analysis to determine the number of differentially expressed genes among conditions. K-means, GO and KEGG enrichment analyses were subsequently performed. The V3V4 regions of the 16S rRNA of the mucus DNA samples from the same animals were 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. Correlations between differentially expressed genes and discriminant bacteria were studied using the corrplot R package., [Results]: RNAseq and discriminant analyses revealed that a total of 2,813 differentially expressed genes significantly separated the three groups (Fig. 1A). K-means analysis divided these genes into four different clusters according to their expression patterns in each group, separating one cluster of 800 genes with higher expression in LD, a second cluster of 1,103 genes with higher expression in MD, a third cluster of 688 genes with higher expression in HD, and a fourth cluster of 222 genes that gradually increased expression with the stocking density (LD, [Conclusions]: Crowding and mild hypoxia had a significant impact on gut health, evidenced by significant changes in host intestinal transcriptome and associated microbial population. These results offer the possibility of developing new tools and approaches for a more precise evaluation of welfare in farmed fish. The ultimate goal is to mitigate the negative impact of stressors related to intensive rearing and climate change on aquaculture production, promoting a more ethical and sustainable production., AQUAEXCEL3.0 (H2020 #871108), EATFISH (H2020 #956697).

Trabajo presentado en la 15th International Conference of Parasitology, celebrada en Copenhague (Dinamarca) del 21 al 26 de agosto de 2022., [Introduction]: Sparicotylosis is a ubiquitous disease affecting mainly gilthead sea bream (GSB; Sparus aurata) across the Mediterranean, and it is caused by the polyopisthocotylean monogenean gill parasite, Sparicotyle chrysophrii (Microcotylidae). The disease involves severe anaemia and white-gill syndrome. This study aims to elucidate how the parasite modulates the GSB health status throughout the infection., [Methods]: Experimental infections were conducted in a recirculating aquaculture system (RAS), exposing naïve GSB (R) to effluent water from S. chrysophrii-infected GSB. An additional tank with unexposed naïve fish (C), was maintained in parallel, with open water flow disconnected from the RAS. Haematological and infection parameters from sampled C and R fish were registered. Plasma samples grouped into 4 categories (high-H, medium-M, low-L and control-C) according to infection intensities, underwent a proteome analysis. Additional assays were performed to validate the proteome analysis findings., [Results]: The differential analysis of the plasma protein abundance revealed a clear separation into 3 groups (H, M-L and C). A pathway analysis was performed with the differentially quantified proteins, indicating that the parasitic infection mainly affected pathways related to homeostasis, complement system and lipid transport. Twenty-two proteins significantly correlated with the infection intensity, highlighting apolipoproteins, globins, biotinidases and complement C3. Validation assays in blood and plasma confirmed these correlations., [Conclusions]: The anaemic condition of severely S. chrysophrii-infected GSB is accompanied by innate immunity impairment, and altered lipid profile and biotin metabolism, besides the evident hypoxia.

Resumen del trabajo presentado al 6th International Symposium on Genomics in Aquaculture, celebrado en Granada (España) del 04 al 06 de mayo de 2022., SAMBA (Scanning Aquaculture Microbiome via Bayesian Approach) is a Guide User Interface application developed in R and powered by a Bayesian network model that indexes the frequencies of the bacterial taxa in a given biological system, correlating microbial architectures with a number of biotic and abiotic factors (e.g. sex, age, season, diet composition, genetic background, etc.). The application identifies conditional dependencies and interrelations among the distinct variables indexed in the network, allowing to predict how the taxa abundance of the pan-microbiome will vary depending on experimental variables. The application permits to predict and assign a metagenome to each inferred microbiome in order to determine its functional profile. SAMBA also permits to interrogate the Bayesian Network model to identify which experimental conditions are optimal for obtaining a given panmicrobiome and associated metagenome. At this stage, a locally run application of SAMBA was used to interrogate the pan-microbiome of farmed gilthead sea bream (a highly valued farmed fish in all the Mediterranean region) with different nutritional and genetic backgrounds across the production cycle. The application can also be used to model the host, metagenome and environment interrelations in a wide range of organisms, including humans. Interfaces of SAMBA were wrapped by Shiny, a framework to build interactive web applications by R. The application uses distinct R dependencies such as 1) bnlearn, for learning the structure of Bayesian Networks and estimate their parameters based on its structure using data from 16s amplicon experiments, 2) visNetwork and bnviewer for network visualization, and 3) PICRUSt2 to infer metagenomic functions from the microbiome population determined by 16s data given specific environmental conditions. SAMBA will be implemented to be accessible as a web server at www.nutrigroup-iats.org, This work was supported by a pre-doctoral research fellowship from Industrial Doctorate of MINECO to BS (Grant 659 DI-17-09134). Additional funding was obtained from National (BreamAquaINTECH, RTI2018-09412-B-I00; RYC2018-024049-I & ESF) and European (AquaIMPACT, H2020 #818367; EATFISH (H2020 #956697) Projects.

