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.     

Communication between the fungus garden and workers of the leaf-cutting ant, Atta sexdens rubropilosa, regarding choice of substrate for the fungus

Bait made from orange peel, containing the fungicide cycloheximide, was initially harvested by workers of Atta sexdens rubropilosa (Forel) and incorporated into the fungus garden as substrate for the fungus. The bait was subsequently rejected by the worker ants days later. Exposure of the fungus to cycloheximide, in laboratory sub-colonies, resulted in the fungus being ‘stressed’. By interchanging normal fungus garden with ‘stressed’ fungus garden, a change in the foraging behaviour of the workers was evident. –Two hypotheses to explain the behavioural changes were tested: a volatile semiochemical is produced by the fungus which affects the foragers directly, or contact between workers (and fungus garden) is necessary for information regarding fungal substrate to be transmitted through the worker force. When pairs of sub-colonies were connected (one colony of each pair exposed to cycloheximide in the bait) and workers were initially prevented from passing from one colony to the other, one colony continued to forage on orange bait while the other did not. When both colonies were allowed to make full contact then both colonies failed to accept orange bait. This discounted the first hypothesis, but supported the second, as a highly volatile chemical should be able to diffuse between colonies. When large foragers were prevented from making contact with the second colony, the information may be communicated by smaller workers.     

The epizootiology of a Metarhizium infection in mini-nests of the leaf-cutting ant Atta sexdens rubropilosa

There is growing interest in the use of entomopathogenic organisms to control leaf-cutting ants (Hymenoptera: Formicidae: Attini). However, the way leaf-cutting ants react as a colony to biohazards is poorly understood. We investigated the effects of Metarhizium anisopliae (Metschnikoff) (Deuteromycotina: Hyphomycetes) applied to the foraging arenas of mini-nests (queenless sub-colonies) of the leaf-cutting ant Atta sexdens rubropilosa (Forel). Dry spores were applied either alone or mixed with citrus powder, at 0.5 g or 0.05 g per mini-nest. The spores were removed four days after application, and all dead ants removed every three days. Ant numbers near the Metarhizium increased as the ants attempted to clean up the biohazard. The ants attempted to place the spores in piles, which they then covered over with other material. They were able to deal with the low doses in this way, but the high doses overwhelmed them. All treated mini-nests suffered increased ant mortality during the first ten days after application. This mortality was particularly high in the media worker caste which had played the major role in attempting to clean up the spores. Foraging activity decreased, as did the health of the fungus gardens. The mini-nests exposed to the low dose of spores mixed with citrus powder then recovered fully. The health of the other treated mini-nests declined gradually until around 26 days after application, when they began deteriorating sharply. However, the decline of these mini-nests after day 26 was not due directly to the pathogenic action of the Metarhizium, nor to the initial ant mortality it had caused. The results suggest that the social stress caused by even such a short-lived Metarhizium epizootic was sufficient to cause the decline and ultimate death of the mini-nests. This has important implications for the control of leaf-cutting ants. It also demonstrates how important the social homeostasis of the colony is to leaf-cutting ants.     

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.     

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.     

The role of fungus in the diet of the leaf-cutting ant Atta cephalotes (L.)

Abstract. 1. The workers and queen of the leaf-cutting ant Atta cephalotes fed on the juice of swollen hyphae (staphylae) produced by their cultivated fungus, but neither obtained sufficient energy from this source for their respiratory needs. The number of staphylae eaten by workers increased with worker size but was not enough to satisfy their energy requirements.
2. Larvae fed on whole staphylae and staphylae previously chewed by workers, and obtained sufficient energy from this source for respiration and growth. No evidence of feeding on fungus hyphae or of trophallaxis between worker and larvae was found. Larvae preferred staphylae to hyphae when fed them artificially and they gained more weight on the former.
3. Worker ants imbibed plant sap during the preparation of plant material for the fungus garden and the uptake of carbohydrate during this process was sufficient to supply their energy needs for approximately 24 h.
4. Staphylae were richer in lipid and carbohydrate, and poorer in protein than ant fungal hyphae.
5. The number of staphylae produced by the fungus gardens of two small nests was comparable with the observed consumption rate but would provide only about 4% of the nest's respiratory requirements.
6. In the light of these findings, a revised view of the role of the fungus in the diet of the ant is discussed.     

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.

