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The present study evaluated the effects of feeding frequency (FF) and dietary protein/carbohydrate (P/CH) ratios on intestinal histomorphology, microbiota profile, and digestive and oxidative stress-related enzyme activities of gilthead seabream (Sparus aurata). To this purpose, two practical diets were formulated: one with 50% P and 10% CH (P50/CH10) and other with 40% P and 20% CH (P40/CH20). Triplicate groups of fish with9.1±0.01 gwere fed these diets for 60 days until visual satiation at a FF of 1, 2, or 3 meals per day. Distal intestine histomorphology was not affected by diet composition or FF. However, the pyloric caeca (PC) of fish fed 1 meal per day presented more gut fold height alterations than the other groups, except in fish fed diet P50/CH10 3 meals per day, where no changes was observed. Fish fed diet P40/CH20 3 meals per day also presented higher PC submucosa cellularity than the other groups. Fish fed diet P40/CH20 presented a higher number of operational taxonomic units, microbial richness, and diversity indices than fish fed diet P50/CH10. Amylase was the only measured digestive enzyme affected by the experimental conditions, presenting higher activity in fish fed diet P50/CH10 once per day. Glucose-6-phosphate dehydrogenase activity was lower in fish fed 2 meals per day than only 1. While catalase activity was lower in fish fed 2 than 3 meals per day. Glutathione reductase activity was the only measured parameter affected both by dietary P/CH ratio and FF, being inferior in fish fed once per day the P50/CH10 diet than the P40/CH20 diet and, also in the P50/CH10 diet, to fish fed 1 than those fed 3 meals per day. Overall, no major interactions was observed between dietary P/CH ratio and FF; however, a P40/CH20 diet fed 2 meals per day might be recommended for gilthead seabream juveniles.

Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1β, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1β, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.

Centro de Investigación en Sanidad Animal (CISA), Research on immunotherapeutic agents has become a focus for the treatment of fish diseases. The ability of algae to produce secondary metabolites of potential interest as immunotherapeutics has been documented. The present research intended to assess antiviral and antibacterial activities of macro- and microalgae extracts against viral and bacterial pathogens and explore their immunomodulatory potential using zebrafish (Danio rerio) larvae as a model organism. The cytotoxicity and antiviral activity of eight methanolic and ethanolic extracts from two macroalgae (Fucus vesiculosus, Ulva rigida) and two microalgae (Nannochloropsis gaditana, Chlorella sp.) were analyzed in established fish cell lines. Six extracts were selected to evaluate antibacterial activity by disk diffusion and growth inhibition assays. The three most promising extracts were characterized in terms of fatty acid composition, incorporated at 1% into a plant-based diet, and evaluated their effect on zebrafish immune response and intestinal morphology in a short-term feeding trial. All extracts exhibited in vitro antiviral activity against viral hemorrhagic septicemia and/or infectious pancreatic necrosis viruses. Methanolic extracts from F. vesiculosus and U. rigida were richer in saturated fatty acids and exhibited in vitro antibacterial action against several bacteria. Most promising results were obtained in vivo with F. vesiculosus methanol extract, which exerted an anti-inflammatory action when incorporated alone into diets and induced pro-inflammatory cytokine expression, when combined with the other extracts. Moreover, dietary inclusion of the extracts improved intestinal morphology. In summary, the results obtained in this study support the potential of algae as natural sources of bioactive compounds for the aquaculture industry., M. Monteiro, A. S. Lavrador, R. A. Santos, and F. Rangel were supported by grants SFRH/BD/114995/2016, ZEBRALGRE_BM_2019-003, SFRH/BD/131069/2017, and SFRH/BD/138375/2018, respectively, from FCT — Foundation for Science and Technology, under the POCI program. A. Couto C. Serra and P. Enes have a scientific employment contract supported by national funds through FCT. E. da Costa and M. R. Domingues are financially supported by FCT/MCTES (Portugal) through CESAM (UIDB/50017/2020 + UIDP/50017/2020), QOPNA (FCT UID/QUI/00062/2019), LAQV/REQUIMTE (UIDB/50006/2020), and RNEM (LISBOA-01–0145-FEDER-402–022125). This research was partially supported by the Strategic Funding to UID/Multi/04423/2019 (POCI-01–0145-FEDER-007621) through national funds provided by FCT under the project PTDC/CVT-WEL/5207/2014.

Aquaculture is responsible for more than 50% of global seafood consumption. Bacterial diseases are a major constraint to this sector and associated with misuse of antibiotics, pose serious threats to public health. Fish-symbionts, co-inhabitants of fish pathogens, might be a promising source of natural antimicrobial compounds (NACs) alternative to antibiotics, limiting bacterial diseases occurrence in aquafarms. In particular, sporeforming Bacillus spp. are known for their probiotic potential and production of NACs antagonistic of bacterial pathogens and are abundant in aquaculture fish guts. Harnessing the fish-gut microbial community potential, 172 sporeforming strains producing NACs were isolated from economically important aquaculture fish species, namely European seabass, gilthead seabream, and white seabream. We demonstrated that they possess anti-growth, anti-biofilm, or anti-quorum-sensing activities, to control bacterial infections and 52% of these isolates effectively antagonized important fish pathogens, including Aeromonas hydrophila, A. salmonicida, A. bivalvium, A. veronii, Vibrio anguillarum, V. harveyi, V. parahaemolyticus, V. vulnificus, Photobacterium damselae, Tenacibaculum maritimum, Edwardsiela tarda, and Shigella sonnei. By in vitro quantification of sporeformers' capacity to suppress growth and biofilm formation of fish pathogens, and by assessing their potential to interfere with pathogens communication, we identified three promising candidates to become probiotics or source of bioactive molecules to be used in aquaculture against bacterial aquaculture diseases.

