Comparative Genomic and Metabolomic Analysis of Termitomyces Species Provides Insights into the Terpenome of the Fungal Cultivar and the Characteristic Odor of the Fungus Garden of Macrotermes natalensis Termites

The symbiosis between macrotermitinae termites and Termitomyces is obligate for both partners and is one of the most important contributors to biomass conversion in the Old World tropic’s ecosystems. To date, research efforts have dominantly focused on acquiring a better understanding of the degradative capabilities of Termitomyces to sustain the obligate nutritional symbiosis, but our knowledge of the small-molecule repertoire of the fungal cultivar mediating interspecies and interkingdom interactions has remained fragmented.

Carbon dioxide levels and ventilation in Acromyrmex nests: significance and evolution of architectural innovations in leaf-cutting ants

Leaf-cutting ant colonies largely differ in size, yet all consume O 2 and produce CO 2 in large amounts because of their underground fungus gardens. We have shown that in the Acromyrmex genus, three basic nest morphologies occur, and investigated the effects of architectural innovations on nest ventilation. We recognized (i) serial nests, similar to the ancestral type of the sister genus Trachymyrmex , with chambers excavated along a vertical tunnel connecting to the outside via a single opening, (ii) shallow nests, with one/few chambers extending shallowly with multiple connections to the outside, and (iii) thatched nests, with an above-ground fungus garden covered with plant material. Ventilation in shallow and thatched nests, but not in serial nests, occurred via wind-induced flows and thermal convection. CO 2 concentrations were below the values known to affect the respiration of the symbiotic fungus, indicating that shallow and thatched nests are not constrained by harmful CO 2 levels. Serial nests may be constrained depending on the soil CO 2 levels. We suggest that in Acromyrmex , selective pressures acting on temperature and humidity control led to nesting habits closer to or above the soil surface and to the evolution of architectural innovations that improved gas exchanges.

Chemical Gradients of Plant Substrates in an Atta texana Fungus Garden

The study of complex ecosystems requires an understanding of the chemical processes involving molecules from several sources. Some of the molecules present in fungus-growing ants’ symbiotic system originate from plants.

Low Virulence of the Fungi Escovopsis and Escovopsioides to a Leaf-Cutting Ant-Fungus Symbiosis

Eusocial insects interact with a diversity of parasites that can threaten their survival and reproduction. The amount of harm these parasites cause to their hosts (i.e., their virulence) can be influenced by numerous factors, such as the ecological context in which the parasite and its host are inserted. Leaf-cutting ants (genera Atta, Acromyrmex and Amoimyrmex, Attini: Formicidae) are an example of a eusocial insect whose colonies are constantly threatened by parasites. The fungi Escovopsis and Escovopsioides (Ascomycota: Hypocreales) are considered a highly virulent parasite and an antagonist, respectively, to the leaf-cutting ants’ fungal cultivar, Leucoagaricus gongylophorus (Basidiomycota: Agaricales). Since Escovopsis and Escovopsioides are common inhabitants of healthy colonies that can live for years, we expect them to have low levels of virulence. However, this virulence could vary depending on ecological context. We therefore tested two hypotheses: (i) Escovopsis and Escovopsioides are of low virulence to colonies; (ii) virulence increases as colony complexity decreases. For this, we used three levels of complexity: queenright colonies (fungus garden with queen and workers), queenless colonies (fungus garden and workers, without queen) and fungus gardens (without any ants). Each was inoculated with extremely high concentrations of conidia of Escovopsis moelleri, Escovopsioides nivea, the mycoparasitic fungus Trichoderma longibrachiatum or a blank control. We found that these fungi were of low virulence to queenright colonies. The survival of queenless colonies was decreased by E. moelleri and fungus gardens were suppressed by all treatments. Moreover, E. nivea and T. longibrachiatum seemed to be less aggressive than E. moelleri, observed both in vivo and in vitro. The results highlight the importance of each element (queen, workers and fungus garden) in the leaf-cutting ant-fungus symbiosis. Most importantly, we showed that Escovopsis may not be virulent to healthy colonies, despite commonly being described as such, with the reported virulence of Escovopsis being due to poor colony conditions in the field or in laboratory experiments.

Queens of the inquiline social parasite Acromyrmex insinuator can join nest-founding queens of its host, the leaf-cutting ant Acromyrmex echinatior

Queens of the inquiline social parasite Acromyrmex insinuator are known to infiltrate mature colonies of Acromyrmex echinatior and to exploit the host’s perennial workforce by producing predominantly reproductive individuals while suppressing host reproduction. Here we report three cases of an A. insinuator queen having joined an incipient colony of A. echinatior that contained only the founding host-queen and her small symbiotic fungus garden. We conjectured that 1:1 host-inquiline co-founding—a phenomenon that has only rarely been reported in ants—may imply that the presence of an A. insinuator queen may incur benefits to the host by increasing survival of its incipient colonies. We observed that the parasite queens neither foraged nor defended the nest against intruders. However, the parasite queens interacted with the host and fungus in a way that could be consistent with grooming and/or with contributing eggs. These observations may help explain why A. insinuator queens have maintained metapleural glands, even though they are smaller than those of host queens, and why A. insinuator has lost the large foraging worker caste but not the small worker caste.

