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[EN] The invention relates to a device placed on the gill cover to monitor the metabolic state and well-being of fish through joint, simultaneous measurements of breathing frequency and physical activity via a plurality of accelerations that are coplanar (x and y axes) and normal to the gill cover (z axis). The substrate is flexible and is covered with an impervious coating, which contains an electronic circuit with an acceleration sensor (208), a control unit (202), a microcontroller (206) configured to execute at least one activity-monitoring routine, a memory (210) and a battery (204). Data are collected via an external unit (201) which can optionally process them in a complete or partial manner. This type of measurement can be processed jointly with another type of measurement, obtained from other sensors of environmental parameters, [ES] Dispositivo emplazado en el opérculo para supervisar el estado metabólico y bienestar de peces a través de medidas conjuntas y simultáneas de frecuencia respiratoria y actividad física mediante una pluralidad de aceleraciones coplanares (ejes x,y) y normales al opérculo (eje z). El sustrato es flexible y está cubierto con un revestimiento impermeable, donde se encuentra un circuito electrónico con sensor de aceleración (208), una unidad de control (202), un microcontrolador (206) configurado para ejecutar al menos una rutina de monitorización de actividad, una memoria (210) y una batería (204). La toma de datos se efectúa a través de una unidad externa (201) que eventualmente puede procesarlos de forma total o parcial. Este tipo de mediciones se pueden procesar de forma conjunta con otro tipo de medidas, obtenidas a partir de otros sensores de parámetros medio-ambientales, [FR] La présente invention concerne un dispositif mis en place sur l'opercule pour surveiller l'état métabolique et le bien-être de poissons au moyen de mesures conjointes et simultanées de la fréquence respiratoire et de l'activité physique avec une pluralité d'accélérations coplanaires (axes x,y) et perpendiculaires à l'opercule (axe z). Le substrat est flexible et couvert d'un revêtement imperméable, où se situe un circuit électronique avec capteur d'accélération (208), une unité de commande (202), un microcontrôleur (206) configuré pour exécuter au moins une routine de surveillance d'activité, une mémoire (210) et une batterie (204). La capture de données est effectuée au moyen d'une unité externe (201) qui peut éventuellement les traiter de manière complète ou partielle. Les mesures de ce type peuvent être traitées de manière conjointe avec un autre type de mesures, obtenues à partir d'autres capteurs de paramètres du milieu ambiant, Consejo Superior de Investigaciones Científicas (España), Universidad de Las Palmas de Gran Canaria, A1 Solicitud de patente con informe sobre el estado de la técnica

