Enrichment and Broad Representation of Plant Biomass-Degrading Enzymes in the Specialized Hyphal Swellings of Leucoagaricus gongylophorus,the Fungal Symbiont of Leaf-Cutter Ants

Leaf-cutter ants are prolific and conspicuous constituents of Neotropical ecosystems that derive energy from specialized fungus gardens they cultivate using prodigious amounts of foliar biomass. The basidiomycetous cultivar of the ants, Leucoagaricus gongylophorus, produces specialized hyphal swellings called gongylidia that serve as the primary food source of ant colonies. Gongylidia also contain plant biomass-degrading enzymes that become concentrated in ant digestive tracts and are deposited within fecal droplets onto fresh foliar material as ants incorporate it into the fungus garden. Although the enzymes concentrated by L. gongylophorus within gongylidia are thought to be critical to the initial degradation of plant biomass, only a few enzymes present in these hyphal swellings have been identified. Here we use proteomic methods to identify proteins present in the gongylidia of three Atta cephalotes colonies. Our results demonstrate that a diverse but consistent set of enzymes is present in gongylidia, including numerous plant biomass-degrading enzymes likely involved in the degradation of polysaccharides, plant toxins, and proteins. Overall, gongylidia contained over three quarters of all biomass-degrading enzymes identified in the L. gongylophorus genome, demonstrating that the majority of the enzymes produced by this fungus for biomass breakdown are ingested by the ants. We also identify a set of 40 of these enzymes enriched in gongylidia compared to whole fungus garden samples, suggesting that certain enzymes may be particularly important in the initial degradation of foliar material. Our work sheds light on the complex interplay between leaf-cutter ants and their fungal symbiont that allows for the host insects to occupy an herbivorous niche by indirectly deriving energy from plant biomass.     

Leucoagaricus gongylophorus Produces Diverse Enzymes for the Degradation of Recalcitrant Plant Polymers in Leaf-Cutter Ant Fungus Gardens

Plants represent a large reservoir of organic carbon comprised primarily of recalcitrant polymers that most metazoans are unable to deconstruct. Many herbivores gain access to nutrients in this material indirectly by associating with microbial symbionts, and leaf-cutter ants are a paradigmatic example. These ants use fresh foliar biomass as manure to cultivate gardens composed primarily of Leucoagaricus gongylophorus, a basidiomycetous fungus that produces specialized hyphal swellings that serve as a food source for the host ant colony. Although leaf-cutter ants are conspicuous herbivores that contribute substantially to carbon turnover in Neotropical ecosystems, the process through which plant biomass is degraded in their fungus gardens is not well understood. Here we present the first draft genome of L. gongylophorus, and, using genomic and metaproteomic tools, we investigate its role in lignocellulose degradation in the gardens of both Atta cephalotes and Acromyrmex echinatior leaf-cutter ants. We show that L. gongylophorus produces a diversity of lignocellulases in ant gardens and is likely the primary driver of plant biomass degradation in these ecosystems. We also show that this fungus produces distinct sets of lignocellulases throughout the different stages of biomass degradation, including numerous cellulases and laccases that likely play an important role in lignocellulose degradation. Our study provides a detailed analysis of plant biomass degradation in leaf-cutter ant fungus gardens and insight into the enzymes underlying the symbiosis between these dominant herbivores and their obligate fungal cultivar.     

Metabolism of Plant Polysaccharides by Leucoagaricus gongylophorus, the Symbiotic Fungus of the Leaf-Cutting Ant Atta sexdens L.

Atta sexdens L. ants feed on the fungus they cultivate on cut leaves inside their nests. The fungus, Leucoagaricus gongylophorus, metabolizes plant polysaccharides, such as xylan, starch, pectin, and cellulose, mediating assimilation of these compounds by the ants. This metabolic integration may be an important part of the ant-fungus symbiosis, and it involves primarily xylan and starch, both of which support rapid fungal growth. Cellulose seems to be less important for symbiont nutrition, since it is poorly degraded and assimilated by the fungus. Pectin is rapidly degraded but slowly assimilated by L. gongylophorus, and its degradation may occur so that the fungus can more easily access other polysaccharides in the leaves.     

