Attine Gardens Research Articles

Broad Escovopsis-inhibition activity of Pseudonocardia associated with Trachymyrmex ants.

Attine ants maintain an association with antibiotic-producing Actinobacteria found on their integuments. Evidence supports these bacteria as auxiliary symbionts that help ants to defend the fungus gardens against pathogens. Using Pseudonocardia strains isolated from Trachymyrmex ants, we tested whether the inhibitory capabilities of such strains are restricted to Escovopsis parasites that infect gardens of this ant genus. Twelve Pseudonocardia strains were tested in in vitro bioassays against Escovopsis strains derived from fungus gardens of Trachymyrmex (n = 1) and leaf-cutting ants (n = 3). Overall, significant differences were observed in the mycelial growth among each Escovopsis strain in the presence of Pseudonocardia. Particularly, Escovopsis from Acromyrmex and Trachymyrmex were the most inhibited strains in comparison to Escovopsis isolated from Atta. This result suggests that Pseudonocardia isolated from Trachymyrmex possibly secrete antimicrobial compounds effective against diverse Escovopsis strains. The fact that Trachymyrmex ants harbour Pseudonocardia strains with broad spectrum of activity and its defensive role on attine gardens are discussed.

Ecology of microfungal communities in gardens of fungus-growing ants (Hymenoptera: Formicidae): a year-long survey of three species of attine ants in Central Texas

We profiled the microfungal communities in gardens of fungus-growing ants to evaluate possible species-specific ant-microfungal associations and to assess the potential dependencies of microfungal diversity on ant foraging behavior. In a 1-year survey, we isolated microfungi from nests of Cyphomyrmex wheeleri, Trachymyrmex septentrionalis and Atta texana in Central Texas. Microfungal prevalence was higher in gardens of C. wheeleri (57%) than in the gardens of T. septentrionalis (46%) and A. texana (35%). Culture-dependent methods coupled with a polyphasic approach of species identification revealed diverse and changing microfungal communities in all the sampling periods. Diversity analyses showed no obvious correlations between the number of observed microfungal species, ant species, or the ants' changing foraging behavior across the seasons. However, both correspondence analysis and 5.8S-rRNA gene unifrac analyses suggested structuring of microfungal communities by ant host. These host-specific differences may reflect in part the three different environments where ants were collected. Most interestingly, the specialized fungal parasite Escovopsis was not isolated from any attine garden in this study near the northernmost limit of the range of attine ants, contrasting with previous studies that indicated a significant incidence of this parasite in ant gardens from Central and South America. The observed differences of microfungal communities in attine gardens suggest that the ants are continuously in contact with a diverse microfungal species assemblage.

Evolutionary patterns of proteinase activity in attine ant fungus gardens.

BackgroundAttine ants live in symbiosis with a basidiomycetous fungus that they rear on a substrate of plant material. This indirect herbivory implies that the symbiosis is likely to be nitrogen deprived, so that specific mechanisms may have evolved to enhance protein availability. We therefore hypothesized that fungal proteinase activity may have been under selection for efficiency and that different classes of proteinases might be involved.ResultsWe determined proteinase activity profiles across a wide pH range for fungus gardens of 14 Panamanian species of fungus-growing ants, representing eight genera. We mapped these activity profiles on an independently obtained molecular phylogeny of the symbionts and show that total proteinase activity in lower attine symbionts peaks at ca. pH 6. The higher attine symbionts that have no known free-living relatives had much higher proteinase activities than the lower attine symbionts. Their total in vitro proteinase activity peaked at pH values around 5, which is close to the pH that the ants maintain in their fungus gardens, suggesting that the pH optimum of fungal proteinases may have changed after the irreversible domestication of evolutionary more derived fungal symbionts. This notion is also supported by buffering capacities of fungus gardens at pH 5.2 being remarkably high, and suggests that the fungal symbiont actively helps to maintain garden acidity at this specific level. Metalloproteinases dominated the activity profiles of lower attine gardens and may thus represent the ancestral type of proteinase production, whereas serine proteinase activity dominated the activity profiles of the higher attine gardens reared by Trachymyrmex and Sericomyrmex, suggesting that there may be trade-offs in the production of these enzyme classes. Remarkably, the single symbiont that is shared by species of the crown group of Atta and Acromyrmex leaf-cutting ants mostly showed metalloproteinase activity, suggesting that recurrent changes in enzyme production may have occurred throughout the domestication history of fungus-garden symbionts.ConclusionsProteinase pH optima and buffering capacities of fungal symbionts appear to have evolved remarkable adaptations to living in obligate symbiosis with farming ants. Although the functional roles of serine and metalloproteinases in fungus gardens are unknown, the differential production of these classes of proteolytic enzymes suggest that substrate specificity may be important and that trade-offs may prevent the simultaneous upregulation of both classes of enzymes.

