Investigation of Physicochemical Indices and Microbial Communities in Termite Fungus-Combs

Termitomyces species are wild edible mushrooms that possess high nutritional value and a wide range of medicinal properties. However, the cultivation of these mushrooms is very difficult because of their symbiotic association with termites. In this study, we aimed to examine the differences in physicochemical indices and microbial communities between combs with Termitomyces basidiomes (CF) and combs without Termitomyces basidiomes (CNF). High-performance liquid chromatography (HPLC), inductively coupled plasma optical emission spectrometry (ICP-OES), gas chromatography equipped with a flame ionization detector (GC-FID), some commercial kits, high-throughput sequencing of the 16s RNA, and internal transcribed spacer (ITS) were used. Humidity, pH, and elements, i.e., Al, Ba, Fe, Mn, Ni, S, Ca, and Mg were higher while amino acids particularly alanine, tyrosine, and isoleucine were lower in CF as compared to CNF. The average contents of fatty acids were not significantly different between the two comb categories. The bacterial genera Alistipes, Burkholderia, Sediminibacterium, and Thermus were dominant in all combs. Brevibacterium, Brevundimonas, and Sediminibacterium were significantly more abundant in CF. Basidiomycota and Ascomycota were also identified in combs. Termitomyces clypeatus, Termitomyces sp. Group3, and Termitomyces sp. were the most dominant species in combs. However, any single Termitomyces species was abundantly present in an individual comb.


INTRODUCTION
Termitomyces is a rare genus among wild edible mushrooms, most abundantly present in (sub)tropical areas of Southeast Asia and Africa (Aanen et al., 2009;Siddiquee et al., 2015). Termitomyces mushrooms are widely consumed because of their taste, flavor, and medicinal benefits (Zhao et al., 2016;Hsieh and Ju, 2018). A wide spectrum of degrading enzymes is produced by Termitomyces, which could be used in industrial manufacturing such as papermaking (Aanen and Eggleton, 2005;da Costa et al., 2018;Banerjee et al., 2019).
Termitomyces appears only on the nests of fungus-growing termites (Macrotermitine) during rainy seasons. The mutualistic association between Termitomyces and termites started 24-34 million years ago in rainforests of sub-Saharan Africa (Aanen and Eggleton, 2005;Mueller et al., 2005;da Costa et al., 2018). Approximately 330 species of fungus-growing termites have been reported so far, which domesticate 30 described species of Termitomyces (Kirk et al., 2008;Makonde et al., 2013). Individual termite colonies have been found to cultivate only single species of Termitomyces (Makonde et al., 2013). In this symbiotic association, old termite workers forage plant materials from the outside environment and store them inside the colony (Poulsen et al., 2014). Young termite workers ingest this stored plant biomass along with Termitomyces nodules containing abundant asexual spores and excrete lignin-rich excrements to build fresh fungus comb (da Costa et al., 2019a). Termitomyces grow on the fresh comb and decompose it efficiently. After 45 days of growth, the comb is mature. Well-decomposed mature combs are consumed by the older workers (Li et al., 2017). Mature combs produce basidiomes during rainy seasons. The evolution of this intricate relationship makes it extremely difficult to cultivate Termitomyces mushrooms artificially, although their mycelium can grow on several nutritive media and substrates (Tibuhwa, 2012;Ono et al., 2016;da Costa et al., 2019b).
Termitomyces rely on some nutrients from combs to fulfill most parts of their life cycle. Some studies investigated the composition of physicochemical properties such as pH, moisture, and soluble protein in combs and termite guts (Gomathi et al., 2009;Zeleke et al., 2013). Amino acids are important nutritional sources for most fungi. They play central roles both as building blocks of proteins and as intermediates in metabolism (Wu, 2009). The fungus comb nodules are enriched with amino acids (Chiu et al., 2019). Bacterial communities in combs also play significant roles. In recent years, some researchers identified various bacteria and fungi in combs Li et al., 2016;Liang et al., 2020). Some strains isolated from combs can stimulate the hyphal growth of Termitomyces (Sawhasan et al., 2012).
In this study, we compared physicochemical parameters including contents in amino acids, elements, sugars, fatty acids, and other basic indices between combs without Termitomyces basidiomes (CNF) and combs with Termitomyces basidiomes (CF) of the fungus-growing termite, Odontotermes formosanus. Microbial communities (bacteria and fungi) were also identified.