Resumen del trabajo presentado en 6th International Symposium on Genomics in Aquaculture, celebrado en Granada (España) del 04 al 06 de mayo de 2022., With the growth of the aquaculture industry, there has been a growing interest in manipulating fish gut microbiota to improve welfare and nutrition since it is critical for many host functions such as digestion, nutrient metabolism, disease resistance, and immune function. Accurate and rapid identification of microbial communities through sequencing of the 16S ribosomal RNA (16S rRNA) gene is an approach widely used since the introduction of high-throughput sequencing technologies (HTS). Nowadays, several affordable methods involving different sequencing technologies exist, being 16S amplicon Illumina sequencing the most widely used. However, the choice of sequencing platform and the downstream analysis are known to yield somewhat different results. Of particular interest, Oxford Nanopore MinION Technology offers a low price, portability, and fast sequencing throughput compared to the Illumina MiSeq and PacBio sequencers. Nevertheless, the error rate of the generated amplicon data is higher, and differences in the experimental conditions for 16S rRNA-based PCR could bias microbiota assessments in the samples. To find a suitable solution that could offer reliable results considering the time, cost, and sequencing throughput, this study aimed to 1) Evaluate the feasibility of MinION sequencing by comparing16S amplicon data of gilthead sea bream intestinal microbiota using the Illumina MiSeq, PacBio (performed with standard procedures by sequencing companies), and MinION (on-site sequencing) platforms; and 2) Standardize the experimental 16S protocol for sample preparation and PCR conditions when sequencing microbiota samples with the Oxford Nanopore MinION Technology. Different 16S gene regions were amplified depending on the sequencing platform (V3-V4 for Illumina MiSeq and full 16S rRNA gene for MinION and PacBio), and bioinformatics pipelines were conducted according to each sequencer amplicon data. Concerning the MinION protocol, five PCR conditions were tested using the Nanopore 16S barcoding kit, combining different temperatures, cycling conditions, and nesting. For all samples, the taxonomic assignment was made using the SILVA (http://www.arb-silva.de) database v. 138. The coming results will provide precise insights into the advantages and disadvantages of this specific sequencing platform, and their suitability depending on the experimental approach, expected output, and cost and time constraints of a given experiment., EATFISH (H2020 #956697), AQUAEXCEL3.0 (H2020 #871108), RYC2018-024049-I & ESF.

Resumen del trabajo presentado en 6th International Symposium on Genomics in Aquaculture, celebrado en Granada (España) del 04 al 06 de mayo de 2022., The use of feed additives has expanded rapidly as an alternative for antibiotics and chemotherapeutics, with also the capacity to modify the composition of gut microbiota. In farmed fish, and in gilthead sea bream in particular, there is evidence that the host genetic background has a major impact on gut microbiota. Thus, families selected for fast growth have a more flexible microbiota capable of exerting a wider nutritionallymediated response with less microbial community changes. However, nutrition and genetic interactions remain poorly explored in fish, and the aim of this study was to unravel how the microbiota of fish selected (GS) and unselected (NGS) for growth is differentially regulated by oil-coated feed additives. The basal diet (CTRL, no feed additives) was a low fish meal/fish containing algae oil, poultry by-products, and plant ingredients. Experimental diets were oil-coated with the additives: organic acids (OA), Bacillus-species probiotics (PROB), or natural plant extracts (PHYTO). Fish were fed to visual satiety with the CTRL diet during two weeks. After this adaptation period, the different supplemented diets were used with a high additive dose (7.5-10 g/kg) during 2 weeks, decreasing thereafter to 2-5 g/kg until the end of the trial (97 days). Then, adherent microbiota was obtained from the anterior intestine. Illumina sequencing of microbiota yielded a mean of 62,594 reads per sample, which were assigned to 1,156 OTUs at 97% identity threshold. A significant lower richness and diversity was found in the GS fish, which was mainly evidenced by a higher abundance of Actinobacteria in GS-PROB. To study in more detail the observed differences on gut microbial populations, supervised partial least-squares discriminant analyses (PLS-DA) were used. When all populations were analysed as a whole (GS vs NGS), dispersal was markedly lower in GS fish. This pattern was further evidenced for fish fed the CTRL, PROB and OA diets but not for the PHYTO additive, explaining the statistically validated PLS-DA models more than 80% of the total variance. This genetically-guided group differentiation was driven by a total of 104 OTUs. Regarding diet and genetic interactions: i) no effect was detected with the PHYTO additive, ii) the OA reshaped the gut microbiota in NGS with a decrease of Photobacterium damselae sp. and an increase of Paracoccus and Acinetobacter genera, and iii) the PROB diet modified the gut microbiota of both GS and NGS fish, with a higher abundance of Kocuria and Bacillus genera, which reflected the establishment of the probiotic bacteria in the mucosal adherent surface, favouring long-term health promoting probiotic effects., AquaIMPACT (H2020 #818367), RYC2018-024049-I & ESF

