| The farming of sea cucumber Apostichopus japonicus was seriously limited by the frequent occurrence of diseases and the abuse of antibiotics.As an important organ of sea cucumber,intestine is closely related to the composition of intestinal microflora and the growth,immunity and metabolism of A.japonicus.Lactic acid bacteria have been widely used in aquaculture,and endogenous strains which can easily occupy the niche in the intestines of host.It is important to explore the action mechanism of endogenous lactic acid bacteria on the intestine of A.japonicus for the application of lactic acid bacteria in the healthy culturing of A.japonicus.In this study,an endogenous Lactobacillus rhamnosus M2-4 isolated from the intestines of healthy A.japonicus was used as supplements for forage.The intestinal microflora,metabolism and related immune genes were determined and analyzed by16S r DNA high-throughput sequencing,non-targeted metabonomics(LC-MS)and RT-PCR technology,which tried to explore the relationship between microflora and metabolism and provide a basis for further analysis of the mechanism of probiotics.The experiment included four groups:T0,T5,T7 and T9(M2-4 was respectively supplemented at 0,10~5,10~7and 10~9CFU/g)and sea cucumbers were cultured for 30days.The results were as follows.(1)Dietary M2-4 did not significantly affect theα-diversity of intestinal and fecal microflora,but it changed the composition of the intestinal and fecal microbiota.The relative abundance of potential probiotics,especially Lactobacillus,in the intestinal microflora was increased with the increasing dietary M2-4,while the potential pathogenic bacteria,especially Vibrio,was decreased with the increasing dietary M2-4.The abundance of Lactobacillus in the fecal microflora was the highest in group T9,while the change of Vibrio in four groups was not obvious.(2)Dietary M2-4 had significant effects on intestinal metabolites and metabolic pathways of A.japonicus.Most differential metabolites were lipid metabolites.The m TOR(T5 vs T0;T7 vs T0)and PPAR signaling pathways(T9 vs T0)were highly enriched.The Glutathione metabolism(T5 vs T0)and Linoleic acid metabolism(T7 vs T0;T9 vs T0)were the most influenced pathways,respectively.And the concentrated differential metabolites Glutathione andγ-Glutamylcysteine(T5)and linoleic acid(T7,T9)were all significantly down-regulated.(3)Procrustes analysis showed that there was a close correlation between intestinal microflora and metabolites.The analysis of Spearman’s correlation showed that differential metabolites significantly correlated with family Flavobacteriaceae.Glutathione andγ-Glutamylcysteine were significantly correlated with Flavicella,Flavobacterium and Sphingobacterium.Linoleic acid was significantly correlated with Flavobacterium,Sphingobacterium,norank Muribaculaceae and Bacteroides.(4)The expression of GPX gene was increased significantly with the increase of dietary M2-4 and reached the peak in group T9(P<0.05).The expression of Lys gene was increased at first and then decreased with the increase of dietary M2-4 and reached the peak in group T5 and was significantly higher than that in control group(P<0.05).In conclusion,dietary M2-4 could significantly improve the structure of intestinal microflora and change the intestinal lipid metabolites of A.japonicus,and the intestinal microflora was significantly correlated with metabolites.A lot of pathways were effected greatly especially pathways related with lipid metabolism,in which m TOR and PPAR signaling pathways were the most enriched,and Glutathione and Linoleic acid metabolism were the most influenced.M2-4 had a certain effect for related intestinal immune genes of A.japonicus.The results of this study provide a reference to furtherly reveal the probiotic mechanism of lactic acid bacteria on the intestinal health of A.japonicus. |
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