Effects Of Different Diets On Intestinal Microflora Succession And Enzyme Activities Of Haliotis Discus Hannai

As an important marine economic shellfish in China,Haliotis discus hannai will undergo diet conversion process in different stages of growth and development,mainly including feeding benthic diatoms to artificial compound feed after early juvenile abalone stripping,and mixing various diets for culture in late juvenile abalone stage,such as artificial compound feed,fresh Laminaria japonica and salted L.japonica.Diet is an important environmental factor affecting the healthy growth and development of H.discus hannai.Diet replacement may affect enzyme activity and intestinal microflora community structure.In this study,samples were collected at the key time points after diet replacement,and the dynamic changes of enzyme activity and intestinal microflora community of H.discus hannai were explored during the weaning period and under different diet conditions,which provides theoretical basis for healthy culture of H.discus hannai.The main research results are as follows:1.Dynamic changes of enzyme activity and intestinal microflora of early juvenile abalone H.discus hannai during weaning periodThe changes of digestive and antioxidant enzyme activities,the structure and diversity of intestinal microflora of H.discus hannai before weaning period(T0),10 days(T10),30 days(T30)and 60 days(T60)after weaning period were analyzed by enzyme activity assay and 16S rDNA high-throughput sequencing.The results showed that the activity of α-amylase(AMS)at T0 was significantly higher than that at T10,T30 and T60;the activity of Lipase(LPS)showed an upward trend,but there was no significant difference among the groups(P>0.05);trypsin activity decreased at first and then increased,which was the lowest at T10 and the highest at T60.The activities of superoxide dismutase(SOD)and catalase(CAT)decreased at first and then increased.The activity of SOD at T0 was significantly higher than that in T10,T30 and T60(P<0.05),while the activity of CAT was the lowest at T10.The results of microbial community analysis showed that Shannon and Chao 1 indexes of intestinal microflora at T0 were the highest,while that at T10 was the lowest.y-proteobacteria,Bacteroides and α-proteobacteria were the dominant strains in the weaning period.At the genera level,the dominant genera in each group during the weaning period were different,and showed specificity on the time scale.Before the weaning period,the dominant bacteria were Pseudomonas,Lactobacillus,Bacteroides and Ahrensia,while Vibrio and Polaribacter were the main bacteria during the weaning period.In addition,Formosa and Psychrilyobacter were the dominant strains of T30 and T60.LEfSe analysis showed that the main differences of intestinal microflora of H.discus hannai during the weaning period were concentrated in T0 and T60.The results of PICRUST2 function prediction showed that the function expression of intestinal microflora of H.discus hannai changed with the weaning period.The main function of intestinal microflora of T0 was related to glycolysis,nucleotide and nucleic acid synthesis and lipid synthesis,while that of T30 and T60 was mainly related to carbohydrate synthesis and amino acid synthesis.2.Effects of different diets on growth,enzyme activity and intestinal microflora of late juvenile abalone H.discus hannaiIn this study,hungry(HG)H.discus hannai was used as the control group,and set other three groups fed with fresh L.japonica(FG),salted L.japonica(SG)and artificial compound diet(AG)for 60 days,respectively.The effects of different diet conditions on the growth and development,the activities of digestive enzymes and antioxidant enzymes,and the structural characteristics of intestinal microflora in H.discus hannai were investigated.The results showed that the shell length,shell width,wet weight and survival rate of H.discus hannai in artificial diet group were the largest,but there was no significant difference between them and fresh L.japonica group and salted L.japonica group(P>0.05).The activities of three digestive enzymes(AMS,LPS and trypsin)and two antioxidant enzymes(SOD and CAT)were the highest in the artificial diet group,among which the activities of AMS and trypsin were increasing continuously,and the activities of LPS were significantly higher than those in the other three groups from 40 days(P<0.05).The activities of digestive enzymes and antioxidant enzymes of late juvenile abalone under long-term hunger stress were lower than those in the other three groups,and the activity of CAT enzyme decreased significantly compared with that at the beginning of the experiment(P<0.05).The results of Alpha diversity of intestinal microflora in late juvenile abalone H.discus hannai showed that the diversity and abundance of intestinal microflora in hungry group were higher than those in other three groups,while the abundance of artificial diet group was inferior to that in hungry group,but the diversity was the lowest.NMDS analysis showed that the samples distance between FG and SG were close,and the gap between them and AG was the largest.y-Proteobacteria,Fusobacteriia and Bacilli were the common dominant bacteria under the four diets,while α-Proteobacteria and Verrucomicrobiae were the unique dominant bacteria in hungry group.At the genera level,the dominant bacteria in the intestinal of H.discus hannai were mainly Psychilyobacter,Mycoplasma and Vibrio,but the relative abundance in different feed groups was different in different sampling periods,among which the growth trend of Psychilyobacter and Vibrio in artificial diet group was the most obvious,and the relative abundance was the highest.In conclusion,the high mortality rate of H.discus hannai in the early stage of weaning period is related to the decrease of enzyme activity and intestinal microflora diversity.The dominant intestinal microflora after weaning is mostly bacteria that can degrade compound feed components,which is of great significance to help H.discus hannai successfully complete weaning period.In the stage of late juvenile H.discus hannai,feeding artificial diet is more conducive to improving the activities of digestive enzymes and antioxidant enzymes,promoting the proliferation of intestinal beneficial bacteria and improving the growth efficiency.