Role of the mutualistic fungus in lignin degradation in the
fungus-growing termite
Macrotermes gilvus
(Isoptera;
Macrotermitinae)
F. Hyodo
a,*, T. Inoue
a, 1, J.-I. Azuma
b, I. Tayasu
b, T. Abe
aa
Center for Ecological Research, Kyoto University, Kamitanakami Hirano-cho, Otsu, Shiga, 520-2113, Japan
b
Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto, 606-8502, Japan
Accepted 3 October 1999
Abstract
In order to investigate the role of the mutualistic fungus, Termitomyces sp., in the fungus-growing termite, Macrotermes gilvus, we applied CP/MAS 13C NMR and selected proximate analyses to fungus comb of dierent ages and degrees of
maturation. We found evidence that lignin degradation took place progressively in the fungus comb. In vitro digestibility of cellulose in old fungus comb, on which the termites feed, was approximately 3-fold higher than that in the fresh part. These results con®rm the `lignin degradation hypothesis' that the role of the mutualistic fungi is to degrade lignin and enhance the digestibility of cellulose for the termites, suggesting the ability of the termite±fungus association to make extremely ecient use of plant material.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Fungus-growing termite; Lignin degradation; Cellulose digestibility;Macrotermes gilvus;Termitomycessp.
1. Introduction
Fungus-growing termites (Isoptera; Termitidae; Macrotermitinae) are abundant in the African and Asian tropics (Wood and Sands, 1978; Abe and Mat-sumoto, 1979). They play a signi®cant role in the de-composition of plant litter, for example consuming more than 90% of dry wood in some arid tropical areas (Buxton, 1981) and directly mineralizing up to 20% of the net primary production in wetter savannas (Wood and Sands, 1978). They have evolved an unique mutualism with basidiomycete fungi of the genus, Ter-mitomyces. The symbiotic fungi grow on a special
cul-ture within the nest maintained by the termites and called `fungus comb'. The fungus comb is made from partly digested foraged plant litter which passes rapidly through the termite's gut. The resulting faecal pellets are pressed together to make a comb-like matrix. As the comb matures, mycelium develops and produces conidial nodules, which together with older, senescent comb are consumed by workers (Sieber and Leuthold, 1981).
Because of the unique symbiotic relationship, many studies have been conducted on the termite±fungus as-sociation (reviews by Sands, 1969; Wood and Thomas, 1989; Darlington, 1994). Several roles have been suggested for the fungal symbiont, for example, the provision of heat and moisture (LuÈsher, 1951), the provision of a concentrated nitrogen source (as coni-dia, Matsumoto, 1976) and the enrichment of nitrogen in foraged foodstus by virtue of the fungal metab-olism (Collins, 1983).
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* Corresponding author. Tel.: 8200; fax: +81-77-549-8201.
E-mail address:[email protected] (F. Hyodo). 1
Plant materials consist mainly of lignocellulose, in which cellulose is protected by lignin against enzymatic attack (Reid, 1989). In terms of the overall processing of lignocellulose by the termite±fungus partnership, most studies have focused on the role of the fungus in assisting cellulose digestion by the termites. Martin and Martin (1978) reported that the fungus-growing termite, Macrotermes natalensis, acquires a component of cellulase from the fungus. This `acquired enzyme hypothesis' is supported by Martin and Martin (1979) in M. natalensis and by Rouland et al. (1988a, 1988b) in M. mulleri, who identi®ed cellulases, apparently of fungal origin, in the termite midgut. However, Veivers et al. (1991) could supply no evidence to support the hypothesis from studies of M. subhyalinus and M. michaelseni, which were said to degrade cellulose en-dogenously. It has been recently recognized that wood and litter feeding termites produce endogenous cellu-lase (Slaytor, 1992; Inoue et al., 1997; Watanabe et al., 1998). Thus, it is dicult to make generalizations about the signi®cance of the fungal cellulase in cellu-lose digestion by fungus-growing termites (Breznak and Brune, 1994).
