| Summary: | The fungus <i>Ascosphaera apis</i>, an obligate fungal pathogen of honey bee brood, causes chalkbrood disease in honey bee larvae worldwide. Biological characteristics of the fungal pathogen and the molecular interactions between <i>A. apis</i> and honey bees have been studied extensively. However, little is known about the effects of <i>A. apis</i> infection on antioxidant enzyme activities and metabolic profiles of the gut of honey bee larvae. In this study, sandwich enzyme-linked immunosorbent assay and LC-MS based untargeted metabolomic analysis were employed to determine the changes in the specific activities of antioxidant enzymes and the metabolomic profiles in gut tissues of <i>A. apis</i>-infected larvae (10<sup>5</sup> <i>A. apis</i> spores per larva) and controls. Results showed that specific activities of superoxide dismutase, catalase and glutathione S-transferase were significantly higher in the guts of the control larvae than in the guts of the <i>A. apis</i>-infected larvae. The metabolomic data revealed that levels of 28 and 52 metabolites were significantly higher and lower, respectively, in the guts of <i>A. apis</i>-infected larvae than in the guts of control larvae. The 5-oxo-ETE level in the infected larvae was two times higher than that in the control larvae. Elevated 5-oxo-ETE levels may act as a potential metabolic biomarker for chalkbrood disease diagnosis, suggesting that <i>A. apis</i> infection induced obvious oxidative stress in the honey bee larvae. The levels of metabolites such as taurine, docosahexaenoic acid, and L-carnitine involved in combating oxidative stress were significantly decreased in the gut of <i>A. apis</i>-infected larvae. Overall, our results suggest that <i>A. apis</i> infection may compromise the ability of infected larvae to cope with oxidative stress, providing new insight into changing patterns of physiological responses to <i>A. apis</i> infection in honey bee larvae by concurrent use of conventional biochemical assays and untargeted metabolomics.
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