Microbial interactions can shape microbiome assembly by limiting invasion by harmful organisms or by preserving beneficial symbionts. In stingless bees (Scaptotrigona depilis), bacteria present in the larval diet have been observed to inhibit potentially pathogenic filamentous fungi while allowing beneficial yeast symbionts to persist. The mechanisms for this selective effect were previously unclear. This study investigated whether bacterial secreted products mediate selective effects on fungal taxa in the bee larval diet.
The authors combined conditioned media assays (cell-free supernatants) with genomic and metabolomic analyses to test how bacterial secreted products affect fungal growth. Conditioned media from prevalent bacterial strains associated with the larval diet were applied to fungal isolates to assess growth responses. Parallel genomic analyses searched for canonical antifungal biosynthetic gene clusters, and metabolomic profiling examined extracellular metabolites present in bacterial supernatants.
Conditioned media from several bacterial taxa significantly affected fungal growth. Across assays, filamentous fungi, including the known pathogen Aspergillus, were consistently inhibited by bacterial secreted products. The inhibition pattern was pronounced among bacteria that are prevalent members of the larval diet microbiota; one highlighted taxon was Apilactobacillus kunkeei, whose secreted products strongly affected fungal growth in vitro.
Part of the inhibitory effect on filamentous fungi was attributable to substrate acidification. Bacterial metabolism produced organic acids that lowered the pH of the medium, and this acidification contributed to reduced growth of filamentous fungi. However, acidification did not fully explain the observed inhibition: experiments indicated additional acidity-independent factors also contributed to antifungal activity. The presence of non-pH-dependent inhibitory activity suggests multiple mechanisms are acting in conditioned media to suppress filamentous fungal growth.
Responses among yeast taxa were more variable than among filamentous fungi. The study compared a non-symbiotic Zygosaccharomyces strain with a symbiotic Zygosaccharomyces lineage that is required for larval development. The non-symbiotic Zygosaccharomyces was inhibited by bacterial metabolites under near-neutral pH conditions, indicating sensitivity to bacterial secreted products independent of acidification. In contrast, the symbiotic Zygosaccharomyces required for larval development was maintained or even promoted when exposed to acid-conditioned media. These contrasting outcomes indicate that bacterial secretions can be selective, suppressing non-symbiotic yeasts while allowing or supporting symbiotic yeasts under the acidic conditions produced by bacterial metabolism.
Genomic analyses of the most prevalent bacteria in the larval diet revealed limited canonical antifungal biosynthetic gene clusters. In other words, classic antifungal biosynthetic pathways commonly identified in other systems were not abundant in these strains. This genomic result suggests that the selective inhibition observed is not primarily driven by well-characterized antifungal biosynthetic gene clusters in these bacterial taxa.
Metabolomic profiling identified extracellular peptide-like compounds across the bacterial strains examined. The presence of these peptide-like metabolites points toward a role for non-canonical secreted products in mediating fungal inhibition. While canonical biosynthetic clusters were limited, these extracellular compounds could represent alternative mechanisms by which bacteria exert selective effects on fungal community members.
Together, the conditioned media experiments, genomic surveys, and metabolomic profiles indicate that bacterial secreted products play a key role in selectively shaping fungal communities in the stingless bee larval diet. Filamentous fungi, including pathogens such as Aspergillus, are suppressed through a combination of substrate acidification driven by organic acids and other acidity-independent factors. Yeasts show variable responses, with symbiotic Zygosaccharomyces tolerated or promoted under acidified conditions while non-symbiotic yeasts are inhibited. Limited detection of canonical antifungal biosynthetic gene clusters alongside detection of extracellular peptide-like compounds suggests non-canonical secreted factors may underlie selective inhibition. These findings provide initial mechanistic insight into how microbial interactions structure the larval diet ecosystem in stingless bees and suggest avenues for further work to define specific compounds and pathways responsible.
Note: The source reported these experimental results and analyses but did not provide exhaustive chemical identification of all inhibitory compounds or detailed molecular mechanisms for the acidity-independent effects within the text provided.