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The synergies between selective breeding and feed additives remain under-explored in farmed fish, despite their sustainability. Reference (REF) and selected gilthead sea bream for growth (GS) were fed with the control (CTRL) diet during 14 days. CTRL diet was oil-coated with three functional additives (PHY: phytogenic based on garlic and medium chain fatty acid; OA: organic acid mixture with a 70% of butyric acid sodium salt; PROB: probiotic based on Bacillus subtilis, pumillus and licheniformes species). These experimental diets were then sequentially administered at high (PHY/OA = 7.5 g/kg, PROB = 2 × 1011 CFU/kg; 2 weeks) and low (PHY = 5 g/kg, OA = 3 g/kg, PROB = 4 × 1010 CFU/kg; 10 weeks) additive doses. The capacity of a given genotype and additive to modify the fish growth performance, gut health and the host interaction with its anterior intestine (AI) microbiota was evaluated as a whole population or individually (9 fish/diet/genetics). GS fish showed a better growth and feed conversion ratio, linked to a reduced individual variability of gut microbial composition. The PHY additive had a major impact upon the intestinal transcriptome of GS-PHY fish, with the up-regulation of markers of epithelial integrity, sphingolipid and cholesterol/bile salt metabolism. With the OA additive, impaired growth performance, reduced AI goblet cell area and enhanced AI granulocyte infiltration were concomitant with a down-regulation of neutrophil degranulation markers associated with a decrease of pathogenic genera (Staphylococcus/Streptococcus/Neisseria), and an over-representation of acetone/butanol/ethanol fermentation and vitamin K biosynthesis inferred pathways. Bacillus establishment and lack of AI inflammation were parallel in PROB fish of both genetic backgrounds. However, GS fish grew and utilized feed better with the additive, whereas a worsening appeared in REF fish. This amelioration was related with a higher abundance of the nitrate-reducer Kocuria, an up-regulat

[Background]: Helminth extracellular vesicles (EVs) are known to have a three-way communication function among parasitic helminths, their host and the host-associated microbiota. They are considered biological containers that may carry virulence factors, being therefore appealing as therapeutic and prophylactic target candidates. This study aims to describe and characterise EVs secreted by Sparicotyle chrysophrii (Polyopisthocotyla: Microcotylidae), a blood-feeding gill parasite of gilthead seabream (Sparus aurata), causing significant economic losses in Mediterranean aquaculture., [Methods]: To identify proteins involved in extracellular vesicle biogenesis, genomic datasets from S. chrysophrii were mined in silico using known protein sequences from Clonorchis spp., Echinococcus spp., Fasciola spp., Fasciolopsis spp., Opisthorchis spp., Paragonimus spp. and Schistosoma spp. The location and ultrastructure of EVs were visualised by transmission electron microscopy after fixing adult S. chrysophrii specimens by high-pressure freezing and freeze substitution. EVs were isolated and purified from adult S. chrysophrii (n = 200) using a newly developed ultracentrifugation-size-exclusion chromatography protocol for Polyopisthocotyla, and EVs were characterised via nanoparticle tracking analysis and tandem mass spectrometry., [Results]: Fifty-nine proteins involved in EV biogenesis were identified in S. chrysophrii, and EVs compatible with ectosomes were observed in the syncytial layer of the haptoral region lining the clamps. The isolated and purified nanoparticles had a mean size of 251.8 nm and yielded 1.71 × 108 particles · mL−1. The protein composition analysis identified proteins related to peptide hydrolases, GTPases, EF-hand domain proteins, aerobic energy metabolism, anticoagulant/lipid-binding, haem detoxification, iron transport, EV biogenesis-related, vesicle-trafficking and other cytoskeletal-related proteins. Several identified proteins, such as leucyl and alanyl aminopeptidases, calpain, ferritin, dynein light chain, 14–3–3, heat shock protein 70, annexin, tubulin, glutathione S-transferase, superoxide dismutase, enolase and fructose-bisphosphate aldolase, have already been proposed as target candidates for therapeutic or prophylactic purposes., [Conclusions]: We have unambiguously demonstrated for the first time to our knowledge the secretion of EVs by an ectoparasitic flatworm, inferring their biogenesis machinery at a genomic and transcriptomic level, and by identifying their location and protein composition. The identification of multiple therapeutic targets among EVs' protein repertoire provides opportunities for target-based drug discovery and vaccine development for the first time in Polyopisthocotyla (sensu Monogenea), and in a fish-ectoparasite model.

