Dynamic Changes Of Bacterial Community In Shellfish Culture And The Immune Function Of CARD-containing Protein In Oyster Crassostrea Gigas

The aquaculture of shellfish is an important part of aquaculture in China.With the maturation of culture technology,the scale of culture is increasing,and its economic and social benefits are becoming increasingly prominent.In recent years,the outbreak of shellfish diseases frequently caused huge economic losses to the shellfish industry,which seriously restricted the sustainable development of shellfish industry.The occurrence of shellfish diseases is the result of interaction among pathogens,hosts and environment.Bacterial infection is one of the main causes leading to the death of shellfish.Therefore,it is of great theoretical and practical significance to study the dynamic changes of bacterial community and host immune genes in shellfish aquaculture waters,and to reveal the causes of shellfish disease from two levels of microecology as well as host.In this study,the dynamic changes of bacterial communities in shellfish aquaculture waters,the internal relationship between bacterial community and environmental factors,changes of shellfish and shellfish diseases were studied by high throughput sequencing,molecular biology and immunological methods,and the immunological function of CARD gene in oyster Crassostrea gigas.The results of study were listed as follows:The temporal and spatial dynamic changes of bacterial community in three regions of the northern Yellow Sea,Q,Z1 and Z2,were analyzed.The results of diversity analysis showed that there were obvious seasonal differences in bacterial community,and the abundance of bacteria in spring and summer were obviously higher than that in autumn and winter.The dominant bacteria in the three regions were similar,including Proteobacteria,Bacteroidetes,Cyanobacteria,Firmicutes,Verrucomicrobia,Actinobacteria and Planctomycetes,which accounted for more than 90% of the total abundance of bacteria in the culture area.At the level of target classification,there are a large number of unclassified and nonculturable bacteria,indicating that there are many potential new bacterial species and shellfish pathogenic bacteria in the shellfish culture system.The cluster analysis based on the classification level of the genus showed that the dominant bacteria groups in the same season of the same breeding area were different,and the dominant bacteria in different sampling sites in the same season also had obvious differences.Redundancy analysis(RDA)of bacterial community and environmental factors showed that the temperature,dissolved oxygen and chlorophyll were the main factors affecting the structure of the bacterial community in the culture environment of shellfish.The change of gene copy number of pathogenic bacteria showed that the number of common pathogenic bacteria Vibrio splendidus and Pseudomonas fluorescens in the three culture areas increased in 5,6,7 months.The analysis of the above data shows that the occurrence of shellfish diseases may be due to the rapid increase in the abundance of pathogenic bacteria caused by changes in the bacterial flora under the influence of environmental factors,thus increasing the probability of the occurrence of shellfish diseases.A new CARD gene(CgCARDCP-1)was identified from the oyster Crassostrea gigas.Its open reading frame(ORF)was 759 bp and encoding a peptide containing 253 amino acids.The molecular weight of the protein is about 28.79 kDa.There is a CARD domain in the N-terminal of CgCARDCP-1 and two PC4 domains at the C-terminal.Multiple sequence alignment analysis showed that CgCARDCP-1 had 30%-46% similarity with caspase-2 from other species.Phylogenetic tree analysis showed that Cg CARDCP-1 was clustered with other invertebrates CARD-containing protein,and the mRNA transcripts of CgCARDCP-1 were found to be widely distributed in all tissues of oyster with the highest expression level in hemocytes and mantle.Subcellular localization analysis showed that CgCARDCP-1 was mainly distributed in the cytoplasm.Moreover,the CgCARDCP-1 mRNA expression level in hemocytes was significantly up-regulated after lipopolysaccharide(LPS)and Vibrio splendidus stimulations.The recombinant CgCARDCP-1 displayed strong binding activity with LPS in vitro.In addition,after transfected into the HEK-293 T cell with luciferase reporter system,CgCARDCP-1 could significantly promote the NF-?B activation(1.29-fold,p < 0.05)compared to that in the control group.The results of this study indicate that CgCARDCP-1 can be used as a new pattern recognition receptor to participate in immune recognition and participate in the regulation of transcriptional activation.