Regulation Mechanism Of Astaxanthin And Vitamin C Improve The Growth And Immune Stress In Coral Trout (Plectropomus Leopardus)

The coral trout(Plectropomus leopardus)is a valuable marine fish.In recent years,the artificial culture of P.leopardus has developed rapidly,however,it is susceptible to environmental stress during the breeding process,resulting in frequent occurrence of various diseases and economic losses.Astaxanthin is a carotenoid with strong antioxidant activity,immunomodulatory activity and disease resistance,and is so far one of the most powerful antioxidants found in nature.Vitamin C,a water-soluble vitamin,is commonly added to feed and water as a natural antioxidant and immune enhancer to improve antioxidant enzyme activity in aquatic animal organisms,alleviate oxidative stress,and enhance fish immunity and disease resistance.In this study,the effects of different concentrations of astaxanthin and vitamin C on the growth performance and immunity of P.leopardus were investigated by adding them to feeds,screening the signaling pathways and factors related to immunity,analyzing their modes of action and elucidating the regulatory mechanisms of immunity,in order to provide a theoretical basis for the development of environmentally friendly compound feeds for P.leopardus and their healthy breeding.The main contents are as follows.1.Effects of astaxanthin on the growth and immunity of P.leopardusFour isonitrogenous and isolipid experimental diets containing 0(control),50(A1),100(A2)and 200(A3)mg/kg of astaxanthin were formulated to examine the growth performance and immune parameters of P.leopardus and transcriptomic and metabolomic studies were conducted on the liver.The results showed that(1)the dietary addition of astaxanthin had no significant effect on the growth performance of l P.leopardus(P>0.05);the activities of amylase,lipase and trypsin in the liver of the A2 group were significantly higher compared with the control group(P<0.05).(2)The activities of catalase(CAT),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and total antioxidant capacity(T-AOC)levels in serum and liver were significantly increased in the A2 group before and after the challenge(P<0.05);the activities of acid phosphatase(ACP)and lysozyme(LZ)in serum and liver in the A2 group after the challenge;The relative expressions of sod-1,sod-2,cat,acp6,akp,lz-c,igm,c3 and c4-b in the liver of A2 group were significantly up-regulated before and after the challenge(P<0.05);the survival rate of Vibrio harveyi infection was significantly higher in A2 group(P<0.05).(3)RNA-Seq screened 87 differentially expressed genes,of which 22 were up-regulated and 65 were down-regulated;KEGG analysis showed that the 87 differentially expressed genes were enriched into 131 pathways;the genes involved in immunity-related genes were mainly biologic clock family genes,which regulated downstream p53,NF-κB,Fox O and other signaling pathways to reduce the inflammatory response of P.leopardus,thus enhancing its immunity.(4)A total of 76 differential metabolites were screened in the non-target metabolome,including 40 up-regulated metabolites and 36 down-regulated metabolites;the KEGG enrichment results showed that 76 differential metabolites were enriched in 69 KEGG pathways,and the arachidonic acid pathway was its specific metabolic pathway.(5)The results of combined transcriptome and metabolome analysis enriched 28 common KEGG pathways,and the KEGG signaling pathways involved in common genes and metabolites were consistent with the results of transcriptome and metabolome.2.Effects of vitamin C on the growth and immunity of P.leopardusFour isonitrogenous and isolipid experimental diets with vitamin C levels of 0(control,207),50(C1,243),100(C2,272)and 200(C3,314)mg/kg were formulated to examine the growth performance and nonspecific immune indexes of P.leopardus,and to perform transcriptomic and metabolomic studies on the liver.The results showed that(1)dietary supplementation with appropriate amounts of vitamin C increased the specific growth rate(SGR)and weight gain rate(WGR),and significantly increased the activities of amylase,lipase and trypsin in the liver of P.leopardus(P<0.05).