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

Physicochemical properties, molecular structure, antioxidant activity, and biological function of extracellular melanin from Ascosphaera apis 病原真菌球囊菌产细胞外黑色素的理化性质、分子结构、抗氧化活性和生物学功能研究 1 2 3 corresp first-author Zhi LI, lizhicqnu@gmail.com Zhi LI, lizhicqnu@gmail.com lizhicqnu@gmail.com https://orcid.org/0000-0003-1611-4873 1 1 1 1 1 4 5 College of Life Sciences, Chongqing Normal University, Chongqing 401331, China College of Life Sciences, Chongqing Normal University, Chongqing 401331, China Chongqing Key Laboratory of Vector Insects, 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 Animal Biology, Chongqing 401331, China Chongqing Key Laboratory of Microsporidia Infection and Control, Chongqing 400715, 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 The State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China 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 C 10 H 6 O 4 N 2 , 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 Fe 3+ . 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 . 目的： 2 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： 2 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： 2 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： 2 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。 Melanin Molecular structure Antioxidant activity Subcellular localization Ascosphaera apis 黑色素 分子结构 抗氧化活性 亚细胞定位 球囊菌 1 Introduction 1 Melanin is a heterogeneous polymer composed of complex polyphenols or steroidal heteropolyaromatic compounds ( Jacobson, 2000 Jacobson, 2000 ). It has been identified in many animals. For example, the ink of a squid consists of a suspension of melanin particles ( Fiore et al., 2004 Fiore et al., 2004 ; Guo et al., 2014 Guo et al., 2014 ), and different types of melanin confer the appearance of black or red hair in animals ( Menon et al., 1983 Menon et al., 1983 ; Césarini, 1990 Césarini, 1990 ) and color feathers in birds ( McGraw et al., 2005 McGraw et al., 2005 ). Many fungal species can also synthesize melanin ( Wheeler and Bell, 1988 Wheeler and Bell, 1988 ; Butler and Day, 1998 Butler and Day, 1998 ). Functionally, melanin contributes to energy transduction ( Dadachova et al., 2007 Dadachova et al., 2007 ) and radiation protection, and can be exploited in edible electronic devices ( Kim et al., 2013 Kim et al., 2013 ). Melanin in melanocytes in the skin provides protection against sunlight and allows melanoma to resist therapeutic radiation ( Hill, 1991 Hill, 1991 ). It shows in vitro activity against human immunodeficiency virus, inhibiting syncytium formation and revealing the cytopathic effects of the virus ( Montefiori and Zhou, 1991 Montefiori and Zhou, 1991 ). Insects can use melanin polymers to block the invasion of microorganisms ( Richman and Kafatos, 1996 Richman and Kafatos, 1996 ; Nappi and Christensen, 2005 Nappi and Christensen, 2005 ). Melanin can protect fungi from severe temperature fluctuations, high osmotic pressure, oxidative stress, ultraviolet (UV) and γ radiation, soil enzymatic lysis, low moisture, and nutrient deficiencies ( Butler and Day, 1998 Butler and Day, 1998 ). Melanins are important for fungal cell wall mechanical strength ( Wang et al., 1995 Wang et al., 1995 ; Gómez et al., 2001 Gómez et al., 2001 ; Nosanchuk and Casadevall, 2003 Nosanchuk and Casadevall, 2003 , 2006 2006 ; Zaragoza et al., 2008 Zaragoza et al., 2008 ; Nosanchuk et al., 2015 Nosanchuk et al., 2015 ). In addition to protection, many studies have found that melanins in pathogenic fungi, such as Aspergillus fumigatus , Aspergillus nidulans , and Cryptococcus neoformans , can assist in invasion ( Kwon-Chung et al., 1982 Kwon-Chung et al., 1982 ; Dixon et al., 1989 Dixon et al., 1989 ), shield the host from recognizing antigens and block the host's immune pathways ( Hernández-Chávez et al., 2017 Hernández-Chávez et al., 2017 ), and chelate environmental metal ions ( Fogarty and Tobin, 1996 Fogarty and Tobin, 1996 ). Research has indicated that fungal melanins can be divided into two main forms: dihydroxyphenylalanine (DOPA)‍-melanins and dihydroxynaphthalene (DHN)‍-melanins ( Langfelder et al., 2003 Langfelder et al., 2003 ). However, the chemical structure of most fungal melanins is unknown because they are not suitable for crystallization or structural study due to their structural diversity, their combination with proteins, carbohydrates or lipids, and the insolubility of amorphous materials. The chemical structure of only a few fungal melanins has been identified ( Ye et al., 2014 Ye et al., 2014 ; Sun et al., 2016a Sun et al., 2016a , 2016b 2016b ; Liu et al., 2018 Liu et al., 2018 ). Therefore, melanins from many important pathogenic fungi remain to be identified. Ascosphaera apis has attracted considerable attention recently. It can cause the deadly chalkbrood disease of bees ( Spiltoir, 1955 Spiltoir, 1955 ; Spiltoir and Olive, 1955 Spiltoir and Olive, 1955 ), inflicting severe worldwide economic losses in beekeeping and seriously undermining the ability of bees to pollinate crops ( Spiltoir, 1955 Spiltoir, 1955 ; Spiltoir and Olive, 1955 Spiltoir and Olive, 1955 ; Aronstein and Murray, 2010 Aronstein and Murray, 2010 ). In the life cycle of A. apis , spores enter the midgut of bee larvae by ingestion and remain dormant ( Morse, 1978 Morse, 1978 ; Wynns et al., 2013 Wynns et al., 2013 ). When the honeybee larvae develop to instars 4‍–‍5, the spores germinate. After the mycelia grow and penetrate the intestinal wall, new spores enter the blood cavity and spread to all the tissues of the larvae. Eventually, the hyphae expand and kill the larvae, resulting in the infected larvae becoming "mummified" ( Bailey, 1968 Bailey, 1968 ; Heath and Gaze, 1987 Heath and Gaze, 1987 ). We recently found that within one week of continued development on in vitro potato dextrose agar (PDA) medium, the opposing mating types of A. apis mycelium turned from white to black and produced newborns containing dark-colored infectious melanized spores at the meeting point. The