| Largemouth bass(Micropterus salmoides)is a typical carnivorous fish,which commonly known as California perch,belongs to perciformes,native to North America.Because of its fast growth,delicious meat and high market value,it has become one of the important breeding varieties in China.However,with the continuous improvement of breeding density and the deterioration of breeding environment,many diseases have occurred.In order to solve the problem of disease,antibiotics and a variety of drugs have been used in aquaculture.Although antibiotics and drugs can reduce the occurrence of disease,the abuse of antibiotics and drugs will lead to excessive residue and increased resistance.In addition,reducing resistance and banning resistance is the theme of the development of modern aquaculture industry.Therefore,it is necessary to develop safe and reliable disease prevention measures to reduce disease occurrence and promote sustainable development of aquaculture industry.β-glucan,as an efficient,safe and non-toxic immune regulatory substance,can promote the growth performance,antioxidant,immunity and antiinfection ability of aquatic animals.In this study,β-glucan was used as an immunomodulator to explore the effects of β-glucan on growth performance,non-specific immunity,glucolipid metabolism and intestinal microbial composition of largemouth bass,and to analyze the possible mechanism of β-glucan enhancing immunity by using transcriptome sequencing technology.This thesis mainly includes the following three aspects:1.Effects of β-glucan on growth,serum biochemistry,liver tissue structure and antiinfection ability of largemouth bassβ-glucan was added 0(Control),100(LA-100),200(MA-200)and 300(HA-300)mg/kg in the basal diets(～53.7% protein and ～15.8% fat),respectively.Each tank contained50 largemouth bass(initial body weight 79.30 ± 0.50 g)and the experiment lasted for 70 days.After the breeding experiment,a 3-day challenge experiment was conducted.The results showed that different levels of β-glucan had no significant effects on growth performance,body indexes and body composition of largemouth bass.Diet supplemented with 300 mg/kg β-glucan can improve the liver tissue structure and relieve the accumulation of liver fat and liver glycogen.With the increase of dietary β-glucan level,the serum lysozyme activity of largemouth bass was significantly increased.After the culture experiment,25 largemouth bass were selected in each tank for Aeromonas Schubertii challenge experiment.The resulted showed that all largemouth bass in the Control group died within 12 hours,and all largemouth bass in the LA-100 group died within 48 hours.All the HA-300 group died within 60 hours.After 60 hours only one fish survived in the MA-200 group.The results showed that β-glucan supplementation could improve the resistance of largemouth bass to Aeromonas schubertii infection.2.Effects of β-glucan on intestinal microbial composition of largemouth bass16S r RNA gene sequencing was used to analyze the intestinal microbial diversity of Control and HA-300 groups of largemouth bass,and to explore the effects of β-glucan on the intestinal microecology of largemouth bass.The results showed that Fusobacteria,Firmicutes and Proteobacteria were the dominant bacteria in largemouth bass intestinal tract.Dietary β-glucan had no significant effect on α-diversity index.β-glucan can increase the relative abundance of Firmicutes and decrease the relative abundance of Proteobacteria in the intestinal tract of largemouth bass,and may activate the immune function as biomarkers.LEf Se analysis showed that β-glucan could increase the relative abundance of Mycoplasma and other beneficial bacteria.The relative abundance of harmful bacteria in Proteobacteria,Gammaproteobacteria,Escherichia-Shigella,Escherichia coli and Bacillus anthracis was decreased(LDA > 2),improve the intestinal microecological environment and maintain intestinal immune function.3.Transcriptome analysis of β-glucan response of largemouth bassIn this part of the experiment,Control and HA-300 group were selected,and transcriptome sequencing technology was used to screen out more regulatory factors and metabolic pathways,and to analyze the response mechanism of largemouth bass to β-glucan.Liver transcriptome sequencing results showed that 22,602 unigenes were obtained after assembly and redundancy removal.After screening the get 1,245 differentially expressed genes(DEGs)(|log2fold change| ≥ 1,q-value ≤ 0.05),which contains 449 DEGs were significantly up-regulated and 796 DEGs were significantly down-regulated.Genes related to immunity and glucolipid metabolism were selected for KEGG enrichment analysis,and20 immune-related metabolic pathways and 21 glucolipid metabolism-related metabolic pathways were significantly enriched(q < 0.05).Selected 9 key DEGs of immune,7 key DEGs of lipid metabolism and 5 key DEGs of carbohydrate metabolism DEGs.The results showed that β-glucan could significantly up-regulate p21 protein(Cdc42/Rac)activated kinase 1(pak1),mitogen-activated protein kinase kinase 7(map2k7),and down-regulated Arrestin β1(arrb1),interleukin 1β(il1β),interleukin 10(il10)and Toll-like receptor9(tlr9)gene expression levels,reduced the inflammatory response and enhance immunity in largemouth bass.Meanwhile,β-glucan significantly up-regulated glutamate-ammonia-ligase(glutamine synthase)b(glulb)gene,isocitrate dehydrogenase(NAD(+))3 catalytic subunitα(idh3a),and significantly down-regulated phosphoglucose mutase 3(pgm3),glucokinase(hexokinase 4)(gck),dihydrothiooctam S-acetyltransferase(pyruvate dehydrogenase E2complex)(dlat).In addition,β-glucan can significantly down-regulate monacylglycerol Oacyltransferase(mogat2),fatty acid synthase(fasn),acyl-Co A synthase long chain family 4a(acsl4a),acetyl-Co A carboxylase α(acaca)and stearyl-Co A desaturase(Delta-9-desaturase)(scd),carnitine palmityl transferase 1B(cpt1b)and liver lipase(lipca)regulate glucolipid metabolism. |
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