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Trabajo presentado en Aquaculture Europe 2020, celebrado en modalidad virtual del 12 al 15 de abril de 2021., [Introduction]: The key effects of intestinal microbiota in animal health led to an increasing interest in manipulating these bacterial populations to improve animal welfare. The aquaculture sector is no exception, and in the later 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 gilthead sea bream (Sparus aurata) aquaculture, genetic selection has been applied to improve growth rates, feed conversion, mortality rates, skeletal deformities, disease resistance and quality (García-Celdrán et al., 2015). Selection for growth has been associated with a more continuous growth across seasons and high intestinal plasticity to maximize nutrient absorption when fed plant-based diets, allowing adaptation to dietary changes with no impact on growth and health (Perera et al., 2019). In the current study, we aimed to determine whether genetic selection in gilthead sea bream influences the intestinal microbial composition, how these bacterial populations are modulated by dietary changes, and the effect on disease resistance., [Methods]: Three different groups of families of gilthead sea bream that were selected for growth (fast: e5e6, intermediate: c2c7 and slow: c4e4) (Perera et al., 2019) were kept together in the same open-flow tanks, and fed a control or a well-balanced plant-based diet during nine months. Twelve animals per group were sacrificed, and the adherent bacteria from the anterior intestinal portion were collected and immediately used for DNA extraction. The V3-V4 region of the 16S rRNA of each individual sample was amplified and sequenced by Illumina MiSeq. After quality filtering, taxonomic assignment was performed with a custom-made pipeline using the RDP database. Alpha diversity was calculated using Phyloseq, and beta diversity using PERMANOVA and PLS-DA models. Metagenome prediction and pathway analysis were performed using Piphillin. In parallel, 30 fish of the fast- and slow-growth groups were experimentally 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. The composition at the level of phylum was also not different, indicating that all families harbour the typical intestinal core microbiota of gilthead sea bream, with Proteobacteria, Firmicutes, Bacteroidetes and Actinobacteria as the most abundant phyla. PERMANOVA and PLS-DA analyses showed significant differences in the microbial composition among families and dietary groups. The plant-based diet significantly changed the microbiota in the intermediate- and slow-growth families, with a much lower effect on the fast-growing group, where only a small percentage of the microbiota was changing (Fig.1). However, when performing pathway analysis from the inferred metagenomes, the small changes detected in the fastgrowing families potentially accounted for more changes at the metabolic level when compared to the other families. This was reflected in the 17, 11 and 4 pathways significantly changing (padj < 0.05, log2 fold change > |1|) in the fast-, intermediate- and slow-growing families, respectively, when fed plant-based diets. Upon parasitic infection, the fastgrowing group showed significantly lower disease signs and parasite intensity and abundance than the slow-growing animals., [Conclusions]: The genetic background, microbiota composition, and physiological plasticity are intricately linked, yielding selection for fast heritable growth more robust individuals. They adapt better to dietary changes, reshape their intestines and organosomatic indexes for an efficient nutrient digestion and absorption, and cope more efficiently with intestinal pathogens. These animals also harbour a plastic microbiota, which effectively adapts to the metabolic challenges induced by dietary changes., Spanish (Bream AquaINTEC, RTI2018-094128-B-100; PROGENSA III) and H2020 EU (AQUAIMPACT 818367) projects. MCP (RYC2018-024049-/AEI/10.13039 /501100011033 & ESF) and EP (IJCI-2016-27637s) were funded by Postdoctoral Research Fellowships.

Amoebic gill disease (AGD) is one of the main diseases affecting Atlantic salmon (Salmo salar L.) mariculture. Hallmarks of AGD are hyperplasia of the lamellar epithelium and increased production of gill mucus. This study investigated the expression of genes involved in mucus secretion, cell cycle regulation, immunity and oxidative stress in gills using a targeted 21-gene PCR array. Gill samples were obtained from experimental and natural Neoparamoeba perurans infections, and sampling points included progressive infection stages and post-freshwater treatment. Up-regulation of genes related to mucin secretion and cell proliferation, and down-regulation of pro-inflammatory and pro-apoptotic genes were associated with AGD severity, while partial restoration of the gill homeostasis was detected post-treatment. Mucins and Th2 cytokines accoun ted for most of the variability observed between groups highlighting their key role in AGD. Two mucins (muc5, muc18) showed differential regulation upon disease. Substantial up-regulation of the secreted muc5 was detected in clinical AGD, and the membrane bound muc18 showed an opposite pattern. Th2 cytokines, il4/13a and il4/13b2, were significantly upregulated from 2 days post-infection onwards, and changes were lesion-specific. Despite the differences between experimental and natural infections, both yielded comparable results that underline the importance of the studied genes in the respiratory organs of fish, and during AGD progression., Irish Research Council (Employment Based Postgraduate Programme Project ID EBPPG/2013/2), European Commission under the TNA programme (project ID AE030036) within AQUAEXCEL2020 project (652831)., CSIC PIE project no. 201740E013.

