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Fungal symbionts that live asymptomatically inside plant tissues (endophytes) can influence plant–insect interactions. Recent work has shown that damage by leaf-cutting ants, a major Neotropical defoliator, is reduced to almost half in plants with high densities of endophytes. We investigated changes in the phenotype of leaves that could influence ants' behavior to result in the reduction of foliar damage. We produced cucumber seedlings with high and low densities of one common endophyte species, Colletotrichum tropicale. We used the leaves in bioassays and to compare chemical and physical leaf characteristics important for ants' food selection. Ants cut about one-third more area of cucumber leaves with lower densities of endophytes and removed c. 20% more paper disks impregnated with the extracts of those leaves compared with leaves and disks from plants hosting the fungus. Colletotrichum tropicale colonization did not cause detectable changes in the composition of volatile compounds, cuticular waxes, nutrients or leaf toughness. Our study shows that endophytes changed leaf chemistry and suggests that compounds with relative low volatility released after leaf wounding are a major factor influencing foraging decisions by ants when choosing between plants with low or high endophyte loads.

In all stages of their life cycle, insects are threatened by a multitude of predators, parasites, parasitoids and pathogens. The lifestyles and feeding ecologies of some hymenopteran taxa render them especially vulnerable to pathogen infestation. Specifically, development in sub‐terranean brood cells, mass provisioning of resources for the offspring and the life of social insects in large communities can enhance the risk of pathogen infestation and/or the spread of disease among conspecifics. To counteract these threats, insects have evolved mechanical, chemical and behavioural defences as well as a complex immune system. In addition to the host's own defences, some Hymenoptera are associated with protective symbionts. Leaf‐cutting ants, solitary digger wasps, bees and bumblebees engage in symbiotic interactions with bacteria that protect the adult host, the developing offspring or the food resources against microbial infections. In the well‐studied cases of ants and wasps, the protective activity is mediated by the production of antimicrobial secondary metabolites. In other symbiotic interactions, however, competitive exclusion and immune priming may also play an important role in enhancing protection. Phylogenetic studies indicate that the defensive associations in Hymenoptera are generally more dynamic than the intimate nutritional mutualisms, with horizontal transfer or de novo uptake of the symbionts from the environment occurring frequently. Mutualistic micro‐organisms can also significantly influence the outcome of host‐parasitoid interactions. Some insects are protected by symbiont‐produced toxins against parasitic wasps. Ichneumonid and braconid parasitoids, on the other hand, are associated with symbiotic viruses that are injected into the caterpillar host during oviposition and suppress its immune system to the advantage of the parasitoid. The increasing affordability of next‐generation sequencing technologies will greatly facilitate the analysis of insect‐associated microbial communities and undoubtedly uncover a plethora of as yet unknown protective symbioses. However, a detailed understanding of the host's natural history is indispensable for elucidating the fitness benefits of the symbionts and the molecular basis of symbiont‐conferred protection.

Leaf-cutting ants combine large-scale herbivory with fungus farming to sustain advanced societies. Their stratified colonies are major evolutionary achievements and serious agricultural pests, but the crucial adaptations that allowed this mutualism to become the prime herbivorous component of neotropical ecosystems has remained elusive. Here we show how coevolutionary adaptation of a specific enzyme in the fungal symbiont has helped leaf-cutting ants overcome plant defensive phenolic compounds. We identify nine putative laccase-coding genes in the fungal genome of Leucocoprinus gongylophorus cultivated by the leaf-cutting ant Acromyrmex echinatior . One of these laccases (LgLcc1) is highly expressed in the specialized hyphal tips (gongylidia) that the ants preferentially eat, and we confirm that these ingested laccase molecules pass through the ant guts and remain active when defecated on the leaf pulp that the ants add to their gardens. This accurate deposition ensures that laccase activity is highest where new leaf material enters the fungus garden, but where fungal mycelium is too sparse to produce extracellular enzymes in sufficient quantities to detoxify phenolic compounds. Phylogenetic analysis of LgLcc1 ortholog sequences from symbiotic and free-living fungi revealed significant positive selection in the ancestral lineage that gave rise to the gongylidia-producing symbionts of leaf-cutting ants and their non–leaf-cutting ant sister group. Our results are consistent with fungal preadaptation and subsequent modification of a particular laccase enzyme for the detoxification of secondary plant compounds during the transition to active herbivory in the ancestor of leaf-cutting ants between 8 and 12 Mya.