(2)Glutathione peroxidase(GSH-Px),catalase(CAT,except in pre-attack serum),superoxide dismutase(SOD)activity and malondialdehyde(MDA)content were significantly enhanced in serum and liver before and after the challenge(P<0.05);lysozyme(LZ)and acid phosphatase(ACP)activities were significantly increased in serum before and after the challenge.The activities of alkaline phosphatase(AKP),LZ,ACP and the levels of complement(C3 and C4)and immunoglobulin M(Ig M)were significantly increased in the liver(P<0.05);the relative expression of sod-1,sod-2,cat,gsh-px1 a,acp6,akp,lz-c,igm,c3 and c4-b in the liver before and after the challenge was significantly increased(P<0.05);and improved the survival rate after V.harveyi infection.(3)RNA-Seq screened a total of 13 differentially expressed genes,of which 6 were up-regulated and 7 were down-regulated;KEGG enrichment analysis showed that 13 differentially expressed genes were annotated to 50 KEGG pathways;gadd45g,dgkz,and fasn were marker differential genes for vitamin C involvement in non-specific immunity.(4)The non-target metabolome was screened for 53 differential metabolites,including 31 up-regulated metabolites and 22 down-regulated metabolites;KEGG enrichment results showed that 53 differential metabolites were enriched into 61 KEGG pathways;vitamin digestion and absorption were the unique metabolic pathways,and L-glutamic acid was its signature differential metabolite.(5)The results of combined transcriptome and metabolome analysis enriched 6 common KEGG pathways,and the KEGG signaling pathways involved in common genes and metabolites were consistent with the results of transcriptome and metabolome.3.Effects of synergistic effect of astaxanthin and vitamin C on growth and immunity of P.leopardusThe diets of control,A2,C2 and AC(A2+C2)were formulated to investigate the synergistic effects of astaxanthin and vitamin C on the growth and immunity of P.leopardus.The results showed that(1)growth and immunity were significantly improved in the AC group compared with the A2 and C2 groups(P<0.05);(2)RNA-Seq screened a total of 230 differentially expressed genes,of which 92 genes were up-regulated and 138 genes were down-regulated;230 differentially expressed genes were enriched into 183 KEGG pathways;the differentially genes enriched into KEGG pathways in the A2 group There were no NF-κB,MAPK,Toll-like signaling pathways among the KEGG pathways enriched by differential genes in group C2;there were no Toll-like signaling pathways among the KEGG pathways enriched by differential genes in group C2 to 50.(3)A total of 83 differential metabolites were screened in the non-target metabolome,including 51 up-regulated metabolites and 32 down-regulated metabolites;83 differential metabolites were enriched to 48 KEGG pathways;compared with both A2 and C2 groups,there was a decrease,but the A2-specific arachidonic acid pathway and C2-specific vitamin uptake and transport were included.(4)The combined transcriptomic and metabolomic analyses enriched 28 common KEGG pathways,and immune-related genes such as ctsk,dgkz,cd38 and xmrk were enriched in the common pathways;vitamin C and astaxanthin played both independent and synergistic roles in the regulation of growth and immunity in P.leopardus.In conclusion,both astaxanthin and vitamin C can improve the growth and immunity of P.leopardus to some extent,and they have synergistic effects.The transcriptional and metabolic pathways of astaxanthin and vitamin C were different in the regulation of immunity in P.leopardus.p53 signaling pathway was the specific pathway of astaxanthin and NF-κB signaling pathway was the specific pathway of vitamin C in the regulation of immunity in P.leopardus.The arachidonic acid pathway is a metabolic pathway specific to the regulation of immunity in P.leopardus by astaxanthin,and vitamin digestion and absorption is a metabolic pathway specific to the regulation of immunity in P.leopardus by vitamin C.The recommended optimal levels of astaxanthin and vitamin C in the diet of P.leopardus were 91.13-116.50 mg/kg and 255.25-314.03 mg/kg,respectively.