Dynamic cellular remodeling and symbiont genetic heterogeneity during early bacterial colonization in the deep-sea mussel Bathymodiolus septemdierum.

Symbiotic relationships between animals and microorganisms are widespread and have driven the adaptation and evolution of animals across diverse environments. The establishment of highly integrated endosymbioses often involves dynamic cellular remodeling and management of symbiont populations. Deep-sea mussels of the subfamily Bathymodiolinae horizontally acquire chemosynthetic bacteria in specialized gill cells called bacteriocytes, primarily at the posterior ends of their gills, where new filaments are continuously formed throughout their lifetime. This group exhibits a wide diversity of symbiotic modes, ranging from extracellular to strictly intracellular, and hosts diverse symbionts, such as methane- and sulfur-oxidizers. To identify the universal mechanisms underlying stable symbiosis from this diversity, it is crucial to investigate how these partnerships are initiated. However, the cellular-level processes underlying the initial symbiont colonization and integration remain elusive in all species of this group. To elucidate this early phase, we employed three-dimensional volume electron microscopy, single-cell amplicon sequencing, and in situ hybridization targeting a specific symbiont genomic locus to examine the initial symbiont colonization process in the deep-sea vent mussel Bathymodiolus septemdierum, focusing on host cell morphology and symbiont population structure. Three-dimensional ultrastructural reconstruction revealed that initial colonization follows a specific and characteristic structural remodeling process distinct from typical phagocytosis, demonstrating how this early cellular reorganization leads to the complex architecture of mature bacteriocytes. Complementing this morphological observation, two independent but mutually supportive approaches-single-cell amplicon sequencing and in situ hybridization-collectively indicated that these early colonizing cells harbor a genetically diverse mixture of symbiont strains. These findings challenge the previously proposed single-cell uptake hypothesis for this species and suggest that host cells initially acquire diverse symbiont subpopulations from the environment. We propose that the clonal symbiont populations observed in mature individual cells may result from an intracellular purification process potentially linked to host cellular remodeling during colonization. The integration of 3D ultrastructural observations and single-cell molecular data provides a foundation for deciphering the universal mechanisms of stable symbiosis, paving the way for a deeper understanding of animal-microbe interactions at the subcellular level.