     

Symbiotic bacteria on the cuticle of the leaf-cutting ant Acromyrmex subterraneus subterraneus protect workers from attack by entomopathogenic fungi

Although only discovered in 1999, the symbiotic filamentous actinobacteria present on the integument of certain species of leaf-cutting ants have been the subject of intense research. These bacteria have been shown to specifically suppress fungal garden parasites by secretion of antibiotics. However, more recently, a wider role for these bacteria has been suggested from research revealing their generalist anti-fungal activity. Here we show, for the first time, evidence for a role of these bacteria in the defence of young worker ants against a fungal entomopathogen. Experimental removal of the bacterial bio-film using an antibiotic resulted in a significant increase in susceptibility of worker ants to infection by the entomopathogenic fungus Metarhizium anisopliae. This is the first direct evidence for the advantage of maintaining a bacterial bio-film on the cuticle as a defensive strategy of the ants themselves and not exclusively for protection of the fungus garden.     

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.     

Substrate distribution in fungus chambers in nests of Atta bisphaerica Forel, 1908 (Hym., Formicidae)

Abstract:  Substrate distribution was studied in three adult colonies of the leaf-cutting ant Atta bisphaerica Forel, 1908 using dye. Some supply holes were mapped using baits made from small plastic straws impregnated with citric pulp. Two holes, equidistant from one another, were then selected in each nest. The colonies were excavated completely 24 h after bait placement. During excavation, fungus chambers were checked for dye. We observed that the dyed baits were distributed in all sectors and at all depths regardless of where the baits had been placed. This supported the hypothesis that toxic baits placed in a single supply hole are uniformly distributed throughout the colony.     

Do Leaf Cutting Ants Cut Undetected? Testing the Effect of Ant-Induced Plant Defences on Foraging Decisions in Atta colombica

Leaf-cutting ants (LCAs) are polyphagous, yet highly selective herbivores. The factors that govern their selection of food plants, however, remain poorly understood. We hypothesized that the induction of anti-herbivore defences by attacked food plants, which are toxic to either ants or their mutualistic fungus, should significantly affect the ants' foraging behaviour. To test this "induced defence hypothesis," we used lima bean (Phaseolus lunatus), a plant that emits many volatile organic compounds (VOCs) upon herbivore attack with known anti-fungal or ant-repellent effects. Our results provide three important insights into the foraging ecology of LCAs. First, leaf-cutting by Atta ants can induce plant defences: Lima bean plants that were repeatedly exposed to foraging workers of Atta colombica over a period of three days emitted significantly more VOCs than undamaged control plants. Second, the level to which a plant has induced its anti-herbivore defences can affect the LCAs' foraging behaviour: In dual choice bioassays, foragers discriminated control plants from plants that have been damaged mechanically or by LCAs 24 h ago. In contrast, strong induction levels of plants after treatment with the plant hormone jasmonic acid or three days of LCA feeding strongly repelled LCA foragers relative to undamaged control plants. Third, the LCA-specific mode of damaging leaves allows them to remove larger quantities of leaf material before being recognized by the plant: While leaf loss of approximately 15% due to a chewing herbivore (coccinelid beetle) was sufficient to significantly increase VOC emission levels after 24 h, the removal of even 20% of a plant's leaf area within 20 min by LCAs did not affect its VOC emission rate after 24 h. Taken together, our results support the "induced defence hypothesis" and provide first empirical evidence that the foraging behaviour of LCAs is affected by the induction of plant defence responses.     

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.     

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.     

Isolation, Growth Characteristics, and Long-Term Storage of Fungi Cultivated by Attine Ants

Seven pure-culture strains of fungi cultivated by attine ants (ant-garden fungi) were isolated from locally maintained leaf-cutting ant colonies. An ant-garden fungus strain obtained from an Atta cephalotes colony, when offered to ants of the colony from which the fungus was isolated, was accepted as their own. Young fungus cultures were harvested and incorporated into the fungus garden, and cultures of intermediate age were used to begin a new fungus garden; old cultures were simply harvested. To facilitate further research on this fungus, growth characteristics of the different isolates were studied under a variety of conditions. They grew better at 24°C than at 30°C, and growth did not occur at an incubation temperature of 37°C. In a broth culture medium, growth was enhanced by aeration of the culture and by addition of yeast extract, olive oil, sesame oil, peanut oil, soybean oil, corn oil, sunflower oil, cottonseed oil, walnut oil, safflower oil, or mineral oil. Glycerol did not noticeably affect growth, but Tween 80 inhibited growth. These fungi were extremely sensitive to cycloheximide, growth being totally inhibited at cycloheximide concentrations ranging from 0.4 to 4.0 μg/ml. To date, the ant-garden fungus isolates have remained viable in long-term mineral oil-overlay storage cultures for up to 4 years.     

