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Abstract Methionine (Met) is one of the most limiting essential amino acids in aquafeeds, and different commercial feed‐grade Met sources are available for diet supplementation, namely L‐Met, DL‐Met, and hydroxy‐Met (OH‐Met). In this work, we compared the bioefficacy of OH‐Met and DL‐Met as diet supplements for gilthead seabream (Sparus aurata) juveniles reared at normal (22°C) and high (29°C) water temperatures. Two isoproteic (50%) and isolipidic (20%) practical diets (24% fish meal; 6% fish oil) were formulated to contain 1% Met and were supplemented with 0.2% DL‐Met (diet DL‐Met) or OH‐Met (diet OH‐Met). The diets were fed to triplicate groups of fish with an initial body weight of 13 g for 78 days. Growth performance and N retention (%) were higher at 29°C. However, feed efficiency, protein efficiency ratio, and hepatic and visceral indexes were higher at 22°C. Hepatic oxidative stress indicators were not affected by dietary Met source. In the muscle, however, oxidative stress index and lipid peroxidation were lower in fish fed the OH‐Met diets. Overall, DL‐Met and OH‐Met proved to be similarly effective as dietary methionine sources for gilthead seabream, while OH‐Met seemed to be more effective in reducing oxidative stress in fish reared at high temperatures.

(1) Background: This study aimed to evaluate the potential of short-chain fatty acids as functional ingredients to improve the immune status and disease resistance of European seabass (Dicentrarchus labrax) juveniles. (2) Methods: For that purpose, triplicate groups of fish with an initial body weight of 15.2 ± 0.03 g were fed isoproteic (43% crude protein) and isolipidic (18% crude lipids) diets supplemented with sodium acetate (SA), sodium propionate (SP), and sodium butyrate (SB) at two inclusion levels: 0.25% and 0.50%. An unsupplemented diet was used as a control. After 56 days of feeding with the experimental diets, fish were intraperitoneally (i.p.) injected with 100 µL of Vibrio anguillarum (1.2 × 107 Colony Forming Units (CFU)/mL) and mortality was recorded for 3 weeks. At the end of the trial, there were no differences in survival between the treatment groups and the control, but survival was higher in fish fed the diet supplemented with SB 0.50 than SP 0.25 (93.3 vs. 66.7%). Compared to the pre-challenge values, and regardless of diet composition, all hematological parameters (hemoglobin, hematocrit, red blood cells, white blood cells) measured decreased after 4 h of bacterial challenge, except for neutrophils which were increased. Independently of diet composition, lysozyme and nitric oxide decreased at 4 and 24 h post infection. Compared to the control, diets supplemented with SA and SP promoted an up-regulation of both pro- and anti-inflammatory cytokines at 4 h after the challenge, while the diets supplemented with SB promoted an up-regulation of pro- and anti-inflammatory cytokines at 24 h after the challenge. (3) Conclusions: Overall, present results suggest that SA and SP provide a fast response to a bacterial challenge in European sea bass juveniles, while SB provides increased survival.

Aquaculture production has been growing consistently over the last few decades to meet the increasing animal protein demand of the human population. However, increased production and rearing intensities raise the challenges of guaranteeing fish health and welfare, which is essential to avoid losses and ensure product quality. Biomarkers can provide insights into the fish’s nutritional, physiological, and health status, and aid in the evaluation of early nutritional and physiological imbalances, distress conditions, and pathological diagnosis. The discovery and validation of biomarkers rely mostly on the use of information provided by different parameters, including biochemical, metabolic, or immunologic, as well as several omics, from genomics and transcriptomics to proteomics and metabolomics. In this review, a summary of the main biomarkers used in aquaculture is provided along with an overview of the main omics technologies available for further biomarker research. This review also highlights the need to develop non-lethal biomarkers that can easily and quickly be measured to provide a prompt response to producers.

This study aimed to evaluate the intestinal interactions between three short-chain fatty acids (SCFA), namely, acetate, propionate, and butyrate, and pathogenic bacteria (Vibrio anguillarum) in intestinal explants of European sea bass (Dicentrarchus labrax) juveniles. The anterior intestine of 12 fish with an average weight of 100 g (killed by excess anesthesia with 2-phenoxyethanol) were sampled and placed in 24-well plates. The experimental treatments consisted of a control medium and a control plus 1 mM or 10 mM of sodium acetate (SA), sodium butyrate (SB), and sodium propionate (SP). After 2 h of incubation, the explants were challenged with Vibrio anguillarum at 1 × 107 CFU/mL for 2 h. After the bacterial challenge, and regardless of the SCFA treatment, the oxidative stress-related genus catalase (cat) and superoxide dismutase (sod) were down-regulated and glutathione peroxidase (gpx) was up-regulated. Furthermore, the immune-related genes, i.e., the tumor necrosis factor (TNF-α), interleukin 8 (IL-8), transforming growth factor (TGF-β), and nuclear factor (NF-Kβ) were also up-regulated, and interleukin 10 (IL-10) was down-regulated. During the pre-challenge, sodium propionate and sodium butyrate seemed to bind the G-protein coupled receptor (grp40L), increasing its expression. During the challenge, citrate synthase (cs) was down-regulated, indicating that the SCFAs were used as an energy source to increase the immune and oxidative responses. Overall, our results suggest that sodium propionate and sodium butyrate may boost European sea bass immune response at the intestine level.

Summary Aquaculture has been one of the fastest‐growing food industry sectors, expanding at the pace of consumers' demands. To promote safe and effective fish growth performance strategies, and to stimulate environmentally friendly solutions to protect fish against disease outbreaks, new approaches are needed to safeguard fish welfare, as well as farmers and consumers interests. Here, we tested the use of cyanobacterial extracellular vesicles (EVs) as a novel nanocarrier system of heterologous proteins for applications in fish. We started by incubating zebrafish larvae with Synechocystis sp. PCC6803 EVs, isolated from selected mutant strains with different cell envelope characteristics. Results show that Synechocystis EVs are biocompatible with fish larvae, regardless of their structural composition, as EVs neither induced fish mortality nor triggered significant inflammatory responses. We establish also that cyanobacteria are amenable to engineering heterologous protein expression and loading into EVs, for which we used the reporter sfGFP. Moreover, upon immersion treatment, we successfully demonstrate that sfGFP‐loaded Synechocystis EVs accumulate in the gastrointestinal tract of zebrafish larvae. This work opens the possibility of using cyanobacterial EVs as a novel biotechnological tool in fish, with prospective applications in carrying proteins/enzymes, for example for modulating their nutritional status or stimulating specific adaptive immune responses.

