Early transcription factor activation distinguishes symbiotic from non-symbiotic bacteria during microbiome processing in a sponge.

Animals that filter-feed on environmental microbes must rapidly discriminate among captured bacteria to maintain beneficial associations while avoiding inappropriate immune activation. In innate immunity, this discrimination is executed through transcription factors (TFs), whose activation and nuclear translocation initiate effector gene expression and shape the nature of the host response. In sponges, bacteria are first physically captured by choanocytes, but the timing and cellular context in which TF-mediated immune discrimination becomes evident remain unclear. Here, we investigate the earliest detectable regulatory responses associated with discrimination between symbiotic and non-symbiotic bacteria in the marine sponge Amphimedon queenslandica. Using a feeding-based design to model post-metamorphic microbiome restructuring, we exposed juvenile sponges that already harbour vertically inherited symbionts to native (symbiont) or foreign (non-symbiont) bacterial communities and assessed early cellular processing and transcriptional responses to bacterial uptake. Symbiotic bacteria were rapidly transported across the epithelium and induced a strong, transient activation of conserved innate immune TFs, including interferon regulatory factor (IRF), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and signal transducer and activator of transcription, together with associated signalling pathways. IRF and NF-κB translocated to the nuclei of amoebocytes that had engulfed symbionts, indicating that discrimination becomes evident shortly after uptake and precedes downstream effector responses. In contrast, foreign bacteria were internalized more slowly, failed to induce coordinated immune TF activation or nuclear translocation, and instead elicited a xenobiotic-dominated transcriptional program. Together, these findings identify TF activation as an early regulatory checkpoint in sponge-microbe interactions and reveal key mechanisms that underpin the initial stages of symbiont discrimination.