Leaf-cutting ants such as Acromyrmex octospinosus live in obligate symbiosis with fungi of the genus Leucoagaricus, which they grow with harvested leaf material. The symbiotic fungi, in turn, serve as a major food source for the ants. This mutualistic relation is disturbed by the specialized pathogenic fungus Escovopsis sp., which can overcome Leucoagaricus sp. and thus destroy the ant colony. Microbial symbionts of leaf-cutting ants have been suggested to protect the fungus garden against Escovopsis by producing antifungal compounds [Currie CR, Scott JA, Summerbell RC, Malloch D (1999) Fungus-growing ants use antibiotic-producing bacteria to control garden parasites. Nature 398:701-704.]. To date, however, the chemical nature of these compounds has remained elusive. We characterized 19 leaf-cutting ant-associated microorganisms (5 Pseudonocardia, 1 Dermacoccus, and 13 Streptomyces) from 3 Acromyrmex species, A. octospinosus, A. echinatior, and A. volcanus, using 16S-rDNA analysis. Because the strain Streptomyces sp. Ao10 proved highly active against the pathogen Escovopsis, we identified the molecular basis of its antifungal activity. Using bioassay-guided fractionation, high-resolution electrospray mass spectrometry (HR-ESI-MS), and UV spectroscopy, and comparing the results with an authentic standard, we were able identify candicidin macrolides. Candicidin macrolides are highly active against Escovopsis but do not significantly affect the growth of the symbiotic fungus. At least one of the microbial isolates from each of the 3 leaf-cutting ant species analyzed produced candicidin macrolides. This suggests that candicidins play an important role in protecting the fungus gardens of leaf-cutting ants against pathogenic fungi.

Understanding the relative evolutionary importance of parasites to different host taxa is problematic because the expression of disease and resistance are often confounded by factors such as host age and condition. The antibiotic-producing metapleural glands of ants are a potentially useful exception to this rule because they are a key first-line defense that are fixed in size in adults. Here we conduct a comparative analysis of the size of the gland reservoir across the fungus-growing ants (tribe Attini). Most attines have singly mated queens, but in two derived genera, the leaf-cutting ants, the queens are multiply mated, which is hypothesized to have evolved to improve colony-level disease resistance. We found that, relative to body size, the gland reservoirs of most attines are similar in size but that those of the leaf-cutting ants are significantly larger. In contrast, the size of the reservoir did not relate with the evolutionary transition from lower to higher attines and correlated at most only slightly with colony size. The results thus suggest that the relationship between leaf-cutting ants and their parasites is distinctly different from that for other attine ants, in accord with the hypothesis that multiple mating by queens evolved to improve colony-level disease resistance.

Leaf-cutting ants cultivate the fungus Leucoagaricus gongylophorus, which serves as a major food source. This symbiosis is threatened by microbial pathogens that can severely infect L. gongylophorus. Microbial symbionts of leaf-cutting ants, mainly Pseudonocardia and Streptomyces, support the ants in defending their fungus gardens against infections by supplying antimicrobial and antifungal compounds. The ecological role of microorganisms in the nests of leaf-cutting ants can only be addressed in detail if their secondary metabolites are known. Here, we use an approach for the rapid identification of established bioactive compounds from microorganisms in ecological contexts by combining phylogenetic data, database searches, and liquid chromatography electrospray ionisation high resolution mass spectrometry (LC-ESI-HR-MS) screening. Antimycins A₁-A₄, valinomycins, and actinomycins were identified in this manner from Streptomyces symbionts of leaf-cutting ants. Matrix-assisted laser desorption ionization (MALDI) imaging revealed the distribution of valinomycin directly on the integument of Acromyrmex echinatior workers. Valinomycins and actinomycins were also directly identified in samples from the waste of A. echinatior and A. niger leaf-cutting ants, suggesting that the compounds exert their antimicrobial and antifungal potential in the nests of leaf-cutting ants. Strong synergistic effects of the secondary meta-bolites produced by ant-associated Streptomyces were observed in the agar diffusion assay against Escovopsis weberi. Actinomycins strongly inhibit soil bacteria as well as other Streptomyces and Pseudonocardia symbionts. The antifungal antimycins are not only active against pathogenic fungi but also the garden fungus L. gongylophorus itself. In conclusion, secondary metabolites of microbial symbionts of leaf-cutting ants contribute to shaping the microbial communities within the nests of leaf-cutting ants.

