Intestinal Microbiota Diversity Analysis Of Trachinotus Ovatus And Nile Tilapia And The Effected Of Intestinal Microbiota Diversity By Woody Forages

Abstract:With the development of aquaculture and the tensions of feed raw,contradictions of aquatic animal feed supply and demand are sharpending.It is urgent to develop new feed raw with high nutritional value to alleviate the contradictions between supply and demand of aquatic feed.The woody plants with high nutritional value are ideal new feed raw,while anti-nutritive factors severely limit the popularization and application of woody plant.The intestinal microbiota play key important role in nutritional metabolism.There is less study about the influence of woody forages on intestinal microbiota diversity and there is no research about the intestinal microbiota diversity of Trachinotus ovatus.Consequencely,the present study first researched the intestinal microbiota diversity of Trachinotus ovatus with high-throughput illumina sequencing and compared the intestinal microbiota diversity of Trachinotus ovatus with the intestinal microbiota diversity of nile tilapia（Oreochromis niloticus）.Furthermore,the researh analyzed the intestial microbiota diversity of the fish fed with new woody forages,isolated the degradation tannin microbiology and determined the fermentation process of Moringa oleifera Lam..The mian research content and results were as followed:1.Intestinal bacteria diversity of Trachinotus ovatus and nile tilapiaThe dominate phylum of the Trachinotus ovatus and nile tilpalia intestinal bacteria were Fusobacteria,Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria with the high-throughput illumina sequencing.Moreover,the Proteobacteria and Firmicutes abundance of Trachinotus ovatus significantly higher than those of nile tipalia（P<0.05）.The Fusobacteria abundance of Trachinotus ovatus were significantly lower than those of nile tipalia（P<0.05）.At genera level,the Cetobacterium abundance of Trachinotus ovatus were significantly lower than those of nile tipalia（P<0.05）.The Lactobacillus abundance of Trachinotus ovatus were significantly higher than those of nile tipalia（P<0.05）.Moreover,Phylogenetic investigation of communities by reconstruction of unobserved states（PICRUSt）showed that the main metabolism pathways of Trachinotus ovatus and nile tilpalia intestinal microbiota were amino acid metabolism,carbon metabolism,energy metabolism,nucleic acid metabolism.Meanwhile,the abundance of arginine and proline metabolism in Trachinotus ovatus intestinal bacteria were significantly higher than those in nile tilapia intestinal bacteria（P<0.05）.The abundance of carbon metabolism,glycolysis/gluconeogenesis and pyruvate metabolism in Trachinotus ovatus intestinal bacteria were significantly lower than those in nile tilapia intestinal bacteria（P<0.05）.2.The influence of woody forages on Trachinotus ovatus intestinal bacteriaThe diets of Moringa oleifera Lam.（MOL）,Broussonetia papyrifera（BP）,Neolamarckia cadamba（NC）and Folium Mori（FM）formulated with 70%of reference（Ref）diet and30%of the four woody plants leaves were fed to Trachinotus ovatus with initial body weight of 34.4±0.5 g for 56 d,respectively.The foregut amylase and midgut amylase of Trachinotus ovatus fed with woody forages were significantly higher than those of Trachinotus ovatus fed with control diet（P<0.05）.Bacteria diversity of Trachinotus ovatus intestine samples were analyzed using illumina sequencing and result indicated that Proteobacteria in BP were significantly higher than those in NC（P<0.05）.Firmicutes in NC were significantly higher than those in BP and FM（P<0.05）.At genera level,Lactobacillus in NC were significantly higher than those in BP,MOL and FM groups（P<0.05）.The principal component and heat map analysis showed that the intestinal microbiota community of Trachinotus ovatus fed with woody forages and reference diet presented higher similarity.PICRUSt showed that the abundance of ABC transporters in BP were significantly higher than those in Ref and FM（P<0.05）.The abundance of glycine,serine and threonine metabolism in FM significantly were significantly higher than those in Ref,BP,NC and MOL（P<0.05）.3.The influence of woody forages on nile tilapia intestinal bacteriaIn this study,Moringa oleifera Lam.