Mantle-Mucus Microbial Communities and Metabolic Responses to Vibrio Stress in the hard clam Meretrix meretrix
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摘要: 文蛤外套膜组织及其粘液在防御病原体中起着重要作用，然而它们自身固有菌群组成和功能与宿主免疫之间的潜在联系尚不清晰。本研究采用16S rRNA基因高通量测序技术，比较分析健康文蛤外套膜组织（M组）与外套膜粘液（N组）的菌群组成及潜在功能，利用非靶向代谢组学（UHPLC-Q-TOF/MS）分析弧菌胁迫下粘液代谢物变化，并通过斯皮尔曼相关性分析联合菌群与代谢物数据，初步探索菌群与宿主免疫的潜在关联。结果显示，外套膜和粘液的菌群存在显著生态位分化：外套膜的菌群丰富度更高，以螺旋体门（Spirochaetota）为绝对优势菌；粘液的菌群均匀度更高，以变形菌门（Proteobacteria）和拟杆菌门（Bacteroidota）为主，并显著富集了弧菌属（Vibrio）、黏着杆菌属（Tenacibaculum）与黄杆菌属（Flavobacterium）等具降解多糖或潜在致病能力的类群。功能预测显示，粘液菌群在半胱氨酸/甲硫氨酸代谢、氧化磷酸化等免疫与能量通路中更为活跃。弧菌胁迫下代谢组结果表明，粘液中存在琥珀酸、丙酸、苯丙氨酸等多种免疫相关代谢物；菌群与代谢物相关性分析揭示了固有微生物与宿主代谢物的紧密联系，如海单胞菌属（Marinomonas）与尿嘧啶强正相关，黄杆菌属与硝酸盐强负相关。综合表明，外套膜-粘液作为一个动态界面微环境，其特定菌群结构可能通过与宿主代谢互作，为机体应对病原体入侵提供了免疫准备。Abstract: The mantle tissue and its mucus of the hard clam Meretrix meretrix play a significant role in defending against pathogens. However, the potential links between the composition and function of their inherent microbiota and host immunity remains unclear. In this study, 16S rRNA gene high-throughput sequencing was used to compare the microbiota composition and potential functions between the mantle tissue (group M) and mantle mucus (group N) in health clams. Non-targeted metabolomics (UHPLC-Q-TOF/MS) was employed to analyze metabolite changes in the mantle mucus under Vibrio stress. Furthermore, spearman correlation analysis was applied to integrate the microbiota and metabolomics data, aiming to preliminarily explore potential associations between the microbiota and host immune metabolism. The results revealed significant niche differentiation between the mantle and mucus microbiota. The mantle microbiota exhibited higher richness and was dominated by the phylum Spirochaetota, while the mucus microbiota showed higher evenness, with Proteobacteria and Bacteroidota as the dominant phyla. The mucus was significantly enriched in taxa with polysaccharide-degrading or potential pathogenic capabilities, such as the genera vibrio, Tenacibaculum and Flavobacterium. Functional prediction indicated that the mucus microbiota was more active in immune- and energy-related pathways, like cysteine and methionine metabolism and oxidative phosphorylation. Metabolomic analysis under Vibrio stress showed significant alterations in various immune-related metabolites in the mucus, including succinate acid, propionate acid, and phenylalanine. Correlation analysis between microbiota and metabolites revealed close associations between resident microbes and host metabolites, such as a strong positive correlation between the genus Marinomonas and uracil, and a strong negative correlation between Flavobacterium and nitrate. Collectively, these findings suggest that the mantle-mucus complex functions as a dynamic interfacial microenvironment. Its specific microbial community structure may interact with host metabolism, providing immunological preparedness for the host to counteract pathogen invasion.
