Generation of Nutrients and Detoxification: Possible Roles of Yeasts in Leaf-Cutting Ant Nests

The possible roles played by yeasts in attine ant nests are mostly unknown. Here we present our investigations on the plant polysaccharide degradation profile of 82 yeasts isolated from fungus gardens of Atta and Acromyrmex species to demonstrate that yeasts found in ant nests may play the role of making nutrients readily available throughout the garden and detoxification of compounds that may be deleterious to the ants and their fungal cultivar. Among the yeasts screened, 65% exhibited cellulolytic enzymes, 44% exhibited pectinolytic activity while 27% and 17% possess enzyme systems for the degradation of protease and amylase, respectively. Galacturonic acid, which had been reported in previous work to be poorly assimilated by the ant fungus and also to have a negative effect on ants’ survival, was assimilated by 64% and 79% of yeasts isolated from nests of A. texana and Acromyrmex respectively. Our results suggest that yeasts found in ant nests may participate in generation of nutrients and removal of potentially toxic compounds, thereby contributing to the stability of the complex microbiota found in the leaf-cutting ant nests.


Introduction
Ants in the tribe Attini maintain a mutualistic association with basidiomycetous fungi cultivated for food [1]. The phylogenetically derived genera in this tribe, Atta and Acromyrmex, known as the leaf-cutting ants, cut and collect fresh leaves and flower parts as substrate for the cultivation of mutualistic fungi [1,2]. This fungus is responsible for the production of extracellular enzymes that help to breakdown the plant substrate found in the fungus gardens [3][4][5][6]. In this process, simple sugars and other nutrients are generated and accumulated in the fungus gardens and it is thought these may in part support development of workers [7].
Because ants provide an optimum environment for fungal growth, additional alien microbes may also find the fungus garden a suitable substrate for development. Thus, it is fundamental for the ants to protect the mutualistic fungus from potential harmful microorganisms. For this reason ants employ several mechanisms such as: (i) massive inoculation of the cultivar onto fresh collected plant substrate [8]; (ii) weeding and grooming behaviors to remove harmful fungi [9]; (iii) secretion of antimicrobial compounds by workers [10][11][12][13][14]; (iv) the use of antibiotic-producing actinobacteria [15][16][17][18][19].
Despite the hygienic strategies adopted by ants to keep their gardens free of alien microbes, members of the fungal genus Escovopsis are frequently found in attine gardens. Escovopsis is considered a specialized parasite that attacks the cultivar hyphae [20] and long-term infections occasionally drive the colony to death [2]. Furthermore, colonies infected with Escovopsis show lesser garden biomass and lower number of workers and brood than uninfected colonies, thus indicating the negative effects of this parasite in the symbiosis [2]. In addition to Escovopsis, several other microorganisms are found in this symbiosis [21][22][23][24][25][26][27][28][29][30][31][32][33][34]. It is likely that many microbes found in the fungus gardens may not play significant roles, but some may provide unknown but important functions.
Several authors have reported the occurrence of yeasts in attine gardens. Craven et al. [22] studied the fungus garden of Atta cephalotes and Acromyrmex octospinosus and reported the first occurrence of yeasts in attine ant nests. Angelis et al. [23] isolated yeasts in gardens of Atta sexdens and Atta laevigata. Carreiro et al. [24] isolated the yeast genera Candida, Cryptococcus, Rhodotorula and Trichosporon from laboratory nests of Atta sexdens rubropilosa. Black yeasts were also isolated from different parts of the ant's integument [25][26][27]. Rodrigues et al. [28] isolated yeasts from fungus garden of A. texana and observed inhibition of the growth of Escovopsis and alien filamentous fungi, suggesting that they may also be involved in defending the fungus garden. In addition, three new yeast species namely Sympodiomyces attinorum [32], Cryptococcus haglerorum [33] and Trichosporon chiarellii [34] were described from this environment.
Preliminary studies suggest that bacteria may act as co-participants with the mutualistic fungus, in the degradation of the plant substrate. Ribeiro [29] and Bacci et al. [30] isolated bacteria from fungus garden of Atta sexdens and found strains that exhibited cellulases, pectinases, amylases and proteases. Recently, Suen et al. [35] revealed that the fungus garden contain a diverse community of bacteria with high lignocellulose-degrading capacity. Pinto-Tomás et al. [36] reported that bacteria in the fungus garden may fix nitrogen and it seems to be an important contribution for the ants. The genus Klebsiella was found as one of the most effective in this task.