Plant feedstuffs are the main ingredients of animal feed. Owing to food–feed competition, increasing the utilization efficiency of these feedstuffs is important for animal nutrition. This can be achieved via solid-state fermentation (SSF). SSF of a plant feedstuff mixture (PFM) (25% rapeseed meal, soybean meal, rice bran, and sunflower meal) by three fungi (Aspergillus ibericus MUM 03.29, Aspergillus niger CECT 2088, and Aspergillus niger CECT 2915) resulted in an increase in protein content by 5%, irrespective of fungi, a reduction in cellulose content by 9 to 11%, and of hemicellulose content by 21 to 34%, relative to unfermented PFM. Enzyme production was measured: the highest cellulase (123.7 U/g), xylanase (431.8 U/g), and beta-glucosidase (117.9 U/g) activity were achieved with A. niger CECT 2088. Principal component analysis showed a positive correlation between all fermented PFMs and enzyme production, protein content, digestibility, and fiber reduction. Bioprocessing of the PFM by SSF increased its nutritional value and digestibility, making it more appealing for animal feeds.

Disease outbreaks are a common problem in aquaculture, with serious economic consequences to the sector. Some of the most important bacterial diseases affecting aquaculture are caused by Gram-negative bacteria including Vibrio spp. (vibriosis), Photobacterium damselae (photobacteriosis), Aeromonas spp. (furunculosis; haemorrhagic septicaemia) or Tenacibaculum maritimum (tenacibaculosis). Lipopolysaccharides (LPS) are important components of the outer membrane of Gram-negative bacteria and have been linked to strong immunogenic responses in terrestrial vertebrates, playing a role in disease development. To evaluate LPS effects in fish, we used a hot-phenol procedure to extract LPS from common fish pathogens. A. hydrophila, V. harveyi, T. maritimum and P. damselae purified LPS were tested at different concentrations (50, 100, 250 and 500 µg mL−1) at 3 days post-fertilisation (dpf) Danio rerio larvae, for 5 days. While P. damselae LPS did not cause any mortality under all concentrations tested, A. hydrophila LPS induced 15.5% and V. harveyi LPS induced 58.3% of zebrafish larvae mortality at 500 µg mL−1. LPS from T. maritimum was revealed to be the deadliest, with a zebrafish larvae mortality percentage of 80.6%. Analysis of LPS separated by gel electrophoresis revealed differences in the overall LPS structure between the bacterial species analysed that might be the basis for the different mortalities observed.

This study aimed to evaluate the utilization by juvenile European sea bass of a SSFed PF mixture with Aspergillus niger CECT 2088. A 22-day digestibility and a 50-day growth trial were performed testing four diets, including 20 or 40% of an unfermented or SSFed PF mixture (rapeseed, soybean, rice bran, and sunflower seed meals, 25% each). SSF of the PF added cellulase and β-glucosidase activity to the diets. Mycotoxin contamination was not detected in any of the experimental diets except for residual levels of zearalenone and deoxynivalenol (100 and 600 times lower than that established by the European Commission Recommendation-2006/576/EC). In diets including 20% PF, SSF did not affect growth but increased apparent digestibility coefficients of protein and energy, feed efficiency, and protein efficiency ratio. On the contrary, in diets including 40% PF, SSF decreased growth performance, feed intake, feed and protein efficiency, and diet digestibility. SSF decreased the intestinal amylase activity in the 40% SSFed diet, while total alkaline proteases decreased in the 20% and 40% SSFed diets. Hepatic amino acid catabolic enzyme activity was not modulated by SSF, and plasma total protein, cholesterol, and triglyceride levels were similar among dietary treatments. In conclusion, dietary inclusion of moderate levels of the SSFed PF, up to 20%, improves the overall feed utilization efficiency without negatively impacting European sea bass growth performance. The replacement of PF with the SSFed PF mixture may contribute to reducing the environmental footprint of aquaculture production.

Abstract Brewer’s spent grain (BSG) is the largest by-product originated from the brewery industry with a high potential for producing carbohydrases by solid-state fermentation. This work aimed to test the efficacy of a carbohydrases-rich extract produced from solid-state fermentation of BSG, to enhance the digestibility of a plant-based diet for European seabass (Dicentrarchus labrax). First, BSG was fermented with A. ibericus to obtain an aqueous lyophilized extract (SSF-BSG extract) and incorporated in a plant-based diet at increasing levels (0—control; 0.1%, 0.2%, and 0.4%). Another diet incorporating a commercial carbohydrases-complex (0.04%; Natugrain; BASF) was formulated. Then, all diets were tested in in vitro and in vivo digestibility assays. In vitro assays, simulating stomach and intestine digestion in European seabass, assessed dietary phosphorus, phytate phosphorus, carbohydrates, and protein hydrolysis, as well as interactive effects between fish enzymes and dietary SSF-BSG extract. After, an in vivo assay was carried out with European seabass juveniles fed selected diets (0—control; 0.1%, and 0.4%). In vitro digestibility assays showed that pentoses release increased 45% with 0.4% SSF-BSG extract and 25% with Natugrain supplemented diets, while amino acids release was not affected. A negative interaction between endogenous fish enzymes and SSF-BSG extract was observed in both diets. The in vivo digestibility assay corroborated in vitro data. Accordingly, the dietary supplementation with 0.4% SSF-BSG increased the digestibility of dry matter, starch, cellulose, glucans, and energy and did not affect protein digestibility. The present work showed the high potential of BSG to produce an added-value functional supplement with high carbohydrases activity and its potential contribution to the circular economy by improving the nutritional value of low-cost and sustainable ingredients that can be included in aquafeeds.

