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

Abstract

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.

Red Cusk-Eel (Genypterus chilensis) Gut Microbiota Description of Wild and Aquaculture Specimens

“…However, it must be noted that the results obtained in this study were obtained analyzing samples of whole intestinal contents, thus corresponding to the total gut microbiota. However, the distribution and percentages described could be different if different tracts of the fish gut were analyzed separately, as previously reported by other species, such as salmonids [60][61][62].…”

Deploying anIn VitroGut Model to Assay the Impact of the Mannan-Oligosaccharide Prebiotic Bio-Mos on the Atlantic Salmon (Salmo salar) Gut Microbiome

“…The biological replicate (the founding inoculum of each SalmoSim run) appears to be a major driver of variations in community composition and structure throughout the experiment. This could be partially explained by the fact that feed used in the in vitro study was sterile, thus the bacterial communities retrieved within the SalmoSim system originated only from real salmon inocula as in a previous experiment involving SalmoSim ( 27 ). The presence of common feed microbes might have reduced inter-individual variability, but in-feed microbes rarely colonize or establish in the gut (e.g., Heys et al, 2020).…”

“…The basal in vitro system feed medium was prepared as described before ( 27 ) by combining the following for a total of 2 liters: 35 g/L of Instant Ocean Sea Salt to generate artificial seawater, 10 g/L of the Fish meal (the same feed as used in [ 27 ]), also as in Kazlauskaite et al, 2021, 1 g/L freeze-dried mucus collected and pooled from the pyloric cecum compartments of multiple adult farmed starved Atlantic salmon (different individuals from the ones used in this study as inocula), 2 liters of deionized water. For the Bio-Mos supplemented feed the basal feed medium was supplemented with 0.4% of Bio-Mos (derived from the outer cell wall of Saccharomyces cerevisiae strain 1026) dissolved in distilled water.…”

“…SalmoSim is a salmon gut simulation system that continuously maintains the microbial communities present in the intestine of marine phase Atlantic Salmon ( Salmo salar ) ( 27 ). The current study deploys a modified version of SalmoSim designed to evaluate the effect of Bio-Mos (Alltech), a commercially available MOS product, on the microbial communities of the Atlantic salmon small intestine (pyloric cecum) in biological triplicate.…”

Assessing Fish Immunotoxicity by Means of In Vitro Assays: Are We There Yet?

“…Typically, cell lines such as the rainbow trout RTgutGC line are cultured on semipermeable supports in order to establish tight, polarized epithelia (196)(197)(198). The realism of these systems can be improved by combining different cell lines, by modifying the culture environment or by including microbial communities (199,200). These in vitro systems have been successfully used to study the impact of toxicants on mucosal biotransformation and oxidative stress pathways (157,158).…”