To investigate the potential of yeasts as co-participants in the degradation of the plant material, we carried out a preliminary screening of cellulase, pectinase, amylase, xylanase and protease enzymes exhibited by yeasts associated with fungus gardens of leaf-cutting ants. By assessing the enzymatic profile of garden yeasts, we can begin to determine the putative contribution of these microorganisms in the degradation of the main plant polysaccharides, protein material and in the assimilation of its hydrolysis products. We show that the yeast community found in fungus gardens may play more important roles than previously thought.
Yeasts isolated from Acromyrmex were first grouped based on phenotypic characteristics. Then, genomic DNA was extracted according to Almeida [37] and subjected to microsatellite-primed PCR (MSP-PCR) with primer (GTG) 5 , following the method of Sampaio et al. [38]. The D1/D2 domains of 26S rDNA of representatives strains (Table S2) were amplified using primers NL1 and NL4 [39]. Forward and reverse sequences were generated using the same amplification primers and using the BigDye Terminator kit on ABI 377 and ABI 3100 (Life Technologies).
Sequences were assembled in contigs and manually edited using BioEdit v.7.0.5.3 [40]. Contigs were queried at the NCBI-GenBank database (National Center for Biotechnology Information) using BLASTn algorithm [41]. Sequences derived from yeast isolated from Acromyrmex were deposited at GenBank (JQ317161-JQ317168). Sequence accessions for yeasts isolated from A. texana are provided in [28].

Assaying Yeasts for Hydrolytic Enzymes
Screenings for cellulase, pectinase, amylase, xylanase and protease enzymes were carried out qualitatively on different media supplemented with a specific polymer as described below. All plates were incubated at 25 °C for seven days for the detection of amylase, protease, and pectinase and 7-14 days for cellulase and xylanase. The formation of hydrolysis halos around the colonies indicated the production of enzymes and consequent degradation of polymers (see Figure S1 for representative examples of halo formation).
Starch hydrolysis was evaluated using the basal medium I described by Looder [42] supplemented with 20 g·L −1 of soluble starch (Mallinckrodt). After incubation, plates were stained with lugol. For the assessment of CMCellulase, pectinase and xylanase a basal medium composed of 6.7 g·L −1 of YNB (Yeast Nitrogen Base, Difco) and 18 g·L −1 of agar supplemented with 5 g·L −1 of carboxymethylcellulose, 10 g·L −1 of polygalacturonic acid or 10 g·L −1 of xylan was used, respectively. The plates were stained with Congo red for CMCellulase [43], ruthenium red for pectinase [44] and lugol for xylanase [45]. Protein hydrolysis was assessed in solid medium containing 100 g·L −1 of Skim Milk medium (Difco). All polymers were supplied by Sigma Chemical Company.

Assimilation of the Hydrolysis Products
In addition to the hydrolytic enzymes, we evaluated the ability of yeasts to assimilate the simple sugars and products resultant of plant polysaccharides hydrolysis: maltose, cellobiose, galacturonic acid and xylose (Sigma Chemical Company). The determination was carried out on solid media containing 6.7 g·L −1 of YNB and 18 g·L −1 of agar supplemented with 5 g·L −1 of each carbon source. The growth of colonies on the simple sugars was compared with negative (medium without any carbon source) and positive controls (medium supplemented with glucose).

Results and Discussion
Several studies reported the occurrence of yeasts in the fungus gardens of both lower and higher attine ants [22][23][24][25][26][27][28][32][33][34]46]. Yeasts may enter the fungus gardens by the foraging activity of workers; specifically for leaf-cutting ants, yeasts may derive from the fresh plant material collected by workers as the phylloplane is considered a rich source of yeast species [47].
Degradation profile and assimilation of hydrolysis products by 82 yeasts isolated from Atta texana and Acromyrmex are shown in Tables 1 and 2, respectively. Specifically for yeasts isolated from fungus gardens of A. texana, 77% showed at least one of the studied enzymes. Among the positive strains, six exhibited only one enzyme whereas the three Cryptococcus laurentii isolates exhibited four enzymes (Table 1 and Table S2 for results of specific isolates). Cryptococcus flavus and Pseudozyma sp. were the only isolates positive for all enzymes evaluated (Table 1 and Table S2 for results of specific isolates). Xylanase was exhibited by 59% of the isolates, amylase, CMCellulase, pectinase and protease activities were observed in 25%, 43%, 20% and 32% of the isolates, respectively ( Figure 1). All the tested yeast isolates assimilated maltose, cellobiose and xylose and 63% assimilated galacturonic acid (Table 1).
