Characterization of Host-Associated Microbiota and Isolation of Antagonistic Bacteria from Greater Amberjack (Seriola dumerili, Risso, 1810) Larvae.

Greater amberjack (Seriola dumerili) is a new species in marine aquaculture with high mortalities at the larval stages. The microbiota of amberjack larvae was analyzed using 16S rDNA sequencing in two groups, one added copepod nauplii (Acartia tonsa) in the diet, and one without copepods (control). In addition, antagonistic bacteria were isolated from amberjack larvae and live food cultures. Proteobacteria was the most abundant phylum followed by Bacteroidota in amberjack larvae. The composition and diversity of the microbiota were influenced by age, but not by diet. Microbial community richness and diversity significantly increased over time. Rhodobacteraceae was the most dominant family followed by Vibrionaceae, which showed the highest relative abundance in larvae from the control group 31 days after hatching. Alcaligenes and Thalassobius genera exhibited a significantly higher relative abundance in the copepod group. Sixty-two antagonistic bacterial strains were isolated and screened for their ability to inhibit four fish pathogens (Aeromonas veronii, Vibrio harveyi, V. anguillarum, V. alginolyticus) using a double-layer test. Phaeobacter gallaeciensis, Phaeobacter sp., Ruegeria sp., and Rhodobacter sp. isolated from larvae and Artemia sp. inhibited the fish pathogens. These antagonistic bacteria could be used as host-derived probiotics to improve the growth and survival of the greater amberjack larvae.

Metaproteome plasticity sheds light on the ecology of the rumen microbiome and its connection to host traits.

The arsenal of genes that microbes express reflect the way in which they sense their environment. We have previously reported that the rumen microbiome composition and its coding capacity are different in animals having distinct feed efficiency states, even when fed an identical diet. Here, we reveal that many microbial populations belonging to the bacteria and archaea domains show divergent proteome production in function of the feed efficiency state. Thus, proteomic data serve as a strong indicator of host feed efficiency state phenotype, overpowering predictions based on genomic and taxonomic information. We highlight protein production of specific phylogenies associated with each of the feed efficiency states. We also find remarkable plasticity of the proteome both in the individual population and at the community level, driven by niche partitioning and competition. These mechanisms result in protein production patterns that exhibit functional redundancy and checkerboard distribution that are tightly linked to the host feed efficiency phenotype. By linking microbial protein production and the ecological mechanisms that act within the microbiome feed efficiency states, our present work reveals a layer of complexity that bears immense importance to the current global challenges of food security and sustainability.

Time to integrate biotechnological approaches into fish gut microbiome research.

Like for other vertebrates, the fish microbiome is critical to the health of its host and has complex and dynamic interactions with the surrounding environment. Thus, the study of the fish microbiome can benefit from the new prospects gained by innovative biotechnological applications in human and other animals, that include manipulation of the associated microbial communities (to improve the health, productivity, and sustainability of fish production), in vitro gut simulators, synthetic microbial communities, and others. Here, we summarize the current state of knowledge on such biotechnological approaches to better understand and engineer the fish microbiome, as well as to advance our knowledge on host-microbes interactions. A particular focus is given to the most recent strategies for fish microbiome manipulation to improve fish health, food safety and environmental sustainability.

In-Situ Biofloc Affects the Core Prokaryotes Community Composition in Gut and Enhances Growth of Nile Tilapia (Oreochromis niloticus).

Biofloc technology is commonly applied in intensive tilapia (Oreochromis niloticus) culture to maintain water quality, supply the fish with extra protein, and improve fish growth. However, the effect of dietary supplementation of processed biofloc on the gut prokaryotic (bacteria and archaea) community composition of tilapia is not well understood. In this study one recirculating aquaculture system was used to test how biofloc, including in-situ biofloc, dietary supplementation of ex-situ live or dead biofloc, influence fish gut prokaryotic community composition and growth performance in comparison to a biofloc-free control treatment. A core gut prokaryotic community was identified among all treatments by analyzing the temporal variations in gut prokaryotes. In-situ produced biofloc significantly increased the prokaryotic diversity in the gut by reducing the relative abundance of dominant Cetobacterium and increasing the relative abundance of potentially beneficial bacteria. The in-situ biofloc delivered a unique prokaryotic community in fish gut, while dietary supplementation of tilapias with 5% and 10% processed biofloc (live or dead) only changed the relative abundance of minor prokaryotic taxa outside the gut core microbiota. The modulatory effect of in-situ biofloc on tilapia gut microbiota was associated with the distinct microbial community in the biofloc water and undisturbed biofloc. The growth-promoting effect on tilapia was only detected in the in-situ biofloc treatment, while dietary supplementation of processed biofloc had no effect on fish growth performance as compared to the control treatment.

