Biology subjects

Pupo, M. T.

Publications and source records attributed to Pupo, M. T..

5 recordsLinked to original sources

bioRxiv2026

Bacterial secreted products selectively inhibit non-symbiotic fungi in bees

Microbial interactions play an important role in shaping microbiome assembly, such as by limiting invasion by harmful organisms that can directly affect the host or disrupt microbiome-associated benefits. Such interactions have been observed across systems, including in the microbiomes of key pollinators such as stingless bees. In Scaptotrigona depilis, bacteria associated with the larval diet inhibit potentially pathogenic filamentous fungi while allowing beneficial yeast symbionts to persist. The mechanisms underlying these effects, however, remain unclear. Here, we combined conditioned media (cell-free supernatant) assays with genomic and metabolomic analyses to investigate whether bacterial secreted products mediate these effects in the bee microbiome. Our results show that bacterial secreted products, particularly from prevalent bacterial taxa such as Apilactobacillus kunkeei, strongly affect fungal growth. Filamentous fungi, including the pathogen Aspergillus, were consistently inhibited, partly through substrate acidification driven by organic acids, but also through additional acidity-independent factors. In contrast, yeast responses were more variable: a non-symbiotic Zygosaccharomyces was inhibited by bacterial metabolites under near-neutral pH, whereas the symbiotic Zygosaccharomyces required for larval development was maintained or promoted under acid-conditioned media. Genomic analyses revealed limited canonical antifungal biosynthetic clusters in the most prevalent bacteria in the larval diet, while metabolomics identified extracellular peptide-like compounds across strains, suggesting a role for non-canonical secreted products. Together, these results show that bacterial secreted products play a key role in selectively shaping fungal communities in the stingless bee larval diet, providing first hints on a mechanistic basis for how microbial interactions structure this ecosystem.

microbiology↗

bioRxiv2026

Ecological Interactions Drive Metabolomic Diversification in Amazonian Pseudonocardia Symbionts

Fungus-growing ants engage in a multipartite symbiosis, including Pseudonocardia bacteria that produce antifungal metabolites to protect their fungal cultivar from the specialized pathogen Escovopsis. While different bioactive metabolites have been reported from ant-associated Pseudonocardia, most studies have focused on a limited number of strains, leaving the extent of chemical diversity across broader ecological contexts less resolved. Here, we investigated the antagonistic potential and metabolomic repertoires of 36 Pseudonocardia strains isolated from Amazonian Paratrachymyrmex ants. Pairwise bioactivity assays against two Escovopsis isolates revealed striking variability, with inhibition generally stronger and more diverse against the pathogenic fungus originating from the same ant genus. Untargeted LC-MS/MS metabolomics coupled with 16S rRNA-based phylogenetic analyses showed that closely related strains harbored highly divergent chemical profiles, underscoring a decoupling between taxonomy and metabolite output. Detailed analyses of selected isolates revealed the production of structurally diverse metabolites, including dentigerumycin analogs, provipeptide A, {beta}-carbolines, and tetracycline-related compounds. Co-culture analysis uncovered metabolites absent in monocultures, including lichenysins, pepstatins, and hallobacillins, as well as conserved attinimicin, whose production was enhanced under pathogen challenge. These results highlight that both strain-specific metabolic repertoires and interaction-induced chemistry contribute to the defensive arsenal of Pseudonocardia. Together, our findings likely demonstrate that ecological pressures and local adaptation, rather than phylogeny alone, drive metabolomic diversification in this defensive symbiosis. Beyond their potential for novel bioactive compound discovery, these results provide insights into the chemical basis of multipartite symbioses, the dynamics of defensive mutualisms, and the ecological forces shaping microbial diversity in underexplored environments such as the Amazon. IMPORTANCEMicrobial symbionts are central to host defense and natural product discovery, yet the factors driving their chemical diversification remain unclear. The fungus-growing ant-Pseudonocardia-Escovopsis system offers a powerful model to study how ecological context shapes microbial metabolism. By systematically characterizing multiple Amazonian Pseudonocardia strains, we show that antagonistic capacity and metabolomic repertoires vary widely, even among strains with highly similar 16S rRNA gene sequences, revealing a pronounced discordance between 16S-based phylogenetic relatedness and specialized metabolite production. These findings highlight the likely importance of ecological pressures and local adaptation in shaping metabolomic output, emphasizing symbiotic actinobacteria as both key ecological players and promising sources of antifungal natural products.

