Study On The Response And Mechanism Of Clostridium Autocthanogenum Protein Used In Litopenaeus Vannamei Feed

In recent years,due to the continuous decline in fish meal production,the contradiction between the growing demand for aquatic feed and the insufficient supply of fish meal has become increasingly acute.Developing new protein sources to replace fish meal has become an urgent issue for the healthy development of the aquatic industry.Litopenaeus vannamei is one of the three main species of shrimp culture,and its feed uses a large amount of fish meal.Therefore,the research and development of new protein sources to replace fish meal is of great significance for the sustainable development of shrimp aquaculture.As one of the new single cell protein sources,Clostridium autocthanogenum protein(CAP)has high application value,but it is currently less used in shrimp feed.This study investigated the response of L.vannamei to the application of CAP in feed under different feed formulations,including growth,body composition,immunity,digestion,intestinal microflora,and transcriptome levels.At the same time,non-targeted metabolomics and proteomics techniques were used to explore the possible mechanisms of action.The specific research results are as follows:1.Effects of replacing fish meal with CAP on growth,body composition,immunity,digestion,intestinal microflora,and transcriptome levels of L.vannameiUsing fish meal,soybean meal,peanut meal,etc.as the main protein source,and a basic diet with a fish meal dosage of 25%as a control,four equal nitrogen and equal fat experimental feeds were prepared using CAP at four different levels(0%,15%,30%,and 45%)instead of fish meal,and were fed to L.vannamei(initial weight 0.30± 0.02g)in four groups(CON,CAP15,CAP30,and CAP45).After an 8-week culture experiment,their growth,body composition,immunity,digestion Changes in intestinal flora and transcriptome levels.The results show that:①CAP substitution for fish meal reduced the weight gain rate(WGR)and specific growth rate(SGR)of L.vannamei(P<0.05),reaching significant levels at 30%and 45%substitution levels,but had no significant impact on the survival rate(SR)and feed conversion rate(FCR)of L.vannamei(P>0.05).②Compared with the control group,the crude fat content of L.vannamei in the CAP30 group was significantly increased.When the proportion of CAP replacing fish meal reached 45%,the content of methionine,proline,and essential fatty acids in the muscle of L.vannamei was significantly higher than that of the control group(P<0.05).③After Vibrio parahaemolyticus infection,the survival rate of shrimp in the CAP30 and CAP45 groups was significantly higher than that in the control group(P<0.05).Compared with the control group,CAP replacing fish meal significantly decreased the activity of acid phosphatase(ACP),alkaline phosphatase(AKP),glutamic oxaloacetic transaminase(GOT),glutamic pyruvic transaminase(GPT),and malondialdehyde(MDA)content in the hepatopancreas of L.vannamei,The activities of superoxide dismutase(SOD),phenol oxidase(PO),and lysozyme(LZM)were significantly increased(P<0.05),but there was no significant effect on the activity of catalase(CAT).Correspondingly,the expression levels of ACP and AKP genes in the hepatopancreas of L.vannamei in all CAP replacement fish meal groups were significantly lower than those in the control group,the expression levels of SOD and PO genes in the CAP30 group were significantly higher than those in the control group,and the expression levels of GOT and GPT genes in the CAP45 group were significantly lower than those in the control group,but there was no significant difference in LZM expression between the groups(P>0.05).④ Compared with the control group,the activities of amylase(AMS),trypsin,and lipase(Lipase)and gene expression in the intestine of L.vannamei in the CAP replacement fish meal group were significantly increased.⑤The results of intestinal microflora analysis showed that replacing fish meal with CAP significantly increased the intestinal microflora Sobs,Shannon,Chao1,ACE,Pielou,and Pd index of L.vannamei,with a significant difference between the CAP 15 group and the control group(P<0.05).In the replacement group,there were significant differences in the relative abundance of multiple dominant species at the phyla,family,and genus levels between the shrimp intestinal microflora and the control group.Tax4fu prediction showed a significant decrease in energy metabolism and amino acid metabolism in the CAP30 and CAP45 groups.