Grasse and Noirot (1958) proposed the `lignin degradation hypothesis' that the symbiotic fungi have the ability to degrade lignin, which makes cellulose more easily attacked by the termites' own cellulase. In support of this, Rohrmann (1978) showed that the lig-nin content decreased in old fungus comb ofM. ukuzii
and M. natalensis. In higher plants, cellulose is encrusted with lignin, which prevents its digestion, so that for enzyme accessibility to the cellulose, the lignin must be disrupted (Kirk and Chang, 1981). Thus, Higashi and Abe (1997) inferred that lignin is an ob-stacle for termites to acquire energy and a carbon source from cellulose.
We examined the `lignin degradation hypothesis' by using CP/MAS 13C NMR to characterize lignin and an estimation of the in vitro digestibility of cellulose in fungus combs from the Southeast Asian fungus-grow-ing termite,M. gilvus.
2. Materials and methods
2.1. Termites and comb-building behavior
Macrotermes gilvus is widely distributed in southern Asia, from Burma to Indonesia and the Philippines. The termite collects grass, leaves and stalks of plants and stores them in the nest before processing the ma-terial to form fungus comb (Roonwal, 1970). To verify that M. gilvus builds the fungus combs as reported in other fungus growers from the genus Macrotermes, a study was conducted at Narathiwat, in Thailand, in December, 1992. Stored food collected from a mound
of M. gilvuswas stained with methylene blue, replaced in situ after drying and the mound sealed. After 3 d, the location of the dye was observed.
2.2. Fungus comb sampling
Fungus combs from M. gilvus were collected from two sites. Fungus combs A were collected from a mound in a dry dipterocarp forest, composed mainly of Shorea spp., in JICA Nursery Station, Nakhon Ratchashima Pref., Thailand, in August, 1997, and fungus combs B were sampled in a rubber plantation at the Narathiwat area in Thailand, in December, 1992.
Comb of progressively increasing age was separated and de®ned as follows (Darlington, 1994): `fresh' comb was the top rim of the comb and freshly made; `old' comb was the bottom of the comb and eaten by the termite workers; `mature' comb was the middle pos-ition between the two parts. Three separate samples of each type of comb were taken from each colony. The separated samples were hand homogenized in a mortar and pestle and used for the chemical analyses and CP/ MAS13C NMR spectroscopic study described below.
2.3. Chemical analyses
For determination of sugars, each sample was hydrolyzed as described by Saeman et al. (1954). Quantitative analysis of monosaccharides including glucosamine was performed by high performance anion exchange chromatography (HPAEC) on a Dio-nex DX-500 system (Sunnyvale, CA) equipped with a CarboPac PA-1 column (4 250 mm) and pulsed
amperometric detector (ED-40), using 1.0 mM NaOH as an elution solvent. Chitin was determined as the amount of glucosamine. Acid-insoluble lignin content was determined based on the Tappi method (T222-om88). Ash was determined by ignition at 6008C for 4 h.
2.4. NMR spectroscopy
2.5. In vitro cellulose digestibility
In vitro cellulose digestibility of the three parts from the fungus combs was estimated by measuring redu-cing sugars produced from each sample after enzy-matic hydrolysis, which was performed at 378C for 48 h at substrate and enzymatic concentrations of 0.5 and 0.2%, respectively, in 0.1 M sodium acetate buer at pH 5.5. One drop of toluene was added as a preserva-tive. The enzyme used was a commercial cellulase preparation (Meicelase CEPB-5042, Meiji Seika Indus-try Co.) prepared from the culture ®ltrate of Tricho-derma viride. The amounts of the reducing sugars were determined by the tetrazolium blue method (Jue and Lipke, 1985) and expressed as glucose equivalents. In vitro digestibility was expressed as hydrolyzable sugar contents in each sample (wt%).
3. Results
3.1. Fungus comb building behavior of M. gilvus
The position on the fungus comb where the stored food was deposited was recognizable by its blue color. Blue colored material was found only on the top of some fungus combs within the mound. This building behavior is consistent with other reports for the genus
Macrotermes.
