SalmoSim: the development of a three-compartment in vitro simulator of the Atlantic salmon GI tract and associated microbial communities

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.

Automated summary

This study developed an in vitro gut model (SalmoSim) to simulate the gut compartments of Atlantic salmon and studied the microbial community dynamics and function in response to different dietary formulations. Results showed that SalmoSim microbiomes were stable and representative, with similar responses to new feed as real salmon. The study contributes to the development of in vitro gut systems for fish nutrition and welfare improvement, serving as guidelines for developing similar systems for other fish species.