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.     

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.     

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.     

The role of the symbiotic fungus in the digestive metabolism of two species of fungus-growing ants

Leaf-cutting ants live in an obligatory symbiosis with a fungus which they grow on fresh leaves harvested by workers. This study attempts to clarify the respective role of ants and fungus in the degradation of plant material, in order to highlight the evolutionary basis of this mutualistic association. The symbiotic system of two ant species, Acromyrmex subterraneus subterraneus and Acromyrmex crassispinus, was investigated. To identify the digestive carbohydrases, a total of 19 specific and synthetic plant material substrates were tested on workers from different castes (major and minor), larvae and fungus. Extracts of A. subterraneus and A. crassispinus workers showed high enzymatic activity particularly on starch, maltose, sucrose and alpha-1,4 glucoside. Larvae degraded starch, sucrose, maltose but also laminarin, and all the detected activities were higher than those found for workers. The symbiotic fungus of A. subterraneus was mostly active on laminarin, xylan and cellulose, while the symbiotic fungus of A. crassispinus was mostly active on laminarin, starch, maltose and sucrose. The enzymatic activities of ants and fungus belonging to the same symbiotic system tended not to overlap, suggesting that the association is highly evolved and of an ancient origin.     

Effects of cycloheximide on the mortality of Atta sexdens leaf-cutting worker ants

Leaf-cutting ants live symbiotically with a fungus that they cultivate on the plant leaves that they cut. The innumerous studies on the plant selection mechanism used by leaf-cutting ants show the researchers’ interest in this issue. Many classical studies propose that plants are selected according to the fungus garden nutritional needs and the absence of potentially harmful substances. This hypothesis is corroborated by behavioral experiments using cycloheximide (fungicide) with citric pulp or forage plants greatly accepted by leaf-cutting ants. According to this hypothesis, under the action of a fungicide, the fungus emits an allomone that informs worker ants that some food is inadequate to its growth. Although some authors state that the cycloheximide “fungicide” used is specific and non toxic to ants, our findings are distinct. In our study, various concentrations of cycloheximide were administered orally to leaf-cutting worker ants in a citric pulp paste diet. After the ingestion period, the ants were isolated and offered the symbiotic fungus for 21 days and the mortality rate was evaluated. As expected, the treatment with 0.01% cycloheximide showed a low mortality rate (8.86%). At 0.1%, the mortality rate was mild (27.85%), and treatment with 1% cycloheximide resulted in moderate mortality (45.57%). In contrast, the positive control with 0.1% sulfluramid showed a high mortality rate (91.14%). Therefore, we concluded that the ingestion of high concentrations of cycloheximide results in a moderate mortality rate in leaf-cutting worker ants.     

Euphorbia cotinifolia (Euphorbiaceae): a promising alternative for leaf cutting ant Atta cephalotes (Hymenoptera: Formicidae) control

Field observations indicate that Euphorbia cotinifolia escapes attack by leaf cutting ants, which are the largest generalist herbivores of the Neotropics. We used controlled bioassays to evaluate the effect of E. cotinifolia on the foraging of the Atta cephalotes ant. In a free-choice trial, to five colonies were offered Mangifera indica leaves with a 10% aqueous E. cotinifolia extract, leaves with distilled water and untreated leaves. The carrying time and leaf area consumed were determined over a five-hour period. The effect of E. cotinifolia on the development of the symbiotic fungus on three sets of five colonies fed the leaves of this plant were compared to the controls fed M. indica and oat flakes, and the effect of the addition of extracts on the culture medium used for the symbiotic fungus isolation was evaluated. Euphorbia leaf consumption was lower than that of the other diets; its consumption as the exclusive foraging resource significantly affected the symbiotic fungus, resulting in changes in colour and texture and an 83.57% decrease in volume that occasionally caused 100% mortality. Although the aqueous extract of E. cotinifolia is not a phagodeterrent for foraging workers, it is evident that E. cotinifolia is not a preferred resource for A. cephalotes due to the negative effect on the growth and viability of the symbiotic fungus.     

Cassia alata and the preclinical search for therapeutic agents for the treatment of opportunistic infections in AIDS patients.

