Symbiosis interaction and localization in the sponge Cymbastela concentrica

Sponge (Porifera) holobionts are complex ecosystems, yet the molecular and spatial mechanisms underpinning their stability and organization remain poorly understood. Here, I applied cutting-edge microscopy techniques to investigate the mechanisms of symbiont recognition and spatial organization within the sponge Cymbastela concentrica. I focus on the role of symbiont-derived eukaryotic-like proteins (ELPs) in mediating recognition and map the community-level distribution of abundant symbionts to infer potential ecological interactions and metabolic niches. Using a combination of fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), I visualized two ELPs and their encoding symbionts: SSA4, an ankyrin-repeat protein from the gammaproteobacterial UBA9145 sp., and ELP5 from the Nitrospirales JAALKI01 sp.. Both symbionts were detected extracellularly and intracellularly, marking the first report of an intracellular Nitrospirales. Notably, host phagocytic cells exhibited selective accumulation of individual ELP-producing symbionts, suggesting specific recognition mechanisms. To dissect molecular interactions, I reconstructed the three-dimensional arrangement of SSA4’s binding to the sponge lectin TJLCc using correlative light and electron microscopy (CLEM). In extracellular UBA9145 sp. cells, SSA4 forms a peri-bacterial shell that binds TJLCc. This shell is proposed to shield the bacterium and camouflage it as a “self” cell of the sponge, potentially establishing stable residency by either promoting cell adhesion and specific-cell phagocytosis, or avoiding immune recognition and subsequent attack. In contrast, this molecular interaction is absent in intracellular bacteria, suggesting SSA4 employs an independent, alternative mechanism to promote intracellular persistence. This highlights the spatial and functional versatility of SSA4 in mediating symbiosis. Finally, to provide community-level context, I performed high-phylogenetic-resolution FISH (HiPR-FISH) to generate the first spatially resolved map of abundant symbionts across the sponge body. The community exhibited vertical stratification and niche partitioning, likely driven by gradients in light and water flow. This spatial structure implies a functional compartmentalization of the holobiont, with distinct metabolic interactions such as nutrient exchange, localized to different tissue layers. Collectively, this work integrates molecular, cellular, and community-level perspectives to advance our understanding of host-symbiont recognition and microbial organization in sponges, linking microbial localization to function in animal holobionts.