Sparicotyle chrysophrii (Microcotylidae) is considered the most threatening pathogen affecting the gilthead seabream (GSB; Sparus aurata) off-shore farming due to its economic impact. This study explores the best experimental conditions to set up an in vivo infection model capable of mimicking the sparicotylosis signs observed in farmed diseased fish. The experimental setup for parasite transmission consisted of a recipient (R) fish tank with naïve GSB receiving water from two S. chrysophrii-infected donor tanks in a recirculating aquaculture system (RAS). Egg collectors, consisting of a polyester mesh in a supporting plastic frame, were placed in the R tank in order to monitor the progression of the parasitosis. An additional tank with control unexposed naïve fish was maintained in parallel, with open water flow and disconnected from the RAS. After a preliminary trial, infective pressure in the R tank was increased by placing an additional egg collector already loaded with entangled parasite eggs, and by maintaining the fish number throughout the experiment. Adult S. chrysophrii parasite load correlated with most of the evaluated biotic and abiotic factors. Haemoglobin and haematocrit significantly dropped around 40 days after exposing GSB to S. chrysophrii. Furthermore, the abundance of eosinophilic granular cells and goblet cells in gill filaments, and splenic melanomacrophagic centres increased. In contrast, hepatic fat was depleted in S. chrysophrii-infected GSB. This study provides an advancement not only for studying S. chrysophrii's biology and its interaction with its host, but also for further studying the disease under experimental conditions in search of treatment alternatives and prophylactic measures.

Sparicotyle chrysophrii (Microcotylidae) is considered the most threatening parasite affecting the gilthead seabream (Sparus aurata, GSB) in sea cage farming. There are no standardised in vivo models fully described in the scientific literature. This study aims to explore the best experimental conditions to set up an in vivo infection model capable of mimicking the sparicotylosis signs observed in diseased farmed fish. A recipient (R) fish tank (200L) with naïve GSB (N=28) received water from two S. chrysophrii-infected donor tanks (N=52; 200L) in a recirculating aquaculture system (RAS). Parasite egg collectors, consisting of a polyester mesh in a supporting plastic frame, were placed in the R tank in order to monitor the progression of the parasitosis. An additional tank with control unexposed naïve fish (C) was maintained in parallel (N=20) disconnected from the RAS. Five C were sampled at 0 days post exposure (dpe), whereas 3 C and 5 R were sampled at 14, 28, 42, 50, 58 dpe. Haemoglobin values were immediately measured and haematocrit values determined by microhematocrit capillary centrifugation. All right gill arches were dissected and infection intensities of juvenile and adult S. chrysophrii determined under a stereomicroscope. The left gill arches, head kidney, spleen and liver samples were fixed in Bouin¿s solution and processed for routine paraffin histology. R fish were successfully infected (100% prevalence after 14 dpe) and the parasite cycle was completed in the experimental setup. Adult parasite load was positively correlated with host biometric factors, temperature, dpe and tank biomass, whereas it negatively correlated with haemoglobin, haematocrit and condition factor. Egg counts peaked at 35 dpe and haemoglobin and haematocrit significantly dropped around 40 dpe. Furthermore, the abundance of eosinophilic granular cells and goblet cells in gill filaments, and splenic melanomacrophagic centres increased, whereas hepatic fat was depleted in infected

