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.     

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.     

Leucoagaricus gongylophorus uses leaf-cutting ants to vector proteolytic enzymes towards new plant substrate

The mutualism between leaf-cutting ants and their fungal symbionts revolves around processing and inoculation of fresh leaf pulp in underground fungus gardens, mediated by ant fecal fluid deposited on the newly added plant substrate. As herbivorous feeding often implies that growth is nitrogen limited, we cloned and sequenced six fungal proteases found in the fecal fluid of the leaf-cutting ant Acromyrmex echinatior and identified them as two metalloendoproteases, two serine proteases and two aspartic proteases. The metalloendoproteases and serine proteases showed significant activity in fecal fluid at pH values of 5–7, but the aspartic proteases were inactive across a pH range of 3–10. Protease activity disappeared when the ants were kept on a sugar water diet without fungus. Relative to normal mycelium, both metalloendoproteases, both serine proteases and one aspartic protease were upregulated in the gongylidia, specialized hyphal tips whose only known function is to provide food to the ants. These combined results indicate that the enzymes are derived from the ingested fungal tissues. We infer that the five proteases are likely to accelerate protein extraction from plant cells in the leaf pulp that the ants add to the fungus garden, but regulatory functions such as activation of proenzymes are also possible, particularly for the aspartic proteases that were present but without showing activity. The proteases had high sequence similarities to proteolytic enzymes of phytopathogenic fungi, consistent with previous indications of convergent evolution of decomposition enzymes in attine ant fungal symbionts and phytopathogenic fungi.     

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.     

Limited impacts of the fungus Syncephalastrum on nests of leaf-cutting ants

Leaf-cutting ants interact naturally with a range of antagonistic microorganisms, among them the soil-borne fungus Syncephalastrum. The antagonism of this fungus to the leaf-cutting ants’ fungal cultivar has been shown in studies without the ant queens. So far, the impacts of this fungus on whole colonies (queenright) of leaf-cutting ants are unknown. We assessed the impacts of Syncephalastrum on queenless and queenright colonies of Acromyrmex subterraneus subterraneus. In general, Syncephalastrum negatively impacted leaf cutting but not midden production or colony weight. This impact was greater in queenless colonies. Nevertheless, it did not compromise the survival of any colony. This indicates that the virulence of this fungus to leaf-cutting ant colonies may be limited in a more realistic set-up than previously reported. We propose that future laboratory studies also use queenright colonies where possible, and that the diverse species of leaf-cutting ants also be considered.     

Comparative analysis of fungal communities in colonies of two leaf-cutting ant species with different substratum preferences

Fungus gardens of leaf-cutting ants harbor diverse alien fungi in addition to their fungal cultivar. Previous work suggested that alien microorganisms are likely derived from the substrata foraged by ant workers and incorporated into the fungus gardens. To test this hypothesis, we sampled 1014 garden fragments from 16 field colonies of Atta sexdens rubropilosa (a dicot-cutting ant) and Atta capiguara (a grass-cutting ant) in Brazil. From a total of 615 fungal isolates recovered, we observed similar diversity of fungi between colonies of both ant species. However, fungal communities differed in composition of taxa between ant colonies. Trichoderma spirale, Trichosporon chiarellii and Penicillium citrinum were prevalent accounting for 18.5%, 12.2% and 11.7% of the total isolates, respectively. As expected, fungal communities clustered in two major groups supporting the hypothesis that plant substratum has an impact on the composition of the alien fungi found in leaf-cutting ant gardens.     

EVOLUTIONARY TRANSITIONS IN ENZYME ACTIVITY OF ANT FUNGUS GARDENS

Fungus‐growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher‐attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher‐attine ants have enhanced activity of protein‐digesting enzymes, whereas gardens of leaf‐cutting ants also have increased activity of starch‐digesting enzymes. However, the enzyme activities of lower‐attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher‐attine and leaf‐cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower‐attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.     