In our search for therapeutic agents from natural sources with potential for the treatment of opportunistic infections in patients afflicted with acquired immunodeficiency syndrome (AIDS), we investigated antibacterial and antifungal activities of water extracts of Cassia alata (C. alata). The extracts are traditionally used in Ivory Coast, West Africa to treat bacterial infections caused by Escherichia coli (E. coli), and fungal infections caused by Candida albicans (C. albicans) and dermatophytes. Our working hypothesis was that the extract contains active ingredient(s) which can be isolated, identified and developed into useful antibacterial/antifungal agents for the treatment of opportunistic infections in patients with AIDS. We used the broth dilution and agar dilution methods. Specifically, we focused on E. coli and C. albicans and the effectiveness of the extracts was evaluated relative to those of standard antibacterial agent chloramphenicol and antifungal agent amphotericin B. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the water extract of C. alata against E. coli were 1.6 mg/ml and 60 mg/ml, respectively; corresponding data for chloramphenicol were 2 micrograms/ml and 10 micrograms/ml. Similarly, the MIC and minimum fungicidal concentration (MFC) for the extract against C. albicans were 0.39 mg/ml and 60 mg/ml in contrast to 0.58 micrograms/ml and 0.98 micrograms/ml for amphotericin B. From the dose-response curve plots, the extract had an IC50 of 31 mg/ml for E. coli and 28 mg/ml for C. albicans. The data suggest that C. alata extracts contain agent(s) which have therapeutic potential and might be useful if isolated and developed for the treatment of opportunistic infections of AIDS patients.     

Cassia alata and the preclinical search for therapeutic agents for the treatment of opportunistic infections in AIDS patients.

In our search for therapeutic agents from natural sources with potential for the treatment of opportunistic infections in patients afflicted with acquired immunodeficiency syndrome (AIDS), we investigated antibacterial and antifungal activities of water extracts of Cassia alata (C. alata). The extracts are traditionally used in Ivory Coast, West Africa to treat bacterial infections caused by Escherichia coli (E. coli), and fungal infections caused by Candida albicans (C. albicans) and dermatophytes. Our working hypothesis was that the extract contains active ingredient(s) which can be isolated, identified and developed into useful antimicrobial/antifungal agents for the treatment of opportunistic infections in patients with AIDS. We used the broth dilution and agar dilution methods. Specifically, we focused on E. coli and C. albicans and the effectiveness of the extracts was evaluated relative to those of standard antibacterial agent chloramphenicol and antifungal agent amphotericin B. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the water extract of C. alata against E. coli were 1.6 mg/ml and 60 mg/ml respectively; corresponding data for chloramphenicol were 2 ug/ml. Similarly, the MIC and minimum fungicidal concentration (MFC) for the extract against C. albicans were 0.39 mg/ml and 60 mg/ml in contrast to 0.58 ug/ml and 0.98 ug/ml for amphotericin B. From the dose-response curve plots, the extract had an IC50 of 31 mg/ml for E. coli and 28 mg/ml for C. albicans. The data suggest that C. alata extracts contain agent(s) which have therapeutic potential and might be useful if isolated and developed for the treatment of opportunistic infections of AIDS patients.     

Evolutionary aspects of ant-fungus interactions in leaf-cutting ants

Leaf-cutting ants are highly successful herbivores because they are able to use a wide variety of plants as food The workers harvest and process plant material to be used as substrate for a fungus on which they feed. New hypotheses concerning the evolution of the ant-fungus relationship have now been proposed. Although the relationship between the ants and the fungus is mutualistic, if may appear that the fungus has little control over the ants. However, evidence suggests that the fungus may be exploiting the ants to provide it with substrate and antimicrobial defence. Furthermore, experimental evidence suggests that the fungus can select its substrate by controlling the foraging behaviour of the ants, by means of an ingenious chemical feedback mechanism.     

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.     

Individual complexity and self-organization in foraging by leaf-cutting ants

Leaf-cutting ants cut vegetation into small fragments that they transport to the nest, where a symbiotic fungus cultivated by the ants processes the material. Since the harvested leaf fragments are incorporated into the fungus garden and not directly consumed by the workers, it is expected that foraging workers select plants by responding to those physical or chemical traits that promote maximal fungal growth, irrespective of the potential direct effects of these leaf features on them. In this paper I summarize experimental work focusing on the decision-making processes that occur at the individual level, and discuss to what extent individual complexity contributes to the emergence of collective foraging patterns. Although some basic features of self-organizing systems, such as the existence of regulatory positive and negative feedback loops, are expected to be involved in the collective organization of leaf-cutting ant foraging, I contend that they are combined with complex individual responses that may result from the integration of local information during food collection with an assessment of colony conditions.     

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.     