Oral vaccines are highly demanded by the aquaculture sector, to allow mass delivery of antigens without using the expensive and labor-intensive injectable vaccines. These later require individual handling of fish, provoking stress-related mortalities.One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores, extremely resistant structures with wide biotechnological applications, including as probiotics, display systems, or adjuvants. Bacterial spores, in particular those of Bacillus subtilis, have been shown to behave as mucosal vaccine adjuvants in mice models. However, such technology has not been extensively explored against fish bacterial disease.In this study, we used a laboratory strain of B. subtilis, for which a variety of genetic manipulation tools are available, to display at its spores surface either a Vibrio antigenic protein, OmpK, or the green fluorescence protein, GFP. When previously vaccinated by immersion with the OmpK- carrying spores, zebrafish survival upon a bacterial challenge with V. anguillarum and V. parahaemolyticus, increased up to 50 - 90% depending on the pathogen targeted. Further, we were able to detect anti-GFP-antibodies in the serum of European seabass juveniles fed diets containing the GFP-carrying spores and anti-V. anguillarum antibodies in the serum of European seabass juveniles fed the OmpK-carrying spores containing diet. More important, seabass survival was increased from 60 to 86% when previously orally vaccinated with in-feed OmpK- carrying spores. Our results indicate that B. subtilis spores can effectively be used as antigen-carriers for oral vaccine delivery in fish.

This study aims to assess the effects of different dietary n-6/n-3 long-chain polyunsaturated fatty acid ratios and CHO content in the immune response of gilthead seabream. For that purpose, gilthead sea bream juveniles (initial body weight = 47.5 g) were fed for 84 days with four isoproteic (47% crude protein) and isolipidic (18% crude lipids) diets with high (20%) or low (5%) level of gelatinized starch (HS or LS diets, respectively) and included approximately 2.4% ARA or DHA. At the end of the trial, the DHA-enriched groups presented increased red blood cell (RBC) count, hemoglobin, plasmatic nitric oxide (NO) content, and antiprotease and alternative complement activities. The ARA groups had increased thrombocyte count, and plasmatic bactericidal activity against Vibrio anguillarum was lower in the fish fed the ARA/LS diet. After the feeding trial, the fish were challenged with an intraperitoneal injection (i.p.) of killed Photobacterium damselae subsp. piscicida (Phdp) and sampled at 4 and 24 h after the challenge. At 4 h after i.p., the ARA groups presented increased plasma total immunoglobulins (Ig) and bactericidal activity against V. anguillarum. In addition, the fish fed the ARA/LS diet presented lower white blood cell (WBC) and alternative complement activity. At 24 h after i.p., the ARA groups presented increased RBC, WBC, and thrombocyte numbers, total IG, plasma peroxidase activity, and casp3 expression in the distal intestine. The HS groups presented increased plasma NO content and bactericidal activity against Phdp and decreased protease, antiprotease activity, and bactericidal activity against V. anguillarum. In conclusion, high dietary DHA levels seemed to improve the immune status of unchallenged gilthead sea bream juveniles, while high dietary ARA levels improved the fish immune response to a bacterial challenge. The energy provided by dietary starch seems to be important to promote a fast response by the fish immune system after a challenge.

Bacillus subtilis has been documented in the past years as an effective probiotic for different aquacultured species, with recognized beneficial effects on water quality, fish growth and immune status. Furthermore, its potential as a vaccine adjuvant has also been explored in different species. In the current work, we have used B. subtilis spores as delivery vehicles for the presentation of the VP2 protein from infectious pancreatic necrosis virus (IPNV). For this, the VP2 gene was amplified and translationally fused to the crust protein CotY. The successful expression of VP2 on the spores was confirmed by Western blot. We then compared the immunostimulatory potential of this VP2-expressing strain (CRS208) to that of the original B. subtilis strain (168) on rainbow trout (Oncorhynchus mykiss) leukocytes obtained from spleen, head kidney and the peritoneal cavity. Our results demonstrated that both strains significantly increased the percentage of IgM+ B cells and the number of IgM-secreting cells in all leukocyte cultures. Both strains also induced the transcription of a wide range of immune genes in these cultures, with small differences between them. Importantly, specific anti-IPNV antibodies were detected in fish intraperitoneally or orally vaccinated with the CRS208 strain. Altogether, our results demonstrate B. subtilis spores expressing foreign viral proteins retain their immunomodulatory potential while inducing a significant antibody response, thus constituting a promising vaccination strategy.

Brewer’s spent grain (BSG) is the main brewery industry by-product, with potential applications in the feed and food industries due to its carbohydrate composition. In addition, the lignocellulosic nature of BSG makes it an adequate substrate for carbohydrases production. In this work, solid-state fermentation (SSF) of BSG was performed with Aspergillus ibericus, a non-mycotoxin producer fungus with a high capacity to hydrolyze the lignocellulosic matrix of the agro-industrial by-products. SSF was performed at different scales to produce a crude extract rich in cellulase and xylanase. The potential of the crude extract was tested in two different applications: -(1) - the enzymatic hydrolysis of the fermented BSG and (2) - as a supplement in aquafeeds. SSF of BSG increased the protein content from 25% to 29% (w/w), while the fiber content was reduced to 43%, and cellulose and hemicellulose contents were markedly reduced to around 15%. The scale-up of SSF from 10 g of dry BSG in flasks to 50 g or 400 g in tray-type bioreactors increased 55% and 25% production of cellulase and xylanase, up to 323 and 1073 U g−1 BSG, respectively. The optimum temperature and pH of maximal activities were found to be 55°C and pH 4.4 for xylanase and 50°C and pH 3.9 for cellulase, cellulase being more thermostable than xylanase when exposed at temperatures from 45°C to 60°C. A Box–Behnken factorial design was applied to optimize the hydrolysis of the fermented BSG by crude extract. The crude extract load was a significant factor in sugars release, highlighting the role of hydrolytic enzymes, while the load of fermented BSG, and addition of a commercial β-glucosidase were responsible for the highest phenolic compounds and antioxidant activity release. The lyophilized crude extract (12,400 and 1050 U g−1 lyophilized extract of xylanase and cellulase, respectively) was also tested as an enzyme supplement in aquafeed for European seabass (Dicentrarchus labrax) juveniles. The dietary supplementation with the crude extract significantly improved feed and protein utilization. The processing of BSG using biological treatments, such as SSF with A. ibericus, led to the production of a nutritionally enriched BSG and a crude extract with highly efficient carbohydrases capable of hydrolyzing lignocellulosic substrates, such as BSG, and with the potential to be used as feed enzymes with remarkable results in improving feed utilization of an important aquaculture fish species.

