| Chitosan oligosaccharides(COS),formed from chitosan by acid hydrolysis or by enzymatic degradation and deacetylation,were mixtures of oligomers of ?-1,4-linked D-glucosamine residues.COS,as an immunopotentiator,could improve the growth performance,immune function and disease resistance of fish.This study was conducted to investigate the effects of COS on immunity,antibacterial infection and intestinal bacterial flora of Russian sturgeon(Acipenser gueldenstaedti).The feasibility of adding COS to diets was analyzed,and the optimum additive amount and feeding time of COS were determined,which would provide the theoretical basis for productive practice and environmental breeding.In addition,study was conducted to investigate the effects of COS on transcriptome in peripheral blood leukocytes of half-smooth tongue sole(Cynoglossus semilaevis),which would provide the basis for exploring how COS affect gene function.Conclusions of this study were as follows:1.Healthy Russian sturgeon(weight 685.0±6.5 g,length 45.5±5.5 cm,estimated age24 months)were randomly selected for experiments.The control group were fed with basal diets,experimental groups were fed with diets supplementing with COS(0.10%,0.30%,0.50%,0.75%,1.00%,1.50%).Each group was set with three replicates and per replicate was selected about 100 fish.ELISA kit was used to detect the activities or concentrations of immune molecules in serum of Russian sturgeon.The results illustrated that: the lysozyme(LYS)activities of experimental groups were proportional to the concentration of COS and feeding time.After 98-day feeding trial,the LYS activity of1.50% group was around 2.40 times as much as the control group.The changing trend of immunoglobulin M(IgM)and complement 3(C3)were similar as LYS.After 98 d,IgM in 1.50% group could reach 1.65 times,and C3 in 1.50% group could reach 1.45 times.The activities of acid phosphatase(ACP)and alkaline phosphatase(AKP)increased with the increase of COS concentration,however,the statistical difference was not significant.All groups were transferred to feed ordinary diets without any COS after 98 d,and the related immune molecules were detected after 130 d.Results showed that the activities of LYS,ACP and the concentrations of IgM,C3 in 1.50% group remained at the level as in90 d.The activity of AKP in 1.50% group remained at the level as in 81 d.2.Then,Russian sturgeon feeding with COS for 98 d were for challenge experiment of Streptococcus dysgalactiae(10×semi-lethal dose),mortality in experimental groups were significantly lower than that in control group.And with the increase of COS concentrations,the mortality was decreased.The gene expression level of Russian sturgeon in control group was set as the standard value,the relative expression of genes(IL-1,IL-2,IL-10,TNF)in the head kidney of the 1.50% group were lower than that in control group.The change of IL-2 was the most obvious,and it was almost not expressed in 1.50% group.The following were the IL-10(0.02),TNF(0.05),IL-1(0.12).The expression of intestinal genes(IL-1,IL-2,IL-10)in 1.50% group were much higher than that in control group,the expression of TNF gene was lower.The change of IL-10(15.74)was the most obvious,the following were the IL-1(6.62),IL-2(1.94);and the TNF decreased by 0.53 as much as that in control group.In addition,the expression of IL-1 and IL-10 in the liver of the 1.50% group were higher than that in control group,however,the expression of IL-2 and TNF were lower.The change of IL-10(5.04)was the most obvious,the following was the IL-1(3.74);the IL-2 decreased by 0.31 and TNF decreased by 0.12 as much as that in control group.3.Based on 16 S rRNA sequence,high-throughput sequencing analysis showed that1,136 species of microorganisms in Russian sturgeon could be identified,which belonged to 42 phyla,81 class,180 order,335 family and 701 genus.COS and infectious symptoms all significantly affected the diversity and richness of intestinal bacterial flora.The phylogenetic tree analysis showed that there were differences in the base sequences of intestinal bacterial flora.The analysis of 16 S function prediction showed that the bacteria had participated in 25 COG pathways and 299 KEGG pathways.There were many microbial species that might be related to metabolic pathways of diseases.4.Healthy half-smooth tongue sole(weight 1,000.0±9.5 g,length 60.5±4.5 cm,estimated age 30 months)were randomly selected for experiments.The control group were injected intraperitoneally with PBS and the experimental group were injected with COS(150 mg/kg).Each group was selected 3 fish.594,000,000 reads were generated by using the Illumina sequencing in peripheral blood leukocytes of semi-smooth tongue soles,which were distributed mostly in the range of 300-400 bp,and the valid reads were analyzed.Then,differentially expressed genes were screened,results showed that 369 unigenes were up-regulated and 240 unigenes were down-regulated in the experimental group.17,671 unigenes were annotated with 1,663 GO terms by Gene Ontology,of which1,589 unigene with differential expression were annotated with the biological process,1,032 unigene were annotated with the cellular component,1,329 unigene were annotated with the molecular function.Lastly,12,054 unigenes were mapped to 229 different pathways by the analysis of KEGG.Collectively,these findings suggested that COS could improve immune function,alter the species of intestinal bacterial flora and enhance survival of Russian sturgeon.And addition ratio of 1.50% was the best,based on feeding cost,it was not necessary to continuously feed the diets added with COS.In addition,COS also affected the transcriptome of half-smooth tongue sole,COS might be a appropriate immunopotentiator. |
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