There are several affordable methods involving different sequencing technologies for microbial characterization. However, the choice of the sequencing platform and the downstream analysis can yield somewhat different results. Here we aimed to examine the strengths and limitations of different sequencing platforms for their use in aquacultured fish gut mucosal samples, using gilthead sea bream (Sparus aurata) as a model. The attention was focused on the portable Oxford Nanopore Technologies (ONT) MinION device, which offers the possibility of fast profiling of mucosal microbial samples. The standard PCR protocol provided for the MinION full-length sequencing of the 16S rRNA was optimized (input material, annealing temperature and number of cycles) for fish mucosal samples and the optimized protocol was validated using a standard mock community with known bacterial taxa and abundances. The optimized PCR (annealing temperature 52 °C, 30 cycles) yielded highly accurate results when sequencing mock community samples (R2 = 0.95) and was chosen for the subsequent analyses. Finally, the sequencing results were compared to those from Illumina MiSeq sequencing of the V3-V4 region of the 16S rRNA gene to determine strengths and weaknesses from both platforms. Our results showed that MinION is a reliable and accurate tool for the assessment of intestinal bacteria communities, yielding similar results to Illumina with correlation coefficients >0.75. However, biologically important but less abundant taxa (e.g. Actinobacteriota and Bacteroidota) were apparently masked when comparisons were made with the Illumina MiSeq results. We conclude that the choice of sequencing platform depends on the type of sample and scientific question. Thus, when evaluating fish gut mucosal samples, where the biological interpretation focuses on taxa related to gut function and metabolism, Illumina MiSeq allows a broader amplification of taxa of interest, while MinION provides good results in terms of

It is yet unknown whether gilthead seabream develops immunologic memory and protection after a first exposure to the gill ectoparasite Sparicotyle chrysophrii, known to feed on its host blood causing severe anemia. For this purpose, experimentally infected gilthead seabream (n=25) were kept during an entire year until they naturally lost the infection. These recovered fish (RE), and additional naïve fish (n=25, NAI), were exposed to S. chrysoprii. At three lethal samplings, blood was taken and hemoglobin and hematocrit values immediately registered, serum samples and gill mucus collected, and biometric and infection intensity data recorded. Before parasite exposure (t0), 5 RE and 5 NAI were sampled. At four- and eightweeks post exposure (wpe) (t1 and t2, respectively), 10 RE and 10 NAI were sampled. ELISAs for total and specific IgM and IgT detection in serum and mucus samples were carried out. Prevalence of infection was 100% in both recipient groups, but in t2, the RE group had significantly lower infection intensity and significantly higher hemoglobin concentration. Gill mucus IgT, total and specific, and specific IgM were significantly higher in RE than in NAI at t0, and were depleted at t2. Gill mucus total IgM of RE increased significantly at t2. Serum IgT was depleted at t2 in both experimental groups, whereas serum IgM, total and specific, was reestablished or increased only in RE at t2. Therefore, specific Ig levels found in the gill mucus of RE seem to mitigate sparicotylosis in gilthead seabream, pointing to a local protective acquired response.

Gut microbiomes of fish species consist of thousands of bacterial taxa that interact among each other, their environment, and the host. These complex networks of interactions are regulated by a diverse range of factors, yet little is known about the hierarchy of these interactions. Here, we introduce SAMBA (Structure-Learning of Aquaculture Microbiomes using a Bayesian Approach), a computational tool that uses a unified Bayesian network approach to model the network structure of fish gut microbiomes and their interactions with biotic and abiotic variables associated with typical aquaculture systems. SAMBA accepts input data on microbial abundance from 16S rRNA amplicons as well as continuous and categorical information from distinct farming conditions. From this, SAMBA can create and train a network model scenario that can be used to (i) infer information of how specific farming conditions influence the diversity of the gut microbiome or pan-microbiome, and (ii) predict how the diversity and functional profile of that microbiome would change under other variable conditions. SAMBA also allows the user to visualize, manage, edit, and export the acyclic graph of the modelled network. Our study presents examples and test results of Bayesian network scenarios created by SAMBA using data from a microbial synthetic community, and the pan-microbiome of gilthead sea bream (Sparus aurata) in different feeding trials. It is worth noting that the usage of SAMBA is not limited to aquaculture systems as it can be used for modelling microbiome–host network relationships of any vertebrate organism, including humans, in any system and/or ecosystem.

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.

[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

[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.

