The larval gut as a mirror: bacterial community composition and functional potential of mayfly larvae reflect site and seasonality differences.

Land use intensification is a major driver of biodiversity loss across ecosystems, yet its consequences for host-associated microbiomes in freshwater food webs remain poorly understood. In this case study, we used the gut microbiome of mayfly larvae (Ephemera danica) as a sensitive biological interface to assess how site-specific adjacent land use types shape microbial community composition and functions in stream ecosystems. Larvae were sampled in summer and autumn from sites adjacent to forest, extensive grassland, and intensive agriculture along the Otterbach stream (Bavarian Forest, Germany). Combining 16S ribosomal RNA (rRNA) amplicon sequencing with long-read metagenomics, we show that site-specific land use, in interaction with seasonality, significantly restructures larval gut bacterial communities without affecting alpha diversity. Rather than introducing distinct agriculturally derived taxa, agricultural land use acted as a selective environmental filter, enriching bacterial groups with specific functional traits. Taxa enriched in the sites adjacent to agricultural sites harboured genes involved in complex carbon and xenobiotic degradation, short-chain fatty acid production, efflux pumps, and stress response. These functional signatures were further supported by 14 metagenome-assembled genomes linked to these enriched taxa. Together, our results reveal that site in combination with seasonality not only reshaped bacterial community composition without affecting alpha diversity but also triggered shifts in the abundance of genes involved in microbial-host interactions and degradation pathways in E. danica larvae. This study also highlights the larval gut microbiome as a sensitive indicator of environmental change, suggesting that environmental microbial shifts may have cascading consequences for freshwater trophic interactions and ecosystem functioning.