Trabajo presentado en la International Conference & Exposition Aquaculture Europe, celebrada en Funchal, Maderia (Portugal) del 04 al 07 de octubre de 2021., [Introduction]: The current drive for aquaculture sustainability has been pushing feed formulators for the design of novel aquafeeds, based on ingredients such as insect meals, by-products of fisheries and animal production, microbial biomasses, and algaebased products. All these are viable alternatives, often with inclusion levels restrictions, to those commonly used up to date, e.g. fish meal, fish oil, soy products. A challenge associated with this transition is ensuring that novel feeds provide all required nutrients, without affecting, and when possible improving farming performance. Effects on growth, health status, feed conversion, environmental impacts, and fish biochemical composition (e.g., n-3 HUFAs) from a consumer perspective, have all to be evaluated. However, these emerging ingredients have mostly been tested by researchers and on a one-by-one basis. Our approach was to test different formulation concepts using combinations of these ingredients, rather than one-by-one. Therefore, the aim was not to test individual emerging ingredients, but rather formulation scenarios that may be relevant in the near future. The GAIN project aims to promote eco-intensification of the European aquaculture, and therefore a more sustainable and efficient use of resources in aquafeeds, including circularity towards zero-waste is paramount. The GAIN aquafeed formulation concept starts in replacing cost-effectively fish meal and fish oil from feedtargeted fisheries, together with commodity ingredients from vegetables such as soy, wheat, and corn. Besides the obvious impact of fisheries, using plant protein and oil sources is an increasing concern, due to pressure on farmland resources, resulting deforestation, negative effects on global food security, EU reliance on imports of major crops, and carbon footprint of transportation of some of the raw materials across continents. The present work summarizes the GAIN project effort in testing several alternative formulation concepts in five farmed species: rainbow trout, Atlantic salmon, gilthead seabream, European seabass, and turbot., [Methods]: A total of nine nutritional trials were performed using 4 main formulation concepts (see Fig. 1). These nutritional trials were performed in optimal rearing conditions for each species, at partner facilities (rainbow trout at FEM, Italy; Atlantic salmon at GIFAS, Norway; gilthead seabream at SPAROS, Portugal, and IATS-CSIC, Spain; European seabass and turbot at AWI, Germany). The feed formulations were developed for each of the target species, taking into their nutritional requirements and tolerance to different ingredients. Feeds were produced by extrusion with oils added by vacuum coating at the SPAROS pilot feed mill (Olhão, Portugal)., [Results and Discussion]: All feed formulations gave good growth performances and feed conversions for salmon, seabream, seabass, and turbot and very good ones for rainbow trout. GAIN formulations reached, in general, very similar values compared to the control feeds. However, some formulations performed slightly worse in terms of feed conversion: MIX and PAP in the case of seabream, and PAP in the case of salmon. Moreover, available results for the other key performance indicators assessed (e.g.: nutrient retention and digestibility; biomarker genes, enzymes, and metabolites; skin/gut histology; organoleptic evaluation) suggest that the novel formulations affect fish physiology, with fish likely driven towards a new allostatic balance, but no detrimental effects to fish health could be identified., [Conclusions]: Overall, these results suggest that the novel GAIN feed formulations tested are viable options shortly, if not immediately. In any case, the major farmed European species can be fed on more sustainable and resource-efficient aquafeeds, towards a zero-waste industry, This project was financed by the European Union’s Horizon 2020 research and innovation programme under grant agreement N° 773330 (GAIN).

Trabajo presentado en el 20th International Conference on Diseases of Fish and Shellfish, celebrado en modalidad virtual del 20 al 23 de septiembre de 2021., [Introduction]: In an era of rising antimicrobial resistance and overfished stocks, aquaculture strives to find cost-effective and environmentally-friendly alternatives for fish meal and chemotherapeutics, while meeting the market demand for high-quality fish products. Use of alternative protein sources and health-promoting additives, such as insect-derived proteins and probiotics, has beneficial effects on fish health at both local and systemic level, although the involved mechanisms are largely unknown., [Methodology]: We performed a six-month farm feeding trial with three groups of gilthead sea bream (Sparus aurata) in duplicate cages using i) feed supplemented with insect protein; ii) feed supplemented with insect protein plus autochthonous intestinal Bacillus spp.; and iii) standard feed (control). For the second treatment group, feeding regime during the whole six-month trial included 2-weeks of Bacillus-supplemented diet administration followed by 2-weeks of feed administration where insect meal was included, i.e. the same diet used for the first treatment group. Twelve fish per treatment group were sampled at 0.5, 1 and 6 months after the start of the trial. Samples were collected for whole-intestine microbiota analysis (MiSeq 16s rRNA), histological analysis (TEM) and gene expression analysis using a customized PCR-array of 44 selected markers of intestinal function and integrity, nutrient transport, and immune response., [Results]: Intestinal epithelium of all treatment groups showed no signs of degenerative or inflammatory changes. Main changes in microbial community composition occurred already within the first month of feeding trial. PLS discriminant analysis of gene expression in respect to the dietary treatment allowed the selection of 17 genes, mostly related to epithelia integrity and immune response that showed high contribution to differences among diets. Heatmap clustering of the selected genes grouped together the experimental diets apart from the control diet. Many of the epithelia integrity markers were up-regulated with insect diet, and probiotic supplementation additionally amplified this up-regulation. Probiotic also reversed the up-regulation of inflammatory/immune-regulatory genes that occured with insect diet., [Conclusions]: In conclusion, probiotic supplementation emerges as beneficial for the preservation of gut homeostasis and innate immunity in fish fed alternative protein sources., This research was funded by Interreg Italy-Croatia project AdriAquaNet – Enhancing Innovation and Sustainability in Adriatic Aquaculture.