The potential benefits of a commercial preparation of heptanoate (NOREL, HEPTON®) were evaluated in an 11‐week gilthead sea bream feeding trial (May–August), using a factorial design with four isoproteic and isoenergetic diets. Fish meal (FM) was added at 200 g/kg in D1–D2 diets and at 50 g/kg in D3–D4 diets, which also contained fish peptones and plant proteins as source of proteins. Heptanoate was added at 3 g/kg in D2 and D4 diets. All fish grew from 13–14 g to 81–84 g with an overall feed efficiency (FE) of 0.91–0.94. An early impairment of FE (weeks 1–4) was found with the standard FM‐based diet (D1), but this detrimental condition was reversed by heptanoate, increasing FE from 0.88 in D1 fish to 0.99 in D2 fish. Further improvements were progressively diluted over time, remaining D2 and D3–D4 fed fish almost undistinguishable through all the trial. Heptanoate supplementation produced higher hepatic glycogen depots, but no signs of histopathological damage were found in liver or intestine. Other lasting heptanoate effects included changes in plasma antioxidant capacity, plasma cortisol and growth hormone levels, and measures of respirometry in swimming performance tests. Altogether, it supports the potential use of heptanoate to speed up adaptive and healthy metabolic states of farmed fish to cope with challenging culture conditions., This work has been carried out with the financial support from NOREL S.A. and BioMar. Additional funding has been received by Spanish MINECO project no. AGL2013‐48560‐R and Generalitat Valenciana (PROMETEO‐FASE II/2014/085). MCP was supported by MINECO through grant FPDI‐2013‐15741 and by CSIC PIE project no. 201740E013. JAMS was supported by a postdoctoral research fellowship (Juan de la Cierva‐Formación, Reference FJCI‐2014‐20161) from MINECO

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., This study was partially supported by the EU H2020 program and by the Spanish Ministry of Economy and Competitiveness through ParaFishControl (634429) and AGL-2013-48560-R research projects, respectively. This publication reflects the views of the authors only; the European Commission cannot be held responsible for any use which may be made of the information contained therein. M.C.P. was contracted under CSIC PIE project no. 201740E013

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., Tis work was supported by the European Commission under the 8th (H2020) Framework Program for Research and Technological Development of the European Union (PARAFISHCONTROL Grant No. 634429) and by the 7th Framework program [NEMO Grant No. PITN-GA-2008–214505].

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 driving the separation. Pathway analysis performed with the inferred metagenome showed significant differences between Y+1 and Y+4, with an increase with age of pathways related to cell death, motility, biofilm formation, environmental response, infection, metabolism of lipids, secondary metabolites, ansamycins biosynthesis and nitrotoluene degradation. Carbohydrate and lipoic acid metabolism and atrazine degradation were down-regulated with age. The effect of host age on the intestinal microbial diversity has been previously studied in other fish species finding an increase in diversity with age (Zhang et al., 2018). However, these studies mainly focused on early life stages of the fish.Nonetheless, it is clear that the intestinal ecological niche matures with age leading to changes in the associated microbiota (Stephens et al., 2016). The dominance of the phyla Proteobacteria, Bacteroidetes, Firmicutes and Actinobacteria has already been reported in several studies on carnivorous marine fish, including GSB (Estruch et al., 2015). The current results highlight a gradual increase of Proteobacteria abundance with age with the consequent decrease of the other phyla. Photobacterium and Vibrio were previously described as the dominant genera in GSB posterior intestine (Piazzon et al., 2017) and it is noteworthy that in the current study these genera are absent in the anterior intestine of Y+1 fish,becoming more dominant with age. These two generainclude many pathogenic and opportunistic species, but others are important symbionts, assisting in the breakdown of dietary components (Egerton et al., 2018). Overall, changes in microbial composition drive changes in metabolic pathways, as the up-regulation of infection-related pathways, nitrotoluene degradation and sphingolipid metabolism, and the down-regulation of carbohydrate metabolism pathways, which seem to indicate an inflammatory profile in the intestines of older fish., This study was funded by Spanish MICIUN (From Nutrition and Genetics to Sea Bream Aquaculture Intensification and Technological Innovation; Bream-AquaINTECH, RTI2018-094128-B-I00). Additional funding was obtained from the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement no. 818367; AquaIMPACT – Genomic and nutritional innovations for genetically superior farmed fish to improve efficiency in European aquaculture.

