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Microalgae are one of the most promising groups of functional ingredients for inclusion in microdiets for Senegalese sole (Solea senegalensis) larvae, supplying components with anti-oxidant effects that may be important during an accelerated stage of development where excessive reactive oxygen substance production leading to oxidative stress may occur. The diatom Phaeodactylum tricornutum complies with such potential since it is rich in fucoxanthin, a carotenoid with demonstrated antioxidant activity. This study aimed at determining the effect of dietary P. tricornutum supplementation on the growth performance, survival and levels of oxidative stress of Senegalese sole postlarvae. Four dietary treatments were tested: a commercial microdiet, used as control, and three experimental variants where P. tricornutum was included (1) whole cells at 5%-WP5, (2) broken cells at 5%-BP5 and (3) broken cells at 10%-BP10. At the end of the trial, no significant differences were found in growth performance of larvae fed diets where P. tricornutum was included at 5% (WP5 and BP5) and the commercial control. However, larvae from the BP10 treatment had a significantly higher dry weight than larvae from the remaining treatments. Moreover, the experimental diets had no significant effects on oxidative stress. Nonetheless, lipid peroxidation levels were significantly higher in smaller larvae fed diets where P. tricornutum whole cells were included, relatively to the commercial control. The results of the present study demonstrate that P. tricornutum broken cell biomass has the potential for inclusion in weaning microdiets for Senegalese sole, increasing larval growth performance at an inclusion level of 10 %. European Union European Commission [613588] info:eu-repo/semantics/publishedVersion

To enhance fish general health, feeds can be supplemented with health-promoting additives, reducing the need to use chemotherapeutics. Incorporation of marine algae biomasses in aquafeeds has been shown to improve fish immune status by enhancing innate immune response. This study evaluated the effects of Phaeodactylum tricornutum incorporation in feed by two different processes, either as freeze-dried biomass or broken cell wall biomass, on fish health status and performance. Triplicate groups of gilthead seabream juveniles (13.3 ± 0.3 g) were either fed a control diet (CTRL) with an extreme (i.e., 0% fishmeal), nutritionally balanced, formulation, or two experimental diets formulated as the CTRL with 1% inclusion of the microalga P. tricornutum at the expense of wheat meal: BC diet contains P. tricornutum broken cells and WC diet microalgae whole cells. After 2 and 12 weeks of feeding, blood was collected for hematological procedures, whereas plasma and mucus were sampled for immune parameters. Head-kidney, liver, and white skeletal muscle were also collected for gene expression measurements. No major differences were observed in hematological nor plasma humoral parameters after 12 weeks irrespective of dietary treatment. Arrays of 29–31 genes were analyzed in the different tissues, revealing an early dietary effect (2 weeks) in a tissue-specific pattern. In the liver, the major effect was found in the GH/IGF axis and in muscle there was a late downregulation of myostatin (mstn) gene, mainly due to WC diet, even though all fish had similar growth performance. Regarding the head-kidney, BC diet led to alpha-2-macroglobulin (a2m) gene upregulation. Also, the same treatment showed increased mucus alternative complement pathway and bactericidal activity at 2 and 12 weeks, respectively. Hence, it seems that BC diet has a potential stimulatory effect that might be relevant as a prophylactic measure before a predictable stressful event., This work has been funded under the EU FP7 by the MIRACLES project No. 613588: Multi-product Integrated bioRefinery of Algae: from Carbon dioxide and Light Energy to high-value Specialties and by national funds through FCT - Foundation for Science and Technology within the scope of UIDB/04423/2020 and UIDP/04423/2020. B. Reis, L. Ramos-Pinto, B. Costas, and S. Engrola were supported by the Portuguese Foundation for Science and Technology (PD/BDE/129262/2017, PD/BDE/114436/2016, IF/00197/ 2015, and IF/00482/2014, respectively).

Background In humans the stress response is known to be modulated to a great extent by psychological factors, particularly by the predictability and the perceived control that the subject has of the stressor. This psychological dimension of the stress response has also been demonstrated in animals phylogenetically closer to humans (i.e. mammals). However, its occurrence in fish, which represent a divergent vertebrate evolutionary lineage from that of mammals, has not been established yet, and, if present, would indicate a deep evolutionary origin of these mechanisms across vertebrates. Moreover, the fact that psychological modulation of stress is implemented in mammals by a brain cortical top-down inhibitory control over subcortical stress-responsive structures, and the absence of a brain cortex in fish, has been used as an argument against the possibility of psychological stress in fish, with implications for the assessment of fish sentience and welfare. Here, we have investigated the occurrence of psychological stress in fish by assessing how stressor controllability modulates the stress response in European seabass (Dicentrarchus labrax). Results Fish were exposed to either a controllable or an uncontrollable stressor (i.e. possibility or impossibility to escape a signaled stressor). The effect of loss of control (possibility to escape followed by impossibility to escape) was also assessed. Both behavioral and circulating cortisol data indicates that the perception of control reduces the response to the stressor, when compared to the uncontrollable situation. Losing control had the most detrimental effect. The brain activity of the teleost homologues to the sensory cortex (Dld) and hippocampus (Dlv) parallels the uncontrolled and loss of control stressors, respectively, whereas the activity of the lateral septum (Vv) homologue responds in different ways depending on the gene marker of brain activity used. Conclusions These results suggest the psychological modulation of the stress response to be evolutionary conserved across vertebrates, despite being implemented by different brain circuits in mammals (pre-frontal cortex) and fish (Dld-Dlv).

