Unlocking the Potential of Green Gravel Production for Efficient Kelp Restoration: How Seeding Density Affects the Development of the Golden Kelp Laminaria ochroleuca
, Inês Amorim Dos Santos 1,2, Isabel Sousa-Pinto 1,2 and Tânia Ribeiro Pereira 1Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
This is a very brief report describing an experimental trial on the laboratory production of the alga Laminaria ochroleuca.
The authors evaluated the influence of the initial density of seedlings on the size and mortality of young sporophytes. Although small, the results are solid and well discussed.
My only reservation concerns the short duration of the trial. 7 weeks of cultivation on these kelp species is too little to assess survival and growth, as other authors have well demonstrated (e.g. Fredriksen, S., Filbee-Dexter, K., Norderhaug, K. M., Steen, H., Bodvin, T., Coleman, M. A., et al. Green gravel: a novel restoration tool to combat kelp forest decline. Sci. Rep., 2020, 10, 1-7).
Also, having already published another short article on this subject, is not cited by the authors, which is difficult to understand (Chemello, S.; Pinto, I.S.; Pereira, T.R. Optimising Kelp Cultivation to Scale up Habitat Restoration Efforts: Effect of Light Intensity on “Green Gravel” Production. Hydrobiology 2023, 2, 347–353.). It would certainly have helped to discuss the results.
Other detailed issues that must be addressed:
Keywords: I would not repeat the same keywords that are stated in the title.
Line 91 – Different irradiances certainly affect algal growth. So, what type of white light? LED or fluorescent? Cold or warm? Is it possible to detail the wavelength?
Line 93 – idem regarding red light
Line 94 to 96 – I understand that the production of a large number of gametes is crucial to the reproductive success of the seaweed, but can you elaborate further? What are the conditions that “produce a larger number of gametes”? Larger than what?
Line 98 – sexual reproduction, right?
Line 102 – provide wavelenght of the lamps
Line 115 – brand, model and city of the stereoscope is missing
Line 120 – what is the suitable size for deployment?
Line 167 – I believe that “however” is not the proper word here. “Self-thinning” is consistent with your results, isn’t it? So, “Therefore”, “In fact” or “Indeed” seem more adequate words.
Conclusions 219 – 221 – do not repeat the same concepts presented in the discussion. I'd say this is better suited to the conclusion.
Comments on the Quality of English Language
Minor english editing is required.
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in track changes in the re-submitted file.
Reviewer's comments:
This is a very brief report describing an experimental trial on the laboratory production of the alga Laminaria ochroleuca.
The authors evaluated the influence of the initial density of seedlings on the size and mortality of young sporophytes. Although small, the results are solid and well discussed.
My only reservation concerns the short duration of the trial. 7 weeks of cultivation on these kelp species is too little to assess survival and growth, as other authors have well demonstrated (e.g. Fredriksen, S., Filbee-Dexter, K., Norderhaug, K. M., Steen, H., Bodvin, T., Coleman, M. A., et al. Green gravel: a novel restoration tool to combat kelp forest decline. Sci. Rep., 2020, 10, 1-7).
Response: Thank you for pointing this out. The goal of this experiment is to produce small individuals that have the potential to grow in natural conditions. Growing them over 1cm would lead to more fragile stipes and less ability to fix substrate. Furthermore, this decreased sturdiness would result in a lesser ability to deal with hydrodynamics, particularly on the coasts strongly affected, such as the Portuguese one.
Also, having already published another short article on this subject, is not cited by the authors, which is difficult to understand (Chemello, S.; Pinto, I.S.; Pereira, T.R. Optimising Kelp Cultivation to Scale up Habitat Restoration Efforts: Effect of Light Intensity on “Green Gravel” Production. Hydrobiology 2023, 2, 347–353.). It would certainly have helped to discuss the results.
Response: Thank you for your feedback. We were trying to avoid shameless self-promotion, but in fact, it would make sense to cite this previous work, as it can provide cumulative knowledge for the improvement of green gravel production. We appreciate the comment and changed it according to the suggestion.
Other detailed issues that must be addressed:
Keywords: I would not repeat the same keywords that are stated in the title.
Response: Agreed. Changed accordingly.
Line 91 – Different irradiances certainly affect algal growth. So, what type of white light? LED or fluorescent? Cold or warm? Is it possible to detail the wavelength?