dead larvae infected by A. apis were blackened and "mummified" ( Li Z et al., 2018 Li Z et al., 2018 ). Our transmission electron microscopy (TEM) observations of subcellular structure showed that the spore wall of mature black spores appeared to have a melanin-producing cyst structure similar to that of other known fungi ( Li et al., 2012 Li et al., 2012 ). However, little is known about the physicochemical properties, chemical structure, or biological activity of pigments from A. apis . Whether the pigment from A. apis can play a role in the A. apis infection process needs further confirmation. This study is the first to isolate and identify melanin from the pathogenic fungus A. apis . The solubility, color value, elemental composition, chemical structure, biological activity, subcellular localization, and antioxidant activity of the melanin were systematically and comprehensively investigated using UV-visible spectroscopy, infrared spectroscopy, Raman spectroscopy, gas chromatography with mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) technology, scanning electron and TEM, and free radical scavenging analysis. In addition, the iron-chelating ability of melanin was further explored, and the relationship between melanin and spore infection ability was analyzed after using tricyclazole to inhibit melanin synthesis. This paper provides an important background reference for studying the nature of melanin in A. apis and its possible role in bee infection. 2 Materials and methods 1 2.1 Isolation and purification of A. apis 2 The A. apis CQ1 isolates in this study were obtained from the College of Life Sciences, Chongqing Normal University, China. A. apis was scraped from the surface of infected larval mummies of honeybees ( Apis mellifera ) and then cultured on PDA. After many generations of incubation, the mature and black spores of A. apis were isolated and purified using discontinuous Percoll gradient centrifugation, as previously described ( Li Z et al., 2018 Li Z et al., 2018 ). 2.2 Extraction and purification of A. apis pigment 2 Melanin was extracted from purified black mature spores of A. apis . Melanin extracted by an alkaline extraction and acid precipitation method was used for the identification of structural, physical, and chemical characteristics, while the melanin "ghost" extraction from an enzymatic digestion method was used for morphological and cell localization observations. For the alkaline extraction and acid precipitation method, the purified A. apis spores were autoclaved at 120 ℃ for 30 min and ultrasonicated in phosphate-buffered saline (PBS) for 30 min using three 1-s bursts. Subsequently, the sample solution was adjusted to pH 10 with 1 mol/L NaOH and kept at 50 ℃ for 3 h. The solution was centrifuged at 12 000 r/min for 10 min to collect the supernatant. Then, melanin was purified by adjusting the pH to 1.0 with 6 mol/L HCl, and the solution was kept at 50 ℃ for 16 h. Melanin, collected after further centrifugation at 12 000 r/min for 20 min, was then successively washed with 0.01 mol/L HCl and distilled water. Finally, the purified melanin was freeze-dried and kept at -20 ℃ until further use. For melanin "ghost" extracted by enzymatic digestion, purified A. apis spores were first mixed with 10 mg/mL novozyme (Sigma-Aldrich, Shanghai, China) and incubated overnight at 30 ℃ to generate protoplasts. The protoplasts were collected after centrifugation at 10 000 r/min for 10 min. After washing three times with PBS, the sediment was suspended in 4.0 mol/L guanidine thiocyanate (Sigma-Aldrich, Shanghai, China) and then incubated overnight at room temperature. The dark-colored particles were collected after centrifugation at 10 000 r/min for 10 min, washed three times with PBS, treated with 1.0 mg/mL proteinase K (Beyotime Biotechnology, Shanghai, China) in reaction buffer (10.0 mmol/L Tris, 1.0 mmol/L CaCl 2 , and 0.5% (5 g/L) sodium dodecyl sulfate (SDS); pH 7.8) and lysing enzymes (containing β‍-glucanase, cellulase, protease, and chitinase; Merck KGaA, Darmstadt, Germany), and then incubated at 37 ℃ for 12 h. The dark-colored particles were washed three times with PBS and boiled in 6.0 mol/L HCl for 1.5 h. Melanin particles were collected after centrifugation at 12 000 r/min for 10 min. Finally, the particles were washed and dialyzed with double-distilled water (ddH 2 O) until the acid was completely removed, and then dried for use. 2.3 Physical and chemical characteristic analyses of A. apis pigment 2 2.3.1　Solubility tests 3 A. apis melanin (10 mg) was weighed and mixed with 10 mL of each of the following solvents at room temperature: ddH 2 O, H 2 O 2 , 1 mol/L NaOH, 1 mol/L HCl, methanol, anhydrous ethanol, ethyl ether, petroleum ether, glacial acetic acid, phosphoric acid, propionic acid, ammonia, glycerol, dimethyl sulfoxide, n -butanol, isoamyl alcohol, acetone, toluene, xylene, and trichloromethane. The solubility of A. apis melanin was observed after leaving the mixture to stand for 3 h. 2.3.2　Determination of the color value 3 The color value of the A. apis pigment was determined as previously described ( Ye et al., 2012 Ye et al., 2012 ; Raman and Ramasamy, 2017 Raman and Ramasamy, 2017 ), and measured at 195 nm using a UV-visible spectrophotometer (Shimadzu 2100, Japan). 2.3.3　UV-visible light absorption spectrum 3 The absorption properties of A. apis melanin were analyzed using UV-visible spectrophotometer (Shimadzu 2100, Japan). Absorption was recorded in the wavelength range of 180–900 nm using 0.1 mol/L NaOH solution as the reference. 2.3.4　Elemental analysis 3 The percentages of C, H, N, O, and S contained in A. apis melanin were determined using an elemental analyzer (Euro Vector EA3000, Milan, Italy) as previously described (de la Rosa et al., 2017). 2.3.5　Infrared spectrum analysis 3 Fourier transform infrared (FTIR) spectroscopy analysis was performed according to the method of Ye et al. (2014) Ye et al. (2014) with slight modifications. Briefly, A. apis melanin powder was ground and mixed with dry KBr (Sigma-Aldrich, Shanghai, China) in a 1:100 mass ratio, and then scanned with a 6700 FTIR spectrometer (Thermo Nicolet Co., Waltham, MA, USA) with a scanning range of 4000–400 cm -1 . 