Trabajo presentado en el 22nd Fish Immunology, celebrado en Wageningen (Países Bajos) del 24 al 28 de abril de 2022., Mucosal (gut, skin, gills) microbial patterns will be assessed in farmed gilthead sea bream (Sparus aurata), combining the use of conventional (Illumina platform) and portable (Oxford Nanopore, MinION) platforms for bacteria sequencing and taxonomic assignment of short- (v3-v4) and long-reads (v1-v9) of 16S rRNA gene, respectively. Changes in microbial patterns in response to biotic (host genetics, pathogen exposure) and abiotic (water temperature, oxygen availability, diet) factors will be assessed to relate changes in the mucosal metagenome and metatranscriptome with a healthy and stress resilience condition. Such data in combination with host transcriptomics (RNA-seq, targeted PCR-arrays), composition and activation of immune cells in gut and gills, and operational welfare indicators based on indicators of growth performance, external tissue damage, and behavioral traits (data-logger measures, activity, and ventilation rates) will serve to fed a meta-analysis system with machine-learning capacity. This will include the combined use of multivariate discriminant analyses (PLS-DA) and Bayesian networks, which will allow correlating microbial taxa among them and with challenging experimental variables. The combination of background knowledge and the previously mentioned tools will allow monitoring changes in the microbiota of gilthead sea bream in response to biotic and abiotic factors and link them to fish health and performance indicators. Thus, filling fish microbiota knowledge gaps by integrating newly generated information will help understand which microbial taxa and genes relate to host health in aquaculture.

This study aimed to highlight the relationship between diet, animal performance and mucosal adherent gut microbiota (anterior intestine) in fish fed plant-based diets supplemented with an egg white hydrolysate (EWH) with antioxidant and antiobesogenic activity in obese rats. The feeding trial with juveniles of gilthead sea bream (Sparus aurata) lasted 8 weeks. Fish were fed near to visual satiety with a fish meal (FM)/fish oil (FO) based diet (CTRL) or a plant-based diet with/without EWH supplementation. Specific growth rate decreased gradually from 2.16% in CTRL fish to 1.88% in EWH fish due to a reduced feed intake, and a slight impairment of feed conversion ratio. Plant-based diets feeding triggered a hyperplasic inflammation of the anterior intestine regardless of EWH supplementation., This work was supported by the EU H2020 Research Innovation Program under the TNA Program (project AE150009) at IATS-CSIC Research Infrastructure within AQUAEXCEL2020 Project (652831). This output reflects only the author’s view and the European Union cannot be held responsible for any use that may be made of the information contained herein. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH and RTI2018–094128-B-I00). MCP was funded by a Ramón y Cajal Postdoctoral Research Fellowship [RYC2018-024049-I/AEI/10.13039/501100011033 cofunded by the European Social Fund (ESF) and ACOND/2020 Generalitat Valenciana].

The present research tested the effects of dietary nisin-producing Lactococcus lactis on growth performance, feed utilization, intestinal morphology, transcriptional response, and microbiota in gilthead sea bream (Sparus aurata). A feeding trial was conducted with fish weighting 70–90 g. Fish were tagged with passive, integrated transponders and distributed in nine 500 L tanks with 40 fish each. Fish were fed for 12 weeks with either a control (diet A) or experimental diets (diets B and C) in triplicate (3 tanks/diet). Extruded pellets of diets B and C were supplemented with a low (2 × 109 CFU/kg) and a high (5 × 109 CFU/kg) dose of probiotic, respectively. No significant differences were found between groups for the feed conversion ratio or specific growth rates. However, the final body weight of fish fed diet C was significantly higher than the control group with intermediate values for fish fed diet B. Histological analysis conducted using a semi-quantitative scoring system showed that probiotic did not alter the morphology of the intestine and did not trigger inflammation. With regard to the transcriptomic response, a customized PCR array layout was designed to simultaneously profile a panel of 44 selected genes. Significant differences in the expression of key genes involved in innate and acquired immunity were detected between fish fed probiotic and control diets. To analyze the microbiota associated to the feeds and the gut autochthonous microbial communities, we used the Illumina MiSeq platform for sequencing the 16S rRNA gene and a metagenomics pipeline based on VSEARCH and RDP databases. The analysis of gut microbiota revealed a lack of colonization of the probiotic in the host’s intestinal mucosa. However, probiotic did modulate the fish gut microbiota, confirming that colonization is not always necessary to induce host modification. In fact, diets B and C were enriched with Actinomycetales, as compared to diet A, which instead showed a higher percentage of Pseudomonas, Sphyngomonas, and Lactobacillus genera. These results were confirmed by the clear separation of gut bacterial community of fish fed with the probiotic from the bacterial community of control fish group in the beta-diversity and PLS-DA (supervised partial least-squares discriminant analysis) analyses., This work was supported by the EU Horizon 2020 Research Innovation Program under the TNA program (project AE130011) at IATS-CSIC research infrastructure within AQUAEXCEL2020 project (652831). This work was also co-funded by the EU Horizon 2020 AquaIMPACT project (Genomic and nutritional innovations for genetically superior farmed fish to improve efficiency in European aquaculture; 818367). This output reflects only the author’s view and the European Union cannot be held responsible for any use that may be made of the information contained herein. MP was funded by a Ramón y Cajal Postdoctoral Research Fellowship [RYC2018-024049-I/AEI/10.13039/501100011033 cofunded by the European Social Fund (ESF) & ACOND/2020 Generalitat Valenciana].

