The discovery of an external bacterial niche reframes sequencing-based microbiomes in a freshwater sponge

Abstract Animals maintain beneficial associations with microbes while avoiding parasitism, often by physically segregating microbes from host tissues. However, sponges (Porifera) are frequently cited as an exception, with bacteria being shown to reside directly within the host mesohyl—an interior space composed of extracellular matrix and migratory cells. Yet this model rests largely on studies of field-collected specimens, where experimental control and spatial resolution are limited. Here, we exploit the experimental advantages of the freshwater sponge Ephydatia muelleri —culturable in the laboratory and highly amenable to confocal imaging—to directly address the spatial organization and transmission of host-associated bacteria. Contrary to expectations from studies of marine sponge systems, and to predictions from microbiome sequencing studies of freshwater sponges, we found limited evidence for diverse bacterial communities inhabiting the mesohyl of E. muelleri . Instead, we identified a novel microbial niche that is external to sponge tissues—a host-secreted matrix that underlies the basal epithelium, coats spicules, and envelops gemmules. In both laboratory cultures and field-collected specimens, matrix-associated microbes were abundant but separated from the sponge interior (mesohyl) by a continuous epithelial barrier. These external matrix-associated communities may contribute to the molecular signatures of stable freshwater sponge microbiomes, even if their functional roles remain unresolved. These findings reframe sequencing-based reports of freshwater sponge microbiomes with confocal imaging, and highlight that spatial context is essential for interpreting animal host–microbe associations. Abstract Figure Graphical Abstract Microbial communities colonize the skeletal matrix during dormancy in E. muelleri . In contrast to interpretations from microbiome sequencing studies, we find limited evidence for a resident microbiome within the mesohyl (shown in green) of the freshwater sponge, Ephydatia muelleri . Instead, we find that microbes are primarily confined to a novel external niche—a host-secreted matrix (shown in brown), which is exposed to the environment during periods of dormancy, and therefore permissive to microbial colonization. This matrix confers attachment to the basal substrate, embeds siliceous spicules, and coats gemmules (stress-resistant propagules maintaining senesced tissues). We find no evidence of bacteria being maintained across dormancy through the interior of gemmules. Instead, the external matrix and associated microbes are physically separated from the host mesohyl by a continuous epithelium (depicted as thick black lines). With spatial context afforded by in vivo imaging, we propose that these external-matrix-resident microbes likely account for previous reports of a stable, internal microbiome in freshwater sponges.