2.3.6　Raman spectroscopy scanning 3 To explore the main functional groups in A. apis melanin, Raman spectroscopy analysis was carried out using a Renishaw Raman RM2000 spectrometer (Renishaw, Gloucestershire, UK) as previously described (de la Rosa et al., 2017; Galván et al., 2018 Galván et al., 2018 ). 2.3.7　 1 H-NMR and 13 C-NMR analyses 3 1 H-NMR analysis was performed using a Bruker Avance AV-400 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) with reference to the method of Ye et al. (2014) Ye et al. (2014) . The conditions were as follows: temperature 300 K (26.85 ℃), delay time 1 s, and observation frequency 500.13 MHz. The sample was dissolved in NaOD, and chemical shifts are given in ppm (1 ppm=1×10 -6 ). 13 C-NMR analysis was performed using a Bruker Avance AV-500 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). The conditions refer to those of Ye et al. (2014) Ye et al. (2014) : resonance frequency 130.255 MHz, using a 4-mm high-resolution magic angle spinning (HRMAS) probe and a spinning rate of 8 kHz, the number of scans 8192, and the delay time 1 s. The samples were dissolved in D 2 O, and chemical shifts are given in ppm. 2.3.8　Electron paramagnetic resonance spectroscopy analysis 3 Electron paramagnetic resonance (EPR) spectral analysis of A. apis melanin was performed at the Analytical and Test Center of Chongqing University, Chongqing, China. Briefly, a 5-mg melanin sample was placed in a quartz EPR tube and frozen in liquid nitrogen. EPR spectra were recorded using a Bruker A300 spectrophotometer (Bruker Biospin GmbH, Rheinstetten, Germany), with the following parameters: microwave bridge frequency 9.85 GHz and power 19.12 mW; sweep width 100 G (1 G=0.1 mT), time 81.92 s and time constant 81.92 ms; modulation frequency 100 kHz and amplitude 1.00 G. 2.3.9　Pyrolysis GC-MS analysis 3 Pyrolysis GC-MS analysis was performed according to a the method of Ye et al. (2012) Ye et al. (2012) . Finally, the samples were analyzed using GC-MS detection (Thermo Trace, Silicon Valley, USA). The parameters and conditions refer to those of a previous study ( Ye et al., 2014 Ye et al., 2014 ) with minor modifications. Briefly, the oven temperature was set to 40 ℃ for 1 min, increased to 300 ℃ at a rate of 5 ℃/min, and then held for 10 min. The helium carrier gas was set to a flow rate of 1 mL/min. The injection volume was 1 μL and the split ratio was 40:1. In the mass spectrometer, the ion source temperature was 230 ℃, and the ionization mode was electron ionization (EI) of 70 eV. 2.4 Biochemical activity of A. apis pigment 2 2.4.1　Total antioxidant capacity assay 3 The ferric-reducing antioxidant power (FRAP) method for the total antioxidant capacity assay was carried out using the FRAP Plus kit (Beyotime Biotechnology, Shanghai, China) as previously described ( Bao et al., 2021 Bao et al., 2021 ). 2.4.2　Superoxide radical scavenging activity assay 3 The pyrogallol oxidation method was used as previously described ( Xu and Guo, 2008 Xu and Guo, 2008 ). Finally, the absorbance of sample ( A sample ) at 318 nm was measured using a UV-visible spectrophotometer (Shimadzu 2100, Japan). Ascorbic acid was used as the control. 2.4.3　Hydroxyl radical scavenging activity assay 3 The antioxidant activity toward hydroxyl radical was detected as previously described ( Chen et al., 2009 Chen et al., 2009 ), with minor modifications. Briefly, 1 mL of 0.75 mol/L 1,‍10-phenanthroline was mixed with 0.2 mmol/L PBS (pH 7.4, 2 mL) and 1 mL of ddH 2 O. Then, 1 mL of 0.75 mmol/L ferrous sulfate (FeSO 4 ) and 1 mL of 0.01% (volume fraction) H 2 O 2 were added to the mixture, followed by shaking and incubation at 37 ℃ for 60 min. Finally, the absorbance of the mixture at 536 nm was measured using a UV-visible spectrophotometer (Shimadzu 2100, Japan). The hydroxyl radical scavenging activity of the extract is expressed as a half maximal inhibitory concentration (IC 50 ) value. 2.4.4　2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay 3 The DPPH radical scavenging activity of A. apis melanin was determined as previously described ( Bersuder et al., 1998 Bersuder et al., 1998 ). The antioxidant activity of the extract is expressed as an IC 50 value, which was defined as the concentration (µg/mL) of extract that inhibits the formation of DPPH radicals by 50%. 2.4.5　Fe 2+ chelating activity assay 3 The Fe 2+ chelating activity assay was performed as previously described ( Ye et al., 2012 Ye et al., 2012 ). The percent chelating rate was calculated according to the following formula: Fe 2+ chelating rate (%)=( A blank - A sample )/ A blank ×100%, where A blank is the absorbance of distilled water as a blank control and A sample is the absorbance of A. apis melanin. 2.5 Preparation of antiserum against A. apis pigment 2 According to a previous method ( Li et al., 2012 Li et al., 2012 ), A. apis melanin antiserum was generated by immunizing mice with purified pigment ghost with appropriate modifications. Briefly, purified melanin ghost was mixed with Freund's adjuvant (1:1 (volume ratio); Sigma-Aldrich, USA) and injected using subcutaneous multipoint injection into three mice. One week after the third injection, the mice were bled and their sera were collected. Another three mice were immunized subcutaneously with multipoint injection of PBS to prepare negative antiserum. 2.6 Subcellular localization observation of A. apis pigment 2 2.6.1　Scanning electron microscopy 3 For scanning electron microscopy (SEM), the extracted melanin "host" particles were treated as previously described ( Li Z et al., 2018 Li Z et al., 2018 ). Finally, the samples were transferred to an Emitech K850 critical point dryer (Emitech, East Sussex, UK) with liquefied carbon dioxide as the transitional fluid. The dried samples were gold-coated and transferred to SEM stubs. SEM investigations were performed with a Hitachi SU3500 scanning electron microscope (Tokyo, Japan). 