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., A customized PCR-array of 28 selected genes was used for assessing the simultaneous expression of the Gh/Igf system and related markers of muscle growth, and lipid and energy metabolism during early life stages of gilthead sea bream (60-127 days posthatching). Also, transcriptional reprogramming by mild hypoxia was assessed in fingerling fish with different history trajectories on O2 availability during the same time window. In normoxic fish, the expression of almost all the genes in the array varied over time with a prompted liver and muscle tissue-specific differentiation, which revealed temporal changes in the relative expression of markers of the full gilthead sea bream repertoire of Gh receptors, Igfs and Igf-binding proteins. Results supported a different contribution through development of ghr and igf subtypes on the type of action of GH via systemic or direct effects at the local tissue level. This was extensive to Igfbp1/2/4 and Igfbp3/5/6 clades that clearly evolved through development as hepatic and muscle Igfbp subtypes, respectively. This trade-off is however very plastic to cope changes in the environment, and ghr1 and igfbp1/3/4/5 emerged as hypoxic imprinting genes during critical early developmental windows leading to recognize individuals with different history trajectories of O2 availability and metabolic capabilities later in life. How this knowledge can contribute to establish robust criteria of larval and juvenile fish quality requires more research, though changes in the expression and the quotient expression ratio of duplicated ghr/igf/ igfbp genes can serve as a measure of developmental progression and metabolic disturbances of the offspring. Besides, growth, health and well-being also depend on the daily synchronization of endogenous biological rhythms, and the expression of selected genes from antiphase clusters can serve to find synchrony indicators of gilthead sea bream during early life., This work was supported by National (Bream-AquaINTECH, RTI2018-094128-B-I00) and European (PerformFISH, H2020-SFS-2016-2017; 727610) projects.

Resumen del trabajo presentado en el XIII Congress of the Iberian Association of Comparative Endocrinology, celebrado en modalidad virtual del 16 al 17 de septiembre de 2021., Gilthead sea bream is a highly cultured marine fish in all the Mediterranean area, but new and strict criteria of welfare are needed to assure that the intensification of production has no negative effects in animal farming. Most welfare indicators are specific of a given phase of the production cycle, but others such as the timing of puberty and/or sex reversal are of retrospective value. This is of particular relevance in the protandrous gilthead sea bream, in which the sex ratio is highly regulated at the nutritional level. Social and environmental factors (e.g. contaminant loads) also alter sex ratio, but the contribution of genetic component remains unclear. To assess this complex issue, five sea bream families representative of slow/intermediate/fast growth were grown out with control or a plantbased diet in a common garden system from early life to completion of their sexual maturity in threeyear old fish. The plant-based diet enhanced the male-to-female sex reversal by more than 50% in average. This occurred in parallel with the progressive impairment of growth performance, which was indicative of changes in nutrient requirements as the result of the different energy demand for growth and reproduction through development. The effect of a different nutritional and genetic background on the reproductive performance was also assessed by measurements of circulating levels of sex steroids during two consecutive spawning seasons, varying plasma levels of 17β-estradiol (E2) and 11-ketotestosterone (11-KT) with age, gender, diet and genetic background. Principal component analysis of three-year-old fish displayed a gradual increase of the E2/11-KT ratio from males to females with the improvement of nutritional/genetic background. Altogether, these results support the use of a reproductive tract scoring system for leading farmed fish towards their optimum welfare condition, contributing to improve the productivity of the current gilthead sea bream livestocks., Bream-AquaINTECH (RTI2018-094128-B-I00) project

