| As an important mariculture species in China,Apostichopus japonicus shows significant growth differences among individuals during the cultivation process,and some individuals show growth retardation or even growth arrest,which seriously hinders the development of its cultivation industry.gut bacterial community and its metabolites play an important role in the growth and development of the host and are potential factors causing differences in the growth of A.japonicus.To dissect the mechanisms underlying the differential effects of gut microbiota on the growth of A.japonicus,in this study,we selected the strains of A.japonicus with significant growth differences in the same family,based on microbiome sequencing technology and transcriptomic and metabolomic analyses,to dissect the structural and functional features of the growth differential A.japonicus gut microbiota,identify the functional bacteria and metabolites that have significant effects on host growth and metabolism,and construct a co metabolic regulatory network between gut microbiota and host,Pinpointing how the gut bacterial community affects the metabolic status of the host through self-metabolism and co metabolism with the host,elucidating the correlation between the three aspects of gut microbiota host co metabolism growth function,the aim is to target the gut flora,optimize the structure of the gut microbiota,and explore the microecological regulation pathways to improve the cultivation yield and provide a certain theoretical basis.The main research content and conclusions are as follows:1.Observation of intestinal tissue section on growth difference of A.japonicusA.japonicus with no difference in body weight in the same family(P > 0.05)were cultured in the same environment.The changes in body weight were observed one year later,and the intestinal tract was dissected under aseptic conditions,and histological studies were conducted after HE staining.The results showed that there were significant differences in specific growth rate(SGR)of A.japonicus after one year of culture.The top 5% of SGR individuals were selected as the fast growing group(FG)and the bottom 5% as the slow growing group(SG).The results showed that the height of microvilli at the tip of intestinal columnar cells in FG group was significantly higher than that in SG group(P<0.05).The height of mucosal layer in FG group ranged from 82.5 to 152.5μm,and that in SG group ranged from 60 to72.5μm.The results showed that there were significant growth differences in A.japonicus under the same culture environment.Increasing the height of mucosal layer in the fast-growing A.japonicus mucosal layer could enhance its absorption capacity of nutrients,so it showed excellent growth performance.2.Study on structural and functional characteristics of gut bacterial community of A.japonicus with growth differences16S r RNA high-throughput sequencing technology and metagenomic sequencing technology were used to analyze the structural and functional characteristics of the gut bacterial community of A.japonicus with growth differences,and screen the specific flora closely related to the growth of A.japonicus.The results showed that FG group(FG)and SG group(SG)had similar gut bacterial community species composition.Bacteroides and Proteobacteria were dominant bacteria in both groups.The first dominant bacterium of group FG was Lutibacter,and the second dominant bacteria were Aquibacter,Sedimentitalea and Bacillus.The dominant bacteria in SG group belong to Sedimentitalea,Bacteroides and Lutibacter.The two groups of bacteria with the biggest difference mainly belong to Bacteroidetes and Epsilonbacteraeota,and both of them are different groups of FG group.At genus level,the diversity of bacteria in group FG was mainly Lutibacter and Bacillus,and in group SG was Ambiguous＿taxa and Pseudomonas.It is worth noting that Lutibacter and Bacillus of FG group in this study have been confirmed to be related to polysaccharide metabolism and lipid metabolism.The results of KEGG metabolic pathway enrichment analysis showed that the gut bacterial community of the two groups obtained 5453 ko,which could be mapped to 307 tertiary metabolic pathways,of which 28 were different tertiary metabolic pathways(LDA > 2)and could be aggregated into 24 secondary metabolic pathways and 5 primary metabolic pathways.Among them,the tertiary metabolic pathways of FG group were mainly starch and sucrose metabolism,sphingolipid metabolism and propionic acid metabolism.The tertiary metabolic pathways in SG group are mainly ABC transport,two-component system and quorum sensing.It can be concluded as a secondary metabolic pathway.The secondary metabolic pathway of FG group is carbohydrate metabolism,polysaccharide biosynthesis and metabolism,lipid metabolism,etc.The different secondary metabolic pathways in SG group were membrane transport,signal transduction and cell motility.The types of primary metabolic pathways enriched in FG and SG groups were the same,but the relative abundance was significantly different.The primary metabolic pathway of FG group was mainly metabolism,while the primary metabolic pathway of SG group was environmental information acquisition.In this study,the gut bacterial community function of Group FG was mainly concentrated in the pathways related to fat,carbohydrate and sugar metabolism.3.Analysis of gene regulation of growth difference of A.japonicusRNA-seq sequencing technology was used to conduct transcriptome sequencing on the intestinal tissues of A.japonicus with growth differences,construct the gene expression profile of A.japonicus with growth differences,and screen the differentially expressed genes and their differential metabolic pathways.In this study,2610 differential genes were obtained by comparison,including 1368 up-regulated genes and 1242 down-regulated genes.KEGG cluster analysis of differentially expressed genes showed that 2610 differentially expressed genes were found in 33 secondary metabolic pathways.Differentially expressed genes were mainly