2.6.2　Transmission electron microscopy 3 For TEM analysis, the A. apis melanin "host" samples were treated as previously described ( Li Z et al., 2018 Li Z et al., 2018 ). Finally, ultrathin sections of 70‍‒‍80 nm were cut with a Reichert Ultracut Ultramicrotome, stained with uranyl acetate followed by lead citrate, and viewed on a transmission electron microscope (FEI Tecnai Spirit, California, USA). 2.6.3　Indirect immunofluorescence assay 3 A. apis melanin was analyzed using indirect immunofluorescence assay (IFA) according to our previous methods ( Li et al., 2012 Li et al., 2012 ). Finally, the spores were examined with an Olympus FluoView FV1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). 2.7 Preparation of melanin-deficient spores of A. apis 2 Spores with functionally defective melanin were prepared by inhibiting melanin synthesis with tricyclazole or incubating spores with melanin antiserum. The preparation methods were as follows. 2.7.1 Tricyclazole inhibitor method 3 Based on our previous culture method of A. apis ( Li Z et al., 2018 Li Z et al., 2018 ), 10 µg/mL tricyclazole was added to PDA (200 g/L peeled potatoes, 20 g/L agar, 10 g/L dextrose, and 5 g/L yeast extract). A. apis was cultured on this PDA medium at (30±5) ℃ and 80% relative humidity (RH). After many generations of incubation and isolation, the spores without melanin were purified using discontinuous Percoll gradient centrifugation, as reported in a previous study ( Li et al., 2012 Li et al., 2012 ). 2.7.2 Melanin antiserum blocking method 3 Purified A. apis wild-type spores (1×10 5 spores/mL) were incubated with A. apis melanin antiserum at 37 ℃ for 3 h at a ratio of 1:100 (volume ratio). Then, the spores were washed with PBS three times to remove excess antiserum and resuspended in PBS solution. 2.8 Rearing and survival analyses of honey bee larvae 2 A frame containing 1st-instar worker larvae from one honey bee colony was brought to the laboratory. Then, 240 1st-instar larvae were collected and raised individually in a standard 48-well cell culture plate (one bee/well), held in a dark incubator at (34.5±0.5) ℃ and 80% RH, and provided with sufficient food (mass fractions: 50% royal jelly, 6% fructose, 6% glucose, 2% yeast extract, and 36% ddH 2 O) once a day. A total of 20 μL of diet was used for 3rd-instar larvae, 30 μL for 4th-instar larvae, 40 μL for 5th-instar larvae, and 50 μL for 6th-instar larvae. The larvae were divided into four groups for the infection experiment: a normal feeding control group, a wild-type spore infection group, a tricyclazole spore group (spores without melanin following tricyclazole culture), and an anti-melanin spore group (spores incubated with melanin antiserum). The detailed procedure was as follows: 4th-instar larvae were deprived of food for 4 h and then orally fed 20 μL of food containing one of the above three types of spores (1×10 5 spores/bee), and the control group was fed normal food. Bees that consumed the entire test solution within 30 min were randomly selected and kept in a standard 24-well cell culture plate (one bee/well), held in a dark incubator at (35±1) ℃ with (65±5)% RH, and provided with sufficient food until they stopped feeding at the 6th instar. The survival of larvae was recorded daily. The mortality of honey bee larvae was analyzed in three replicates. According to Abbott's formula, mortality data were corrected and subjected to statistical survival analysis using the Kaplan-Meier survival model with a log-rank test for overall comparison, using Statistical Package for Social Sciences (SPSS) v23.0 (SPSS Inc., Chicago, IL, USA). 2.9 Statistical analysis 2 The results were analyzed using SPSS v20.0 for Windows. All the data are expressed as the mean±standard error of the mean (SEM) from three independent biological repeats, with each composed of three technical repeats. Student's t -test was used to compare the means, and the values were considered significant at P < 0.05. 3 Results 1 3.1 Optimum conditions for extracting A. apis melanin 2 The extraction of melanin depends on its solubility in alkaline solution and the formation of precipitates in acidic solution. Hydrolysis in acidic solutions can remove the proteins, carbohydrates, and lipids bound to melanin. In this study, A. apis melanin was extracted by the alkaline extraction and acid precipitation method. The maximum rate of production of A. apis melanin was 7.67%. The optimum hydrolysis temperature was 40‒60 ℃ (Fig. S1a), and the optimum solid to liquid ratio was 1 g:‍10 mL (Fig. S1b). The extraction rate was positively correlated with the hydrolysis time of NaOH (Fig. S1c) and the concentration of HCl, but there was almost no change after the concentration of HCl increased to 6 mol/L (Fig. S1d). 3.2 Physicochemical properties and molecular structure of A. apis melanin 2 3.2.1　Solubility and colority 3 A. apis melanin was easily soluble in alkaline solutions such as NaOH and ammonia, and had relatively high solubility in formic acid, phosphoric acid, and dimethyl sulfoxide. It was insoluble in common organic solvents such as chloroform, ethyl acetate, ethanol, propionic acid, acetic acid, ether, hexane, and acetone, but could be precipitated in mineral acid solutions such as HCl (Fig. S2). The colority of A. apis melanin was 273, which is close to that reported for melanin ( Ye et al., 2012 Ye et al., 2012 ). 3.2.2　UV-visible light absorption spectrum 3 UV-visible absorption spectrum analysis showed that the optical density of A. apis melanin gradually decreased with increasing wavelength. A. apis melanin had a maximum absorbance at 195 nm (Fig. S3). No absorption peaks between 260 and 280 nm were observed in the spectrum, suggesting that A. apis melanin did not contain nucleic acids, proteins, or lipids. 