Introduction

Colonies of leaf cutting ants may contain as many as 8 million individuals, imparting a capacity to cut sizeable amounts of fresh foliage every day. Leaf cutting ants do not necessarily eat the plant material directly, but may convert the foliage into a substrate that is suitable to sustain the growth of fungal colonies within the nest (the fungal garden). The interaction between ants and fungi is extremely complex, with both organisms benefiting from the association. Apart from providing a constant source of biomass for the fungi, ants of the genera Atta and Acromyrmex secrete enzymes that are essential for fungal growth (Bass and Cherret 1995), remove waxes from the surface of leaves thus facilitating fungal colonisation, and secrete antibiotic compounds that destroy or inhibit the attack of other micro-organisms (Bass and Cherret 1994). The fungus serves as a primary source of nutrients for the larvae and, since no trophallaxis from worker ants to larvae occurs, maintenance of the fungal garden is of paramount importance for the survival of the colony (Wilson 1971).

Two mechanisms have been proposed to explain how ants eliminate non-mutualistic fungi and preserve a parasite-free colony. The first mechanism implicates a mutualistic association between ants and filamentous bacteria (Actinomycetes) of the genus Streptomyces located on many places of the cuticle in addition to the lateral-cervical portion of the thorax of the worker ant (Currie et al. 2006). These bacteria produce antibiotic compounds that are deleterious to the parasitic fungi Escovopsis (Ascomycota: anamorphic Hypocreales; Currie et al. 1999). The benefits and disadvantages of this mutualistic association are not, however, completely understood since the nature of the bacterial substrate and the effects of the antibiotics subsequently secreted by the ants have yet to be elucidated (Poulsen et al. 2003). The second mechanism involves the production and secretion of antibiotics by specialised exocrine glands present in the leaf cutting ants, which have an important function in the maintenance of the fungus garden hygiene (Wilson 1971; Bot et al. 2001).

The mandibular gland secretions of leaf cutting ants contain low molecular weight alcohols and ketones that induce alarm behaviour and mediate processes related to inter-colony recognition (Brown et al. 1970). The alarm pheromone in Atta bisphaerica, Atta capiguara, Atta robusta, Atta sexdens and Atta opaciceps is 4-methyl-3-heptanone, a compound that is characteristic of the genus (Blum et al. 1968; Parry and Morgan 1979; Hernandez et al. 1999; Hughes et al. 2001; Francelino et al. 2006, 2008; North et al. 1997), whereas the main component of the alarm pheromone in Acromyrmex versicolor and Acromyrmex octospinosus is 3-octanone (Crewe and Blum 1972).

The mixture excreted by leaf cutting ants mandibular glands also possesses antimicrobial activity with a significant fungicidal effect (Colle et al. 1975; Brough 1983; Knapp et al. 1994; Marsaro et al. 2001; North et al. 1997). Thus, the secretion produced by the mandibular gland of Atta sexdens rubropilosa inhibits the germination of Botrytis cinerea, a fungus that attacks crops of economic importance including wheat, soy, rice, cotton, sun flower, beans, corn, sorghum, carrots, tomatoes, peas and onions (Marsaro et al. 2001). Of the main fungicidal compounds, namely, citral, 2-heptanone and 4-methyl-3-heptanone, produced by this gland, citral was found to be more active against phytopathogenic fungi than the co-occurring ketones (Colle et al. 1975).

Metapleural (metasternal or metathoracic) glands, which occur only in ants, produce a carboxylic acid-rich secretion. In worker ants of the sub-family Myrmicinae (including A. sexdens), the main constituent of the secretion released by these glands is phenylacetic acid (Maschwitz 1974), although other acids (indolacetic, 3-hydroxyhexanoic, 3-hydroxyoctanoic and 3-hydroxydecanoic acids) also occur in A. sexdens rubropilosa (Schildknecht and Koob 1971). Additionally, the secretion of Crematogaster deformis (Myrmicinae) contains 3-propyl- and 3-pentyl-phenol, and 5-propyl- and 5-pentyl-resorcinol together with related coumarins (Attygalle et al. 1989). Further analysis of the secretions of the metapleural glands of A. sexdens rubropilosa and A. cephalotes (workers and soldiers) and of A. octospinosus (workers) revealed a protein component together with a volatile acid fraction in the form of an ionised salt (Do Nascimento et al. 1993). The major component of the acid fraction was identified as phenylacetic acid, with lesser amounts of 3-hydroxydecanoic (myrmicacin), 3-hydroxydodecanoic and indolacetic acids, the proportions of which varied quantitatively between the species studied. More recently, Ortius-Lechner et al. (2000) detected 20 additional polar compounds in these glandular secretions by direct GC–MS analysis (on a column containing the polar stationary phase polyethylene glycol) of sectioned posterolateral regions of the ants. The main compounds found were short chain (acetic, valeric, hexanoic, heptanoic, octanoic, nonanoic, decanoic and dodecanoic) and long chain (myristic, pentadecanoic, palmitic, oleic and linoleic) carboxylic acids, indolacetic acid, lactones (γ-octalactone and γ-decalactone) and oxyacids (4-oxo-octanoic, 4-oxo-decanoic and β-hydroxydecanoic acid).

