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The authors have answers nicely all concern raised by the reviewers.
[# PeerJ Staff Note - this decision was reviewed and approved by James Reimer, a PeerJ Section Editor covering this Section #]
Dear Authors,
As you can see the manuscript has been evaluated by 3 reviewers.
I encourage you to resubmit the manuscript as all three find the work to be interesting.
Please see comments mainly from reviewers 2 & 3.
[# PeerJ Staff Note: Please ensure that all review comments are addressed in a response letter and any edits or clarifications mentioned in the letter are also inserted into the revised manuscript where appropriate. It is a common mistake to address reviewer questions in the response letter but not in the revised manuscript. If a reviewer raised a question then your readers will probably have the same question so you should ensure that the manuscript can stand alone without the response letter. Directions on how to prepare a response letter can be found at: https://peerj.com/benefits/academic-rebuttal-letters/ #]
This is a well planned experimental work putting together an interesting biological model-coral-dinoflagellate endosymbiosis-with the use of a series of morphological and molecular techniques. The manuscript is well written and illustrated, with results that contribute to a better knowledge of lipid biogenesis in a natural in vivo context.
The work was well planned and executed. The materials and methods sections details all basic information on coral husbandry, light and electron microscopy analysis of the lipid bodies, transcriptomic analysis related to genes involved in the lipid body biogenesis as well as from the endoplasmic reticulum and bioinformatic analysis.
The results obtained shows clearly the lipid body formation in the gastrodermal tissue layer of the coral, the rhythmicity in the size and distribution of lipid bodies. It is convincingly shown that the lipid bodies are formed in the endoplasmic reticulum, as previous shown in other cell types. Transcriptomic analysis support the morphological observations.
Congratulations for this interesting piece of work where an excellent natural in vivo model was used to follow the biosynthesis of lipid bodies.
The paper is well writen throught, the structure is clear in general, although there are a few weaknesses in the figures and captions (please see "comments").
Research well design. Methods explained in enough detail. Please see "comments".
Please see comments section.
The paper by Chen et al examines lipid body content, size and localisation in the host cells of symbiotic corals (in the species Euphyllia glabrescens). The authors have also studied the diel cycle dynamic in the distribution of lipid bodies across the coral host gastrodermal region, and differential regulation of genes and enzyme potential involved in lipogenesis in E. glabrescens.
This paper is topical and the authors have done careful experiments, including improving their protocol for collection of tentacle samples for TEM analysis. Overall, I think this work provides interesting elements to forward our understanding of lipid body formation, daily dynamic and potentially lipid synthesis.
I would suggest, however, that a few aspects that need to be better clarified before publication of this work:
- Lines 232-236 and Figure 1: Although I appreciate the improvement of the method that the authors have achieved to examine the ultrastructure of the tentacle by the clamping technique I do not think that Figure 1 is justified as part of the results section. This display and related description should be better placed in the Supplemental section.
- I find that the photographs shown in Figure 2 do not unequivocally support the statements made in lines 237-246, particularly because it is not really possible to discern many features from the displays (for example in panels a and b). The authors should in the first place clarify how they come to the conclusion that the structures that they point to are in fact lipid bodies. This is critical for the further revision of the rest of the manuscript, which is strongly based on a potential close associated between the LB and the RE. For instance, the discussion of potential interaction with other intracellular structures (lines 313-321) should be revised accordingly to clarify how that is extrapolated from the data shown in Figure 2. I wonder if clarity in this figure can be at least partially improved by (i) labelling other components in the photographs and (ii) positioning the arrows more specifically (particularly for example the identification of LBs in panels a and b are confusing).
- I struggled to see why the authors explain the expression of BiP protein as a response to ER- stress. As I see from the M&M the corals were kept under control environmental conditions and corals should be fully acclimated to the light environment. Is the argument that the daily fluctuations in ROS due to the photosynthesis of the symbiont results in ER stress within the host cells also in fully acclimated corals? Can the authors explain that more in detail?
- Figure 5: Could the authors clarify what are the different sizes of the LBs that the images show are? Is there any correspondence of these to the LB sizes that were classified in Figure 3 or are the different sizes a result of the purification step? I’m trying to understand the results presented since in panel Ai there seems to be more “LB” than are recognised by either the lipid marker (Aii) or the ER-tracker (Aiii). Also, panel D in that figure is not clear.
- Apologies if it is my omission but I could not find where the transcriptome data has been deposited.
The report matched categories for basic reporting.
The report matched categories for experimental design.
I only have some concerns on the general interpretation & presentation of the data, which are included in the "general comments for the authors".
The paper presents an interesting phenomenon, in which the formation of lipid body in coral during coral-dinoflagellate endosymbiosis, represented by LB size and distributions fluctuate by diel rhythmicity. The study also revealed critical transcriptomic regulation of lipid biosynthesis. The experiments are well-executed, with experimental procedures described in much detail in the Methods section. However, some concerns about the results need to be addressed before this manuscript can be accepted for publication in PeerJ.
Line 254: Please specify the method to count the LB on mesoglea side or coelenteron side (What percentage of the LB falling into a certain side is considered counted as mesoglea or coelenteron?)
Line 277-280: On Figure 4, it appears that PERK and IRE gene expression increased by one log, which means 2 folds, not one fold as stated in this sentence.
I encourage the author to deposit the data of their RNA-Seq analysis. The supplementary data only contained a small number of genes (BIP, PERK, IRE1, etc.). A GO analysis of the RNA-Seq data could reveal even more patterns in gene expression during diel fluctuations.
Figure 5A: the author should perform an analysis of colocalization between Bodipy and ER Tracker signals. This can be done with ImageJ. A merged image of these 2 channels should also be included.
The authors claimed micro-LBs fused to form larger LBs. I assumed that
However, looking at micro-LB, can the authors provide an explaination why the number of Micro-LB did not really change much between light to dark phase. (a one-way ANOVA analysis of samples near mesoglea would be able to distinguish if there is any difference in Micro-LB number over time)
Figure 2: Panels in the second and third columns appeared to be at different scale compated to the corresponding rows in the first column. For example: 2I scale bar is larger than 2G,H even though they are at the same magnification. Working with images at the inaccurate scale could lead to inaccurate quantification of the LB size. I encourage the authors to double check their raw data/images to confirm this.
Figure 5B: what was the time-point chosen for this Western Blot?
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