ARTICLE IN PRESS Microbiological Research 161 (2006) 299—303 0944-5013/$ - s doi:10.1016/j. �Correspond E-mail addr www.elsevier.de/micres Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants A. Silvaa,b,�, M. Bacci Jra,c, F.C. Pagnoccaa,c, O.C. Buenoa,d, M.J.A. Heblinga aCentro de Estudos de Insetos Sociais, Universidade Estadual Paulista (UNESP), Rio Claro, São Paulo, Brazil bDepartamento de Produc-ão Vegetal/Defesa Fitossanitária, UNESP, Botucatu, São Paulo, Brazil cDepartamento de Bioquı́mica e Microbiologia, UNESP, Rio Claro, São Paulo, Brazil dDepartamento de Biologia, UNESP, Rio Claro, São Paulo, Brazil Accepted 28 November 2005 KEYWORDS Leucoagaricus gon- gylophorus; Atta sexdens; Enzymes induction; Leaf-cutting ants; Amylase ee front matter & 2005 micres.2005.11.001 ing author. Centro de E ess: enila@estadao.com Summary Leucoagaricus gongylophorus, the symbiotic fungus of the leaf-cutting ants, degrades starch, this degradation being supposed to occur in the plant material which leafcutters forage to the nests, generating most of the glucose which the ants utilize for food. In the present investigation, we show that laboratory cultures of L. gongylophorus produce extracellular a-amylase and maltase which degrade starch to glucose, reinforcing that the ants can obtain glucose from starch through the symbiotic fungus. Glucose was found to repress a-amylase and, more severely, maltase activity, thus repressing starch degradation by L. gongylophorus, so that we hypothesize that: (1) glucose down-regulation of starch degradation also occurs in the Atta sexdens fungus garden; (2) glucose consumption from the fungus garden by A. sexdens stimulates degradation of starch from plant material by L. gongylo- phorus, which may represent a mechanism by which leafcutters can control enzyme production by the symbiotic fungus. Since glucose is found in the fungus garden inside the nests, down-regulation of starch degradation by glucose is supposed to occur in the nest and play a part in the control of fungal enzyme production by leafcutters. & 2005 Elsevier GmbH. All rights reserved. Elsevier GmbH. All rights reserved. studos de Insetos Sociais, Universidade Estadual Paulista (UNESP), Rio Claro, São Paulo, Brazil. .br (A. Silva). www.elsevier.de/micres ARTICLE IN PRESS A. Silva et al.300 Introduction The fungus Leucoagaricus gongylophorus is the symbiotic partner of several species of leaf-cutting ants (Silva-Pinhati et al., 2004), living on cut leaves inside the nests, to form the so-called fungus garden (Weber, 1955). There, the fungus produces enzymes that degrade foliar polysaccharides into assimilable nutrients for the ants. Among these nutrients, glucose produced from plant material in the fungus garden seems to be the main food source supporting the ants (Silva et al., 2003). Glucose could also be shown to support L. gongylophorus growth in laboratory cultures (Siqueira et al., 1998). Martin and Weber (1969) have proposed cellu- lase, which is produced by the symbiotic fungus, as the main operating enzyme to generate nutrients for the ants from cellulose degradation. This theory has been accepted for many years and was reinforced by findings that show L. gongylophorus to be able to degrade cellulose (Bacci et al., 1995). However, additional studies have indicated that cellulase is poorly produced by the symbiotic fungus compared to other polysaccharidases, such as pectinases, amylases and xylanases (Siqueira et al., 1998). Therefore, we have proposed starch degradation by L. gongylophorus as the main source of glucose in the fungus garden (Silva et al., 2003). In the present investigation, we show new insights in starch metabolism by L. gongylophorus, by characterizing growth rate and a-amylase, maltase and glucose production by this fungus cultured on different carbon sources. Our results confirmed the production of extracellular glucose from starch by L. gongylophorus and showed that glucose represses the production of fungal a- amylase and maltase and, ultimately, starch de- gradation by the fungus. We propose that such repression is a mechanism by which leafcutters control enzyme production by the symbiotic fungus inside the nests. Material and methods Fungal cultivation The strain B1-97 of L. gongylophorus Möller (Singer), isolated from Atta sexdens Linnaeus nest, was cultured at 25 1C in slanted medium