It is known that high concentrations of myrmicacin and other hydroxy acids inhibit fungal growth, exhibit herbicide activity (Schildknecht and Koob 1971) and prevent the growth of gram-negative bacteria (Do Nascimento et al. 1996), whilst indolacetic acid inhibits the growth of hyphae (Schildknecht and Koob 1971) and the production of fungal spores (Do Nascimento et al. 1996; Bot et al. 2002). Additionally, phenylacetic acid exerts strong inhibitory activity against gram-negative and gram-positive bacteria (Maschwitz et al. 1970; Do Nascimento et al. 1996). The antimicrobial effects of these compounds on the leaf cutting ants fungal culture may result from individual or synergistic action since both hyphae and spores are sensitive. Moreover, it is possible that the short chain carboxylic acids, which have variable antimicrobial activity, may play a secondary role by increasing the effectiveness of the secretion (by decreasing its pH) against non-mutualistic bacteria and fungi (Ortius-Lechner et al. 2000).

Yeasts which belong to the genus Candida is widely distributed in colonies of leaf cutting ants, such as A. sexdens rubropilosa (Carreiro et al. 1997). However, it is not clear if these yeasts are contaminants found in their nests or if they play an additional role in the association of these ants with its symbiotic fungus. In a similar manner, gram-positive and gram-negative bacteria were also found to be present in leaf cutting ant nests (Bacci et al. 1995; Craven et al.1970; Serzedelo and Tauk 1974) and according to these authors, these bacteria are potentially able to degrade the structural biopolymers of plant material, which are assumed not to be directly taken up by the ants.

Considering the reports given in the literature which claims that the function of the mandibular and metapleural gland secretions of leaf cutting ants is to preserve the mutualistic relationship between ants and fungi by protecting the colony against invading micro-organisms and also that the micro-organisms used in this study may co-habit with ants, the components of the secretions were investigated for potential activities against human pathogenic micro-organisms, and particularly those that are resistant to conventional antibiotics.

Materials and methods

Bacteria and fungal strains

Five strains of the bacteria Staphylococcus aureus and Escherichia coli and 10 isolates of the fungus Candida albicans, were used in the experiments (Table 1). Four bacterial strains were resistant to at least two common antibiotics, whilst the fungal isolates were resistant to at least one commercial fungicide. Bacteria and fungi were maintained, respectively, on solid Mueller–Hinton (Merck®) and Sabouraud media (Merck®) at 37°C in the dark.

Table 1 Micro-organisms employed in the assays
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Compounds assayed

The major constituents (where commercially available: Table 2) of the secretions released by the mandibular and metapleural glands of leaf cutting ants were assayed for their antimicrobial activities (Blum et al. 1968; Schildknecht and Koob 1971; Do Nascimento et al. 1993; Hernandez et al. 1999; Ortius-Lechner et al. 2000; Hughes et al. 2001; Francelino 2006, 2008). 4-methyl-3-heptanone, an important component of the mandibular gland secretion, was unavailable at the time of study hence the corresponding alcohol (4-methyl-3-heptanol), which is also present in the secretion, and the isomeric ketone (4-methyl-2-heptanone) were employed instead. A total of 11 compounds were assayed at concentrations selected on the basis of earlier literature reports of their activities against different sets of micro-organisms (see Table 2). Hexane and water were used as controls.

Table 2 Compounds present in the glandular secretions of leaf cutting ants assayed for antimicrobial activity
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Determination of antimicrobial activity

The samples were assayed according to the technique of Bauer et al. (1966) in which an assessment is made of the inhibition of superficial growth on medium around a filter paper disc impregnated with the analyte or control.

Bacterial and fungal strains were cultured in liquid maintenance media (Mueller–Hinton and Sabouraud, respectively) for 18 h at 37°C in the dark. Inoculums were prepared from fresh liquid cultures, and the turbidity of each was standardised to 0.5 on the MacFarland scale (equivalent to 108 colonies/ml).

Sterile Petri dishes of diameters 140 or 100 mm containing, respectively, 72 ml of Mueller–Hinton (Merck®) or 54 ml of Sabouraud solid medium (Merck®), were prepared, sealed with plastic film and placed into a bacteriological incubator at 32°C for 24 h prior to the assays in order to test for potential contamination. After this time, standardised inoculums of bacterial or fungal cultures were spread uniformly over the surface of the appropriate contaminant-free medium with the aid of sterile swabs.

