Abstract
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-picrylhyclrazyl (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.
概要
目的
提取和纯化球囊菌黑色素, 并系统研究其理化性质、分子结构、抗氧化活性和生物学功能, 为病原真菌黑色素的鉴定提供完整的流程标准, 同时为后续研究该黑色素在球囊菌感染蜜蜂中的作用提供参考。
创新点
首次提取和鉴定球囊菌黑色素, 并建立了一套系统的鉴定真菌黑色素的流程。
方法
通过酸水解结合碱熔解法提取和纯化黑色素, 并综合采用紫外-可见光谱、红外光谱、拉曼光谱、气相色谱、核磁共振技术、扫描电子和透射电镜、自由基清除等分析法, 系统、全面地研究黑色素的溶解度、颜色值、元素组成、化学结构、生物活性、亚细胞定位和抗氧化活性。同时, 采用三环唑抑制黑色素的合成研究黑色素与孢子感染能力之间的关系。
结论
球囊菌黑色素属于真黑色素, 定位于孢子壁, 具有抗氧化活性, 且能螯合金属离子, 但是它不能增强孢子毒力。
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Acknowledgments
This work was supported by the Science and Technology Project of the Chongqing Municipal Education Commission (No. KJZD-K202100502), the Natural Science Foundation Project of Chongqing (No. cstc2021jcyj-msxmX0422), the Higher Education Teaching Reform Research Project of the Chongqing Municipal Education Commission (Nos. 213132 and KJ173061), the Postgraduate Education and Teaching Reform Research Project of Chongqing Normal University (No. xyjg21012), the Creation & Research Team in College and Universities of Chongqing Municipal Education Commission (No. CXQT21013), the College Student Innovation and Entrepreneurship Training Program Project of the Chongqing Municipal Education Commission (No. 202110637013), and the Natural Science Foundation Project of Chongqing Normal University (No. 13XLB009), China.
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Zhi LI and Zeyang ZHOU conceived the research and designed experiments. Zhi LI, Hui HENG, and Qiqian QIN performed the experiments and analysis. Zhi LI, Hui HENG, Lanchun CHEN, and Yuedi WANG interpreted the data and wrote the paper. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Zhi LI, Hui HENG, Qiqian QIN, Lanchun CHEN, Yuedi WANG, and Zeyang ZHOU declare that they have no conflict of interest.
The animal experimental processes were approved by the Ethnic Committee of College of Life Sciences, Chongqing Normal University and conducted in strict accordance with the standard of the Guide for the Care and Use of Laboratory Animals published by the Ministry of Science and Technology of the People’s Republic of China in 2006.
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Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis
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Li, Z., Heng, H., Qin, Q. et al. Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis. J. Zhejiang Univ. Sci. B 23, 365–381 (2022). https://doi.org/10.1631/jzus.B2100718
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DOI: https://doi.org/10.1631/jzus.B2100718