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: In the animal production sector, there is an increasing interest in manipulating intestinal microbiota due to their undeniable key effects on host health and welfare. To that aim, numerous studies have been conducted in order to define microbial populations and changes in fish intestine under different conditions. However, most of the studies conducted so far are focused on describing the presence and abundance of bacterial populations based on DNA sequencing technologies. In the current study, we aimed to define the actual metabolic potential of fish intestinal resident microbiota, by studying the gene expression of all gut microbes instead of just defining presence/absence of bacteria. To that aim, we used two families of gilthead sea bream (Sparus aurata) selected for heritable growth (fast- and slow-growth families) and fed control or plant-based diets. In previous studies, the fast-growth family showed a more continuous growth across seasons, genetically regulated intestinal plasticity to maximize nutrient absorption when fed plant-based diets, improved resilience to intestinal parasites, and a plastic microbiota that efficiently adapted to the metabolic challenges induced by diet changes (Perera et al., 2019; Piazzon et al., 2020). Upon diet changes, only a small percentage of the adherent bacterial populations changed in the fast-growth family, but these small changes seemed to account for higher metabolic changes (inferred metagenome and pathway analysis) when compared to the slow-growth family. Here, we compare the transcriptional profile of the total microbial populations in fast- and slow-growth families in order to validate and have further details on the previous observations., [Methods]: Two gilthead sea bream families, selected for fast- (e6e2) and slow-growth (c4c3) were kept together in the same open-flow tanks and fed a control or a well-balanced plant-based diet during nine months. Eight animals per group were sacrificed and the adherent microbiota from the anterior intestinal portion was collected and used for RNA extraction. RNA samples were pooled in groups of two to yield a total of four samples per group. After rRNA removal, RNA was sequenced (Illumina, 150PE). Reads were quality filtered and the transcriptome was reconstructed using Trinity. For annotation, the obtained unigenes were aligned with Bacteria, Fungi, Archaea and Viruses sequences from the NCBI’s NR database using Diamond. Partial least squares discriminant analyses (PLS-DA) and DESeq2 were used to determine group differences and differentially expressed genes among groups. Gene ontology analysis was performed using GOSeq., [Results]: The total number of annotated transcripts found in this study was 35,144, from which 232 (0.7%) were Archaea, 17,516 (49.8%) Bacteria, 15,703 (44.7%) Fungi, and 1,693 (4.8%) Virus. Regarding the level of expression of the transcripts per group and organism, no differences were detected between families, but differences were found between dietary groups. Plant-based diets increased the number of Fungi transcripts and decreased bacteria transcripts. Diet and genetic background were responsible for changing the expression of 425 and 329 transcripts, respectively. PLS-DA analysis showed a significant separation between fast- and slow-growth families, and within the fast-growth family the different diets showed a significantly different profile. However, diet driven differences were not detected in the slow growth family (Fig. 1). This was supported by the DESeq2 analysis which showed a change in 271 transcripts in the fast-growth family when fed different diets, whereas only 40 differentially expressed transcripts were found in the slow-growth group. GOSeq analyses revealed that plant-based diets were upregulating genes involved in anatomic structure morphogenesis and development, and downregulating genes involved in several metabolic pathways, lipid localization, cytolysis and killing of cells of other organisms, and response to stress in the fast-growth group. Downregulation of vitamin metabolism and upregulation of carbohydrate metabolism, cytokine production, movement and entry into host, and cell proliferation was found in the slow growth group., AquaIMPACT (H2020 #818367); Bream-AquaINTECH (RTI2018–094128-B-I00); RYC2018- 024049-I/AEI/10.13039/501100011033.