Three phylogenetically distinct and culturable diazotrophs are perennial symbionts of leaf‐cutting ants

The obligate mutualistic basidiomycete fungus, Leucocoprinus gongylophorus, mediates nutrition of leaf‐cutting ants with carbons from vegetal matter. In addition, diazotrophic Enterobacteriales in the fungus garden and intestinal Rhizobiales supposedly mediate assimilation of atmospheric nitrogen, and Entomoplasmatales in the genus Mesoplasma, as well as other yet unidentified strains, supposedly mediate ant assimilation of other compounds from vegetal matter, such as citrate, fructose, and amino acids. Together, these nutritional partners would support the production of high yields of leafcutter biomass. In the present investigation, we propose that three phylogenetically distinct and culturable diazotrophs in the genera Ralstonia, Methylobacterium, and Pseudomonas integrate this symbiotic nutrition network, facilitating ant nutrition on nitrogen. Strains in these genera were often isolated and directly sequenced in 16S rRNA libraries from the ant abdomen, together with the nondiazotrophs Acinetobacter and Brachybacterium. These five isolates were underrepresented in libraries, suggesting that none of them is dominant in vivo. Libraries have been dominated by four uncultured Rhizobiales strains in the genera Liberibacter, Terasakiella, and Bartonella and, only in Acromyrmex ants, by the Entomoplasmatales in the genus Mesoplasma. Acromyrmex also presented small amounts of two other uncultured Entomoplasmatales strains, Entomoplasma and Spiroplasma. The absence of Entomoplasmatales in Atta workers implicates that the association with these bacteria is not mandatory for ant biomass production. Most of the strains that we detected in South American ants were genetically similar with strains previously described in association with leafcutters from Central and North America, indicating wide geographic dispersion, and suggesting fixed ecological services.     

The fungal cultivar of leaf‐cutter ants produces specific enzymes in response to different plant substrates

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf‐cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants’ fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L. gongylophorus to leaves, flowers, oats or a mixture of all three. Across all treatments, our analysis identified and quantified 1766 different fungal proteins, including 161 putative biomass‐degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of subcolonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase and β‐glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate‐specific enzymatic response of the fungal cultivar allows leaf‐cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores.     

Instability of novel ant-fungal associations constrains horizontal exchange of fungal symbionts

One of the more fascinating features of fungus-gardening ants (Attini: Formicidae) is their fidelity to their lineage-specific fungal symbionts. Among the derived higher-attine ants (leafcutter ants and close relatives), it is thought that most leaf-cutting ants grow Attamyces fungus whereas most Trachymyrmex ants grow ‘Trachymyces’ fungus, but there exist exceptions to this clade-to-clade correspondence between ants and fungi. The exceptions are inconsistent with strict one-to-one coevolution, which suggests that ants sometimes are able to switch to novel fungi. Such switches appear to be largely constrained and ants are generally faithful to their species-specific fungi. Prior experiments demonstrated no clear fitness consequences of growing novel fungi over the short-term when the ant Trachymyrmex septentrionalis was symbiont-switched by forcing it to grow Attamyces leaf-cutter fungus. We hypothesized that long-term ant-fungal fidelity is constrained either by physiological differences among fungal species or by garden diseases that symbiont-switched ants cannot control. Repeat experiments in a different location show that T. septentrionalis colonies switched to grow Attamyces exhibit sudden declines in garden biomass and consequent fitness reductions due to garden destruction by pathogens, whereas control colonies (Trachymyrmex ants cultivating Trachymyces fungus) do not show parallel garden declines. These patterns are mirrored in symbiont-switch experiments conducted on colonies in Trachymyrmex turrifex. Disease microbes selecting on ant-cultivar combinations therefore can constrain switches to novel cultivars and maintain combinations that are more resistant to disease.     

