基于中肠代谢组学意大利蜜蜂工蜂枣花病的发病机理研究

Study on the pathogenesis of jujube flower disease in Apis mellifera ligustica workers based on midgut metabolomics

【目的】利用代谢组学分析患枣花病意大利蜜蜂Apis mellifera ligustica工蜂中肠内代谢物变化,挖掘出与蜜蜂枣花病相关的重要代谢通路,旨在揭示蜜蜂枣花病发病机制,为枣花病靶向药物的研发提供理论依据。【方法】分别利用液相色谱-质谱联用(liquid chromatography-mass spectrometry,LC-MS)和气相色谱-质谱联用(gas chromatography-mass spectrometry,GC-MS)技术对患枣花病和健康意大利蜜蜂工蜂进行中肠非靶向代谢组检测;通过主成分分析(principal component analysis,PCA)和正交偏最小二乘法判别分析(orthogonal partial least squares discriminant analysis,OPLS-DA),以变量投影重要性(variable projection importance,VIP)>1.0,差异倍数(fold change,FC)>2.0和P<0.05为标准筛选差异代谢物,并对其进行KEGG注释和通路富集;利用生化方法测定意大利蜜蜂工蜂中肠中超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)的活性,并通过qRT-PCR测定抗氧化基因Sod1,Sod 2和CAT及免疫基因Defensin,Hymenoptaecin,Apidaecin和Abaecin在中肠内的表达量。【结果】基于LC-MS代谢组检测,正离子和负离子模式下分别获得患枣花病和健康意大利蜜蜂工蜂间差异代谢物49和43种,其中分别有33和30种能够在HMDB和KEGG数据库中鉴定到。基于GC-MS代谢组检测,患枣花病和健康意大利蜜蜂工蜂间筛选到22种挥发性差异代谢物,其中11种能够在HMDB和KEGG数据库中鉴定到。与健康意大利蜜蜂工蜂相比,患枣花病意大利蜜蜂工蜂中肠内经鉴定的73种差异代谢物中,28种含量上升,45种含量下降;其中,京尼平苷酸、吲哚-3-乙酸、喹啉和棉子糖在患枣花病意大利蜜蜂工蜂中肠内大量积累,而乌索酸、L-苏糖酸、葡萄糖酸、D-半乳糖酸、苏糖酸和芦丁的含量下降最为明显。KEGG代谢通路富集分析结果表明,患枣花病和健康意大利蜜蜂工蜂间差异代谢物显著富集在抗坏血酸和醛酸代谢、半乳糖代谢及丁酸甲酯代谢这3条碳水化合物代谢通路。酶活性测定及qRT-PCR结果显示,与健康意大利蜜蜂工蜂相比,患枣花病意大利蜜蜂工蜂中肠中SOD和CAT酶活显著降低,Sod1,Sod2,CAT,Defensin,Hymenoptaecin和Apidaecin的表达量均呈下调趋势。【结论】利用LC-MS和GC-MS非靶向代谢组学均能够有效地分析枣花病影响下蜜蜂中肠内代谢物的变化差异。研究结果表明,意大利蜜蜂采集枣花后其中肠内碳水化合物代谢发生异常,引起肠道抗氧化和免疫能力显著下降,推测肠道功能障碍是导致蜜蜂死亡的主要原因。该研究为蜜蜂枣花病的发病机理提供了新见解。

【Aim】Changes in midgut metabolites of the Italian honeybee,Apis mellifera ligustica workers suffering jujube flower disease were analyzed using metabolomics and important metabolic pathways related to jujube flower disease were excavated,so as to reveal the pathogenesis of jujube flower disease and provide a theoretical basis for the research and development of targeted medicine for jujube flower disease.【Methods】Liquid chromatography-mass spectrometry(LC-MS)and gas chromatography-mass spectrometry(GC-MS)were used respectively to perform midgut non-targeted metabolomics detection on jujube flower disease-suffered and healthy A.m.ligustica workers.Through principal component analysis(PCA)and orthogonal partial least squares discriminant analysis(OPLS-DA),the differential metabolites were screened by variable projection importance(VIP)>1.0,fold change(FC)>2.0,and P<0.05.KEGG annotation and pathway enrichment were conducted to all differential metabolites screened.The activities of superoxide dismutase(SOD)and catalase(CAT)in the midgut of A.m.ligustica workers were determined by biochemical methods,and the expression levels of antioxidant genes Sod1,Sod 2 and CAT,and immune genes Defensin,Hymenoptaecin,Apidaecin and Abaecin in the midgut of A.m.ligustica workers were determined by qRT-PCR.【Results】According to the metabolomics detection by LC-MS,49 and 43 differential metabolites between jujube flower disease-suffered and healthy A.m.ligustica workers were obtained,of which 33 and 30 could be identified in HMDB and KEGG databases under the positive ion mode and negative ion mode,respectively.Based on GC-MS metabolomics detection,22 volatile differential metabolites between jujube flower disease-suffered and healthy A.m.ligustica workers were screened,among which 11 were identified in HMDB and KEGG databases.The contents of 28 of the 73 differential metabolites identified in the midgut of jujube flower disease-suffered A.m.ligustica workers increased,and those of 45 decreased as compared to those in healthy A.m.ligustica workers.Among them,geniposidic acid,indole-3-acetic acid,quinoline and raffinose accumulated in large quantities in the midgut of the jujube flower disease-suffered A.m.ligustica workers,while the contents of ursolic acid,L-threonic acid,gluconic acid,D-galactonic acid,threonic acid and rutin decreased most significantly.The analysis result of KEGG metabolic pathway enrichment showed that differential metabolites between jujube flower disease-suffered and healthy A.m.ligustica workers were significantly enriched in three carbohydrate metabolic pathways,namely ascorbate and aldarate metabolism,galactose metabolism,and butanoate metabolism.Enzyme activity determination and qRT-PCR results indicated that the activities of SOD and CAT in the midgut of jujube flower disease-suffered A.m.ligustica workers were significantly decreased,and the expression levels of Sod1,Sod2,CAT,Defensin,Hymenoptaecin and Apidaecin showed a downward trend as compared with those of healthy A.m.ligustica workers.【Conclusion】Both LC-MS and GC-MS non-targeted metabolomics can effectively analyze the changes of metabolites in the midgut of honeybees under the influence of jujube flower disease.The research results indicate that the abnormal carbohydrate metabolism in the honeybee midgut after collecting jujube flower caused a significant decline in the intestinal antioxidant and immune capacities,speculating that intestinal dysfunction may be the main cause of honeybee death.The research provides new insights into the pathogenesis of honeybee jujube flower disease.