First record of Cyphoderus innominatus Mills, 1938 (Collembola: Paronellidae) in Early Colonies of the Leaf-cutting Ant Atta sexdens

Cyphoderus innominatus Mills, 1938 (Collembola: Paronellidae) was first observed in early colonies of Atta sexdens leaf-cutting ants (Hymenoptera: Formicidae). The colonies were collected on February 6, 2019, from a transition area between the Atlantic Forest and the Cerrado, located in the municipality of Botucatu, São Paulo, Brazil. A total of four colonies collected had an average population density of 227 ± 212 C. innominatus individuals, and most of the latter were found in peripheral areas inside the fungus-garden-growing chamber of the colony. In addition, we observed a possible defensive behavior on the part of workers when C. innominatus individuals were present in the fungus garden chamber. Thus, this is the first record of C. innominatus living in association with early colonies of A. sexdens. Cyphoderus innominatus Mills, 1938 (Collembola: Paronellidae) foi observado pela primeira vez nas primeiras colônias de formigas cortadeiras Atta sexdens rubropilosa , Forel, 1908 (Hymenoptera: Formicidae). Essas colônias foram coletadas em 6 de fevereiro de 2019, em uma área de transição entre a Mata Atlântica e o Cerrado, localizada no município de Botucatu, São Paulo, Brasil. Um total de quatro colônias coletadas teve uma densidade populacional média de 227, de C. innominatus , ea maioria dos últimos foram encontrados em áreas periféricas dentro da câmara de cultivo do jardim de fungo da colônia. Além disso, observamos um possível comportamento defensivo por parte dos trabalhadores quandoC. innominatus estava presente presentes na câmara do jardim de fungos. Assim, este é o primeiro registro de C. innominatus vivendo em associação com colônias iniciais de A. sexdens rubropilosa .

Burkholderia from fungus gardens of fungus-growing ants produce antifungals that inhibit the specialized parasite Escovopsis

Within animal associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants’ fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus Leucoagaricus spp. converting plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus Escovopsis. Here, we examine the potential role of Burkholderia spp. that occur within ant fungus gardens in inhibiting Escovopsis. We isolated members of the bacterial genera Burkholderia spp. and Paraburkholderia spp. from 50% of the 52 colonies sampled, indicating that family Burkholderiaceae are common fungus garden inhabitants of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one Escovopsis strain with a zone of inhibition greater than 1 cm. Genomic assessment of fungus-garden associated Burkholderiaceae indicated that isolates with strong inhibition all belonged to the genus Burkholderia and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed these compounds as responsible for antifungal activity that inhibit Escovopsis but, at equivalent concentrations, not Leucoagaricus spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of the fungus-growing ant colony. IMPORTANCE Many organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect Leucoagaricus spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, Escovopsis spp. The ants take multiple approaches, including weeding their fungus gardens to remove Escovopsis spores, as well as harboring Pseudonocardia that produce antifungals that inhibit Escovopsis. In addition, a genus of bacteria commonly found in fungus gardens, Burkholderia spp., is known to produce secondary metabolites that inhibit Escovopsis spp. In this study, we isolated Burkholderia spp. from fungus-growing ants, assessed the isolates’ ability to inhibit Escovopsis spp., and identified two compounds responsible for inhibition. Our findings suggest that Burkholderia spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite Escovopsis.

Habitat Quality Determines Dispersal Decisions and Fitness in a Beetle – Fungus Mutualism

Delayed dispersal of sexually mature offspring is a fundamental component of cooperative breeding. In ambrosia beetles, female offspring temporarily remain in their natal nest and refrain from reproduction, instead investing in alloparental care. Previous work has demonstrated a link between helping behaviour and the increased need for pathogen defence, arising from their close association with fungal cultivars. In the ambrosia beetle Xyleborinus saxesenii, mature female offspring can effectively fight pathogen infections and manage the microbial composition within the nest by adjusting the frequency of different hygienic and nest maintenance behaviours. This suggests a potential to respond flexibly to the ecology of their nest, which calls for a better understanding of the connection between behaviour and the microbial community thriving within their nests. Here, we studied the significance of the mutualistic fungus garden composition for the beetles’ nest ecology and fitness by experimentally varying substrate quality. We found that the vertically transmitted ambrosia fungus garden is composed of at least two fungus mutualist species and a wide variety of other microbes varying in their relative abundance. This is strongly affected by the moisture content of the substrate, which in nature depends on the age and type of wood. We found that the mutualist fungi complement each other in terms of dryness-resistance, allowing the beetles to utilise a broad range of substrates over prolonged time during which the wood gradually desiccates. Under suboptimal humidity conditions, the interaction between host and multiple fungus species has important ramifications for the behaviour of philopatric helpers, including their alloparental investment, sibling cannibalism and the timing of dispersal. Rearing five generations of beetles consecutively in dry substrate resulted in transgenerational effects on philopatry and alloparental care, probably mediated through the dominance of a particular fungus species that was driven by the experimental habitat condition. Interestingly, the nests of these selection lines produced much more offspring after five generations than any first-generation nest, which may have reflected increased egg laying by non-dispersing daughters. Our study highlights the importance of considering the interactions between the microbial community and their insect hosts for understanding social evolution in cooperatively breeding beetles.