Leaf-cutting ant fungi produce cell wall degrading pectinase complexes reminiscent of phytopathogenic fungi

Background

Leaf-cutting (attine) ants use their own fecal material to manure fungus gardens, which consist of leaf material overgrown by hyphal threads of the basidiomycete fungus Leucocoprinus gongylophorus that lives in symbiosis with the ants. Previous studies have suggested that the fecal droplets contain proteins that are produced by the fungal symbiont to pass unharmed through the digestive system of the ants, so they can enhance new fungus garden growth.

Results

We tested this hypothesis by using proteomics methods to determine the gene sequences of fecal proteins in Acromyrmex echinatior leaf-cutting ants. Seven (21%) of the 33 identified proteins were pectinolytic enzymes that originated from the fungal symbiont and which were still active in the fecal droplets produced by the ants. We show that these enzymes are found in the fecal material only when the ants had access to fungus garden food, and we used quantitative polymerase chain reaction analysis to show that the expression of six of these enzyme genes was substantially upregulated in the fungal gongylidia. These unique structures serve as food for the ants and are produced only by the evolutionarily advanced garden symbionts of higher attine ants, but not by the fungi reared by the basal lineages of this ant clade.

Conclusions

Pectinolytic enzymes produced in the gongylidia of the fungal symbiont are ingested but not digested by Acromyrmex leaf-cutting ants so that they end up in the fecal fluid and become mixed with new garden substrate. Substantial quantities of pectinolytic enzymes are typically found in pathogenic fungi that attack live plant tissue, where they are known to breach the cell walls to allow the fungal mycelium access to the cell contents. As the leaf-cutting ant symbionts are derived from fungal clades that decompose dead plant material, our results suggest that their pectinolytic enzymes represent secondarily evolved adaptations that are convergent to those normally found in phytopathogens.

     

An Insect Herbivore Microbiome with High Plant Biomass-Degrading Capacity

Herbivores can gain indirect access to recalcitrant carbon present in plant cell walls through symbiotic associations with lignocellulolytic microbes. A paradigmatic example is the leaf-cutter ant (Tribe: Attini), which uses fresh leaves to cultivate a fungus for food in specialized gardens. Using a combination of sugar composition analyses, metagenomics, and whole-genome sequencing, we reveal that the fungus garden microbiome of leaf-cutter ants is composed of a diverse community of bacteria with high plant biomass-degrading capacity. Comparison of this microbiome''s predicted carbohydrate-degrading enzyme profile with other metagenomes shows closest similarity to the bovine rumen, indicating evolutionary convergence of plant biomass degrading potential between two important herbivorous animals. Genomic and physiological characterization of two dominant bacteria in the fungus garden microbiome provides evidence of their capacity to degrade cellulose. Given the recent interest in cellulosic biofuels, understanding how large-scale and rapid plant biomass degradation occurs in a highly evolved insect herbivore is of particular relevance for bioenergy.     

Hydrolytic enzymes of leaf-cutting ant fungi

The production of enzymes and the colonization of leaves by Leucoagaricus gongylophorus were investigated to further understand the digestive interactions of leaf-cutting ant colonies. The enzymes detected were indicative of a saprophytic origin of this fungus, producing all the enzymes necessary for plant tissue breakdown. Enhanced activities of certain enzymes in the fungus garden extracts may be due to the particular behaviour of the adult worker ants that concentrate fungal acquired enzymes in the rectal fluid and subsequently defaecate these enzymes onto the leaves. The production of chitinases by the fungus may be an ancestral vestige of lower attines, and may have a role as agonists of invading microbes. Growth of the fungus on plant cell wall medium resulted in highest enzyme activity against pectin, reflecting the fact that polygalacturonans comprise the main matrix of the primary plant cell wall. SEM shows that L. gongylophorus does not form specialized structures for cell wall penetration, but gains access to the inner plant tissue at the cut edges of the leaf fragments. Enzymes secreted by the fungus were compared to those seen in larval and adult leaf-cutting ants, demonstrating the inter-dependence of the symbiotic relationship between the ants and their fungi.     