Colony Dehydration and Water Collection by Specialized Caste in the Leaf-Cutting Ant <Emphasis Type="Italic">Atta Sexdens Rubropilosa</Emphasis>

Social organization enables leaf-cutting ants to keep appropriate micro-ecological nest conditions for the fungus garden (their main food), eggs, larvae and adults. To maintain stability while facing changing conditions, individual ants must perceive destabilising factors and produce a proper behavioral response. We investigated behavioral responses to experimental dehydration in leaf-cutting ants to verify if task specialization exists, and to quantify the ability of ant sub-colonies for water management. Our setup consisted of fourteen sub-colonies, ten of which were randomly assigned to different levels of experimental dehydration with silica gel, whereas the remaining four were controls. The ten experimental sub-colonies were split into two groups, so that five of them had access to water. Diverse ant morphs searched for water in dehydrated colonies, but mainly a caste of small ants collected water after sources had been discovered. Size specialization for water collection was replicable in shorter experiments with three additional colonies. Ants of dehydrated colonies accumulated leaf-fragments on the nest entrance, and covering the fungus garden. Behaviors that may enhance humidity within the nests were common to all dehydration treatments. Water availability increased the life span of dehydrated colonies.     

Prevalence and impact of a virulent parasite on a tripartite mutualism

The prevalence and impact of a specialized microfungal parasite (Escovopsis) that infects the fungus gardens of leaf-cutting ants was examined in the laboratory and in the field in Panama. Escovopsis is a common parasite of leaf-cutting ant colonies and is apparently more frequent in Acromyrmex spp. gardens than in gardens of the more phylogenetically derived genus Atta spp. In addition, larger colonies of Atta spp. appear to be less frequently infected with the parasite. In this study, the parasite Escovopsis had a major impact on the success of this mutualism among ants, fungi, and bacteria. Infected colonies had a significantly lower rate of fungus garden accumulation and produced substantially fewer workers. In addition, the extent of the reduction in colony growth rate depended on the isolate, with one isolate having a significantly larger impact than two others, suggesting that Escovopsis has different levels of virulence. Escovopsis is also spatially concentrated within parts of ant fungus gardens, with the younger regions having significantly lower rates of infection as compared to the older regions. The discovery that gardens of fungus-growing ants are host to a virulent pathogen that is not related to any of the three mutualists suggests that unrelated organisms may be important but primarily overlooked components of other mutualistic associations.     

The effects of leaf deprivation on leaf-cutting ants and their mutualistic fungus

1. When leaf-cutting ants were deprived of leaves for 5 days, they increased their consumption rates of 'staphylae', the nutritive bodies produced by their mutualistic fungus. They also pruned their fungus garden more intensively, presumably stimulating increased staphyla production.
2. Depriving fungus gardens of leaves for 5 days led to greater rates of staphyla production compared with control gardens. The rate of substrate exhaustion also increased, possibly because of this increased staphyla production, which led to greater removal of fungal resources by the ants.
3. The availability of leaf substrate directly affected staphyla production by the fungus garden. When leaf fragments were inserted into fungus garden samples, staphyla production was depressed, suggesting that the fungus can allocate resources either to hyphal colonization of fresh substrate or to staphyla production.
4. Under conditions of leaf deprivation, food production for the ants effectively increased, due to the combined effects of increased pruning by the ants and the response of the fungus to lack of substrate. This will allow nests to survive for long periods in the field when forage availability is reduced.
5. Exposing parts of the colony to the stress of leaf deprivation showed that there is a high level of flexibility in the fungus garden's ability to produce staphylae. This may be important during the cyclic production of broods, when demands for larval food will fluctuate widely. Under normal conditions, the optimal strategy may be for the fungus garden to have a moderate rate of production with a low rate of turnover, unless demand for staphylae increases dramatically.     

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.

     

Use of leaf resources by howling monkeys (Alouatta palliata) and leaf-cutting ants (Atta cephalotes) in the tropical rain forest of Los Tuxtlas,Mexico

Use of leaf resources by a troop of howling monkeys and two colonies of leaf cutting ants was studied for an annual cycle in the rain forest of Los Tuxtlas, Mexico. Howling monkeys spent half their annual foraging time feeding on leaves; leaf-cutting ants spent at least 80% of their recorded foraging time harvesting leaves. Both herbivores preferred young leaves over nature ones, and chemical analysis showed that the protein: fibre ratio of the leaves used was correlated with these preferences. Howling monkeys used 34 tree species as leaf sources. Leaf-cutting ants used 40 plant species of which 38 were trees. Eighteen species used by Alouatta were also used by Atta; species of Moraceae and Lauraceae were among the most important in their foraging preferences. The plant species used by monkeys and ants occurred at low densities (? 4.0 ind/ha). The seasonal production of leaves, the high density of leaf-cutting ant colonies at the study site, and the high amounts of young foliage harvested by the ants from tree species, and individual trees used by howling monkeys as sources of young leaves suggest that the foraging activities of Atta may represent a significant pressure upon leaf resources available to Alouatta.     

