Identification of 14 cell types in the gill of deep-sea symbiotic mussel Gigantidas platifrons. A: Overall experimental scheme describing the deep-sea in situ transplant experiment, the sample preparation procedures and the single-cell analysis and validation pipeline. Three G. platifrons samples were included in the present study: ‘Fanmao,’ Starvation and Reconstitution. The cell nucleus was extracted from each sample, which included a pool of gill posterior tip of three mussels. The snRNA-seq libraries were constructed according to the BD Rhapsody singlenuclei 3′ protocol. Cell population-specific markers were validated by WISH and ISH. B: The image shows the posterior end of the gill of G. platifrons. C: UMAP representation of G. platifrons gill single cells. Cell clusters are coloured and distinctively labelled. D: Heat map profile of markers in each cluster. The colour gradient represents the expression level of every single cell.

Supportive cell populations of G. platifrons gill. A: UMAP representation of the four supportive cell populations. B: Expression profiles of the cell markers that are specific or enriched in the supportive cell populations. The sizes of the circles represent the percentages of cells in those clusters that expressed a specific gene. Genes shown in red were validated by WISH or double FISH. C and C’: Schematics demonstrating the overall structural (panel C) and supportive cell distribution (C’). D, F, H, and J: WISH characterisation of the selected representative cell population markers. E, G, I, and K: Double FISH characterisation of the selected representative cell population markers. The white arrowheads in G indicate the BMC1 cells locates at the otter rim of gill slice. Scale bar: 50µm.

Ciliary cell populations of G. platifrons gill. A: UMAP representation of the four ciliary cell populations and potential smooth muscle cell population. B: Expression profiles of the cell markers that are specific or enriched in the ciliary cell populations. The sizes of the circles represent the percentages of cells in those clusters that expressed a specific gene. The genes shown in red were validated by WISH or double FISH. C-E, G and I-J: WISH characterisation of the selected representative cell population markers. F and H: SEM analysis of the ciliary cells of G. platifrons gill. K: Schematic of the water flow agitated by different ciliary cell types. The coluor of arrowheads corresponds to water flow potentially influenced by specific types of cilia, as indicated by their colour code in Figure 3A.

Proliferation cell populations of G. platifrons gill. A: UMAP representation of the three proliferation cell populations. B: Expression profiles of the cell markers that are specific or enriched in the supportive cell populations. The sizes of the circles represent the percentages of cells in those clusters that expressed a specific gene. Genes shown in red were validated by WISH or double FISH. C and D: Photographic and schematic analyses of the spatial position of the three Proliferation cell populations. E, F and J: FISH and WISH characterisation of the selected population markers. The marker genes confirmed by ISH or WISH in the current study are indicated in red. Scale bar: 50 µm.

Characterisation of the bacteriocytes of G. platifrons. A: Schematic of the overall structure of G. platifrons gill filaments. B: Whole-mount FISH analyses of the overall distribution of bacteriocytes on the G. platifrons gill filament. C: SEM analysis of the bacteriocytes. D: TEM analysis of a bacteriocyte. E: UMAP representation of G. platifrons bacteriocytes. F: Expression profiles of the cell markers that are specific or enriched in the bacteriocytes. The sizes of the circles represent the percentages of cells in those clusters that expressed a specific gene. Double FISH validated the genes shown in red. I: Schematic of the host-symbiont interaction based on the single-cell transcriptome of G. platifrons bacteriocytes. The marker genes confirmed by ISH in the current study are indicated in red. Scale bar in panel G and H: 25 µm.

Analysis of cell population-specific DEGs. A: UMAP representation of the impact of deep-sea in situ transplant treatments on the gene expression pattern of each cell population. The cells from different treatments were labelled with different colours. The dashed line encircled bacteriocyte populations have a considerably altered expression profile. B: Histogram of cross-state distances between the centroids of the Fanmao, Starvation and Reconstitution groups per cell type on UMAP. The black dashed lines indicate the bacteriocyte populations whose expression profile was remarkably altered. C: Visualization of bacteriocytes onto the pseudo time map using monocle. The black lines indicate the main path of the pseudotime ordering of the cells. D: Bifurcation of selected gene expression along two branches in response to environmental perturbation. Genes are clustered hierarchically into two groups, illustrating up- (cluster 1) and down- (cluster2) regulated genes in the starvation state compared with Fanmao. Genes in red colour were discussed in Section 4. The heat map showing the gene expression profiles of all bacteriocytes’ DEGs is shown in supplementary Fig. S7; E: Proposed model for the molecular mechanisms of host-symbiont interactions in response to environmental changes.