Effects Of Oyster Shell-loading On Water Variables And Bacterial Community In Shrimp (Litopenaeus Vannamei) Culture System

Litopenaeus vannamei is one of the most important aquaculture shrimp species worldwide.However,its aquaculture is threatened by environmental pollution and various diseases in recent years,which has seriously affected the development of shrimp aquaculture.The application of biofilm can remove toxic ammonium and nitrite and reduce the risk of disease outbreak by decreasing water exchange in shrimp aquaculture.Among the many biofilm carriers,oyster(Crassostrea gigas)shells are good carriers for biofilm because of their chemical stability,large specific surface areas,local abundance,and economic availability.Microbes in oyster shell biofilm play important roles in improving aquaculture water quality,but the process and mechanisms of this remain elusive.Hence,based on the 16 S r RNA gene amplicon sequencing,this study focused on the effects of oyster shell-loading on bacterial communities in both shrimp nursery and on growing culture system.We also analyzed the effects of oyster shell-loading on bacterial nitrogen and phosphorus metabolism potential by using PICRUSt2 analysis.The main results are as following:(1)In the experiment of oyster shell-loading on growing shrimp culture,the concentration of ammonium,nitrite and chlorophyll-a decreased significantly.Shrimp survival rate of oyster shellloading group(Treatment group)reached 97.2%,which was significantly higher than 92.5% in control group.The relative abundance of Alphaproteobacteria increased significantly(P < 0.05)in treatment group,whereas the relative abundances of Actinobacteria,Deltaproteobacteria,and Cyanobacteria decreased significantly.Water temperature,salinity,ammoniu,and chlorophyll-a were the most important water variables driving bacterial community variation in treatment group.The bacterial co-occurrence networks in treatment group contained 361 nodes linked by 2639 edges.Node properties including the degree and betweenness centrality were significantly higher than those in control group,while closeness centrality values were lower in treatment group.The co-occurrence networks of both groups were mainly occupied by Flavobacteriaceae and Rhodobacteraceae.The bacterial community assembly mechanisms were analyzed by Sloan neutral model,finding that neutral processes dominated the bacterial community assembly,and85.5% and 84.5% of the operational taxonomic units(OTUs)occurred neutrally in control and treatment group,respectively.(2)In the experiment of oyster shell-loading(two treatment groups,1% and 2% of water body)on shrimp nursery,the concentration of ammonium and nitrite decreased significantly,while shrimp larval survival rate increased.The effects of 2% loading group were better than 1% loading group.Oyster shell-loading increased the diversity of bacterial communities in water and significantly affected bacterial community composition,for instance,the relative abundances of Flavobacteriaceae and Halomonadaceae increased significantly.Bacterial co-occurrence networks in oyster shell biofilm were more complicate than those in water,and bacterial co-occurrence networks in water could be increased by oyster shell,which may be beneficial for the stability of water bacterial community.Based on the Sloan neutral model analysis,the results showed that the importance of neutral processes decreased in treatment groups in water bacterial community assembly.About 86.3%,79.2% and 79.0% of the water bacterial OTUs distributed neutrally in control,1% and 2% loading group,respectively.For shrimp bacterial community,about 76.9%OTUs occurred in neutral in 2% loading group,which were higher than 73.8% in control.(3)The main genes related to nitrogen metabolism in water of on growing shrimp culture were NADH dehydrogenase,NADH quinone oxidoreductase and denitrification genes.The genes related to phosphorus metabolism were GAPDH,his C,pls C and ppx-gpp A.The genes of nitrogen metabolism in the water of shrimp larvae culture were the genes encoded nitrite and nitrous oxide reductase.For phosphorus metabolism in the water of shrimp larvae culture,the genes were GAPDH,his C,thi E,pls C and ppx-gpp A.Oyster shells-loading in both shrimp larvae and on growing shrimp culture had significant impacts on the denitrification,nitrite and nitrate assimilation and dissimilation in water,which affected the succession of genes related to nitrogen and phosphorus metabolism remarkably.The genes of nitrogen metabolism in biofilm of shrimp larvae culture were also the genes encoded nitrite and nitrous oxide reductase.The main genes related to phosphorus metabolism were GAPDH,his C,pls C,ppx-gpp A and pst S.The denitrification and nitrite dissimilation processes were significant different in two oyster shellloading groups indicating that oyster shell contents could affect nitrogen metabolism in biofilm.In summary,this study showed that oyster shell-loading can significantly improve water quality and survival rate of shrimp.Further,the succession patterns and assembly mechanisms of bacterial communities in water,shrimp larvae and biofilm were deeply investigated,and the correlation between water variables,bacterial communities in water,shrimp larvae and biofilm,and shrimp survival rate were also clearly elucidated.These results were valuable to reveal the mechanisms of oyster shell-loading in regulating shrimp culture in terms of microbial ecological processes,providing scientific supports for oyster shell application in the whole aquaculture cycle.In addition,the use of PICRUSt2 further revealed the changes of nitrogen and phosphorus metabolism potential,which is of great significance for elucidating the effect of oyster shellloading on bacterial community in shrimp culture.