The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. The maximized replacement of fish meal (FM) and fish oil (FO) with plant ingredients has been extensively studied in the economically important species gilthead sea bream (Sparus aurata). Recently, major progress has been done with other alternative raw materials, though some non-pathological inflammatory response persisted with feed formulations that increased the circularity of resource utilization. In the present study, we evaluated the effects on growth performance, gene expression, intestinal microbiota and disease resistance of a FM-free diet (NoPAP SANA), based on plant ingredients, aquaculture by-products, algae oil, insect meal and bacterial fermentation biomasses as main dietary oil and protein sources, and supplemented with a commercially available health-promoting feed additive (SANACORE®GM). Juveniles of 21 g initial body weight were fed control or NoPAP SANA diets for 34 days, and head kidney, liver and posterior intestine were collected for gene expression analyses using customized PCR-arrays. Each tissue-specific PCR covered 96 genes in total and included markers of growth performance, lipid and energy metabolism, antioxidant defence, immune system, and intestinal function and integrity. From the same fish, the adherent bacteria of the posterior intestine were studied by Illumina sequencing of the V3-V4 region of the 16S rRNA. The remaining fish were challenged with the intestinal parasite Enteromyxum leei for 78 days and sampled for parasite diagnosis. Both control and NoPAP SANA fish grew efficiently considering gilthead sea bream standards. Before parasite challenge, the NoPAP SANA group showed differential expression of 17, 2 and 4 genes in liver, head kidney and posterior intestine, respectively. The intestinal bacterial composition showed no major differences in diversity or at the phylum level. However, 29 abundant OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differentially expressed genes in the different target tissues. Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge did not significantly vary between dietary treatments, and infected fish from both groups showed similar disease outcome. Altogether, these results indicate that the NoPAP SANA diet promoted optimal growth and a healthy condition in gilthead sea bream without affecting susceptibility against the tested intestinal parasite, as often observed with alternative diets following current industry formulations., This project has received funding from the European Union's Horizon 2020 Research and Innovation Programme under grant agreement No 773330 (GAIN, Green Aquaculture Intensification). This publication reflects the views only of the authors, and the European Commission cannot be held responsible for any use which may be made of the information contained therein. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH, RTI2018–094128-B-I00, AEI/FEDER, UE). M.C·P was funded by a Ramón y Cajal Postdoctoral Research Fellowship (RYC2018-024049-I, co-funded by the European Social Fund & ACOND/2020 Generalitat Valenciana).

Trabajo presentado en el 6th International Symposium on Genomics in Aquaculture, celebrado en Granada (España) del 05 al 07 de mayo de 2021., The aquaculture industry continues to grow faster than any other food production sector. This growth was also convoyed by increased consumer environmental awareness and knowledge. Hence, the necessity to make aquaculture as sustainable as possible became more obvious day-byday. This work is part of GAIN project and aims to rise solutions to this need. For that four diets were formulated for in gilthead seabream (Sparus aurata) rich in animal protein (PAP), without animal protein (NOPAP) and a combination of diverse emerging ingredients (MIX), against a control diet. The selected ingredients were defined based on circularity principles, maximizing resource efficiency. The immune parameters analyzed (bactericidal, IgM, protease and antiprotease activity) suggest that fish fed with NOPAP diet showed a slight stimulation of innate immunity. This is corroborated by the head kidney gene expression results, which also showed that il-8 was up-regulated in NOPAP, PAP and MIX groups. The PAP group presented a strong proinflammatory profile, evidenced not only by the up regulation of il-8 but also other cytokines (il1β, tnf-α), chemokines (ck8) and chemokine receptors (ccr3). The same pattern was found for the T-cell markers cd3x, cd4-full and cd8a, whereas the expression of the mucosal igt-m was consistently down-regulated. Both MIX- and NOPAP-fed fish showed a more attenuated response, since compared to the control diet, only a reduced number of genes was differentially expressed, il8 in NOPAP-fed fish, and il-8, il-1β and ck8 in the MIX diet group. These results support the hypothesis that these new formulations are viable options for seabream feed, in particular the NOPAP.