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 effect of partially replacing fishmeal (FM) by black soldier fly larvae meal (Hermetia meal--HM) in meagre (Argyrosomus regius) diet was evaluated for nutrient digestibility and digestive enzyme activity. For that purpose, triplicate groups of fish (18.0 ± 0.02 g) were fed during 48 days either a control diet (CTR), without HM, or one of three diets including 100, 200 and 300 g/kg of HM, replacing 17, 35 and 52% of FM, respectively. Apparent digestibility coefficients (ADCs) of dry matter, energy, protein, lysine, isoleucine, leucine, phenylalanine, alanine, glutamate, glycine and serine presented a decreasing response with increased HM inclusion. Chitin ADC was null, independent of dietary HM inclusion. Total alkaline protease activity increased while trypsin activity decreased with dietary HM inclusion. No intestinal chitinolytic activity was detected. Intestinal alkaline protease zymogram revealed nine bands with proteolytic activity against casein, with molecular weights ranging between 15 and 75 kDa. Anti-protease activity in the intestine was not affected by dietary inclusion of HM compared to the CTR diet. Overall, it is concluded that replacement of up to 17% FM with HM (100 g/kg HM inclusion level) in meagre diets has no major adverse effects in diet digestibility and digestive enzyme activity.

In recent years, the alkyl-quinolone molecular framework has already provided a rich source of bioactivity for the development of novel anti-infective compounds. Based on the quorum-sensing signalling molecules 4-hydroxy-2-heptylquinoline (HHQ) and 3,4-dihydroxy-2-heptylquinoline (PQS) from the nosocomial pathogen Pseudomonas aeruginosa , modifications have been developed with markedly enhanced anti-biofilm bioactivity towards important fungal and bacterial pathogens, including Candida albicans and Aspergillus fumigatus. Here we show that antibacterial activity of HHQ against Vibrionaceae is species-specific and it requires an exquisite level of structural fidelity within the alkyl-quinolone molecular framework. Antibacterial activity was demonstrated against the serious human pathogens Vibrio vulnificus and Vibrio cholerae as well as a panel of bioluminescent squid symbiont Allivibrio fischeri isolates. In contrast, Vibrio parahaemolyticus growth and biofilm formation was unaffected in the presence of HHQ and all the structural variants tested. In general, modification to almost all of the molecule except the alkyl-chain end, led to loss of activity. This suggests that the bacteriostatic activity of HHQ requires the concerted action of the entire framework components. The only exception to this pattern was deuteration of HHQ at the C3 position. HHQ modified with a terminal alkene at the quinolone alkyl chain retained bacteriostatic activity and was also found to activate PqsR signalling comparable to the native agonist. The data from this integrated analysis provides novel insights into the structural flexibility underpinning the signalling activity of the complex alkyl-quinolone molecular communication system.

The Water Framework Directive (WFD) is used to evaluate the water quality of aquatic ecosystems. Phytoplankton is the only biological element considered in the reservoirs water quality assessment. In this study, we aimed to assess the use of the bacterioplankton community as an indicator of water quality, using a culture-independent assay (denaturing gradient gel electrophoresis, DGGE). Four Portuguese reservoirs (Miranda, Pocinho, Aguieira and Alqueva) were analysed in four periods (autumn 2018, spring and autumn 2019, and spring 2020). Bacterial total abundance had similar values for Miranda, Pocinho and Aguieira, and generally lower values for Alqueva. Diversity and richness values did not show a clear trend. Negative correlations were observed between some nutrients and the bacterial community. Overall, members of Actinobacteria, Bacteroidetes, Cyanobacteria, Nitrospirae and Proteobacteria were identified in all sampling sites. In Alqueva, no spatial, temporal or water body quality relationships with bacterial community were observed, which may be due to its higher size, low water velocity rate and higher residence times. However, in Miranda, Pocinho and Aguieira, a strong spatial and temporal bacterial community dynamic was observed. Furthermore, the presence of some species (e.g., Acinetobacter sp.) may reflect the poor water quality that was not detected by the WFD approach.

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.

