皱纹盘鲍在转口期阶段肠道菌群结构演替研究

Succession of the intestinal microflora structure of Haliotis discus hannai during the weaning period

关键词:  皱纹盘鲍  转口期  肠道菌群  多样性  功能预测

Key words:  Haliotis discus hannai  Weaning period  Intestinal microflora  Diversity  Function prediction

摘要:

转口期是皱纹盘鲍(Haliotis discus hannai)早期育苗的重要时期，本研究结合16S rDNA高通量测序和生物信息学技术，分析皱纹盘鲍转口前(T0)、转口后第4天(T4)、第10天(T10)、第35天(T35)和第40天(T40)的肠道菌群结构和多样性。结果显示，皱纹盘鲍T35和T40的肠道菌群多样性低于T0和T4。主成分分析(PCA)显示，T0~T4、T35~T40肠道菌群结构相似度高，T10属于转口期肠道菌群演替变化的过渡阶段。鲍转口期肠道内优势菌门为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes)；优势菌属在转口期不同阶段呈现明显的变化，到后期逐渐趋于稳定。Lentilitoribacter是T0和T4的主要优势菌属；到了T10，弓形菌属(Arcobacter)和弧菌属(Vibrio)的相对丰度增加，Lentilitoribacter减少；在转口后期(T35和T40)，弧菌属和Formosa则占绝对优势。LEfSe分析共获得59个生物标志物，各个组的生物标志物与其阶段的优势菌群相对应。共发生网络图结果显示，T35阶段的网络关系最为复杂，皱纹盘鲍肠道菌群之间的关系随着转口期的进行发生改变。Tax4Fun功能预测显示，随着转口期的进行，稚鲍肠道菌群所编码的基因多与代谢有关。本研究首次以微生物学的角度揭示了转口期皱纹盘鲍对饵料的适应机制，为皱纹盘鲍的健康养殖奠定了理论基础。

Abstract:

Pacific abalone Haliotis discus hannai is an economically important aquaculture species in China, whose production accounts for approximately 93% of world abalone aquaculture production. Its weaning phase is a vulnerable life stage associated with high mortality, which has seriously impeded the sustainable development of Chinese abalone aquaculture. Previous research has shown that the intestinal microflora in farmed abalone is affected by numerous abiotic and biotic factors. The weaning post-settlement of diatom-fed abalone on artificial feed may alter the natural succession of microflora in their guts. To study the succession of the intestinal microbiota in the weaning of Pacific abalone, we collected Pacific abalone at the weaning period days 0 (T0), 4 (T4), 10 (T10), 35 (T35), and 40 (T40) by 16S rRNA high-throughput sequencing. Results showed no significant differences in alpha diversity (Shannon, Simpson, ACE, and Chao1) between T0, T4, and T10 and between T35 and T40. The ACE and Chao1 indices tended to decrease with sampling time. Both Good’s coverage values exceeded 99.50%, indicating that the sequence libraries covered most of the microbial community in these samples. A total of 3609 OTUs were identified across all samples after pre-processing, and the unique OTUs tended to decline in the weaning phase, varying from 419 OTUs in T4 to 169 OTUs in T40. The beta diversity of intestinal microbiota showed that the T0/T4 and T35/T40 samples were clustered separately in principal coordinate analysis, with overlaps between T0 and T4, T35 and T40; T10 was in the transitional stage of intestinal microflora succession during the weaning period. In terms of the composition and structure at the phylum level, the dominant bacterial groups in diatom-fed abalone and weaning abalone were relatively consistent, including Proteobacteria, Bacteroidetes, and Firmicutes. The ratio of Proteobacteria decreased, while that of Bacteroidetes increased with the time of weaning. The composition of the dominant genera during the diatom feeding and weaning stages differed significantly at the genus level. The dominant genera of the diatom-feeding stage (T0) included Lentilitoribacter (14.18%), Dinoroseobacter (9.90%), and Neptuniibacter (9.86%). During the weaning stage, Lentilitoribacter (19.70%), Pseudoalteromonas (9.86%), and Arcobacter (5.52%) were the dominant genera in the T4 group; Lentilitoribacter (6.95%), Arcobacter (8.15%), and Vibrio (7.50%) were the dominant genera in the T10 group; and Formosa and Vibrio were the most dominant genera in the T35 and T40 groups. To study the impact of diet change on the microbial communities of the weaning abalone, linear discriminant analysis (LDA) was used to analyze differences in taxon composition among the five sampling groups. A total of 59 biomarkers were identified (LDA>4.0, P<0.05). From T0 to T40, 9, 11, 17, 9, and 13 biomarkers were found, revealing that the dominant species of the intestinal microflora varied significantly over time. The intestinal microflora co-occurrence networks based on robust and significant correlations were constructed to explore synergetic relationships in the samples from T0, T4, T35, and T40. The values of the network topological characteristics, including node, edge, average degree, clustering coefficient, average path length, and modularity, were distinct at different sampling stages. The values at T0 and T35, except modularity, were higher than those in the other groups, indicating that microbial interaction may be more intensive in the T0 and T35 groups. The co-occurrence (positive) and co-exclusion (negative) patterns of microbial genera were distinct between these four groups, especially between T0 and T35/T40. Tax4Fun function predictions showed that the genes encoded by the intestinal microflora of T0 and T4 were mostly related to diseases and cell processing, while T10, T35, and T40 were mostly related to metabolic functions, indicating that the intestinal microbes were involved in various molecular metabolisms, assisting juvenile abalone in adapting to the nutrient-rich artificial feed. This study revealed the adaptation mechanism of H. discus hannai to diet change during the weaning stage from a microbiology perspective for the first time, laying a theoretical foundation for healthy abalone breeding.