Impact of early-life rearing history on gut microbiome succession and performance of Nile tilapia.

BACKGROUND: Fish gut microbial colonisation starts during larval stage and plays an important role in host's growth and health. To what extent first colonisation could influence the gut microbiome succession and growth in later life remains unknown. In this study, Nile tilapia embryos were incubated in two different environments, a flow-through system (FTS) and a biofloc system (BFS); hatched larvae were subsequently cultured in the systems for 14 days of feeding (dof). Fish were then transferred to one common recirculating aquaculture system (RAS1, common garden, 15-62 dof), followed by a growth trial in another RAS (RAS2, growth trial, 63-105 dof). In RAS2, fish were fed with two types of diet, differing in non-starch polysaccharide content. Our aim was to test the effect of rearing environment on the gut microbiome development, nutrient digestibility and growth performance of Nile tilapia during post-larvae stages. RESULTS: Larvae cultured in the BFS showed better growth and different gut microbiome, compared to FTS. After the common garden, the gut microbiome still showed differences in species composition, while body weight was similar. Long-term effects of early life rearing history on fish gut microbiome composition, nutrient digestibility, nitrogen and energy balances were not observed. Still, BFS-reared fish had more gut microbial interactions than FTS-reared fish. A temporal effect was observed in gut microbiome succession during fish development, although a distinct number of core microbiome remained present throughout the experimental period. CONCLUSION: Our results indicated that the legacy effect of first microbial colonisation of the fish gut gradually disappeared during host development, with no differences in gut microbiome composition and growth performance observed in later life after culture in a common environment. However, early life exposure of larvae to biofloc consistently increased the microbial interactions in the gut of juvenile Nile tilapia and might possibly benefit gut health.

Exogenous enzymes and probiotics alter digestion kinetics, volatile fatty acid content and microbial interactions in the gut of Nile tilapia.

Sustainable aquafeed production requires fishmeal replacement, leading to an increasing use of plant-derived ingredients. As a consequence, higher levels of antinutritional substances, such as non-starch polysaccharides and phytate, are present in aquafeeds, with negative effects on fish performance, nutrient digestibility and overall gut health. To alleviate these negative effects, providing exogenous digestive enzymes and/or probiotics can be an effective solution. In this study, we tested the effect of dietary supplementation of enzymes (phytase and xylanase) and probiotics (three strains of Bacillus amyloliquefaciens) on nutrient digestion kinetics and volatile fatty acid content along the gut, and the distal gut microbiome diversity in Nile tilapia. Chyme volatile fatty content was increased with probiotic supplementation in the proximal gut, while lactate content, measured for the first time in vivo in fish, decreased with enzymes along the gut. Enzyme supplementation enhanced crude protein, Ca and P digestibility in proximal and middle gut. Enzymes and probiotics supplementation enhanced microbial interactions as shown by network analysis, while increased the abundance of lactic acid bacteria and Bacillus species. Such results suggest that supplementation with exogenous enzymes and probiotics increases nutrient availability, while at the same time benefits gut health and contributes to a more stable microbiome environment.

Isolation of Phaeobacter sp. from Larvae of Atlantic Bonito (Sarda sarda) in a Mesocosmos Unit, and Its Use for the Rearing of European Seabass Larvae (Dicentrarchus labrax L.).

The target of this study was to use indigenous probiotic bacteria in the rearing of seabass larvae. A Phaeobacter sp. strain isolated from bonito yolk-sac larvae (Sarda sarda) and identified by amplification of 16S rDNA showed in vitro inhibition against Vibrio anguillarum. This Phaeobacter sp. strain was used in the rearing of seabass larvae (Dicentrarchus labrax L.) in a large-scale trial. The survival of seabass after 60 days of rearing and the specific growth rate at the late exponential growth phase were significantly higher in the treatment receiving probiotics (p < 0.05). Microbial community richness as determined by denaturing gradient gel electrophoresis (DGGE) showed an increase in bacterial diversity with fish development. Changes associated with the administration of probiotics were observed 11 and 18 days after hatching but were not apparent after probiotic administration stopped. In a small challenge experiment, seabass larvae from probiotic treatment showed increased survival (p < 0.05) after experimental infection with a mild pathogen (Vibrio harveyi). Overall, our results showed that the use of an indigenous probiotic strain had a beneficial impact on larval rearing in industry-like conditions.