The interest in using marine organisms as a source of novel bioactive compounds has increased recently, reinforcing the need to find efficient and cost-effective methods to extract these biocompounds. Thus, the present study has focused on evaluating four solvent systems and three extraction methods to obtain extracts of three macroalgae (Gracilaria sp., Fucus vesiculosus, and Ulva rigida) and two microalgae (Chlorella sp. and Nannochloropsis gaditana). The extracts were evaluated regarding yield, phenolic content, and potential antioxidant activity. Results showed that a lower organic solvent:water ratio increased macroalgae, but not microalgae, biomass extraction efficiency. Levels of total phenolics, ortho-diphenols and flavonoids were highly influenced by algae material and solvent system applied. Strong 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity was obtained with ethanol:water, 80:20 (E80:20) microalgal extracts, while no visible trend was detected for macroalgae extracts. However, methanolic extracts were the most effective in scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH). In macroalgae, a high positive correlation was observed between phenolic content and antioxidant capacity while in microalgae, the opposite was verified, suggesting that in microalgae, phenolic compounds are not the main contributors to the observed antioxidant capacity.

The gastrointestinal microbiota plays a critical role on host health and metabolism. This is particularly important in teleost nutrition, because fish do not possess some of the necessary enzymes to cope with the dietary challenges of aquaculture production. A main difficulty within fish nutrition is its dependence on fish meal, an unsustainable commodity and a source of organic pollutants. The most obvious sustainable alternatives to fish meal are plant feedstuffs, but their nutritive value is limited by the presence of high levels of non-starch polysaccharides (NSP), which are not metabolized by fish. The composition of fish-gut microbial communities have been demonstrated to adapt when the host is fed different ingredients. Thus, we hypothesized that a selective pressure of plant-based diets on fish gut microbiota, could be a beneficial strategy for an enrichment of bacteria with a secretome able to mobilize dietary NSP. By targeting bacterial sporulating isolates with diverse carbohydrase activities from the gut of European sea bass, we have obtained isolates with high probiotic potential. By inferring the adaptive fitness to the fish gut and the amenability to industrial processing, we identified the best two candidates to become industrially valuable probiotics. This potential was confirmed in vivo, since one of the select isolates lead to a better growth and feed utilization efficiency in fish fed probiotic-supplemented plant-based diets, thus contributing for sustainable and more cost-effective aquaculture practices.

Legume stovers may be an important feedstuff resource for animals, especially in the Mediterranean production systems. However, these stovers contain antinutritional factors, such as lignin, that may affect the growth performance of the animals. Studies with white-rot fungi showed their ability to increase the nutritional value of several agricultural wastes through changes in lignin structure facilitating the access to structural polysaccharides that are potentially digestible. This contributes to a better utilization of the diet’s nutrients and, consequently, to a better animal performance and health. The present work aimed at evaluating the effect of cowpea stover (Vigna unguiculata), untreated (US) or treated (TS) with Pleurotus citrinopileatus, on performance, digestibility, health and meat quality of growing rabbits. The TS treatment resulted from the inoculation of cowpea stover with Pleurotus citrinopileatus mycelium under solid-state fermentation for 22 days. Five experimental diets incorporating 0 (C), 50 (US50) and 100 g/kg (US100) of US and 50 (TS50) and 100 g/kg (TS100) of TS were used. Trials were conducted using 80 rabbits with 35 days old. The animals were randomly assigned to one of the 5 treatments and were slaughtered at 63 days of age. The incorporation of cowpea stover did not affect animal growth performances (mean daily weight gain of 44.4 g/kg; mean feed intake of 154 g/day; mean feed conversion rate of 3.46) or digestibility (mean dry matter digestibility of 564.8 g/kg; mean neutral detergent fibre digestibility of 369.6 g/kg). The orthogonal contrasts showed that the animals fed with US had a final live weight 5.0% lower (P = 0.04) than the ones fed TS (2214 vs. 2323 g). The TS diet allowed a 17.4% reduction (P = 0.03) in blood cholesterol levels. No influence (P > 0.05) of treatments was detected for carcass traits (mean chilled dressing out percentage of 59%) and meat quality parameters. There was a decrease in the caecal microbiota similarity index (P = 0.01) between US and TS diets. The incorporation up to 100 g/kg of treated cowpea stover with Pleurotus citrinopileatus in the rabbits’ compound feed showed potential to overcome the detrimental effect that the incorporation of the US has on the final live weight of growing rabbits. Further research on the specific effect of TS diets on cholesterol blood levels and shifts on the microbial population of the gastrointestinal tract should be assessed.

Here, we present the genome sequences of two environmental Bacillus strains with broad hydrolytic capacity toward different nonstarch polysaccharides (NSPs) that were isolated from the gut of marine fish fed NSP-rich diets. Several genes that may contribute to the NSP-degrading behavior were identified through in silico analysis.

Here, we present the genome sequences of two environmental Bacillus strains with broad hydrolytic capacity toward different nonstarch polysaccharides (NSPs) that were isolated from the gut of marine fish fed NSP-rich diets. Several genes that may contribute to the NSP-degrading behavior were identified through in silico analysis.

