Bacterial community structure and stable predicted functional profiles in the renal sac fluid of the cuttlefish Acanthosepion pharaonis

Cephalopods have high metabolic rates and rely heavily on amino acid catabolism, producing large amounts of ammonia that must be efficiently excreted to avoid physiological stress. The cephalopod renal organ, consisting of renal appendages enclosed within an acidic and ammonia-rich fluid-filled renal sac, plays a central role in nitrogen waste elimination. Yet, the existence and biological meanings of renal sac microbiota have never been investigated. In this study, we characterized the renal sac bacterial community of the pharaoh cuttlefish Acanthosepion pharaonis using PacBio full-length 16S rRNA sequencing. Renal sac fluid was collected from wild-caught individuals from northern Taiwan and from laboratory-reared individuals raised from eggs under controlled conditions. ASV-based analysis showed that bacterial communities were detectable in all renal sac fluid samples. Laboratory-reared individuals showed relatively consistent bacterial compositions dominated by Vibrionaceae, whereas wild-caught individuals showed strong inter-individual variation and no single dominant bacterial family shared across samples. The wild-caught and laboratory-reared groups showed a significant group-associated difference in overall community composition, while multivariate dispersion was significantly greater among wild-caught individuals. To further examine the functional potential of these bacterial communities, we performed functional prediction using PICRUSt2 and Tax4Fun2. Predicted pathways converged on several metabolic themes, including tryptophan-derived metabolism, aromatic compound degradation, amino acid metabolism, and related metabolic processes. Family-level correlation analysis identified candidate bacterial taxa associated with these predicted pathways, including Burkholderiaceae, Microbacteriaceae, Muribaculaceae, and Flavobacteriaceae. These findings suggest that the renal sac fluid may act as a chemically selective host-associated niche, shaping bacterial communities at the level of metabolic potential rather than enforcing a conserved taxonomic composition. This study provides the first systematic characterization of cephalopod renal sac fluid microbiota and shows how a chemically robust host-associated environment may support functional convergence among taxonomically variable bacterial communities.