Trabajo presentado en 3rd International Conference on Fish and Shellfish Immunology (ISFSI), celebrado en Gran Canaria (España), del 16 al 20 de junio de 2019, 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 fish and their relevance for the human immune system. Additionally, new results on the effects of environmentally relevant concentration of exogenous estradiol exposure during European sea bass (Dicentrarchus labrax) development will be presented with regard to oestrogen's ability to trigger effects on immunocompetence, contributing to fill the gaps on vertebrate immunotoxicology.

[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., This work has been carried out with financial support from the European Union, through the Horizon H2020 research and innovation programme under grant agreement 634429 (ParaFishControl). MCP was contracted under CSIC PIE project no. 201740E013. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

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

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

Trabajo presentado en el XVI Congreso Nacional de Acuicultura, celebrado en Zaragoza (España), del 3 al 5 de octubre de 2017, [EN] This project promotes technology developments for gilthead sea bream industrial sector of Spain, to facilitate and normalize the implementation of genetic selection schemes in this species, by defining new technological traits of commercial interest, with new standardized and extensible technological developments that permit, in an economical manner, normalized measuring methods, to study genetic parameters (growth,morphology, carcass and resistance disease), genotype-environment interaction to extend the Spanish production, and guarantee the production levels to the whole production chain. At the same time, improving the breeders management under industrial conditions, the validation of feeding efficiency during the selection processes and location of biomarkers linked and applied in genomic selection., [ES] Con la presente propuesta se pretende desarrollar metodología transferible al sector industrial de dorada en España que facilite y normalice la implementación de esquemas de selección genética en esta especie, mediante la definición de caracteres de interés comercial con nuevos desarrollos tecnológicos que estandaricen y hagan extensible a todo el sector, de manera económica, la medición de caracteres a escala industrial, el estudio de parámetros genéticos para caracteres de interés comercial (crecimiento, morfología, rendimiento y resistencia a enfermedades) en stocks de reproductores de la industria española, la estimación de la interacción genotipo-ambiente para permitir de manera garante la expansión de los rendimientos productivos de la industria Española, la gestión de reproductores bajo los propios condicionantes de la industria, así como la validación de la eficiencia alimenticia de los procesos de selección y búsqueda de biomarcadores asociados y aplicables en los modelos de selección., Este trabajo ha sido financiado a través de las Ayudas a la investigación de los Planes Nacionales de Acuicultura 2016 por el Ministerio de Agricultura y Pesca, Alimentación y Medioambiente (MAPAMA) y el Fondo Europeo Marítimo y de Pesca (FEMP).

Trabajo presentado en la 18th International Conference on Diseases of Fish and Shellfish, celebrada en Belfast, del 4 al 8 de septiembre de 2017, Teleost fish express three different immunoglobulin isotypes: IgM, IgD and the teleost specific IgT, which is attributed a more mucosal role similar to mammalian IgA. In this study, we describe the complete sequence of gilthead sea bream IgM and IgT in both, their soluble (s) and membrane bound (m) forms and study their differential expression in different tissues and upon infections of different etiology, vaccination and dietary interventions. Gilthead sea bream IgM and IgT show the prototypical structure known for teleost and, while sIgM was the overall highest expressed isoform, mIgT was highest expressed in mucosal tissues such as gills and intestine. IgM and IgT were differentially regulated upon infection. IgT was highly upregulated locally upon infection with the intestinal parasite Enteromyxum leei or systemically after Nodavirus infection. Long-term intestinal parasitic infections increased the serum titer of both isotypes. Mucosal vaccination against Photobacterium damselae subsp. piscicida finely regulated the Ig response inducing a systemic increase of IgM titers in serum and a local IgT response in skin mucus when animals were exposed to the bacterial pathogen by bath challenge. Interestingly, when fish were fed for a long term with plant-based diets, the significant upregulation of sIgT observed in parasite infected intestines and head kidneys of control fed fish was completely inhibited. Fish fed plant-based diets also presented a worse disease outcome reflecting the importance of this immunoglobulin in the maintenance of intestinal health. All these results corroborate the mucosal role of IgT and emphasize the importance of a finely tuned regulation of Ig isotypes upon infection, which could be of special interest in vaccination studies. They also highlight the importance of the dietary conditions as significant regulators of the immune response and disease resistance, This work has been carried out with financial support from by the Spanish MINECO under projects AGL2013-48560-R to JP-S and AS-B and AGL2014-51773-C3-3-R to EG-C. Additional funding was provided by the European Union, through the Horizon H2020 research and innovation program under grant agreement 634429 (ParaFishControl) and through the FP7 under grant 311993 (TargetFish).

