Microbial, dietary insect, and pathogen communities in fresh and decomposing guano of anthropic little brown bat (Myotis lucifugus) maternity colonies

The guano of insectivorous bats holds ecological information that can be assessed non-invasively to characterize the gut microbiome and diet, alongside environmental microbes and pathogens of conservation concern. Despite this potential, how guano communities change during decomposition remains understudied, particularly inside anthropic roosts rather than caves. In this study, guano from little brown bat (Myotis lucifugus) colonies was sampled monthly across the summer maternity season from three sites across two locations in Connecticut, USA, at fresh deposition and at 4, 8, and 12 weeks following deposition. Resolving these cross-kingdom signals required five workflows: short-read 16S and ITS2 amplicon sequencing (Illumina) for bacterial and fungal profiling, long-read CO1 metabarcoding (Oxford Nanopore) for arthropod diet in fresh samples, long-read shotgun metagenomics for viral identification in aged samples, and targeted qPCR for organisms of bat, human, and forest-health concern. Fresh guano generated a consistent bacterial signal across sites, whereas fresh fungal communities differed by site. Responses to decomposition depended on roost setting: exterior sites lost fungal diversity and shifted toward environmental aerobes over time, while the interior roost retained the fresh sample profile. Dietary composition varied temporally, was dominated by Diptera, and included the invasive emerald ash borer (Agrilus planipennis). Pseudogymnoascus destructans, the causal agent of white-nose syndrome, occurred in fresh and aged samples at all three sites but persisted for 12 weeks only at the interior roost, where antifungal bacterial taxa were depleted. Long-read shotgun metagenomics of aged guano recovered roughly 100 viral species, predominantly bacteriophages, alongside non-bacteriophage mastadenoviruses associated with humans, bats, and other mammals. These results show that anthropic structures influence the trajectory of guano microbiome succession, and that maternity colony guano enables non-invasive assessment of environmental pathogens, bat diet, and bacterial and fungal communities.