Metagenomic and metaproteomic insights into bacterial communities in leaf-cutter ant fungus gardens

Herbivores gain access to nutrients stored in plant biomass largely by harnessing the metabolic activities of microbes. Leaf-cutter ants of the genus Atta are a hallmark example; these dominant neotropical herbivores cultivate symbiotic fungus gardens on large quantities of fresh plant forage. As the external digestive system of the ants, fungus gardens facilitate the production and sustenance of millions of workers. Using metagenomic and metaproteomic techniques, we characterize the bacterial diversity and physiological potential of fungus gardens from two species of Atta. Our analysis of over 1.2 Gbp of community metagenomic sequence and three 16S pyrotag libraries reveals that in addition to harboring the dominant fungal crop, these ecosystems contain abundant populations of Enterobacteriaceae, including the genera Enterobacter, Pantoea, Klebsiella, Citrobacter and Escherichia. We show that these bacterial communities possess genes associated with lignocellulose degradation and diverse biosynthetic pathways, suggesting that they play a role in nutrient cycling by converting the nitrogen-poor forage of the ants into B-vitamins, amino acids and other cellular components. Our metaproteomic analysis confirms that bacterial glycosyl hydrolases and proteins with putative biosynthetic functions are produced in both field-collected and laboratory-reared colonies. These results are consistent with the hypothesis that fungus gardens are specialized fungus–bacteria communities that convert plant material into energy for their ant hosts. Together with recent investigations into the microbial symbionts of vertebrates, our work underscores the importance of microbial communities in the ecology and evolution of herbivorous metazoans.     

Survival of Atta sexdens workers on different food sources

Leaf-cutting ants belonging to the tribe Attini are major herbivores and important agriculture pests in the neotropics, these ants being thought to feed on the sap which exudes from the plant material which they cut and also on the mycelium of a symbiotic fungus that grows on plant material inside their nests in what is called "the fungus garden". However, we have found that the survival of Atta sexdens worker ants on leaves, on mycelium of the ants' symbiotic fungus, Leucoagaricus gongylophorus, or on plant polysaccharides was the same as that of starved A. sexdens, while, conversely, significantly longer survival was achieved by ants fed on the fungus garden material or on some of the products (especially glucose) of the hydrolysis of plant polysaccharides. We found that the fungus garden contained glucose at a higher concentration than that found in leaves or fungal mycelium, and that this glucose was consumed by the ant to the extent that it was probably responsible for up to 50% of the nutritional needs of the workers. The fungus garden contained polysaccharide degrading enzymes (pectinase, amylase, xylanase and cellulase) in proportions similar to that observed in laboratory cultures of L. gongylophorus. It thus appears that A. sexdens workers obtain a significant part of their nutrients from plant polysaccharide hydrolysis products produced by the action of extracellular enzymes released by L. gongylophorus. In this paper we discuss the symbiotic nutrition strategy of A. sexdens workers and brood and the role played by plant polysaccharides in the nutrition of attine ants.     

Rapid shifts in <Emphasis Type="Italic">Atta cephalotes</Emphasis> fungus-garden enzyme activity after a change in fungal substrate (Attini,Formicidae)

