Deciphering deep-sea chemosynthetic symbiosis by single-nucleus RNA- sequencing

Reviewer #1 (Public Review):

Wang et al have constructed a comprehensive single nucleus atlas for the gills of the deep sea Bathymodioline mussels, which possess intracellular symbionts that provide a key source of carbon and allow them to live in these extreme environments. They provide annotations of the different cell states within the gills, shedding light on how multiple cell types cooperate to give rise to the emergent functions of the composite tissues and the gills as a whole. They pay special attention to characterizing the bacteriocyte cell populations and identifying sets of genes that may play a role in their interaction with the symbiotes.

Wang et al sample mussels from 3 different environments: animals from their native methane-rich environment, animals transplanted to a methane-poor environment to induce starvation, and animals that have been starved in the methane-poor environment and then moved back to the methane-rich environment. They demonstrated that starvation had the biggest impact on bacteriocyte transcriptomes. They hypothesize that the upregulation of genes associated with lysosomal digestion leads to the digestion of the intracellular symbiont during starvation, while the non-starved and reacclimated groups more readily harvest the nutrients from symbiotes without destroying them.

Strengths:
This paper makes available a high-quality dataset that is of interest to many disciplines of biology. The unique qualities of this non-model organism and the collection of conditions sampled make it of special interest to those studying deep sea adaptation, the impact of environmental perturbation on Bathymodioline mussels populations, and intracellular symbiotes. The authors do an excellent job of making all their data and analysis available, making this not only an important dataset but a readily accessible and understandable one.

The authors also use a diverse array of tools to explore their data. For example, the quality of the data is augmented by the use of in situ hybridizations to validate cluster identity and KEGG analysis provides key insights into how the transcriptomes of bacteriocytes change.

The authors also do a great job of providing diagrams and schematics to help orient non-mussel experts, thereby widening the audience of the paper.

Weaknesses:
One of the main weaknesses of this paper is the lack of coherence between the images and the text, with some parts of the figures never being referenced in the body of the text. This makes it difficult for the reader to interpret how they fit in with the author's discussion and assess confidence in their analysis and interpretation of data. This is especially apparent in the cluster annotation section of the paper.

Another concern is the linking of the transcriptomic shifts associated with starvation with changes in interactions with the symbiotes. Without examining and comparing the symbiote population between the different samples, it cannot be concluded that the transcriptomic shifts correlate with a shift to the 'milking' pathway and not other environmental factors. Without comparing the symbiote abundance between samples, it is difficult to disentangle changes in cell state that are due to their changing interactions with the symbiotes from other environmental factors.

Additionally, conclusions in this area are further complicated by using only snRNA-seq to study intracellular processes. This is limiting since cytoplasmic mRNA is excluded and only nuclear reads are sequenced after the organisms have had several days to acclimate to their environment and major transcriptomic shifts have occurred.

Reviewer #2 (Public Review):

Wang, He et al. shed insight into the molecular mechanisms of deep-sea chemosymbiosis at the single-cell level. They do so by producing a comprehensive cell atlas of the gill of Gigantidas platifrons, a chemosymbiotic mussel that dominates the deep-sea ecosystem. They uncover novel cell types and find that the gene expression of bacteriocytes, the symbiont-hosting cells, supports two hypotheses of host-symbiont interactions: the "farming" pathway, where symbionts are directly digested, and the "milking" pathway, where nutrients released by the symbionts are used by the host. They perform an in situ transplantation experiment in the deep sea and reveal transitional changes in gene expression that support a model where starvation stress induces bacteriocytes to "farm" their symbionts, while recovery leads to the restoration of the "farming" and "milking" pathways.

A major strength of this study includes the successful application of advanced single-nucleus techniques to a non-model, deep-sea organism that remains challenging to sample. I also applaud the authors for performing an in situ transplantation experiment in a deep-sea environment. From gene expression profiles, the authors deftly provide a rich functional description of G. platifrons cell types that is well-contextualized within the unique biology of chemosymbiosis. These findings offer significant insight into the molecular mechanisms of deep-sea host-symbiont ecology, and will serve as a valuable resource for future studies into the striking biology of G. platifrons.