Trabajo presentado en Aquaculture Europe 2020, celebrado en modalidad virtual del 12 al 15 de abril de 2021., [Introduction]: A bioactive egg white hydrolysate (EWH) treated with pepsin has demonstrated potent in vitro and in vivo antioxidant and anti-inflammatory properties, improving oxidative stress and inflammation biomarkers on genetically and diet induced obese rats (Requena et al., 2017). However, the effects of protein hydrolysates and bioactive food-derived peptides on gut microbiome remain relatively poorly studied in mammals and fish in particular. Thus, the aim of this study was to unravel the main effects on fish performance, histopathological scoring and mucosal adherent gut microbiota of EWH supplementation in a fish fed a formulation with a high replacement of marine feedstuffs by alternative plant ingredients, using gilthead sea bream as a farmed fish model. [Methods]: The feeding trial lasted 8 weeks (May-July) under natural photoperiod and temperature conditions. Juvenile fish (20-24 g initial body weight, 4.8-4.9 kg/m3) were fed near to visual satiety with control (CTRL) or low fish meal (FM)/fish oil (FO) diets with/without egg white hydrolysate (EWH) supplementation (7.5%). DNA from the adherent bacteria of the anterior intestine was collected and the V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq. 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 partial least-squares discriminant analysis (PLSDA) models. Metagenome prediction and pathway analysis were performed using Piphillin., [Methods]: The feeding trial lasted 8 weeks (May-July) under natural photoperiod and temperature conditions. Juvenile fish (20-24 g initial body weight, 4.8-4.9 kg/m3) were fed near to visual satiety with control (CTRL) or low fish meal (FM)/fish oil (FO) diets with/without egg white hydrolysate (EWH) supplementation (7.5%). DNA from the adherent bacteria of the anterior intestine was collected and the V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq. 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 partial least-squares discriminant analysis (PLSDA) models. Metagenome prediction and pathway analysis were performed using Piphillin., [Results]: Daily specific growth rates (SGR) varied significantly from 2.16 in CTRL fish to 1.88 in EWH fish as a result of a reduced feed intake. A slight impairment of feed conversion ratio, from 1.03 to 1.10, was also observed. Intermediate values on growth performance parameters were reported with the low FM/FO diet without EWH supplementation. No changes in total plasma antioxidant capacity, and faecal concentrations of lactic acid and short-chain fatty acids were found among dietary groups. The dietary replacement of FM/FO triggered a hyperplasic inflammation of the anterior intestine submucosa that was not alleviated by EWH supplementation. Conversely, alterations on the staining pattern and amount of goblet cells at the level of anterior intestine were reversed in EWH fish, together with a decreased accumulation of lipid vacuoles in the epithelium of posterior intestine, a high abundance of hepatic melanomacrophage centers, and depletion of hepatocyte lipid depots until the restoration of CTRL fish values. Illumina sequencing reads were assigned to 2,117 OTUs and a significantly lower richness was found in the EWH group. Indeed, at the phylum level, Proteobacteria reached the highest proportion in CTRL and EWH fish, whereas Firmicutes were decreased and Actinobacteria increased with the replacement of FM/FO. The proportion of Actinobacteria was restored to CTRL values with the dietary EWH supplementation. Additionally, EWH triggered the highest amount of Bacteroidetes and Spirochaetes phyla. Detailed differences in microbiota composition were analysed with a statistically validated PLS-DA which clearly separated CTRL fish from fish fed low FM/FO diets along x-axis (component 1, 37.4%), whereas component 2 (43.2%) separated the low FM/FO diets with/without EWH along y-axis (Fig. 1). This analysis disclosed 165 OTUs discriminating among diets (VIP ≥ 1), with 46 OTUs representing at least the 1% in one of the groups. For these abundant bacteria, a first type of response was mediated by 17 OTUs that were increasing with the FM/FO replacement and decreasing again in EWH fish. In this group, Neisseriaceae family and species of Ralstonia, Lactobacillus, Streptococcus, Corynebacterium and Nocardioides genera were included. A group of 14 OTUs were present in high proportion in the CTRL group, but decreased in fish fed the two low FM/FO diets. In this case, the dietary plant ingredients drove the decrease of the Comamonadaceae family and Mesorizhobium, Brochotrix, Bacillus, Clostridium sensu stricto and Exiguobacterium genera. The remaining 15 OTUs increased their proportion in fish fed the EWH diet, being in a very low proportion in the other two dietary groups. This response triggered the presence of Bacteroidetes phylum, Rhodospirilalles order and Granucatella, Bradyrizhobium, Propionibacterium and Streptophyta genera. Inferred metagenome results showed two pathways corresponding to primary bile acid biosynthesis and steroid degradation consistently underrepresented in the microbiota of EWH fish when compared to the other two groups, [Conclusions]: These results reinforce the central role of gut microbiota in the regulation of host metabolism and lipid metabolism in particular (Hegyi et al., 2018), supporting a main role of the EWH as an anti-obesity and satiety factor in fish as suggested in rat models of obesity. The potential use of this functional food ingredient in finishing diets, and the role of gut microbiota in tuning fillet fatty acid composition of marketable fish merits further research., This work was funded by the TNA programme (AE150009) within H2020 AQUAEXCEL2020 project (652831) to GAWP for accessing to IATS-CSIC facilities.

Trabajo presentado en el XII Conference of the Iberian Association for Comparative Endocrinology (AIEC), celebrado en Faro (Portugal), del 26 al 28 de septiembre de 2019, With the use of electronic tracking technology, data on fish movements, physiology and environmental parameters can be collected from each tagged individual. The perfect tag - small size, lasting activity, long distance data transmission (e.g. radiotransmitters, acoustic transmitters, pop-up satellite archival tags) - does not exist, and the choice of telemetry technology and tagging method (external, intracelomic, intragastric) depends on fish species, life stage and research question. The proposed solution within the AQUAEXCEL2020 EU project is a stand-alone, small and light (1 g) device (AEFishBIT), composed of a tri-axial accelerometer, a microprocessor, a battery and an RFID tag for quick-smart identification. The device is externally attached to the operculum to monitor physical activity by mapping accelerations in x- and y-axes, while operculum beats (z-axis) serve as a measurement of respiratory frequency. Initial functional validation has been made with gilthead sea bream and European sea bass juveniles in swimming test chambers, highlighting a high correlation of oxygen consumption and swimming activity with the calculated AEFishBIT records (MartosSitcha et al., 2019; Frontiers in Physiology 10:667). Current studies with free-swimming fish reveal that the attachment protocol does not have a negative impact on growth performance in active feeding fish, offering a wide range of new information based on individual behaviour: i) the switch from nocturnal to diurnal rhythms of activity across season, ii) the different energy partitioning for growth and maintenance, iii) the asynchrony of movements as an indirect measure of aging and adaptability to culture environment, and iv) the different coping behaviour of fish challenged with low water oxygen concentrations. AEFishBIT also provides reliable information of disease outcome in fish parasitized with the intestinal myxozoan Enteromyxum leei, emerging as the ideal complement for sensing the quality of the environment and improving selective breeding protocols., AQUAEXCEL2020 EU Project, GA 652831