3.2. Chemical characteristics
The overall chemical characteristics of the fungus comb are listed in Table 1. Lignin, neutral carbo-hydrates and chitin concentration showed signi®cant dierences among the dierent age combs at both study sites (P < 0.05, Kruskal±Wallis test). Lignin
concentration clearly decreased with ripening with an accompanying increase of neutral carbohydrates and chitin. These trends were the same at both study sites, although there were dierences in the trend in ash con-centration and the values of chemical components between the two sites. The reason for the dierence between the two study sites is not certain, but may be due to the dierence in the primary plant materials at the two study sites. There were signi®cant dierences in the relative concentrations of neutral carbohydrate components among the dierent age combs (P< 0.05, Kruskal±Wallis test), except for arabinose and gluco-samine of fungus comb A and rhamnose of fungus comb B (Table 2). Relative concentrations of glucose and glucosamine apparently increased, while those of almost all the other neutral carbohydrates decreased. These data indicate the conversion of carbohydrates from plant tissue into those of fungal cell wall.
3.3. CP/MAS 13C NMR spectroscopic study
The spectra of three parts of fungus combs A are shown in Fig. 1. The comparison of spectra showed that, as the fungus comb ripens, the signal at 21.5 ppm due to acetate groups in hemicellulose decreased. The signal at 33 ppm due to alkyl-C increased, suggesting the preser-vation of aliphatic compounds (Norden and Berg, 1990).
Signals around 110±160 ppm, especially at 154 ppm, corresponding to aromatic-C in lignin, decreased as the ripeness advanced. The signal at 56 ppm, assigned to aryl methoxyl carbons of lignin, was clearly
Table 1
Chemical composition of fungus comb ofM. gilvus(as ash-free dry weight %)a
Fresh Mature Old Pb
Fungus comb A
Neutral carbohydrate 32.822.5 37.621.1 42.020.9 < 0.05 Lignin 32.621.1 18.921.2 15.021.3 < 0.05 Chitin 0.420.04 0.620.03 0.720.04 < 0.05
Ash 14.021.2 11.421.1 15.520.9 NS
Fungus comb B
Neutral carbohydrate 43.820.8 46.521.2 53.420.8 < 0.05 Lignin 34.620.5 16.520.5 53.420.8 < 0.05 Chitin 0.620.04 1.120.04 1.620.01 < 0.05 Ash 5.720.8 9.321.5 11.221.2 < 0.05
a
Values are means of three determinations2S.D. NS=not signi®-cant.
b
Kruskal±Wallis test was used for comparisons among the dier-ent age combs.
Table 2
Relative neutral carbohydrate composition of each part of fungus comb A and B (relative weight %)a
Fresh Mature Old Pb
Fungus comb A
Arabinose 5.820.01 5.220.47 4.320.31 NS Rhamnose 1.920.08 1.220.00 1.020.04 < 0.05 Galactose 5.220.08 3.620.19 3.120.08 < 0.05 Glucosamine 1.220.06 3.620.11 1.720.09 NS Glucose 52.220.05 57.921.47 62.620.14 < 0.05 Xylose 3.020.20 27.420.84 24.220.27 < 0.05 Mannose 3.620.08 3.020.07 3.020.03 < 0.05
Fungus comb B
Arabinose 7.520.06 6.620.05 5.720.23 < 0.05 Rhamnose 1.120.05 1.120.05 1.320.05 NS Galactose 4.620.04 3.720.03 3.620.06 < 0.05 Glucosamine 1.320.07 2.420.06 3.120.03 < 0.05 Glucose 49.220.16 53.720.08 63.320.48 < 0.05 Xylose 33.620.14 29.620.06 20.320.14 < 0.05 Mannose 2.820.10 2.920.02 2.620.07 < 0.05
a
Values are means of three determinations2S.D. NS=not signi®-cant.
b
reduced. These results indicate that lignin degradation progressed in the fungus comb with aging. Fungus combs B also showed the same spectroscopic patterns as those from the fungus combs A (not shown).
