Antibiotics Modulate Intestinal Regeneration

Round 1

Reviewer 1 Report

Using the sea cucumber, the authors describe the effects of antibiotics on intestinal regeneration. As they describe nicely, the sea cucumber is a perfect model for this study. The figures are clear and associated with good statistical analyses. This article will have a nice impact on the communities of scientists working on either regeneration and or microbiomes.

The authors first tested the effects of specific antibiotics in the regenerating gut after evisceration. However, the toxicity of these antibiotics were tested on primary muscle cell culture and muscle tissue explants and not directly on the regenerating gut. The authors should test the toxicity directly on the regenerating gut, or explain in the manuscript why it couldn’t be done if there is a technical issue with this experiment. The correlation between the previous figures and this analysis will then be more direct and accurate.

A similar issue concerns the effect of the antibiotics on the microbiome of the gut. From my understanding, this analysis was done using the intestinal detritus from a normal gut. Will it be possible instead to test the effects of the antibiotics on the microbiome of a regenerating gut? (Incubating the eviscerated sea cucumber with antibiotics like previously done, and comparing the microbiome of this regenerating gut with the microbiome of a regenerating gut obtained in absence of antibiotics)

Minor comments:

In figure 4, the authors could use arrows to point the different areas that they are analyzing: coelomic epithelium versus connective tissue

In the discussion, the authors describe nicely the cellular events that take place during intestinal regeneration. This paragraph could be moved to the introduction, so the readers will better understand the regular process before going into the result section describing how antibiotics affect this process.

Author Response

Dear Reviewer 1:

Please see the attachment.

LMDD

Reviewer 2 Report

In this manuscript, Diaz-Diz et al. evaluate four antibiotics' impact on intestinal regeneration in H. glaberrima, emphasizing two possible models for the observed antibiotic effect.  This well-written and nicely illustrated article will be of broad interest to readers of the biology journal. However, some items need to be addressed before publication:

1. My primary is Figure 1. The SW group in Figure 1A has the same shape as SW in Figure 3A, which was labeled with anti-collagen (yellow) and DAPI staining (magenta). The authors should clarify it. 
2. Page 3, Line 141-142: It is essential to explain why penicillin-streptomycin to all experimental groups in the materials and methods. It makes the readers much easy to follow. 
3. The authors used GraphPad Prism to plot the bar graphs for Figures 1-3. It would nice if authors can use consistent border color and symbol shape among them. 

Author Response

Dear Reviewer 2:

Please see the attachment.

LMDD

Author Response File: Author Response.docx