3.2.3　Elemental composition 3 The elemental composition (mass fraction) of A. apis melanin was 42.18% C, 2.65% H, 6.40% N, 12.12% O, and 0.86% S (Fig. S4). The corresponding molar ratios of C:H, C:O, and C:N were 1.0:1.3, 4.6:1.0, and 7.7:1.0, respectively. Based on the defining characteristics of eumelanin (0.09% S) and phaeomelanin (9.78% S), DOPA-melanin (6%‍–‍11% N) and DHN-melanin (0% N), A. apis melanin contained 0.86% S and 6.40% N, indicating that A. apis melanin was primarily a DOPA-eumelanin. Moreover, the higher C:N ratio indicated that A. apis melanin did not contain amino groups, and the lower C:H ratio suggested that the pigment contained more heterocyclic structures ( Schweitzer et al., 2009 Schweitzer et al., 2009 ). 3.2.4　EPR spectroscopy analysis 3 The EPR spectrum of pigment particles obtained from A. apis had a slightly asymmetric singlet with a full width at half maximum of the linewidth of EPR signal ∆ B pp =4 G and the spectroscopic splitting factor g =2.005. The signal peak appeared at 3510 G of the EPR spectrum ( Fig. 1 Fig. 1 ), indicating that A. apis melanin contained a stable free radical compound, the defining feature of almost all reported melanins. 3.2.5　Infrared spectra 3 The results of infrared spectra analysis were shown in Fig. S5. Similar to other natural melanins, A. apis melanin had a wide range of broad absorption bands of 3500.0–3300.0 cm -1 and a strong absorption peak at 3439.9 cm -1 , corresponding to the –OH and –‍NH 2 groups of the indole ring, respectively. Three typical sharp peaks common to fungal melanins were also found in A. apis melanin at 2920.2, 1631.5, and 1075.1 cm -1 , corresponding to the C‍–‍H group of quinone, C=C or C‍–‍C, and C=O and C‍–‍O of quinone neighboring the carboxyl group. Another stretching vibration at 2851 cm -1 was attributed to the –CH 2 or –CH 3 group neighboring the quinone. The absorption at 2361 cm -1 may indicate the stretching vibration of –‍R 3 NHX‍– (R=H or NH). Moreover, three medium-strong characteristic absorption peaks at 1737, 1631, and 1399 cm -1 were attributed to the stretching vibrations of the aromatic skeleton, –‍COC‍–, and C=O groups, respectively. Overall, the molecular structure of A. apis melanin has fingerprint peaks of the common functional group characteristics of eumelanin, and contains a typical indole structure. 3.2.6　Raman spectroscopy 3 Raman spectroscopic analysis revealed that A. apis melanin showed three strong absorption bands at 1152, 1508, and 1005 cm -1 , attributed to the C=C and C–C stretching vibrations in aromatic compounds and the C‍–‍N stretching vibration of hydroxyl groups, respectively ( Fig. 2 Fig. 2 ). These three bands can be considered fingerprints specific to A. apis melanin. In addition, another band with minor intensity was observed in the range of 2824–2467 cm -1 , attributed to the C‍–H stretching vibration of lipid-based branching. Moreover, another signal peak was observed at 3497 cm -1 , which can be attributed to free radicals. 3.2.7　NMR analysis 3 1 H-NMR and 13 C-NMR spectral analyses revealed that A. apis melanin has a series of broad absorption peaks, reflecting the complexity of the chemical structure and the characteristics of macromolecular compounds ( Fig. 3 Fig. 3 ). In the 1 H-NMR spectrum ( Fig. 3 Fig. 3 a), the HC=C resonance of the indole was observed at 7.1 ppm. The peaks with chemical shifts of 4.5‍‒5.4 ppm indicated a C=C‍–‍H aromatic nucleus contained within the pigment structure. A‍ –‍CH 2 might cause the peaks between 3.1 and 4.2 ppm or a –CH 3 group connected to an N or O. The peaks between 2.2 and 2.5 ppm showed an NH group linked to the indole. The peaks between 0.8 and 1.5 ppm were caused by the –CH 3 group in –CH 2 CH 3 . In addition to the 1 H-NMR spectrum, the 13 C-NMR spectral analysis revealed that A. apis melanin has indole-based aromatic carbons and other long chains ( Fig. 3 Fig. 3 b). The peaks at 63 and 204 ppm, between 76 and 101 ppm, and between 125 and 137 ppm were assigned to the carbon atoms of the CHO or CH 2 O, carboxylic acid, and CH–N or CH–S group, respectively, in the indole aromatic structure. A resonance from the indole carbon skeleton appeared at 128 ppm, and a signal peak from a phenol structure at 136 ppm. However, the absence of resonances within the range of 10‍‒‍40 ppm indicated no carbon atoms in methyl (–CH 2 CH 3 ) or methylene (–CH 2 –) group. 3.2.8　GC-MS analysis 3 Based on the quasi-molecular ion peaks of [M] + and [M+H] + at mass-to-charge ratio ( m / z ) 236.03 and 409.01, the molecular weight of A. apis melanin was determined to be 409 Da (Fig. S6). Combined with the results of elemental compound and chemical structure analysis (FTIR, 1 H-NMR, 13 C-NMR, and GC-MS), the molecular formula of A. apis melanin was speculated to be C 10 H 6 O 4 N 2 , and the possible chemical structure formula could be inferred (Fig. S7). 3.3 Biological activity of A. apis melanin 2 3.3.1　Antioxidant activity 3 The results of FRAP antioxidant analysis showed that compared with the ascorbic acid control, the antioxidant value of A. apis melanin increased with an increasing concentration of melanin ( Fig. 4 Fig. 4 a) and exhibited a dose-dependent relationship ( Y =0.2478 X +0.07531, R 2 =0.9981). When the pigment concentration was 5.0 mg/mL, the total antioxidant capacity of A. apis melanin was equivalent to the antioxidant capacity of 0.52 mmol/L ascorbic acid, and presented high total antioxidant activity. 3.3.2　Superoxide radical scavenging activity 3 The superoxide radical scavenging activity is considered a good indicator of the antioxidant activity of an extract. In this study, the superoxide radical scavenging activity of A. apis melanin increased with increasing concentration in a dose-dependent manner ( Y =0.1029 X +0.2193, R 2 =0.9829) ( Fig. 4 Fig. 4 b). At 5.0 mg/mL, the superoxide radical scavenging rates of A. apis melanin reached 71.2%. The IC 50 value of A. apis melanin was 26.23 mg/mL compared to the standard antioxidant ascorbic acid. These results indicated that A. apis melanin has a strong superoxide radical scavenging ability. 