The farming ant Sericomyrmex amabilis nutritionally manages its fungal symbiont and its social parasite

1. When parasites exploit mutualisms involving food exchange, they can destabilise the partnership with costs to interacting partners. For instance, the ant Sericomyrmex amabilis farms fungal symbionts to produce food, but, in so doing, attracts parasitic Megalomyrmex symmetochus guest ants that infiltrate fungus‐farming ant societies and live with their hosts their entire lives. 2. The present study examined whether host foraging in parasitised colonies shifts towards nutritional requirements of the parasitic guest ants as inferred from the parasite's elemental content (%C, %N, and C:N). 3. Laboratory feeding experiments with nutritionally defined diets indicated that S. amabilis ants harvest protein‐biased substrate, and more total substrate when hosting M. symmetochus relative to when provisioning their fungus gardens and nestmates. 4. Field surveys further showed that parasitised colonies incur reductions in fungus garden nutritional quality and quantity, brood mass, and host worker body condition. And yet these costs appear manageable across growing seasons, as parasitised fungal cultivars appear to provide sufficient nutrition for stable populations of host ants. 5. The approach developed here shows how behavioural strategies for nutrient regulation can extend beyond the needs of the individual to entire fungus‐farming systems, and implies that S. amabilis dynamically adjusts collective foraging strategies when parasitised to enhance long‐term symbiotic stability.     

Fungus gardens of the leafcutter ant Atta colombica function as egg nurseries for the snake Leptodeira annulata

Attine ants are well known for their mutualistic symbiosis with fungus gardens, but many other symbionts and commensals have been described. Here, we report the discovery of two clusters of large snake eggs in neighboring fungus gardens of a mature Atta colombica colony. The eggs were completely embedded within the fungus garden and were ignored by the host ants, even when we placed them into another, freshly excavated fungus garden of the same colony. All five eggs contained embryos and two snakes eventually hatched, which we identified as being banded cat eyed snakes Leptodeira annulata L. Ant fungus gardens are likely to provide ideal climatic conditions for developing snake eggs and almost complete protection from egg predation. Our observations therefore indicate that mature banded cat eyed snakes are able to enter and oviposit in large and well defended Atta colonies without being attacked by ant soldiers and that also newly hatched snakes manage to avoid ant attacks when they leaving their host colony. We speculate that L. annulata might use Atta and Acromyrmex leafcutter ant colonies as egg nurseries by some form of chemical insignificance, but more work is needed to understand the details of this interaction. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Forage collection,substrate preparation,and diet composition in fungus‐growing ants

1. Variation and control of nutritional input is an important selective force in the evolution of mutualistic interactions and may significantly affect coevolutionary modifications in partner species. 2. The attine fungus‐growing ants are a tribe of more than 230 described species (12 genera) that use a variety of different substrates to manure the symbiotic fungus they cultivate inside the nest. Common ‘wisdom’ is that the conspicuous leaf‐cutting ants primarily use freshly cut plant material, whereas most of the other attine species use dry and partly degraded plant material such as leaf litter and caterpillar frass, but systematic comparative studies of actual resource acquisition across the attine ants have not been done. 3. Here we review 179 literature records of diet composition across the extant genera of fungus‐growing ants. The records confirm the dependence of leaf‐cutting ants on fresh vegetation but find that flowers, dry plant debris, seeds (husks), and insect frass are used by all genera, whereas other substrates such as nectar and insect carcasses are only used by some. 4. Diet composition was significantly correlated with ant substrate preparation behaviours before adding forage to the fungus garden, indicating that diet composition and farming practices have co‐evolved. Neither diet nor preparation behaviours changed when a clade within the paleoattine genus Apterostigma shifted from rearing leucocoprinous fungi to cultivating pterulaceous fungi, but the evolutionary derived transition to yeast growing in the Cyphomyrmex rimosus group, which relies almost exclusively on nectar and insect frass, was associated with specific changes in diet composition. 5. The co‐evolutionary transitions in diet composition across the genera of attine ants indicate that fungus‐farming insect societies have the possibility to obtain more optimal fungal crops via artificial selection, analogous to documented practice in human subsistence farming.     

Starch metabolism in Leucoagaricus gongylophorus, the symbiotic fungus of leaf-cutting ants

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 -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 -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. gongylophorus, 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.     