Paper discs (6 mm diameter, 30 ± 4 mg/cm2) were sterilised in an autoclave at 121°C for 15 min and then impregnated with 20 μl aliquots of water solutions of the analytes (Table 2) or with a similar volume of hexane or water as control. Each of the seeded Petri dishes received 12 impregnated discs (including a control discs), placed equidistant on the surface of the medium. Following incubation for 18 h at 37°C in the dark, the diameter of the halos of inhibition formed around the paper discs were measured using a ruler calibrated in mm. For all of the bacterial strains, and for C. albicans isolates CA1007 and CA22, the tests were repeated six times, whilst for isolates CA01–08 the tests were repeated twice.

Statistical analyses

Assays were conducted in random sequence and the means (±standard errors) calculated. Mean values were compared using the parametric F test (comparison of two means) and the Tukey test (comparison of more than two means). Differences between mean values were considered significant for P ≤ 0.05.

Results

Antibacterial activities

Except for 4-methyl-2-heptanone and citronellol, all of the components of the mandibular secretions assayed proved to be active against the bacterial strains employed (Fig. 1a). Citral and 4-methyl-3-heptanol were significantly more active against S. aureus, particularly strain ATCC6538, than the other compounds. Inhibitory activity against E. coli varied, but 4-methyl-3-heptanol was significantly more effective than the other analytes.

Fig. 1
figure 1

Diameters of inhibition halos (means ± SE) exhibited by compounds present in the secretions of mandibular (a) and metapleural (b) glands of leaf cutting ants. The compounds tested were: Staphylococcus aureus (strains: ATCC 6538, IC311, IC133, IC-multiresistant) and Escherichia coli (strain: IC08). The concentrations of analytes employed are shown in Table 2. Hexane and water, used as controls showed no inhibition halos. With respect to each of the micro-organisms tested, bars bearing different superscript letters represent mean values that are significantly different at P ≤ 0.05 (Tukey test)

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Components of the metapleural secretions were all active against the bacterial strains tested (Fig. 1b) and formed inhibition halos of between 15 and 30 mm diameter. In general, hexanoic acid exhibited a significantly higher antibacterial activity compared with the other compounds, but in the case of the multiresistant strain of S. aureus, the activity of hexanoic and indolacetic acids were somewhat similar. The used controls, hexane and water formed no inhibition halos.

Antifungal activities

To verify the existence of antifungal activity initially, the antifungal assays were performed using only C. albicans isolates CA22 and CA1007. Similar to the results of the antibacterial assay, all of the components of the leaf cutting ants mandibular secretions exhibited antifungal activities with the exception of 4-methyl-2-heptanone and citronellol (Fig. 2a). Geraniol presented the highest activity, followed by citral and 2-heptanone. All of the tested constituents of the metapleural secretions showed antifungal activities against isolates CA22 and CA1007 (Fig. 2b), with hexanoic and octanoic acids being significantly more active than the other compounds.

Fig. 2
figure 2

Diameters of inhibition halos (means ± SE) exhibited by compounds present in the secretions of mandibular (a) and metapleural (b) glands of leaf cutting ants on two isolates of Candida albicans. The strains tested were: CA 1007 and CA 22. The concentrations of analytes employed are shown in Table 2. Hexane and water, used as control showed no inhibition halos.With respect to each of the micro-organisms tested, bars bearing different superscript letters represent mean values that are significantly different at P ≤ 0.05 (Tukey test)

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Based on these results, further assays were performed to verify the variation in the antifungal activity among strains of the same fungus, using all compounds (except for 4-methyl-2-heptanone) and eight different clinical isolates of C. albicans (CA01–08).

The results demonstrated that all isolates were susceptible to the compounds tested since the diameters of the inhibition halos varied between 8 and 41 mm (Table 3). The used controls, hexane and water formed no inhibition halos.

Table 3 Susceptibility of Candida albicans isolates CA01–8 towards compounds present in the glandular secretions of leaf cutting ants
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Comparison between antibacterial and antifungal activities

The mean diameters of the halos of inhibition of S. aureus, E. coli and C. albicans (CA22 and CA1007) that formed around the discs impregnated with the tested analytes are presented in Fig. 3. It can be seen that the majority (80%) of the compounds assayed (i.e. citral, 2-heptanone, 3-octanone, geraniol, indolacetic acid, hexanoic acid and octanoic acid) presented stronger antifungal activities in comparison with their antibacterial activities, and this was particularly noticeable for citronellol. The remaining analytes (i.e. 4-methyl-3-heptanol and phenylacetic acid) exhibited antibacterial activities that were stronger than their corresponding antifungal activities.