Collection and Preparation of Fungus Combs
Six healthy fungal combs of O. formosanus were collected from a forested area of Hangzhou City, Zhejiang Province, P. R. China (N 30 • 18 , E 120 • 5 ). There were three CNF and three CF. Samples were wrapped in plastic bags separately and transported to the laboratory within 6 h of excavation. For each sample, the pH and moisture contents were estimated immediately, and the rest of the combs were stored at −80 • C for subsequent experiments.

Basic Chemical Compositions of Fungus Combs
Moisture content and pH were tested as described by Zeleke et al. (2013) with some modifications. Approximately, 1 g of each sample was ground and dissolved with 5 ml distilled water, centrifuged at 8,000 × g for 10 min; the supernatants were used for the determination of pH. Moisture content was estimated by drying the comb to a constant weight. Contents of total soluble protein, total reducing sugar, and polysaccharides including lignin, hemicellulose, and cellulose were determined by using commercial kits (Comin Biotechnology Co., Ltd., Suzhou, China).

Element Content Determination
Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to quantify elements. The procedure for determining the element contents from the samples was similar to that of Toyama-Kato et al. (2003) with minor modifications. Around 0.1 g comb was dried at 40 • C, digested, heated to dryness, dissolved with 20 ml HNO 3 (1 M), and filtered using a membrane filter (pore size 0.22 µm). Then, 1 ml filtered solution was diluted 10 times with 1 M HNO 3 for the detection of calcium and magnesium separately. The operating conditions were shown in Supplementary Table S1.

Determination of Amino Acids
The extraction procedure: Approximately 0.05 g dried comb with 5 ml HCl (6 mol/L, containing 0.1% phenol) was ground and transferred to an Eppendorf tube. After hydrolysis for 20 h at 100 • C, 1 ml hydrolysate was collected in a petri dish and cooled down to room temperature and blown to almost dryness using a nitrogen blowing instrument (NBI, Organomation, United States). Then, 1 ml HCl (0.1 mol/L) was added to redissolve, and the turbid liquid was filtered.
Samples and standards derivatization: 200 µl comb extract from the extraction procedure mentioned above and 200 µl standard solution of 17 amino acids were transferred to Eppendorf tubes with 20 µl norleucine as an internal standard. Then, 200 µl triethylamine acetonitrile and 100 µl phenyl isothiocyanate-acetonitrile were added. After mixing, tubes were stable for 1 h at 25 • C. Finally, 400 µl n-hexane was added in each tube, left for 10 min after shaking, and then the lower solutions were collected and diluted five times, then filtered (pore size 0.45 µm). Finally, a 10-µl sample was injected for each analysis.
The amino acid analysis was carried out with a highperformance liquid chromatography (HPLC) system (Rigol L3000, Beijing RIGOL Technology Co., Ltd., China) equipped with a UV detector (HPLC-UV), and the column was Kromasil C18 (250 mm × 4.6 mm, 5 µm). The chromatographic conditions were: a flow rate at 1 ml/min, mobile phase A was sodium acetateacetonitrile solution and phase B was 80% acetonitrile solution.
The gradient was: 100% A for 15 min, 90% A with 10% B for 10 min, 70% A with 30% B for 8 min, 55% A with 45% B for 1 s, 100% B for 5 min, and 100% A for the last 7 min.
Approximately 1 g sample was weighed and homogenized with 5 ml ethyl alcohol for 3 min, then kept at room temperature and shaken occasionally for 10 min. The suspension was transferred into an Eppendorf tube and centrifuged for 20 min at 6,000 rpm. The internal standard (2-ethylbutyric acid solution) was added according to Zhao et al. (2006), and the supernatant was injected into a gas chromatography system equipped with a flame ionization detector (GC-FID) for analysis.
The capillary column (30 m × 320 µm × 1.80 µm) was used, and nitrogen was applied at a flow rate of 15.0 ml/min as the carrier gas. The initial oven temperature was 100 • C, maintained for 5 min, then raised to 200 • C at 20 • C/min and held for 5 min. Hydrogen, air, and nitrogen were the makeup gases at the flow rates of 35, 350, and 30 ml/min, respectively. The volume of the injected sample was 1 µl, and the run time for each solution was 15 min.