Fungus gardens of the basidiomycete Leucocoprinus gongylophorus sustain large colonies of leaf-cutting ants by degrading the plant material collected by the ants. Recent studies have shown that enzyme activity in these gardens is primarily targeted toward starch, proteins and the pectin matrix associated with cell walls, rather than toward structural cell wall components such as cellulose and hemicelluloses. Substrate constituents are also known to be sequentially degraded in different sections of the fungus garden. To test the plasticity in the extracellular expression of fungus-garden enzymes, we measured the changes in enzyme activity after a controlled shift in fungal substrate offered to six laboratory colonies of Atta cephalotes. An ant diet consisting exclusively of grains of parboiled rice rapidly increased the activity of endo-proteinases and some of the pectinases attacking the backbone structure of pectin molecules, relative to a pure diet of bramble leaves, and this happened predominantly in the most recently established top sections of fungus gardens. However, fungus-garden amylase activity did not significantly increase despite the substantial increase in starch availability from the rice diet, relative to the leaf diet controls. Enzyme activity in the older, bottom sections of fungus gardens decreased, indicating a faster processing of the rice substrate compared to the leaf diet. These results suggest that leaf-cutting ant fungus gardens can rapidly adjust enzyme activity to provide a better match with substrate availability and that excess starch that is not protected by cell walls may be digested by the ants rather than by the fungus-garden symbiont.     

Production of Polysaccharidases in Different Carbon Sources by Leucoagaricus gongylophorus Möller (Singer), the Symbiotic Fungus of the Leaf-Cutting Ant Atta sexdens Linnaeus

Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens, produces polysaccharidases that degrade leaf components by generating nutrients believed to be essential for ant nutrition. We evaluated pectinase, amylase, xylanase, and cellulase production by L. gongylophorus in laboratory cultures and found that polysaccharidases are produced during fungal growth on pectin, starch, cellulose, xylan, or glucose but not cellulase, whose production is inhibited during fungal growth on xylan. Pectin was the carbon source that best stimulated the production of enzymes, which showed that pectinase had the highest production activity of all of the carbon sources tested, indicating that the presence of pectin and the production of pectinase are key features for symbiotic nutrition on plant material. During growth on starch and cellulose, polysaccharidase production level was intermediate, although during growth on xylan and glucose, enzyme production was very low. We propose a possible profile of polysaccharide degradation inside the nest, where the fungus is cultured on the foliar substrate.     

The dynamics of plant cell-wall polysaccharide decomposition in leaf-cutting ant fungus gardens

The degradation of live plant biomass in fungus gardens of leaf-cutting ants is poorly characterised but fundamental for understanding the mutual advantages and efficiency of this obligate nutritional symbiosis. Controversies about the extent to which the garden-symbiont Leucocoprinus gongylophorus degrades cellulose have hampered our understanding of the selection forces that induced large scale herbivory and of the ensuing ecological footprint of these ants. Here we use a recently established technique, based on polysaccharide microarrays probed with antibodies and carbohydrate binding modules, to map the occurrence of cell wall polymers in consecutive sections of the fungus garden of the leaf-cutting ant Acromyrmex echinatior. We show that pectin, xyloglucan and some xylan epitopes are degraded, whereas more highly substituted xylan and cellulose epitopes remain as residuals in the waste material that the ants remove from their fungus garden. These results demonstrate that biomass entering leaf-cutting ant fungus gardens is only partially utilized and explain why disproportionally large amounts of plant material are needed to sustain colony growth. They also explain why substantial communities of microbial and invertebrate symbionts have evolved associations with the dump material from leaf-cutting ant nests, to exploit decomposition niches that the ant garden-fungus does not utilize. Our approach thus provides detailed insight into the nutritional benefits and shortcomings associated with fungus-farming in ants.     

Susceptibility of the ant-cultivated fungus Leucoagaricus gongylophorus (Agaricales: Basidiomycota) towards microfungi

The aim of this study was to select virulent strains of microfungi against Leucoagaricus gongylophorus, a symbiotic fungus cultivated by leaf-cutting ants. The results from in vitro assays showed that microfungal strains had a variable and significant impact on the colony development of L. gongylophorus. Specifically, Trichoderma harzianum, Escovopsis weberi CBS 810.71 and E. weberi A088 were more effective, inhibiting the L. gongylophorus colonies by 75, 68 and 67%, respectively (P < 0.05) after 15 days. Strain E. weberi A086 and Acremonium kiliense were less effective: 43 and 26%, respectively (P < 0.05). In spite of the current negative perspective of a microbiological control approach for these ants, the present work discusses the possibility of using mycopathogenic fungi for the control of these insects, and points out the importance of encouraging more studies in this area.     