This study aimed to determine the effects of dietary inclusion of Tenebrio molitor larvae (yellow worms) meal (TM) on meagre fish (Argyrosomus regius) whole-body fatty acids (FA) profile and hepatic and intestine oxidative status. For that purpose, fish were fed for 9 weeks a fishmeal-based diet (control) or diets including 10%, 20%, or 30% TM. With the increase in dietary TM level, whole-body oleic acid, linoleic acid, monounsaturated FA, and n−6 polyunsaturated FA (PUFA) increased while saturated FA (SFA), n−3 PUFA, n−3 long chain-PUFA, SFA:PUFA ratio, n3:n6 ratio, and FA retention decreased. Hepatic superoxide dismutase (SOD), glucose-6-phosphate dehydrogenase (G6PDH), and glutathione reductase (GR) activities increased and catalase (CAT) and glutathione peroxidase (GPX) activities decreased with dietary TM inclusion. Hepatic total and reduced glutathione were lower in fish fed 20% TM. Intestinal CAT activity and oxidized glutathione increased and GPX activity decreased with dietary TM inclusion. Intestine SOD, G6PDH, and GR activities increased and malondialdehyde concentration decreased in fish fed the diets with lower TM inclusion levels. Liver and intestine oxidative stress index and liver malondialdehyde concentration were unaffected by dietary TM. In conclusion, to avoid major whole-body FA changes or antioxidant status imbalances, it is recommended to limit TM to 10% inclusion in meagre diets.

The present study was conducted to investigate the effects of dietary fish oil replacement with a mixture of vegetable oils and probiotic supplementation on plasma biochemical parameters, oxidative stress, and antioxidant ability of Seriola dumerili. Specimens with an initial weight of 175 g were used. Four feeds were formulated with 0% (FO-100), 75% (FO-25), and 100% (FO-0 and FO-0+ with the addition of Lactobacillus probiotics) substitution of fish oil with a mixture of linseed, sunflower, and palm oils. After 109 days, no significant differences were observed in the activity of antioxidant enzymes in the liver, foregut, and hindgut, only glucose-6-phosphate dehydrogenase activity in the liver was higher in the fish fed the FO-100 diet than in those fed the FO-0 diet. No significant differences were observed in the total, reduced, and oxidized glutathione and the oxidative stress index in the liver. In addition, lipid peroxidation in the liver and red muscle values were higher in the fish fed the FO-100 diet than in the fish fed the FO-0+ diet, however, the foregut of the fish fed the FO-100 diet presented lower values than that of the fish fed the FO replacement diet, with and without probiotics. There were significant differences in cholesterol levels in the FO-100 group; they were significantly higher than those observed with the fish diets without fish oil. To sum up, fish oil can be replaced by up to 25% with vegetable oils in diets for Seriola dumerili juveniles, but total fish oil substitution is not feasible because it causes poor survival. The inclusion of probiotics in the FO-0+ diet had no effects on the parameters measured.

Tryptophan (Trp) is an essential amino acid, commercially available as a feed-grade product, and is a precursor to serotonin and melatonin, which are both important molecules in stress mitigation. Meagre have a high potential for marine aquaculture diversification but are highly susceptible to stressful conditions. This study aimed to assess the potential role of dietary tryptophan supplementation in meagre juveniles in order to minimize the deleterious effect of potential stress conditions. For this, a growth trial was performed wherein meagre juveniles were fed four isoproteic (45%DM) and isolipidic (16%DM) diets; namely, a control diet, and three diets similar to the control diet but supplemented with varying levels of tryptophan, graded according to the resulting percentage in each diet’s dry matter (Control, 0.25%Trp, 0.5%Trp, and 1%Trp), corresponding to a total dietary tryptophan of 1.06, 1.70, 2.08, and 3.24 g 16 g−1 N, respectively. Diets were tested in triplicate, and fish were fed twice a day, six days a week, for eight weeks. Five days after the end of the growth trial, a time-course blood sampling was performed at 0 h, 1 h, 3 h, 6 h, and 12 h after feeding. At the 6 h sampling point, the liver was also collected. Overall, our results indicate that 1Trp supplementation (total dietary Trp 3.24 g 16 g−1 N) may be harmful to fish, decreasing growth performance and feed utilization, although doses up to 0.5Trp do not influence these parameters. Voluntary feed intake lineally decreased with the increase in the level of dietary Trp. Whole-body lipid content decreased at the highest tryptophan inclusion, whereas no changes were observed in protein levels. After 12 h from feeding, plasma glucose levels were lower in all dietary treatments supplemented with tryptophan compared to those observed in the control. Hepatic enzyme activity of protein catabolism decreased with dietary Trp inclusion. Overall, our results suggested that while a dietary Trp level increase of up to 2.08 g 16 g−1 N did not affect growth performance and feed efficiency, both these parameters were severely compromised with a Trp level of 3.24 g 16 g−1 N.