The present work assessed the effects of dietary ratios of essential fatty acids, arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), on liver and intestine oxidative status, intestinal histomorphology and gut microbiota of gilthead sea bream. Four isoproteic and isolipidic plant-based diets were formulated containing a vegetable oil blend as the main lipid source. Diets were supplemented with ARA/EPA/DHA levels (%DM) equivalent to: 2%:0.2%:0.1% (Diet A); 1.0%:0.4%:0.4% (Diet B); 0%:0.6%:0.6% (Diet C); 0%:0.3%:1.5% (Diet D) and tested in triplicate groups for 56 days. Lipid peroxidation was higher in fish fed diets C and D while no differences were reported between diets regarding total, oxidized, and reduced glutathione, and oxidative stress index. Glutathione reductase was higher in fish fed diet A than diets C and D. No histological alterations were observed in the distal intestine. Lower microbiota diversity was observed in intestinal mucosa of fish fed diet C than A, while diets C and D enabled the proliferation of health-promoting bacteria from Bacteroidetes phylum (Asinibacterium sp.) and the absence of pathogenic species like Edwardsiella tarda. Overall, results suggest that a balance between dietary ARA/EPA + DHA promotes gilthead sea bream juveniles’ health however higher dietary content of n-3 LC-PUFA might limited the presence of microbial pathogens in intestinal mucosa.

This study aimed to evaluate the effect of diets including 10%, 20%, and 30% of black soldier fly larvae (Hermetia illucens) meal (HM) (diets HM10, HM20, and HM30, respectively), replacing 17%, 35%, and 52% of fishmeal (FM), on growth performance, whole-body composition and fatty acid (FA) profile, plasma biochemistry, and intermediary metabolism of meagre (Argyrosomus regius) juveniles. For that purpose, triplicate groups of 18.0 ± 0.02 g fish were fed a control diet (CTR) without HM, and the experimental diets for 9 weeks. Growth, feed efficiency, protein efficiency ratio, nitrogen and energy retentions linearly decreased with the increase of dietary HM level. With the exception of ash, whole-body composition was not linearly affected by dietary HM inclusion. Regarding whole-body FA profile, total saturated fatty acids (SFA), SFA: polyunsaturated fatty acids (PUFA) ratio, and total n-6PUFA linearly increased with increasing levels of dietary HM, while total n-3PUFA, n-3 long chain PUFA (LC-PUFA), n-3:n-6 ratio, and unsaturation index linearly decreased with increasing levels of HM in the diets. Docosahexaenoic acid (DHA) whole-body retention linearly decreased while lauric acid (12:0) retention increased with the increase of HM in the diet. Plasma glucose linearly increased, while plasma triglycerides and total lipids decreased with the dietary level of HM. Hepatic amino acid catabolic (alanine aminotransferease and aspartate aminotransferease), and lipogenic (glucose-6-phosphate dehydrogenase and malic enzyme) enzymes activities were unaffected by diet composition, whereas lipid catabolic (3-hydroxyacyl-CoA dehydrogenase) enzyme activity linearly decreased with dietary HM level. Glutamate dehydrogenase, an amino acid catabolic enzyme, was lower in fish fed the HM20 diet than the other HM-including diets. Overall, 10% of HM, corresponding to 17% of FM replacement, might be included in meagre diets without major adverse effects on growth, feed utilization, whole-body composition and FA profile.

Probiotics benefits in fish farming have been usually inferred appraising the effects observed on the host and not through the direct assessment of probiotic dynamics in the host gut microbiota. To overcome this gap, quantitative PCR (qPCR) can be a powerful approach to study the bacterial dynamics in fish gut microbiota. The presented work proposes four B. licheniformis-specific DNA markers and details a qPCR method to track putative probiotics B. licheniformis on fish gut. The four B. licheniformis-specific DNA markers - BL5B (hypothetical protein BL00303), BL8A (serA2), BL13C (rfaB) and BL18A (ligD) - were selected and validated by PCR and multiplex-PCR with 20 B. licheniformis isolates and a broad range of non-target bacteria. To assess the dynamics of B. licheniformis in the digesta of farmed fish, a qPCR was validated using markers BL8A and BL18A and calibration curves obtained for both markers with digesta samples spiked with B. licheniformis cells showed a high correlation (R2 > 0.99) over 6 log units (CFU/reaction), and a limit of detection (LOD) as low as 247 CFUs/reaction. Furthermore, the consistent qPCR repeatability and reproducibility underline the specificity and reliability of the qPCR proposed. Ultimately, the possibility to monitor the dynamics of B. licheniformis probiotics in the gut microbiota of farmed fish might be instrumental to optimize best practices in aquaculture.

A trial was conducted to evaluate the effect of dietary exogenous enzymes supplementation on apparent digestibility of macronutrients, energy and amino acids, and on digestive enzymes activity and gut microbiota of turbot (Scophthalmus maximus) juveniles fed diets rich in plant feedstuffs. For that purpose, a control diet was formulated to contain circa half protein from fishmeal and the other half from plant feedstuffs (dried distillers' grains with solubles, soybean meal, corn gluten, and wheat gluten). Two other diets were formulated similar to the control but including exogenous enzyme complexes: Synergen® (Alltech; product of solid state fermentation of Aspergillus niger) or Natugrain®TS (BASF; non-starch polysaccharides degrading enzymes complex). Both exogenous enzyme complexes increased the apparent digestibility coefficient (ADC) of dry matter, while the ADC of protein, lipid, and energy were only increased with Natugrain®TS. Moreover, the ADC of methionine, isoleucine, and aspartic acid were increased with both exoenzymes supplementation, while the ADC of lysine and glycine were increased with Synergen® and the ADC of arginine and threonine were increased with Natugrain®TS. Dietary supplementation with Synergen® or Natugrain®TS did not affect intestinal pH but increased the activity of lipase and protease in the posterior intestine, while amilase activity was increased only with Natugrain®TS. Microbiota was also affected by both exoenzymes complexes, increasing its richness and diversity. Present results indicate that diet supplementation with exogenous enzymes complexes, Synergen® or Natugrain®TS, may be a feasible strategy to enhance the digestibility of plant feedstuff-rich diets for turbot juveniles.