Digestive capacities of leaf-cutting ants and the contribution of their fungal cultivar to the degradation of plant material

Leaf-cutting ants (tribe Attini) are a unique group of ants that cultivate a fungus that serves as a main source of their food. The fungus is grown on fresh leaves that are harvested by workers. We examine the respective contribution of ants and their symbiotic fungus in the degradation of plant material by examining the digestive capacities of seven Attini species in the genera Atta and Acromyrmex. The results show that both, the ants and their mutualistic fungi, have complementary enzymatic activities. Ants are specialized in the degradation of low molecular weight substrates (oligosaccharides and heterosides) whereas the fungus displays high polysaccharidase activity. The two genera Atta and Acromyrmex are not distinguished by a specific enzymatic activity. The seven different mutualistic associations examined display a similar enzymatic profile but have quantitative differences in substrate degradation activities. The respective contribution of ants and the fungus garden in plant degradation are discussed.     

Yeasts found on an ephemeral reproductive caste of the leaf-cutting ant Atta sexdens rubropilosa

Winged males of leaf-cutting ants are considered an ephemeral reproductive caste only produced before the mating flight season. Although much is known about the yeast diversity found in fungus gardens of attine ants, no study has focused on the yeasts associated with males of leaf-cutting ants. Here, we surveyed the yeasts on the integuments of males of Atta sexdens rubropilosa and assessed their potential role in the attine ant-microbe symbiosis. Using culture-dependent techniques, we found yeasts to be abundant on the integuments of males (54.5 %, n = 200 alates). A total of 242 yeast strains were obtained representing six orders, ten genera and 25 species. Strains of Aureobasidium, Cryptococcus, Hannaella and Rhodotorula were prevalent on the integuments and likely originated from the fungus garden of the parental nest or from the soil. The majority of strains (87.1 %) produced at least one of the evaluated enzymes: pectinase, polygalacturonase, cellulase, xylanase, ligninases and lipase. Aureobasidium pullulans accounted for the highest number of strains that produced all enzymes. In addition, yeasts showed the ability to assimilate the resulting oligosaccharides, supporting observations of other studies that yeasts may be involved in the plant biomass metabolism in the fungus gardens. Because winged males harbor several yeasts with putative functional roles, these fungi may take part and be beneficial in the microbial consortia of the new incipient nest.     

Fitness consequences of nest infiltration by the mutualist‐exploiter Megalomyrmex adamsae

1. Fungus‐growing ants are obligate mutualists. Their nutrient‐rich fungus garden provides a valuable food store that sustains the ant hosts, but can also attract social parasites. 2. The ‘guest ant' Megalomyrmex adamsae Longino parasitises the fungus‐growing Trachymyrmex zeteki Weber queen just after nest founding. The parasitic queen infiltrates the incipient nest, builds a cavity in the fungal garden, and lays eggs that develop into workers and reproductive males and females. 3. This study compared young parasitised and non‐parasitised laboratory colonies by measuring garden growth and biomass, and the number of host workers and reproductives. Host queen survival and parasite colony growth were also monitored. 4. Parasitised Trachymyrmex colonies had reduced host worker and alate numbers, as well as lower garden biomass, compared with non‐parasitised control colonies, confirming that M. adamsae is a xenobiotic social parasite. Host queen survival was not significantly different between parasitised and control colonies. 5. This is the first study that experimentally infects host colonies with a xenobiotic social parasite to measure fitness cost to the host. The natural history of M. adamsae and the fungus‐growing ant mutualism are evaluated in the context of three general predictions of (Bronstein, Ecology Letters, 4 , 277–287, 2001a) regarding the cost of mutualism exploiters.     