Trabajo presentado en el 31st European Society of Comparative Biochemistry and Physiology Congress (ESCBPC), celebrado en Oporto (Portugal), del 9 al 12 de septiembre de 2018, The farming of fish inflicts various stresses upon the animals, which may impact their immune system. A reduced capacity to fight pathogenic agents may entail important losses in aquaculture production. Oestrogens are well-known endocrine disruptors that are present in natural forms in fish farms because non-degraded hormones, such as oestradiol, are released by the fish into the water body. Oestradiol is involved in the regulation of numerous physiological functions, including the modulation of immune system function and performance. Hence, we studied the long-term consequences of exposure to environmentally relevant concentrations of oestradiol on fish immunocompetence. Fingerlings of 47 and 60 days post-hatch were exposed to 20ng/L of oestradiol for 7 days and one month, respectively. Water samples were collected and analyzed by radioimmunoassays. Following exposure, each stage was sampled for gene expression analysis to assess the impacts of oestradiol on the ontogenesis of the thymus, which is the primary lymphoid organ responsible for T cell differentiation. After depuration phase, the fish were challenged by intracoelomic injection with Vibrio harveyi and cumulative mortality was recorded. Exposure to oestradiol, which was detected in the exposure water, from 47 to 54 days post-hatch caused an increase in sea bass mortality following bacterial challenge. By contrast, oestradiol exposure from 60 to 90 days post-hatch improved the fish resistance to vibriosis. As in mammals, oestradiol seems to have immunostimulating or immunosuppressive effects depending on the critical window of development. The relation between these immunomodulatory effects, thymus development and associated peripheral T cells is currently investigated.

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: In a sustainable aquafeed scenario, processed animal proteins (PAP), insect, microbial and algae-products appear as suitable replacers of fish meal (FM) in practical fish diets (Glencross et al., 2020; Basto et al., 2021). However, such new and emerging ingredients have been tested one-by-one rather than using different formulation combinations. In this regard, a previous study in gilthead sea bream (Sparus aurata) pointed out that graded levels of sustainable PAP- and NoPAPbased feed formulations are able to support optimal growth performance when nutrient requirements are met (Fernandes et al., 2021). However, PAP-based feed formulations caused a slight impairment of feed conversion ratio (FCR) associated with a down-regulation of the hepatic insulin-growth factor-I and the up-regulation in head-kidney of pro-inflammatory cytokines, chemokines and T-cell markers. Here, we focused on gut health indicators to assess the main effects of PAP and NoPAP- based diets on the mucosal adherent microbiota of the anterior intestine (AI) and its impact on changes in the host transcriptomic profile of selected markers of intestinal function and integrity., [Methods]: Quadruplicate groups of gilthead seabream were fed ad libitum daily with three different diets in a 77 days feeding trial. The control diet (CRTL) followed a commercial type formulation. In both NoPAP and PAP diets, insect meal, fish by-products, microbial and yeast biomasses were used as FM and vegetable protein replacers. The PAP diet also comprised several PAP ingredients such as poultry meal, feather meal hydrolysate, and porcine blood meal. The NoPAP diet included Spirullina and Chlorella meal as additional protein sources. At the end of the feeding trial, RNA from the AI was collected and run through a PCR-array to profile the expression of a panel of 44 genes, including markers of epithelial integrity, interleukins and immunoglobulins, among others. DNA from the adherent bacteria of the AI was collected and the V3-V4 region of the 16S rRNA of each sample was amplified and sequenced by Illumina MiSeq. Taxonomic assignment was performed against the RDP database. Alpha diversity was calculated using Phyloseq, and beta diversity using partial least-squares discriminant analysis (PLS-DA) models. Metagenome prediction and pathway analysis were performed using Piphillin. Differentially expressed (DE) genes and discriminant OTUs were correlated using the corrplot R package., [Results]: The gene expression analysis revealed 13 DE genes (out of 44) (P < 0.1) in response to the experimental diets. Markers of epithelial integrity (pcna) and pro-inflammatory genes (alpi, il8 and igm) were significantly up-regulated in fish fed PAP diet. Fish fed NoPAP diet presented an up-regulation of the anti-inflammatory cytokine il10. In the microbiota analysis, Illumina sequencing reads were assigned to 2,180 OTUs and a significantly lower richness and alpha diversity were found in the NoPAP group in comparison to CTRL fish. Detailed differences in microbiota composition were analysed with a statistically validated PLS-DA which clearly separated CTRL fish from fish fed PAP and NoPAP diets, with 135 OTUs mainly driving this separation (VIP ≥ 1). Inferred metagenome results showed a differential regulation of 34 pathways. Both NoPAP and PAP groups showed, among others, the up-regulation of routes tailoring immune response and inflammation (C-type lectin receptor, VEGF, TNF and NFκ-β signalling pathways), with a lower degree of activation in fish fed NoPAP diet. Fish fed PAP and NoPAP diets also displayed an exclusive type of response at this level, with the differential regulation of 14 and 10 inferred pathways, respectively. Correlation tests disclosed a remarkable amount of DE intestinal genes (12) involved in 38 significant (P < 0.01) correlations with 29 of the discriminant OTUs (Fig. 1). This highlights the relation between host gene expression and changes in the gut microbiota. In addition, 11 out of the 29 correlated OTUs were also associated with changes in the relative expression of liver and head-kidney genes, retrieved from previous studies performed in the same fish., [Conclusions]: Even though growth performance was not affected by the experimental diets, these results disclosed a pro-inflammatory response to PAP-based formulation in terms of gene expression and intestinal microbiota. This inflammatory condition seems to be ameliorating using NoPAP-based feed formulation, which becomes especially interesting as an alternative eco-efficient feed for seabream. At the same time, a remarkable correlation between changes in gut microbial population and gene expression was unravelled at local and systemic levels. This highlights the potential action of the gut microbiome as a “second genome”, probably being involved in the regulation of the transcriptomic response of this marine farmed fish when fed innovative diet formulations based on increased circularity and resource utilization principles., GAIN (EU-H2020 #773330); AQUAEXCEL2020 (EU-H2020 #652831, TNA project AE150004); BreamAquaINTECH (RTI2018–094128-B-I00); RYC2018-024049-I/AEI/10.13039/501100011033.