This deposit is composed by the main article plus the supplementary materials of the publication. Prokaryote genomes are the result of a dynamic flux of genes, with increases achieved via horizontal gene transfer and reductions occurring through gene loss. The ecological and selective forces that drive this genomic flexibility vary across species. Bacillus subtilis is a naturally competent bacterium that occupies various environments, including plant-associated, soil, and marine niches, and the gut of both invertebrates and vertebrates. Here, we quantify the genomic diversity of B. subtilis and infer the genome dynamics that explain the high genetic and phenotypic diversity observed. Phylogenomic and comparative genomic analyses of 42 B. subtilis genomes uncover a remarkable genome diversity that translates into a core genome of 1,659 genes and an asymptotic pangenome growth rate of 57 new genes per new genome added. This diversity is due to a large proportion of low-frequency genes that are acquired from closely related species. We find no gene-loss bias among wild isolates, which explains why the cloud genome, 43% of the species pangenome, represents only a small proportion of each genome. We show that B. subtilis can acquire xenologous copies of core genes that propagate laterally among strains within a niche. While not excluding the contributions of other mechanisms, our results strongly suggest a process of gene acquisition that is largely driven by competence, where the long-term maintenance of acquired genes depends on local and global fitness effects. This competence-driven genomic diversity provides B. subtilis with its generalist character, enabling it to occupy a wide range of ecological niches and cycle through them. Fundação para a Ciência e a Tecnologia grants: (PTDC/EBB-BIO/119006/2010, PEst-OE/EQB/LA0004/2011, SFRH/BPD/89907/2012, SFRH/BD/29397/06); FEDER grant: (LISBOA-01-0145-FEDER-007660). info:eu-repo/semantics/publishedVersion

This study aimed at assessing the effect of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS), and galactooligosaccharides (GOS) on European sea bass juveniles gut microbiota. Four practical diets were formulated with fish meal (FM) and plant feedstuffs (PF) as protein sources (circa 30:70 of protein from FM:PF) and to include 1% of α-cellulose (control diet, CTR), or 1% of scFOS, XOS, or GOS (diets FOS, XOS, and GOS, respectively). Triplicate groups of fish with 79 g were fed with the experimental diets during 30 days. Gut content was sampled at days 7 and 30 for allochthonous microbiota characterization. Lactobacillus, Pseudomonas, Vibrio, and Burkholderia were the main genera found in fish gut. Gut allochthonous microbiota presented an increased number of operational taxonomic units (OTUs) and a higher Margalef index of bacterial richness in fish fed scFOS and XOS. Gut allochthonous microbial OTUs, Margalef species richness index, Shannon’s diversity index, and SIMPER similarity were unaffected by sampling day. In conclusion, scFOS and XOS modulated European sea bass gut microbial community and this effect persisted throughout time. These changes in gut microbiota composition contribute to explain the positive effects on growth performance, immune status, and lipid and glucose metabolism previously reported in European sea bass fed with scFOS and XOS.

[EN] A digestibility trial was conducted to evaluate the effect of fishmeal (FM) replacement with meat and bone meal on diets digestibility, digestive enzymes activity, and microbiota modulation in gilthead seabream (Sparus aurata) juveniles fed diets (45% CP; 20% CL) including 0, 50, and 75% of protein from MBM (diets MBM0; MBM50; MBM75). The ADC of protein was high and unaffected by dietary MBM level, whereas the ADC of energy was higher with diet MBM50 than with MBM0. The ADC of essential amino acids was also high and not affected by diet composition, except for the ADC of phenylalanine and tyrosine, which were lower in diet MBM75 than in the other diets. Pepsin, trypsin, chymotrypsin, lipase, and total alkaline proteases activities were all unaffected by diet composition. Dietary inclusion of MBM modulated gastrointestinal tract microbiota, decreasing the average number of operational taxonomic units and microbial richness. Dietary MBM inclusion promoted an increase of Vibrio, Bacillus, and Mycobacterium genera, whereas colonization by Staphylococcus and Corynebacterium decreased. Overall, present results indicate that up to 75% of FM protein can be replaced by MBM protein in diets for gilthead seabream juveniles without major adverse effects on diet digestibility and digestive function. However, gastrointestinal microbiota was modulated, and further research should be conducted to evaluate the impact of gastrointestinal microbiota modulation on immune and health status of gilthead seabream., This research was partially supported by the Strategic Funding UID/Multi/04423/2013 through national funds provided by FCT - Foundation for Science and Technology and European Regional Development Fund (ERDF), in the framework of the programme PT2020. C.R. Serra was supported by grant SFRH/BPD/101038/2014, from FCT.

