| As an important aquaculture prawn species,Penaeus chinensis is widely cultivated in northern China due to its high-added value and delicious taste.At present,multi-species polyculture is the main culture mode of Penaeus chinensis along the Liaoning coast.The microbial community in the aquaculture environment is an important part of the ecosystem,and closely related to the health of the aquaculture species and the environmental quality.A healthy microecological status is of positive significance for preventing and controlling diseases and ensuring a smooth aquaculture process.A healthy microbial community has a certain degree of functional redundancy,which can drive the decomposition of organic matter such as bait and feces and water purification through the carbon,nitrogen,and sulfur metabolism based on functional genes,and control the spread of antibiotic resistance genes(ARGs)simultaneously.This dissertation uses high-throughput amplicon and metagenomic sequencing technology to compare six different polyculture modes of Penaeus chinensis,analyzes the structure and function of microbial communities in the culture environment,and investigates the "microecological health" status of different polyculture modes.Aiming at the better polyculture mode,the microecological characteristics at different times and in different environmental media during the whole aquaculture cycle,as well as the existence form and change of functional genes and ARGs were studied.The main research contents are as follows:Firstly,using the "microecological health" status to evaluate the different polyculture modes of Penaeus chinensis.The composition of the sediment microbial community structure of six different polyculture modes was compared using bioinformatics analysis.α-diversity analysis showed that richness and diversity of the microbial communities in the ponds decreased as the following order: Penaeus chinensis-Rhopilema esculentum-Ruditapes philippinarum> Penaeus chinensis-Portunus trituberculatus>Penaeus chinensis-Stichopus japonicus-Penaeus monodon>Penaeus chinensis-Rhopilema esculentum-Sinonovacula constricta>Penaeus chinensis-Rhopilema esculentum-Ruditapes philippinarum-Penaeus monodon>Penaeus chinensis-Rhopilema esculentum-Sinonovacula constricta-Paralichthys olivaceus.In all the investigated polyculture environments,the dominant bacteria phyla were Proteobacteria and their abundances were all higher than 47.4%.Except for the polyculture model of Penaeus chinensis-Rhopilema esculentum-Ruditapes philippinarum,other polyculture models may have the risk of producing excessive hydrogen sulfide or methane.The abundance of pathogenicity-related genes in the polyculture model of Penaeus chinensis-Rhopilema esculentum-Ruditapes philippinarum-Penaeus monodon,Penaeus chinensis-Portunus trituberculatus,and Penaeus chinensis-Rhopilema esculentum-Sinonovacula constricta-Paralichthys olivaceus polyculture are all higher than3%,indicating that these polyculture modes present disease risk.Taking the richness,diversity,functional microorganisms and pathogenic bacteria abundances of the microbial community as the key indicators to measure the "microecological health" of the polyculture environment of Penaeus chinensis,the "microecological health" status of the polyculture of Penaeus chinensis and benthic invertebrates or shellfish(Ruditapes philippinarum,Sinonovacula constricta,Rhopilema esculentum)is significantly better than other polyculture modes.Secondly,in order to clarify the correlation between "microecological health" status and aquaculture time,the polyculture model of Penaeus chinensis-Penaeus monodon-Stichopus japonicus was selected to analyze the time correlation of aqueous microbial community structure.Cluster analysis found that the species richness and diversity of the aqeous microbial communities in the first half of aquaculture(from June to July)and the second half of aquaculture(from August to September)are significantly different.The main bacterial genera enriched in the water in July include Pelagibacter and Pseudoalteromonas,with abundances of 23.0% and 7.8%,respectively.In August samples,higher abundance of Pseudomonas and Clostridium was found.The abundances of them were 4.5% and 11.1%,respectively.And the abundance of Clostridium further increased to 14.6% in September.The increase in the abundance of Pseudomonas and Pseudoalteromonas helped to inhibit the growth and outbreak of algae.The enrichment of Pseudomonas and Clostridium was closely related to the ammonia and nitrite removal in water.The second half of the "microecological health" of the polyculture model of Penaeus chinensis-Penaeus monodon-Stichopus japonicus is better than that of the first half,which needs to be controlled during the whole aquaculture cycle.Thirdly,the differences of the microbial communities’ composition in water and sediments over the full cycle of the polyculture model of Penaeus chinensis-Rhopilema esculentum-Sinonovacula constricta were investigated.The results showed that through the aquaculture process,the diversity and richness of microbial communities in the two environmental media showed an upward trend,and the richness and diversity of microbial communities in the aqueous phase was significantly lower than that of sediment samples.The aqueous microbial communities are more susceptible to change in the external environment.However,the impact of environmental factors on the microbial communities in water and sediments is not significant.Function annotation of microbial community found that the function of microbial community in sediments is mainly related to diseases and basic metabolism,and there is a risk of disease outbreaks.It is necessary to strengthen the monitoring and control of the environmental quality of the sediments in aquaculture ponds.Fourthly,all the information of microbial functional genes in water and sediment samples in the polyculture environment of Penaeus chinensis-Rhopilema esculentum-Sinonovacula constricta were obtained using metagenomic sequencing in months.Analyzing the abundance and types of functional genes showed that the microbial communities in water and sediments are quite different and the microbial functional genes present seasonal characteristics.Genes related to nitrogen assimilation,organic nitrogen decomposition,and dissimilated nitrate reduction are distributed in both water and sediments,but the types and abundances are significantly different.Genes related to nitrate reduction/nitrite oxidation,denitrification and nitrogen fixation mainly exist in sediments.The abundance of Arnon cycle-related functional genes accounts for the highest proportion of all carbon fixation pathways,suggesting that it may be the main carbon fixation process in polyculture environments.Sulfate assimilation,sulfide assimilation and sulfur oxidation process related genes are widely present in water and sediments,whereas the types and abundances of specific functional gene are quite different.The sulfate reduction process mainly occurs in sediments,among which Desulfatitalea,Desulfobacter and Desulfofustis are the main sulfate-reducing bacteria,and dsr AB,qmo ABC and phs C are the main functional genes.Through the above analysis,the complete carbon,nitrogen,and sulfur metabolic function network driven by microorganisms in the polyculture environment is clarified.Fifthly,the antibiotic resistome in the polyculture pond of Penaeus chinensis-Rhopilema esculentum-Sinonovacula constricta was studied,and the changes in the types and abundance of ARGs during the entire culture cycle were obtained.The results showed that the total amount of ARGs in the water changed greatly during the whole aquaculture cycle,while those in the sediments were relatively stable.The ARGs with the highest content in water and sediment are EF-Tu and ala S,respectively.Analyzing the correlation between the microbial species and the ARGs types found that the Actinomycota are the main hosts of ARGs,and an abundance of less than 1% can contribute to 8% of ARGs.The co-occurrence analysis of functional genes and ARGs revealed that the main ARGs in the polyculture environment are closely related to some of the sulfur cycle functional genes.Therefore,focusing on the control of Actinomycota and sulfate-reducing bacteria in sediments can effectively prevent and control the spread of ARGs,which is beneficial to maintaining the ecological health of the aquaculture environment. |
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