| After millions of years of co-evolution,the symbiotic microbiota of aquaculture animals have become increasingly close to their hosts and play an important role in the health,immunity,and metabolism of their hosts.Understanding the characteristics of the symbiotic microbiota at these sites in different health states can facilitate the identification of health-and disease-specific bacteria,monitoring host health,understanding pathogenesis and screening effective probiotics.At present,there are few studies on the characteristics of symbiotic microbiota after host disease,and its composition,structure and function in the host disease state are also unclear.In this study,based on the high-throughput technology of 16 S rRNA gene,we compared the differences in the composition,structure,and predicted function,including exactly intestine,hemolymph,hepatopancreas and gills of Chinese mitten crab(Eriocheir sinensis)and intestine of yellow catfish(Pseudobagrus fulvidraco)in healthy and diseased states.Meanwhile,performed proteomic analysis of the hepatopancreas of Chinese mitten crab.The main findings are presented as follows:1.Characteristics of hemolymph,hepatopancreas and intestinal symbiotic microbiota of healthy Chinese mitten crab:Results showed no significant differences in microbial diversity in healthy hemolymph and hepatopancreas(p > 0.05),but they were significantly higher than in the intestine,and distinct differences were found in the structure among three sites(p < 0.05).The composition and abundance of symbiotic microbiota in the hemolymph and hepatopancreas were extremely similar,and no significant difference in functional prediction was found(p < 0.05),but both were significantly different in the intestine(p > 0.05).The dominant phyla in three sites were Proteobacteria,Firmicutes,Bacteroidota and Acidobacteriota.The distribution of the microbiota in the hemolymph was relatively uniform at the genus level,with relative abundance below 5%;the hepatopancreas were mainly composed of Candidatus Hepatoplasma,Shewanella and Aeromonas;the dominant genus in the intestine were Candidatus Bacilloplasma,Dysgonomonas and Rosemary.Compared with hemolymph and intestine,the differences in hemolymph were smaller among individuals,mainly reflected in the more uniform abundance of major bacterial taxa,smaller coefficient of variation,and the highest proportion of shared genera.All 12 samples shared 177 genera common to the three anatomical sites,mainly were Candidatus Hepatoplasma,unclassified＿Alphaproteobacteria and Acinetobacter.Network complexity varied greatly among the three sites,the hepatopancreas microbiota was the most complex,followed by the hemolymph microbiota,and the intestinal microbiota had the simplest network.The functional group function prediction results revealed that xenobiotics biodegradation and metabolism,energy metabolism,and terpenoid and polyketide metabolism were significantly enriched in the hemolymph compared with the intestine and hepatopancreas.Moreover,porphyrin and chlorophyll metabolism were significantly enriched in the hepatopancreas whereas environmental information processing,glycan biosynthesis and metabolism,human disease,and bacterial infectious disease were significantly enriched in the intestine(p < 0.05).2.Characteristics of gill and hepatopancreas microbiota and hepatopancreas protein expression in Chinese mitten crab with “shui-bie-zi” disease: The structure of the hepatopancreas and gill microbiota were significantly different compared with healthy crabs(p < 0.05),but not in the microbial diversity(p > 0.05).The relative abundance of Proteobacteria were significantly increased and Firmicutes were significantly decreased in the gills and hepatopancreas of diseased crabs.Further analysis revealed that 22 OTUs in the hepatopancreas of diseased crabs were significantly changed,in which the relative abundance of Spiroplasma was significantly reduced and Pseudomonas was significantly increased;8 OTUs were significantly changed,including the significant decrease of Spiroplasma and Albimonas.In addition,a total of 29 differentially expressed proteins were found in the hepatopancreas of healthy and diseased crabs,of which 6were up-regulated and 23 were down-regulated.Differentially expressed protein annotation results show down-regulated of protein expression related to metabolic and immune functions in hepatopancreas.The relative abundance value of some potential opportunistic pathogens,such as Pseudomonas,Brucella,Brevundimonas,Vibrionimonas and Stenotrophomonas were negatively correlated with most of the differentially expressed proteins.3.Research on the influence of “milk disease” on the composition,structure and predicted function of the digestive system microbiota of Chinese mitten crab: The structure of the hepatopancreas and gill microbiota were significantly changed compared with healthy crabs(p <0.05),but no significant differences in microbial diversity(p > 0.05).At phyla level,the relative abundance of Firmicutes were significant increased,and Proteobacteria were significant decreased in the hepatopancreas;the relative abundance of Firmicutes and Bacteroidota were significant decreased,and Proteobacteria were significant increased in the intestine compared with the healthy group.At genus level,the relative abundance of Achromobacter and Pedobacter were significant decreased,and Candidatus Bacilloplasma were significant increased in the hepatopancreas;the relative abundance of ZOR0006 and Candidatus Bacilloplasma were significant decreased in the intestine compared with the healthy group.Co-occurrence network results showed that the negative correlation of interaction network of digestive system microbiota in diseased group was significantly increased as compared with that in healthy group.Further functional predictions of the microbiota showed a significant decrease in the abundance of hepatopancreas microbiota with metabolic and immune-related functions and a significant increase in the abundance of repair-related functions compared with healthy crabs,and a significant decrease in the abundance of intestinal microbiota of diseased crabs with metabolism of cofactors and vitamins,biosynthesis of other secondary metabolites,and a significant enrichment in functions such as xenobiotics biodegradable and metabolism,signal transduction and cancer overvie.4.Characterization of the gut microbiota of yellow catfish(Pelteobagrus fulvidraco)infected with Edwardsiella ictaluri: The gut microbial richness and diversity of the diseased fish were significantly lower than those of healthy fish,and the microbial community structure was significantly changed(p < 0.05).The composition and predicted function of yellow catfish intestine microbiota were drastically altered after infection by Edwardsiella ictaluri.Fusobacteriota,Proteobacteria,and Firmicutes were the predominant bacterial phyla in the intestine of the healthy fish,while Proteobacteria was the dominant phylum in the intestine of the diseased fish.At the genus level,the intestine of healthy fish was dominated by Cetobacterium,Plesiomonas,and Romboutsia,while the intestine of diseased fish was overwhelmed by the pathogenic Edwardsiella ictaluri(99.22% ± 0.85%),and Cetobacterium,Plesiomonas,and Romboutsia disappeared.The same sequence of Edwardsiella ictaluri was detected from the intestine of the healthy and diseased fish and from the liver of the diseased fish.In addition,Bug Base phenotype prediction results showed,the anaerobic and gram-positive bacteria were significantly decrease,and the abundance of pathogenicity,biofilm-forming ability,and mobile elements were significantly increased.Functional prediction results showed,the digestive system,immune system,and metabolic functions of the intestine microbiota were significantly reduced in the diseased fish intestine. |
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