Abstract:Pacific white shrimp, , is the most popular shrimp species cultured worldwide. Previous studies have indicated that high stocking densities negatively affect growth and survival rates, production, water quality, and immune parameters but promote disease outbreaks. Bacteria play a key role in organic contamination. However, little is known about the stress of stocking density on the rearing pond microbial community. Accordingly, the purpose of this investigation was to reveal the effect of shrimp stocking density on microbial community structure in intensive ponds as part of a recirculating aquaculture system. Water samples were collected from a high-density pond (200 ind/m2) and a low-density pond (133 ind/m2) every 15 days during the rearing period and filtered through 0.22-μm polycarbonate filters to collect bacterial cells. Environmental genomic DNA was extracted from the membrane filters. The microbial communities were profiled by high-throughput sequencing of the V4-V5 hypervariable region of 16S rRNA gene using the MiSeq Illumina sequencing platform. The results showed that 1264 operational taxonomic units (OTUs) were obtained via high-throughput sequencing and bioinformatics analyses. The mean number of bacterial OTUs was higher in the high-density pond (549.4) than that in the low-density pond (431.8). Clear differences in relative abundance among bacterial phyla were detected in different samples. Five phylogenetic subgroups were the most dominant in the entire bacterial community, including Proteobacteria, Cyanobacteria, Bacteroidetes, Actinobacteria, and Planctomycetes, accounting for > 98% of all OTUs. The mean Shannon-Wiener diversity index for the high-density pond (4.36) was higher than that for the low-density pond (3.51), and bacterial diversity increased gradually over timein both rearing ponds. This result suggests that new dominant bacterial populations appeared fromthe high nutrient input during the later rearing period, leading to higher bacterial diversity at a higher nutrient level. The relative abundance of Planctomycetes was significantly higher in the high-density pond ( < 0.01) during the initial rearing period than that in the low-density pond, which may have contributed to degradation of more residual feed in the high-density pond. The multidimensional scaling plot and cluster analysis demonstrated that all samples grouped into three clusters, indicating clear differences in the bacterial community structure between the high- and low-density ponds during the initial 50 days of rearing. However, the bacterial communities in the two ponds were more similarduringthe late stage compared with the marked differences during the initial stage, suggesting an initial stage (days 0-50) and a late stage (days 50-80), based on bacterial community structure. The effect of different stocking densities on the bacterial community decreased during the late stage.