| Epinephelus fuscoguttatus is an important culture species recently developed in South China.However,diseases frequently broke out and caused huge losses to its culture industry.Herbal immunostimulants have the advantages of being environmental friendly,producing no-drug resistance,no drug residues.It is getting more and more attention in fish disease prevention studies.However,the mechanism of herb immunostimulants' immune regulatory effects on fish is still not clear.Previous in vitro study indicated that Phellodendron amurense(H),Spatholobus suberectus(J)and Eclipta prostrata(M)were three potential fish immunostimulants that could significantly improve the macrophage respiratory burst activity of leucocytes in peripheral blood.To further study the immune-stimulant effects of these herbs on fish and its function mechanism,in vivo feeding experiments were conducted and the effects of herb supplement on the growth,non-specific immune parameters and disease resistance of E.fuscoguttatus were investigated.Transcriptome sequencing on the headkidney of E.fuscoguttatus fed with above herbs was conducted and large amount of immune-relative genes and pathways were identified.Further more,Digital Gene Expression(RNA-Seq Quantification)technique was applied and the differentially expressed genes and pathways were identified in the headkidney of E.fuscoguttatus fed with different herbs.This study will not only help with screening optimal herbal immunostimulants for E.fuscoguttatus,but also provide new information for our understanding of the molecular response mechanism of E.fuscoguttatus immune system after herb stimulation.The main study results are as following:1.The effect of three herbs on the growth,non-specific immune parameters and disease resistance of E.fuscoguttatusAfter 56 days of herb supplement,no significant differences were observed in the weight gain rate,specific growth rate,hepticsomatic index,spleensomatic index in any of the experimental groups.The headkidney macrophage respiratory burst activity were significantly increased in J group at 7th day and in H group at 28th day.As for non-specific immune parameters,only H group showed significant increases in CH50 at 14th day and in AKP at 56th day.Most non-specific immune parameters were either not significantly different or significantly declined in other herb supplemented groups.Vibrio harveyi challenge test showed that after 7 days of herb supplement,M could significantly improved the disease resistance of E.fuscoguttatus.After 14 days,all three herbs significantly decreased the cumulative mortality of E.fuscoguttatus.After 28 days,only H group's cumulative mortality was slightly lower than control group.After 56 days,only M could significantly decrease the cumulative mortality of E.fuscoguttatus.Above results imply that all three herbs could effectively improve the disease resistance of E.fuscoguttatus in different degrees.The disease resistance improving effects varied with different continual herb-supplement time period.2.Transcriptome sequencing and analysis of E.fuscoguttatus headkidneyi ranscriptome sequencing ana analysis were conauctea on the pooled RNA extracted from headkidneys of E.fuscoguttatus in all experimental groups using the Illumina Hiseq2000 sequencing system.A total of 80014 Unigenes with an average length of 694 bp and N50 of 1092 were assembled.For functional annotation analysis,all unigenes were searched against the NR,NT,Swiss-Prot,KEGG,COG and GO databases using BLASTX.49901 Unigenes were annotated altogether,which is about 62.37%of all Unigenes assembled.And the numbers of Unigenes annotated were 39026,46937,33616,27457,11700 and 22738 for NR,NT,Swiss-Prot,KEGG,COG and GO,respectively.The 27457 Unigenes annotated in KEGG were assigned to 258 known metabolic or signal transduction pathways.Further analysis showed that of level-two pathways,those annotated with the largest number of Unigenes were all related to immune functions.They include Immune systems(5121),Signal transduction(4789),Infectious diseases-Viral(4193),Infectious diseases-Bacterial(3952),et al.In addition,most level-three pathways with the largest number of Unigenes were also somehow associated with immunity,confirming the important role headkidney plays in E.fuscoguttatus.KEGG annotation also identified many immune related pathways and genes,including 42 genes in the MAPK pathway,40 genes in the Fc gamma R-mediated phagocytosis pathway and 25 genes in the JAK-STAT pathway.By transcriptome sequencing the headkidney of E.fuscoguttatus fed with different herbs,we obtained large amount of sequence information of candidate immune relative genes and pathways.Our study will provide helpful background information to understanding the immune regulation mechanism of herbs on E.fuscoguttatus.3.Digital Gene Expression analysis of E.fuscoguttatus headkidney fed with different herbs.Using E.fuscoguttatus headkidney transcriptome sequencing data as reference data base,Digital Gene Expression(using RNA-Seq quantification technique)analysis was conducted to find changes in gene expression after feeding with different herbs.Screening of differently expressed genes(DEGs)showed that the main regulatory pattern was downward in H group(180/51,down/up)and M group(132/54,down/up),and upward in J group(47/97,down/up).Comparing to control,M and H group share largest number of DEGs(59).M and J are the second,sharing 40 common DEGs.H and J share the least number of DEGs(29).The GO enrichment analysis of DEGs indicated that largest number of DEGs were enriched in 12 major GO terms,and in all these terms H and M group showed downward regulatory pattern while J was the opposite.The KEGG enrichment analysis of DEGs showed that largest number of DEGs were enriched in pathways like infective diseases,immune diseases and immune system,implying the major regulatory effect of H,M and J on E.fuscoguttatus were immune related.Further data mining resulted in more specific genes and pathways involved in the regulatory function of herbs on E.fuscoguttatus.They include IgG-CD45-Src-Myosin genes on the Fc gamma r mediated phagocytosis pathway,MAPK pathway,IgG-BCR and TLR5 gene,SOCS1&PIM1 genes on the JAK-STAT pathway and COX-2 gene.The identification of these pathways and genes could help with our understanding of the molecular responding mechanism of E.fuscoguttatus immune system after herb stimulation. |
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