3.4. In vitro cellulose digestibility
In vitro cellulose digestibility values were signi®-cantly dierent between the dierent age combs (P<
0.05, Kruskal±Wallis test) and clearly increased with the aging in the two study sites (Fig. 2). In the old comb, cellulose digestibility was 2.6 (fungus combs A) and 3.1 (fungus combs B) times higher than that of the fresh part.
4. Discussion
From both the behavioral and the chemical study, we suggest that M. gilvus selectively utilizes the old part of the fungus comb, where lignin degradation has occurred and cellulose is therefore more digestible. Thus, this study con®rmed the `lignin degradation hy-pothesis' of Grasse and Noirot (1958) that the role of the symbiotic fungi is to overcome the lignin obstacle and to increase the digestibility of cellulose for the ter-mites. However, it is necessary to note that T. viride, rather than termite cellulase, was used to make this assessment.
Our results also indicate that the fresh part of the fungus combs contained a higher amount of carbo-hydrate relative to lignin (lignin-to-carbocarbo-hydrate ratio is 0.99 and 0.77 in fungus comb A and B, respectively) compared to faecal-derived nest structures of non fun-Fig. 1. CP/MAS13C NMR spectra of three parts (fresh, mature, old) of fungus combs fromM. gilvus.
gus-associated termites where the ratio ranges between 1.64 and 5.39 (Lee and Wood, 1971). Assuming that fresh faeces are almost equivalent to the fresh part of the comb, we conclude thatM. gilvusdid not, or could not, digest cellulose and other carbohydrates exten-sively in the ®rst passage of food through the gut. This is consistent with previous information that fresh faeces are macerated and undigested plant materials (Darlington, 1994). In terms of deligni®cation, the high carbohydrate content in the primary faeces may be very important, since white-rot fungi which include
Termitomyces spp. are not able to use lignin alone as growth substrate, so that other carbon and energy sources, such as glucose and cellulose, are required (Kirk et al., 1976).
The occurrence of lignin and polysaccharide degra-dation in the fungus comb during aging is predicted to be accompanied by nitrogen enrichment by eliminating carbon from the substrate. However, there is no evi-dence to support this prediction. In fact, the published results from M. subhyalinus (Abo-Khatwa, 1977), M. natalensis and M. ukuzii (Rohrmann, 1978) revealed that the nitrogen concentration in the old comb is lower than that in the fresh comb, or unchanged. Nitrogen depletion during the aging might be caused by the uptake of nitrogen by conidia in the mature comb (Rohrmann, 1978). Thus, it is unlikely that the fungus-growing termites selectively consume the old comb on the basis of their nitrogen requirement alone, although the nitrogen concentration of the whole fun-gus comb is high, relatively to the plant litter collected by the termites, and furthermore, fungus comb pro-duces conidia rich in nitrogen which is consumed to support larvae and nymphs (Collins, 1983). Therefore, the old comb is suggested to serve as the most suitable food, because it combines general nitrogen enrichment with speci®c accessibility of cellulose for digestion.
Compared with other termites, for example those with protozoa and bacteria as mutualists or those with intestinal bacteria only, it is unclear whether the fun-gus-growing termites can utilize cellulose in plant ma-terials with greater overall eciency, because white-rot fungi also consume cellulose for metabolism. From the ecosystem point of view, however, we can note that by associating with the lignin decomposer, the fungus-growing termites make it possible to utilize lignocellu-lose nearly completely, re¯ected in the small volume of their ®nal faeces (Darlington, 1994) and therefore to play a dominant role in decomposition processes in many parts of the tropics (Abe, 1980; Buxton, 1981).
Acknowledgements
We thank Dr. N. Kirtibutr, Dr. Y. Takematsu and Mr. C. Klangkaew for their various assistance in this
study and Dr. M. Higashi and Dr. A. Ushimaru for their helpful comments. We are also grateful to National Research Council of Thailand (NRCT) for the permission of this research at Sakaerat Environ-mental Research Station, and the stas of Royal For-est Department (RFD) for their kind cooperation. The study was carried out within the framework of an inte-grated study on biodiversity conservation under global change and bio-inventory management system sup-ported by a Grant-in-Aid for Creative Basic Research of the Japanese Ministry of Education, Science and Culture.
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