Microbial Community Characterizations in Fungus Combs
DNA was extracted from approximately 0.5 g of each comb using the DNeasy PowerSoil kit (Qiagen, GER) for bacteria and omega M5635-02 kit (Omega, United States) for fungi separately, according to manufacturer's instructions. The primers 341F (5 -CCTACGGGRBGCASCAG-3 ) and 806R (5 -GGACTACNNGGGTATCTAAT-3 ) were selected to amplify the V3-V4 variable regions of the bacterial 16S r RNA gene Liang et al., 2020). ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ) were used to amplify the fungal ITS region (Makonde et al., 2017). The amplification reaction for bacteria was performed in 25 µl final mixture [14.75 µl dd H 2 O, 2 µl dNTP (2.5 mM), 5 µl 5 × Reaction buffer, 1 µl of each primer (10 µM), 1 µl DNA template, and 0.25 µl Fast pfu DNA Polymerase (NEB, United States)], and the conditions for PCR were 98 • C for 5 min, followed by 25 cycles of 98 • C for 30 s, 52 • C for 30 s, and 72 • C for 45 s, with a final extension at 72 • C for 5 min. The fungal amplification reaction was the same as that for bacteria, and the conditions for PCR were 98 • C for 5 min, followed by 28 cycles of 98 • C for 30 s, 52 • C for 45 s, and 72 • C for 45 s, with a final extension at 72 • C for 5 min. The final target PCR products were visualized with agarose gel electrophoresis, then extracted and purified from the gel. DNA concentrations were quantified using Quant-iT PicoGreen ds DNA Assay Kit (Invitrogen, CA) and Microplate reader (BioTek, United States). Finally, samples were sequenced commercially (Personalbio Biotech, Shanghai, China) on Illumina MiSeq platform (paired-end) for bacteria and Pacbio Sequel platform (single-molecule real-time) for fungus.

Statistical Analysis
Differences in physicochemical parameters were tested with oneway ANOVA analysis and Student's t-test using SPSS, v.20.0 at alpha = 0.05. Pyrotag reads were denoised using the DADA2 method according to Benjamini and Hochberg (1995). Raw sequences were quality-filtered according to Koljalg et al. (2013) using the QIIME Release 8.0 software. Silva (Release132) 1 with 16S r RNA gene sequences of cockroach (Quast et al., 2013) and UNITE (Release 8.0) 2 (Koljalg et al., 2013) were used as reference databases for bacteria and fungi, respectively. Packages (stat, ape, Venn diagram, pheatmap, and ggplot2) of R software 3 were used to map and compare the relative abundance of bacteria and fungi at different levels. QIIME2 (2019.4) was used to calculate every index of alpha diversity including Chao1 (Chao, 1984), Observed species, Shannon (Shannon, 1948), Simpson (Simpson, 1949), Pielou's evenness (Pielou, 1966), and Good's coverage (Good, 1958). Kruskal-Wallis test (H-test) and Dunn test were applied to test for differences in alpha diversity.

Basic Parameters of Combs
Lignin was significantly (P < 0.01) lower in CF (229.84 ± 25.12) as compared to CNF (277.67 ± 45.82), whereas pH (P < 0.05) and moisture (P < 0.01) were relatively higher in CF as compared to CNF. The contents in total soluble protein, total reducing sugar, hemicellulose, and cellulose were not significantly different between CF and CNF (P > 0.05; Table 1).

Concentration of Elements in Combs
Eighteen elements in each sample were determined by ICP-OES. The average concentration of each element was different between CF and CNF. All elements (except Cd, Co, Na, P, and Pb) were higher in CF as compared to CNF ( Table 2). Cd and Pb were lower in CF than in CNF.

Amino Acid Analysis
We compared 17 amino acids between CF and CNF by HPLC-UV (Supplementary Figure S1 and Supplementary Table S2).

