| Bacillus strains used in aquaculture can be endogenous or exogenous,benefiting fish health and growth.However,indigenous Bacillus strains isolated from the gut or surrounding water are considered uniquely beneficial probiotics because the bacteria isolated from the intestinal tract of fish can adjust to the same environment and obviate competitive refusal,thus multiplying more efficiently on the host than other bacteria.This study aims to investigate the effect of Bacillus aryabhattai(LSG3-7)and Bacillus mojavensis(LSG3-8)were isolated from the intestine of Rhynchocypris lagowskii(Dybowski,1869)on growth performance,intestine health,microbial community,antioxidant,immunity,and metabolomics of this fish.The rearing experiment was conducted at the Aquaculture Laboratory of Jilin Agricultural University(Changchun City,Jilin Province,China).Firstly,the biosafety of Bacillus aryabhattai(LSG3-7)and Bacillus mojavensis(LSG3-8)isolated from the Rhynchocypris lagowskii(Dybowski,1869)were tested in vitro.Secondly,A 630 healthy fish(10.76±0.05)were randomly divided into six groups:control group(A)was fed the basal diet,groups(B and C)were supplemented with LSG 3-7 and LSG3-8(1×10~8 CFU/g),whereas group(D)was supplemented with a mixture of both bacteria(0.5×10~8 CFU/g each),and group(E)was supplemented with LSG3-7 0.75×10~8 CFU/g+LSG3-8 0.25×10~8 CFU/g,and group(F)supplemented with LSG3-7 0.25×10~8 CFU/g+LSG3-8 0.75×10~8 CFU/g.The experiment lasted 60 days.Finally,a 14-day challenge trial by Aeromonas hydrophila was performed after the breeding experiment.The results of this study were divided into four chapters,each chapter showing a set of measurements related to each other,which will be explained as follows:Chapter one:Characterization and assessment of B.aryabhattai and B.mojavensis isolated from the intestines of Rhynchocypris lagowskii as a potential probiotic.The strains showed a high percentage of co-aggregation,more than 70%,with Aer.hydrophile,Staph.epidermidis,and Klebsiella aerogenes.The two strains showed high hydrophobicity activity with toluene and xylene.The strains showed safety properties,non-hemolytic,and sensitivity to many antibiotics.The strains also showed bile salt hydrolase activity and biofilm formation,allowing them to tolerate stressful conditions.Conclusion:Based on the B.aryabhattai and B.mojavensis characteristics and features,they could be considered promising candidate probiotics and can be used as anti-pathogenic,especially in aquaculture.Chapter two:Effects of Bacillus aryabhattai and Bacillus mojavensis on growth performance,intestine health,and microbial diversity of Rhynchocypris lagowskiiThe measurements were growth performance,expression of growth-related genes,digestive enzyme activity,morphometric assessment of intestinal,expression of mucus-related genes,expression of the tight junction-related genes,and microbial diversity.The results of growth performance,including final weight(FW),weight gain(WG),weight gain ratio(WGR),specific growth rate(SGR),hepatosomatic index(HSI),viscerosomatic index(VSI),and condition factor(CF),showed nonsignificant(p<0.05)effect after 30 days,while after 60 days showed a significant increase(p<0.05)in treatment groups compared to the control group,except CF and HSI showed increased in treatments without significant(p<0.05).In the same context,expression of growth-related genes:insulin-like growth factor 1(IGF-1),targets of the rapamycin(TOR),and ribosomal protein S6 kinase 1(S6K-1)showed significant upregulation(p<0.05)in treatment groups,especially groups D and E,compared to the control group.Results of the digestive enzyme(trypsin,amylase,and lipase)were significantly increased(p<0.05)in the treatment groups compared to the control group after 30 and 60days.However,groups D and E showed the highest values.Results of the morphometric assessment of the intestinal(villi length,villi width,muscle layer,and diameter)in intestine parts(proximal intestine[PI],mid intestine[MI],and distal intestine[DI])showed improvement and increase(p<0.05)in villi length,villi width,and muscle layer in all parts of the intestine,especially PI segment as a result of treated by B.aryabhattai and B.mojavensis.However,group E showed the highest values of morphometric intestine measurements.Expression of mucus-related genes(Muc2,Muc5B,and Muc5ac)showed significant upregulation(p<0.05)in the groups supplemented by B.aryabhattai and B.mojavensis compared to the control group.The highest values are shown in group E.Expression of tight injection-related genes:Claudin-1,Occludin,and zonula Occludin-1(ZO-1)showed a significant increase(p<0.05)in treatment groups compared to the control group.However,ZO-1 showed upregulation more than claudin-1 and Occludin.Results of microbial diversity showed the ability of B.aryabhattai and B.mojavensis to modify the microbial community in the intestine of R.lagowskii.The treatments showed increasing in the genre helpful bacteria such as Bacillus,Rhodobacter,Oceanicaulis,Burkholderia,Pseudomonas,and Blautia.In contrast,the genre of harmful bacteria such as Aeromonas and shigella decreased.Chapter three:Effects of Bacillus aryabhattai and Bacillus mojavensis on antioxidants,immunity,and disease resistance of Rhynchocypris lagowskiiThe results of antioxidant enzymes include Catalase(CAT),superoxide dismutase(SOD),glutathione(GSH)content,and glutathione peroxidase(GSH-Px)in the liver and intestine.Results showed