Full Text:   <318>

Summary:  <9>

Suppl. Mater.: 

CLC number: 

On-line Access: 2022-05-13

Received: 2021-08-16

Revision Accepted: 2022-01-04

Crosschecked: 2022-05-13

Cited: 0

Clicked: 345

Citations:  Bibtex RefMan EndNote GB/T7714


Zhi LI


-   Go to

Article info.
Open peer comments

Journal of Zhejiang University SCIENCE B 2022 Vol.23 No.5 P.365-381


Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis

Author(s):  Zhi LI, Hui HENG, Qiqian QIN, Lanchun CHEN, Yuedi WANG, Zeyang ZHOU

Affiliation(s):  College of Life Sciences, Chongqing Normal University, Chongqing 401331, China; more

Corresponding email(s):   lizhicqnu@gmail.com

Key Words:  Melanin, Molecular structure, Antioxidant activity, Subcellular localization, Ascosphaera apis

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]. Journal of Zhejiang University Science B, 2022, 23(5): 365-381.

@article{title="Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis",
author="Zhi LI, Hui HENG, Qiqian QIN, Lanchun CHEN, Yuedi WANG, Zeyang ZHOU",
journal="Journal of Zhejiang University Science B",
publisher="Zhejiang University Press & Springer",

%0 Journal Article
%T Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis
%A Zhi LI
%A Qiqian QIN
%A Lanchun CHEN
%A Yuedi WANG
%A Zeyang ZHOU
%J Journal of Zhejiang University SCIENCE B
%V 23
%N 5
%P 365-381
%@ 1673-1581
%D 2022
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B2100718

T1 - Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis
A1 - Zhi LI
A1 - Hui HENG
A1 - Qiqian QIN
A1 - Lanchun CHEN
A1 - Yuedi WANG
A1 - Zeyang ZHOU
J0 - Journal of Zhejiang University Science B
VL - 23
IS - 5
SP - 365
EP - 381
%@ 1673-1581
Y1 - 2022
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B2100718

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.




Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article


[1]AisenP, EnnsC, Wessling-ResnickM, 2001. Chemistry and biology of eukaryotic iron metabolism. Int J Biochem Cell Biol, 33(10):940-959.

[2]AkoumianakiT, KyrmiziI, ValsecchiI, et al., 2016. Aspergillus cell wall melanin blocks LC3-associated phagocytosis to promote pathogenicity. Cell Host Microbe, 19(1):79-90.

[3]al KhatibM, HarirM, CostaJ, et al., 2018. Spectroscopic characterization of natural melanin from a Streptomyces cyaneofuscatus strain and comparison with melanin enzymatically synthesized by tyrosinase and laccase. Molecules, 23(8):1916.

[4]AronsteinKA, MurrayKD, 2010. Chalkbrood disease in honey bees. J Invertebr Pathol, 103(S1):S20-S29.

[5]ArunG, EyiniM, GunasekaranP, 2015. Characterization and biological activities of extracellular melanin produced by Schizophyllum commune (Fries). Indian J Exp Biol, 53(6):380-387.

[6]BaileyL, 1968. Honey bee pathology. Annu Rev Entomol, 13:191-212.

[7]BaoT, ZhangM, ZhouYQ, et al., 2021. Phenolic profile of jujube fruit subjected to gut microbiota fermentation and its antioxidant potential against ethyl carbamate-induced oxidative damage. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 22(5):397-409.

[8]BersuderP, HoleM, SmithG, 1998. Antioxidants from a heated histidine-glucose model system. I: Investigation of the antioxidant role of histidine and isolation of antioxidants by high-performance liquid chromatography. J Am Oil Chem Soc, 75(2):181-187.

[9]ButlerMJ, DayAW, 1998. Fungal melanins: a review. Can J Microbiol, 44(12):1115-1136.

[10]CamachoE, VijR, ChrissianC, et al., 2019. The structural unit of melanin in the cell wall of the fungal pathogen Cryptococcus neoformans. J Biol Chem, 294(27):10471-10489.

[11]CésariniJP, 1990. Hair melanin and hair color. In: Orfanos CE, Happle R (Eds.), Hair and Hair Diseases. Springer, Berlin, Heidelberg, p.165-197.

[12]ChenYG, ShenZJ, ChenXP, 2009. Evaluation of free radicals scavenging and immunity-modulatory activities of Purslane polysaccharides. Int J Biol Macromol, 45(5):448-452.

