Study On The Diversity Of Coelomic Fluid Bacterial Community In Apostichopus Japonicus

The sea cucumber Apostichopus japonicus,a highly economically valuable echinoderm,is mainly cultured along the northern coast of China.In recent years,the rising water temperature and the constant outbreak of diseases,such as skin ulcer syndrome(SUS),have imposed formidable threats on the healthy development of the A.japonicus farming industry.The bacterial community in the organism plays an extremely important role in the maintenance of normal growth and development,metabolism and immunity of the organism.The coelomic fluid of A.japonicus contains coelomocytes and a variety of immunologically active factors all considerably affect the response to environmental stimuli,intestinal regeneration and immune defense.Due to the characteristics of intestine regeneration and aestivation of the A.japonicus,studies on their symbiotic microorganisms mainly concentrate on intestinal bacteria.Therefore,little is known about the community diversity and function of coelomic fluid bacteria of A.japonicus.The preliminary data indicate that there is an abundant bacterial community in the coelomic fluid of A.japonicus.Hence,the current study focuses on the coelomic fluid bacterial community of A.japonicus with the application of 16 s r RNA sequencing technology.Firstly,the structure and composition of the bacterial community in the coelomic fluid of A.japonicus was identified through the establishment of a recovery model after antibiotic treatment.Subsequently,efforts are made to investigate the temporal changes of the bacterial community structure in coelomic fluid after evisceration and their potential functions in the metabolic homeostasis during intestine regeneration.We then applied 16 s r RNA sequencing technology was then employed to detect the coelomic fluid bacterial community structure in response to heat stimulation for the sake of further investigation of the potential adaptation mechanism to temperature change.Finally,an we established a A.japonicus SUS model by V.splendidus infection,measured immune response and the changes in coelomic fluid bacterial community structure with the development of SUS was built to explore the possible pathogenic mechanism of SUS.These results of the current study will:elucidate for the first time the coelomic fluid bacterial community composition and its diversity from A.japonicus for the first time;reveal coelomic fluid bacterial community dynamics under multiple environmental stimulations;figure out the correlation between coelomic fluid bacterial communities.The main findings are as follows:(1)Reconstruction of coelomic fluid bacterial community in A.japonicus after antibiotic treatmentIn this section,the presence of bacterial community in the coelomic fluid of A.japonicus was firstly determined by plate testing and bacterial copy counting.The bacterial community was then eliminated by antibiotic treatment,and then cultured in normal seawater to investigate the recovery process of bacterial population,bacterial community diversity,species distribution and community structure during in the coelomic fluid.The results showed that the bacterial population was gradually recovered after the A.japonicus was cultured 96 h in normal seawater,while the bacterial community diversity and richness were also close to the control group.At 24 h post treatment,the bacterial community had the lowest diversity index and greatest difference from the control.The abundance of probiotics Rhodobacteraceae and Flavobacteriaceae plunged significantly at 24 h,while the abundance of pathogenic bacteria Streptococcus and Enterobacteriaceae surged considerably.The structure of bacterial communities in other groups exhibited similar trends which differed greatly from the control group at 24 h,and then gradually converged to the control group at 96 h.In addition,LDA analysis illuminated that the abundance of Bacillus sp.was significantly higher than the other groups at 24 h,indicating that Bacillus sp.may potentially affect in the recovery and maintenance of the bacterial communities.Together,the above results suggested that the bacterial community in the coelomic fluid was high stabilized and has the capability to recover after being disrupted.