| In recent years,the shrimp industry has suffered serious losses due to diseases such as white spot syndrome and hepatopancreas necrosis.In the traditional breeding process,chemicals and antibiotics are used as the common ways for disease prevention and treatment.However,usage of chemicals and antibiotics on a large scale has caused environmental pollution and will threaten human health.Therefore,one of the current and future development directions of the shrimp aquaculture industry and the entire aquatic industry is to produce healthy and green products by developing safe,environmentally friendly,and effective feed or veterinary drugs.In this study,using two substances,zymosan A and unmethylated cytosine-phosphate-guanosine(CpG)-oligodeoxynucleotide(ODN,CpGODN)as additives in the feed,the performance,disease resistance and immunity,enzyme activity,intestinal flora and transcriptome were analyzed to study the response of Litopenaeus vannamei to them.The specific research results are as follows:1.Zymosan A were added into the basic feed formula at the doses of 0,1,5,25,125 and 625 mg/kg respectively to prepare 6 kinds of test feeds,feeding L.vannamei(initial body weight 0.41 ± 0.05 g)as 6 groups(Z0,Z1,Z5,Z25,Z125,Z625).After 8 weeks of culture,the effect of Zymosan A on L.vannamei is studied from the aspects of growth performance,disease resistance,immunity,enzyme activity,intestinal flora and transcriptome.The results showed that:(1)The end weight of L.vannamei in the Z125 group was significantly higher than that of the control group,and the feed coefficient(FCR)was significantly lower than that of the control group(P < 0.05).(2)The activities of lysozyme(LZM),phenol oxidase(PO),acid phosphatase(ACP),superoxide dismutase(SOD),alkaline phosphatase(AKP)and catalase(CAT)in group Z125 were significantly higher.In the control group(P < 0.05).The activities of malondialdehyde(MDA)and alanine aminotransferase assay kit(ALT)in the Z125 group were significantly lower than those in the control group(P < 0.05).The aspartate aminotransferase assay kit(AST)activity of Z125 and Z625 groups was significantly lower than that of the control group(P < 0.05).The activities of trypsin,amylase and lipase in shrimp intestines in Z125 group were significantly higher than those in control group(P < 0.05)(3)The survival rate of shrimps infected with white spot syndrome virus(WSSV)in the Z25 group was significantly higher than that of the control group(P < 0.05).(4)The results of intestinal flora showed that the relative abundance of conditional pathogenic bacteria in the intestine of L.vannamei decreased after adding Zymosan A.(5)Transcriptome sequencing results showed that differentially expressed genes(DEGs)4,040(3,430 up-regulated,610 down-regulated),4,967(2,942 up-regulated,2,034 down-regulated)were identified between the Z1,Z5,Z25,Z125 and Z625 groups and the control Z0,4,040(3,211 up,829 down),6,572(3,644 up,2,928 down)and 4,857(2,995 up,1,862 down).The GO analysis of differentially expressed genes(DEG)shows that the secondary classification of GO annotation includes 24 subtypes involved in biological processes(BP),18 subtypes belong to cell components(CC),and 10 subtypes have molecular functions.(MF),among which the number of genes involved in biological processes is the largest.The DEGS enrichment analysis results of KEGG showed that each group of differential genes were significantly enriched in the PI3K-Akt signaling pathway.And the expression levels of akt1,mapk1,myd88 and ap1 in the addition group were significantly lower than those in the control group(P < 0.05).The expression levels of Map2k7,pi3kr1,tab2,cact and traf6 in the Z1 and Z5 groups were significantly lower than those in the control group 5.In summary,the addition of Zymosan A in feed can significantly improve the growth performance of L.vannamei,increase its serum non-specific immune enzyme and intestinal digestive enzyme activities,increase the survival rate against WSSV,and reduce the conditional pathogenic bacteria in the intestine Relative abundance regulates the expression of genes related to Toll-like receptor signaling pathway.Based on this,it can be speculated that Zymosan A can act as a ligand of TLR2 in the Toll-like receptor signaling pathway to stimulate the expression of related genes in the pathway,thereby affecting the growth performance and disease resistance of L.vannamei.2.CpG ODN were added into the basic feed formula at the doses of 0,0.1,0.4,1.6,6.4and 25.6 mg/kg respectively to prepare 6 kinds of test feeds,feeding L.vannamei(initial body weight 0.41 ± 0.05 g)as 6 groups(C0,C0.1,C0.4,C1.6.C6.4 and C25.6).After 8weeks of culture,the effect of CpG ODN on L.vannamei is studied from the aspects of growth performance,disease resistance,immunity,enzyme activity,intestinal flora and transcriptome.The results showed that:(1)The growth performance of L.vannamei was not significantly affected by CpG ODN added in feed.(2)The activities of LZM,SOD,CAT,AKP,PO and ACP in C25.6 group were significantly higher than those in control group C0.4,C1.6,C6.4 and C25.6 group(P < 0.05).The ALT activity of C1.6,C6.4 and C25.6groups was significantly lower than that of the control group(P < 0.05).The ALT activity of C0.4 group was significantly higher than that of other groups(P < 0.05).The activities of amylase and trypsin in the intestine of L.vannamei were significantly higher than those in the control group(P < 0.05).The activity of intestinal lipase in C1.6 and C6.4 groups was significantly higher than that in the control group(P < 0.05).(3)After white spot syndrome virus infection,the survival rate of shrimp in C25.6 group was significantly higher than that in C0 group(P < 0.05).(4)The different amount of CpG ODN can make the shrimp intestinal microbial population of different quantity and species,but the microbial diversity index has no significant difference.(5)The results of transcriptional sequencing showed that Differentially expressed genes(DEGs)3,200(2,612 up-regulated,588 down-regulated)were identified between the C0.1,C0.4,C1.6,C6.4,and C25.6 groups and the control group C0,respectively.629(680 upwards,949 downwards),2,015(1,154 upwards,861downwards),8,294(3,081 upwards,5,213 downwards),and 1,606(806 upwards,800downwards).Go analysis showed that the second-grade classification of GO annotation of differential genes includes 24 subtypes involved in biological process(BP),17 sub classes belong to cell component(CC),10 sub classes have molecular function(MF),among which the number of genes involved in biological process is the most.The analysis of the enrichment of DEGs in KEGG showed that there was more significant enrichment of DEGs and metabolism in the control group than in the control group.In conclusion,CpG ODN added to feed had no significant effect on the growth performance of L.vannamei,but it could improve the serum nonspecific immune enzyme and intestinal digestive enzyme activity,improve the survival rate of anti WSSV,and affect the differential expression of immune and metabolic related genes.It is suggested that CpG ODN can improve the growth performance and disease resistance immunity of L.vannamei by regulating the metabolism process of prawn,but its long-term continuous stimulation may weaken the activation of downstream signal pathway,which will bring adverse effects. |
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