Intestinal microbiome analysis and change of immune response in olive flounder supplemented with probiotics

Abstract

This study was carried out to investigate the effects of probiotics supplementation on intestinal microbiome and change of immune response in olive flounder, and compared and analyzed the effects of probiotics supplementation according to species and physiological conditions. Feeding trial was conducted by supplying fishmeal (FM), low-fishmeal (LFM), and probiotic (Pro) diets, respectively, and Chao1, ACE, and Jackknife, which are richness estimates were significantly increased in the Pro group. Operational taxonomic units (OTUs) showed the largest value in the Pro group (611 ± 227 OUTs), and 432 OTUs existed only in the Pro group. The most abundant phylum in all groups was Proteobacteria. The second abundant phylum in the FM and LFM groups was Firmicutes, while in the Pro group was Bacteroidetes. As a result of predicting the potential function of intestinal microorganisms through PICRUSt analysis, the Pro group had a significantly higher proportion of the biosynthesis of secondary metabolites, glycine, serine, and threonine metabolism, and glutathione metabolism pathways than the other groups. In immune-related gene expression analysis, IL-10 and IL-1β were significantly increased in the LFM group, and IL-10, IL-6 and IL-1β were significantly increased in the Pro group. These results suggest that intestinal microbial diversity, composition, and cytokine gene expression were clearly different depending on the diet provided. In particular, probiotic supplementation changed intestinal microbial diversity and composition significantly more within the same period. As a supplementary effect according to the probiotics species, experiments were conducted on two strains Bacillus sp. SJ-10 (ProB) and Lactobacillus plantarum (ProL), which have been proven to have probiotic effects in olive flounder. The estimated intestinal microbial richness (Chao1, ACE) and diversity (Shannon) demonstrated a significant abundance in the ProB group compared to the ProL and control groups. Analysis of intestinal microbial abundance and beta diversity at the phylum and genus levels showed the largest change in intestinal microbial composition in ProB. Trypsin and lipase activities were elevated in both the ProB and ProL groups compared to the control, but amylase was only higher in the ProB group. The length of villi and microvilli of probiotic-fed olive flounder was increased but was not significantly different from the control group. In immune-related gene expression analysis, TNF-α, IL-10, IL-6, and IL-1β were significantly increased in the ProB group, and IL-10 and IL-1β were significantly increased in the ProL group. In an in vivo challenge experiment with Streptococcus iniae, the survival rates of the ProB and ProL groups were 29.17% and 12.50%, respectively, when control mortality reached 100%. In this study, intestinal microbial diversity, digestive enzyme activity, intestinal immune-related gene expression, and the alteration of intestinal structure in olive flounder clearly differed according to the probiotic species. In this experimental condition, it was confirmed that the addition of Bacillus sp. SJ-10 induces a positive change compared to the Lactobacillus plantarum. The effect of probiotics supplementation according to the physiological processes was investigated using spores (SS) and vegetative cell supplementation (VCS) of the spore-forming strain Bacillus sp. SJ-10. At the end of the experiment, significant differences were found in terms of weight gain and specific growth rate between the probiotics supplement group and control groups. Respiratory burst, superoxide dismutase activity, and lysozyme activity, which are innate immune indicators, were observed with positive alterations in SS compared to controls. Intestinal microbial diversity analysis showed that the SS group had an increased richness estimate (Chao 1, ACE, and Jackknife) compared with the control group. Diversity estimates (Shannon and reverse Simpson) revealed that the VCS group was increased compared with the control group. Analyzing the similarity between groups through beta-diversity and microbial abundance the control and VCS groups were similar, and the SS group was different. IL-1β and TNF-α expression levels in the liver and spleen were increased in the SS group compared with the control group. In growth-related gene expression analysis, only growth hormone increased in the VCS group compared with the control group. In this study, supplementation of olive flounder with Bacillus of different physiological processes showed differences in growth, nonspecific immune response, gut microbial composition, and growth-and immune-related gene expression. The addition of vegetative cells increased the growth performance and growth hormone expression level, and the addition of spores affected the innate immune response, gut microbial composition, and immune-related gene expression in the olive flounder. These results show that probiotics supplementation affect the intestinal microbial composition and immune response in olive flounder, and may have different effects depending on the species and physiological conditions of probiotics.