Study On The Influence Mechanisms Of Clostridium Autoethanogenum Protein Fubstituting Fish Meal On The Growth And Immunity Of Pacific White Shrimp(Litopenaeus Vannamei)

The application of Clostridium autoethanogenum protein as a new protein source in aquatic feed has emerged in the past years,but few reports were found in shrimps.In this study,Pacific white shrimp(Litopenaeus vannamei)was used as the experimental shrimp,and the effects of Clostridium autoethanogenum protein on the growth and immunity were explored through growth performance,hemolymph immune enzyme activity,intestinal structure and transcriptomics.In addition,amino acids and Eucommia ulmoides leaf extracts were added in diets to improve the growth and immunity of shrimp.The findings will provide valuable data for the application of Clostridium autoethanogenum protein in shrimp feed and for the low fish meal shrimp diet.1.Dietary Effects of Clostridium Autoethanogenum Protein Substituting Fish Meal on Growth,Intestinal Histology and Immunity of Pacific White Shrimp(Litopenaeus Vannamei)Based on Transcriptome AnalysisThe study investigated the dietary effects of Clostridium autoethanogenum protein(CAP)substituting fish meal on the growth,intestinal histology,serum immune indexes and transcriptome of Pacific white shrimp,Litopenaeus vannamei.Four isonitrogenous and isolipidic diets were designed as the control diet(CON)containing 560 g/kg fish meal,and three fish meal-substituted diets in which 30%(CAP-30),45%(CAP-45)and70%(CAP-70)fish meal were replaced with CAP,respectively.The four diets were fed to shrimp with initial body weight of 2.8g for 8 weeks.The results showed that the weight gain,feed intake,survival and intestinal villus height in CAP-45 and CAP-70 groups were lower than those of the control and CAP-30 groups(P<0.05).In addition,the serum aspartate aminotransferase and phenol oxidase activities in all fish mealsubstituted groups,and the lysozyme activity in CAP-45 and CAP-70 groups were increased,while the total protein content in CAP-45 and CAP-70 groups was decreased when compared with the control(P<0.05).Transcriptome profiling of hepatopancreas indicated that high inclusion of CAP negatively affected the protein synthesis and the utilization of nutrients by regulating pancreas secretion,protein digestion and absorption,ribosome pathways,and disturbed the immune system and metabolic processes by phagosomes and lysosomes pathways,thereby affecting the growth performance and immune function of shrimp.In conclusion,CAP could substitute 30%fish meal in a diet containing 560 g/kg fish meal without adverse effects on the growth,intestinal histology and immunity of Pacific white shrimp.Excessive CAP could affect the growth and immunity of shrimp by regulating signaling pathways such as pancreatic secretion,protein digestion and absorption,phagosome,lysosome and so on.2.Dietary Effects of Clostridium Autoethanogenum Protein Substituting Fish Meal on Growth,Intestinal Histology,Hemolymph Immune Index and Hepatopancreatic Transcriptomicsof Pacific White Shrimp(Litopenaeus Vannamei)Based on Amino Acid BalanceOn the basis of Experiment 1,tryptophan,arginine,histidine,phenylalanine,taurine and cholesterol were supplemented in the CAP-30,CAP-45 and CAP-70 diets to reach the same level as the control group(CON).Pacific white shrimp with an initial body weight of 2.8g were fed the four diets for 8 weeks.The results showed that the CAPA-45 and CAPA-70 groups had significantly lower weight gain,specific growth rate,feed intake,survival,villus height,and villus number than the control and CAPA-30 groups(P<0.05).The activities of AST and PO in CAP-30,CAP-45 and CAP-70 groups were significantly higher than those in the control group(P<0.05),while the LZM activity of CAPA-70 group was significantly higher than that of the other experimental groups(P<0.05).The hepatopancreatic transcriptomics analysisshowed that the pancreatic secretion and protein digestion and absorption pathways were influenced in the high-proportion substitution group,thereby affecting the nutrients utilization andgrowth ofshrimp.In addition,the immune-related genes such as lysozyme(LZM),C-type lectins(CLEC17A,CLEC4 F,CLEC4E),γ-interferon-induced lysosomal sulfhydryl reductase(GILT)and hemocyanin-C(hemocyanin-C)were significantly down-regulated in CAPA-70 group.In conclusion,CAP could substitute 30% fish meal in a diet containing 560 g/kg fish meal without adverse effects on the growth,intestinal histology and immunity of Pacific white shrimp,even amino acids were supplemented.Excessive CAP still affected the growth of shrimp through pancreatic secretion,protein digestion and absorption or other pathways and affected the innate immune response by inhibiting the expression of immune genes such as lysozyme,C-type lectin and so on.3.Dietary Effects of Clostridium Autoethanogenum Protein Replacing Fish Meal and Eucommia Ulmoides Leaf Extracts Addition in Practical Feed on Growth and Immunity of Pacific White Shrimp(Litopenaeus Vannamei)This study investigated the dietary effects of Clostridium autoethanogenum protein replacing fish meal and Eucommia ulmoides leaf extracts addition in practical feed on growth and immunity of Pacific white shrimp.The basal diet was designed to contain200 g/kg FM(control,FM-20),and then,FM was reduced to 100 g/kg(FM-10)and 0g/kg(FM-0)by the CAP inclusion of 100 g/kg and 200 g/kg,respectively.Meanwhile,the FM-10 diet was added with 1g/kg Eucommia ulmoides leaf extracts(EL).Then,the four diets were fed to shrimp with initial body weight of 1.5g for 8 weeks.The results showed that there were no significant differences in weight gain,feed conversion ratio and specific growth rate among the four groups(P>0.05),but the villus width of EL group was significantly higher than that of the other groups(P<0.05).The total protein(TP)content of FM-20 group and the alkaline phosphatase(AKP)activity of the EL group were significantly higher than those of the other groups(P<0.05).In the genes expression in hepatopancreas,the chymotrypsin(CTRL)expression in FM-10,FM-0and EL groups,the activated protein kinase C receptor 1(RACK1)expression in FM-0and EL groups,and the hemocyanin(HC-C)expression in FM-10 group were significantly higher than those in FM-20 group(P<0.05).In addition,EL group showed significantly higher expression of LZM than the other groups(P<0.05).After the feeding trial,Vibrio parahemolyticus was injected into the shrimp,and the cumulative mortality of shrimp in FM-10 group was significantly higher than FM-20 and EL groups on the fourth day(P<0.05).In conclusion,in a basal diet containing 200 g/kg FM,CAP could completely replace FM without negative effects on the growth of shrimp,but negatively affect the the mortality of shrimp infected with Vibrio Parahemolyticus.Moreover,the supplementation of 1 g/kg Eucommia ulmoides leaf extracts could increase the villus width of shrimp and reduce the mortality of shrimp infected with Vibrio Parahemolyticus.