Response: Agree. The illumination was done with LED lights, AQUABAR T Series, ultra-daylight. We added details in the text.
Line 93 – idem regarding red light
Response: Agree. We used a cinematographic filter that reduced light to 700nm wavelength only and added this information to the text.
Line 94 to 96 – I understand that the production of a large number of gametes is crucial to the reproductive success of the seaweed, but can you elaborate further? What are the conditions that “produce a larger number of gametes”? Larger than what?
Response: Thank you for your comment. For Laminaria ochroleuca, female gametophytes are often initially unicellular and produce only one gamete per gametophyte. In contrast, multicellular female gametophytes can produce a larger number of gametes, with the total number depending on their size. Larger gametophytes generally have the capacity to produce more gametes, which is crucial for reproductive success. This concept is discussed in more detail in Pereira et al. (2011), which may provide additional context.
Pereira, T. R., Engelen, A. H., Pearson, G. A., Serrao, E. A., Destombe, C., & Valero, M. (2011). Temperature effects on the microscopic haploid stage development of Laminaria ochroleuca and Sacchoriza polyschides, kelps with contrasting life histories. CBM-Cahiers de Biologie Marine, 52(4), 395.
Line 98 – sexual reproduction, right?
Response: Thank you for the question. Yes, unlike other algae species, the reproduction of Laminaria species is exclusively sexual.
Line 102 – provide wavelenght of the lamps
Response: Agree. We added the details in the text.
Line 115 – brand, model and city of the stereoscope is missing
Response: Agreed. Information was added in the text.
Line 120 – what is the suitable size for deployment?
Response: Thank you for pointing this out. We defined the optimum size as visible to the naked eye but smaller than 1cm. The information was added to the text, as suggested.
Line 167 – I believe that “however” is not the proper word here. “Self-thinning” is consistent with your results, isn’t it? So, “Therefore”, “In fact” or “Indeed” seem more adequate words.
Response: Agreed. We changed the wording in the text.
Conclusions 219 – 221 – do not repeat the same concepts presented in the discussion. I'd say this is better suited to the conclusion.
Response: We agree. We deleted this portion from the discussion.
Reviewer 2 Report
Comments and Suggestions for Authors
Dear Authors,
the manuscript reports convincing data on the possibility of using this method for the reforestation of Laminaria ochroleuca populations and on the growth of juveniles. Germination and growth experiments report data that show a high level of success, and these are interesting data for researchers working on this topic. Considering this after minor revisions (reported in attached file), the manuscript can be accepted for publication.
Comments for author File: 
Comments.pdf
Author Response
Thank you very much for taking the time to review this manuscript. Please find the suggested revisions highlighted in track changes in the re-submitted file.
Reviewer 3 Report
Comments and Suggestions for Authors
This study aims to evaluate the effect of seeding density, focusing on golden kelp Laminaria ochroleuca, with the aim of establishing green gravel technique which is considered to be effective in creating kelp forests. Accumulation of knowledge is important for the future dissemination of this technique. However, the research content of this paper is poor, and there are many vague expressions in the description, so it is thought that the manuscript will need to be significantly revised in order to be accepted for publication in this journal. I would like you to refer to the points below and make appropriate revisions.
General point
If the purpose of this study is as stated above, the contents of the manuscript are scarce. In particular, the experimental method lacks specificity, and the experimental items are insufficiently set. Please refer to "Specific points" for details.
Specific points
L36-38: Changes in the distribution range of herbivores and their increased feeding ability must also be influenced by climate change.
L55-58 & L59-60: Are there any contradictions in the description?
L79-81: If what is described here is the purpose of this study, the experimental content described in this paper seems extremely poor as a research paper.
L81-85: I don't understand this. Please clearly state its relationship to this study.
L90-92: How long was it maintained?
L92-94: How long was maturation suppression applied?
L96: How long ago was it specifically?
L96-98: What specific operation was used to cut them finely? Also, how much gametophyte was used, and how large were the cut finely fragments?
L99-101: The general reader does not understand why this was used as a substrate. In addition, why were experiments conducted with only these two seeding densities? Was choosing one of these two densities a proposition in previous studies? If the purpose of this study is to evaluate the effect of seeding density, wouldn't a more detailed experimental setup be necessary?