Aquaculture requires new, economical, and eco-friendly protein sources to replace traditional fisheries and plant ingredients. Using agriculture by-products as protein sources would reduce land-based feed production pressure and waste production, promoting a circular economy and sustainable aquaculture. Distiller’s dried grains with solubles (DDGS) is the main by-product of bioethanol production. Corn DDGS has a high protein level, but its high fiber content limits its use as a feed ingredient, particularly for carnivorous fish. Solid-state fermentation (SSF) uses lignocellulosic-rich substrates, such as DDGS, for microbial growth in the near absence of water, promoting enzyme production that degrades the lignocellulosic matrix, increasing free reducing sugars, protein, and antioxidant levels of the substrate. In the present work, the SSF of corn DDGS with Aspergillus carbonarius, A. ibericus, and A. uvarum was tested. Then, the digestibility of the most promising fermented DDGS (in terms of upgraded nutritional composition) was tested by including it in a reference diet (70% of a reference diet; 48% crude protein; 15% crude lipids) for European seabass (Dicentrarchus labrax) juveniles (171 g averaged weight; trial duration of 52 days). Among the fungi tested, Aspergillus ibericus led a generally higher upgrading of the DDGS nutritional composition, leading to a high amount of protein (from 42.7 to 49.7 g N/kg DM), phenolic compounds (1.49 to 4.86 mg/g caffeic acid equivalents), free sugars (9.5 to 31.9 mg/g), and enzyme production (45 U/g and 68 U/g of cellulase and xylanase, respectively), and a high reduction in acid detergent fiber and neutral detergent fiber content (up to 29 and 43%, respectively). Compared to the unfermented DDGS, fermented DDGS presented increased protein, lipids, starch, and energy digestibility, while phosphorous digestibility was similar. Compared to the reference diet, dietary inclusion of unfermented or fermented DDGS increased trypsin and chymotrypsin activities. The activity of digestive enzymes was not affected by the inclusion of fermented DDGS, except for amylase activity, which was lower with the fermented DDGS than with the unfermented DDGS diet. In conclusion, SSF of DDGS enhanced its nutritional value, increasing DDGS digestibility when included in diets for European seabass juveniles.

Abstract A healthy gastrointestinal microbiota is essential for host fitness, and strongly modulated by host diet. In aquaculture, a current challenge is to feed carnivorous fish with plant-feedstuffs in substitution of fish meal, an unsustainable commodity. Plants have a limited nutritive value due to the presence of non-starch polysaccharides (NSP) which are not metabolized by fish. In this work we assessed the effects of NSP-enriched diets on European seabass gut microbiota and evaluate the selective pressure of plant feedstuffs towards gut microbes with NSP-hydrolytic potential, i.e. capable to convert indigestible dietary constituents in fish metabolites. Triplicate groups of European seabass juveniles were fed a fish meal-based diet (control) or three plant-based diets (SBM, soybean meal; RSM, rapeseed meal; SFM, sunflower meal) for 6 weeks, before recovering intestinal samples for microbiota analysis, using the Illumina’s MiSeq platform. Plant-based diets impacted differently digesta and mucosal microbiota. A decrease (p = 0.020) on species richness, accompanied by a decline on the relative abundance of specific phyla such as Acidobacteria (p = 0.030), was observed in digesta samples of SBM and RSM experimental fish, but no effects were seen in mucosa-associated microbiota. Plant-based diets favored the Firmicutes (p = 0.01), in particular the Bacillaceae (p = 0.017) and Clostridiaceae (p = 0.007), two bacterial families known to harbor carbohydrate active enzymes and thus putatively more prone to grow in high NSP environments. Overall, bacterial gut communities of European seabass respond to plant-feedstuffs with adjustments in the presence of transient microorganisms (allochthonous) with carbohydrolytic potential, while maintaining a balanced core (autochthonous) microbiota.

The aquaculture industry is one of the fastest-growing sectors in animal food production. However, farming of carnivorous fish strongly relies on the use of wild fish-based meals, a practice that is environmentally and economically unsustainable. Insect-based diets constitute a strong candidate for fishmeal substitution, due to their high nutritional value and low environmental footprint. Nevertheless, data on the impact of insect meal (IM) on the gut microbiome of farmed fish are so far inconclusive, and very scarce in what concerns modulation of microbial-mediated functions. Here we use high-throughput 16S rRNA gene amplicon sequencing and quantitative PCR to evaluate the impact of different IMs on the composition and chitinolytic potential of the European sea bass gut digesta- and mucosa-associated communities. Our results show that insect-based diets of distinct origins differently impact the gut microbiota of the European sea bass (Dicentrarchus labrax). We detected clear modulatory effects of IM on the gut microbiota, which were more pronounced in the digesta, where communities differed considerably among the diets tested. Major community shifts were associated with the use of black soldier fly larvae (Hermetia illucens, HM) and pupal exuviae (HEM) feeds and were characterized by an increase in the relative abundance of the Firmicutes families Bacillaceae, Enterococcaceae, and Lachnospiraceae and the Actinobacteria family Actinomycetaceae, which all include taxa considered beneficial for fish health. Modulation of the digesta community by HEM was characterized by a sharp increase in Paenibacillus and a decrease of several Gammaproteobacteria and Bacteroidota members. In turn, a mealworm larvae-based diet (Tenebrio molitor, TM) had only a modest impact on microbiota composition. Further, using quantitative PCR, we demonstrate that shifts induced by HEM were accompanied by an increase in copy number of chitinase ChiA-encoding genes, predominantly originating from Paenibacillus species with effective chitinolytic activity. Our study reveals an HEM-driven increase in chitin-degrading taxa and associated chitinolytic activity, uncovering potential benefits of adopting exuviae-supplemented diets, a waste product of insect rearing, as a functional ingredient.

Fortifying fish feeds with bioactive compounds, such as enzymes and antioxidants, has been an adopted strategy to improve feed nutritional quality and sustainability. However, feed additives can lose activity/effectiveness during pelleting and storage processes. This work aimed to monitor functional activity stability in feeds supplemented with a bioactive extract, including cellulases, xylanases, and antioxidants. This bioactive extract (FBE) was produced by Aspergillus ibericus under solid-state fermentation of olive mill and winery by-products. Two isoproteic and isolipidic diets were formulated and unsupplemented or supplemented with lyophilized FBE (0.26% w/w). Both diets were stored at room temperature (RT) or 4 °C for 4 months. Results showed that feed storage at 4 °C enhanced the stability of the enzymes and cellulase was more stable than xylanase. Compared to RT, storage at 4 °C increased cellulase and xylanase half-life by circa 60 and 14%. Dietary FBE supplementation increased antioxidant activity and storage at 4 °C reduced antioxidant activity loss, while in the unsupplemented diet, antioxidant activity decreased to the same level in both storage temperatures. Dietary supplementation with FBE reduced lipid peroxidation by 17 and 19.5% when stored at 4 °C or RT, respectively. The present study is a step toward improving the storage conditions of diets formulated with bioactive compounds.