The authors' conclusions are generally well-supported by their results. However, I recognize that the difficulty of obtaining deep-sea specimens may have impacted experimental design. In this area, I would appreciate more in-depth discussion of these impacts when interpreting the data.

Because cells from multiple individuals were combined before sequencing, the in situ transplantation experiment lacks clear biological replicates. This may potentially result in technical variation (ie. batch effects) confounding biological variation, directly impacting the interpretation of observed changes between the Fanmao, Reconstitution, and Starvation conditions. It is notable that Fanmao cells were much more sparsely sampled. It appears that fewer cells were sequenced, resulting in the Starvation and Reconstitution conditions having 2-3x more cells after doublet filtering. It is not clear whether this is due to a technical factor impacting sequencing or whether these numbers are the result of the unique biology of Fanmao cells. Furthermore, from Table S19 it appears that while 98% of Fanmao cells survived doublet filtering, only ~40% and ~70% survived for the Starvation and Reconstitution conditions respectively, suggesting some kind of distinction in quality or approach.

There is a pronounced divergence in the relative proportions of cells per cell type cluster in Fanmao compared to Reconstitution and Starvation (Fig. S11). This is potentially a very interesting finding, but it is difficult to know if these differences are the expected biological outcome of the experiment or the fact that Fanmao cells are much more sparsely sampled. The study also finds notable differences in gene expression between Fanmao and the other two conditions- a key finding is that bacteriocytes had the largest Fanmao-vs-starvation distance (Fig. 6B). But it is also notable that for every cell type, one or both comparisons against Fanmao produced greater distances than comparisons between Starvation and Reconstitution (Fig. 6B). Again, it is difficult to interpret whether Fanmao's distinctiveness from the other two conditions is underlain by fascinating biology or technical batch effects. Without biological replicates, it remains challenging to disentangle the two.

Reviewer #3 (Public Review):

Wang et al. explored the unique biology of the deep-sea mussel Gigantidas platifrons to understand the fundamental principles of animal-symbiont relationships. They used single-nucleus RNA sequencing and validation and visualization of many of the important cellular and molecular players that allow these organisms to survive in the deep sea. They demonstrate that a diversity of cell types that support the structure and function of the gill including bacteriocytes, specialized epithelial cells that host sulfur-oxidizing or methane-oxidizing symbionts as well as a suite of other cell types including supportive cells, ciliary, and smooth muscle cells. By performing experiments of transplanting mussels from one habitat which is rich in methane to methane-limited environments, the authors showed that starved mussels may consume endosymbionts versus in methane-rich environments upregulated genes involved in glutamate synthesis. These data add to the growing body of literature that organisms control their endosymbionts in response to environmental change.

The conclusions of the data are well supported. The authors adapted a technique that would have been technically impossible in their field environment by preserving the tissue and then performing nuclear isolation after the fact. The use of single-nucleus sequencing opens the possibility of new cellular and molecular biology that is not possible to study in the field. Additionally, the in-situ data (both WISH and FISH) are high-quality and easy to interpret. The use of cell-type-specific markers along with a symbiont-specific probe was effective. Finally, the SEM and TEM were used convincingly for specific purposes in the case of showing the cilia that may support water movement.

The one particular area for clarification and improvement surrounds the concept of a proliferative progenitor population within the gill. The authors imply that three types of proliferative cells within gills have long been known, but their study may be the first to recover molecular markers for these putative populations. The markers the authors present for gill posterior end budding zone cells (PEBZCs) and dorsal end proliferation cells (DEPCs) are not intuitively associated with cell proliferation and some additional exploration of the data could be performed to strengthen the argument that these are indeed proliferative cells. The authors do utilize a trajectory analysis tool called Slingshot which they claim may suggest that PEBZCs could be the origin of all gill epithelial cells, however, one of the assumptions of this analysis is that differentiated cells are developed from the same precursor PEBZC population.

However, these conclusions do not detract from the overall significance of the work of identifying the relationship between symbionts and bacteriocytes and how these host bacteriocytes modulate their gene expression in response to environmental change. It will be interesting to see how similar or different these data are across animal phyla. For instance, the work of symbiosis in cnidarians may converge on similar principles or there may be independent ways in which organisms have been able to solve these problems.