Selection criteria for sperm cryopreservation are highly relevant in zebrafish since sperm quality is particularly variable in this species. Successful cryopreservation depends on high-quality sperm, which can only be ensured by the selection of breeders. Consequently, male selection and management are a priority to improve cryopreservation, and therefore, this study aimed to characterize optimal age and sperm collection frequency in zebrafish. For this purpose, males from wild type (AB) and from a transgenic line [Tg(runx2:eGFP)] were sampled at 6, 8, 12, and 14 months. For each age, sperm were collected at time 0 followed by samplings at 2, 7, and 14 days of rest. Sperm quality was assessed according to motility and membrane viability parameters. Quality assessment showed that Tg(runx2:eGFP) displayed significantly higher motility than AB and younger males showed higher motility in both lines. Sperm collection frequency affected membrane viability. While AB fish recovered sperm viability after 14 days of rest, Tg(runx2:eGFP) could not recover. Consequently, it may be important to study the sperm quality of each zebrafish line before sperm cryopreservation. Taking into consideration the results achieved in both lines, sperm collection should be performed between 6 and 8 months of age with a minimum collection interval of 14 days. N730984, EBB-EAPA_501/2016, PEst-C/MAR/LA0015/2011 info:eu-repo/semantics/publishedVersion

© 2021 by the authors., AAs have become interesting feed ingredients to be used in functional fish feeds as not only are they protein building blocks, but they also participate in several other key metabolic processes. In the present study, a comprehensive analysis of transcriptomics, hematology, and humoral immune parameters (plasma and skin mucus) were measured twice over the course of the feeding trial (four weeks). Plasma antiprotease activity increased in fish fed Thr compared to those fed the CTRL and Tau treatments, regardless of sampling time. The bactericidal activity in skin mucus decreased in fish fed Tau and His treatments compared to those fed the CTRL diet after two weeks. The membrane IgT (mIgT) was upregulated in fish fed Tau after four weeks, while C-type lectin domain family domain 10 member (clec10a) was downregulated in fish fed Thr after two weeks of feeding. By comparing the molecular signatures of head-kidney by means of a PLS-DA, it is possible to visualize that the main difference is between the two sampling points, regardless of diet. Altogether, these results suggest that dietary supplementation with these AAs at the tested levels causes mild immune-modulation effects in gilthead seabream, which should be further studied under disease challenge conditions., This work was partially supported by Projects ALISSA (reference ALG-01-0247- FEDER-3520), UIDB/04423/2020, UIDP/04423/2020 and IF/00482/2014, financed by Portugal and the European Union through FEDER, COMPETE 2020, and CRESC Algarve 2020, in the framework of Portugal 2020, and through the COMPETE and Operational Human Potential Programs and national funds through Fundação para a Ciência e a Tecnologia (FCT, Portugal). This study was also partially funded by project ATLANTIDA (ref. NORTE-01-0145-FEDER-000040), supported by the Norte Portugal Regional Operational Program (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement and through the European Regional Development Fund (ERDF). L. Ramos-Pinto, and B. Costas were supported by FCT, Portugal (PD/BDE/114436/2016 and IF/00197/2015, respectively).

This data article is associated with the research article "Evaluating the impact of methionine-enriched diets in the liver of European seabass through label-free shotgun proteomics". Here it is described the data obtained from proteomic analysis of 36 European seabass juveniles (3 fish x 3 replicate tanks) after 18 days of feeding with experimental diets containing four inclusion levels of methionine (Met): 0.77%, 1%, 1.36% and 1.66% Met (w/w). We analysed this dataset and compared it with that obtained during the long-term feeding period i.e., 85 days. Fish liver proteins were digested with trypsin and purified peptides were analysed by LC-MS/MS. Proteins were identified with at least two peptides at 0.1% Decoy false discovery rate (FDR). In this dataset, we present the analysis of the differential abundant proteins (DAP) with significant differences across treatments after 18 days of feeding (One-Way ANOVA, p < 0.05). Treatment's comparisons were also performed between the 18- and 85-days feeding trials through Two-Way ANOVA (p < 0.05). MS/MS raw data are available via ProteomeXChange with identifiers PXD019610 and 10.6019/PXD019610 (18-days dataset); and PXD019622 and 10.6019/PXD019622 (85-days dataset). This dataset corresponds to fish sampled after 18-days of experimental trial and is made available to support the study conducted in the afore-mentioned article, by performing the analysis during a short-term period of feeding. The data presented may be further used in other nutritional studies e.g., addressing hepatic changes mediated by Met. ALG-01-0247-FEDER-3520/ UIDB/04326/2020/ IF/00482/2014/CP1217/CT0005 info:eu-repo/semantics/publishedVersion

