循环水养殖系统中凡纳滨对虾肠道微生物对三种复合益生菌制剂的响应

Effects of three biological agents on intestinal microbiota of white shrimp (Litopenaeus vannamei) in a water-recirculating cultivation system

摘要:

[目的]在循环养殖系统中应用不同的复合益生菌制剂，探讨凡纳滨对虾肠道菌群结构特征及免疫水平发生的变化。[方法]30 d养殖周期结束后，通过平板计数法分析肠道细菌总数和弧菌总数；基于高通量测序技术分析肠道样品V3+V4区菌群特征；采用实时荧光定量PCR技术分析免疫相关因子TLR1和Dorsal基因表达水平，阐述益生菌制剂应用的意义。[结果]益生菌制剂的应用降低了凡纳滨对虾肠道中细菌总数，抑制弧菌的生长，间接预防疾病的发生。不同益生菌制剂从不同程度上优化了凡纳滨对虾肠道菌群结构，提高高质量序列和有效OTU数量，Chao1、Simpson、Shannon指数显示了丰富度和多样性变化，复合益生菌制剂3效果较好。同时，菌群结构得到优化，其中益生菌制剂1组对拟杆菌门含量百分比产生显著影响。Toll受体TLR1和Toll通路中的Dorsal基因mRNA表达受到益生菌制剂的影响，1、3组促进了TLR 1表达，1、2、3组都促进了Dorsal基因表达。[结论]在循环水养殖系统中添加益生菌制剂可优化凡纳滨对虾肠道菌群特征，提高免疫水平，为病害防控和健康养殖提供理论依据。

Abstract:

[Objective] We studied the effects of three biological agents on intestinal microbiota and immunity of white shrimp (Litopenaeus vannamei) in a water-recirculating cultivation system.[Methods] The composition of three biological agents were Bacillus subtilis, Candida utilis, nitrifying bacteria and photosynthetic bacteria (treatment 1); Proteinase, Acidophilus spp., Bacillus subtilis and Faecalis spp. (treatment 2); and Protease, yeast, Bacillus subtilis and Lactobacillus acidophilus (treatment 3). After feeding 30 days, the total numbers of bacteria and Vibrio in the intestine of L. vannamei were calculated using plate counting. The high throughput sequencing of 16S rDNA gene's V3+V4 region was used to determine diversity and composition of bacterial communities. Real time fluorescence quantitative PCR was used to analyze the expression levels of Toll-like receptor 1 (TLR1) and Dorsal.[Results] After the application of biological agents, the total number of bacteria in the intestine of L. vannamei was reduced to 7.22 CFU/g, and the growth of Vibrio was inhibited (5.01 CFU/g) in treatment 3. In treatment 3, the percentage of high sequences and Operational Taxonomic Unit (OTU) were increased to 98.8% and 326, Chao1, Simpson, Shannon was 581.34, 0.94, 5.52 respectively, which displayed the changes of bacterium abundance and diversity in intestine. Furthermore, the community structure of intestinal microbiota was optimized. Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, Firmicutes, Candidatus Saccharibacteria and Cyanobacteria were the main dominant phyla. However, the proportion of Bacteroidetes was significantly increased (40.7%) in the treatment 1 (P<0.05). The different expression levels of Toll-like receptor 1 and Dorsal were changed in different probiotics. In treatment 3, the expression level of Toll-like receptor 1 (TLR1) was 3.2 (P<0.05), which was 220% higher than control, meanwhile, the expression level of Dorsal was 4.7 (P<0.05), which was 370% higher than control.[Conclusion] Our study revealed that biological agents played an active role in the recirculating system, which could optimize the structure of intestinal microbiota and improve the expression levels of immune genes. In this case, these biological agents could serve as candidates for diseases prevention and healthy aquaculture. And the treatment 3 was the better.