Branched-chain amino acid assimilation promotes mixotrophy of ammonia-oxidizing archaeal sponge symbionts

Ammonia-oxidizing archaea (AOA) frequently form symbiotic associations with marine sponges. While free-living AOA are generally considered metabolically constrained chemolithoautotrophs, sponge-associated AOA encode for a branched-chain amino acid (BCAA) transporter, suggesting mixotrophic potential. Here, we test the unusual mixotrophic lifestyle of sponge-associated AOA by tracing the assimilation of ¹³ C- and ¹⁵ N-labeled BCAA in the sponge holobiont Ianthella basta . We demonstrate that BCAA degradation fuels ammonia oxidation and quantify BCAA uptake at the single-cell level by combining stable isotope probing, catalyzed reporter deposition fluorescence in situ hybridization, and nanoscale secondary ion mass spectrometry. Our results reveal that sponge-associated AOA are mixotrophic, assimilating BCAA as an additional carbon and nitrogen source. This metabolic adaptation may modulate BCAA availability in the holobiont, potentially regulating the host’s mTOR pathway. Collectively, our study reveals a novel nutritional interaction in sponge holobionts and challenges the perception of constrained metabolic capacities of AOA.