[13]CunhaMML, FranzenAJ, AlvianoDS, et al., 2005. Inhibition of melanin synthesis pathway by tricyclazole increases susceptibility of Fonsecaea pedrosoi against mouse macrophages. Microsc Res Tech, 68(6):377-384.

[14]DadachovaE, BryanRA, HuangXC, et al., 2007. Ionizing radiation changes the electronic properties of melanin and enhances the growth of melanized fungi. PLoS ONE, 2(5):e457.

[15]de la RosaJM, Martin-SanchezPM, Sanchez-CortesS, et al., 2017. Structure of melanins from the fungi Ochroconis lascauxensis and Ochroconis anomala contaminating rock art in the Lascaux Cave. Sci Rep, 7:13441.

[16]DixonDM, PolakA, ConnerGW, 1989. Mel- mutants of Wangiella dermatitidis in mice: evaluation of multiple mouse and fungal strains. J Med Vet Mycol, 27(5):335-341.

[17]EnochsWS, NilgesMJ, SwartzHM, 1993. A standardized test for the identification and characterization of melanins using electron paramagnetic resonance (EPR) spectroscopy. Pigment Cell Res, 6(2):91-99.

[18]FanQL, ChengK, HuX, et al., 2014. Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging. J Am Chem Soc, 136(43):15185-15194.

[19]FioreG, PoliA, di CosmoA, et al., 2004. Dopamine in the ink defence system of Sepia officinalis: biosynthesis, vesicular compartmentation in mature ink gland cells, nitric oxide (NO)/cGMP-induced depletion and fate in secreted ink. Biochem J, 378(3):785-791.

[20]FogartyRV, TobinJM, 1996. Fungal melanins and their interactions with metals. Enzyme Microb Technol, 19(4):311-317.

[21]GalvánI, Araujo-AndradeC, MarroM, et al., 2018. Raman spectroscopy quantification of eumelanin subunits in natural unaltered pigments. Pigment Cell Melanoma Res, 31(6):673-682.

[22]GeisslerC, SinghM, 2011. Iron, meat and health. Nutrients, 3(3):283-316.

[23]GómezBL, NosanchukJD, 2003. Melanin and fungi. Curr Opin Infect Dis, 16(2):91-96.

[24]GómezBL, NosanchukJD, DíezS, et al., 2001. Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection. Infect Immun, 69(9):5760-5767.

[25]GuoT, HouCL, WeiL, et al., 2010. Antioxidant activities of extracts and sub-fractions from Tuber indicum. Mycosystema, 29(4):569-575 (in Chinese).

[26]GuoX, ChenSG, HuYQ, et al., 2014. Preparation of water-soluble melanin from squid ink using ultrasound-assisted degradation and its anti-oxidant activity. J Food Sci Technol, 51(12):3680-3690.

[27]HeathL, GazeBM, 1987. Carbon dioxide activation of spores of the chalkbrood fungus Ascosphaera apis. J Apicult Res, 26(4):243-246.

[28]Hernández-ChávezMJ, Pérez-GarcíaLA, Niño-VegaGA, et al., 2017. Fungal strategies to evade the host immune recognition. J Fungi, 3(4):51.

[29]HillHZ, 1991. Melanins in the photobiology of skin cancer and the radiobiology of melanomas. In: Wilson SH (Ed.), Cancer Biology and Biosynthesis. Telford Press, Caldwell, p.31-53.

[30]HongL, SimonJD, 2007. Current understanding of the binding sites, capacity, affinity, and biological significance of metals in melanin. J Phys Chem B, 111(28):7938-7947.

[31]HuangL, LiuMY, HuangHY, et al., 2018. Recent advances and progress on melanin-like materials and their biomedical applications. Biomacromolecules, 19(6):1858-1868.

[32]JacobsonES, 2000. Pathogenic roles for fungal melanins. Clin Microbiol Rev, 13(4):708-717.

[33]KimYJ, WuW, ChunSE, et al., 2013. Biologically derived melanin electrodes in aqueous sodium-ion energy storage devices. Proc Natl Acad Sci USA, 110(52):20912-20917.

[34]Kwon-ChungK, PolacheckI, PopkinTJ, 1982. Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol, 150(3):1414-1421.

[35]LangfelderK, StreibelM, JahnB, et al., 2003. Biosynthesis of fungal melanins and their importance for human pathogenic fungi. Fungal Genet Biol, 38(2):143-158.

[36]LiC, JiCM, TangBP, 2018. Purification, characterisation and biological activity of melanin from Streptomyces sp. FEMS Microbiol Lett, 365(19):fny077.