3.3.3　Hydroxyl radical scavenging activity 3 The hydroxyl radical scavenging activity was investigated at various concentrations (0‒5.0 mg/mL) of A. apis melanin, and the results are shown in Fig. 4 Fig. 4 c. The hydroxyl radical scavenging activity of A. apis melanin increased with increasing concentration, exhibited a dose-effect relationship ( Y =0.1086 X -0.05596, R 2 =0.9582), and showed good hydroxyl radical scavenging activity with an IC 50 value of 5.467 mg/mL and good scavenging rates (47.1%) at a concentration of 5.0 mg/mL ( Fig. 4 Fig. 4 c). These results demonstrated that A. apis melanin has a strong free radical scavenging ability. 3.3.4　DPPH radical scavenging activity 3 The radical scavenging activity of A. apis melanin against stable DPPH was determined spectrophotometrically. The results indicated that the DPPH scavenging activity of A. apis melanin increased with increasing concentration and showed significant radical scavenging activity with an IC 50 value of 74.49 mg/mL ( Fig. 4 Fig. 4 d). When the pigment concentration was 4 mg/mL, the DPPH scavenging rate of A. apis melanin reached 69.3%, slightly lower than that of ascorbic acid (87.2%). These results indicated that A. apis melanin has a notable effect on DPPH radical scavenging activity. 3.3.5　Chelating effect on ferrous ions 3 The ability of A. apis pigment to bind ferric ions increased with increasing concentration in a dose-dependent manner ( Fig. 4 Fig. 4 e). The IC 50 value of A. apis melanin was 58.79 mg/mL compared to 0.83 mg/mL ethylenediaminetetraacetic acid disodium salt (EDTA-2Na). At 5 mg/mL, the Fe 2+ chelating rate of A. apis melanin was 49.6%. These results suggested that A. apis melanin has significant chelating activity against Fe 2+ . 3.4 Subcellular localization characteristics of A. apis melanin 2 SEM, TEM, and IFA were performed to determine the precise location of melanin in the A. apis spores. The pigment extracted by enzymatic hydrolysis formed granules of the polymer. The granules retained the spherical shape of the spores as viewed with the electron microscope (Fig. S8), and represented the melanin "ghost." Under SEM, the A. apis melanin ghost had an average size of 2 µm×1 µm, consistent with that of normal spores in this study (Figs. S8a and S8b) and our previous study ( Li Z et al., 2018 Li Z et al., 2018 ). The most striking feature of the melanin ghosts was their concavity. They appeared to be empty shells that had lost the endoplasm (Fig. S8b). However, the surface of the pigment ghosts was not as smooth as that of normal wild-type spores (Figs. S8a and S8b). TEM showed that the melanin granular particles were clustered and formed two electron-dense layers, filling the endospore (about 20 nm thick) and exospore (about 50 nm thick) of the normal spore (Figs. S8c and S8e). However, the pigment of the ghosts appeared to be more concave, flatter, and smaller (about 1.5 µm×0.5 µm) than that of normal spores (about 2.0 µm×1.0 µm) (Figs. S8d and S8f). In addition, the surface of the ghosts was covered with granular particles that were 50‒300 nm thick, and looser than that of the melanin distributed on the normal spores (Figs. S8c and S8d). The IFA showed that the spore coat displayed a bright green fluorescence signal ( Fig. 5 Fig. 5 ) after incubation with the A. apis melanin antiserum, while no such signal was observed in the negative control. The results suggested that melanin was localized on the spore wall of A. apis . 3.5 No apparent toxic effects of melanin-deficient spores on bee 2 Spores with functionally defective melanin were successfully prepared by inhibiting melanin synthesis with tricyclazole ( Fig. 6 Fig. 6 a) or incubating spores with melanin antibody ( Fig. 6 Fig. 6 b). Then, the defective spores were fed to bee larvae for infection experiments. Survival analysis showed that the mortality of bee larvae fed spores without melanin as a result of tricyclazole inhibition or incubation with melanin antibody was not significantly different from that of the normal spore infection group ( Fig. 6 Fig. 6 c). The results showed that this melanin-deficient spore was still pathogenic to bee larvae, indicating that melanin did not affect the virulence of A. apis spores-infecting bees. 4 Discussion 1 Melanins are divided into three main types: eumelanin, phaeomelanin, and allomelanin. Eumelanin is a brown or black nitrogen-containing heterogeneous macromolecular compound derived from 5,6-dihydroxyindole-2-carboxylic acid (DHICA) and 5,6-dihydroxyindole (DHI). DHICA and DHI are derived from DOPA and DOPA quinone ( Tadokoro et al., 2003 Tadokoro et al., 2003 ; Sansinenea and Ortiz, 2015 Sansinenea and Ortiz, 2015 ). Some of the key physicochemical properties and chemical structures of eumelanin have been clearly defined and are used as the standard for melanin identification: (1) melanin is insoluble in acidic solutions and common organic reagents ( Butler and Day, 1998 Butler and Day, 1998 ); (2) melanin has a 6%–11% N content, low S content, and a low molar ratio of S/N in elemental composition ( Tu et al., 2009 Tu et al., 2009 ; Sun et al., 2016a Sun et al., 2016a , 2016b 2016b ; Huang et al., 2018 Huang et al., 2018 ); (3) melanin is a macromolecule of DHI and DHICA, which is primarily formed from catechol monomers, and especially contains an indole in its chemical structure ( Meredith and Sarna, 2006 Meredith and Sarna, 2006 ). Melanin is a complex mixture that is difficult to separate into monomers. Therefore, multiple analytical techniques need to be combined to confirm its identification. Melanin contains a unique and stable population of organic free radical compounds ( Enochs et al., 1993 Enochs et al., 1993 ; Prados-Rosales et al., 2015 Prados-Rosales et al., 2015 ), resulting in characteristic EPR behavior and inducing a unique signal peak near 3500 G in the EPR spectrum ( Suwannarach et al., 2019 Suwannarach et al., 2019 ). EPR spectroscopy is thus considered the most reliable technique for distinguishing the characteristics of melanins ( Enochs et al., 1993 Enochs et al., 1993 ). In this study, we successfully extracted dark-colored pigments produced by A. apis using alkali extraction, acid hydrolysis, and repeated precipitation. Its chemical structure was comprehensively characterized by