Our results indicated that the yeast species composition differed between gardens of A. texana and the various Acromyrmex (Tables 1 and 2). Cryptococcus was the only genus shared by both ant genera. This yeast genus is widely distributed in the environment and has been shown to be prevalent among the yeast communities found on the phylloplane [47][48][49], but is also commonly found in soils from grasslands, pastures, and tundra [49]. Because of its widespread occurrence, Cryptococcus is expected to be in contact with workers and be transported to nests through the plant material collected by workers [24]. Previous studies on attine ants also recorded this genus as member of the microbiota of attine ants [24,25,33,34,46]. Table 1. Enzymatic activity and assimilation profile of yeasts and yeast-like fungi isolated from fungus gardens of Atta texana.   Interestingly, Trichosporon chiarellii [34] was found in three nests, one of Ac. heyeri and two of Ac. lundi located at different sites (Table S1). This species was described from fungus gardens of Myrmicocrypta camargoi, a recently described lower attine [50]. So far, there are no reports of the isolation of T. chiarellii from sources other than attine gardens.
Although differences between the yeast communities from the target ant genera were evident, the degradation profile of plant polysaccharides by yeasts was similar between the two communities ( Figure 1). In nature, yeasts are associated with specific habitats [51]; one might expect that their presence and abundance in gardens of attine ants may not be coincidental. The acidic pH and nutrient availability [52] may allow some yeast species to be selected and multiply in attine gardens. Thus, species found in such environment rich in readily available nutrients, may not only use the nutrients for their development, but also to exhibit enzymes that aid in the hydrolysis of the plant tissues, resulting in increased nutrient availability for the microbiota and facilitating substrate colonization by the mutualistic fungus.
Pectinolytic enzymes seem to play a key role in the fungus gardens because the mutualistic fungus of leaf-cutting ants exhibit such enzymes in large quantities [3,4]. The mutualistic fungus of Atta sexdens rubropilosa exhibits pectinase 7 times higher than amylase and more than 24 times higher than xylanase and 38 times higher than CMCellulase [3]. Pectinolytic enzymes are important because they promote the degradation of the pectic substances present in the middle lamella of plant cells, facilitating the colonization of the plant substrate and access to other cell wall components [53]. This seems to explain the large amounts of pectinolytic enzymes exhibited by the mutualistic fungus.
The proportion of yeasts that degrade pectin was 20% and 71% of isolates recovered from A. texana and Acromyrmex, respectively (Tables 1 and 2). This would result in a further increase in pectin breakdown in the garden. However, despite the large amount of pectinolytic enzymes exhibited by the fungus cultivar, it does not present good growth in the presence of galacturonic acid, the most important hydrolysis product of pectin [3]. In addition, Silva et al. [7] experimentally demonstrated that galacturonic acid does not contribute to the nutrition of ants, but rather decrease the survival of Atta sexdens workers. Thus, it is expected that galacturonic acid would accumulate in the fungus gardens. In contrast to what was observed for the mutualistic fungus and workers, galacturonic acid was assimilated by 64% (Table 1) and 79% (Table 2) of the yeasts isolated from nests of A. texana and Acromyrmex, respectively. In addition, Carreiro [54] working with fungus gardens and refuse dumps of laboratory nests of A. sexdens rubropilosa recovered a very different yeast community profile when compared to our findings. However, considering these distinct communities, 22% of the isolates (n = 93 yeasts) found in Carreiro's study also assimilated galacturonic acid. Thus, the results of two independent studies suggest that yeasts may perform a detoxification (filtering) process in attine gardens by consuming galacturonic acid. It was also observed that the assimilation of galacturonic acid was not restricted to a specific group of yeast; instead, this ability was distributed among the majority of the taxa (Tables 1, 2 and S2). Thus, the utilization of galacturonic acid may be an advantage for the survival of yeasts recently introduced into the gardens and at same time it would be a remarkable contribution to the maintenance of the nest homeostasis.