Polyene-Producing Streptomyces spp. From the Fungus-Growing Termite Macrotermes barneyi Exhibit High Inhibitory Activity Against the Antagonistic Fungus Xylaria

Fungus-growing termites are engaged in a tripartite mutualism with intestinal microbes and a monocultivar (Termitomyces sp.) in the fungus garden. The termites are often plagued by entomopathogen (Metarhizium anisopliae) and fungus garden is always threatened by competitors (Xylaria spp.). Here, we aim to understand the defensive role of intestinal microbes, the actinomycetes which were isolated from the gut of Macrotermes barneyi. We obtained 44 antifungal isolates, which showed moderate to strong inhibition to Xylaria sp. HPLC analysis indicated that different types of polyenes (tetraene, pentene, and heptaene) existed in the metabolites of 10 strong antifungal Streptomyces strains. Two pentene macrolides (pentamycin and 1′14-dihydroxyisochainin) were firstly purified from Streptomyces strain HF10, both exhibiting higher activity against Xylaria sp. and M. anisopliae than cultivar Termitomyces. Subsequently, tetraene and heptaene related gene disruption assay showed that the mutant strains lost the ability to produce corresponding polyenes, and they also had significantly decreased activities against Xylaria sp. and M. anisopliae compared to that of wild type strains. These results indicate that polyene-producing Streptomyces from the guts of M. barneyi have strong inhibition to competitor fungus and polyenes contribute to inhibitory effects on Xylaria sp.

Burkholderia from fungus gardens of fungus-growing ants produce antifungals that inhibit the specialized parasite Escovopsis

ABSTRACTWithin animal associated microbiomes, the functional roles of specific microbial taxa are often uncharacterized. Here, we use the fungus-growing ant system, a model for microbial symbiosis, to determine the potential defensive roles of key bacterial taxa present in the ants’ fungus gardens. Fungus gardens serve as an external digestive system for the ants, with mutualistic fungi in the genus Leucoagaricus spp. converting plant substrate into energy for the ants. The fungus garden is host to specialized parasitic fungi in the genus Escovopsis. Here, we examine the potential role of Burkholderia spp. that occur within ant fungus gardens in inhibiting Escovopsis. We isolated members of the bacterial genera Burkholderia spp. and Paraburkholderia spp. from 50% of the 52 colonies sampled, indicating that the family Burkholderiaceae are common fungus garden inhabitants of a diverse range of fungus-growing ant genera. Using antimicrobial inhibition bioassays, we found that 28 out of 32 isolates inhibited at least one Escovopsis strain with a zone of inhibition greater than 1 cm. Genomic assessment of Burkholderiaceae isolates indicated that isolates with strong inhibition all belonged to the genus Burkholderia and contained biosynthetic gene clusters that encoded the production of two antifungals: burkholdine1213 and pyrrolnitrin. Organic extracts of cultured isolates confirmed these compounds as responsible for antifungal activity that inhibit Escovopsis but, at low concentrations, not Leucoagaricus spp. Overall, these new findings, combined with previous evidence, suggest that members of the fungus garden microbiome play an important role in maintaining the health and function of the fungus-farming ant colony.IMPORTANCEMany organisms partner with microbes to defend themselves against parasites and pathogens. Fungus-growing ants must protect Leucoagaricus spp., the fungal mutualist that provides sustenance for the ants, from a specialized fungal parasite, Escovopsis spp. The ants take multiple approaches, including weeding their fungus gardens to remove Escovopsis spores, as well as harboring Pseudonocardia that produce antifungals that inhibit Escovopsis. In addition, a genus of bacteria commonly found in fungus gardens, Burkholderia spp., is known to produce secondary metabolites that inhibit Escovopsis spp. In this study, we isolated Burkholderia spp. from fungus-growing ants, assessed the isolates’ ability to inhibit Escovopsis spp., and identified two compounds responsible for inhibition. Our findings suggest that Burkholderia spp. are often found in fungus gardens, adding another possible mechanism within the fungus-growing ant system to suppress the growth of the specialized parasite Escovopsis.