combining UV, elemental composition, FTIR, Raman spectroscopy, NMR, EPR, and GC-MS techniques. First, we showed that the A. apis pigment is insoluble in common organic solvents and precipitates in HCl (Fig. S2), consistent with the typical solubility characteristics of eumelanin ( Gómez and Nosanchuk, 2003 Gómez and Nosanchuk, 2003 ). Second, we found that the A. apis pigment has elemental composition characteristics similar to those of eumelanin from Streptomyces sp. ( Li C et al., 2018 Li C et al., 2018 ), especially the low S content (mass fraction: 0.86%) and low S/N ratio (0.13) (Fig. S4). Third, through FTIR and NMR analyses, we showed that the A. apis pigment has a typical indole structure. The characteristic absorption of the indole structure occurred at 2361 cm -1 and 804–572 cm -1 in the FTIR spectrum (Figs. S5 and 3). The CH=C resonance of the indole appeared at 7.1 ppm in the 1 H-NMR spectrum. In the 13 C-NMR spectrum, signal peaks from the indole-based aromatic carbons appeared from 125 to 137 ppm. In addition, a resonance from the indole carbon skeleton appeared at 128 ppm, and no signal peak from a phenol structure was observed at 136 ppm. In addition, two typical eumelanin signature C=C and C‍–‍C groups were present in the aromatic indole structure ( Fig. 2 Fig. 2 ), thus proving that there is an indole moiety in the A. apis pigment. Combined with GC-MS data, this showed that the phenyl and pentacyclic rings linked to the indole group reported in most melanin structures were replaced by carboxyl, amino, and ester groups in the A. apis pigment (Fig. S6). Fourth, and most importantly, a stable free radical compound (generating a signal at 3510 G in the EPR spectrum; Fig. 1 Fig. 1 ) reported in most fungal melanin structures ( Morris-Jones et al., 2003 Morris-Jones et al., 2003 ; Nosanchuk and Casadevall, 2003 Nosanchuk and Casadevall, 2003 ; al Khatib et al., 2018; Camacho et al., 2019 Camacho et al., 2019 ), which is considered a unique feature of melanin, was also found in the A. apis pigment. By comparing the solubility characteristics, elemental composition, chemical structure, and functional groups of the typical melanins reported above, the A. apis pigment in this study was identified as a DOPA-eumelanin. The location of melanin in cells determines its corresponding biological functions and has thus received widespread attention. Some fungal melanins have been found to be part of the spore wall or spore extracellular matrices. For instance, melanins of C. neoformans are arranged neatly and bridged to each other through chitin or polysaccharides, forming an electron-dense layer from the cell wall to the plasma membrane ( Nosanchuk and Casadevall, 2003 Nosanchuk and Casadevall, 2003 ). Functional studies have shown that this type of melanin is generally involved in the virulence of invasion and protection against oxidant stress, ionizing radiation, and antifungal drugs ( Dixon et al., 1989 Dixon et al., 1989 ; Nosanchuk and Casadevall, 2003 Nosanchuk and Casadevall, 2003 ; Hernández-Chávez et al., 2017 Hernández-Chávez et al., 2017 ). In addition, cell wall melanization can also increase the mechanical strength and rigidity of cells, changing cell permeability and turgor forces ( Money et al., 1998 Money et al., 1998 ). In contrast, melanin in Candida is externalized from spores in the form of electron-dense melanosomes that are free or usually loosely bound to the outside of the cell wall, and which cannot form an electron-dense layer to protect the spores ( Walker et al., 2010 Walker et al., 2010 ). There are also a few species of melanin located in special vacuoles or intracellular vesicles, functioning as "virulence bags" or involved in cell autolysis ( Rodrigues et al., 2008 Rodrigues et al., 2008 ; Smith and Casadevall, 2019 Smith and Casadevall, 2019 ). Our previous study found that the spore wall consists of an electron-dense outer surface layer (exospore), an electron-lucent layer, and an electron-dense inner plasma membrane. The exospore and endospore together form a spore coat with a uniform thickness that helps spores resist various environmental pressures ( Li Z et al., 2018 Li Z et al., 2018 ). In this study, the spore wall was observed with SEM and TEM. The results showed that A. apis melanin clustered to form an electron-dense layer extending throughout the entire cell wall in immature and mature spores (Figs. S8 and 5). Therefore, A. apis melanin probably has similar functions to the melanins mentioned above in fungal species. Moreover, considering that in the invasion process, the A. apis mycelium can overcome the gut wall barrier and eventually penetrate the entire honeybee larva, melanin located within the spore wall is likely similar to the melanin of Gaeumannomyces graminis var. graminis ( Money et al., 1998 Money et al., 1998 ), which can promote spores to generate enormous turgor pressures and provide the necessary force to enable penetration by spore mycelia. A. apis melanin within the cell wall probably also helps spores survive in drought or high osmotic pressure environments, resulting in A. apis being able to survive and remain infectious, even in beehives that have been unused and contain mummified larvae that have been dead for a long time. Antioxidant capacities refer to DPPH radical scavenging, superoxide radical scavenging, hydroxyl radical scavenging, nitric oxide scavenging, reducing power and metal chelating abilities. These antioxidant capacities are the basic functions and characteristics of almost all reported melanins and have become an important index of melanin identification. Melanins from Lachnum YM-346 ( Ye et al., 2012 Ye et al., 2012 ), Actinoalloteichus sp. ( Manivasagan et al., 2013 Manivasagan et al., 2013 ), Schizophyllum commune ( Arun et al., 2015 Arun et al., 2015 ), Streptomyces sp. ( Arun et al., 2015 Arun et al., 2015 ), and Actinomycetes sp. ( Manivasagan et al., 2013 Manivasagan et al., 2013 ) have strong antioxidant activity, which is helpful in protecting cells from oxidative damage caused by various