| Sea urchin Mesocentrotus nudus is mainly distributed in the northern waters of China and is an important economic species in the coastal areas of Liaoning and Shandong.In recent years,the fishing of natural sea urchin populations has been unable to meet the rapidly growing market demand for sea urchins in various regions,and therefore,aquaculture of M.nudus is playing an increasingly important role in alleviating the contradiction between market demand and overfishing of natural sea urchin populations.However,the problems of frequent disease outbreaks during sea urchin farming,especially bacterial infectious diseases can lead to massive mortality of juvenile or adult individuals on farms,causing huge economic losses to the sea urchin farming industry.On the other hand,sea urchins,as a model animal to study the molecular regulation of innate immunity,are commonly used for immune system defense studies in some stressful environments such as heavy metals,hypoxia and acidification.However,the immune regulatory mechanisms of sea urchins against bacterial pathogen infections are not known.Improving disease resistance through selective breeding is an important means to prevent and control various diseases in M.nudus during culture,but only if the integrated immune responses of M.nudus to the corresponding pathogens and the underlying molecular mechanisms of M.nudus in defense against pathogenic infection are elucidated.For this reason,the present study used M.nudus as a model organism to study the immune regulatory mechanism of sea urchins against Vibrio infection.Using techniques and methods such as bacterial genomics,transcriptomics,proteomics,and fluorescence quantitative PCR,the present study firstly comprehensively analyzed the pathogenicity of Vibrio coralliilyticus to M.nudus;secondly,we systematically described the physiological responses of M.nudus during V.coralliilyticus infection and the intrinsic link between oxidative stress,apoptosis and immune regulation of coelomocytes based on physiological and molecular levels;finally,key immune pathways and immune genes were screened from the m RNA and protein levels,the lysosome-phagosome pathway mediates immune regulatory mechanisms in M.nudus against V.coralliilyticus infection was comprehensively interpreted.The main results are listed as follows:(1)The strain V.coralliilyticus G2-37 was isolated and identified from individual sea urchins with obvious disease symptoms.Bacterial infection experiments confirmed the high pathogenicity of V.coralliilyticus G2-37 for M.nudus.Histopathological observations showed that the intestine,gonad and peristomal membrane of the diseased M.nudus had obvious symptoms.Bacterial drug sensitivity tests reflected multiple drug resistance of V.coralliilyticus to some antibiotics such as penicillins,suggesting that the susceptibility of the bacterium to some antibiotics of cephalosporins,macrolides,phenicols,and fluoroquinolones can be used to prevent and control M.nudus disease in actual sea urchin culture.(2)Flow cytometry experiments demonstrated that V.coralliilyticus G2-37 could significantly induce the apoptosis of M.nudus coelomocytes.The immunoenzyme activity analysis of M.nudus tissues at different times of infection indicated that the complex immune responses induced by V.coralliilyticus infection were tissue-specific and time-dependent.Principal component analysis(PCA)showed that the physiological states of the intestine,coelomocyte lysate supernatant(CLS),and cellfree coelomic fluid(CCF)from M.nudus were significantly affected with increasing duration of infection.Integrated biomarker response(IBR)analysis proved that M.nudus exposed to the pathogen may have a physiologically stable period at midinfection,however,long-term pathogen infection would reduce the immune capacity and increase the oxidative damage of M.nudus.(3)By Illumina sequencing,10,861,15,201 and 8,809 differentially expressed genes(DEGs)were obtained in the I20,I60 and I100 infection comparison groups of M.nudus,respectively.GO enrichment analysis showed that cellular anatomical entity,organelle membrane and intracellular membrane-bounded organelle were the most abundant and significantly enriched in the above three infection comparison groups,respectively,and the oxidative stress response in M.nudus was regulated by a large number of DEGs in the middle and late stages of infection.KEGG enrichment analysis showed that the majority of DEGs in M.nudus at the early stage of infection were associated with immune-related pathways,and those in the middle and late stages of infection were mainly concentrated in different disease pathways.The expression analysis of oxidative stress and apoptosis related genes demonstrated that the immune regulation of M.nudus against V.coralliilyticus infection was mediated by differential regulation of antioxidants and promoted oxidative stress,which induced apoptosis in coelomocytes.(4)By tandem mass spectrometry labeling(TMT)quantitative proteomics sequencing,2,188,2,386 and 2,516 differentially expressed proteins(DEPs)were obtained in the I20,I60 and I100 infection comparison groups of M.nudus,respectively.GO classification showed roughly identical functional annotations in the three infection comparison groups,mainly related to cellular process,cellular anatomical entity,binding and catalytic activity.Subcellular localization prediction indicated that DEPs were mainly localized in the cytoplasmic region,with the number of cytoplasmic DEPs varied between infection periods.GO enrichment analysis showed that up-regulated DEPs were significantly enriched in the categories of membrane,cellular component,cellular anatomical entity and molecular function regulator,and down-regulated DEPs were significantly enriched in the categories of oxidoreductase activity,catalytic activity,dioxygenase activity and various metabolic processes.KEGG enrichment analysis revealed that up-regulated DEPs were significantly enriched in multiple immune and disease pathways and down-regulated DEPs were significantly enriched in multiple metabolic pathways.i Path metabolic analysis revealed that carbohydrate metabolism,lipid metabolism,energy metabolism,amino acid metabolism,and nucleotide metabolism were the major biological processes and showed down-regulated expression.The expression of DEPs associated with oxidative phosphorylation was largely upregulated in the middle stage of infection.Comprehensive protein function analysis suggested that M.nudus under pathogenic infection reduced their metabolic processes to balance oxidative stress responses and promote organismal immune regulation.(5)Comparative analysis of transcriptome and proteome revealed a relatively low correlation between transcriptome and proteome,suggesting that post-transcriptional and post-translational regulation may play an important role in the immune regulatory mechanism of M.nudus.The joint GO classification of DEGs and DEPs showed that the binding and catalytic activity in molecular function may play an important role in M.nudus defense against pathogenic infection.KEGG pathway enrichment analysis showed that most upregulated DEGs and DEPs were involved in immune strategies and the lysosome and phagosome were the most important enrichment pathways at the m RNA and protein levels and were activated throughout the infection process.The significant increase in phagocytosis of infected M.nudus coelomocytes further demonstrated that the lysosome-phagosome pathway played an important immunological role in M.nudus resistance to pathogenic infection.Key gene expression profiles and protein?protein interaction analysis showed that cathepsin family(CLSL and CLSZ)and V-ATPase family(ATPe V0 D,ATPe VS1,ATPe V1 E and ATPe V1C)genes may play a central role in the lysosome-phagosome pathway.The q RT-PCR experiments verified the expression patterns of key immune genes,and the different expression trends of candidate genes reflected,to some extent,the regulatory mechanism of immune homeostasis mediated by the lysosome-phagosome pathway in M.nudus against pathogenic infection.In summary,the present study demonstrated the high pathogenicity of V.coralliilyticus to M.nudus,and further revealed the comprehensive physiological responses and immune regulatory mechanisms of M.nudus against V.coralliilyticus infection at levels of tissue physiology and molecular immunity.The findings add new knowledge to our understanding of the immune regulatory mechanisms of sea urchins under pathogenic stress and provide new information for the construction of the sea urchin integrative omics data platform and the optimization of disease prevention and control strategies in sea urchin farming. |