L106-108: What is ”appropriate water flow”? Please state the reason and the specific water flow value.
L119-120: What is ”a suitable size for deployment in the field”? Please state the reason and the specific thallus size.
L129-: The density of seedlings attached to the substrate (e.g. individuals/cm２) is important in promoting the content of this study. It should be stated in the results or M&M.
L132-134&fig1: What does this "length" mean?
L161-165: What does "three times the size" mean in this study, which lacks specificity about the method and has few evaluation criteria?
L165-167: Are the individuals that were treated at high density really dead? How about describing this in detail, including the process?
L186-193: I don't understand the relationship between this description and the contents of this study.
L194-196: Please clarify the basis by citing references, etc.
L196-199: The "affordability" part, including the description in the introduction, is confusing to readers. Please clarify this by calculating the specific costs, etc.
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in track changes in the re-submitted file.
Reviewer's comment:
This study aims to evaluate the effect of seeding density, focusing on golden kelp Laminaria ochroleuca, with the aim of establishing green gravel technique which is considered to be effective in creating kelp forests. Accumulation of knowledge is important for the future dissemination of this technique. However, the research content of this paper is poor, and there are many vague expressions in the description, so it is thought that the manuscript will need to be significantly revised in order to be accepted for publication in this journal. I would like you to refer to the points below and make appropriate revisions.
General point
If the purpose of this study is as stated above, the contents of the manuscript are scarce. In particular, the experimental method lacks specificity, and the experimental items are insufficiently set. Please refer to "Specific points" for details.
Specific points
L36-38: Changes in the distribution range of herbivores and their increased feeding ability must also be influenced by climate change.
Response: We agree. We changed the text accordingly.
L55-58 & L59-60: Are there any contradictions in the description?
Response: Thank you for pointing that out. The description was:
“Green gravel was proposed as an economic and sustainable alternative to current restoration efforts, which were limited by the need for high investment or by the difficulty in both investment and logistics for scaling them up [2,9]. Comparatively, lower investment is needed to produce green gravel, whose systems have a great potential for scaling up”
In our view, the two phrases convey the same essential point and may be somewhat redundant. We agree that the fact that green gravel requires lower investment does not imply that it has no investment needs or cannot be optimized. If that was your concern, we appreciate the opportunity to clarify.
However, we deleted the second sentence and put an economic reference to address another commentary about the affordability of the method.
L79-81: If what is described here is the purpose of this study, the experimental content described in this paper seems extremely poor as a research paper.
Response: Thank you for your comment. We would like to clarify that this manuscript is intended as a brief report rather than a full research paper. The primary goal is to enhance methods for green gravel production. We agree that more extensive experimental research would be necessary if this were a different type of publication.
L81-85: I don't understand this. Please clearly state its relationship to this study.
Response: Thank you for your feedback. We apologize for any confusion. The section in question discusses the potential ecological impact of using gametophytes from natural populations to sow green gravel. Specifically, the reproductive tissue removed for this purpose could reduce the availability of spores in the natural environment, which in extreme cases, might compromise the population density and affect its dynamics. While green gravel has the advantage of not requiring the removal of whole individuals, it's important to consider the potential effects of reduced spore availability on reproductive success.
We hope this clarification helps to better explain the relationship to the study.
L90-92: How long was it maintained?
Response: Thank you for your question. The duration can vary widely, taking anywhere from 2 days to a month, depending on the condition of the donor individuals, which necessitates constant observation. In this particular case, it took 1 week. We have added this specific information to the text for clarity.
L92-94: How long was maturation suppression applied?
Response: Thank you for your question. Maturation suppression was applied long enough to achieve multicellular gametophytes. The time required for this process can vary significantly depending on the conditions of the donor population, so there is no standard duration that can be universally applied. In this specific case, it took 3 months. We have added this information to the text for clarification.
L96: How long ago was it specifically?
Response: Thank you for your question. The action was taken immediately before the next steps in the process. We believe the previous changes to the manuscript have addressed this point and provided the necessary clarity.
L96-98: What specific operation was used to cut them finely? Also, how much gametophyte was used, and how large were the cut finely fragments?