Experimental infection model with the bivalvulid Enteromyxum leei (Myxidiidae) in the sharpsnout seabream, Diplodus puntazzo (Sparidae), and evaluation of the antiparasitic efficacy of a functional diet.

An infection model for sharpsnout seabream Diplodus puntazzo (Walbaum) challenged with the myxosporean Enteromyxum leei (Diamant, Lom et Dyková, 1994), resembling the natural infection conditions, was used to evaluate the antiparasitic efficacy of a functional diet. Fish of an average weight of 12.5 ± 1.2 g were delivered either a functional (included as feed supplement at 0.3% levels) or a control extruded diet. After four weeks of administration of the experimental diets, fish were challenged with the parasites (cohabitation with infected donors; donor: recipient ratio 1 : 1). The experiment was terminated four weeks after the start of the challenge. At the end of the experiment, growth and feeding (specific growth rate and feed efficiency), as well as immunological parameters (respiratory burst activity, antibacterial activities, hemoglobin concentration, anti-protease activity and ceruloplasmin activity) were measured along with cumulative mortality and total parasitic count in the gut. No significant difference was evident with regard to growth and feeding performance, mortality, gut parasitic load or immunological parameters as the parasitical challenge significantly affected both the performance of the control and functional diet fed fish. However, there was a less prominent impact on antibacterial, anti-protease and ceruloplasmin activity in fish fed with the functional diet. Overall, the present study validated the experimental cohabitation infection model and evaluated the efficacy of a functional ingredient as an antiparasitic agent, showing some potential effects on the fish immune response.

Antibiotic effect and microbiome persistence vary along the European seabass gut.

The constant increase in aquaculture production has led to extensive use of antibiotics as a means to prevent and treat diseases, with adverse implications on the environment, animal health and commensal microbes. Gut microbes are important for the host proper functioning, thus evaluating such impacts is highly crucial. Examining the antibiotic impact on gut segments with different physiological roles may provide insight into their effects on these microhabitats. Hence, we evaluated the effect of feed-administrated antibiotics on the composition and metabolic potential of the gut microbiome in the European seabass, an economically important aquaculture species. We used quantitative PCR to measure bacterial copy numbers, and amplicon sequencing of the 16S rRNA gene to describe the composition along the gut, after 7-days administration of two broad-range antibiotic mixtures at two concentrations. While positive correlation was found between antibiotic concentration and bacterial abundance, we showed a differential effect of antibiotics on the composition along the gut, highlighting distinct impacts on these microbial niches. Moreover, we found an increase in abundance of predicted pathways related to antibiotic-resistance. Overall, we show that a high portion of the European seabass gut microbiome persisted, despite the examined antibiotic intake, indicating high stability to perturbations.

Stochasticity constrained by deterministic effects of diet and age drive rumen microbiome assembly dynamics.

How complex communities assemble through the animal's life, and how predictable the process is remains unexplored. Here, we investigate the forces that drive the assembly of rumen microbiomes throughout a cow's life, with emphasis on the balance between stochastic and deterministic processes. We analyse the development of the rumen microbiome from birth to adulthood using 16S-rRNA amplicon sequencing data and find that the animals shared a group of core successional species that invaded early on and persisted until adulthood. Along with deterministic factors, such as age and diet, early arriving species exerted strong priority effects, whereby dynamics of late successional taxa were strongly dependent on microbiome composition at early life stages. Priority effects also manifest as dramatic changes in microbiome development dynamics between animals delivered by C-section vs. natural birth, with the former undergoing much more rapid species invasion and accelerated microbiome development. Overall, our findings show that together with strong deterministic constrains imposed by diet and age, stochastic colonization in early life has long-lasting impacts on the development of animal microbiomes.

Core gut microbial communities are maintained by beneficial interactions and strain variability in fish.

The term core microbiome describes microbes that are consistently present in a particular habitat. If the conditions in that habitat are highly variable, core microbes may also be considered to be ecological generalists. However, little is known about whether metabolic competition and microbial interactions influence the ability of some microbes to persist in the core microbiome while others cannot. We investigated microbial communities at three sites in the guts of European seabass under four dietary conditions. We identified generalist core microbial populations in each gut site that are shared across fish, present under multiple diets and persistent over time. We found that core microbes tend to show synergistic growth in co-culture, and low levels of predicted and validated metabolic competition. Within core microbial species, we found high levels of intraspecific variability and strain-specific habitat specialization. Thus, both intraspecific variability and interspecific facilitation may contribute to the ecological stability of the animal core microbiome.

A heritable subset of the core rumen microbiome dictates dairy cow productivity and emissions.

A 1000-cow study across four European countries was undertaken to understand to what extent ruminant microbiomes can be controlled by the host animal and to identify characteristics of the host rumen microbiome axis that determine productivity and methane emissions. A core rumen microbiome, phylogenetically linked and with a preserved hierarchical structure, was identified. A 39-member subset of the core formed hubs in co-occurrence networks linking microbiome structure to host genetics and phenotype (methane emissions, rumen and blood metabolites, and milk production efficiency). These phenotypes can be predicted from the core microbiome using machine learning algorithms. The heritable core microbes, therefore, present primary targets for rumen manipulation toward sustainable and environmentally friendly agriculture.

Transcriptome Analysis Reveals Common and Differential Response to Low Temperature Exposure Between Tolerant and Sensitive Blue Tilapia (Oreochromis aureus).

Tilapias are very important to the world's aquaculture. As befitting fish of their tropical origin, their distribution, and culture practices are highly affected by low temperatures. In this study, we used genetic and genomic methodologies to reveal pathways involved in the response and tolerance of blue tilapia (Oreochromis aureus) to low temperature stress. Cold tolerance was characterized in 66 families of blue tilapia. Fish from cold-tolerant and cold-sensitive families were sampled at 24 and 12°C, and the transcriptional responses to low-temperature exposure were measured in the gills and liver by high-throughput mRNA sequencing. Four hundred and ninety four genes displayed a similar temperature-dependent expression in both tolerant and sensitive fish and in the two tissues, representing the core molecular response to low temperature exposure. KEGG pathway analysis of these genes revealed down-regulation of focal-adhesion and other cell-extracellular matrix (ECM) interactions, and up-regulation of proteasome and various intra-cellular proteolytic activities. Differential responses between cold-tolerant and cold-sensitive fish were found with genes and pathways that were up-regulated in one group and down-regulated in the other. This reverse response was characterized by genes involved in metabolic pathways such as glycolysis/gluconeogenesis in the gills and biosynthesis of amino-acids in the liver, with low temperature down-regulation in tolerant fish and up-regulation in sensitive fish.

Short- and long-term low-salinity acclimation effects on the branchial and intestinal gene expression in the European seabass (Dicentrarchus labrax).

The European seabass (Dicentrarchus labrax) is a teleost remarkably adapted to a wide range of water salinity, through osmoregulatory mechanisms, mainly operating in the gills and the intestine. As an important aquaculture species, its rearing in low-salinity conditions offers benefits for its inland culture. However, this demands a full comprehension of the European seabass osmoregulatory mechanisms and its response to acclimation protocols. The purpose of this study was to evaluate different acclimation protocols in terms of osmoregularity and stress response, following transferring of European seabass juveniles from seawater to freshwater. In addition, nutrient absorption was also examined since drinking rates are sensitive to salinity. The acclimation challenge was applied through three protocols: direct transfer (0 h) to freshwater, gradual transfer during 3 h and during 72 h. The short- (1 h after complete change to freshwater) and long-term effects (after 2 months) of each acclimation protocol were evaluated by assessing the expression of 1. The osmoregulatory genes: Na+/K+-ATPase α1, Na+/K+/2Cl- 1 co-transporter, aquaporins 1 and 3, and the cystic fibrosis transmembrane conductance regulator; 2. The heat shock protein 70 gene; 3. The peptide transporter genes corresponding to PepT1a, PepT1b and PepT2. The short-term acclimation response was pronounced in both gills and the intestine affecting stress-, osmoregulatory- and nutrient-related gene expression. Long-term effects were only evident in the intestine. Direct transfer in freshwater mainly induced a long-term stress response, while the short-term effect was more pronounced in the 3 h-transfer, potentially due to handling. Our results suggest that although the European seabass can withstand direct transfer to low-salinity conditions, a gradual transfer is recommended to prevent long-term stress effects.

Host genetic selection for cold tolerance shapes microbiome composition and modulates its response to temperature.

The hologenome concept proposes that microbes and their host organism are an independent unit of selection. Motivated by this concept, we hypothesized that thermal acclimation in poikilothermic organisms, owing to their inability to maintain their body temperature, is connected to their microbiome composition. To test this hypothesis, we used a unique experimental setup with a transgenerational selective breeding scheme for cold tolerance in tropical tilapias. We tested the effects of the selection on the gut microbiome and on host transcriptomic response. Interestingly, we found that host genetic selection for thermal tolerance shapes the microbiome composition and its response to cold. The microbiomes of cold-resistant fish showed higher resilience to temperature changes, indicating that the microbiome is shaped by its host's selection. These findings are consistent with the hologenome concept and highlight the connection between the host and its microbiome's response to the environment.

Dietary salt levels affect digestibility, intestinal gene expression, and the microbiome, in Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is the world's most widely cultured fish species. Therefore, its nutritional physiology is of great interest from an aquaculture perspective. Studies conducted on several fish species, including tilapia, demonstrated the beneficial effects of dietary salt supplementation on growth; however, the mechanism behind these beneficial effects is still not fully understood. The fish intestine is a complex system, with functions, such as nutrient absorption, ion equilibrium and acid-base balance that are tightly linked and dependent on each other's activities and products. Ions are the driving force in the absorption of feed components through pumps, transporters and protein channels. In this study, we examined the impact of 5% increase in dietary NaCl on protein, lipid, ash and dry matter digestibility, as well as on the expression of intestinal peptide transporters (PepTs) and ion pumps (Na+/K+-ATPase, V-H+-ATPase, N+/H+-Exchanger) in Nile tilapia. In addition, effects on the gut microbiome were evaluated. Our results show that dietary salt supplementation significantly increased digestibility of all measured nutritional components, peptide transporters expression and ion pumps activity. Moreover, changes in the gut microbial diversity were observed, and were associated with lipid digestibility and Na+/K+-ATPase expression.

Effects of graded dietary levels of soy protein concentrate supplemented with methionine and phosphate on the immune and antioxidant responses of gilthead sea bream (Sparus aurata L.).

The effects of a dietary soy protein concentrate (SPC) as a fish meal (FM) substitute, on selected innate immune responses, the oxidative status, hepatic and intestinal morphology of gilthead sea bream, Sparus aurata, were evaluated after a three-month feeding trial. Isonitrogenous (45% crude protein) and isoenergetic (23 kJ/g gross energy) diets with 20% (SPC20), 40% (SPC40) and 60% (SPC60) of SPC inclusion, supplemented with methionine and phosphate, were evaluated against a diet containing FM as the sole protein source. Diets were allocated in triplicate groups of 26-g fish (8 kg m-3/tank) and administered for three months. Immune responses were evaluated by performing immunological assays in blood (respiratory burst activity) and serum (myeloperoxidase content, bacteriolytic and lysozyme activity), as well as by gene expression analysis of immune-associated genes (MHCIIα, ß2m, CSF-1R, NCCRP-1, TGF-ß1, HSP70) in the head kidney and distal intestine. In addition, oxidative stress was evaluated by measuring the activity of liver enzymes associated with the antioxidant system. The respiratory burst activity of blood was significantly decreased in the SPC40 group, while serum myeloperoxidase content and bacteriolytic and lysozyme activities were affected. Significantly higher expression levels of NCCRP-1 and HSP70 were found in SPC60 head kidneys, while increased intestinal MHCIIα and NCCRP-1 transcripts were observed in SPC40. Hepatic antioxidant enzyme activity of glutathione reductase and glutathione-S-transferase was significantly enhanced in the SPC40 and SPC60 groups, while superoxide dismutase activity was increased only in the SPC40 group. Moreover, increased lipid accumulation in the enterocytes of the distal intestine was observed in the SPC60 group. Overall, a three-month feeding period with diets over 40% of dietary SPC inclusion as a FM substitute, indicated increases on immune and antioxidant enzyme responses, suggesting the dietary SPC levels that gilthead sea bream can tolerate.