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. Tolerance to fish meal (FM) and fish oil replacement in the economically important gilthead sea bream (Sparus aurata) is being extensively studied with many products emerging as alternative feed ingredients. It has been demonstrated that alternative diets influence the composition of intestinal adherent microbial populations, which have a key role on host metabolism, health and disease resistance. In addition, low fish meal diets showed an increased susceptibility to enteric parasites (Piazzon et al., 2017). Clearly, differences in diet have an impact on the overall health and metabolism of the fish and many parameters have to be taken into account when studying alternative diets for their use in aquaculture. In this study we evaluated the effect of a novel feed formulation (NoPAP SANA) with total replacement of FM by insect meal and bacterial fermentation biomass, and supplemented with the health-promoter additive SANACORE®GM (Palenzuela et al., 2020), on growth performance, gene expression, intestinal microbiota and disease resistance in gilthead sea bream., [Methods]: Tagged gilthead sea bream of mean weight 21.3 g were distributed in two open-flow tanks (160 fish/tank) and fed ad libitum during 34 days with control or NoPAP SANA diets. Twelve fish/diet were sacrificed and head kidney (HK), liver (L) and posterior intestine (PI) were taken for RNA extraction. From the same fish, the adherent bacteria of PI were collected and immediately used for DNA extraction. RNA from HK, L and PI was used to run three customized PCR-arrays including genes of interest for each tissue, with markers of performance and metabolism (L), immune system (HK and PI), epithelial integrity, nutrient transport and mucins (PI). Using the bacterial DNA, 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. Differential gene expression and OTU presence and abundance correlations were studied using the corrplot R package. From the remaining fish, 70 fish/group were challenged with the intestinal parasite Enteromyxum leei by effluent exposure and the remaining fish were used as controls. The challenge lasted 78 days, including a non-lethal diagnosis sampling at day 40. At the end of the challenge all fish were sampled for histological and molecular diagnosis. Biometric values from all fish were taken in all sampling points., [Results]: A slight decrease in condition factor and specific growth rate was detected in the NoPAP SANA group. However, all fish grew efficiently considering gilthead sea bream standards. NoPAP SANA group showed differential expression of 17 out of 44 genes in L, two out of 29 in HK, and 4 out of 44 in PI. The bacterial composition at the PI showed no major differences in diversity or at the phylum level. However, 29 abundant (>1%) OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differential expression of genes in the different tissues, highlighting Pseudoxanthomonas which was positively correlated with the expression of seven L genes, or Actinomyces, significantly correlated with the expression of L and HK genes (Fig. 1). Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge was not significantly different between diets. In fact, infected fish from both groups showed similar recovery rates., [Conclusions]: NoPAP SANA promoted good growth parameters and efficient conversions arising as a good alternative for a FMbased diet in gilthead sea bream diets. This diet modulated the expression of several genes in L showing the capacity to reduce lipogenesis, mitochondrial activity and the risk of oxidative stress and, at the same time, promoting an antiinflammatory gene expression profile in HK and PI. Changes were also detected in the adherent bacterial populations of PI, with significant changes of OTUs that could potentially account for significant metabolic alterations. The correlations between presence and abundance of intestinal bacteria with changes in gene expression of different tissues, together with the pathway analysis results, show that microbiota changes can have an impact on host metabolism at a systemic level, and vice versa. Clearly, the changes induced by this novel FM-free diet supported an accelerated growth with an overall feed conversion ratio close to 1 and no increased susceptibility against this intestinal parasite, as often observed in studies when replacing a FM-based diet., GAIN (EU-H2020 #773330); RYC2018-024049-I/AEI/10.13039/501100011033