The present study aimed to unravel the effects of partial (50%; 50LSAqua) and total (100%; 100LSAqua) replacement of fish meal (FM) by a commercial protein source (LSAqua SusPro) made of bacterial and processed animal proteins (PAP) in farmed juveniles of gilthead sea bream (Sparus aurata). The trial lasted 8 weeks, and the feasibility of replacement was assessed in terms of growth performance, histopathological scoring and composition of mucosal adherent microbiota from anterior intestine (AI). Specific growth rates (SGR) of 50LSAqua fish were undistinguishable from the CTRL group, whereas a slight but significant growth impairment was found with the total replacement. Histological signs of inflammation across the intestine were more evident at the highest level of FM replacement. Illumina sequencing of gut mucosal microbiota yielded a mean of 130,439 reads per sample assigned to 1,567 OTUs at 97% identity threshold. The bacterial richness was similar in all groups, but a significantly higher Simpson diversity index was found in 100LSAqua fish. At the phylum level, Proteobacteria were the most abundant in all groups, whereas Firmicutes decreased and Actinobacteria increased with the FM replacement. The inferred metagenome analysis suggested that these discriminant bacteria could be implicated in a counter-regulatory anti-inflammatory response. It also pointed to an over-representation of mucosal microbes that can potentially be involved in the natural production of antimicrobials in fish fed the experimental diets.

[Background]: Sparicotylosis is an enzootic parasitic disease that is well established across the Mediterranean Sea. It is caused by the polyopisthocotylean monogenean Sparicotyle chrysophrii and affects the gills of gilthead sea bream (GSB; Sparus aurata). Current disease management, mitigation and treatment strategies are limited against sparicotylosis. To successfully develop more efficient therapeutic strategies against this disease, understanding which molecular mechanisms and metabolic pathways are altered in the host is critical. This study aims to elucidate how S. chrysophrii infection modulates the plasma proteome of GSB and to identify the main altered biological processes involved. [Methods]: Experimental infections were conducted in a recirculating aquaculture system (RAS) in which naïve recipient GSB ([R]; 70 g; n = 50) were exposed to effluent water from S. chrysophrii-infected GSB (98 g; n = 50). An additional tank containing unexposed naïve fish (control [C]; 70 g; n = 50) was maintained in parallel, but with the open water flow disconnected from the RAS. Haematological and infection parameters from sampled C and R fish were recorded for 10 weeks. Plasma samples from R fish were categorised into three different groups according to their infection intensity, which was based on the number of worms fish−1: low (L: 1–50), medium (51–100) and high (H: > 100). Five plasma samples from each category and five C samples were selected and subjected to a SWATH-MS proteome analysis. Additional assays on haemoglobin, cholesterol and the lytic activity of the alternative complement pathway were performed to validate the proteome analysis findings. [Results]: The discriminant analysis of plasma protein abundance revealed a clear separation into three 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 haemostasis, the immune system and lipid

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.

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

The present study aimed to unravel the effects of partial (50%; 50LSAqua) and total (100%; 100LSAqua) replacement of fish meal (FM) by a commercial protein source (LSAqua SusPro) made of bacterial and processed animal proteins (PAP) in farmed juveniles of gilthead sea bream (Sparus aurata). The trial lasted 8 weeks, and the feasibility of replacement was assessed in terms of growth performance, histopathological scoring and composition of mucosal adherent microbiota from anterior intestine (AI). Specific growth rates (SGR) of 50LSAqua fish were undistinguishable from the CTRL group, whereas a slight but significant growth impairment was found with the total replacement. Histological signs of inflammation across the intestine were more evident at the highest level of FM replacement, and the total concentration of short chain fatty acids (SCFA) in stripped feces decreased in a dose dependent manner. Illumina sequencing of gut mucosal microbiota yielded a mean of 130,439 reads per sample assigned to 1,567 OTUs at 97% identity threshold. The bacterial richness was similar in all groups, but a significantly higher Simpson diversity index was found in 100LSAqua fish. At the phylum level, Proteobacteria were the most abundant in all groups, whereas Firmicutes decreased and Actinobacteria increased with the FM replacement. At a closer look, pro-inflammatory Gammaproteobacteria of the genus Psychrobacter, and Acinetobacter decreased with FM replacement, whereas the anti-inflammatory Paracoccus, Arthrobacter, and Actinomycetales increased, with a remarkable presence of the Propioniciclava genus in LSAqua groups. The inferred metagenome analysis suggested that these discriminant bacteria could be implicated in a counter-regulatory anti-inflammatory response. It also pointed to an over-representation of mucosal microbes that can potentially be involved in the natural production of antimicrobials in fish fed the experimental diets.