A (Pagnocca et al., 1990) for 30 days, after which two loopful fungal mycelia were transferred to 250mL flasks containing 50mL YNB-glucose culture medium (75mM citrate-phosphate buffer pH 5.0, 0.67 g 100mL�1 Yeast Nitrogen Base (Difco, United States, catalog no. 100690) and 0.50 g 100mL�1 glucose) and the flasks were statically incubated at 25 1C for 30 days. Mycelial mass was then harvested with a loop and suspended in 150mL sterile water and mixed with a Potter device. Then, 1.0mL of cell suspension (0.870.02mg cell mass, dry weight) was transferred to 125mL flasks with 50mL YNB medium containing one (0.5 g100mL�1) or two (0.25 g100mL�1 each) of the following carbohydrates: (a) glucose (J.T. Baker, Mexico, catalog no. 1916-01); (b) starch (Sigma, Japan, catalog no. S 9765); (c) maltose (Sigma, Germany, catalog no. M 9171); (d) starch and maltose; (e) starch and glucose; (f) maltose and glucose. Twenty-four flasks of each culture medium were statically incubated at 25 1C, six of each were collected at each of the incubation times (10, 15, 20 and 30 days). Culture media (50mL) were then filtered through a 0.45 mm filter, and 0.1mL samples were used for glucose assay (see below). The remaining filtrate was dialyzed against distilled water (24 h, 6–9 1C, replacing the water after 12 h), concentrated by liophylization, and dissolved in 1.0mL 75mM citrate-phosphate buffer (pH 5.0), in order to form what we called the crude sample, which was glucose-free and utilized for enzymatic assays. The mycelium, which was collected from each of the 50mL culture media, was used to determine cell mass, which was expressed in milligrams of dry weight. The experiment described here was carried out in duplicate. Enzyme assay a-amylase was assayed with 1.0mL of 2.0 g 100mL�1 starch solution in 75mM citrate- phosphate buffer (pH 5.0) containing 2mM calcium chloride. A similar procedure was used for maltase assay, with 2.0 g 100mL�1 maltose as the substrate. Starch or maltose solutions were mixed with 1mL 1/10 (v/v) crude sample and the reaction mixture was kept at 30 1C for 60min. At zero, 15, 30, 45 and 60min, two 0.1mL samples were collected from the reaction mixture, boiled for 3min and sub- mitted to glucose analysis (maltase determination) or starch analysis (amylase determination). Glucose was assayed with 0.1mL sample, 0.9mL water and 1.0mL of glucose oxydase reagent (Labtest, Brazil, catalog no. 34-E). After 15min at 37 1C, the reaction mixture had its optical density (OD) at 505 nm determined and glucose concentration in the culture media was calculated by comparison with a glucose standard curve. Starch was assayed with 0.1mL sample and 1.0mL iodine reagent, this ARTICLE IN PRESS Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants 301 reagent was prepared with 0.25 g 100mL�1 iodine (Merck, Germany, catalog no. 4761) and 0.16 g 100mL�1 potassium iodide (Mallinckrodt, France, catalog no. 1127), according to Bernfeld (1955). Starch concentration was determined by OD at 620 nm and a starch standard curve. Glucose or starch concentrations were plotted against the incubation time and the resulting curve slope was used for calculating enzyme activity. One amylase unit (UA) corresponded to a decrease of 1 mg starchmin�1mL�1 of culture medium. One maltase unit (UM) corresponded to the production of 1 mMol glucosemin�1mL�1 of culture medium. Statistics In order to detect differences in amylase or maltase production in distinct carbon sources, enzyme activity values obtained in the five culture media (n ¼ 48 values for each culture medium for each enzyme) were compared to each other (a) 0 10 20 30 40 50 0 10 15 20 30 0 10 15 20 30 0 10 15 20 30 U 0 1 2 3 4 5 6 1 2 3 4 5 (c) 0 10 20 30 40 50 U 0 1 2 3 4 5 6 (e) 0 10 20 30 40 50 Days U 0 1 2 3 4 5 6 Figure 1. Units (U) of a-amylase (&) or maltase (’) and co Leucoagaricus gongylophorus in different carbon sources: (a) (e) starch and glucose and (f) maltose and glucose. Values of incubation times corresponded to the means obtained from 1 through the Tukey test (95% significance level) (Zar, 1996), which was also used for comparing cell mass production (n ¼ 48 per culture medium). Results The fungus L. gongylophorus was cultured for 30 days at 25 1C in YNB broth containing glucose, starch, maltose, starch and maltose, starch and glucose, or maltose and glucose. The mycelial mass (mg of dry weight) produced was 2271.6, 1571.2, 1571.3, 1670.5, 1972.0 and 1970.6, respec- tively, values which are not significantly distinct from each other. The fungus growing on media containing starch, maltose, or starch and maltose produced greater amounts of a-amylase (Fig. 1b–d) than those grown on culture media containing glucose, starch and glucose or maltose and glucose, indicating that glucose repressed a-amylase activity slightly 0 10 15 20 30 0 10 15 20 30 0 10 15 20 30 (b) 0 0 0 0 0 0 0 1 2 3 4 5 6 G lu co se ( m g/ m L ) (d) 0 10 20 30 40 50 0 1 2 3 4 5 6 G lu co se ( m g/ m L ) (f) 0 10 20 30 40 50 Days 0 1 2 3 4 5 6 G lu co se ( m g/ m L ) ncentration (mg/mL) of glucose (m) during cultivation of glucose, (b) starch, (c) maltose, (d) starch and maltose, enzyme activity or glucose concentration for each of the 2 replicates. ARTICLE IN PRESS Table 1. a-amylase (UA) or maltase (UM) units (mean7SD, n ¼ 48) produced by Leucoagaricus gongylo- phorus on different carbon sources over entire period of growth Carbon source UA UM Glucose 7.5 (4.9)a,b� 0.0c Starch 9.6 (4.2)a 12.7 (9.8)a Maltose 11.2 (3.9)a 3.6 (1.3)b Starch and maltose 13.1 (3.4)a 3.4 (1.5)b Starch and glucose 5.0 (2.6)b 0.0c Maltose and glucose 5.7 (3.7)b 0.0c �Values followed by distinct letters are significantly different from each other (Tukey test). A. Silva et al.302 (Fig. 1a, e and f). Similarly, starch was the best carbon source supporting maltase production, followed by maltose and starch plus maltose (Fig. 1b–d), as no maltase activity could be detected in media containing glucose, starch plus glucose nor maltose plus glucose (Fig. 1a, e and f). These results indicate that glucose severely represses maltase production by L. gongylophorus. The higher sensitivity of maltase to glucose was characterized during growth on YNB containing glucose (Fig. 1a), during which no maltase was detected, whereas a-amylase production was very low during the first 10 days of incubation, and increased after 20 days, when glucose concentra- tion decreased to 3.470.4mgmL�1. When L. gongylophorus was cultured on starch, glucose was produced in the culture medium, resulting in a dramatic decrease of maltase activity from the 10th to the 20th incubation day, as a-amylase activity increased in this period of time (Fig. 1b). The total production of a-amylase over the entire period of growth indicated that starch or maltose, sole or mixed, are the best carbon source to induce a-amylase production by L. gongylophorus. For maltase production, starch was the best substrate (Table 1). Discussion In a previous work (Silva et al., 2003) it had been shown that glucose is the main nutrient supporting the survival of workers of A. sexdens, these workers are able to consume glucose which is generated apparently from plant material in the fungus garden. In the present investigation we found that L. gongylophorus, the fungus symbiotic with leaf- cutting ants, is able to grow on starch, maltose, glucose, or combinations of these carbon sources. During growth on starch or maltose, glucose was generated in the culture media, suggesting that glucose may be produced by L. gongylophorus through the degradation of the starch occurring in the plant material which is introduced in the nest by forager ants. These findings suggest that glucose production from plant material, through starch hydrolysis by fungal extracellular a-amylase and maltase, is a continuous process inside the ants’ nest by which L. gongylophorus contributes to the ants’ nutrition on starch. This process may be especially important for the ants, which apparently are not able to feed directly on starch, but readily utilize glucose (Silva et al., 2003). The results also point to regulatory character- istics of enzyme production by L. gongylophorus, which produced a-amylase in all the carbon sources analyzed in the present investigation, indicating that a-amylase is constitutively produced by the fungus. However, this enzyme activity decreased when glucose was one of the carbon sources of the culture medium or when glucose was generated in the culture medium by the hydrolysis of maltose or starch, indicating that amylase can be somewhat repressed by glucose. A similar reduction in enzyme activity upon glucose production was also observed with maltase, but, contrarily to a-amylase, maltase was not produced