Analysis Of Microbial Diversity In Shrimp And Mollusc Polyculture Pond Environment Based On High-throughput Sequencing Technology

In recent years,multi-species polyculture farming has gradually become an emerging aquaculture mode with the rapid development of aquaculture,which could improve the water quality deterioration and the slow growth caused by long-term singlevariety and high-density farming.Microorganisms play a vital role in polyculture ecosystems,and they are closely related to water quality management,feed nutrition,and disease prevention and control.In this study,we collected 6 kinds of components,including deep-mud(20 cm below surface mud),shallow-mud(surface mud),oyster,shrimp,plankton,and water in the "shrimp and mollusc polyculture" pond.Prokaryotic and eukaryotic microorganisms in the samples were amplified by the 16 S and 18 S universal primers,and then PCR libraries were constructed for high-throughput sequencing.Bioinformatics analysis began after obtaining the rawdata.In order to learn the microbial diversity in the polyculture environment,a variety of tools and methods for analysis were used in this study.The main results were shown as follows:1.Exploration of methods for eukaryotic microorganisms in Litopenaeus vannamei and Crassostrea hongkongensis——Development of a blocking primer to inhibit the PCR amplification of the 18 S rDNA sequences of L.vannamei and C.hongkongensis.The blocking primer(X-BP2-DPO)designed for shrimp(L.vannamei)is a reliable,simple and efficient primer for inhibiting the amplification of 18 S rDNA sequences of shrimp and oyster host.The inhibition rate is 99% for the amplification of shrimp rDNA,and 17% for the amplification of oyster rDNA.In the case of a large amount of host DNA in the sample,the use of blocking primer greatly increases the number and variety of eukaryotic microorganisms detected in shrimp and oyster,and 0.6 ?M is the most suitable concentration for X-BP2-DPO to work.The development of blocking primers provided a valid method to study the composition and characteristics of eukaryotic microorganisms in shrimp and oyster guts,thus providing a good tool for the study of feed nutrition,disease prevention and control in aquaculture.2.The community structure and characteristics of microorganisms in the environment of "shrimp and mollusc polyculture" pond.A total of 4,095,794 sequences,6398 OTUs were obtained from 16 S sequencing,and 56 phyla and 1,570 genera were annotated.A total of 8,879,369 sequences and 1510 OTUs were obtained from 18 S sequencing,and 11 phyla and 552 genera were annotated.The dominant prokaryotic and eukaryotic species in s-mud,d-mud and plankton in the "shrimp and mollusc polyculture" pond are close,with high diversity;shrimp and oyster contain similar microbial composition with low diversity.The microbial community in the water contains complex composition with low prokaryotic diversity and high eukaryotic diversity,which is different from other components.The prokaryotic and eukaryotic groups with the most significant difference in each component were obtained by LEfSe(LDA Effect Size)analysis,which served as biomarkers for the community.3.Analysis of the relationship and interaction of microorganisms in "shrimp and mollusc polyculture" pond ecosystem based on co-occurrence network.Based on co-occurrence network analysis,it can be obtained that: There are close relations between prokaryotes and eukaryotes,most of which are positively correlated in the pond.The OTUs with low abundance could play an important role in the ecosystem.The network structure of microbes in s-mud and water is complex,followed by d-mud,plankton,shrimp and oyster.The microorganisms in different phyla are ubiquitous in the co-occurrence networks of various components,but the degree of association in different components is different.Based on the analysis of "bray-curtis similarity" method,it can be known that the microbial communities in d-mud and s-mud have a high degree of mutual influence;The interaction between oyster and shrimp is significant,and the influence of oyster on shrimp has time fluctuation;Microbes in plankton are affected by other components;And in water,microorganisms are more stable and less susceptible to other components.