[37]LiYW, XieYJ, WangZ, et al., 2016. Structure and function of iron-loaded synthetic melanin. ACS Nano, 10(11):10186-10194.

[38]LiZ, PanGQ, LiT, et al., 2012. SWP5, a spore wall protein, interacts with polar tube proteins in the parasitic microsporidian Nosema bombycis. Eukaryot Cell, 11(2):229-237.

[39]LiZ, YouXL, WangLL, et al., 2018. Spore morphology and ultrastructure of an Ascosphaera apis strain from the honeybees (Apis mellifera) in southwest China. Mycologia, 110(2):325-338.

[40]LiuQM, XiaoJJ, LiuBT, et al., 2018. Study on the prepar

[41]ation and chemical structure characterization of melanin from Boletus griseus. Int J Mol Sci, 19(12):3736.

[42]ManivasaganP, VenkatesanJ, SenthilkumarK, et al., 2013. Isolation and characterization of biologically active melanin from Actinoalloteichus sp. MA-32. Int J Biol Macromol, 58:263-274.

[43]McGrawKJ, SafranRJ, WakamatsuK, 2005. How feather colour reflects its melanin content. Funct Ecol, 19(5):816-821.

[44]MenonIA, PersadS, HabermanHF, et al., 1983. A comparative study of the physical and chemical properties of melanins isolated from human black and red hair. J Invest Dermatol, 80(3):202-206.

[45]MeredithP, SarnaT, 2006. The physical and chemical properties of eumelanin. Pigment Cell Res, 19(6):572-594.

[46]MoneyNP, Caesar-TonThatT, FrederickB, et al., 1998. Melanin synthesis is associated with changes in hyphopodial turgor, permeability, and wall rigidity in Gaeumannomyces graminis var. graminis. Fungal Genet Biol, 24(1-2):240-251.

[47]MontefioriDC, ZhouJY, 1991. Selective antiviral activity of synthetic soluble L-tyrosine and L-dopa melanins against human immunodeficiency virus in vitro. Antiviral Res, 15(1):11-25.

[48]Morris-JonesR, YoungchimS, GomezBL, et al., 2003. Synthesis of melanin-like pigments by Sporothrix schenckii in vitro and during mammalian infection. Infect Immun, 71(7):4026-4033.

[49]MorseRA, 1978. Honey Bee Pests, Predators, and Diseases. Comstock Publishing Associates, Division of Cornell University Press, Ithaca and London, p.430.

[50]NappiAJ, ChristensenBM, 2005. Melanogenesis and associated cytotoxic reactions: applications to insect innate immunity. Insect Biochem Mol Biol, 35(5):443-459.

[51]NosanchukJD, CasadevallA, 1997. Cellular charge of Cryptococcus neoformans: contributions from the capsular polysaccharide, melanin, and monoclonal antibody binding. Infect Immun, 65(5):1836-1841.

[52]NosanchukJD, CasadevallA, 2003. The contribution of melanin to microbial pathogenesis. Cell Microbiol, 5(4):203-223.

[53]NosanchukJD, CasadevallA, 2006. Impact of melanin on microbial virulence and clinical resistance to antimicrobial compounds. Antimicrob Agents Chemother, 50(11):3519-3528.

[54]NosanchukJD, StarkRE, CasadevallA, 2015. Fungal melanin: what do we know about structure? Front Microbiol, 6:1463.

[55]Peltroche-LlacsahuangaH, SchnitzlerN, JentschS, et al., 2003. Analyses of phagocytosis, evoked oxidative burst, and killing of black yeasts by human neutrophils: a tool for estimating their pathogenicity? Med Mycol, 41(1):7-14.

[56]Prados-RosalesR, ToriolaS, NakouziA, et al., 2015. Structural characterization of melanin pigments from commercial preparations of the edible mushroom Auricularia auricula. J Agric Food Chem, 63(33):7326-7332.

[57]RamanNM, RamasamyS, 2017. Genetic validation and spectroscopic detailing of DHN-melanin extracted from an environmental fungus. Biochem Biophys Rep, 12:98-107.

[58]RecalcatiS, MinottiG, CairoG, 2010. Iron regulatory proteins: from molecular mechanisms to drug development. Antioxid Redox Signal, 13(10):1593-1616.

[59]RichmanA, KafatosFC, 1996. Immunity to eukaryotic parasites in vector insects. Curr Opin Immunol, 8(1):14-19.

[60]RodriguesML, NimrichterL, OliveiraDL, et al., 2008. Vesicular trans-cell wall transport in fungi: a mechanism for the delivery of virulence-associated macromolecules? Lipid Insights, 2:27-40.