Bacterial Community Analysis
Between 21,566 and 24,198 high-quality sequences were obtained from each comb. The Good's coverage for every comb was more than 99.6% (Supplementary Table S5). The microbial diversity indices (Shannon and Simpson), estimated community richness (Chao1 and Observed species), and evenness (Pielou's evenness) were not statistically different between CF and CNF (Student's t-test; P > 0.05; Figure 3A). The unweighted pair-group method with arithmetic means (UPGMA) clustering analysis grouped all combs into two distinct groups, i.e., CF and CNF ( Figure 3B).
The Venn diagram showed that CNF and CF occupied 532 (41.30%) and 441 (31.91%) operational taxonomic units (OTUs), respectively. The OTU 345 (26.79%) was common between both comb categories ( Figure 3C). The abundance of various bacterial groups at different taxonomic levels (phylum, family, and genus) was investigated. A total of 20 bacterial phyla were found and compared ( Figure 4A) Table S6).

Fungal Community Analysis
Between 4,008 and 5,854 high-quality sequences were obtained from every comb. The coverage for every comb was closed to 100% (Supplementary Table S9). Alpha diversity indices (Shannon,Simpson,Chao1,Observed species,and Pielou's evenness) were not significantly different between comb types (Student's t-test; P > 0.05).

DISCUSSION
The present study showed differences in physicochemical properties and microbial communities between CF and CNF. Most fungi grow well at acidic pH and low humidity (Hesse, 1955;Gomathi et al., 2009), and we indeed found low moisture and pH in all combs (Table 1). However, we disagree with the previous study reporting that moisture content decreased after basidiome formation (Sidde Gowda and Rajagopal, 1990). Less lignin was recorded from combs with basidiomes as compared to combs without basidiomes (Table 1), indicating that Termitomyces degrades lignin during the comb maturation period (Hyodo et al., 2000;Bashir et al., 2015).
Our results showed the variation tendency of 18 elements including 16 metal and two non-metal elements in combs ( Table 2). Li et al. (2012) also found metal ions in fungus combs. Element contents were relatively higher in CF as compared to CNF. Mushrooms can absorb large amounts of water and minerals from their substrates (Koutrotsios et al., 2020). The bioaccumulation capacity of mushroom is influenced by the substrate, fungal lifestyle, age of the fruiting body, species, type of element, and environment (Campos et al., 2009;Lee et al., 2009;Koutrotsios et al., 2020). We assume that the high concentrations of elements in combs are associated with lignocellulose degradation and fungal growth. Some studies demonstrated that elements are important for fungal metabolic functions related to enzyme production and lignocellulose degradation (Li et al., 2012;Koutrotsios et al., 2020), but less information is available on the significant roles of elements in fungal growth. Cuero et al. (2003) reported that various metal ions caused some phenotypic or genotypic influences in fungus and could stimulate fungal growth. Moreover, some elements play a crucial role in enzymatic reactions regarding nitrogen fixation (Hoffman et al., 2014). For example, molybdenum, iron, and magnesium are essential for the structure formation of metal clusters in nitrogenase and MgATP energy supply (Hoffman et al., 2014), which may strengthen nitrogen fixation.
Amino acids were higher in CNF as compared to CF. This may be due to the uptake of amino acids in basidiomes from combs. It has been reported that amino acids are transported into a fungal cell from the substrate (Whitaker, 1976). Termitomyces mushrooms are capable of transforming amino acids or synthesizing amino acids from non-protein nitrogen (Botha and Eicker, 1992;Deacon, 2006). In termitefungus association, the amino acids are accumulated in matured fungus combs and fungal nodules (Botha and Eicker, 1992). Chiu et al. (2019) compared 18 amino acids between fungus combs and fungal nodules of Termitomyces. They allocated 7% amino acids to nodules and 93% to combs. Moreover, it has also been reported that amino acids are important for fungal growth and reproduction (Steinberg, 1950;Yang et al., 2020). However, knowledge on the role of amino acids on fungal growth is less satisfactory.
All fatty acids were found in our experiment except formic acid (Figure 2 and Supplementary Table S4). Some short-chain fatty acids such as acetic acid, propionic acid, and butyric acid were also reported previously both in termite guts and fungus combs . It has been reported that fatty acids are produced by microorganisms living in termite guts during the fermentation of fiber and starch (Odelson and Breznak, 1983;Thorburn et al., 2014;Ciarlo et al., 2016). The bacterial communities in fungus combs are involved in lignocellulose degradation, nutrient supplementation, nitrogen fixation, and antibiotic production , but whether these bacteria play significant roles in the appearance of basidiomes from the comb remains to be proven. We found that the major bacterial lineages were similar among all combs, but there were marked changes in their abundance (Figures 4, 5 and Supplementary Tables S6-S8). Actinobacteria, Bacteroidetes, Cyanobacteria, Firmicutes, and Proteobacteria were plentifully found in all combs ( Figure 3A and Supplementary Table S6). We are consistent with the previous studies on fungus combs Liang et al., 2020). It has been reported that some members of Bacteroidetes and Firmicutes can play important roles in the metabolism of carbohydrates (Poulsen et al., 2014;Delmont et al., 2018). Proteobacteria along with Bacteroidetes and Firmicutes are related with nitrogen input, which may help meet the nutritional needs of Termitomyces (Poulsen et al., 2014;Delmont et al., 2018). The nitrogenase reductase nifH genes have been identified in Proteobacteria (Delmont et al., 2018). Liang et al. (2020) suggested that Proteobacteria in fungus comb assist fungus in atmospheric nitrogen fixation. Our findings also indicated that Cyanobacteria and Actinobacteria were more abundant in CF than in CNF (Figure 3A and Supplementary Table S6). Biologists suggested that many species of Cyanobacteria found in fungus combs can fix N 2 (Stewart, 1980;Makonde et al., 2015). It has been described that the C/N ratio can maximize fungal growth performance in vitro (Shik et al., 2016). Actinobacteria have also been isolated from fungus combs previously (Visser et al., 2012;Benndorf et al., 2018;Yin et al., 2019). They are generally known as defensive symbionts because they produce antibiotic compounds to suppress invading pathogens (Visser et al., 2012;Benndorf et al., 2018;Yin et al., 2019). Based on these results, we believed that some bacteria from combs are important to create a suitable growth condition for Termitomyces by maintaining the C/N balance or inhibiting the infectious microbes.
Fungal communities of Basidiomycota and Ascomycota were also identified in combs; however, the members of Termitomyces were dominant in all combs ( Table 3). These results were the same as reported previously by Shinzato et al. (2005) that Termitomyces was the only visible fungus on the nests of termites, and the individual nest has been found to contain only single species of Termitomyces within the fungus garden.

