Programmed Cell Death in Sea Urchins: A Review

Round 1

Reviewer 1 Report

This can be an interesting review for JMSE, but I would like to be improved taking into account the audience of this journal.

I recommend to include a new section explaining the different species of sea urchins and their geographic distribution. This is important because depending on the region different species are used as models, and this section will help to understand the different species cited later. It is important to describe where they live also in terms of coastal waters with rock substrate, etc. 

I will also recommend to the authors that for the different pollutants they specify where they are discharged, and if the studies have been developed only in laboratories (theoretic studies) or if they are part of monitoring programs. 

In the discussion section, please, include the role of sea urchins in coastal waters monitoring programs.

I believe that in general along the manuscript more references are needed, there are many paragraphs without a single reference and this is a review paper.

Regarding temperature, please eliminate this paragraph: "This process brings with it many consequences: increased acidification of the oceans, melting of sea ice in polar regions, unpredictability and violence of storms, and an increase in tropical and subtropical regions [80,81]." It is not relevant in this context, completely unnecessary. 

When talking about diatoms, it is not clear if they are talking about diatom blooms or diatom in "normal" abundances, this needs to me explained much better. 

About emerging pollutants I think it is too short section, and some more examples are needed, for instance sunscreen filters.

Author Response

R. This can be an interesting review for JMSE, but I would like to be improved taking into account the audience of this journal.

A. We thank the reviewer for his/her comments and suggestions.

R. I recommend to include a new section explaining the different species of sea urchins and their geographic distribution. This is important because depending on the region different species are used as models, and this section will help to understand the different species cited later. It is important to describe where they live also in terms of coastal waters with rock substrate, etc. 

A. To address this suggestion, we have added in a new section a table listing the different species of sea urchins used for the studies reported in our paper, with their geographical distribution and habitat.

R. I will also recommend to the authors that for the different pollutants they specify where they are discharged, and if the studies have been developed only in laboratories (theoretic studies) or if they are part of monitoring programs.

A. We have added information related to the different pollutants as suggested.

R. In the discussion section, please, include the role of sea urchins in coastal waters monitoring programs.

A. We have added information on the role of sea urchins in coastal waters monitoring programs In the discussion section.

R. I believe that in general along the manuscript more references are needed, there are many paragraphs without a single reference and this is a review paper.

A. We have added more reference where needed

R. Regarding temperature, please eliminate this paragraph: "This process brings with it many consequences: increased acidification of the oceans, melting of sea ice in polar regions, unpredictability and violence of storms, and an increase in tropical and subtropical regions [80,81]." It is not relevant in this context, completely unnecessary. 

A. We have deleted the sentence as suggested

R. When talking about diatoms, it is not clear if they are talking about diatom blooms or diatom in "normal" abundances, this needs to me explained much better. 

A. We have modified the text to make this topic clearer, also underlining that the studies reported in this review only refer to investigations conducted on pure molecules to identify the molecular mechanisms underlying the effect of these natural compounds.

R. About emerging pollutants I think it is too short section, and some more examples are needed, for instance sunscreen filters.

A. We have improved the section on emerging pollutants including recent studies on the effect of sunscreen filters on sea urchin embryos.

Reviewer 2 Report

 Many well-known stress factors may induce both necrosis and apoptosis]. Unlike necrosis, apoptosis results in cellular changes under normal physiological conditions. The activation of caspases is considered to be the most specific marker of apoptosis. Apoptosis has been detected in all eukaryotes, from protozoa to highly multicellular animals.   An unusually large number of caspase genes and some novel pro-apoptotic proteins were revealed in sea urchins. Apoptosis has previously been detected using in situ hybridization, TUNEL assay, binding of fluorescently labeled general inhibitors of caspase, histochemistry,  electron microscopy, and flow cytometry. Environmental pollutants (toxicants, heavy metals) can activate death programs in both immune andnnon-immune cells of echinoderms. So, the  relevance of a  topic could hardly be questioned. I have a suggestion – you must estimate the main methods of apoptotic detection.  As an additional marker of plasma membrane alterations, you could use Yellow PRO® 1 iodide (Molecular Probes, USA) in combination with DAPI. It is difficult to distinguish post-apoptotic dead cells and cells that died through non-apoptotic processes after any stress. Thus, the use of FLICA binding and YO-PRO-1 staining together can more accurately detect apoptosis in sea urchin cells.

Author Response

Many well-known stress factors may induce both necrosis and apoptosis]. Unlike necrosis, apoptosis results in cellular changes under normal physiological conditions. The activation of caspases is considered to be the most specific marker of apoptosis. Apoptosis has been detected in all eukaryotes, from protozoa to highly multicellular animals.   An unusually large number of caspase genes and some novel pro-apoptotic proteins were revealed in sea urchins. Apoptosis has previously been detected using in situ hybridization, TUNEL assay, binding of fluorescently labeled general inhibitors of caspase, histochemistry, electron microscopy, and flow cytometry.

Environmental pollutants (toxicants, heavy metals) can activate death programs in both immune andnnon-immune cells of echinoderms. So, the relevance of a  topic could hardly be questioned.

R. I have a suggestion – you must estimate the main methods of apoptotic detection.  As an additional marker of plasma membrane alterations, you could use Yellow PRO® 1 iodide (Molecular Probes, USA) in combination with DAPI. It is difficult to distinguish post-apoptotic dead cells and cells that died through non-apoptotic processes after any stress. Thus, the use of FLICA binding and YO-PRO-1 staining together can more accurately detect apoptosis in sea urchin cells.

A. We thank the reviewer for his suggestions. We further examined the literature to include information on additional markers of plasma membrane alterations, e.g. Yellow PRO® 1 iodide, in combination with DAPI or the use of FLICA to assess the activation of Caspases, but for the best of our knowledge, there aren’t studies in the literature using these methods to detect apoptosis in sea urchins. The main method used for apoptotic detection has been underlined in the discussion section. We also added as suggestion for future investigations, the use of FLICA binding and YO-PRO-1 to detect apoptosis in sea urchins.

Reviewer 3 Report

The manuscript by Di Tuccio and colleagues addresses a fundamental problem of cell death in response to stress and in normal development. The topic of the work is extremely relevant in connection with unresolved problems of a fundamental and applied nature. The strength of the work must be recognized as a multimodal consideration of the problem in the light of morphological and molecular data in the context of environmental problems of pollution and global warming. Chemical, physical and physiological inducers of autophagy and apoptosis have been holistically analyzed in a very interesting and productive organismal model.

The manuscript is very well and logically written, giving a comprehensive presentation on the stated topic.

Author Response

The manuscript by Di Tuccio and colleagues addresses a fundamental problem of cell death in response to stress and in normal development. The topic of the work is extremely relevant in connection with unresolved problems of a fundamental and applied nature. The strength of the work must be recognized as a multimodal consideration of the problem in the light of morphological and molecular data in the context of environmental problems of pollution and global warming. Chemical, physical and physiological inducers of autophagy and apoptosis have been holistically analyzed in a very interesting and productive organismal model.

The manuscript is very well and logically written, giving a comprehensive presentation on the stated topic.

We are grateful to the Reviewer for his/her positive comments on our manuscript.