that antioxidant parameters were significantly increased after30 and 60 days in the supplemented groups compared to the control group.However,the intestine showed more significance than the liver in all measurements.In contrast,the gene expression related-antioxidants(CAT,GPX,Cu Zn-SOD,and Nrf2)showed significant upregulation in treated groups in the liver and intestine compared to the control group.The same was observed in antioxidant enzymes and gene-related antioxidants after the challenge test by Aer.hydrophila for 14 days;the treatment groups were superior to the negative and positive controls.After 30,60 days,and a challenge test,the results of immune serum parameters:immunoglobulin M(Ig M),lysozyme(LZM),complement component 3(C3),complement component 4(C4),and alkaline phosphatase(AKP)showed a significant increase in treated groups than the control group.However,the treatments supplemented by the mixture of Bacillus aryabhattai and Bacillus mojavensis showed more improvement than other groups.The results of expression of immune-related genes include Ig M:immunoglobin M;NF-κB:nuclear factor kappa B;TLR1:Toll-like receptor 1;TLR2:Toll-like receptor2;My D88:myeloid differentiation factor 88;IL-1β:interleukin-1β;TNF-α:tumor necrosis factor-αin the liver and muscle after 30,60 days and challenge test were recorded.Results of Ig M,TLR-1,TLR-2,and My D88 after 30 days showed significant upregulation(p<0.05)in treatment groups than those in the control group.Meanwhile,the gene expression level of NF-κB showed nonsignificant upregulation(p<0.05)compared to the control group.In contrast,after 60 days and a challenge test Ig M,TLR-1,TLR-2,NF-κB,and My D88 showed increased upregulation(p<0.05)in treated groups compared to the control group.On the other hand,the pro-inflammatory cytokines IL-1 and TNF-αm RNA levels showed downregulation(p<0.05)in supplemented groups compared to the control group after trial and challenging periods.However,the gene expression in the liver was greater than muscle.Survival rate result after the challenge test was observed after the fish injection by Are.hydrophila and showed that the mortality ratio increased between days 3 to 7,then decreased till the end of the test.The treatment groups showed high survival,especially groups F and E,compared to the positive control.Chapter four:Effects of Bacillus aryabhattai and Bacillus mojavensis on the metabolomics of Rhynchocypris lagowskiiThe present study showed 182 significant metabolic compounds in treatments compared to the control group.Multivariate statistical analysis of metabolites,including principal component analysis(PCA),partial least squares discriminant analysis(PLS-DA),and discriminant analysis with orthogonal projection to latent structures(OPLS-DA),were performed.The results showed perfect discrimination between treatments and the control group,indicating a significant change in the metabolite profiles after the probiotic supplementation.In contrast,the model was tested by 7-round cross-validation and permutation tests to confirm the validity model.The results showed that the original model did not have the overfitting phenomenon,and the model was stable.Results of univariate statistical analysis of metabolites showed 67 metabolites in the negative ions and 117 in the positive ions in the treatment groups compared with the control group.Eighty-four metabolic compounds showed upregulation,while 98compounds showed downregulation.These results were presented in volcano plot charts.Bioinformatics analysis of differential metabolites includes cluster analysis,Hierarchical clustering heatmap,correlation analysis,and path analysis.The results showed a difference in the metabolic results between the control and the treated groups.This resulted in activity in some essential metabolic pathways,impacting the fish’s growth and health.The activated pathways due to the treatments include biosynthesis of unsaturated fatty acids,vitamin digestion and absorption,alpha-linolenic acid metabolism,phototransduction,arginine,and proline metabolism,pyrimidine metabolism,neuroactive ligand-receptor interaction,lysosome,ABC transporters,and HIF-1 signaling pathway.Activation of many critical metabolic pathways resulted in production of many metabolites,such as linolenic acid,linoleic acid,eicosapentaenoic acid,pyridoxal,retinene,deoxycytidine,5-methylcytosine,thymine,D-mannose 6-phosphate,and L-(+)-lactic acid;these showed affected positively on growth,immunity,and nutritional value of R.lagowskii.In conclusion,Bacillus aryabhattai and Bacillus mojavensis isolated from the intestine of R.lagowskii fish showed probiotic properties and the ability to be applied in aquaculture.The addition of Bacillus aryabhattai and Bacillus mojavensis in R.lagowskii diets has been shown to improve growth performance,intestine health,modified microbial community with increased beneficial bacteria compared to harmful bacteria,enhancement of the antioxidants and immunity system,ability to decrease the side effects and mortality after infection by Aer.hydrophila,activate the essential metabolic pathways,which lead to increased crucial metabolic compounds and positively affect the growth and health of R.lagowskii fish.Furthermore,the combined groups were better than the single groups.Bacillus aryabhattai showed a slight improvement compared to Bacillus mojavensis without significance. |
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