Leaf-cutting ants and avoided plants: Defences against Atta texana attack

Summary Leaf-cutting ants (Formicidae; Attini) characteristically never attack some common plant species in their habitats. These plants may be defended against the ants in several ways. In Texas, mature leaves of Sapindus saponaria (Sapindaceae) and Celtis reticulata (Ulmaceae) are unpalatable to Atta texana Buckley foragers, while mature leaves of Berberis trifoliata (Berberidaceae) are palatable to the ants, but are too tough to cut. Young Celtis leaves and and young Berberis leaves are palatable and can be cut by the ants, however. These young leaves may escape attack by remaining palatable a brief amount of time (new Celtis leaves), or by occurring patchily in space and time (new Berberis leaves).     

Mycelial growth inhibition of plant pathogenic fungi by extracts of South African plant species

Eleven plant species were collected from the Blyde River Canyon area, Mpumalanga, South Africa. Methanolic crude extracts of the plant samples were concentrated to dryness, dissolved in sterile distilled water and bioassayed in the laboratory for their fungicidal potential against seven plant fungal pathogens, namely Botrytis cinerea, Fusarium oxysporum, Sclerotium rolfsii, Rhizoctonia solani, Verticillium dahliae, Botryosphaeria dothidea and Pythium ultimum, at equal concentrations. A modified agar dilution method was used to determine the inhibitory effect of the plant extracts on the mycelial radial growth of the plant pathogens. Antifungal activity was confirmed in all the plants, but not in all plant parts and also not against all the test organisms. The crude extract of Eucomis autumnalis performed best of all the plant extracts tested, as it showed significant antifungal activity against all seven of the plant pathogenic test organisms and compared favourably to the inhibition of the mycelial growth by a broad spectrum synthetic fungicide (carbendazim/ difenoconazole). The crude extract of Schrebera alata came out second best, in the sense that at least one plant part inhibited the mycelial radial growth of four of the seven test organisms by at least 50%.     

Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, to determine oral acute toxicity of plant extracts

Artemia salina L. (Artemiidae), the brine shrimp larva, is an invertebrate used in the alternative test to determine toxicity of chemical and natural products. In this study the Medium Lethal Concentrations (LC50 value) of 20 plant extracts, Aloe vera (L.) Burm. F. (Aloeaceae), Artemisia absinthium L. (Asteraceae); Citrus aurantium L. (Rutaceae); Cymbopogon citratus (DC. Ex Nees) Stapf (Poaceae); Datura stramonium L. (Solanaceae); Justicia pectoralis Jacq. (Acanthaceae); Musa x paradisiaca L. (Musaceae); Ocimum basilicum L.; O. gratissimum L.; O. tenuiflorum L. (Lamiaceae); Pimenta dioica (L.) Merr. (Myrtaceae); Piper auritum Kunth (Piperaceae); Plantago major L. (Plantaginaceae); Plectranthus amboinicus (Lour.) Spreng. (Lamiaceae); Ruta graveolens L. (Rutaceae); Senna alata (L.) Roxb. (Fabaceae); Stachytarpheta jamaicensis (L.) Vahl (Verbenaceae); and Thuja occidentalis L. (Cupressaceae), were determined using Artemia salina L. (Artemiidae), with the objective of relating the results to the LD50 values reported in mice (tested at three concentrations: 10, 100, and 1000 microg/mL, for each extract). We found good correlation between the in vivo and the in vitro tests (r = 0.85 p < 0.05), and this method is a useful tool for predicting oral acute toxicity in plant extracts.     

A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for >60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in ～30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds.     

Interactions among leaf toughness, chemistry, and harvesting by attine ants

Abstract. 1. Young and mature leaves of a tropical legume, Inga edulis var. minutula Schery, are strikingly different in secondary chemistry, especially condensed tannins, and leaf toughness.
2. Bioassays with the two different leaf types indicate that leaf cutter ants, Atta cephalotes (L.), always find mature leaves relatively more acceptable than young leaves when selection was based on chemical cues.
3. Since extracts of young leaves show greziter inhibition of fungal pectinases we suggest that leaf-cutter ants are capable of distinguishing which leaf types are most suitable for the growth of their symbiotic fungus.
4. However, mature leaves are 3 times tougher than young leaves and this prevents leaf-cutter ants from harvesting the more suitable mature leaves.
5. Consequently, bioassays which require cutting before leaf removal indicate that some colonies actually harvest more from the less suitable young leaves.
6. We suggest that the quality of a colony's habitat may indicate whether a colony will harvest more of the less suitable young leaves. Colonies which are harvesting from highly suitable host plants avoid the tropical legume I.edulis while those in poorer habitats accept I.edulis but, because of leaf toughness, mostly harvest the less suitable young leaves.     

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.     

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 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.