Several studies have found yeast species associated with fungus gardens of leaf-cutting ant colonies (Carreiro et al. 1997; 2004; Little and Currie 2007; 2008; Masiulionis and Pagnocca 2016; Melo et al. 2014; Middelhoven et al. 2003; Rodrigues et al. 2009). However, it is interesting that yeast composition is variable among Atta species. In a pioneering study, Carreiro et al. (1997) found 34 yeast species associated with Atta sexdens rubropilosa, grouped in seven genera: Candida, Cryptococcus, Rhodotorula, Sporobolomyces, Tremella, Trichosporon and Pichia. Recently, Arcuri et al. (2014) updated this information by finding a low number of orders (six) and species (25) but increasing the number of genera (10). Our results coincide with those of these authors, revealing that there is an unequal distribution of yeasts in the different elements of the colony (e.g. foliage, new fungus, old fungus, workers' bodies, nest floor, and waste material) and that some occur frequently, while others are intermittent.
Also, the results of the present study are also similar to that of Carreiro et al. (1997) in recording the genera Candida, Trichosporon and Pichia and the species C. homilentoma (now Hyphopichia homilentoma) as prevalent yeasts. It is important to mention that similar to what was recorded in this study, these authors found the species C. homilentoma, C. robusta, C. sake, C. valida, Candida sp., Pichia anomala (now Wickerhamomyces anomalus) and Trichosporon beigelii, on the integument of workers. Therefore, both studies reinforce the hypothesis that yeasts are not only found in the fungus gardens but can also be part of the microbiota of workers, most likely fulfilling certain physiological functions.
When investigating the yeasts associated with the integuments, pellet and infrabucal pocket of queens of A. capiguara and A. laevigata, Pagnocca et al. (2008) found Aureobasidium pullulans, Rhodotorula glutinis, Cryptococcus laurentii (now Papiliotrema laurentii) and Candida parapsilosis. Similarly, Angelis et al. (1983) and later Carreiro et al. (1997), isolated 13 yeast species from the nests of A. sexdens rubropilosa and A. laevigata from the genera Candida, Torulopsis, Rhodotorula and Kloeckera. Also, Middelhoven et al. (2003) and Carreiro et al. (2004) found Cutaneotrichosporon haglerorum and Blastobotrys attinorum, respectively, associated with leafcutter ants, while Little and Currie (2007; 2008) found the black yeast Phialophora sp. related to Apterostigma sp. However, none of these species are coincident with those found in the present investigation. It should be noted that none of aforementioned authors sampled the workers considering caste separation.
Although Rhodotorula was found in attine ants by Angelis et al. (1983) and later on by Carreiro et al. (1997) and Pagnocca et al. (2008), this genus was not found in the workers of A. cephalotes in our study. On the other hand, Candida was present consistently in all the investigations mentioned, and presented a large variation and diversity of species, which allows us to infer a possible persistent association between
In the present study, the fact that genera and species were found exclusively in the garden caste, including Candida intermedia, Candida spp., C. fermentati, Trichosporon coremiiforme, Yamadazyma mexicana, H. burtonii and D. hansenii has special relevance. The isolation of large numbers of yeasts from the garden workers justifies the study of these organisms as being potentially associated with different processes in the colony. Also, it is necessary to deepen these investigations to understand whether these microorganisms are directly associated with the bodies of the ants or if they are exclusively found in the fungus garden and then transported by the workers when they come in contact with the mutualistic fungus.
Carreiro (2000) hypothesized that both yeasts and ants may benefit from their relationships. They posit that while yeasts are taking advantage of the nutrients available in the fungus garden, ants might benefit from the enzymes that yeasts secrete into the fungus garden, as they would contribute to the digestion of the plant substrate collected by the ants. These enzymes may also contribute to the degradation of plant tissue by the fungal cultivar by breaking down structural plant polysaccharides such as pectin and hemi-cellulose (Carreiro 2000; Pagnocca et al. 2010; Mendes et al. 2012). The elucidation of the pathways of these processes (and if yeasts in fact participate on those) may render great potential biotechnological applications. However, the identification of species biodiversity, which is a basic preliminary step, has not been completed in our study. Further studies are required to elucidate the role of isolated yeast species in the framework of the complex mutualistic system hitherto described.
At least 17 species of yeasts have ecologically diverse associations with ants (Rosa and Gábor 2006). Many are mutualistic but not completely understood (Suh et al. 2008) while others, called killer yeasts, are capable of secreting protein metabolites of variable molecular weights named killer toxins, which are capable of inhibiting other microorganisms by altering cell walls, membranes, or vulnerable cell nuclei (Buzzini et al. 2007), which in the case of the killer phenotype of Torulaspora globosa strain 1S112 in the Atta genus is under in vitro and in vivo evaluations (Robledo-Leal et al. 2016). Among the yeasts identified, there were several species reported as antagonistic or effective as potential killer yeasts for the control of fungi that cause plant deterioration. These yeasts might have great potential for biotechnological applications in diverse areas, especially in agriculture, where better results might be obtained in the biological control of phyto-pathogens (Ahansal et al. 2008; Rosa et al. 2010). For instance, the fungus Botrytis cinerea, one of the most harmful pathogens in grape and strawberry crops, was inhibited using a partially purified strain of Pichia membranifaciens CYC 1106 (Santos and Marquina 2004). This same fungus was later inhibited using strains of Pichia anomala and Debaryomyces hansenii, increasing the biological control arsenal for this pathogen (Santos et al. 2004). Strains of Issatcehnkia orientalis, Candida guilliermondii (now Meyerozyma guilliermondii), P. ohmeri and Torulaspora globosa, among others, have been reported to successfully inhibit important plant pathogenic fungi, such as Aspergillus cabonarius, A. niger, Penicillium expansum and Colletotrichum sublineolum in grape, pear, apple, and sorghum crops, respectively (Bleve et al. 2006; Coelho et al. 2009; Rosa et al. 2010). In Mexico, Hernández-Montiel et al. (2011) reported a significant reduction of Geotrichum citriaurantii in limes post-harvest, using epiphytic D. hansenii yeasts.
Several authors (Currie et al. 1999; 2003; Poulsen et al. 2003; Santos et al. 2004) previously stated the need to challenge the misconception of an axenic culture (monoculture) of the mutualistic fungus of leaf-cutting ants. They also suggested that symbiotic interactions cannot be viewed as bipartite associations and alleged the presence of a third additional organism. Our results support a well-conceptualised idea that the fungus gardens are home of a diverse and complex microbiota. Furthermore, the fact that we found abundant and dominant yeasts associated with the worker caste highlights the importance of the presence of these neglected but potential symbiotic organisms in the ancestral Atta cephalotes-fungal cultivar mutualism. Hence, the mutualistic network within this system could be more complex than previously thought. Confirmation of this issue is challenging, although with the advent of novel technologies more precise and detailed identification of the yeasts is achievable. Knowing the species identity might provide valuable information that helps to elucidate the role of these microorganisms as poly-associates, as the composition of many of these associations depends on the microbial diversity of the environment their colonies inhabit. Future work on A. cephalotes or other leaf-cutting ants should focus on the extent and similarities of microbiota and also should address whether this biota has a role in the division of the labour within their colonies.