Trabajo presentado en la 18th International Conference on Diseases of Fish and Shellfish, celebrada en Belfast, del 4 al 8 de septiembre de 2017, Enteromyxum leei is the causative agent of a parasitic emaciative disease (enteromyxosis) in multiple cultured marine fish species worldwide. In the Mediterranean, this parasite seriously hampers the production of sharpsnout sea bream (Diplodus puntazzo) or red porgy (Pagrus pagrus). The main economic impact on gilthead sea bream (GSB) aquaculture results from arrested growth and difficulties to reach commercial size of infected stocks, but epizootic outbreaks are not rare under some conditions. The lack of effective anti-parasitic therapies in Mediterranean aquaculture has stimulated the search for natural feed additives, which can be incorporated in functional feeds aiming at minimizing the impact of parasitic outbreaks on productivity. Empirical field evidence suggests certain efficacy on the mitigation of health problems including parasites such as E. leei of a functional feed additive (Sanacore® GM, Nutriad). These effects, however, are seldom validated in the laboratory. This work explores the effect of this functional feed additive on GSB enteromyxosis under controlled experimental laboratory conditions. Groups of GSB fed with additive - supplemented or control diets for 5 weeks were challenged with E. leei by anal intubation, and then maintained for 10 further weeks with the different diets. Data on food intake and fish growth were taken throughout the study. In the final sampling (10 wks post challenge), when fish fed the control diet showed the clinical signs of enteromyxosis, intestinal samples were taken for molecular and histological parasite diagnosis. The total parasite load per fish was calculated using a qPCR test targeting the E. leei rDNA gene. The anterior, middle and posterior intestinal segments were differentially examined in the histopathological study. Quantitative analyses of biometrical, growth performance, and parasite load of each group were conducted. The results showed significant differences between the additive-treated groups and the control group, in terms of increased growth performance and food intake, and decreased infection prevalence and parasite abundance. These results provide some experimental support for the consideration of preventive strategies using functional feeds to mitigate some chronic disease problems, although the mechanisms involved are not yet elucidated, This study was partially supported by EU H2020 program through the ParaFishControl Project (634429).

We recently reported on the functional characterization of carp Il10. We showed that carp Il10 is able to downregulate proinflammatory activities by carp phagocytes and promote B cell proliferation, differentiation and antibody production as well as proliferation of memory T cells. Taking advantage of the recent annotation of the carp genome, we completed the sequence of a second il10 paralogue, named il10b, the presence of which was expected owing to the recent (8 million years ago) fourth round of whole genome duplication that occurred in common carp. In the present study we closely compared the two Il10 paralogues and show that Il10a and Il10b have almost identical gene structure, synteny, protein sequence as well as bioactivity on phagocytes. Although the two il10 paralogues show a large overlap in tissue expression, il10b has a low constitutive expression and is highly upregulated upon infection, whereas il10a is higher expressed under basal conditions but its gene expression remains constant during viral and parasitic infections. This differential regulation is most likely due to the observed differences in their promoter regions. Altogether our results demonstrate that gene duplication in carp, although recent, led to sub-functionalization and expression divergence rather than functional redundancy of the Il10 paralogues, yet with very similar protein sequences, Authors were supported by the European Community's 7th Framework Programme (FP7/2007–2013) and by the 8th (H2020) Framework Programs for Research and Technological Development: MCP under Grant FISHIL10 (PIEF-GA-2011-302444); MF and GFW under Grant TARGETFISH (311993) and IMAQUANIM (FOOD-CT-2005-007103); GFW and ASW were also supported under grant PARAFISHCONTROL (634429). MF was also supported by the Netherlands Organisation for Scientific Research (NWO) under Veni Grant number 11200.