This study aimed to assess the effect of Hermetia illucens meal (HM) dietary inclusion on meagre oxidative status. Thus, fish were fed a fishmeal-based diet (CTR diet) and three other diets with increasing levels of HM inclusion, namely 10%, 20%, and 30% (diets HM10, HM20, and HM30, respectively). At the end of the trial, hepatic and intestine superoxide dismutase, catalase, and glucose-6-phosphate dehydrogenase activities and malondialdehyde concentration were unaffected by the diet composition. Liver glutathione peroxidase activity was higher in the fish fed the HM20 diet than in the fish fed the CTR and HM30 diets, and glutathione reductase activity linearly increased with the dietary HM level. The hepatic total glutathione and reduced glutathione contents were significantly lower in fish fed the HM20 diet than in fish fed the CTR and HM10 diets. In the intestine, the oxidized glutathione (GSSG) content and oxidative stress index linearly increased with the increase in dietary HM level, with the GSSG content of fish fed the HM20 diet being significantly higher than of fish fed the CTR diet. In conclusion, 30% HM might be included in meagre diets without negatively affecting hepatic and intestine oxidative status.

The present study aimed to evaluate the effects of feeding or feed deprivation on the orexigenic and anorexigenic responses at the central (whole brain) and peripheral (anterior and posterior intestine, stomach, and liver) system levels in European seabass. For this purpose, a group of fish (208 g) was fed a single meal daily for 8 days (fed group) and another group was feed-deprived for 8 days (unfed group). Compared to the fed group, in the whole brain, feed deprivation did not induce changes in npy, agrp1, and cart2 expression, but increased agrp2 and pomc1 expression. In the anterior intestine, feed deprivation increased cck expression, while in the posterior intestine, the npy expression increased and pyyb decreased. In the stomach, the ghr expression decreased regardless of the feeding status. The hepatic lep expression increased in the unfed fish. The present results suggest a feed intake regulation mechanism in European seabass similar to that observed in other teleosts.

Insect meal (IM), recently authorized for use in aquafeeds, positions itself as a promising commodity for aquafeed inclusion. However, insects are also rich in chitin, a structural polysaccharide present in the exoskeleton, which is not digested by fish, resulting in lower fish performance. Through the application of a dietary pressure, this study aimed to modulate European sea bass gut microbiota towards the enrichment of chitinolytic bacteria to allow the isolation of novel probiotics capable of improving the use of IM-containing diets, overcoming chitin drawbacks. Five isoproteic (44%) and isolipidic (18%) diets were used: a fish meal (FM)-based diet (diet CTR), a chitin-supplemented diet (diet CHIT5), and three diets with either 25% of Hermetia illucens and Tenebrio molitor larvae meals (HM25 and TM25, respectively) or H. illucens exuviae meal (diet HEM25) as partial FM substitutes. After an 8-week feeding trial, the results showed a clear modulatory effect towards spore-forming bacteria by HM25 and HEM25 diets, with the latter being responsible for the majority of the chitinolytic fish isolates (FIs) obtained. Sequential evaluation of the FI hemolytic activity, antibiotic resistance, total chitinolytic activity, sporulation, and survival in gastrointestinal-like conditions identified FI645 and FI658 as the most promising chitinolytic probiotics for in vivo application.

The range of metabolic pathways that are dependent on a proper supply of specific amino acids (AA) unveils their importance in the support of health. AA play central roles in key pathways vital for immune support and individual AA supplementation has shown to be able to modulate fish immunity. In vitro trials are important tools to evaluate the immunomodulatory role of AA, and the present study was conceived to evaluate methionine and tryptophan roles in immune-related mechanisms aiming to understand their effects in leucocyte functioning and AA pathways. For that purpose, head-kidney leucocytes were isolated and a primary cell culture established. The effect of methionine or tryptophan surplus on cell viability was assessed. Medium L-15 10% FBS without AA addition (0.5mM of L-methionine, 0.1 mM of L-tryptophan) was used as control. To that, L-methionine or L-tryptophan were supplemented at 1 and 2 times (M1x or M2x, and T1x or T2x). Nitric oxide, ATP, total antioxidant capacity, and immune-related genes were evaluated in response to lipopolysaccharides extracted from Photobacterium damselae subsp. piscicida or UV-inactivated bacteria). Moreover, caspase 3 activity and apoptosis-related genes were evaluated in response to the apoptosis-inducing protein, AIP56. Distinct roles in leucocytes’ immune response were observed, with contrasting outcomes in the modulation of individual pathways. Methionine surplus improved cell viability, polyamine production, and methionine-related genes expression in response to an inflammatory agent. Also, methionine supplementation lowered signals of apoptosis by AIP56, presenting lower caspase 3 activity and higher il1β and nf-κb expression. Cells cultured in tryptophan supplemented medium presented signals of an attenuated inflammatory response, with decreased ATP and enhanced expression of anti-inflammatory and catabolism-related genes in macrophages. In response to AIP56, leucocytes cultured in a tryptophan-rich medium presented lower resilience to the toxin, higher caspase 3 activity and expression of caspase 8, and lower expression of several genes, including nf-κb and p65. This study showed the ability of methionine surplus to improve leucocytes’ response to an inflammatory agent and to lower signals of apoptosis by AIP56 induction, while tryptophan attenuated several cellular signals of the inflammatory response to UV-inactivated bacteria and lowered leucocyte resilience to AIP56.