Fish meal replacement by plant-protein sources is a priority in aquaculture feeds. Within this framework, dietary supplementation with essential amino acids (EAA), as tryptophan (TRP), is strategic to ensure that the individual nutritional needs are met, besides promoting enhanced immunological status. The purpose of this study was to examine the beneficial effects of TRP incorporation in plant-protein source diets on fish growth performance and nutritional status. We tested diets with 20% lower (LTRP) and 27% higher (HTRP) of the putative requirements of TRP for seabream (Sparus aurata) and assessed its impact on fish physiology and liver metabolism and proteome. After 12 weeks, growth performance, body proximate, hepatic composition and liver metabolic profiling were similar between diets. Nevertheless, liver proteome analysis indicated a higher accumulation of proteins involved in acute-phase responses, typically triggered by infection, inflammation or trauma, in fish fed with HTRP diet as compared with those fed with LTRP. The overall results obtained suggest a potential beneficial effect of TRP supplementation in terms of immune stimulation, without compromising growth or feed intake. Moreover, proteomics and metabolic profiling demonstrate to be valuable tools in this endeavour. Significance: Nutritional needs are hard to assess in aquaculture fisheries, and many times controversial depending on the methodology employed. The estimated amino acid requirements depend on both fish species and stage development, making it extremely hard to standardise. On the other hand, the substitution of fish-based to plant-based protein sources diets towards a sustainable aquaculture, may imbalance these requirements, being necessary further studies to assess the impact on fish growth and development. Finally, the incorporation of crystalized amino acids such as TRP into diets aims global better performance both at fish health/immune condition and growth development. This work focused on the potential beneficial effects of TRP supplementation into diets with a plant-based protein source, addressing the effects on the liver metabolism and proteome, and on growth performance of Gilthead seabream juveniles, a species with special relevance and economical importance in the Mediterranean region. The present study by employing proteomics together with metabolic profiling shows that TRP supplementation at the tested doses, does not compromise growth performance, and seems to stimulate the immune system. Our findings can contribute to the development of new feed formulations for Gilthead seabream species, therefore, reinforcing the resilience and competitiveness of the on-growing aquaculture industry and impact directly the sustainability of living resources with the decrease of the fisheries needs to fulfil the human search for quality proteins consume. European Union through FEDER [ALG-01-0247-FEDER3520] European Union through COMPETE 2020 [ALG-01-0247-FEDER3520] European Union through CRESC Algarve 2020 [ALG-01-0247-FEDER3520] Portuguese Foundation for Science and Technology (FCT)Portuguese Foundation for Science and Technology [UIDB/04326/2020] Portuguese Foundation for Science and Technology (FCT) through COMPETE Programme Portuguese Foundation for Science and Technology (FCT) through Operational Human Potential Programme info:eu-repo/semantics/publishedVersion

European aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 ± 0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29–31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15–24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R) and predicted (Q) with four components up to 93% and 78% of total variance, respectively. Component 1 (R = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period., This work was partially supported by Projects ALISSA (reference ALG-01-0247-FEDER-3520), UID/Multi/04423/2019, IF/00197/2015 and IF/00482/2014, financed by Portugal and the European Union through FEDER, COMPETE 2020 and CRESC Algarve 2020, in the framework of Portugal 2020, and through the COMPETE and Operational Human Potential Programmes and national funds through Fundação para a Ciência e a Tecnologia (FCT, P ortugal). L. Ramos-Pinto, S. Engrola and B. Costas were supported by FCT, Portugal (PD/BDE/114436/2016, IF/00482/2014 and IF/00197/2015, respectively).

This deposit is composed by the main article plus the supplementary materials of the publication. The occurrence of emotions in non-human animals has been the focus of debate over the years. Recently, an interest in expanding this debate to non-tetrapod vertebrates and to invertebrates has emerged. Within vertebrates, the study of emotion in teleosts is particularly interesting since they represent a divergent evolutionary radiation from that of tetrapods, and thus they provide an insight into the evolution of the biological mechanisms of emotion. We report that Sea Bream exposed to stimuli that vary according to valence (positive, negative) and salience (predictable, unpredictable) exhibit different behavioural, physiological and neuromolecular states. Since according to the dimensional theory of emotion valence and salience define a two-dimensional affective space, our data can be interpreted as evidence for the occurrence of distinctive affective states in fish corresponding to each the four quadrants of the core affective space. Moreover, the fact that the same stimuli presented in a predictable vs. unpredictable way elicited different behavioural, physiological and neuromolecular states, suggests that stimulus appraisal by the individual, rather than an intrinsic characteristic of the stimulus, has triggered the observed responses. Therefore, our data supports the occurrence of emotion-like states in fish that are regulated by the individual's perception of environmental stimuli. European Commission grant: (FP7–KBBE-2010-4 Contract n°: 265957 Copewell); Fundação para a Ciência e Tecnologia grants: (SFRH/BD/80029/2011, SFRH/BPD/72952/2010). info:eu-repo/semantics/publishedVersion