Diel transcriptional dynamics of a marine sponge and its microbiome in a natural environment

Marine sponges are ecologically critical animals that host diverse microbial communities, forming complex symbiotic systems crucial to nutrient cycling and ecosystem functioning in the ocean. While several studies have started to delineate the interactions between sponges and their microbiomes, whether and how these interactions are influenced by night and day cycles remains unclear, particularly in natural environments. Here, we analyzed the in situ transcriptional changes of the demosponge species Aplysina aerophoba and its microbiome over a diel cycle, sampling four specimens each at four time points within a 24-hour period. At the global metatranscriptome level, microbiome activity clustered by animal subject, rather than time of sampling. However, diel transcriptional patterns were observed for a limited number of microbial genes, primarily involved in secondary metabolite biosynthesis and antibiotic efflux pumps. At the individual microbial lineage level, we identified diel patterns in cyanobacterial and alphaproteobacterial symbionts. Cyanobacterial symbionts exhibited canonical circadian regulation, with daytime expression of photosynthesis-related genes. At night upregulation of ammonium assimilation via glutamine synthetase coincided with induction of the oxidative pentose phosphate pathway and respiratory genes, indicating reliance on host-derived carbohydrates to generate ATP, NADPH, and the 2-oxoglutarate carbon backbone required for glutamate synthesis in the dark. This pattern contrasts with free-living cyanobacteria, where nitrogen assimilation is typically day-active and fueled by photosynthesis-derived energy and reducing power. The sponge host transcriptome displayed distinct diel regulation of key circadian genes (cry2 and PAR-bZIP), in addition to daytime upregulation of genes involved in sulfur metabolism and oxidative stress defense, patterns mirrored by alphaproteobacterial symbionts that together contribute to glutathione-based detoxification (sponge Glo2, bacterial GstB) and H2S management (SQOR). Our study demonstrates that diel environmental fluctuations modulate the transcriptome of the sponge hosts and only select microbial lineages - primarily Cyanobacteria and heterotrophic Alphaproteobacteria – highlighting targeted rather than community-wide diel transcriptional responses within the holobiont. Together, these lineage-specific responses reveal mechanistic links between nitrogen assimilation, sulfur metabolism, and oxidative stress detoxification. These findings provide novel insights into the metabolic integration and functional stability of one of the earliest evolved animal-microbe symbiotic systems.