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 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 29th Conference of European Comparative Endocrinologists (CECE), celebrado en Glasgow, del 19 al 22 de agosto de 2018, Ghrelin is a peptide hormone that regulates different physiological processes in vertebrates (feeding, metabolism, intestinal activity and growth hormone (Gh) secretion). However, specific actions of this hormone have been reported to change among species. The objective of this work was to characterize preproghrelin and Ghrelin receptors (ghsrs) sequences, and to study their responses to fasting and refeeding in gilthead sea bream (Sparus aurata) juveniles. Structural and phylogenetic analysis of the S. aurata preproghrelin was accomplished, and its expression analyzed in main tissues. Fish were fasted for 21 days and then refed and sampled at 2, 5, 24 h, and 7 days afterwards. Plasma levels of Ghrelin, Gh and insulin-like growth factor (Igf-1), and preproghrelin, ghsrs and members of the Gh/Igf-1 axis gene expression were determined in key tissues. preproghrelin and its receptors presented a good conservation among fish, being highly expressed in stomach and pyloric caeca, and in the pituitary and brain, respectively. Fasting caused a growth recession while increased Ghrelin plasma levels, which rapidly recovered at 5 h postprandial. Gh plasma levels were also increased by fasting, and slowly recovered after refeeding. Contrarily, circulant Igf-1 followed an inverse pattern, suggesting important changes in the Gh/Igf ratio by nutritional status. In agreement with the plasma levels, fasting up-regulated gh expression in pituitary until 1 day after refeeding, when lowest igf-1 expression in the liver was observed. Moreover, the expression profile of hepatic Gh receptors resulted to be inverse to that if circulating Gh levels. Altogether, this study demonstrates the important role of Ghrelin concerning nutritional status, suggesting an orexigenic function in gilthead sea bream, and an interaction with pituitary Ghsrs and Gh/Igf-1 axis to promote optimal growth in this species, Supported by funds from the MINECO (AGL2014-57974-R and AGL2015-70679-R) and Generalitat de Catalunya (2014SGR-01371 and XRAq)

Trabajo presentado en el 18th International Symposium on Fish Nutrition and Feeding (ISFNF), celebrado en Las Palmas de Gran Canaria (España), del 3 al 07 de junio de 2018, The production of environmentally sustainable aquaculture feeds starts with the selection of high-quality raw materials that may need to be supplemented with specific nutrients. Nevertheless, different competences for developing tools and methodologies are needed to assess and predict nutrient requirements or status of fish, going further than just identifying differences in growth parameters. Thus, the challenge is to screen and make the best use of a given tool, but also to integrate new knowledge arising from transcriptomics, proteomics, metagenomics and metabolomics in order to define a reliable healthy fish phenotype. Of particular value are biomarkers that precede the onset of metabolic disturbances or those that predict the capacity to cope with dietary, environmental and age-related stresses. Hence, changes in the intestinal transcriptome, integrity of intestinal barrier, intestinal mucus proteome and gut microbiota mostly reflect a pro-inflammatory condition in fish fed plant-based diets, but dietary butyrate helps to restore the wild phenotype resulting in improved diseases outcomes in fish challenged with bacteria and enteric parasites. Currently, a new promising omics approach is metabolomics, and more than 15,000 m/z ions have been detected in the serum metabolome of juvenile fish with around 850 highly discriminant features between fed and short-term fasted fish, using ultra-high performance liquid chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS). When comparing the serum metabolome of fish fed marine and plant-based diets, several lipid related compounds, including phosphocholines, lysophosphocholines and sphingolipids raised as highly discriminatory compounds. A number of exogenous compounds (cysteinolic acid, tauropine, trimethylamine N-oxide, arsenobetaine, hercynin) also contributed to discriminate fish with different nutritional backgrounds. However, from a functional point of view, it is of special relevance that the abundance of N-aryl amino acids, with repair properties in epithelial mucosa, is consistently reduced in fish fed plant-based diets. Conversely, circulating levels of pyrimidine and related nucleosides, especially markers of DNA degradation (deoxycytidine) and methylation (methylcytosine) were increased by plant-based diets, which is viewed as part of a pro-inflammatory condition and/or overall cellular DNA instability. This opens new research issues to alleviate or mitigate the drawback effects of plant based diets in marine carnivorous farmed fish.

