Abstract:  【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, Sod2 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.

Key words: Apis mellifera ligustica, jujube flower disease, midgut, metabolomics, antioxidant, immune