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Key words:
- Meretrix meretrix /
- mantle /
- mucus /
- bacterial community /
- metabolomics
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图 1 文蛤外套膜（M）和粘液（N）菌群的聚类及β多样性分析
A. 所有样品的稀释曲线；B. 所有样品的Shannon-Wiener曲线；C. 所有样品的等级-丰度曲线；D. 所有样本的物种积累曲线；E. PCoA分析; F. NMDS分析
Fig. 1 Clustering and β diversity analysis of the mantle (M) and mucus (N) microbiota in M. meretrix
A. Rarefaction curves of all samples; B. Shannon-Wiener curves of all samples; C. Rank-abundance curves of all samples; D. Species accumulation curves of all samples; E. PCoA analysis; F. NMDS analysis
图 2 文蛤外套膜（M）和粘液（N）细菌群落的α-多样性指数
Fig. 2 The α -diversity index of the bacterial communities in the mantle (M) and mucilage (N) in M. meretrix
图 3 文蛤外套膜（M）和粘液（N）菌群在各分类水平的平均相对丰度分布情况
A. 门水平上分类组成；B. 科水平上分类组成；C. 属水平上分类组成
Fig. 3 Average relative abundance distribution of the microbial communities in the mantle（M） and mucus（N） at different taxonomic levels in M. meretrix
A．Taxonomic composition at the phylum level; B. Taxonomic composition at the family level; C. Taxonomic composition at the genus level
图 4 文蛤外套膜和粘液菌群在属水平上的组间方差分析柱状图
“**”表示差异极显著 （P<0.01）
Fig. 4 Bar chart of intergroup analysis of variance (ANOVA) of the microbiota in the mantle and mucus of M. meretrix at the genus level
“**” indicates the significant difference in the level of P<0.01
图 5 文蛤外套膜和粘液菌群LEfSe分析结果
A.LDA值分布直方图; B. 进化分枝图
Fig. 5 LEfSe analysis results of the mantle and mucus bacterial communities of M. meretrix
A. Cladogram; B. Histogram of LDA value distribution.
图 6 文蛤外套膜和粘液菌群的KEGG代谢途径分析
A. 二级功能水平；B. 三级功能水平
Fig. 6 KEGG metabolic pathway analysis of the mantle and mucus microbiota in M. meretrix.
A. Secondary functional level; B. Tertiary functional level
图 7 文蛤弧菌胁迫后外套膜组织切片
A，B. 外套膜区域Masson染色；C，D. 外套膜外褶区域AB-PAS染色。图A、C为对照组，图B、D为副溶血弧菌感染组。CFS：胶原纤维结构，CM：肌纤维和胞质，VS：液态结构，MV：微绒毛，AM：酸性黏液细胞；比例尺为100 μm
Fig. 7 Histological sections of mantle tissues after Vibrio infection in M. meretrix.
A, B. Masson's trichrome staining of the mantle region; C, D. Alcian blue-periodic acid-Schiff (AB-PAS) staining of the outer fold region of the mantle. A and C were the control groups, and B and D were the Vibrio infected groups. CFS: collagen fiber structure, CM: cytoplasmic matrix and muscle fibers, VS: liquid (-phase) structure, MV: microvilli, AM: acidic mucus; Scale bar = 100 μm.
图 8 基于UHPLCQ-TOF/MS的12份粘液样本和3份QC样本分析
A. 基于正离子模式的主成分分析图；B. 基于正离子模式的样本相关性热图；C. 基于正离子模式的偏最小二乘法判别分析的评分图；D. 基于负离子模式的主成分分析图；E. 基于负离子模式的样本相关性热图；F. 于负离子模式的偏最小二乘法判别分析的评分图
Fig. 8 12 mucus and 3 quality control (QC) sample analysis based on UHPLC-Q-TOF/MS
A. Principal component analysis (PCA) score plot in positive ion mode; B. Sample correlation heatmap in positive ion mode; C. Partial least squares-discriminant analysis (PLS-DA) score plot in positive ion mode; D. PCA score plot in negative ion mode; E. Sample correlation heatmap in negative ion mode; F. PLS-DA score plot in negative ion mode
图 9 基于正、负离子模式Top20代谢物的聚类热图
C1-C6为对照组；E1-E6为实验组
Fig. 9 Clustered heatmap of the Top 20 metabolites.
C1-C6 are the control group；E1-E6 are the experimental group
图 10 正、负离子模式下差异代谢物的KEGG富集气泡图（A）和MetPA拓扑分析（B）
Fig. 10 KEGG enrichment bubble chart (A) and MetPA topological analysis (B) of differential metabolites in positive and negative ion modes
图 11 文蛤外套膜粘液优势菌属与代谢产物的相关性热图
“*”表示差异显著 （P<0.05），“**”表示差异极显著 （P<0.01），“***”表示差异极显著 （P<0.001）
Fig. 11 Correlation heatmap between the dominant bacterial genera and metabolites in the mucus of M. meretrix.
“*” indicates the significant difference in the level of P<0.05, “**” indicates the significant difference in the level of P<0.01, and “***” indicates the significant difference in the level of P<0.001
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