This study aimed to evaluate the effects of including microalgae Chlorella sp. or Nannochloropsis sp. in plant-based diets on antioxidant mechanisms of European sea bass (Dicentrarchus labrax) juveniles. For this purpose, three isoproteic (50%) and isolipidic (19%) diets were formulated: a practical diet, containing 15% fish meal (FM) and plant ingredients as the protein source and a mixture of fish oil and vegetable oils (40: 60) as lipid source (control diet); and two diets identical to the control but with the FM replaced by Nannochloropsis sp. or Chlorella sp. (diets Nanno and Chlo, respectively). The diets were offered to quadruplicate groups of 25 fish (initial body weight: 24 ± 1 g) for 11 weeks and then enzymatic and non-enzymatic antioxidant mechanisms and lipid oxidative biomarkers were assessed in the liver and intestine of these fish. Results showed that the antioxidant response was tissue-dependent, with the liver exhibiting lower glutathione peroxidase and glucose-6-phosphate dehydrogenase (only in Chlo group) activities, and intestine lower superoxide dismutase activity with the diets including microalgae compared to control diet. An increase of oxidized glutathione content was also observed in the intestine of fish fed the microalgae diets. Catalase and glutathione reductase activities, oxidative stress index, and total and reduced glutathione, were unaffected by dietary treatments in both tissues. Overall, the lipid peroxidation status was not compromised by the replacement of FM by microalgae.

This study investigated, for the first time, the integrated effects of dietary protein source and protein/carbohydrate (P/CH) ratio on gilthead seabream gut histomorphology, microbiota composition, digestive enzymes activity, and immunological and oxidative stress-related gene expressions. Four isolipidic diets: two fishmeal-based (FM) and two plant feedstuff (PF)-based diets, with P/CH ratios of 50/10 or 40/20 each (FM-P50/CH10; FM-P40/CH20; PF-P50/CH10; PF-P40/CH20), were tested. PF-based diets lead to more histomorphological alterations than FM-based diets. P/CH ratio had no relevant effect on gut histomorphology. Gut mucosa of fish fed PF-based diets presented a higher number of operational taxonomic units, and richness and diversity indices, while the P/CH ratio did not affect those parameters. The α-amylase activity was lower in fish fed with PF-based diets and in fish fed the P40/CH20 diets. Regarding the immune-related genes, only cyclooxygenase-2 was affected, being higher in fish fed the P50/CH10 diets than the P40/CH20 diets. Fish fed the FM-based diets presented higher expression of glutathione reductase and glutathione peroxidase, while fish fed the P50/CH10 diet had higher expression of superoxide dismutase. In conclusion, PF-based diets can compromise gut absorptive and digestive metabolism, but decreasing the dietary P/CH ratio had little effect on the parameters measured.

Aquaculture is among the industries growing at the fastest rate in the world. This industry has been recognized to play a critical role in food production for a continuously expanding world population. However, despite various technological innovations and improvements in production techniques, this sector is still associated with misperceptions and negative opinions hampering its implementation and wide consumption of its products. The integrated multi-trophic aquaculture (IMTA) concept was developed as a way to increase the sustainability of intensive aquaculture systems, using an ecosystem-based approach. In this study, following this sustainable aquaculture concept, a closed recirculation IMTA system, at laboratorial scale, was developed and tested with the simultaneous production of fish, sea urchin and seaweed for 70 days. Based on this proof of concept, a hands-on experimental activity was developed to teach and communicate recent scientific advances in environmental sustainability and value of aquaculture products to young students and the general public. This experimental activity was tested and evaluated with students (n = 60) of basic and high-school (secondary) learning cycles. A quantitative assessment was carried out through a short questionnaire provided to the students before and after the experimental activity. After the experimental activity, a qualitative assessment was also performed through questions expressed without preconceived categories or hypotheses. Results indicated that the overall frequency of students who consider the ocean to be “very important” and “extremely important” increased from 68 to 81% after performing the experimental activity. Moreover, the percentages of correct answers to the questions related to IMTA concepts also increased significantly after the experimental activity. In the discussion of the experimental activity results, the students stated that they appreciated the opportunity to develop a hands-on experimental activity, which allowed them to increase their knowledge and obtain information on aquaculture and the quality of its products.

Marine algae are recognised sources of bioactive compounds that have attracted great interest as nutritional supplements for aquaculture fish. Intensive rearing conditions often expose fish to husbandry-related stressors, rendering fish more susceptible to disease and reducing production yields. The present work evaluated the potential of two marine algae extracts (Fucus vesiculosus and Nannochloropsis gaditana) as nutritional supplements to mitigate stress effects in meagre (Argyrosomus regius) exposed to an acute handling stress (AS). A plant-based diet was used as a control, and three other diets were prepared, which were similar to the control diet but supplemented with 1% of each algal extract or a combination of the two extracts (0.5% each). The effects of supplemented diets on stress biomarkers, antioxidant enzyme activities, and immune response were analysed in fish exposed to AS after 4 weeks of feeding. Supplemented diets did not affect growth performance but the inclusion of F. vesiculosus promoted higher feed efficiency, as compared to the control group. Dietary algal extracts supplementation reduced plasma glucose levels, increased white blood cell counts, and reduced the expression of pro-inflammatory genes when compared with the control. N. gaditana supplementation led to a reduction in hepatic antioxidant enzyme activity and glutathione levels, while F. vesiculosus supplementation increased muscle glutathione reductase activity and reduced lipid peroxidation. These findings support the potential of algal extracts as nutraceuticals in aquafeeds to enhance the ability of fish to cope with husbandry-related stressful conditions and ultimately improve fish health and welfare.