concentrated in lipid metabolism,amino acid metabolism,carbohydrate metabolism,signal transduction and immune system.In the metabolic signaling pathway,the significantly up-regulated genes of lipid metabolism include pyruvate kinase(PK),cytochrome P450 enzyme(CYP2J3),glycerin triphosphate dehydrogenase(PGP),etc.Carbohydrate metabolism includes down-regulated phosphoenolpyruvate carboxykinase 1(PCK1)and phosphoenolpyruvate carboxykinase 2(PCK2),and upregulated hexokinase(HK).Amino acid metabolism significantly up-regulated genes include homocysteine S-methyltransferase 1(bhmt)and ornithine decarboxylase 1(ODC1).The signaling pathway contains significantly up-regulated genes insulin-like growth factor 1(IGF-1)and fibroblast growth factor 4(FGFR4).The results showed that PK,HK,IGF-1 and FGFR4 had positive regulatory effects on growth,glucose metabolism and lipid metabolism of A.japonicus,and could be screened as the key genes for growth differences.4.Metabolic phenotype analysis of growth difference of A.japonicusBased on non-targeted metabolomics,the metabolic pathways of intestinal metabolites and enrichment in growth differential of A.japonicus were analyzed,and the metabolic network of growth differential was constructed to explore the intestinal metabolic phenotypic characteristics of growth differential of A.japonicus.A total of155 differential metabolites were screened,among which 135 metabolites were upregulated and 20 were down-regulated.Metabolites such as arachidonic acid(AA),oleic acid,diglycerol(DG),dihydroxyacetone phosphate,fructose 6-phosphate and dextrin were significantly up-regulated.P-hydroxyphenylhexanoic acid,2-phenylacetamide and 5-phenyl-1,3-oxazinan-2,4-dione were significantly upregulated in SG group.Based on the annotation of KEGG Pathway database,a total of 17 differential metabolic pathways were selected.Among them,8 differential metabolites were enriched in glycerophospholipid metabolism,7 in arachidonic acid metabolism,and 5 in sphingolipid metabolism.In this study,the intestinal differential metabolites in group FG mainly focus on growth function,and the differential expression of metabolites makes group FG exhibit better growth performance,which can lay a theoretical foundation for the study on the correlation between gut bacterial community and host co-metabolism and growth function of A.japonicus.5.Study on intestinal microbiota-host co-metabolism-growth function of A.japonicus with growth differencesCombined with multi-omics data,the correlation between gut bacterial community,differential genes and metabolites was calculated,and functional bacteria and expressed genes that significantly affected the growth and metabolism of the host were identified,and the correlation between gut bacterial community,host cometabolism and growth function was explained.The results showed that Lutibacter and Bacillus were significantly correlated with PK,HK,PCK1 and IGF-1,the key regulatory genes of host growth.Differential metabolites(fructose 6-phosphate,dihydroxyacetone phosphate,arachidonic acid,oleic acid and other unsaturated fatty acids)were significantly correlated with differential genes(PCK1,PK,IGF-1,HK,etc.).Differential genes and differential metabolites are involved in related metabolic pathways of metabolism and cellular processes,and the metabolism-related pathways are mainly concentrated in lipid metabolism and carbohydrate metabolism.There were 8 metabolites and 100 genes involved in phosphosphingolipid metabolism.Seven metabolites and 76 genes were involved in arachidonic acid metabolism.Five metabolites and 45 genes were involved in the biosynthesis of unsaturated fatty acids.Studies have shown that the key candidate genes related to the intestinal differential flora of Group FG are related to metabolic signal transduction pathways,which can mediate PI3K-Akt signaling pathway,glucose metabolism and lipid metabolism pathway by influencing the expression of key genes,thus affecting the metabolic phenotype of the A.japonicus,and ultimately resulting in differences in growth performance.6.Living transplantation of intestinal fecal flora of A.japonicusIn order to establish the causal relationship between gut bacterial community and body weight of A.japonicus,the feasibility of living transplantation of intestinal fecal flora of A.japonicus was innovatively discussed.600 young A.japonicus with the body weight of 5g were selected and divided into 2 groups on average.Larvae of A.japonicus from group FG group were collected and fed in the diet regularly,and were set as enterofecal flora transplantation group(BMG).Larvae of A.japonicus without feces in group FG were fed as control group(MG).After feeding under the same conditions for 5 weeks,there was no significant difference in body weight between A.japonicus(MG)and A.japonicus(MG)groups.16 S r RNA high-throughput sequencing results showed that the two groups of dominant bacteria belonged to Proteobacteria,Firmicutes,Bacteroidota and Actinobacteriota.The sum of relative abundance accounts for more than 95%.The dominant bacteria genera in the two groups are similar,with Raistonia,Escherichia-Shigella and Lactobacillus.The different flora between the two groups mainly belonged to Proteobacteria,which was represented by Ralstonia.According to KEGG database annotation,there was no significant difference in the tertiary metabolic pathway between the two groups.In this study,enterofecal microflora transplantation experimental group did not show high growth performance of A.japonicus,and no differential microflora was detected in the intestinal tract of group FG.The reason for the failure of enterofecal microflora transplantation may be that the key growth promoting microflora of Group FG has been inactivated after in vitro,or the concentration of the bacteria has not reached the content that can be successfully colonized in the new receptor.The means and approaches of enterofecal transplantation of A.japonicus need to be further explored. |
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