The design of transport paths in consuming entities that use routes to access food should be under strong selective pressures to reduce costs and increase benefits. We studied the adaptive nature of branching angles in foraging trail networks of the two most abundant tropical leaf-cutting ant species. We mathematically assessed how these angles should reflect the relative weight of the pressure for reducing either trail maintenance effort or traveling distances. Bifurcation angles of ant foraging trails strongly differed depending on the location of the nests. Ant colonies in open areas showed more acute branching angles, which best shorten travel distances but create longer new trail sections to maintain than a perpendicular branch, suggesting that trail maintenance costs are smaller compared to the benefit of reduced traveling distance. Conversely, ant colonies in forest showed less acute branching angles, indicating that maintenance costs are of larger importance relative to the benefits of shortening travel distances. The trail pattern evident in forests may be attributable to huge amounts of litterfall that increase trail maintenance costs, and the abundant canopy cover that reduces traveling costs by mitigating direct sunlight and rain. These results suggest that branching angles represent a trade-off between reducing maintenance work and shortening travel distances, illustrating how animal constructions can adjust to diverse environmental conditions. This idea may help to understand diverse networks systems, including urban travel networks.

Fungus garden material from recently established Atta sexdens rubropilosa colonies (6–12 months old) was sampled to detect antibiotic producing microorganisms that inhibited the growth of pathogens of insects and of the fungus gardens but did not affect their mutualistic fungus. A bacterium with activity against the entomopathogenic fungus Beauveria bassiana was isolated from 56% of the gardens tested ( n = 57) and identified from its biochemical profile and from 16S and 23S ribosomal DNA sequences as a member of the genus Burkholderia. The ant-associated Burkholderia isolates secreted a potent, anti-fungal agent that inhibited germination of conidia of the entomopathogenic fungi B. bassiana, Metarhizium anisopliae, of the saprophytic Verticillium lecanii, and also of a specialist fungus garden Escovopsis weberi. Growth of the ant’s mutualist fungus was unaffected.

Chemical communication is a dominant method of communication throughout the animal kingdom and can be especially important in group-living animals in which communicating threats, either from predation or other dangers, can have large impacts on group survival. Social insects, in particular, have evolved a number of pheromonal compounds specifically to signal alarm. There is predicted to be little selection for interspecific variation in alarm cues because individuals may benefit from recognizing interspecific as well as conspecific cues and, consequently, alarm cues are not normally thought to be used for species or nestmate recognition. Here, we examine the composition of the alarm pheromones of seven species of fungus-growing ants (Attini), including both basal and derived species and examine the behavioral responses to alarm pheromone of Acromyrmex leaf-cutting ants, the sister genus to the highly studied Atta leaf-cutting ants. We find surprisingly high interspecific variation in alarm pheromone composition across the attine phylogeny. Interestingly, the active component of the alarm pheromone was different between the two leaf-cutting ant genera. Furthermore, in contrast to previous studies on Atta , we found no differences among morphological castes in their responses to alarm pheromone in Acromyrmex but we did find differences in responses among putative age classes. The results suggest that the evolution of alarm communication and signaling within social insect clades can be unexpectedly complex and that further work is warranted to understand whether the evolution of different alarm pheromone compounds is adaptive.