An additional possible contribution of yeasts is the degradation of cellulose. This polysaccharide is the most abundant component of plant cell wall [55]. It was formerly assumed that cellulose was the main source of carbohydrates for the ant cultivar [56]. This assumption was reinforced by Bacci et al. [57], who showed that the mutualistic fungus exhibits cellulolytic enzymes. However, further studies demonstrated that the ability of the mutualistic fungus to degrade cellulose is limited when compared with other polysaccharidases as pectinases, amylases and xylanases [3,4]. In addition, Abril and Bucher [58] suggested that the mutualistic fungus does not exhibit cellulase, showing that the cellulolytic activity by the cultivar does not seem to be relevant. Recently, Suen and colleagues [35] determined the amount of cellulose present in the fungus gardens. The authors discovered low amounts of cellulose in mature garden parts when compared to younger portions where newly plant material is added by the ants, suggesting that cellulose degradation does occur in the fungus gardens. Using cultivation-independent techniques, the authors also showed that the fungus garden harbors a community of cellulose-degrading bacteria [35].
Our data shows that yeasts can also contribute to the degradation of cellulose present in the fungus gardens. The CMCellulose was degraded by 43% and 89% of the yeasts isolated from A. texana and Acromyrmex, respectively (Tables 1 and 2). These data indicate that yeasts associated with fungus gardens may contribute, along with cellulose-degrading bacteria [35], to the production of easily assimilated sugars, especially glucose, for other members of the symbiosis, and also facilitate the colonization of plant tissues by the mutualistic fungus. On the other hand, cellobiose, the disaccharide resulting from the hydrolysis of cellulose, was assimilated by all yeast isolates from Atta texana (Table 1) and by 61% of yeasts from Acromyrmex ( Table 2), revealing that this sugar may support the growth of yeasts. Also, there is evidence that Atta sexdens workers cannot utilize this sugar for survival [7] and the mutualistic fungus only grows at an intermediate rate on this carbon source [3]. Thus, cellobiose resulting from cellulose breakdown would be available for yeasts, thereby providing for their survival without competing with the other members of the symbiosis as also observed in respect to galacturonic acid.
The yeast participation in the generation of assimilable compounds from starch seems to be as important as that observed for the other plant polysaccharides. We found that 25% of the yeasts isolated from gardens of A. texana exhibited amylase, while only 8% of yeasts isolated from Acromyrmex exhibited this enzyme. The hydrolysis products of starch are mainly glucose and maltose. Glucose is the carbon source that best supports the development of the mutualistic fungus [3] and the survival of workers of Atta sexdens [7]. According to Silva et al. [59] glucose resultant from the degradation of starch by the ant cultivar is present in the fungus garden. Although the proportion of yeast capable of starch degradation is not high, they may also contribute to the hydrolysis of starch in gardens, generating easily assimilable sugars for their growth and the other members of the symbiosis. On the other hand, except for Galactomyces candidus (differentiated from G. geotrichum by growth at 35 °C according to [60]) all yeast species assimilated maltose (Tables 1 and 2). These data indicate that the degradation of starch is essential for the maintenance of workers [7,59] and is also important for the yeasts present in nests.
Few studies have explored the use of nitrogen sources by the ants and their associated microbes in comparison to carbon sources. Abril and Bucher [61] reported that the ant cultivar grows on inorganic sources of nitrogen, but does not grow in media containing protein (peptone) as a nitrogen source. In addition, Silva et al. [7] reported that workers of Atta sexdens are not able to use peptone and suggest that ants are not able to feed on plant proteins. Recently, Pinto-Tomás et al. [36] observed nitrogen enrichment in the fungus gardens of attine ants. In the present study, the production of proteolytic enzymes was observed in 32% and 16% of the yeasts isolated from A. texana and Acromyrmex, respectively. The community of yeasts and the proteolytic bacteria associated with nests [29,30] could contribute to the degradation of proteins present in the plant material and generate available nitrogen, which together with the nitrogen incorporated by the action of nitrogen-fixing bacteria [36] would support the development of the mutualistic fungus, ants and other microbes. Xylanolytic enzymes were exhibited by 59% of the yeasts isolated from A. texana gardens. Xylanase is the second most abundant enzyme exhibited by the mutualistic fungus [3]. Both ants and the cultivar may have mutual development supported by xylose [3,7], the hydrolysis product of xylan. Xylan-degrading yeasts may contribute to the degradation of hemicellulose present in the plant material used by the fungal cultivar. Moreover, all yeast isolates screened in the present study were able to assimilate xylose (Tables 1 and 2), suggesting that this carbon source would also be important in the nutrition of yeasts.
It seems that the degradation of polymers present in the fungus gardens is the result of a complementary action of enzymes exhibited by the mutualistic fungus, bacteria and also yeasts that are found in the fungus garden. Such microbial consortium would generate readily available nutrients that may help to sustain the homeostasis of the attine ant-microbe symbiosis.