Fish genetically selected for growth (GS) and reference (REF) fish were fed with CTRL (15% FM, 5–7% FO) or FUTURE (7.5% FM, 10% poultry meal, 2.2% poultry oil + 2.5% DHA-algae oil) diets during a 12-months production cycle. Samples from initial (t0; November 2019), intermediate (t1; July 2020) and final (t2; November 2020) sampling points were used for Illumina 16S rRNA gene amplicon sequencing of the adherent microbiota of anterior intestine (AI). Samples from the same individuals (t1) were also used for the gene expression profiling of AI by RNA-seq, and subsequent correlation analyses with microbiota abundances. Discriminant analyses indicated the gut bacterial succession along the production cycle with the proliferation of some valuable taxa for facing seasonality and different developmental stages. An effect of genetic background was evidenced along time, decreasing through the progression of the trial, namely the gut microbiota of GS fish was less influenced by changes in diet composition. At the same time, these fish showed wider transcriptomic landmarks in the AI to cope with these changes. Our results highlighted an enhanced intestinal sphingolipid and phospholipid metabolism, epithelial turnover and intestinal motility in GS fish, which would favour their improved performance despite the lack of association with changes in gut microbiota composition. Furthermore, in GS fish, correlation analyses supported the involvement of different taxa with the down-regulated expression of pro-inflammatory markers and the boosting of markers of extracellular remodelling and response to bacterium. Altogether, these findings support the combined action of the gut microbiome and host transcriptionally mediated effects to preserve and improve gut health and function in a scenario of different growth performance and potentiality.

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

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 us

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 analy

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 es

The gut microbiota is now recognised as a key target for improving aquaculture profit and sustainability, but we still lack insights into the activity of microbes in fish mucosal surfaces. In the present study, a metatranscriptomic approach was used to reveal the expression of gut microbial genes in the farmed gilthead sea bream. Archaeal and viral transcripts were a minority but, interestingly and contrary to rRNA amplicon-based studies, fungal transcripts were as abundant as bacterial ones, and increased in fish fed a plant-enriched diet. This dietary intervention also drove a differential metatranscriptome in fish selected for fast and slow growth. Such differential response reinforced the results of previously inferred metabolic pathways, enlarging, at the same time, the catalogue of microbial functions in the intestine. Accordingly, vitamin and amino acid metabolism, and rhythmic and symbiotic processes were mostly shaped by bacteria, whereas fungi were more specifically configuring the host immune, digestive, or endocrine processes.

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

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

Passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that gilthead sea bream (GSB, Sparus aurata) that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific IgM in serum. Thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in GSB and to study in detail the nature of these protective antibodies. Serum from a pool of resistant (SUR) or naïve (NAI) animals was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). Effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. In parallel, we performed an immunoglobulin repertoire analysis of the fish generating SUR and NAI sera. The results showed that, fish injected with parasite-specific antibodies (spAbs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. Repertoire analysis revealed that E. leei induced a polyclonal expansion of diverse IgM and IgT subsets that could be in part an evasion strategy of the parasite. Nonetheless, GSB was able to produce sufficient levels of parasite-spAbs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. These results highlight the crucial