The effect of wilting on the selection of leaves by the leaf-cutting ant Atta laevigata

Using field assays of leaf preference, we tested the hypothesis that wilting affects the selection of leaves by the leaf-cutting ant Atta laevigata (Fr. Smith). Detached leaves were left to air-dry until noticeably wilted. The area removed by the ants from wilted leaves was significantly greater than the area removed from fresh leaves, this effect being observed in several plant species, in leaves of different age, and in assays with different ant colonies. Leaves collected from water-stressed plants were also preferred to leaves from non-stressed plants. A. laevigata was found to employ a two-stage, size-related, strategy when cutting plants. Larger workers climbed the plant stem and dropped whole leaves to the ground by severing their petioles; smaller workers cut the lamina of the dropped leaves. The ants frequently left dropped leaves on the ground, until the next foraging day or even later, when they were harvested in a wilted condition in preference to newly-dropped leaves.It is possible that during wilting some repellent substances evaporate or become less effective, thus enhancing leaf palatability. Alternatively or in addition, changes in nutrient and water content may have rendered wilted leaves more palatable to leaf-cutting ants.     

Specificity and stability of the Acromyrmex–Pseudonocardia symbiosis

The stability of mutualistic interactions is likely to be affected by the genetic diversity of symbionts that compete for the same functional niche. Fungus‐growing (attine) ants have multiple complex symbioses and thus provide ample opportunities to address questions of symbiont specificity and diversity. Among the partners are Actinobacteria of the genus Pseudonocardia that are maintained on the ant cuticle to produce antibiotics, primarily against a fungal parasite of the mutualistic gardens. The symbiosis has been assumed to be a hallmark of evolutionary stability, but this notion has been challenged by culturing and sequencing data indicating an unpredictably high diversity. We used 454 pyrosequencing of 16S rRNA to estimate the diversity of the cuticular bacterial community of the leaf‐cutting ant Acromyrmex echinatior and other fungus‐growing ants from Gamboa, Panama. Both field and laboratory samples of the same colonies were collected, the latter after colonies had been kept under laboratory conditions for up to 10 years. We show that bacterial communities are highly colony‐specific and stable over time. The majority of colonies (25/26) had a single dominant Pseudonocardia strain, and only two strains were found in the Gamboa population across 17 years, confirming an earlier study. The microbial community on newly hatched ants consisted almost exclusively of a single strain of Pseudonocardia while other Actinobacteria were identified on older, foraging ants in varying but usually much lower abundances. These findings are consistent with recent theory predicting that mixtures of antibiotic‐producing bacteria can remain mutualistic when dominated by a single vertically transmitted and resource‐demanding strain.     

β-eudesmol-induced aggression in the leaf-cutting ant Atta sexdens rubropilosa

Leaf‐cutting ants are highly polyphagous insects, but some plants escape their attack due to the presence of secondary metabolites that are toxic to the ant–fungus symbiosis. Previous studies have demonstrated that the terpenoid β‐eudesmol extracted from Eucalyptus species (Myrtaceae) is responsible for the deleterious behavior in colonies of leaf‐cutting ant species. The objective of this study was to evaluate the effect of β‐eudesmol on workers of the leaf‐cutting ant Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae). This chemical caused behavioral modification in the colonies, leading to mutilation and death of workers. It is suggested that β‐eudesmol interferes with colony nestmate recognition. As a consequence, colony cohesion may be disrupted by β‐eudesmol what could be used as an additional control tactic against this important pest ant.     

Humidity preference for fungus culturing by workers of the leaf-cutting ant Atta sexdens rubropilosa

Summary: The hygropreference of gardening workers of the leaf-cutting ant Atta sexdens rubropilosa was investigated in the laboratory using a gradient of relative humidity. Gardening workers were placed, together with pieces of fungus garden, in small, interconnected nest chambers offering four different relative humidities: 33 %, 75 %, 84 % and 98 % RH. Workers were allowed to move freely between them and to relocate the fungus following their humidity preference. While workers distributed themselves randomly in the nest chambers, they located the fungus gardens in the chamber with the highest humidity. These results indicate that gardening workers are able to sense differences in relative humidity, and that this ability is shown when they are engaged in fungus culturing. Humidity is discussed as one of the relevant variables that probably underlay the evolution of regulatory responses for the control of fungus growth in leaf-cutting ants.