[61]Romero-MartinezR, WheelerM, Guerrero-PlataA, et al., 2000. Biosynthesis and functions of melanin in Sporothrix schenckii. Infect Immun, 68(6):3696-3703.

[62]SamokhvalovA, LiuY, SimonJD, 2004. Characterization of the Fe(III)-binding site in Sepia eumelanin by resonance Raman confocal microspectroscopy. Photochem Photobiol, 80(1):84-88.

[63]SansineneaE, OrtizA, 2015. Melanin: a photoprotection for Bacillus thuringiensis based biopesticides. Biotechnol Lett, 37(3):483-490.

[64]SargentPJ, FarnaudS, EvansRW, 2005. Structure/function overview of proteins involved in iron storage and transport. Curr Med Chem, 12(23):2683-2693.

[65]SchweitzerAD, HowellRC, JiangZW, et al., 2009. Physico-chemical evaluation of rationally designed melanins as novel nature-inspired radioprotectors. PLoS ONE, 4(9):e7229.

[66]SmithDFQ, CasadevallA, 2019. The role of melanin in fungal pathogenesis for animal hosts. Curr Top Microbiol Immunol, 422:1-30.

[67]SolanoF, 2017. Melanin and melanin-related polymers as materials with biomedical and biotechnological applications—cuttlefish ink and mussel foot proteins as inspired biomolecules. Int J Mol Sci, 18(7):1561.

[68]SpiltoirCF, 1955. Life cycle of Ascosphaera apis (Pericystis apis). Am J Bot, 42(6):501-508.

[69]SpiltoirCF, OliveLS, 1955. A reclassification of the genus Pericystis Betts. Mycologia, 47(2):238-244.

[70]SunSJ, ZhangXJ, SunSW, et al., 2016a. Production of natural melanin by Auricularia auricula and study on its molecular structure. Food Chem, 190:801-807.

[71]SunSJ, ZhangXJ, ChenWX, et al., 2016b. Production of natural edible melanin by Auricularia auricula and its physicochemical properties. Food Chem, 196:486-492.

[72]SuwannarachN, KumlaJ, WatanabeB, et al., 2019. Characterization of melanin and optimal conditions for pigment production by an endophytic fungus, Spissiomyces endophytica SDBR-CMU319. PLoS ONE, 14(9):e0222187.

[73]TadokoroT, KobayashiN, ZmudzkaBZ, et al., 2003. UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin. FASEB J, 17(9):1177-1179.

[74]TuYG, SunYZ, TianYG, et al., 2009. Physicochemical characterisation and antioxidant activity of melanin from the muscles of Taihe Black-bone silky fowl (Gallus gallus domesticus Brisson). Food Chem, 114(4):1345-1350.

[75]WalkerCA, GómezBL, Mora-MontesHM, et al., 2010. Melanin externalization in Candida albicans depends on cell wall chitin structures. Eukaryot Cell, 9(9):1329-1342.

[76]WangY, AisenP, CasadevallA, 1995. Cryptococcus neoformans melanin and virulence: mechanism of action. Infect Immun, 63(8):3131-3136.

[77]WheelerMH, BellAA, 1988. Melanins and their importance in pathogenic fungi. Curr Top Med Mycol, 2:338-387.

[78]WynnsAA, JensenAB, EilenbergJ, 2013. Ascosphaera callicarpa, a new species of bee-loving fungus, with a key to the genus for Europe. PLoS ONE, 8(9):e73419.

[79]XuY, GuoZJ, 2008. Study on antioxidant activity of extracts from Aconitum taipeicum. Res Pra Chin Med, 22(1):38-40 (in Chinese).

[80]YeM, WangY, GuoGY, et al., 2012. Physicochemical characteristics and antioxidant activity of arginine-modified melanin from Lachnum YM-346. Food Chem, 135(4):2490-2497.

[81]YeM, GuoGY, LuY, et al., 2014. Purification, structure and anti-radiation activity of melanin from Lachnum YM404. Int J Biol Macromol, 63:170-176.

[82]ZaragozaO, ChrismanCJ, CastelliMV, et al., 2008. Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival. Cell Microbiol, 10(10):2043-2057.

Open peer comments: Debate/Discuss/Question/Opinion


Please provide your name, email address and a comment

Journal of Zhejiang University-SCIENCE, 38 Zheda Road, Hangzhou 310027, China
Tel: +86-571-87952783; E-mail: cjzhang@zju.edu.cn
Copyright © 2000 - 2022 Journal of Zhejiang University-SCIENCE