when glucose was added to culture media, which indicates that maltase is more severely repressed by glucose. Regulation of enzyme production by L. gongylo- phorus is similar to that seen in some Aspergillus species, in which the production of a-amylase on starch or maltose was higher than that on glucose (Lachmund et al., 1993; Nahas and Waldemarin, 2002). Also, regulation of enzyme production in L. gongylophorus resembles that seen in Sulfolobus solfataricus, in which glucose inhibits the produc- tion of a-amylase (Haseltine et al., 1996), as well as that seen in some species of Termitomyces, the fungi cultured by termites, in which starch induces the production of a-amylase and maltase (Mora and Rouland, 1995). Glucose down-regulation of its own production from starch by L. gongylophorus is also expected to occur in the A. sexdens fungus garden, which was found to contain glucose (Silva et al., 2003). In addition, glucose was found to be consumed from the fungus garden by worker ants (Silva et al., 2003), so that this consumption is expected to stimulate a-amylase and maltase production by L. gongylophorus and, ultimately, starch degradation of plant material. Therefore, it is possible that leafcutters can control the amount of enzyme produced by their symbiotic fungus through glucose consumption from the fungus garden. ARTICLE IN PRESS Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants 303 Acknowledgements This work was supported by FAPESP – The State of São Paulo Research Foundation (Fundac-ão de Am- paro à Pesquisa do Estado de São Paulo), CAPES – Higher Level Professional Enhancement Coordinating Foundation (Coordenac-ão de Aperfeic-oamento de Pessoal de Nı́vel Superior) and CNPq – National Council for Scientific and Technological Develop- ment (Conselho Nacional de Pesquisa). References Bacci Jr., M., Anversa, M.M., Pagnocca, F.C., 1995. Cellulose degradation by Leucocoprinus gongylo- phorus, the fungus cultured by the leaf-cutting ant Atta sexdens rubropilosa. Braz. J. Med. Biol. Res. 28, 79–82. Bernfeld, P., 1955. In: Colowick, S.P. (Ed.), Methods in Enzymology, vol. I. Academic Press, New York, USA, pp. 149–158. Haseltine, C., Rolfsmeier, M., Blum, P., 1996. The glucose effect and regulation of alpha-amylase synthesis in the hyperthermophilic archaeon Sulfolobus solfatar- icus. J. Bacteriol. 178, 945–950. Lachmund, A., Urmann, U., Minol, K., Wirsel, S., Ruttkowski, E., 1993. Regulation of alfa-amylase formation in Aspergillus oryzae and Aspergillus nidu- lans transformants. Curr. Microbiol. 26, 47–51. Martin, M.M., Weber, N.A., 1969. The cellulose-utilizing capability of the fungus cultured by the attini ant Atta colombica tonsipes. Ann. Entomol. Soc. Am. 62, 1386–1387. Mora, P., Rouland, C., 1995. Comparison of hydrolitic enzymes produced during growth on carbohydrate substrates by Termitomyces associates of Pseuda- canthotermes spininger and Microtermes subhyalinus (Isoptera: Termitidae). Sociobiology 26 (1), 39–53. Nahas, E., Waldemarin, M.M., 2002. Control of amylase production and growth characteristics of Aspergillus ochraceus. Rev. Latin Am. Microb. 44 (1), 5–10. Pagnocca, F.C., Silva, A.O., Hebling-Beraldo, M.J.A., Bueno, O.C., Fernandes, J.B., Vieira, P.C., 1990. Toxicity of sesame extracts to the symbiotic fungus of leaf-cutting ants. Bul. Entomol. Res. 80, 349–352. Silva, A., Bacci Jr., M., Siqueira, C.G., Bueno, O.C., Pagnocca, F.C., Hebling, M.J.A., 2003. Survival of Atta sexdens on different food sources. J. Insect Physiol. 49, 307–313. Silva-Pinhati, A.C., Bacci Jr., M., Hinkle, M.L., Sogin, M.L., Pagnocca, F.C., Martins, V.G., Bueno, O.C., Hebling, M.J.A., 2004. Low variation in ribossomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae). Braz. J. Med. Biol. Res. 37, 1463–1472. Siqueira, C.G., Bacci Jr, M., Pagnocca, F.C., Bueno, O.C., Hebling, M.J.A., 1998. Metabolism of plant polysac- charides by Leucoagaricus gongylophorus, the symbio- tic fungus of leaf-cutting ant Atta sexdens L. Appl. Environ. Microbiol. 64 (12), 4820–4822. Weber, N.A., 1955. Pure cultures of fungi produced by ants. Science 121, 109. Zar, J.H., 1996. Bioestatistical Analysis, 3rd ed. Prentice- Hall, NJ, USA. Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of �leaf-cutting ants Introduction Material and methods Fungal cultivation Enzyme assay Statistics Results Discussion Acknowledgements References