CONCLUSION
The current study investigated the differences in physicochemical factors (pH, moisture, total soluble protein, total reducing sugar, cellulose, hemicellulose, lignin, amino acids, metal ions, and fatty acids) and microbial communities (bacteria and fungi) between CF and CNF. A noticeable increase of humidity, pH, and elements (Al, Ba, Fe, Mn, Ni, S, Ca, and Mg) and a decrease of some amino acids (alanine, tyrosine, and isoleucine) were recorded in CF as compared to CNF. This study also revealed a high level of bacterial diversity in fungal combs. The majority of the bacterial communities might assist Termitomyces in lignocellulose degradation and maintenance of the C/N ratio. The possible role of physicochemical characteristics and bacterial communities on the onset of basidiome development by Termitomyces remains unknown, and further in-depth studies are needed.

DATA AVAILABILITY STATEMENT
The datasets were submitted to the Sequence Read Archive of NCBI (http://www.ncbi.nlm.nih.gov; BioProject PRJNA66 2821).

AUTHOR CONTRIBUTIONS
GY and JM: conceptualization, project administration, and funding acquisition. GY: methodology. GY and FA: software, writing-original draft preparation, and visualization. GY, QZ, and MG: validation. GY and SL: formal analysis and data curation. GY and MG: investigation. GY and QZ: resources. FA, HG, and JM: writing-review and editing. JM: supervision.
All authors have read and agreed to the published version of the manuscript.

FUNDING
This work was financially supported by the National Natural Science Foundation of China (No. 31770686) and a project supported by the Scientific Research Fund of Zhejiang Provincial Education Department (Y201941400).