COEVOLUTION BETWEEN ATTINE ANTS AND ACTINOMYCETE BACTERIA: A REEVALUATION

We reassess the coevolution between actinomycete bacteria and fungus-gardening (attine) ants. Actinomycete bacteria are of special interest because they are metabolic mutualists of diverse organisms (e.g., in nitrogen-fixation or antibiotic production) and because Pseudonocardia actinomycetes are thought to serve disease-suppressing functions in attine gardens. Phylogenetic information from culture-dependent and culture-independent microbial surveys reveals (1) close affinities between free-living and ant-associated Pseudonocardia, and (2) essentially no topological correspondence between ant and Pseudonocardia phylogenies, indicating frequent bacterial acquisition from environmental sources. Identity of ant-associated Pseudonocardia and isolates from soil and plants implicates these environments as sources from which attine ants acquire Pseudonocardia. Close relatives of Atta leafcutter ants have abundant Pseudonocardia, but Pseudonocardia in Atta is rare and appears at the level of environmental contamination. In contrast, actinomycete bacteria in the genera Mycobacterium and Microbacterium can be readily isolated from gardens and starter-cultures of Atta. The accumulated phylogenetic evidence is inconsistent with prevailing views of specific coevolution between Pseudonocardia, attine ants, and garden diseases. Because of frequent acquisition, current models of Pseudonocardia-disease coevolution now need to be revised. The effectiveness of Pseudonocardia antibiotics may not derive from advantages in the coevolutionary arms race with specialized garden diseases, as currently believed, but from frequent recruitment of effective microbes from environmental sources. Indeed, the exposed integumental structures that support actinomycete growth on attine ants argue for a morphological design facilitating bacterial recruitment. We review the accumulated evidence that attine ants have undergone modifications in association with actinomycete bacteria, but we find insufficient support for the reverse, modifications of the bacteria resulting from the interaction with attine ants. The defining feature of coevolution--reciprocal modification--therefore remains to be established for the attine ant-actinomycete mutualism.

Microfungal “Weeds” in the Leafcutter Ant Symbiosis

Leafcutter ants (Formicidae: tribe Attini) are well-known insects that cultivate basidiomycete fungi (Agaricales: Lepiotaceae) as their principal food. Fungus gardens are monocultures of a single cultivar strain, but they also harbor a diverse assemblage of additional microbes with largely unknown roles in the symbiosis. Cultivar-attacking microfungi in the genus Escovopsis are specialized parasites found only in association with attine gardens. Evolutionary theory predicts that the low genetic diversity in monocultures should render ant gardens susceptible to a wide range of diseases, and additional parasites with roles similar to that of Escovopsis are expected to exist. We profiled the diversity of cultivable microfungi found in 37 nests from ten Acromyrmex species from Southern Brazil and compared this diversity to published surveys. Our study revealed a total of 85 microfungal strains. Fusarium oxysporum and Escovopsis were the predominant species in the surveyed gardens, infecting 40.5% and 27% of the nests, respectively. No specific relationship existed regarding microfungal species and ant-host species, ant substrate preference (dicot versus grass) or nesting habit. Molecular data indicated high genetic diversity among Escovopsis isolates. In contrast to the garden parasite, F. oxysporum strains are not specific parasites of the cultivated fungus because strains isolated from attine gardens have similar counterparts found in the environment. Overall, the survey indicates that saprophytic microfungi are prevalent in South American leafcutter ants. We discuss the antagonistic potential of these microorganisms as "weeds" in the ant-fungus symbiosis.

Garden sharing and garden stealing in fungus-growing ants.

Fungi cultivated by fungus-growing ants (Attini: Formicidae) are passed on between generations by transfer from maternal to offspring nest (vertical transmission within ant species). However, recent phylogenetic analyses revealed that cultivars are occasionally also transferred between attine species. The reasons for such lateral cultivar transfers are unknown. To investigate whether garden loss may induce ants to obtain a replacement cultivar from a neighboring colony (lateral cultivar transfer), pairs of queenright colonies of two Cyphomyrmex species were set up in two conjoined chambers; the garden of one colony was then removed to simulate the total crop loss that occurs naturally when pathogens devastate gardens. Garden-deprived colonies regained cultivars through one of three mechanisms: joining of a neighboring colony and cooperation in a common garden; stealing of a neighbor's garden; or aggressive usurpation of a neighbor's garden. Because pathogens frequently devastate attine gardens under natural conditions, garden joining, stealing and usurpation emerge as critical behavioral adaptations to survive garden catastrophes.

The agricultural pathology of ant fungus gardens.

Gardens of fungus-growing ants (Formicidae: Attini) traditionally have been thought to be free of microbial parasites, with the fungal mutualist maintained in nearly pure "monocultures." We conducted extensive isolations of "alien" (nonmutualistic) fungi from ant gardens of a phylogenetically representative collection of attine ants. Contrary to the long-standing assumption that gardens are maintained free of microbial pathogens and parasites, they are in fact host to specialized parasites that are only known from attine gardens and that are found in most attine nests. These specialized garden parasites, belonging to the microfungus genus Escovopsis (Ascomycota: anamorphic Hypocreales), are horizontally transmitted between colonies. Consistent with theory of virulence evolution under this mode of pathogen transmission, Escovopsis is highly virulent and has the potential for rapid devastation of ant gardens, leading to colony mortality. The specialized parasite Escovopsis is more prevalent in gardens of the more derived ant lineages than in gardens of the more "primitive" (basal) ant lineages. Because fungal cultivars of derived attine lineages are asexual clones of apparently ancient origin whereas cultivars of primitive ant lineages were domesticated relatively recently from free-living sexual stocks, the increased virulence of pathogens associated with ancient asexual cultivars suggests an evolutionary cost to cultivar clonality, perhaps resulting from slower evolutionary rates of cultivars in the coevolutionary race with their pathogens.