Dispositivo emplazado en el opérculo para supervisar el estado metabólico y bienestar de peces a través de medidas conjuntas y simultáneas de frecuencia respiratoria y actividad física mediante una pluralidad de aceleraciones coplanares (ejes x,y) y normales al opérculo (eje z). El sustrato es flexible y está cubierto con un revestimiento impermeable, donde se encuentra un circuito electrónico con sensor de aceleración (208), una unidad de control (202), un microcontrolador (206) configurado para ejecutar al menos una rutina de monitorización de actividad, una memoria (210) y una batería (204). La toma de datos se efectúa a través de una unidad externa (201) que eventualmente puede procesarlos de forma total o parcial. Este tipo de mediciones se pueden procesar de forma conjunta con otro tipo de medidas, obtenidas a partir de otros sensores de parámetros medio-ambientales, Consejo Superior de Investigaciones Científicas (España), Universidad de Las Palmas de Gran Canaria, A1 Solicitud de patente con informe sobre el estado de la técnica

Trabajo presentado en el 5th International Symposium on Genomics in Aquaculture, celebrado en Albufeira (Portugal) del 21 al 23 de marzo de 2018

Trabajo presentado en el 6th International Symposium on Genomics in Aquaculture, celebrado en Granada del 5 al 7 de mayo de 2021., Hypoxia is a common stressor in aquatic environments, and fish reduce feed intake and reorganize its metabolism to limit the tissue O2 demand. This allows to preserve aerobic metabolism by means of a restricted mitochondrial respiration and a shift in substrate preferences as part of the adaptive response of the skeletal muscle to hypoxia exposure. Herein, we aimed to underline new insights on the mild-hypoxia driving force for metabolic and muscle transcriptional reprogramming of gilthead sea bream juveniles. For this purpose, on-growing juveniles of gilthead sea bream were acclimated for 45 days to mild-hypoxia (M- HYP, 40-60% O2 saturation), whereas normoxic fish (85-90% O2 saturation) constituted two different groups depending if they were fed to visual satiety (N; control fish) or pair-fed to M-HYP fish (N-PF). Following the hypoxia conditioning period, all fish were maintained in normoxia and continued to be fed until visual satiation for 3 weeks. The time course of hypoxia-induced changes was assessed by changes in blood metabolic landmarks and muscle transcriptomics before and after exhaustive exercise in a swim tunnel respirometer. Maximumfeed intake was reduced by MHYP pre-conditioning, and both N-PF and M-HYP experienced an improved feed conversion during the normoxia recovery period. M-HYP conditioning reduced circulating levels of free fatty acids and lactate as part of the hypo- metabolic response to face a reduced O2 availability. In exercise tests, M-HYP group showeda higher critical swimming speed (Ucrit) that was preserved along the normoxia recovery period. Changes of circulating metabolites and hormones evidenced an enhanced aerobic ATPproduction in M-HYP fish at the end of the conditioning period, whereas anaerobic metabolism was primed at the end of the normoxia recovery period. Heatmap clustering of muscle differentially expressed (DE) genes, after filtering by ANOVA (P < 0.05) and PLS- DA (VIP > 1), grouped together N-PF and M-HYP after the mild-hypoxia conditioning. This yielded 222 differentially expressed genes that were increased up to 421 after exercise exhaustion, although this divergent expression pattern was reduced thereafter to 180 genes at the end of the normoxia recovery period. Gene enrichment analysis and protein interaction plots also highlighted a higher contribution of lipid metabolism and, thus, of aerobic metabolism to whole energy supply, shifting towards a higher anaerobic fitness following normoxia restoration. Despite of these changes in substrate preference, MHYP fish shared a persistent improvement of swimming performance with a higher critical speed at exercise exhaustion. The machinery of muscle contraction and protein synthesis and breakdown was also largely altered by mild-hypoxia conditioning, contributing this metabolic re-adjustmentto the positive regulation of locomotion and to the catch-up growth response during the normoxia recovery period. Altogether, these results reinforce the large phenotypic plasticityof gilthead sea bream, becoming mild-hypoxia a promising prophylactic measure beforepredictable stressful events.