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.     

Acromyrmex octospinosus (Hymenoptera: Formicidae) management: effects of TRAMILs fungicidal plant extracts

Leaf-cutting ants, Acromyrmex octospinosus (Reich), are considering among the most important pest species of the New World. Until now, the main insecticides used for controlling these ants were synthetic chemicals. Leaf-cutting ants live in obligate symbiosis with abasidiomycete fungus, Leucocoprinus gongylophorus (Heim) Moeller. The crucial role of this symbiotic partner in the nest of leaf-cutting ants has prompted us to focus on A. octospinosus management through the use of fungicides in our study. Five parts of plants identified for their antifungal potential through TRAMIL ethnopharmacological surveys were tested: 1) bulbs of Allium cepa L.; 2) seed pods of Allium sativum L.; 3) green fruits of Lycopersicon esculentum L.; 4) leaves of Manihot esculenta Crantz; and 5) leaves of Senna alata (L.) Roxburgh. One plant extract with strong fungicidal activity (S. alata) against L. gongylophorus was found. The other extracts had lesser fungistatic or fungicidal effects depending on the concentrations used. The data presented in this study showed that TRAMILs fungicidal plant extracts have potential to control the symbiotic fungus of leaf cutting ants, in particular a foliage extract of S. alata.     

Observation of leaf-cutting ants foraging on wild mushrooms

This study reports on the observation of an unusual behavior in leaf-cutting ants: foraging on wild mushrooms. A colony of Acromyrmex lundi in Buenos Aires (Argentina) was observed intensively harvesting basidiomes (mushroom fructifications) of wild Agrocybe fungus developing on a tree bark. Another colony maintained for a month in laboratory conditions also accepted Agrocybe mushroom and incorporated the cut bits into the fungus garden in the same way as they do with leaves. We recorded these events confident that they open a new perspective on the study of the feeding habits of leaf-cutting ants as well as on the relationship between their fungus garden and other organisms.     

Spatial and Temporal Variation of Plant Fragment Removal by Two Species of <Emphasis Type="Italic">Atta</Emphasis> Leaf-Cutting Ants

Leaf-cutting ants are responsible for much of living plant fragment removal in Neotropical regions, causing major damage in both in natural and agricultural systems. To understand the variation in plant fragment removal by leaf-cutting ants and the influence that temporal and spatial factors have on such variation, we evaluated plant fragments collected by two ant species, Atta laevigata and A. sexdens rubropilosa in different seasons (rainy and dry) and in different forest systems (natural area of cerrado sensu stricto and monoculture of Eucalyptus sp.) in 2014 in Goianésia, Goiás, Brazil. Heavier plant fragments were removed during the dry season and in monoculture areas; A. laevigata removed heavier fragments than A. sexdens rubropilosa. Furthermore, we observed a positive association between the weight of collected fragments and the weight of worker ants and higher weights in A. laevigata. The study shows that the weight of plant fragments removed by leaf-cutting ants is conditioned temporally and spatially, and by the ant species involved. More detailed studies of each of these factors are need to better understand the dynamics of these foraging ants and how abiotic and biotic factors affect this process.     

Regulation and specificity of antifungal metapleural gland secretion in leaf-cutting ants

Ants have paired metapleural glands (MGs) to produce secretions for prophylactic hygiene. These exocrine glands are particularly well developed in leaf-cutting ants, but whether the ants can actively regulate MG secretion is unknown. In a set of controlled experiments using conidia of five fungi, we show that the ants adjust the amount of MG secretion to the virulence of the fungus with which they are infected. We further applied fixed volumes of MG secretion of ants challenged with constant conidia doses to agar mats of the same fungal species. This showed that inhibition halos were significantly larger for ants challenged with virulent and mild pathogens/weeds than for controls and Escovopsis-challenged ants. We conclude that the MG defence system of leaf-cutting ants has characteristics reminiscent of an additional cuticular immune system, with specific and non-specific components, of which some are constitutive and others induced.