[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 famil

In gilthead sea bream Sparus aurata, infection by Enteromyxum leei produces a cachectic syndrome with anorexia, weight loss, severe epaxial muscle atrophy and, eventually, death. Currently, there are neither vaccines nor effective prescription medicines to control this infection. Nutraceutical approaches are raising interest in the aquaculture industry, responding to the lack of therapeutic tools for the management of insidious chronic losses due to parasites. In this study, the effect of a commercially available health-promoting feed additive (SANACORE® GM) at 2 different doses was tested in comparison with a basal diet without the additive during a laboratory-controlled challenge with E. leei. Group performance and biometrical values were monitored, and an in-depth parasitological diagnosis, quantification of parasite loads and histopathological examination were carried out at the end of the trial. Supplemented diets mitigated the anorexia and growth arrestment observed in challenged fish fed the basal diet. This mitigation was maximum in the highest dose group, whose growth performance was not different from that of unchallenged controls. Treated groups also presented lower prevalence of infection and a lower parasite load, although the differences in the mean intensity of infection were not statistically significant. Although the decrease in parasite levels was similar with both doses of additive tested, the pathogeny of the infection was mostly suppressed with the higher dose, while only mitigated with the lower dose. The mechanisms involved in the effects obtained remain to be investigated, but the results point to a modulation of the immunopathological response to the infection.

Mammalian macrophages can adopt polarization states that, depending on the exact stimuli present in their extracellular environment, can lead to very diferent functions. Although these diferent polarization states have been shown primarily for macrophages of humans and mice, it is likely that polarized macrophages with corresponding phenotypes exist across mammals. Evidence of functional conservation in macrophages from teleost fsh suggests that the same, or at least comparable polarization states should also be present in teleosts. However, corresponding transcriptional profles of marker genes have not been reported thus far. In this study we confrm that macrophages from common carp can polarize into M1- and M2 phenotypes with conserved functions and corresponding transcriptional profles compared to mammalian macrophages. Carp M1 macrophages show increased production of nitric oxide and a transcriptional profle with increased pro-infammatory cytokines and mediators, including il6, il12 and saa. Carp M2 macrophages show increased arginase activity and a transcriptional profle with increased anti-infammatory mediators, including cyr61, timp2b and tgm2b. Our RNA sequencing approach allowed us to list, in an unbiased manner, markers discriminating between M1 and M2 macrophages of teleost fsh. We discuss the importance of our fndings for the evaluation of immunostimulants for aquaculture and for the identifcation of gene targets to generate transgenic zebrafsh for detailed studies on M1 and M2 macrophages. Above all, we discuss the striking degree of evolutionary conservation of macrophage polarization in a lower vertebrate.

Endocrine disruptors in general and oestrogenic compounds in particular have been widely investigated in view of their effects on several physiological processes and, therefore, their ecotoxicologic relevance. The modulation of the immune system by oestrogens has increasingly sparked interest in the research community, that previously had been mainly centered on the reproductive effects of these hormones. In fact, since the industrialization an increasing variety of endocrine disruptors, such as oestrogenic endocrine disruptors, are retrieved in the environment. These oestrogenic endocrine disrupting chemicals (EEDCs) have also been suggested to increase the prevalence of autoimmune diseases and cancer. With regard to the high degree of similarities in the immune system of jawed vertebrates as well as the conserved immunomodulatory roles of oestrogen, environmental EEDCs possibly have the capacity to alter immune system functions of teleost fish, which may impair their capacity to fight infectious diseases and eventually may contribute, together with overfishing, to wild stock losses. Importantly, the most deleterious effects of EEDCs, both in mammals and teleosts, appear to arise when the exposure occurs during specific periods of the immune system ontogenesis, commonly referred as critical windows of exposure. However, in mammals and especially in teleost fish, these stages of the immune system development as well as the EEDC-action remain to be fully identified and characterised. The concept and the importance of developmental immunotoxicity is presented by addressing the mechanisms of oestrogenic regulation and the mode of action of EEDCs from an immunological perspective. Emphasis is given to the critical windows of development of the immune system during which EEDCs may alter the immune system development and function with long-term consequences on immunocompetence. Results from different classes of vertebrates are compiled, highlighting studies on teleost fis

