Mechanism Analysis Of Chlorella Sorokiniana Meal And Tenebrio Molitor Meal Replacing Fish Meal On Growth And Immunity Of Litopenaeus Vannamei

The application of Chlorella Sorokiniana and Tenebrio molitor as new protein sources in aquafeeds has been a hot topic in recent years,but most studies in shrimps have focused on the biochemical level,and few studies have investigated the mechanism of their substitution for fishmeal on shrimp growth and immunity at the molecular level.In this study,based on the previous studies on the growth performance of Litopenaeus vannamei by replacing fishmeal with two protein sources,transcriptomic analysis was performed by RNA-seq sequencing technology to obtain the differential gene expression of the two protein sources with different proportions of fishmeal replacement.Through GO function enrichment and KEGG enrichment analysis,the related information was integrated,and the mechanism of Chlorella Sorokiniana and Tenebrio molitor affecting the growth and immunity of Litopenaeus vannamei was discussed,which provided theoretical basis for the rational utilization of Chlorella Sorokiniana and Tenebrio molitor in shrimp feed.Expt.1 Affecting mechanism of Chlorella Sorokiniana meal replacing fish meal on growth and immunity of Litopenaeus vannamei based on transcriptome analysisThe purpose of this study was to analyze the affecting mechanism of Chlorella Sorokiniana meal replacing fish meal on the growth performance and immune function of Pacific white shrimp,Litopenaeus vannamei by transcriptomics.A control diet was formulated to contain 560 g/kg fish meal,then chlorella was used to replace 0%(C0),20%(C20),40%(C40),60%(C60),80%(C80)and 100%(C100)of dietary fish meal to form six iso-nitrogenous and iso-lipidic diets.The six diets were used to feed white shrimp(1.37±0.02g)for 8 weeks.Compared to the control group,the final weight,specific growth rate and feed efficiency of C20 group were significantly improved(P<0.05).There was no significant difference in growth performance between C40 group and C0 group(P>0.05).When the replacement level of fish meal by chlorella was ≥ 60 %,the average final weight,specific growth rate and feed efficiency of shrimp decreased significantly(P<0.05).In the analysis of hepatopancreatic transcriptome,the amount of differentially expressed genes(DEGs)in C20,C40,C60,C80 and C100 groups were 322(176 up-regulated and 146 down-regulated),482(246 up-regulated and 236 downregulated),804(487 up-regulated and 317 down-regulated),1080(684 up-regulated and396 down-regulated)and 1262(827 up-regulated and 435 down-regulated),respectively,when compared to the control group(C0).GO Term and KEGG pathway enrichment analysis of DEGs showed that DEGs between the C20 and C0 groups were significantly enriched in chitin binding and inositol metabolism processes.The enrichment was also observed in multiple immune-related signaling pathways such as C-type lectin receptor signaling pathway,complement and coagulation cascades,b-cell receptor signaling pathway,and most of these genes were up-regulated in the C20 group.Compared with the C0 group,in the C40,C60,C80 and C100 groups,the DEGs were significantly enriched in lysosomal,protein digestion and absorption signaling pathways.In addition,in the C80 and C100 groups,the DEGs were also significantly enriched in lipid catabolism process.The results indicated that the high replacement of fish meal with chlorella affected the absorption and utilization of nutrients by regulating protein digestion and absorption signaling pathways,and aggravated the body ’s lipid catabolism,thereby affecting the growth of shrimp.The high replacement of fish meal may affect the homeostasis of shrimp cells and reduce immunity through the lysosomal signaling pathway.In summary,in a diet with fish meal content of 560 g/kg,the replacement of 20%of dietary fish meal with Chlorella Sorokiniana significantly improved the growth performance of L.vannamei,which might be realized through activating the C-type lectin receptor signaling pathway,and enhancing the immune function of the body.The high replacement of fish meal(≥ 60 %)reduced the growth performance and immunity by affecting the lysosomal signaling pathway.Expt.2 Effects of Supplementing Amino Acids and Functional Additives on Growth,Immunity and Related Gene Expression of Litopenaeus vannamei in Chlorella High Substitution GroupIn order to avoid the negative effects of chlorella on the organism of Litopenaeus vannamei,a nutritional balance scheme after high proportion substitution was sought.On the basis of experiment 1,the group was selected as the positive control group(PC group)with the most suitable replacement ratio of chlorella meal at 40% and the group with significantly lower growth at 80% as the negative control group,and supplemented with essential amino acids(histidine,isoleucine,lysine and methionine)and cholesterol to the level of the positive control group(AC group)on the basis of the negative control group.On the basis of AC group,12.0 g/kg of micro-encapsulated hydroxyproline and 12.0 g/kg of micro-encapsulated creatine(AC-H,AC-C and AC-H+C groups)were added respectively and jointly to form six iso-nitrogenous and iso-lipidic diets.The six diets were used to feed white shrimp(initial body weight 1.20 ± 0.02 g)for 8 weeks.The preliminary results showed that the growth performance of shrimps could be improved by supplementing restrictive essential amino acids,cholesterol and adding microencapsulated hydroxyproline and