stresses inside and outside cells. In this study, FRAP, DPPH, superoxide and hydroxyl radical scavenging analyses showed that A. apis melanin also has a strong antioxidant capacity ( Fig. 4 Fig. 4 ). The antioxidant ability is probably helpful for enabling A. apis spores to survive in harsh environments. Melanin can also bind directly with metal ions such as Fe 2+ , exhibiting a high affinity and high capacity ( Guo et al., 2010 Guo et al., 2010 ; Li et al., 2016 Li et al., 2016 ). This is because the aromatic components and functional groups in melanin, such as hydroxyl, carboxyl, amine, and phenol groups, provide a chemical basis for the interaction of melanin with many organic and inorganic molecules ( Solano, 2017 Solano, 2017 ). For example, Mg 2+ , Ca 2+ , and Zn 2+ preferentially bind to the carboxyl group ( Fan et al., 2014 Fan et al., 2014 ), Cu 2+ binds to the hydroxyl group ( Samokhvalov et al., 2004 Samokhvalov et al., 2004 ), and Fe 2+ prefers to coordinate with hydroxyl groups, amines, imines, and acetate groups ( Hong and Simon, 2007 Hong and Simon, 2007 ) of melanin. In this study, the FTIR and Raman data indicated that A. apis melanin has amino, hydroxyl, carboxyl, and other potential fatty side chain groups (Figs. S5 and 2). Therefore, A. apis melanin can also chelate to iron due to the presence of these high-affinity groups. The FRAP antioxidant activity analysis showed that A. apis melanin could bind to Fe 3+ and reduce Fe 3+ to Fe 2+ ( Fig. 4 Fig. 4 ). Many studies have confirmed that metal elements such as iron play an important role in the metabolism of cells ( Aisen et al., 2001 Aisen et al., 2001 ; Recalcati et al., 2010 Recalcati et al., 2010 ; Geissler and Singh, 2011 Geissler and Singh, 2011 ). Obtaining iron and other metal elements from the environment is an important strategy for multicellular or single-cell microorganisms ( Sargent et al., 2005 Sargent et al., 2005 ). For A. apis , melanin can chelate iron and potentially bind other metals, providing a good tool for spores to absorb essential metal elements from host bees and other environments. Fungal melanin usually contributes to spore pathogenicity and survival in their host. For example, A. fumigatus melanin can inhibit nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent LC3-associated phagocytosis (LAP) activation in phagocytes, thus protecting spores from being engulfed by host phagocytes ( Akoumianaki et al., 2016 Akoumianaki et al., 2016 ). C. neoformans melanin can transfer or store electrons to alter the fungal cell surface charge, thus interfering with the transfer of the host electron transfer chain, which may contribute to inhibiting phagocytosis by the host ( Wang et al., 1995 Wang et al., 1995 ; Nosanchuk and Casadevall, 1997 Nosanchuk and Casadevall, 1997 ). The ability of melanin to enhance pathogenicity is also commonly found in other fungi, such as Sporothrix schenckii ( Romero-Martinez et al., 2000 Romero-Martinez et al., 2000 ), Fonsecaea pedrosoi ( Cunha et al., 2005 Cunha et al., 2005 ), and Exophiala spp. ( Peltroche-Llacsahuanga et al., 2003 Peltroche-Llacsahuanga et al., 2003 ). This ability of fungal melanin rests on two major premises, spore wall localization and the ability to be a highly effective scavenger of free radicals, which are consistent with the results in this paper. Therefore, A. apis melanin could also contribute to the pathogenicity of spores. However, the survival rate of the larvae did not increase significantly after using melanin-deficient spores ( Fig. 6 Fig. 6 ) to infect bee larvae. This result indicated that A. apis melanin did not promote the pathogenicity of spores. Nonetheless, we cannot rule out the possibility that A. apis , similar to A. fumigatus , uses the redox functions of melanin to interfere with the host's life activities ( Akoumianaki et al., 2016 Akoumianaki et al., 2016 ) or that C. neoformans uses melanin to interfere with the host's electron transfer ( Wang et al., 1995 Wang et al., 1995 ). These effects promoting infection probably affect only the metabolism of the host rather than its survival. This possibility needs further analysis by transcriptomics and metabolomics. Note that this study investigated only melanin produced from spores, whereas previous studies normally used mycelium cultures to prepare melanin. A. apis sporangiophores, sporophores or sporocysts, and spores contain melanin. The location of melanin in organisms usually determines its corresponding biological function. Melanin from the other three sources is likely to differ in its chemical structure, physicochemical properties, and biological activity. Therefore, future research should compare these three melanins to provide a more comprehensive understanding of fungal melanins. 5 Conclusions 1 This paper is the first report on melanin produced by A. apis . A. apis melanin is a eumelanin with a typical indole structure. It is located on the spore wall, has strong antioxidant activity, can potentially chelate to metal ions, and does not enhance the virulence of A. apis spores. This work provides valuable information for understanding the architecture and function of melanin in pathogenic fungi. Future studies will focus on the functional analysis of melanin in A. apis in relation to resistance to environmental stresses. References Aisen P , Enns C , Wessling-Resnick M , 2001 . 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Cell Microbiol , 10 ( 10 ): 2043 - 2057 . https://doi.org/10.1111/j.1462-5822.2008.01186.x https://doi.org/10.1111/j.1462-5822.2008.01186.x Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival 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. 10.1631/jzus.B2100718 1673-1581（2022）05-0365-17 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. 2021-08-16 2022-01-04 2022-05-15 2022-12-27 2022-05-11 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2022 2022 浙江大学出版社 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. Compliance with ethics guidelines Supplementary information 1 Figs. S1–S8 浙江大学学报（英文版）（B辑：生物医学和生物技术） 5 23