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

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 (MGEs). 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 was found, that were mainly enriched in functions related to genome transposition, immune response and response to stimulus. These duplications and gene family expansions could be related in part to the activity of MGEs. 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, This work was financed by Spanish (Intramural-CSIC, 1201530E025; MICINN, BreamAquaINTECH, RTI2018-094128-B-I00) and European Union (AQUAEXCEL2020, 652831) projects. BS was supported by a predoctoral research fellowship (Doctorados industriales, DI-17-09134) from Spanish MINECO.

This is the 10th volume of Advances in Comparative Endocrinology, a series of books that collect the communications presented at the congresses of the Iberian Association for Comparative Endocrinology (AIEC). This round number marks roughly 20 years since the foundation of AIEC and the beginning of the biennial meetings organized by AIEC affiliated groups. In 2005, our Comparative Endocrinology and Integrative Biology group CEIB-CCMAR organized the 5th AIEC meeting and it was a pleasure to welcome the 12th AIEC Congress back again to Faro, Portugal, in 2019 (26-28 September). Volume X includes contributions resulting from plenary, keynote, oral or poster presentations, and authored by almost 150 senior or young scientists and students, which show the latest advances in the fields of reproduction, energy metabolism, genomics, evolution, stress, immune response and growth in both vertebrates and invertebrates. Studies range from fundamental questions on endocrinology to environmental challenges, animal production and welfare, or the mechanisms of disease, and reach beyond the Iberian borders. In addition to Portugal and Spain, the science presented here comes from institutions in Canada, Chile, France, Norway and USA. About 30% of the communications result from international cooperation, and over 55% were direct collaborations between institutions. AIEC aims to promote collaborations and its congresses are a privileged stage for networking., Supported by funds from the Ministerio de Ciencia, Innovación y Universidades (MICIUN) (AGL2017-89436-R and RTI2018- 100757-B-I00), 2017SGR-1574 and XRAq. M.P-A and S.B-P are recipients of a FPI fellowship from the MICIUN (BES-2016-078697 and PRE2018-085580 respectively).

Biosensor technology for tracking individual challenged fish behaviour has the potential to revolutionize aquaculture, allowing farmers and breeders to orientate selective breeding towards more robust and efficient fish or improve culture conditions for a more sustainable and ethical production. The proposed solution within the AQUAEXCEL2020 EU project is a stand-alone, small and light (1 g) device (AEFishBIT), based on a tri-axial accelerometer and a microprocessor. It 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. The conducted operculum attachment protocol does not show signs of tissue damage or growth impairment in active feeding gilthead sea bream. AEFishBIT offers a wide range of new information based on individual behaviour, allowing to point out the asynchrony of movements as an indirect measure of aging and adaptability to farming environment, as well as to discriminate different coping behaviour (proactive or reactive) of gilthead sea bream challenged with low water oxygen concentrations. AEFishBIT also provides reliable information of disease outcome in fish parasitized with an intestinal myxozoan, emerging as a powerful tool for sensing the quality of the environment and improving selective breeding protocols., The study has received funding from the European Union’s Horizon 2020 research and innovation programme, GA no 652831 (AQUAEXCEL2020).

The present study evaluated, in an 18-month gilthead sea bream trial, the time course effects of genetics on individual size variation and growth compensation processes in families selected by heritable growth in the PROGENSA®breeding program. Families categorized as fast, intermediate, and slow growing had different growth trajectories with a more continuous growth in fast growth families. This feature was coincident with a reduced size variation at the beginning of the trial that clustered together the half-sib families sharing the same father. Regression analysis evidenced that the magnitude of compensatory growth was proportional to the initial size variation with no rescaling of families at this stage. By contrast, the finishing growth depensation process can mask, at least partially, the previous size convergence. This reflects the different contribution across the production cycle of genetics in growth. How early-life experiences affect growth compensation at juvenile stages was also evaluated in a separate cohort, and intriguingly, a first mild-hypoxia pulse at 60–81days post-hatching (dph) increased survival rates by 10%, preventing growth impairment when fish were exposed to a second hypoxia episode (112–127dph). The early hypoxia experience did not have a negative impact on growth compensatory processes at juvenile stages. By contrast, a diminished capacity for growth compensation was found with repeated or late hypoxia experiences. All this reinforces the use of size variation as a main criterion for improving intensive fish farming and selective breeding.