Response: Thank you for your question. To separate the healthy gametophyte mass into smaller fractions, we used a handheld electric blender, as referred to in the text “healthy gametophyte mass was separated into smaller fractions using a handheld electric blender”. Due to the tendency of gametophytes to stick together, it is not feasible to count the exact number of gametophytes within the mass before blending. This is why we performed the counting only afterward. Additionally, since each gametophyte can produce several gametangia, the number of gametophytes alone is less informative. After blending, we considered each fragment as a gametophyte, as it is likely to include only one gametangium, reflecting the functional role it plays in the life cycle.
L99-101: The general reader does not understand why this was used as a substrate. In addition, why were experiments conducted with only these two seeding densities? Was choosing one of these two densities a proposition in previous studies? If the purpose of this study is to evaluate the effect of seeding density, wouldn't a more detailed experimental setup be necessary?
Response: Thank you for your insightful questions. Granite was chosen as a substrate because it is extremely common on rocky shores, particularly along the Portuguese coast, and it is an economical and readily available material, easily sourced from construction material stores. We have added this detail to the manuscript for clarity.
Regarding the seeding densities, we used only two due to logistical limitations. We agree that a more detailed experimental setup with a broader range of densities would be beneficial. However, as this study represents an initial exploration into the effects of seeding density, it was published as a brief report. Further, more comprehensive studies would be necessary to fully evaluate the impact of varying seeding densities.
L106-108: What is ”appropriate water flow”? Please state the reason and the specific water flow value.
Response: Thank you for your comment. By "appropriate water flow," we mean a flow rate that is sufficient to circulate the water effectively without displacing the rocks. In our setup, which is dependent on the tank size, the water flow was set to 950 L/h, as specified by the water pump used. We have added this specific detail to the manuscript for clarity.
L119-120: What is “a suitable size for deployment in the field”? Please state the reason and the specific thallus size.
Response: Thank you for your question. We defined a "suitable size for deployment" as a thallus that is visible to the naked eye but smaller than 1 cm. This size was chosen to ensure that the recruits are small enough to securely attach to the substrate while still being transferred to natural conditions early enough to develop sturdiness under natural hydrodynamic forces. In previous experiments, we observed that larger recruits tend to dislodge and detach from the gravel during deployment, which is why we selected this specific size range. We have added this information to the manuscript for clarity.
L129-: The density of seedlings attached to the substrate (e.g. individuals/cm２) is important in promoting the content of this study. It should be stated in the results or M&M.
Response: Thank you for your comment. We understand the importance of reporting the density of seedlings attached to the substrate. However, since our study focuses on the density of recruits, we have presented and discussed this specific metric in the results. If there is a different aspect of density that you believe should be addressed, we would appreciate further clarification.
L132-134&fig1: What does this "length" mean?
Response: Thank you for your comment. The "length of L. ochroleuca recruits" refers to the measurement from the base to the tip of the blade. We have added this clarification to the methods section for better understanding.
L161-165: What does "three times the size" mean in this study, which lacks specificity about the method and has few evaluation criteria?
Response: Thank you for your comment. We were comparing the results of recruit length between treatments. Specifically, the phrase "three times the size" refers to the average length of recruits in the high-density treatment being nearly three times greater than those in the low-density treatment. If further clarification is needed, we are happy to provide additional details.
L165-167: Are the individuals that were treated at high density really dead? How about describing this in detail, including the process?
Response: Thank you for your question. Laminaria ochroleuca does not have the ability to survive as a free-floating species, unlike Macrocystis. Therefore, by monitoring the number of individuals in a specific area over time, we can infer mortality based on their disappearance. We have relied on this method to assess the survival rate in the high-density treatment.
L186-193: I don't understand the relationship between this description and the contents of this study.
Response: Thank you for your comment. The goal of this brief report is to contribute to the optimization of protocols for scaling up restoration efforts. We included this description to highlight the broader context and significance of our study, specifically why these restoration efforts are necessary and how they align with common restoration plans within the European Union. We believe this context is relevant to understanding the importance of our study.
L194-196: Please clarify the basis by citing references, etc.
Response: Thank you for your feedback. We have added relevant references to support the basis of the information in the revised manuscript.
L196-199: The "affordability" part, including the description in the introduction, is confusing to readers. Please clarify this by calculating the specific costs, etc.
Response: Thank you for your comment. We have added a reference to support our claim regarding affordability. While a detailed economic evaluation was not within the scope of this brief report, we have provided an economic reference in the introduction to offer additional context.