We report a draft genome assembly for the teleost gilthead sea bream (Sparus aurata), reconstructed by combination of short- and long-read high-throughput sequencing, and genetic linkage maps. The assembly comprised 5,039 scaffolds that span 1.24 Gb of the expected 1.59 Gb complete genome size, with 932 scaffolds (~732 Mb) anchored to 24 chromosomes. This sequencing strategy resulted in a high quality fish genome assembly, as supported by the high N50 (1.07 Mb) and lower L50 (227) values. These long and continuous reads allowed the annotation of a large number of coding genes (55,423), non-coding RNA genes (2,991) and 345 Mb of mobile genetic elements. Synteny analysis revealed a high level of homology between gilthead sea bream genes. Phylogenomics relationships with other fish species were established to locate temporal speciation and to assign duplication events. A large degree of recent and lineage-specific gene duplications were found, that were mainly enriched in functions related to genome transposition, immune response and response to stimulus. These duplication and gene family expansions could be related in part to the activity of mobile genetic elements. This fact makes gilthead sea bream an interesting model for the study of gene repertoire expansion strategies complementary to fish whole genome duplication events. Transcriptional analyses across six different tissues (anterior and posterior intestine, skeletal muscle, liver, gills, spleen) indicated that gene duplication preferentially affected genes expressed in two or more tissues. These findings highlight the genomic complexity of this species and the importance of selective gene duplications in the adaptation of fishes to challenging environment.

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 enviro

Passive immunization is used in humans for treating or preventing some infectious diseases, but it also constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. Intracoelomically-injected antibodies can be detected in fish sera within the first 8 h and their half-life ranges from 7 to 22 days post-injection, depending on the species. The fish models studied so far used fast-acting pathogens such as virus, bacteria or ciliate parasites. The current work aimed to determine if passive immunization could help to prevent enteromyxosis in gilthead sea bream (GSB, Sparus aurata). Enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. We have previously demonstrated that GSB that survive E. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific serum antibodies. In the current study, we evaluated whether injection with sera from resistant animals would protect naïve fish when challenged by effluent exposure to the parasite. Serum from a pool of resistant (R) and naïve (N) animals (intact or heat inactivated, 10 ¿l/g BW) was intracoelomically injected 24 h prior to the E. leei-effluent challenge and at 9 days post-challenge (dpc). At 23 dpc, the different groups were allocated in separate tanks and the effluent exposure was terminated. A non-lethal parasite diagnosis was performed at 56 dpc. At the final sampling (100 dpc), blood, serum and tissues were collected for hematology, circulating antibodies, histological and molecular diagnosis and gene expression. Groups injected with R sera had lower prevalence and intensity of infection than those with N sera, both in the intermediate and final samplings. At 100 dpc, the prevalence of infection in the PBS and N groups was 70%, whereas in R group it onl

Intestinal microbiota is key for many host functions, such as digestion, nutrient metabolism and absorption, 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 (Egerton et al., 2018). However, a long road lies ahead to establish the baseline parameters to guide this manipulation. 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 (GSB, Sparus aurata) from three groups of age maintained under the same conditions.

Intestinal microbiota is key for many host functions, such as digestion, nutrient metabolism and absorption, 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 (Egerton et al., 2018). However, a long road lies ahead to establish the baseline parameters to guide this manipulation. 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 (GSB, Sparus aurata) from three groups of age maintained under the same conditions. One-, 2- and 4-year old GSB (Y+1, Y+2, Y+4) were kept in the same open-flow system and fed the same diet for more than 6 months. After 2-days of fasting, 10 fish per group were sacrificed and the anterior intestinal portion was dissected, opened and washed to remove non-adherent bacteria. Intestinal mucus was scrapped off 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. All Y+1 individuals were males, while the ones belonging to Y+2 and Y+4 age classes were females. A total of 686461 high quality reads (22882/sample) were assigned to 846 OTUs (97% identity). Almost 30% of the OTUs were classifiedup to the level of species. Microbiota diversity and richness did not differ among age groups; however bacterial composition did , highlighting the presence of Photobacterium and Vibrio only after 2 years of age and a higher presence of Staphylococcusand Corynebacterium in Y+1 animals. The core microbiota was defined by 14 OTUs and the groups that showed more OTUs in common were Y+2 and Y+4. PLS-DA analysis showed a clear separation by sex (component 1) and age (component 2), with bacteria belonging to the phyla Firmicutes, Proteobacteria and Actinobacteria drivi