Fig. 3
figure 3

Antifungal ( ) and ( ) antibacterial activities of compounds present in the secretions of mandibular and metapleural glands of leaf cutting ants. With respect to each of the analytes tested, bars bearing different superscript letters represent mean values that are significantly different at P ≤ 0.05 (F test)

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Discussion

In the present work, it has been demonstrated that the main constituents of the mandibular and metapleural gland secretions of leaf cutting ants possess strong inhibitory activities, against bacteria and fungi, including those that are resistant to conventional antibiotics. These compounds contain oxygen atoms in their molecules, which present the possibility of binding to the micro-organisms membrane via hydrogen bonds, and thus promoting the breakdown of the membrane structure.

The antibacterial activities of citral have been fully reported in the literature. Essential oils that are rich in citral (a monoterpene with a strong smell of lemon) are known to inhibit the growth of S. aureus, E. coli, Pseudomonas aeruginosa and Klebsiella pneumoniae (Hayes and Marckovic 2002). Saleem et al. (2003), for example, demonstrated that the essential oil of Cymbopogon citrates, in which citral is the major component, was inhibitory to enzymes responsible for the synthesis of peptidoglycan in the bacterial cell wall. Although 4-methyl-3-heptanol is not present in the secretion of ants, its activity against the micro-organisms tested suggests that chemical related compounds may also show inhibitory activity.

The mechanism of action of citral, 4-methyl-3-heptanol and 2-heptanone requires clarification since branched ketones such as 4-methyl-2-heptanone were not active against the bacteria tested in the present study.

The antimicrobial activities of the secretions produced by metapleural glands of leaf cutting ants have been previously attributed to the presence of phenylacetic acid, indolacetic acid and lactones (Do Nascimento et al. 1996; Bot et al. 2002). In the present study, however, such compounds exhibited low antibacterial activities compared with hexanoic and octanoic acids. The strong antibiotic activity of medium chain carboxylic acids of this type has previously been reported by Koidsumi (1957). However, the inhibitory effects of phenylacetic and indolacetic acids may be variable or influenced by the presence of other compounds, such as short chain acids, which acidify the environment and render it unsuitable for the growth of bacteria (Schildknecht and Koob 1971). Indeed, Kreisel (1972) observed that the removal of ants from the fungal garden led to a gradual increase in pH of the culture followed by invasion by opportunistic bacteria and fungi.

The components presenting the strongest antifungal activities against various isolates of C. albicans were citral, geraniol, 2-heptanone, 4-methyl-3-heptanol and citronellol. The antifungal activities of plant essential oils have also been well recorded. For example, Colle et al. (1975) investigated the activities of 4-methyl-3-heptanone, 2-heptanone and citral, and reported that the latter (in the concentration range 100–1,000 ppm or 0.1–1 mg/ml) was particularly efficient against a number of fungal species including Rhizoctonia solani, Fusarium oxysporum and Penicillium spp., whereas the activities of the ketones were lower than that of citral and varied according to the fungus tested. Wolken et al. (2002) demonstrated that citral and, to a lesser extent, nerol, geraniol and geranic acid were highly toxic to the spores of Penicillium digitatum ATCC2067. Citral, geraniol, citronellol, eugenol and menthol have also been found to exhibit strong growth inhibitory activities against B. cinerea and, more especially, Monilinia fructicola (Tsao and Zhou 2000). Nakahara et al. (2003) demonstrated that agar plates that had been treated with the vapour of the essential oil of Cymbopogon nardus (250 mg/l or 0.025%), of which the major components are citral (36.9%), geraniol (35.7%), citronellal (5.8%) and citronellol (4.6%), inhibited the growth of Aspergillus sp., Penicillium sp. and Eurotium sp. With respect to the inhibition of C. albicans, Wilkinson et al. (2003) reported that the minimum inhibitory concentration of the essential oil of Backhousia citriodora (containing 80–90% citral) was 0.3% (v/v), although the susceptibility of different clinical isolates of the fungus varied. The authors attributed this variation to discrepancies in the composition of the essential oil and to different growth conditions of the micro-organisms.

Because the solutions of compounds used in this study were prepared based on data from the literature, their variation in concentration were not taken into consideration, however, further studies should be conducted, using solutions of compounds with similar concentrations to find out which compound is more effective and also if more diluted solutions of the tested compounds is as effective as the conventional antibiotics.

Since the prevalence of human candidiasis has increased over the years, primarily because of the widespread use of immunosuppressive agents (Hirasawa and Takada 2004), it is clearly of particular importance to investigate alternative drugs to treat this condition.

The role and relative importance of the components of the mandibular and metapleural glands of leaf cutting ants in the preservation of the ant colony have been highlighted, and the results may contribute to our knowledge of the organisation and function of this complex society. However, despite the evidence of antibiotic activity, further studies need to be performed in order to understand the relation between the compound structure and its antibacterial and antifungal activities, the effect of the compound’s concentration on the antibiotic activity, as well as toxicological and in vivo assays aiming their application.