microbiology↗

bioRxiv2023

Further evidences of an emerging stingless bee-yeast symbiosis

Symbiotic interactions between microorganisms and social insects have been described as crucial for the maintenance of these multitrophic systems, as observed for the stingless bee Scaptotrigona depilis and the yeast Zygosaccharomyces sp. The larvae of S. depilis ingest fungal filaments of Zygosaccharomyces sp. to obtain ergosterol, which is the precursor for the biosynthesis of ecdysteroids that modulate insect metamorphosis. In this work we verified that nutritional fungal symbioses also occur in other species of stingless bees. We analyzed brood cell samples from 19 species of stingless bees collected in Brazil. The osmophilic yeast Zygosaccharomyces spp. was isolated from eight bee species, namely Scaptotrigona bipuctata, S. postica, S. tubiba, Tetragona clavipes, Melipona quadrifasciata, M. fasciculata, M. bicolor and Partamona helleri. These yeasts form pseudohyphae and also accumulate ergosterol in lipid droplets, similar to the pattern observed for S. depilis. The phylogenetic analyses including various Zygosaccharomyces revealed that strains isolated from the brood cells formed a branch separated from the previously described Zygosaccharomyces species, suggesting that they are new species of this genus and reinforcing the symbiotic interaction with the host insects. ImportanceBenefits exchanged in insect-fungus mutualisms include nutrition, protection, and dispersal. Fungal nutritional roles are well described for some eusocial insects, such as fungus growing ants and termites, but similar interaction in stingless bees was so far observed just in Scaptotrigona depilis. Here we expand the knowledge of yeast-bee symbiosis by analyzing the presence, cell morphologies, lipid accumulation and phylogenetic relationships of fungi isolated from brood cells and other locations of bee colonies. Zygosaccharomyces isolates were recovered from 42% of the bee species assessed, and probably represent new species showing pseudohyphae formation and lipid accumulation similar to S. depilis associated Zygosaccharomyces strains. The phylogenetic analyses suggested an evolutionary adaptation of Zygosaccharomyces spp. to the brood cell environment to provide nutritional benefits for the developing insect. Stingless bees play important ecosystem services, and our results raise the concern that fungicidal agents used in agriculture could disrupt this symbiosis, impacting bee health.

microbiology↗

bioRxiv2021

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 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.

microbiology↗

bioRxiv2018

Metagenomics reveals diet-specific specialization in fungus gardens of grass- and dicot-cutter ants

Leaf-cutter ants in the genus Atta are dominant herbivores in the Neotropics. While most species of Atta cut dicots to incorporate into their fungus gardens, some species specialize on grasses. Here we examine the bacterial community associated with the fungus gardens of grass- and dicot-cutter ants to examine how changes in substrate input affect the bacterial community. We sequenced the metagenomes of 12 Atta fungus gardens, across four species of ants, with a total of 5.316 Gbp of sequence data. We show significant differences in the fungus garden bacterial community composition between dicot- and grass-cutter ants, with grass-cutter ants having lower diversity. Reflecting this difference in community composition, the bacterial functional profiles between the fungus gardens are significantly different. Specifically, grass-cutter ant fungus garden metagenomes are particularly enriched for genes responsible for amino acid, siderophore, and terpenoid biosynthesis while dicot-cutter ant fungus gardens metagenomes are enriched in genes involved in membrane transport. These differences in bacterial community composition and functional capacity show that different substrate inputs matter for fungus garden bacteria, and sheds light on the potential role of bacteria in mediating the ants transition to the use of a novel substrate.

microbiology↗