（MOL）diet,Broussonetia papyrifera（BP）diet,Neolamarckia cadamba（NC）diet and Folium Mori（FM）diet formulated with 70%of reference（Ref）diet and 30%of the four woody plants leaves,respectively.And each diet was fed to nile tilapia with initial body weight of 100.0±10.0 g.After 45 d,microbiota diversity of nile tilapia intestine samples were analyzed using high-throughput illumina sequencing.The results showed that Fusobacteria,Proteobacteria,Firmicutes and Bacteroidetes presented no significant difference in FM,MOL,NC,BP and Ref groups（P>0.05）.Cetobacterium presented no significant differences among FM,MOL,NC,BP and Ref groups（P>0.05）.The principal component and heat map analysis indicated that the intestine content microbiota community of nile tilapia were higher similarity.PICRUSt showed that the intestinal content microbiota dominate metabolism pathways were biosynthesis of amino acids and carbon metabolism and without significant difference（P>0.05）.4.The intestinal microbiota difference analysis of fishes fed with woody foragesThe abundance of Bacteroidetes in the intestine of Trachinotus ovatus fed with MOL and BP were significantly lower than those in the intestine of nile tilapia fed with MOL and BP（P<0.05）.The abundance of Fusobacteria in the intestine of nile tilapia fed with MOL and BP were significantly higher than those in the intestine of Trachinotus ovatus fed with MOL and BP（P<0.05）.Meanwhile,the abundance of Cetobacterium in the intestine of nile tilapia fed with MOL,BP,NC and FM were significantly higher than those in the intestine of Trachinotus ovatus fed with MOL,BP,NC and FM（P<0.05）.5.Screening the degradation tannin microbiology.The study successfully screening two degradation tannin microbiology from nile tilapia intestinal content and the soil of Moringa oleifera Lam.tree roots,respectively.And the degradation tannin microbiogly were Aspergillus niger and Aspergillus sydowii with the molecular biological identification and morphology observation.The Aspergillus niger and Aspergillus sydowii could significantly degradate tannin of Moringa oleifera Lam.（P<0.05）.Moreover,Aspergillus sydowii presented better ability to degradate tannin of Moringa oleifera Lam.than Aspergillus niger.6.Optimization of Moringa oleifera Lam.fermentation processThe ferment times,temperature and inoculation quantity were optimized with L₉（3×3）orthogonal design.The results showed that the optimal combination of Aspergillus sydowii fermentation Moringa oleifera Lam.were A1B3C3（the temperature was 30℃,the inoculum quantity was 5%,the fermentation times were 9 d）.The tannin content in fermentative Moringa oleifera Lam.were significantly lower than those in Moringa oleifera Lam.（P<0.05）.The crude protein in fermentative Moringa oleifera Lam.were significantly higher than those in Moringa oleifera Lam.（P<0.05）.The degradation rate of tannin in fermentative Moringa oleifera Lam.were 67.1%and the crude protein in fermentative Moringa oleifera Lam.increased 17.6%.Moreover,the aspartic acid,serine and threonine in fermentation Moringa oleifera Lam.were significantly higher than those in Moringa oleifera Lam.（P<0.05）.The ferment times,temperature,inoculation quantity and mixed bacteria proportion were optimized with L₉（3×3）orthogonal design.The results indicated that the optimal combination of Aspergillus sydowii and Bacillus subtilis fermentation Moringa oleifera Lam.were A1B3C2D3（the temperature was 30℃,the inoculum quantity was 5%,the fermentation times were 9 d,the proportion of Aspergillus sydowii and Bacillus subtilis were 2:1）.The tannin content in fermentative Moringa oleifera Lam.were significantly lower than those in Moringa oleifera Lam.（P<0.05）.The crude protein in fermentative Moringa oleifera Lam.were significantly higher than those in Moringa oleifera Lam.（P<0.05）.The degradation rate of tannin in fermentative Moringa oleifera Lam.were46.2%and the crude protein in fermentative Moringa oleifera Lam.increased 24.1%.Moreover,the valine in fermentative Moringa oleifera Lam.were significantly higher than those in Moringa oleifera Lam.（P<0.05）.Based on the tannin,crude protein and amino acid content,the optimal combination were the temperature was 30℃,the inoculum quantity was 5%and the fermentation times were 9 d with Aspergillus sydowii at present study.