creatine.On this basis,the activity of immune enzymes in each group was determined.The results showed that there was no significant difference in serum immune enzyme activity among groups except PC group(P > 0.05).ACP activity of hepatopancreas in AC-C group was significantly higher than that in AC group(P < 0.05).PO activity in AC-C group was significantly higher than that in PC group(P < 0.05).There was no significant difference in SOD activity in hepatopancreas among all groups(P > 0.05).There was no significant difference in cumulative mortality of Vibrio parahaemolyticus infection among the groups(P > 0.05).Five growth-related genes(DPP1,Actin-5C,PKB,Trypsin-1,CPB)and three immune-related genes(HSC70,LZM,CTL4F)were selected on the basis of experiment1.The results showed that the expression of Trypsin-1 gene in AC group was significantly higher than that in NC group(P < 0.05),and the rest genes were compared with NC group.The relative expression levels of Actin-5C,PKB and CTL4 F in AC-H group were significantly higher than those in AC group and NC group(P < 0.05),while the expression levels of Trypsin-1,CPB and HSC70 were significantly lower than those in AC group(P< 0.05),among which Actin-5C was significantly higher than that in PC group(P < 0.05),and PKB and Trypsin-1 were significantly higher.The expressions of DPP1 and CPB in AC-C group and AC-H+C group were significantly lower than those in AC group(P <0.05),and there was no significant difference between them and PC group(P > 0.05).The expression of LZM gene in AC-H+C group was significantly higher than that in AC group and PC group(P < 0.05).In conclusion,the supplementation of 110 g/kg of fishmeal and 506.5 g/kg of chlorella meal with 12 g/kg of microencapsulated hydroxyproline and creatine significantly promoted the expression of genes related to shrimp growth,and the addition of hydroxyproline also significantly up-regulated the expression of immune enzyme activity and related genes,but failed to offset the effect of the high percentage of chlorella flour on the expression of genes related to shrimp growth.The addition of hydroxyproline also significantly up-regulated the expression of immune enzymes and related genes,but did not offset the negative effects of replacing fishmeal with a high proportion of chlorella.Expt.3 Affecting mechanism of Tenebrio molitor meal replacing fish meal on growth and immunity of Litopenaeus vannamei based on transcriptomics analysisThe aim of this study was to analyze the affecting mechanism of Tenebrio molitor(TM)meal replacing fish meal on the growth performance and immune function of Litopenaeus vannamei by transcriptomics.In the previous experiment,a control diet was formulated to contain 560 g/kg fish meal,then Tenebrio molitor meal was used to replace0%(TM0),20%(TM20),40%(TM40),60%(TM60),80%(TM80)and 100%(TM100)of dietary fish meal to form six iso-nitrogenous and iso-lipidic diets.The six diets were used to feed white shrimp(1.36 ± 0.10 g)for 8 weeks.The results showed that with the increase of TM substitution ratio,the weight gain rate of each group decreased significantly step by step(P < 0.05).Compared with the control group,there was no significant difference in survival rate between TM40 group and the control group(P >0.05),and it decreased significantly from TM60 group(P < 0.05),and the survival rate of TM80 group was the lowest.On this basis,the immune related indexes of each group were determined.The results showed that there was no significant difference in serum SOD activity among all groups(P > 0.05).With the increase of TM substitution ratio,the serum AKP and PO activities in each group showed a downward trend,while ACP activity and NO content showed an upward trend,all of which changed significantly from TM60 group(P < 0.05),but there was no significant difference between TM20 group and TM40 group compared with the control group(P > 0.05).Compared with the control group,the AKP activity of hepatopancreas in TM40 group increased significantly(P < 0.05),while the SOD activity and NO content decreased significantly(P < 0.05).There was no significant difference in PO activity between TM20 groups and the control group(P > 0.05).The cumulative mortality of TM80 group was the highest after infection with Vibrio parahaemolyticus.Meanwhile,the control group,TM40 group and TM80 group were selected for transcriptomics analysis.The results showed that compared with the control group,the differentially expressed genes in TM40 group and TM80 group were significantly enriched in signal pathways such as arginine and proline metabolism,choline metabolism in tumor,lysine biosynthesis and folic acid biosynthesis.In TM40 group,the expression levels of folate biosynthesis-related genes and gluconeogenesis-related genes were significantly down-regulated,while the expression levels of immune-related gene acylCo A binding protein gene in PPAR signaling pathway were up-regulated.In TM80 group,the expression of genes related to metabolism and biosynthesis of 9-cis retinoic acid was significantly down-regulated,and glutathione S-transferase 1-like gene related to cytochrome P450 detoxification was significantly up-regulated.In conclusion,the substitution of 40% fishmeal by Tenebrio molitor meal in the basal diet with a fishmeal content of 560 g/kg affected the synthesis and metabolism of amino acids and folate in L.vannamei,and the substitution of 80% fishmeal activated the detoxification mechanism of the organism and decreased the immunity of the organism.