This study aimed at evaluating the effects of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS) and galactooligosaccharides (GOS) on growth performance, hepatic metabolism, gut microbiota and digestive enzymes activities of white sea bream juveniles. Four diets were formulated: a control diet with fish meal (FM) and plant feedstuffs (PF) (30FM:70PF) and three test diets similar to control but supplemented with 10 g of scFOS, XOS or GOS per kilo diet, which were fed to fish during 12 weeks. Prebiotics had no effect on growth, feed efficiency or gut microbiota. Plasmatic triglycerides were lower in fish fed XOS than FOS and GOS diets. Malic enzyme activity was lower in fish fed XOS than FOS diet. Fish fed XOS diet had lower fatty acid synthetase (FAS), a key lipogenic enzyme and higher alanine aminotransferase activities. Fifteen days after the start of the trial, an enhancement of total alkaline protease, trypsin and lipase activities was observed in fish fed prebiotics, but such effect disappeared at 12 weeks. In conclusion, scFOS, XOS or GOS seem to have limited applicability in white sea bream feed.

This study aimed to evaluate the effect of including 10%, 20% and 30% of mealworm (Tenebrio molitor, TM) larvae meal (diets TM10, TM20 and TM30, respectively), replacing 25%, 50% and 75% of fishmeal (FM) in a isoproteic (50%) and isolipidic (19%) FM-based diet (CTR) for meagre (Argyrosomus regius). The diets were fed for 9 weeks to triplicate groups of fish with 18.0 g initial body weight and results were evaluated for growth performance; whole-body composition; amino acid (AA) catabolism and digestive enzymes activity; nutrients and chitin digestibility. A digestibility trial was also simultaneously conducted for 48 days. Growth and feed utilization parameters decreased linearly as dietary TM inclusion level increased, while mortality, hepatosomatic and visceral indexes, and AA catabolism enzymes activity were unaffected by the dietary treatments. Whole-body protein was reduced in fish fed TM20 and TM30 diets, with the latter group also having lower energy content than fish fed the CTR diet. The apparent digestibility coefficients (ADC) of dry matter, energy, protein, and AA (except leucine, valine and phenylalanine), decreased as dietary TM inclusion level increased. The lower ADC of protein was related to a linear decrease in trypsin activity. Lipase and total alkaline protease activities were also lower in fish fed the TM30 diet than in the other groups. A quadratic response of total alkaline protease with the increase of dietary TM meal was similar to that observed in the intensity of zymograms bands corresponding to proteases other than trypsin or chymotrypsin-like. Chitin ADC was null and no chitinolytic activity was found in the intestine. Overall, meagre presented a limited capacity to utilize TM, with a 10% TM dietary inclusion already resulting in significant impairment of fish digestive capacity and growth performance, while higher TM dietary levels further negatively affecting fish whole-body composition.

This study aimed to evaluate the effect of replacing fish meal by poultry by-product meal (PBM) in actual commercial standard diets for gilthead seabream juveniles on growth performance, diet digestibility, digestive function, intestinal microbiota, and provide an economic analysis of using PBM in practical diets. Six isoproteic and isolipidic diets were formulated including increasing levels (from 0 to 37.5%) of PBM in replacement of fish meal, corresponding to a fish meal protein replacement in the diets up to 83%. Each diet was randomly assigned to triplicate groups of fish (IBW 63 g) and the growth trial lasted 70 days. No differences between groups were observed on growth performance, voluntary feed intake, feed efficiency, nitrogen utilization, and whole-body composition. Digestive enzymes activity and the ADC of protein, phosphorus, and energy were also not affected by dietary treatment. Regarding gut microbiota, fish fed diet PBM22.5 showed higher species richness and diversity of digesta microbiota, but no differences between groups were observed in mucosa OTUs, richness, and diversity indices. The cost of diet formulation decreased with the dietary replacement of FM by PBM, highlighting the potential of poultry by-products inclusion in commercial diets. Overall, this study indicates that dietary FM could be replaced at least up to 83% with PBM in diets for gilthead seabream juveniles without affecting growth performance and feed utilization while improving the economic efficiency of these diets.

For an increased incorporation of plant ingredients in aquafeeds at the expense of fish meal (FM) and fish oil (FO), more knowledge is needed on the effects at the intestine level of dietary vegetable oils (VO) and carbohydrates (CH), and of possible interactions. For that purpose, in this study, the activities of digestive pancreatic enzymes (amylase, lipase, total alkaline proteases), gut microbiota, and histomorphology were assessed in gilthead sea bream (IBW 71.0 ± 1.5 g) fed four diets differing in lipid source (FO or a blend of VO) and carbohydrate content (0% or 20% gelatinized starch) for 81 days. No major changes in digestive enzyme activities were noticed in fish fed the experimental diets. Dietary VO, but not CH content, modified intestinal microbial profile, by increasing the similarity of bacterial communities. Especially when combined with CH, dietary VO promoted abnormal enterocyte architecture. Liver histology was also accessed, and an increased cytoplasmic vacuolization of hepatocytes was related with dietary CH inclusion, being only significantly different in fish fed FO-based diets. Overall, nutritional interactions between dietary lipid source and carbohydrate content were not observed on digestive enzyme activities and microbial profile. However, the intestine histological modifications observed in fish fed the VOCH+ diet suggest a negative interaction between dietary VO and CH. This requires a more in depth assessment in future studies as it can have negative consequences at a functional level.

