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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.

Caffeine (CAF) exposures have been shown to cause several pharmacological and biological effects in target and non-target organisms. Although there are already several ecotoxicological studies with CAF in non-target organisms, they are focused on marine organisms, with relevant concentrations in these ecosystems, therefore, less ecologically relevant to freshwater ecosystems (the main ecoreceptor of this type of anthropogenic contaminant). The present study aimed to assess the chronic effects (28 days) of sub-lethal and environmentally relevant concentrations of CAF (0.16, 0.42, 1.09, 2.84, 7.40, 19.23, and 50 μg/L) in Danio rerio. Biochemical endpoints as biomarkers of antioxidant defense, biotransformation, lipid peroxidation, energy sources, and neurotransmission were assessed. CAF exposure induced alterations in antioxidant defenses (superoxide dismutase and glutathione reductase activities, and glutathione content) preventing lipid peroxidation. Lactate dehydrogenase activity decreased in all the concentrations tested, while acetylcholinesterase activity was only affected by the highest concentrations tested (19.23 and 50 μg/L). We also utilized a multi-biomarker approach (Integrated Biomarker Response version 2, IBRv2) to investigate the effects of CAF in the dispersion scope of individual biochemical responses of D. rerio. IBRv2 showed that the concentration of 50 μg/L promotes the highest stress. However, the results showed that CAF induced disturbances in the metabolic pathways studied in D. rerio. These results demonstrated the toxic effects of CAF on freshwater fish, compromising their physiological functions and evidencing the need for monitoring the residues of CAF released into the inland aquatic environments. Furthermore, this research evidence that phylogenetically and physiologically different species may present different biological responses with concern for ecologically relevant environmental conditions. In this sense, the present study generated ecotoxicologically relevant data, that can be considered by environment regulators, since the here-endpoints evaluated showed sensitivity and consistency in the evaluation of caffeine risks in freshwater environments.

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.

The tadpole shrimp Triops longicaudatus is a freshwater crustacean with fast embryonic and larval development, short life cycle, and high fecundity. They are very active swimmers of a reasonable size, easy to spot and record. Such characteristics make it a promising candidate as an experimental model in ecotoxicology to evaluate the effects of aquatic pollutants, particularly using its locomotor behavior as an endpoint. To evaluate the sensitivity of T. longicaudatus and develop endpoints of interest, we conducted exposure experiments with lethal and sub-lethal concentrations of potassium dichromate, a compound known for its ecotoxicological importance and as a hexavalent chromium source. The endpoints evaluated were mortality, growth, sexual maturation, reproductive output, cholinesterase activity and locomotor/swimming behavior. The 96 h median lethal concentration was found to be 65 µg/L. Furthermore, exposure to potassium dichromate at higher concentrations had a significant negative impact on the growth rate of T. longicaudatus in terms of both body mass and length. The time for maturation was also delayed at higher concentrations. In addition, locomotor behavior allowed for the discrimination of all tested chromium concentrations and the control group and from each other, proving to be the most sensitive endpoint. Overall, the data support the potential of T. longicaudatus as a model for ecotoxicity testing, using apical endpoints with impact at the population level; in particular, results suggest that behavior assessments in this species might be useful for detecting hazardous compounds in environmental monitoring of freshwater ecosystems.

Triops longicaudatus is a crustacean typically inhabiting temporary freshwater bodies in regions with a Mediterranean climate. These crustaceans are easily maintained in the laboratory and show a set of biological features that make them good candidates for diagnosing environmental quality and health. However, information about their responses to environmental contamination is scarce. This study characterised the locomotor responses of juvenile and adult/mature T. longicaudatus to low concentrations of five model toxicants upon a very short 1.5 h exposure: tributyltin, mercury, lindane, sodium hypochlorite and formaldehyde. A video-tracking system was used to record the locomotor behaviour. The data were analysed with an artificial neural network to identify distinct behaviours, followed by Chi-square and Correspondence analysis to characterise the response to each toxicant. The results showed that T. longicaudatus is sensitive to aquatic contamination, particularly sodium hypochlorite. Six behaviour types were defined, which allowed for the characterisation and discrimination of the test toxicants. The results support the need for more investigation into this species and its behaviour types as an alternative to animal testing and the more apical and often invasive endpoints commonly recommended in standard guidelines.

Tributyltin (TBT) is a biocide introduced in the 1960s in antifouling paints. Despite legislation banning its use, its persistence in the environment still causes significant harm to organisms. Tributyltin is a ligand of retinoid X receptors (RXR) and ecdysteroid receptors (EcRs), which in arthropods act as homologs of RXR. Focusing on Metazoan species, this study used genomic and proteomic information from different sources to compare their three-dimensional structure, phylogenetic distribution, and amino acid sequence alterations. The objective was to identify possible patterns that relate organisms’ sensitivity to TBT using the species Triops longicaudatus as the basis for the comparisons. The results showed great conservation of this protein across several species when comparing the interaction amino acids described to RXR (an EcR analog) in Homo sapiens. The three-dimensional comparison of RXR showed little conformational variation between different sequences by maintaining the interaction pocket. As for the Species Sensitivity Distribution (SSD) curve, an HC05 = 0.2649 [0.0789–0.7082] µg/L was obtained with no specific distribution between the different taxa. Protein-ligand docking analysis was then used to confirm the SSD curve ranking of species. Still, the results showed an opposite trend that may be related, for example, to differences in the LC50 values used in the calculations. This study serves as the first step for applying bioinformatics techniques to produce information that can be used as an alternative to animal or cellular experimentation. These techniques could be adapted to various chemicals and proteins, allowing for observations in a shorter timeframe and providing information on a broader spectrum.

(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. [ABSTRACT FROM AUTHOR]

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.

Objective: To present prenatal diagnosis and cytogenetic characterization of a unique pattern of partial tetrasomy 18 mosaicism. Case report: A 34-year-old woman underwent amniocentesis at 25 weeks of gestation due to anomalies detected in obstetric ultrasound. It revealed a de novo supernumerary partial isochromosome 18 in 11 of 37 metaphases of cultured amniocytes. The karyotype was 47,XX,+idic(18) (q12.3)[11]/46,XX[26]. Elective cesarean section was performed at 33 weeks of gestational age due to anhydramnios. A female symmetric small for gestational age baby with dysmorphic features and an Apgar score of 9/10/10 was born. She had a good clinical outcome during hospitalization. Postnatal peripheral blood karyotype was normal. Interphase fluorescence in situ hybridization in a sample of the oral mucosa confirmed the prenatal diagnosis. At three months of corrected age she had a normal psychomotor development. Conclusion: To the best of our knowledge, this is the first reported case of mosaic partial tetrasomy 18 including segments of the long arm. This newborn's relatively mild phenotype highlights the challenges of prenatal genetic counselling in mosaic cases with fetal anomalies.

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.