The disruption of pathogen communication or quorum-sensing (QS) via quorum-quenching (QQ) molecules has been proposed as a promising strategy to fight bacterial infections. Bacillus spp. have recognizable biotechnology applications, namely as probiotic health-promoting agents or as a source of natural antimicrobial molecules, including QQ molecules. This study characterized the QQ potential of 200 Bacillus spp., isolated from the gut of different aquaculture fish species, to suppress fish pathogens QS. Approximately 12% of the tested Bacillus spp. fish isolates (FI). were able to interfere with synthetic QS molecules. Ten isolates were further selected as producers of extracellular QQ-molecules and their QQ capacity was evaluated against the QS of important aquaculture bacterial pathogens, namely Aeromonas spp., Vibrio spp., Photobacterium damselae, Edwardsiela tarda, and Shigella sonnei. The results revealed that A. veronii and E. tarda produce QS molecules that are detectable by the Chr. violaceum biosensor, and which were degraded when exposed to the extracellular extracts of three FI isolates. Moreover, the same isolates, identified as B. subtilis, B. vezelensis, and B. pumilus, significantly reduced the pathogenicity of E. tarda in zebrafish larvae, increasing its survival by 50%. Taken together, these results identified three Bacillus spp. capable of extracellularly quenching aquaculture pathogen communication, and thus become a promising source of bioactive molecules for use in the biocontrol of aquaculture bacterial diseases.

High rearing densities are typical conditions of both inland and onshore intensive aquaculture units. Despite obvious drawbacks, this strategy is nonetheless used to increase production profits. Such conditions inflict stress on fish, reducing their ability to cope with disease, bringing producers to adopt therapeutic strategies. In an attempt to overcome deleterious effects of chronic stress, Senegalese sole, Solea senegalensis, held at low (LD) or high density (HD) were fed tryptophan-supplemented diets with final tryptophan content at two (TRP2) or four times (TRP4) the requirement level, as well as a control and non-supplemented diet (CTRL) for 38 days. Fish were sampled at the end of the feeding trial for evaluation of their immune status, and mortalities were recorded following intra-peritoneal infection with Photobacterium damselae subsp. piscicida. Blood was collected for analysis of the hematological profile and innate immune parameters in plasma. Pituitary and hypothalamus were sampled for the assessment of neuro-endocrine-related gene expression. During the feeding trial, fish fed TRP4 and held at LD conditions presented higher mortalities, whereas fish kept at HD seemed to benefit from this dietary treatment, as disease resistance increased over that of CTRL-fed fish. In accordance, cortisol level tended to be higher in fish fed both supplemented diets at LD compared to fish fed CTRL, but was lower in fish fed TRP4 than in those fed TRP2 under HD condition. Together with lower mRNA levels of proopiomelanocortin observed with both supplementation levels, these results suggest that higher levels of tryptophan might counteract stress-induced cortisol production, thereby rendering fish better prepared to cope with disease. Data regarding sole immune status showed no clear effects of tryptophan on leucocyte numbers, but TRP4-fed fish displayed inhibited alternative complement activity (ACH50) when held at LD, as opposed to their HD counterparts whose ACH50 was higher than that of CTRL-fed fish. In conclusion, while dietary tryptophan supplementation might have harmful effects in control fish, it might prove to be a promising strategy to overcome chronic stress-induced disease susceptibility in farmed Senegalese sole.

Insect meals are good candidates to replace fishmeal as new protein sources in aquafeeds. This study evaluated the effects of fishmeal replacement with different dietary inclusion levels of a partially defatted Tenebrio molitor (L.) larva meal (TM) on rainbow trout (Oncorhynchus mykiss Walbaum) growth, diet digestibility, and hepatic intermediary metabolism. A 154-day growth trial was performed with 252 rainbow trout (78.3 ± 6.24 g) randomly divided into twelve tanks and fed four experimental diets containing increasing levels of TM: 0% (TM0), 25% (TM25), 50% (TM50), and 100% (TM100) of fishmeal substitution, corresponding to TM dietary inclusion levels of 0%, 5%, 10%, and 20%, respectively. A digestibility trial was performed feeding 180 rainbow trout (94.6 ± 7.31 g) with the experimental diets used in the growth trial. The growth parameters were not affected by TM dietary inclusion. Regarding the evaluated apparent digestibility coefficients (ADC), only the ADC of crude protein was affected, showing the following trend: TM0 = TM25 > TM50 > TM100. The activities of key hepatic amino acid catabolic and lipogenic enzymes were not affected by the dietary composition. The results suggest that a partially defatted TM could totally replace fishmeal in commercial rainbow trout diets without negative effects on fish performance.

Methionine and tryptophan appear to be fundamental in specific cellular pathways involved in the immune response mechanisms, including stimulation of T-regulatory cells by tryptophan metabolites or pro-inflammatory effects upon methionine supplementation. Thus, the aim of this study was to evaluate the immunomodulatory effect of these amino acids on the inflammatory and neuroendocrine responses in juveniles of European seabass, Dicentrarchus labrax. To achieve this, goal fish were fed for 14 days methionine and tryptophan-supplemented diets (MET and TRP, respectively, 2× dietary requirement level) or a control diet meeting the amino acids requirement levels (CTRL). Fish were sampled for immune status assessment and the remaining fish were challenged with intraperitoneally injected inactivated Photobacterium damselae subsp. piscicida and sampled either 4 or 24 h post-injection. Respiratory burst activity, brain monoamines, plasma cortisol, and immune-related gene expression showed distinct and sometimes opposite patterns regarding the effects of dietary amino acids. While neuroendocrine intermediates were not affected by any dietary treatment at the end of the feeding trial, both supplemented diets led to increased levels of plasma cortisol after the inflammatory insult, while brain monoamine content was higher in TRP-fed fish. Peripheral blood respiratory burst was higher in TRP-fed fish injected with the bacteria inoculum but only compared to those fed MET. However, no changes were detected in total antioxidant capacity. Complement factor 3 was upregulated in MET-fed fish but methionine seemed to poorly affect other genes expression patterns. In contrast, fish fed MET showed increased immune cells numbers both before and after immune challenge, suggesting a strong enhancing effect of methionine on immune cells proliferation. Differently, tryptophan effects on inflammatory transcripts suggested an inhibitory mode of action. This, together with a high production of brain monoamine and cortisol levels, suggests that tryptophan might mediate regulatory mechanisms of neuroendocrine and immune systems cooperation. Overall, more studies are needed to ascertain the role of methionine and tryptophan in modulating (stimulate or regulate) fish immune and neuroendocrine responses.

Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy.