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Background The aquaculture sector now accounts for almost 50% of all fish for human consumption and is anticipated to provide 62% by 2030. Innovative strategies are being sought to improve fish feeds and feed additives to enhance fish performance, welfare, and the environmental sustainability of the aquaculture industry. There is still a lack of knowledge surrounding the importance and functionality of the teleost gut microbiome in fish nutrition. In vitro gut model systems might prove a valuable tool to study the effect of feed, and additives, on the host’s microbial communities. Several in vitro gut models targeted at monogastric vertebrates are now in operation. Here, we report the development of an Atlantic salmon gut model, SalmoSim, to simulate three gut compartments (stomach, pyloric caecum, and midgut) and associated microbial communities. Results The gut model was established in a series of linked bioreactors seeded with biological material derived from farmed adult marine-phase salmon. We first aimed to achieve a stable microbiome composition representative of founding microbial communities derived from Atlantic salmon. Then, in biological triplicate, the response of the in vitro system to two distinct dietary formulations (fishmeal and fishmeal free) was compared to a parallel in vivo trial over 40 days. Metabarcoding based on 16S rDNA sequencing qPCR, ammoniacal nitrogen, and volatile fatty acid measurements were undertaken to survey the microbial community dynamics and function. SalmoSim microbiomes were indistinguishable (p = 0.230) from their founding inocula at 20 days and the most abundant genera (e.g., Psycrobacter, Staphylococcus, Pseudomonas) proliferated within SalmoSim (OTUs accounting for 98% of all reads shared with founding communities). Real salmon and SalmoSim responded similarly to the introduction of novel feed, with majority of the taxa (96% Salmon, 97% SalmoSim) unaffected, while a subset of taxa (e.g., a small fraction of Psychrobacter) was differentially affected across both systems. Consistent with a low impact of the novel feed on microbial fermentative activity, volatile fatty acid profiles were not significantly different in SalmoSim pre- and post-feed switch. Conclusion By establishing stable and representative salmon gut communities, this study represents an important step in the development of an in vitro gut system as a tool for the improvement of fish nutrition and welfare. The steps of the system development described in this paper can be used as guidelines to develop various other systems representing other fish species. These systems, including SalmoSim, aim to be utilised as a prescreening tool for new feed ingredients and additives, as well as being used to study antimicrobial resistance and transfer and fundamental ecological processes that underpin microbiome dynamics and assembly.

In this paper we report the results of the impact of a prebiotic (alpha-MOS supplementation) on microbial communities, using anin vitrosimulator of the gut microbial environment of the Atlantic salmon. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production by the microbiota from salmon pyloric ceca and correlates with a significant shift in microbial community composition with observed increase in lactic acid producingCarnobacterium.

Alpha mannose-oligosaccharide (MOS) prebiotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and their associated metabolism. Bio-Mos is a commercially available prebiotic currently used in the agri-feed industry, but studies show contrasting results of its effect on fish performance and feed efficiency. Thus, detailed studies are needed to investigate the effect of MOS supplements on the fish microbiome to enhance our understanding of the link between MOS and gut health. To assess Bio-Mos for potential use as a prebiotic growth promoter in salmonid aquaculture, we have modified an established Atlantic salmon

Mannose-oligosaccharide (MOS) pre-biotics are widely deployed in animal agriculture as immunomodulators as well as to enhance growth and gut health. Their mode of action is thought to be mediated through their impact on host microbial communities and the associated metabolism. Bio-Mos is a commercially available pre-biotic currently used in the agri-feed industry. To assess Bio-Mos for potential use as a prebiotic growth promotor in salmonid aquaculture, we have modified an established Atlantic salmonin vitrogut model, SalmoSim, to evaluate its impact on the host microbial communities. Inoculated from biological triplicates of adult farmed salmon pyloric caeca compartments, the microbial communities were stabilised in SalmoSim followed by a twenty-day exposure to the prebiotic and in turn followed by an eight day ‘wash out’ period. Dietary inclusion of MOS resulted in a significant increase in formate (p=0.001), propionate (p=0.037) and isovalerate (p=0.024) levels, correlated with increased abundances of several, principally, anaerobic microbial genera (Fusobacterium,Agarivorans,Pseudoalteromonas). DNA metabarcoding with the 16S rDNA marker confirmed a significant shift in microbial community composition in response to MOS supplementation with observed increase in lactic acid producingCarnobacterium. In conjunction with previousin vivostudies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggests that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role ofin vitrogut models to augmentin vivotrials of microbiome modulators.ImportanceIn this paper we report the results of the impact of prebiotic (MOS supplementation) on microbial communities within recently developed Atlantic salmon gut microbiomein vitrosimulator. Our data suggest that Bio-Mos may be of value in salmonid production as it enhances volatile fatty acid production in the Atlantic salmon gut and correlates with a significant shift in microbial community composition with observed increase in lactic acid producingCarnobacterium. In conjunction with previousin vivostudies linking enhanced volatile fatty acid production alongside MOS supplementation to host growth and performance, our data suggest that Bio-Mos may be of value in salmonid production. Furthermore, our data highlights the potential role ofin vitrogut models to augmentin vivotrials of microbiome modulators.

Atlantic salmon are a species of major economic importance. Intense innovation is underway to improve salmon feeds and feed additives to enhance fish performance, welfare, and the environmental sustainability of the industry. Several gut models targeted at monogastric vertebrates are now in operation. Here we report progress in the development of an Atlantic salmonin vitrogut model, SalmoSim, to simulate three gut compartments (stomach, pyloric caecum and mid gut) and associated microbial communities. The artificial gut model was established in a series of linked bioreactors seeded with biological material derived for adult marine phase salmon. In biological triplicate, the response of thein vitrosystem to two distinct dietary formulations (fish meal and fish meal free) was compared to a parallelin vivotrial over forty days. 16S rDNA sequencing, qPCR, ammoniacal nitrogen and volatile fatty acid measurements were undertaken to survey microbial community dynamics and function. SalmoSim communities were indistinguishable (p=0.230) from their founding inocula at 20 days and most abundant genera (e.g.Psycrobacter, Staphylococcus, Pseudomonas) proliferated thein vitrosystem. Real salmon and SalmoSim responded similarly to the introduction of the novel feed, with most taxa (96% Salmon, 97% SalmoSim) unaffected, while a subset of taxa was affected non-identically across both systems. Consistent with a low impact of the novel feed on microbial community function, VFA profiles were not significantly different in SalmoSim pre and post the switch feed. This study represents an important first-step in the development of anin vitrogut system as a tool for the improvement of salmon nutrition.

Lacking a peptidoglycan cell wall, mycoplasmas are the smallest self-replicating life forms. Members of this bacterial genus are known to parasitise a wide array of metazoans including vertebrates. Whilst much research has been significant targeted at parasitic mammalian mycoplasmas, very little is known about their role in other vertebrates. In the current study, we aim to explore the biology and evolution of Mycoplasma in salmonids, including cellular niche, genome size structure and gene content. Using Fluorescence in-situ hybridisation (FISH), mycoplasmas were identified in epithelial tissues across the digestive tract (stomach, pyloric caecum and midgut) during the developmental stages (eggs, parr, subadult) of farmed Atlantic salmon (Salmo salar), showing a high abundance in acidic compartments. With high throughput sequencing from subadults farmed Atlantic salmon, we assembled a nearly complete genome (~0.57 MB) via shotgun-metagenomics. The phylogenetic inference from the recovered genome revealed successful taxonomic proximity to Mycoplasma penetrans (~1.36 Mb) from the recovered genome. Although, no significant correlation between genome size and its phylogeny was observed, we recovered functional signatures, especially, riboflavin encoding genes pathway and sugars transporters, suggesting a symbiotic relationship between Mycoplasma and the host. Though 247 strains of Mycoplasma are available in public databases, to the best of our knowledge, this is the first study to demonstrate ecological and functional association between Mycoplasma and Salmo salar which delineates symbiotic reductive evolution and genome erosion primarily and also serves as a proxy for salmonid health in aquaculture processes (cell lines, in vitro gut models).

A growing number of studies have examined variation in the microbiome to determine the role in modulating host health, physiology, and ecology. However, the ecology of host microbial colonization is not fully understood and rarely tested. The continued increase in production of farmed Atlantic salmon, coupled with increased farmed-wild salmon interactions, has accentuated the need to unravel the potential adaptive function of the microbiome and to distinguish resident from transient gut microbes. Between gut compartments in a farmed system, we found a majority of operational taxonomic units (OTUs) that fit the neutral model, with Mycoplasma species among the key exceptions. In wild fish, deterministic processes account for more OTU differences across life stages than those observed across gut compartments. Unlike previous studies, our results make detailed comparisons between fish from wild and farmed environments, while also providing insight into the ecological processes underpinning microbial community assembly in this ecologically and economically important species., In recent years, a wealth of studies has examined the relationships between a host and its microbiome across diverse taxa. Many studies characterize the host microbiome without considering the ecological processes that underpin microbiome assembly. In this study, the intestinal microbiota of Atlantic salmon, Salmo salar, sampled from farmed and wild environments was first characterized using 16S rRNA gene MiSeq sequencing analysis. We used neutral community models to determine the balance of stochastic and deterministic processes that underpin microbial community assembly and transfer across life cycle stage and between gut compartments. Across gut compartments in farmed fish, neutral models suggest that most microbes are transient with no evidence of adaptation to their environment. In wild fish, we found declining taxonomic and functional microbial community richness as fish mature through different life cycle stages. Alongside neutral community models applied to wild fish, we suggest that declining richness demonstrates an increasing role for the host in filtering microbial communities that is correlated with age. We found a limited subset of gut microflora adapted to the farmed and wild host environment among which Mycoplasma spp. are prominent. Our study reveals the ecological drivers underpinning community assembly in both farmed and wild Atlantic salmon and underlines the importance of understanding the role of stochastic processes, such as random drift and small migration rates in microbial community assembly, before considering any functional role of the gut microbes encountered. IMPORTANCE A growing number of studies have examined variation in the microbiome to determine the role in modulating host health, physiology, and ecology. However, the ecology of host microbial colonization is not fully understood and rarely tested. The continued increase in production of farmed Atlantic salmon, coupled with increased farmed-wild salmon interactions, has accentuated the need to unravel the potential adaptive function of the microbiome and to distinguish resident from transient gut microbes. Between gut compartments in a farmed system, we found a majority of operational taxonomic units (OTUs) that fit the neutral model, with Mycoplasma species among the key exceptions. In wild fish, deterministic processes account for more OTU differences across life stages than those observed across gut compartments. Unlike previous studies, our results make detailed comparisons between fish from wild and farmed environments, while also providing insight into the ecological processes underpinning microbial community assembly in this ecologically and economically important species.

Mycoplasmas are the smallest autonomously self-replicating life form on the planet. Members of this bacterial genus are known to parasitise a wide array of metazoans including vertebrates. Whilst much research has been significant targeted at parasitic mammalian mycoplasmas, very little is known about their role in other vertebrates. In the current study, we aim to explore the biology of mycoplasmas in Atlantic Salmon, a species of major significance for aquaculture, including cellular niche, genome size structure and gene content. Using fluorescent in-situ hybridisation (FISH), mycoplasmas were targeted in epithelial tissues across the digestive tract (stomach, pyloric caecum and midgut) from different development stages (eggs, parr, subadult) of farmed Atlantic salmon (Salmo salar), and we present evidence for an intracellular niche for some of the microbes visualised. Via shotgun metagenomic sequencing, a nearly complete, albeit small, genome (~0.57 MB) as assembled from a farmed Atlantic salmon subadult. Phylogenetic analysis of the recovered genome revealed taxonomic proximity to other salmon derived mycoplasmas, as well as to the human pathogen Mycoplasma penetrans (~1.36 Mb). We annotated coding sequences and identified riboflavin pathway encoding genes and sugar transporters, the former potentially consistent with micronutrient provisioning in salmonid development. Our study provides insights into mucosal adherence, the cellular niche and gene catalog of Mycoplasma in the gut ecosystem of the Atlantic salmon, suggesting a high dependency of this minimalist bacterium on its host. Further study is required to explore and functional role of Mycoplasma in the nutrition and development of its salmonid host., Highlights • Mycoplasmas colonize the epithelial niche of the Atlantic salmon GI tract through its different development stages. • A nearly complete, albeit small, genome (~0.57 MB) assembled from Atlantic salmon subadult suggests genome reduction. • The lack of mobile genes and virulence factors in the mycoplasma genome could support a non-pathogenic lifestyle. • Mycoplasma’s genome content suggests role for host nutrient provisioning relevance to aquaculture. • Shotgun metagenomics, phylogenetics, and fluorescence in situ hybridization are relevant methods in aquaculture research.