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017, The aim of this study was to evaluate the potential benefits of a commercial preparation of the medium-chain fatty acid (MCFA) heptanoate sodium salt (NOREL, HEPTON®) in a factorial design (2 x 2) with standard (20%, D1 and D2) or low (5%, D3 and D4) inclusion levels of fish meal (FM) as dietary protein source in an 11-weeks gilthead sea bream feeding trial. Heptanoate was added at 0.3% in D2 and D4 diets. Results of growth performance from 13-14 g to 81-84 g indicated a fast (SGR=2.2-2.3%) and efficient (FE=0.91-0.94) growth, with no changes in plasma IGF-I levels among fish fed the four experimental diets. Even so, an early impairment of FE, reversed by heptanoate supplementation, was observed in D1 FM-based diet. No signs of histopathological damage were found in any group, although higher hepatic glycogen depots were observed in fish fed both heptanoate-supplemented diets at the end of the trial. Other lasting heptanoate effects included changes on plasma antioxidant capacity, circulating cortisol and growth hormone (GH) levels, as well as in metabolic capabilities assessed by measures of oxygen consumption (MO2, mgO2/kg/h) and critical swimming speed (Ucrit) in swim performance tests. The pattern of respirometry metabolism closely resembled that of early FE, and the predictive value of plasma GH for different critical swimming speeds at fish exhaustion is suggested. Altogether, these results support the potential use of heptanoate to improve the healthy metabolic condition of farmed fish under sub-optimal rearing conditions., This work has been carried out with financial support from NOREL S.A. and BioMAR. Additional funding has been received by Spanish MINECO project no. AGL2013-48560-R and Generalitat Valenciana (PROMETEO-FASE II/2014/085). M.C.P. was supported by MINECO through grant FPDI-2013-15741 and by CSIC PIE project no. 201740E013. J.A.M.-S. is supported by a Postdoctoral Research Fellowship (Juan de la CiervaFormación, Reference FJCI-2014-20161) from MINECO.

Trabajo presentado en la 18th International Conference on Diseases of Fish and Shellfish, celebrada en Belfast, del 4 al 8 de septiembre de 2017, The study of T cell responses in fish has been hampered by the lack of specific antibodies and molecular tools. Nowadays, several T cell specific antibodies have been developed for a few fish species. However, our knowledge on fish T cell responses is mainly derived from gene expression analyses of signature genes characterized as a result of the recent availability of genomic and transcriptomic information. Enteromyxum leei is a myxozoan intestinal parasite that produces a slow-progressing intestinal disease leading to anorexia, cachexia and mortalities. This parasite invades the paracellular space of the intestinal epithelium and progresses from the posterior to the anterior intestinal segment. In this study, we define the T cell populations in different intestinal segments and in immune relevant tissues of gilthead sea bream after E. leei infection by anal inoculation. Data mining of the gilthead sea bream transcriptomic database (Nutrigroup-IATS) allowed the construction of a PCR-array composed of 20 different T cell signature transcription factors, cytokines and effector molecules. Our results show that upon E. leei infection, the transcription of the pan T cell markers zap70 and cd3¿, the helper T cell (Th) specific cd4 and the cytotoxic T cell (Tc) specific cd8¿ and cd8ß decreased in head kidney and increased in anterior intestine. The parasite induced a shift from a Th2 to a Th1 response in infected anterior intestines, characterized by down-regulation of the transcription factor gata3 and up-regulation of t-bet, respectively. Significant up-regulation of gzmA and prf1 expression also supported the increase of Tc cells in infected intestines. However, the posterior intestine showed a mixed profile skewed to the anti-inflammatory side, characterized by up-regulation of foxp3, gata3, il10 and il6, still with significant presence of t-bet and ifn¿ transcripts. The expression of il17A/F was significantly up-regulated in infected posterior intestines. Analyses of the overall expression of these signature molecules separated groups of fish by their infection status. In conclusion, E. leei infection activated different T cell phenotypes depending on the intestinal segment and the intensity of infection. This could be due to the chronology of the parasite establishment along the intestinal segments., This work was funded by EU H2020 program through ParaFishControl Project (634429) and Spanish MINECO through project AGL2013-48560 and partially supported by the “Generalitat Valenciana” (PROMETEOII/2014/085). M.C.P. was contracted under CSIC PIE project no. 201740E013 and FPDI-2013-15741 and I.E. under APOSTD/2016/037 grant by the “Generalitat Valenciana”.

