Ascosphaera apis spores containing a dark-colored pigment infect honeybee larvae, resulting in a large-scale collapse of the bee colony due to chalkbrood disease. However, little is known about the pigment or whether it plays a role in bee infection caused by A. apis. In this study, the pigment was isolated by alkali extraction, acid hydrolysis, and repeated precipitation. Ultraviolet (UV) analysis revealed that the pigment had a color value of 273, a maximum absorption peak at 195 nm, and a high alkaline solubility (7.67％) and acid precipitability. Further chemical structure analysis of the pigment, including elemental composition, Fourier transform infrared (FTIR) spectroscopy, Raman spectroscopy, mass spectrometry, and nuclear magnetic resonance (NMR), proved that it was a eumelanin with a typical indole structure. The molecular formula of melanin is C10H6O4N2, and its molecular weight is 409 Da. Melanin has hydroxyl, carboxyl, amino, and phenolic groups that can potentially chelate to metal ions. Antioxidant function analyses showed that A. apis melanin had a high scavenging activity against superoxide, hydroxyl, and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and a high reducing ability to Fe3+. Indirect immunofluorescence assay (IFA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analyses showed that A. apis melanin was located on the spore wall. The spore wall localization, antioxidant activity, and metal ion chelating properties of fungal melanin have been suggested to contribute to spore pathogenicity. However, further infection experiments showed that melanin-deficient spores did not reduce the mortality of bee larvae, indicating that melanin does not increase the virulence of A. apis spores. This study is the first report on melanin produced by A. apis, providing an important background reference for further study on its role in A. apis.

Zhi LI;Hui HENG;Qiqian QIN;Lanchun CHEN;Yuedi WANG;Zeyang ZHOU

College of Life Sciences,Chongqing Normal University,Chongqing 401331,China;Chongqing Key Laboratory of Vector Insects,Chongqing 401331,China;Chongqing Key Laboratory of Animal Biology,Chongqing 401331,China;Chongqing Key Laboratory of Microsporidia Infection and Control,Chongqing 400715,China;The State Key Laboratory of Silkworm Genome Biology,Southwest University,Chongqing 400715,China

浙江大学学报（英文版）（B辑：生物医学和生物技术）

[1]Zhi LI;Hui HENG;Qiqian QIN;Lanchun CHEN;Yuedi WANG;Zeyang ZHOU-.Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis)[J].浙江大学学报（英文版）（B辑：生物医学和生物技术）,2022(05):365-381,中插1-中插4

chalkbrood,precipitability,eumelanin,C10H6O4N2

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