Round 2
Reviewer 3 Report
Comments and Suggestions for Authors
General point
Once again, I would like to point out the thinness of the content of this study. The authors state, "We would like to clarify that this manuscript is intended as a brief report rather than a full research paper. The primary goal is to enhance methods for green gravel production." However, I do not think that the content of this study meets that requirement. To be published in an academic journal, even a short paper should be informative to the readers.
If the reason for choosing the substrate is that it is common in Portugal, and if the poor setting of the seedling densities are due to logistical limitations, then the manuscript should be submitted to a domestic journal in Portugal.
Specific points
L136-: The authors state that "as this study represents an initial exploration into the effects of seeding density," but it is strange that the density of seedlings attached to the substrate was not counted. I understand that the focus is on the density of recruits, but considering the above-mentioned research objective (The primary goal is to enhance methods for green gravel production), it is important to discuss the dropout and death of seedlings, and it is essential to include the counted values.
L172-176: Just because the seedlings have fallen off does not mean they have died. Careful observation and careful discussion are necessary.
Author Response
Dear Reviewer,
Thank you for your continued feedback on our manuscript. We appreciate your time and effort in reviewing our work. We carefully considered your comments from the latest review round and would like to address the concerns you raised.
General Comments:
You mentioned:
"Once again, I would like to point out the thinness of the content of this study. The authors state, 'We would like to clarify that this manuscript is intended as a brief report rather than a full research paper. The primary goal is to enhance methods for green gravel production.' However, I do not think that the content of this study meets that requirement. To be published in an academic journal, even a short paper should be informative to the readers."
We understand your interest in more comprehensive information. However, we think that the scope of our manuscript is consistent with the intended purpose of a brief report. Our primary aim was to enhance methods for green gravel production, and we believe the results provided align with this objective. Moreover, the amount of information included is comparable to other research papers on green gravel production, several of which are cited in our manuscript. In this context, we are unsure what additional information should be included or which is the reference point to assert our report is not informative enough.
You also commented:
"If the reason for choosing the substrate is that it is common in Portugal, and if the poor setting of the seedling densities are due to logistical limitations, then the manuscript should be submitted to a domestic journal in Portugal."
We respectfully disagree with this assessment. While there were, of course, other options to choose from, the reason we chose this particular substrate instead of other common ones was its availability and cost, which we argue in our response, while also considering its adequacy to our particular location, which we don’t find takes any value from the results. Granite is, of course, a widely used and natural substrate on rocky shores, not at all limited to Portugal. Moreover, in our literature review on green gravel, we have never encountered a requirement to justify the use of common and natural rocky substrates, especially one that has been widely used, particularly by the pioneers of the green gravel technique (Fredriksen et al., 2020).
Regarding the reviewer’s point on the “poor setting of the seeding densities”, we would argue that similar experiments published in peer-reviewed, high-impact journals typically test between two and three seeding densities (e.g. Alsuwaiyan et al. 2022), and have been cited in our manuscript. It is thus unclear what the basis of the reviewer is to state that our approach is inadequate.
Specific Comments:
You raised the following point:
"L136: The authors state that 'as this study represents an initial exploration into the effects of seeding density,' but it is strange that the density of seedlings attached to the substrate was not counted. I understand that the focus is on the density of recruits, but considering the above-mentioned research objective (The primary goal is to enhance methods for green gravel production), it is important to discuss the dropout and death of seedlings, and it is essential to include the counted values."
We respectfully disagree with this point, as the manuscript does include data on the density of seedlings attached to the substrate. These results are presented in the figures and discussed in detail within the text. We would be grateful for specific guidance on any areas you feel require further elaboration.
Additionally, you mentioned:
"L172-176: Just because the seedlings have fallen off does not mean they have died. Careful observation and careful discussion are necessary."
As we explained in our previous response, Laminaria species, unlike Macrocystis, do not survive once they are dislodged. This is a well-documented aspect of their biology. Our conclusions are based on this understanding, and we do not have additional observational data to address this point further.
While we value your feedback and have carefully reviewed your comments, we currently do not have additional data to comply with your requests. We hope the explanations provided clarify our approach and address your concerns to the extent possible.
Given these circumstances, we respectfully leave the final decision regarding the suitability of our manuscript for publication to the editor.