⑥Transcriptome analysis showed that 841 differentially expressed genes(DEGs)were identified between the CAP15,CAP30,and CAP45 groups and the CON group(401 up,440 down),1917(1005 up,912 down),and 546(249 up,297 down),respectively.KEGG enrichment analysis showed that DEGs in the CAP 15 and CON groups were mainly concentrated in signal pathways such as oxidative phosphorylation,Parkinson’s disease,and thermogenesis,while DEGs in the CAP30 and CON groups were mainly concentrated in signal pathways such as oxidative phosphorylation,metabolism,Parkinson’s disease,and carbon metabolism,while DEGs in the CAP45 and CON groups were mainly concentrated in signal pathways such as glycolysis/glycosylation,pyruvate metabolism,TCA cycle,and oxidative phosphorylation.⑦The personal association analysis of the oxidative phosphorylation signaling pathway genes and the family level species selected by the intestinal flora indicator analysis showed that the expression of oxidative phosphorylation pathway related genes was positively correlated with the Rubitalaceae family,and negatively correlated with the Rubinisphaeraceae family.In summary,when the proportion of CAP replacing fish meal in the feed of L.vannamei does not exceed 15%,the growth performance of L.vannamei has no significant impact,but when the proportion of CAP replacing fish meal is too high,the growth performance of L.vannamei significantly decreases;Replacing fish meal with CAP in feed can significantly improve shrimp disease resistance,enhance nonspecific immunity,promote digestion,regulate intestinal microflora structure,and affect multiple signal pathways,including oxidative phosphorylation.2.Effects of CAP as the only protein source other than fish meal on growth,body composition,immunity,digestion,intestinal microflora,and transcriptome levels of L.vannameiIn the basic diet with a fish meal dosage of 15%,CAP was used as the only source of egg white except for fish meal,and five kinds of equal fat diets with protein levels of 32%,36%,40%,44%,and 48%were prepared.The shrimp were fed in five groups(CAP32,CAP36,CAP40,CAP44,and CAP48)(initial body weight 0.30 ±0.02g).After eight weeks of cultivation experiments,the changes in growth,body composition,immunity,digestion,intestinal flora,and transcriptome levels were analyzed.The results show that:①The SR of L.vannamei in each group was not high,and was significantly higher in the CAP48 group than in other groups.The FW,WGR,SGR,and FCR of L.vannamei all showed a trend of first increasing and then decreasing with the increase of protein level in the feed,and all reached their maximum values in the CAP40 group.Using SGR as a criterion and using a broken line model,it was found that under the condition that CAP was the only protein source except fish meal,the optimal level of protein addition in the feed of L.vannamei was 39.44%.②The increase in protein levels in the diet significantly increased the content of total shrimp crude fat,muscle threonine,tyrosine,and essential amino acids in L.vannamei,but had no significant impact on the total shrimp crude protein and ash,total amino acids in muscle,total non essential amino acids,and amino acid content in L.vannamei.③When the protein level in the diet was 44%,the anti Vibrio parahaemolyticus infection ability of L.vannamei was the strongest,significantly higher than that of the CAP32 group.With the increase of protein levels in the diet,the activities of GOT,GPT,and LZM in the hepatopancreas of L.vannamei showed a trend of first increasing and then decreasing,all of which were the highest in the CAP40 group,significantly higher than other groups,while the content of MDA showed a trend of first decreasing and then increasing,with the highest in the CAP40 group,significantly lower than the CAP32 and CAP40 groups.In the CAP32 group,the SOD activity in the hepatopancreas of L.vannamei was significantly lower than that of other groups(P<0.05).The gene expression results showed that the expression levels of GOT,GPT,and SOD genes in the hepatopancreas of L.vannamei in the CAP40 group were significantly higher than those in other groups.