Background. The increased demand for fish protein has led to the intensification of aquaculture practices which are hampered by nutritional and health factors affecting growth performance. To solve these problems, antibiotics have been used for many years in the prevention, control and treatment against disease as well as growth promoters to improve animal performance. Nowadays, the use of antibiotics in the European Union and other countries has been completely or partially banned as a result of the existence of antibiotic cross-resistance. Therefore, a number of alternatives, including enzymes, prebiotics, probiotics, phytonutrients and organic acids used alone or in combination have been proposed for the improvement of immunological state, growth performance and production in livestock animals. The aim of the present study was to evaluate two commercially available feed additives, one based on medium-chain fatty acids (MCFAs) from coconut oil and another with a Bacillus-based probiotic, in gilthead sea bream (GSB, Sparus aurata), a marine farmed fish of high value in the Mediterranean aquaculture. Methods. The potential benefits of adding two commercial feed additives on fish growth performance and intestinal health were assessed in a 100-days feeding trial. The experimental diets (D2 and D3) were prepared by supplementing a basal diet (D1) with MCFAs in the form of a sodium salt of coconut fatty acid distillate (DICOSAN R ; Norel, Madrid, Spain), rich on C-12, added at 0.3% (D2) or with the probiotic Bacillus amyloliquefaciens CECT 5940, added at 0.1% (D3). The study integrated data on growth performance, blood biochemistry, histology and intestinal gene expression patterns of selected markers of intestinal function and architecture. Results. MCFAs in the form of a coconut oil increased feed intake, growth rates and the surface of nutrient absorption, promoting the anabolic action of the somatotropic axis. The probiotic (D3) induced anti-inflammatory and anti-oxidant effects with changes in circulating cortisol, immunoglobulin M, leukocyte respiratory burst, and mucosal expression levels of cytokines, lymphocyte markers and immunoglobulin T. Discussion. MCFA supplementation showed positive effects on GSB growth and intestinal architecture acting mainly in the anterior intestine, where absorption takes place. The probiotic B. amyloliquefaciens CECT 5940 exhibited key effects in the regulation of the immune status inducing anti-inflammatory and anti-oxidant effects which can be potentially advantageous upon infection or exposure to other stressors. The potential effects of these feed additives in GSB are very promising to improve health and disease resistance in aquaculture., Spanish MINECO: AGL2013-48560-R. ., Generalitat Valenciana: PROMETEO-FASE II/2014/085., MINECO: FPDI-2013-15741., CSIC PIE: 201740E013

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 duplications coupled to tissue expression diversification in the evolution of gilthead sea bream genome during its successful adaptation to a changing and pathogen-rich environment. This issue also underscores a role of evolutionary routes for rapid increase of the gene repertoire in teleost fish that are independent of polyploidization. Since gilthead sea bream has a well-recognized plasticity, the current study will advance our understanding of fish biology and how organisms of this taxon interact with the environment., This work was financed by Spanish (Intramural CSIC, 1201530E025; MICINN BreamAquaINTECH, RTI2018-094128-B-I00) and European Union (AQUAEXCEL2020, 652831) projects to JP-S. BS was supported by a predoctoral research fellowship (Doctorados Industriales, DI-17-09134) from Spanish MINECO.

Trabajo presentado en 3rd International Conference on Fish and Shellfish Immunology (ISFSI), celebrado en Gran Canaria (España), del 16 al 20 de junio de 2019, 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 only reached 55%. Condition factor (CF) and specific growth rate (SGR), key parameters affected by enteromyxosis, were higher in R group. There was a significant correlation between prevalence of infection and SGR and CF. Immunohistochemistry and gene expression studies will reveal whether this partial protection was due to higher presence of specific antibodies or specific cell populations. These results show that, even with this long term disease, passive immunization can confer some degree of protection. The administration of specific antibodies during exposure, probably provided fish with time to activate the specific defenses before the parasite proliferated., This work was funded by EU H2020 program through ParaFishControl Project (634429) and Spanish MINECO project AGL2013-48560. M.C.P. was contracted under CSIC PIE project no. 201740E013 and FPDI-2013-15741 and I.E. under APOSTD/2016/037 grant by the “Generalitat Valenciana”.