A Brazilian Population of the Asexual Fungus-Growing Ant Mycocepurus smithii (Formicidae,Myrmicinae, Attini) Cultivates Fungal Symbionts with Gongylidia-Like Structures

Attine ants cultivate fungi as their most important food source and in turn the fungus is nourished, protected against harmful microorganisms, and dispersed by the ants. This symbiosis evolved approximately 50–60 million years ago in the late Paleocene or early Eocene, and since its origin attine ants have acquired a variety of fungal mutualists in the Leucocoprineae and the distantly related Pterulaceae. The most specialized symbiotic interaction is referred to as “higher agriculture” and includes leafcutter ant agriculture in which the ants cultivate the single species Leucoagaricus gongylophorus. Higher agriculture fungal cultivars are characterized by specialized hyphal tip swellings, so-called gongylidia, which are considered a unique, derived morphological adaptation of higher attine fungi thought to be absent in lower attine fungi. Rare reports of gongylidia-like structures in fungus gardens of lower attines exist, but it was never tested whether these represent rare switches of lower attines to L. gonglyphorus cultivars or whether lower attine cultivars occasionally produce gongylidia. Here we describe the occurrence of gongylidia-like structures in fungus gardens of the asexual lower attine ant Mycocepurus smithii. To test whether M. smithii cultivates leafcutter ant fungi or whether lower attine cultivars produce gongylidia, we identified the M. smithii fungus utilizing molecular and morphological methods. Results shows that the gongylidia-like structures of M. smithii gardens are morphologically similar to gongylidia of higher attine fungus gardens and can only be distinguished by their slightly smaller size. A molecular phylogenetic analysis of the fungal ITS sequence indicates that the gongylidia-bearing M. smithii cultivar belongs to the so-called “Clade 1”of lower Attini cultivars. Given that M. smithii is capable of cultivating a morphologically and genetically diverse array of fungal symbionts, we discuss whether asexuality of the ant host maybe correlated with low partner fidelity and active symbiont choice between fungus and ant mutualists.     

Microbial Community Structure of Leaf-Cutter Ant Fungus Gardens and Refuse Dumps

Background

Leaf-cutter ants use fresh plant material to grow a mutualistic fungus that serves as the ants'' primary food source. Within fungus gardens, various plant compounds are metabolized and transformed into nutrients suitable for ant consumption. This symbiotic association produces a large amount of refuse consisting primarily of partly degraded plant material. A leaf-cutter ant colony is thus divided into two spatially and chemically distinct environments that together represent a plant biomass degradation gradient. Little is known about the microbial community structure in gardens and dumps or variation between lab and field colonies.

Methodology/Principal Findings

Using microbial membrane lipid analysis and a variety of community metrics, we assessed and compared the microbiota of fungus gardens and refuse dumps from both laboratory-maintained and field-collected colonies. We found that gardens contained a diverse and consistent community of microbes, dominated by Gram-negative bacteria, particularly γ-Proteobacteria and Bacteroidetes. These findings were consistent across lab and field gardens, as well as host ant taxa. In contrast, dumps were enriched for Gram-positive and anaerobic bacteria. Broad-scale clustering analyses revealed that community relatedness between samples reflected system component (gardens/dumps) rather than colony source (lab/field). At finer scales samples clustered according to colony source.

Conclusions/Significance

Here we report the first comparative analysis of the microbiota from leaf-cutter ant colonies. Our work reveals the presence of two distinct communities: one in the fungus garden and the other in the refuse dump. Though we find some effect of colony source on community structure, our data indicate the presence of consistently associated microbes within gardens and dumps. Substrate composition and system component appear to be the most important factor in structuring the microbial communities. These results thus suggest that resident communities are shaped by the plant degradation gradient created by ant behavior, specifically their fungiculture and waste management.