The endospore-forming Bacillus subtilis has been used as probiotics over the last 50 years. However, little is known on how Bacillus spp act in the gut compared to other well-characterized probiotics such as lactic acid bacteria. It is believed that the competitive exclusion of pathogens results from different mode of action notably the production of antimicrobial compounds such as bacteriocins. Here, we report the characterization of the unexpected ability of a gut-associated B. subtilis BSP1 to synthetize the cyclic bacteriocin subtilosin A at high level. Our findings suggest that the BSP1 phenotype could be related, at least in part, to a subsequent increased expression level of the subtilosin A biosynthetic gene cluster sbo-alb in response to a higher activity of the stationery and sporulation master regulator Spo0A. ©2013 Science Publication.

The effects of dietary short chain fructooligosaccharides (scFOS) incorporation on hematology, fish immune status, gut microbiota composition, digestive enzymes activities, and gut morphology, was evaluated in gilthead sea bream (Sparus aurata) juveniles reared at 18 °C and 25 °C. For that purpose, fish with 32 g were fed diets including 0, 0.1, 0.25 and 0.5% scFOS during 8 weeks. Overall, scFOS had only minor effects on gilthead sea bream immune status. Lymphocytes decreased in fish fed the 0.1% scFOS diet. Fish fed the 0.5% scFOS diet presented increased nitric oxide (NO) production, while total immunoglobulins (Ig) dropped in those fish, but only in the ones reared at 25 °C. Red blood cells, hemoglobin, bactericidal activity and NO were higher at 25 °C, whereas total white blood cells, circulating thrombocytes, monocytes and neutrophils were higher at 18 °C. In fish fed scFOS, lymphocytes were higher at 18 °C. Total Ig were also higher at 18 °C but only in fish fed 0.1% and 0.5% scFOS diets. No differences in gut bacterial profiles were detected by PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) between dietary treatments. However, group's similarity was higher at 25 °C. Digestive enzymes activities were higher at 25 °C but were unaffected by prebiotics incorporation. Gut morphology was also unaffected by dietary prebiotic incorporation. Overall, gut microbiota composition, digestive enzymes activities and immunity parameters were affected by rearing temperature whereas dietary scFOS incorporation had only minor effects on these parameters. In conclusion, at the tested levels scFOS does not seem worthy of including it in gilthead sea bream juveniles diets., This research was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Programme and national funds through FCT Foundation for Science and Technology, under the projects "PEst-C/MAR/LA0015/2013 and UID/Multi/04423/2013". I. Guerreiro, P. Enes, A. Couto and B. Costas were supported by grants (SFRH/BD/76139/2011; BPD/39688/2007, BD/47495/2008 and BPD/77210/2011, respectively) from FCT, Portugal. C. Serra and A. Salvador were recipient of grants within the Project AQUAIMPROV (reference NORTE-07-0124-FEDER-000038) and Project BioEnv (REF. NORTE-07-0124-FEDER-000048), co-financed by the North Portugal Regional Operational Programme (ON.2 - O Novo Norte). We would like to express our thanks to Pedro Correia for the assistance during the growth trial. Authors would also thank to Jefo Species-specific additives France, for providing the prebiotic.

The impact of replacing circa 70 % fish oil (FO) by a vegetable oil (VO) blend (rapeseed, linseed, palm oils; 20:50:30) in diets for European sea bass juveniles (IBW 96 ± 0.8 g) was evaluated in terms of activities of digestive enzymes (amylase, lipase, alkaline phosphatase, trypsin and total alkaline proteases) in the anterior (AI) and posterior (PI) intestine and tissue morphology (pyloric caeca—PC, AI, PI, distal intestine—DI and liver). For that purpose, fish were fed the experimental diets for 36 days and then liver and intestine were sampled at 2, 6 and 24 h after the last meal. Alkaline protease characterization was also done in AI and PI at 6 h post-feeding. Dietary VO promoted higher alkaline phosphatase activity at 2 h post-feeding in the AI and at all sampling points in the PI. Total alkaline protease activity was higher at 6 h post-feeding in the PI of fish fed the FO diet. Identical number of bands was observed in zymograms of alkaline proteases of fish fed both diets. No alterations in the histomorphology of PC, AI, PI or DI were noticed in fish fed the VO diets, while in the liver a tendency towards increased hepatocyte vacuolization due to lipid accumulation was observed. Overall, and with the exception of a higher intestine alkaline phosphatase activity, 70 % FO replacement by a VO blend in diets for European sea bass resulted in no distinctive alterations on the postprandial pattern of digestive enzyme activities and intestine histomorphology.