④When the protein level in the diet was 40%,the activity and gene expression of Amylase,Trypsin,and Lipase in the intestine of shrimp were significantly higher than those of other groups(P<0.05).⑤The results of intestinal flora analysis showed that the Sobs,ACE,Chao1,Shannon,Simpson,Pielou,and Pd indices of the intestinal flora of L.vannamei in the CAP48 group were significantly lower than those in the CAP32 and CAP40 groups(P<0.05).In the three groups,there were significant differences in the relative abundance of multiple dominant species of shrimp intestinal microflora at the level of phyla,family,and genus.Tax4fu prediction shows that the function of intestinal flora is mostly related to carbohydrate metabolism,amino acid metabolism,vitamin metabolism,energy metabolism,and nucleotide metabolism.⑥Transcriptome analysis showed that 2168 DEGs were identified between the CAP32 and CAP40 groups(1103 up-regulated,1065 down-regulated),277 DEGs were identified between the CAP40 and CAP48 groups(198 up-regulated,79 down-regulated),and 205 DEGs were identified between the CAP32 and CAP48 groups(138 up-regulated,67 down-regulated).The results of KEGG enrichment analysis showed that the DEGs between CAP40 and CAP32 groups were mainly enriched in signal pathways such as ribosomes,eukaryotic ribogenesis,and Alzheimer’s disease.The DEGs between CAP40 and CAP48 groups were mainly enriched in signal pathways such as amino sugar and nucleotide sugar metabolism,tryptophan metabolism,metabolic pathways,lysine biosynthesis,and glycosaminoglycan degradation.The DEGs between CAP32 and CAP48 groups were mainly enriched in folate synthesis On signal pathways such as the mutual conversion of pentose and glucuronic acid and the biosynthesis of amino acids.In summary,when using CAP as the sole protein source except fish meal,the protein level in the feed has a significant impact on the growth performance of L.vannamei.When the protein level in the feed is 39.44%,L.vannamei achieves the best growth performance.The protein level in the diet has a significant impact on the body composition,anti infective ability,non specific immunity,digestive ability,intestinal microflora structure,and transcriptional level of L.vannamei.3.Effects of adding CAP to feed on L.vannamei at high protein levelsBased on previous research,using fish meal as the sole source of egg white to prepare a feed with a protein level of 48%as a control,and using CAP as the sole source of egg white except fish meal to prepare an experimental feed with a protein level of 48%on the basis of a fish meal content of 15%.They were divided into FM48 and CAP48 groups and fed with vqnnamei(initial weight 0.30 ± 0.02g).After eight weeks of cultivation experiments,their changes in growth,body composition,nonspecific immunity,and digestive ability were analyzed,Using non-targeted metabolomics and proteomics techniques,the possible mechanisms of influence were explored.The results showed that at a protein level of 48%,the addition of CAP as the only protein source in the diet except fish meal had a significant negative impact on the growth performance of L.vannamei.After V.parahaemolyticus infection,the survival rate of L.vannamei in CAP48 group was higher than that in FM48 group,but there was no significant difference.Compared with the control group,the activities of ACP and SOD in the hepatopancreas of L.vannamei in the CAP48 group significantly increased,the content of MDA significantly decreased,and the activities of amylase and trypsin in the intestine significantly increased.The metabolomic analysis identified pyruvic acid as a significantly different metabolite in both positive and negative ion modes,mainly concentrated in signal pathways such as protein digestion and absorption,and amino acid biosynthesis and metabolism.Proteomic analysis showed that there were a large number of differentially expressed proteins(DEPs)related to carbon metabolism,energy metabolism,and amino acid functions.Correlation analysis of differential metabolites and differential proteins revealed significant changes in signal pathways such as protein digestion and absorption,TCA cycle,and amino acid biosynthesis.The key factor for changes in the signal pathways of protein digestion and absorption is the ribosome catalyzed synthesis of proteases.