Gilthead sea bream is an economically important fish species that is remarkably well-adapted to farming and changing environments. Understanding the genomic basis of this plasticity will serve to orientate domestication and selective breeding toward more robust and efficient fish. To address this goal, a draft genome assembly was reconstructed combining short- and long-read high-throughput sequencing with genetic linkage maps. The assembled unmasked genome spans 1.24 Gb of an expected 1.59 Gb genome size with 932 scaffolds (~732 Mb) anchored to 24 chromosomes that are available as a karyotype browser at www.nutrigroup-iats.org/seabreamdb. Homology-based functional annotation, supported by RNA-seq transcripts, identified 55,423 actively transcribed genes corresponding to 21,275 unique descriptions with more than 55% of duplicated genes. The mobilome accounts for the 75% of the full genome size and it is mostly constituted by introns (599 Mb), whereas the rest is represented by low complexity repeats, RNA retrotransposons, DNA transposons, and non-coding RNAs. This mobilome also contains a large number of chimeric/composite genes (i.e., loci presenting fragments or exons mostly surrounded by LINEs and Tc1/mariner DNA transposons), whose analysis revealed an enrichment in immune-related functions and processes. Analysis of synteny and gene phylogenies uncovered a high rate of species-specific duplications, resulting from recent independent duplications rather than from genome polyploidization (2.024 duplications per gene; 0.385 excluding gene expansions). These species-specific duplications were enriched in gene families functionally related to genome transposition, immune response, and sensory responses. Additionally, transcriptional analysis of liver, skeletal muscle, intestine, gills, and spleen supported a high number of functionally specialized paralogs under tissue-exclusive regulation. Altogether, these findings suggest a role of recent large-scale gene duplicat

[Background] Monogenean flatworms are the main fish ectoparasites inflicting serious economic losses in aquaculture. The polyopisthocotylean Sparicotyle chrysophrii parasitizes the gills of gilthead sea bream (GSB, Sparus aurata) causing anaemia, lamellae fusion and sloughing of epithelial cells, with the consequent hypoxia, emaciation, lethargy and mortality. Currently no preventive or curative measures against this disease exist and therefore information on the host-parasite interaction is crucial to find mitigation solutions for sparicotylosis. The knowledge about gene regulation in monogenean-host models mostly comes from freshwater monopysthocotyleans and almost nothing is known about polyopisthocotyleans. The current study aims to decipher the host response at local (gills) and systemic (spleen, liver) levels in farmed GSB with a mild natural S. chrysophrii infection by transcriptomic analysis., [Results] Using Illumina RNA sequencing and transcriptomic analysis, a total of 2581 differentially expressed transcripts were identified in infected fish when compared to uninfected controls. Gill tissues in contact with the parasite (P gills) displayed regulation of fewer genes (700) than gill portions not in contact with the parasite (NP gills) (1235), most likely due to a local silencing effect of the parasite. The systemic reaction in the spleen was much higher than that at the parasite attachment site (local) (1240), and higher than in liver (334). NP gills displayed a strong enrichment of genes mainly related to immune response and apoptosis. Processes such as apoptosis, inflammation and cell proliferation dominated gills, whereas inhibition of apoptosis, autophagy, platelet activation, signalling and aggregation, and inflammasome were observed in spleen. Proteasome markers were increased in all tissues, whereas hypoxia-related genes were down-regulated in gills and spleen., [Conclusions] Contrasting forces seem to be acting at local and systemic levels. The splenic down-regulation could be part of a hypometabolic response, to counteract the hypoxia induced by the parasite damage to the gills and to concentrate the energy on defence and repair responses. Alternatively, it can be also interpreted as the often observed action of helminths to modify host immunity in its own interest. These results provide the first toolkit for future studies towards understanding and management of this parasitosis.