Study On The Ecological Relationship Between Different Tissues And Microorganisms And Their Molecular Responses In Mytilus Coruscus

Mussel is one of the most important shellfish with economic value in China,and it is also the fourth largest cultured mussel in China.Previous studies have shown that mussels are generally highly adaptable to the environment,including greater tolerance to disease,suggesting that mussels must have a strong innate immune system to ensure the survival of the mussel in a marine environment rich in a variety of microorganisms.Although the research on mussel innate immune system has been done deeply,the ecological relationship between mussel and microorganism and the immune response process and mechanism induced by microorganism in different tissues of mussels are still poorly understood.Mytilus Coruscus was used to study the interaction between M.coruscus and the microorganisms in different tissues.In order to study the differences of microbial community structure in different tissues of mussel,we collected 7 tissues of M.coruscus cultured in Shengsi breeding area.These tissues included hemolymph,digestive glands,kidneys,gills,mantles,gonads and feet.Through the 16 S r RNA gene amplicon sequencing targeting the V3-V4 variable region on the Illumina Mi Seq platform,we characterized the community structure and distribution of microbiota.On average,a total of 36,860high-quality reads were obtained from 21 samples of M.coruscus.The highest number of OTU(1,237)was detected in hemolymph,followed by digestive glands and kidneys(1,014 and 1,015 OTUs),and the gonad was the lowest(only 553 OTUs).Despite the number of OTU in hemolymph was the highest,there was only 9 unique OTUs in hemolymph.The kidneys had a maximum of 172 unique OTUs,followed by digestive glands(144 unique OTUs).Compositional distribution showed that Proteobacteria,Bacteroidetes and Verrucomicrobia were the main phyla in all tissues.The Bray-Curtis distance results indicated that the bacterial composition of hemolymph,digestive gland,kidney and foot were clustered and the bacterial community from gills,gonads and mantle had higher similarity.Analysis of alpha diversity indicated that the microbiota in hemolymph,digestive glands and kidneys were more diverse than in other tissues.The results indicated that there was a strong tissue specificity in the microbial community of different tissues of mussel,which indicated that there were differences in microbial enrichment and colonization among different tissues of mussel,it also suggests that different tissues of mussels may have different immune response mechanisms to microorganisms.In order to further understand the immune response process and molecular mechanism of different tissues of M.coruscus to different microorganisms,the gill and gonad tissues of M.coruscus were collected,and the transcriptome analysis of gill and gonad tissues was carried out after different microbial induction.Considering that the mussel tissues are naturally rich in a large number of microorganisms,low concentration of mixed antibiotics was used to remove the influence of these background microorganisms on the follow-up experiments,the test mussels were pretreated with antibiotics.The results showed that low concentration of mixed antibiotics could effectively remove microorganisms in mussel tissues,and had no significant effect on blood cell counts.The antibiotic treated mussels were further induced by Micrococcus luteus and Vibrio parahaemolyticus,representing Gram-positive bacteria and Gram-negative,respectively.The gill and gonad of M.coruscus were collected at 0.5 and 2 hours after induction,and a total of 36 c DNA libraries were sequenced and 245.01 Gb clean data was acquired with an average of 5.91 GB and Q30 >94% for each library.The filtered high-quality reads were assembled into 91,850 transcripts,which after clustering resulted in 59,206 unigenes.Overall,19,783 differentially expressed genes(DEGs)were detected from the pair-wise comparisons among the gill samples,and12,655 DEGs among the gonad samples.Comparing with the gonad,the gill showed strong immune-related responses towards bacteria challenge,with abundant immune-related signaling pathway being disturbed,highlighting the important roles of gill in mussel immune system and the tissue specificity of mussel immune responses.The gonad showed that the DEGs induced by bacteria were mainly enriched in regulatory pathways related to cell growth and proliferation.In addition,both gill and gonad presented rapid activation towards M.luteus infection comparing with that of V.parahemolyticus,indicating a more sensitivity of mussel to Gram-positive bacteria.Our data revealed different immune responses in different mussel tissues under different bacteria challenges,and thus provided clues for exploring the mechanism of mussel immune system.