(2)Temporal dynamics of bacterial community in the coelomic fluid of A.japonicus after eviscerationIn this section,a model of A.japonicus evisceration by intracelomic injection of 0.35 M KCl was established to study the coelomic fluid bacterial community dynamics during intestine regeneration.It is found that the structure of bacterial community in coelomic fluid of A.japonicus changed significantly with lower diversity and total bacterial load at 7 dpe(days post evisceration)and 14 dpe.The bacterial community tended to restore at 28 dpe.In particular,the relative abundances of Bacteroidetes and Rubritaleaceae,which were reported to be involved in degradation of polysaccharides and lipid,were significantly increased at 7dpe(p < 0.05),and returned to the original level at 28 dpe.In addition,the functional prediction of bacterial community by Kyoto Encyclopedia of Genes and Genomes(KEGG)also demonstrated that the bacteria associated with metabolism pathways of amino acid,lipid and carbohydrate,were significantly increased at 7 dpe.These results together suggested that the bacterial community in coelomic fluid of A.japonicus is highly dynamic and can rebuild a stable community structure after evisceration.The enriched metabolic related beneficial bacteria at early stage may function after evisceration in terms of decomposing polysaccharides and lipid to provide energy.(3)Structural and functional analysis of coelomic fluid bacterial community in response to heat stress of A.japonicusThis study aimed to investigate the effects of heat stress on the coelomic fluid bacterial community composition and function of the sea cucumber with the help of high-throughput sequencing.The results indicated that coelomic fluid bacterial communities responded significantly to heat,as indicated by apparent reduction in the richness and diversity of coelomic fluid bacterial community(P < 0.05).Several potential pathogens,such as Staphylococcus,Vibrio and Pseudomonas,were found to increase in terms of their dominance,which might aggravate the risk of disease.The functional profiling prediction based on the Clusters of Orthologous Groups(COGs)database showed that the bacterial community related to inorganic ion transport and metabolism,cellular signaling processes,information storage and processing,cell motility,and carbohydrate transport and metabolism were enhanced,which may be conducive to adapt to the high temperature.In the KEGG pathway analysis,44 pathways were found to be significantly altered after heat stress.The main differences in the KEGG pathways were bacterial community that enriched in metabolism,cell motility,and environmental information processing pathways.These results demonstrated that heat stress might contribute to the pathogenesis of A.japonicus by increasing the pathogenic bacteria and the bacterial community might adapt to the high temperature by changing its structure and group behavior,particularly on functional proteins and metabolic pathways.(4)Changes of coelomic fluid bacterial community during the pathogenesis of SUS in A.japonicusIn the present study,sea cucumber SUS was induced by the immersion of seawater with V.splendidus.This study collected the coelomic fluid from A.japonicus at different status based on the severity of SUS(SUS-I and SUS-II).16 S r RNA sequencing results revealed significant differences in the bacterial phylotypes and community structure between the coelomic fluid of healthy and diseased A.japonicus,which were correlated with the severity of SUS.Notably,probiotics Bifidobacterium and pathogenic bacteria Fusobacterium displayed opposite trends in healthy and SUS groups,with higher abundance of Fusobacterium and lower Bifidobacterium in SUS groups.However,Vibrio spp.were found to have relative low abundance in SUS groups.Psychrobacter,which belongs to Pseudomonadales,was the keystone species among all disease samples that had the most complex network relationships,which was especially high in SUS-II groups.KEGG enrichment further illustrated that bacteria enriched in bacterial-mediated infectious diseases and signal transduction pathways were more abundant in SUS groups compared to control group.Overall,this study suggested that changes in coelomic fluid bacterial communities were closely associated with the severity of SUS,in which Psychrobacter might be a potential indicator taxon for the evalutation of the health status of A.japonicus.In summary,this thesis had established several models and taken the advantage of 16 s r RNA high-throughput sequencing technology as well as the application of multiple molecular biology techniques to study the dynamics and functions of coelomic fluid bacterial community of A.japonicas.Firstly,it identified the structure,composition,and stability of coelomic fluid bacterial community through antibiotic treatment.Secondly,we investigated the dynamic changes of the bacterial community in the coelomic fluid after stimulated by different environmental factors(regeneration,high temperature and V.splendidus),and explored the potential functions of bacterial community in different conditions.Results of this study will contribute to providing new insights into the relationship between coelomic fluid bacterial community and the health status of A.japonicus,which will lay theoretical foundation for the development of new strategies of disease prevention and probiotic application that are beneficial to healthy aquaculture development.