Comparison Of Gut Bacteria Composition Between Healthy And Diseased Shrimp And Source Tracker Analysis At Different Growth Stages

Litopenaeus vannamei is one of the largest farming scale and the highest production aquacultured shrimp species globally,with an annual value of 24 billion dollars.In recent years,due to the immoderate pursuit of high yield,the culture model has been changed to high density and excessive feeding of bait,leading to the increased frequency and newly emerged diseases.Recent studies have shown that gut microbiome contributes important roles in affecting the colonization of pathogens and improving the adaptability of the host,thereby determining the health status of shrimp.For this reason,a large number of studies have focused on the environmental factors that affecting the shrimp gut microbiota.By contrast,we know little about the impact of biological factors,such as environmental and internal sources,on the gut microbiota.In this context,we compared the differences in gut bacterial communities between healthy and Enterocytozoon hepatopenaei(EHP)infected shrimp.Meanwhile,the environment and gut commensals of juvenile were used as the sources of adult gut microbiota.Source Tracker analysis was used to quantitatively calculate the relative contributions of external and internal sources to the shrimp gut microbiota.To achieve these purpose,healthy and EHP infected shrimp,as well as rearing sediment and water samples at shrimp juvenile and adult stages were collected at Hongye farm,Taizhou in 2019 and to analyze the bacterial community compositions.Meanwhile,we analyzed shrimp growth traits,digestive and immune enzyme activities.The mainly findings were as followings:(1)The body size and weight of EHP shrimp were significantly lower than these of healthy shrimp.In addition,the activities of immune enzyme,such as lysozyme and superoxide dismutase,were significantly stimulated in EHP shrimp compared with healthy cohorts.(2)To obtain gut and hepatopancreas tissues,healthy and diseased shrimp samples were dissected under sterile condition.The gut tissue was used for DNA extraction and the hepatopancreas was used to measure digestive and immune enzyme activities.Using amplicon sequencing technology,the compositions of gut bacterial community of healthy and diseased shrimp were analyzed.Structural equation model was used to explore the interrelationship among the gut bacterial community,growth traits,digestive and immune enzyme activities of healthy and EHP infected shrimp.The effects of disease on the interspecies interactions and potential functions of gut bacteria were also evaluated.Results showed that the α-diversity of gut bacterial community in healthy shrimp were significantly higher than that in shrimp infected with EHP disease.The structures of gut bacterial community formed different clusters and significant different between health status.The gut bacterial community and enzyme activities under the influence of EHP disease directly affected the growth traits of shrimp.By analyzing the interspecies interaction relationship of gut microbiota of healthy and diseased shrimps,we found that the average path(6.34)and modularity(0.79)of EHP infected shrimp were lower,and the average clustering coefficient(0.23)and average degree(3.34)were higher than these in healthy shrimp,indicating that interspecies interaction was more complex and better connected in EHP infected shrimp.Using PICRUSt to predict the potential function of shrimp gut microbiota,it was found that multiple metabolic pathways,cell growth and death,digestive and immune system in EHP infected shrimp were significantly decreased compared with healthy ones.(3)In order to analyze the sources of bacterial community in shrimp gut,we collected shrimp gut,rearing sediment and water samples of the culture environment at juvenile and adult stages.The indicator taxa among the three habitats(gut,rearing water and sediment)at each stage were screened by indicator value method.The dominant and ubiquitous taxa in different habitats were revealed by using the ternary plot(taxonomy operation unit level).Results demonstrated that the dominant(relative abundance > 1% at least in one group)phyla were Proteobacteria,Bacteroidetes,Tenericutes,Actinobacteria,and Cyanobacteria in three habitats.Relative abundances of these dominant phyla were significantly different among the three habitats.Consistent change trends were observed among the three habitats at juvenile and adult stages.By contrast,for a given dominant phylum,its relative abundance was relative stable in each habitat between juvenile and adult stages.Given that habitat was a fundamental factor in affecting the bacterial communities,we screened indicator populations that were responsible for the differences among the three habitats.As a result,12 indicator families were identified,which displayed different distribution patterns across habitats.There were significant difference in the bacterial community structure among shrimp gut,sediment and rearing water,and between the two shrimp life stages.The bacterial α-diversity was the highest in sediment at both stages,the α-diversity of bacteria community in the gut was significantly lower than that in rearing water at the juvenile stage.However,at the adult stage,the diversity was comparable between shrimp gut and rearing water.Only three(Alphaproteobacteria,Cyanobacteria,Bacteroidetes)and two dominant taxa were shared among the three habitats at the juvenile and adult stages,respectively.In contrast,more diverse taxa were unique in each habitat at both stages.(4)The sources of shrimp gut bacterial community at the juvenile and adult stages were quantitatively analyzed by Source Tracker analysis.The structural equation model was used to investigate the interaction among shrimp gut,sediment and water bacterial communities at the two stages.Results indicated that the contributions of bacterioplankton communities afforded minor sources to shrimp gut commensals,contributing 1.7% in juvenile and 3.0% in adult.In contrast,the bacterial community in sediment and the gut microbiota of juvenile were the main sources for adult gut bacteria,contributing 18% and 66.7%,respectively.Therefore,the gut microbiota of juvenile individuals were the main sources of adult shrimp,which has not been recognized in previous studies.