Tenacibaculum maritimum, the aetiological agent for marine tenacibaculosis, is one of the most significant pathogens that threaten Senegalese sole, Solea senegalensis (Kaup), aquaculture. Because no immersion challenge with T. maritimum has been reported previously for this flatfish species, this study aimed to optimize bacterial yields as well as to establish a challenge model for tenacibaculosis induction. Several approaches were performed to optimize bacterial culture conditions, including treatment with non-ionic surfactants, detergents, cellulase hydrolysis and strong shaking. A prolonged bath challenge was performed for 24 h under two different temperatures, 16 and 23 °C. Moreover, mucus and plasma bactericidal activities against T. maritimum were also assessed. Culturing bacteria with strong shaking and continuous shaking provided suitable culture conditions to obtain higher bacterial yields without aggregation and fluctuation, contrary to most other treatments that showed a huge amount of bacterial aggregates. A prolonged bath method for 24 h, without skin or gill scarification, was considered suitable for disease induction with high mortality rates. Moreover, data regarding mucus and plasma bactericidal activities suggested that there is a lack of host innate immune response against T. maritimum or that this particular pathogen presents evading strategies against Senegalese sole.

The display of proteins such as feed enzymes at the surface of bacterial spore systems has a great potential use for animal feed. Feed enzymes increase the digestibility of nutrients, leading to greater efficiency in the manufacturing of animal products and minimizing the environmental impact of increased animal production. To deliver their full potential in the gut, feed enzymes must survive the harsh conditions of the feed preparation and the gastrointestinal tract. The well-documented resistance of spores to harsh environments, together with the ability to use proteins that compose the spore as carriers for the display of passenger proteins, suggests that spores could be used as innovative tools to improve the formulation of bioactive molecules. Although some successful examples have been reported, in which abundant structural proteins of the Bacillus subtilis spore outer-coat layer were used as carriers for the display of recombinant proteins, only one convincing example resulted in the display of functional enzymes. In addition, no examples are available about the use of an inner-coat protein for the display of an active passenger enzyme. In our study, we show that the inner-coat oxalate decarboxylase (OxdD) can expose an endogenous phytase, a commonly used feed enzyme for monogastric animals, in an active form at the spore surface. Importantly, despite the higher abundance of CotG outer-coat protein, an OxdD-Phy fusion was more represented at the spore surface. The potential of OxdD as a carrier protein is further documented through the spore display of a bioactive heterologous passenger, the tetrameric β-glucuronidase enzyme from Escherichia coli .

This study aimed to evaluate the effects of dietary lipid source and carbohydrate content on the oxidative status of European sea bass (Dicentrarchus labrax) juveniles. For that purpose, four diets were formulated with fish oil (FO) and vegetable oils (VO) as the lipid source and with 20 or 0 % gelatinised starch as the carbohydrate source, in a 2×2 factorial design. Liver and intestine antioxidant enzyme activities (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PD)), hepatic and intestinal lipid peroxidation (LPO), as well as hepatic oxidative stress index (OSI), were measured in fish fed the experimental diets for 73 d (n9 fish/diet). Carbohydrate-rich diets promoted a decrease in hepatic LPO and OSI, whereas the lipid source induced no changes. Inversely, dietary lipid source, but not dietary carbohydrate concentration, affected LPO in the intestine. Lower intestinal LPO was observed in VO groups. Enzymes responsive to dietary treatments were GR, G6PD and CAT in the liver and GR and GPX in the intestine. Dietary carbohydrate induced GR and G6PD activities and depressed CAT activity in the liver. GPX and GR activities were increased in the intestine of fish fed VO diets. Overall, effects of diet composition on oxidative status were tissue-related: the liver and intestine were strongly responsive to dietary carbohydrates and lipid sources, respectively. Furthermore, different metabolic routes were more active to deal with the oxidative stress in the two organs studied.

Bacillus subtilis is a Gram-positive, rod-shaped, spore-forming bacterium. We present the genome sequence of an undomesticated strain, BSP1, isolated from poultry. The sequence of the BSP1 genome supports the view that B. subtilis has a biphasic lifestyle, cycling between the soil and the animal gastrointestinal tract, and it provides molecular-level insight into the adaptation of B. subtilis to life under laboratory conditions., SCOPUS: ar.j, info:eu-repo/semantics/published

Bacillus subtilis is considered a soil organism for which endospore formation provides a means to ensure long-term survival in the environment. We have addressed here the question of what happens to a spore when ingested. Spores displaying on their surface a heterologous antigen, tetanus toxin fragment C (TTFC), were shown to generate anti-TTFC responses not to the antigen contained in the primary oral inoculum but to those displayed on spores that had germinated and then resporulated. We then used reverse transcription-PCR to determine expression of vegetative genes and sporulation-specific genes in the mouse gut following oral dosing with spores. Significant levels of germination and sporulation were documented. Using natural isolates of B. subtilis that could form biofilms, we showed that these strains could persist in the mouse gut for significantly longer than the laboratory strain. Moreover, these isolates could grow and sporulate anaerobically and exhibited a novel phenomenon of being able to form spores in almost half the time required for the laboratory isolate. This suggests that spores are not transient passengers of the gastrointestinal tract but have adapted to carry out their entire life cycle within this environment. This is the first report showing an intestinal life cycle of B. subtilis and suggests that other Bacillus species could also be members of the gut microflora.