Artificial Reefs Reduce Morbidity and Mortality of Small Cultured Sea Cucumbers Apostichopus japonicus at High Temperature

Round 1

Reviewer 1 Report

It is an interesting research that deserves consideration after some altrerations and explanations. 

The major comments are:

1. you need to include in the title that it is a bioassay (in the lab) with cultured specimens.

2. You need to raise some hypothesis at the end of the Introduction section.

3. Experimental design is the major issue here. You have several interferences that were not evaluated. how did you test the effect of the artifact? you should use these small artifical habitats in the field. You have a lot of intereferences that could bias your data: acclimatation, confined space, different temperature from the natural habitat, transportation. You need to evaluate this. You need to explain how did you deal with these potential external interference

4. did you compare this experiment in winter months to see the temperature plus handling effect? this is the way to confirm your hypothesis.

5. these two stressful effects need to be separated: handling and air exposure. Then you will know if one or both are responsible for your results. Besides, if the individuals are in the beginning of the disease and able to crawl (to the artificial reefs), then they will be in contact with the healthy ones and disseminate the disease, no?

6. You need to improve your discussion, have a robust comparative analysis. 

7. Please, include the weaknesses of your study at the beginning of your discussion.

 

Comments for author File: Comments.pdf

Author Response

General comments:

It is an interesting research that deserves consideration after some altrerations and explanations.

Response:

We appreciate your comments, which were very helpful for us to improve the ms. Based on your comments, we carefully revised the whole manuscript. Based on your comments, we carefully revised the whole manuscript, especially the Abstract, Introduction, Materials and methods, and Discussion. Further, we asked Wei Tang, a professional translator who works at Alibaba, to edit this manuscript to ensure the English quality of the manuscript. You can find our endeavor through the revised manuscript. We believe the current version of the ms is now ready for publication in Journal of Marine Science and Engineering. Or, we shall be happy to take any further revisions if necessary.

 

Comment 1: you need to include in the title that it is a bioassay (in the lab) with cultured specimens

Response:

Corrected. The current title is “Artificial reef reduces morbidity and mortality of small cultured sea cucumbers Apostichopus japonicus at high temperature”. Please see lines 1–2.

 

Comment 2: You need to raise some hypothesis at the end of the Introduction section.

Response:

We added the suggested content in the manuscript. Please see lines 75–77.

 

Comment 3: Experimental design is the major issue here. You have several interferences that were not evaluated. how did you test the effect of the artifact? you should use these small artificial habitats in the field. You have a lot of intereferences that could bias your data: acclimatation, confined space, different temperature from the natural habitat, transportation. You need to evaluate this. You need to explain how did you deal with these potential external interference

Response:

We sincerely appreciate your advice. Here we explain some of your concerns. Related information was added in the revised ms.

The effect of the artifact was performed before the experiment. Artificial reef consists of seven plastic cylinders, in which only one sea cucumber is accessible. Related information was added in the revised ms.

The main aim of this experiment is based on the aquaculture of sea cucumbers in land-based factories (seed production). So far we have only done laboratory experiment, not field experiment.

The natural habitat and the laboratory are at the same latitude and in close proximity, so the effects of traffic and temperature on sea cucumbers are not that large. After being transported to the laboratory, sea cucumbers are cultured for two weeks to adapt to the experimental environment. Please see lines 94–95.

We have added the information of the culture density in the manuscript. Please see lines 91–92. In order to reduce the effect of high density on the experiment, lower culture density (400 g/m²) was adopted in the present study (Pei et al., 2012; Xu et al., 2017).

[1] Pei, S., Dong, S., Wang, F., Tian, X., Gao, Q., 2012. Effects of density on variation in individual growth and differentiation in endocrine response of Japanese sea cucumber (Apostichopus japonicus Selenka). Aquaculture 356–357, 398–403. http://dx.doi.org/10.1016/j.aquaculture.2012.04.032

[2] Xia, B., Ren,Y., Wang, Y., Sun, Y., Zhang, Z., 2017. Effects of feeding frequency and density on growth, energy budget and physiological performance of sea cucumber Apostichopus japonicus (Selenka). Aquaculture 466, 26–32. http://dx.doi.org/10.1016/j.aquaculture.2016.09.039

 

Comment 4: did you compare this experiment in winter months to see the temperature plus handling effect? this is the way to confirm your hypothesis

Response:

Thank you for this comment. It would have been interesting to explore this aspect. However, in our study, the main objective is to solve the survival of sea cucumbers at high temperature in summer, so winter is not considered.

 

Comment 5: these two stressful effects need to be separated: handling and air exposure. Then you will know if one or both are responsible for your results. Besides, if the individuals are in the beginning of the disease and able to crawl (to the artificial reefs), then they will be in contact with the healthy ones and disseminate the disease, no?

Response:

We apologize for the unclearness. In experiment I, sea cucumber was only affected by air exposure and high temperature. Relevant content was revised in the manuscript.

Only one sea cucumber can get into each crevice in the artificial reef. When unaffected sea cucumbers entered the crevice, those in the early stages of disease could only reach the outside of the reef, unable to enter the crevice.

 

Comment 6: You need to improve your discussion, have a robust comparative analysis.

Response:

Thank you for the comments. We carefully revised the whole Discussion. Especially, we clarified the significant findings. Artificial reefs significantly improved survival and fitness-related of small A. japonicus after air exposure and disease outbreak at high temperature. Sea cucumbers with better movement probably actively enter inside artificial reefs, thereby reducing frequent physical contact between healthy and problematic individuals, and decreasing the likelihood of the disease transmission.

 

Comment 7: Please, include the weaknesses of your study at the beginning of your discussion.

Response:

Specific content has been updated. Please see line 352–353.

Reviewer 2 Report

The manuscript “Artificial reef reduces morbidity and mortality of small sea cucumbers Apostichopus japonicus at high temperature” presents a relevant topic dealing with a particular problem on the viability of early life stages, which is crucial for a successful and optimized rearing of this species. However, I have several major concerns, mainly regarding the methodology that is, currently, not reproducible. Many of the procedures and quantifications are not described and other I am not sure were conducted (please check the comments below). The part of the objectives, that aim to assess disease transmission, are also not addressed in the experiments. The authors mention disease transmission (one of the objectives) and the whole experiment is based on the control of contact between healthy and affected sea cucumbers (Exp II), but the authors never state what is the condition “disease” and how was it determined. Never once the authors addressed and assessed disease transmission, as indicated in the objectives of the study. The manuscript also needs a deep revision in English because many of the ideas and sentences regarding procedures are confusing.

 

General comments

 

 

Abstract – needs to be re-written. It should have a sequential structure, with intro, objective, m&m (essencials) results and conclusions. This abstract has a confusing structure and goes back and forth in results and conclusions. Also, as is, the interpretations and conclusions are speculative, but that is mainly related to the issues the study has (see below). Besides, the English needs a deep revision.

 

 

Introduction

 

Since temperature is the central issue in this article, the introduction must describe why high temperature is a problem for early A. japonicus juveniles and what is considered a high temperature for this species. What are the common constraints and resulting problems? Hatcheries cannot control room/water temperature in the summer? If the authors do not provide more information it feels like a random factor to be analysed.

 

 

Specific comments

 

Introduction

Ln42 – replace stresses by stressors.

 

Throughout the text replace “problematic”, which is a wrong term for this, by something disease related, like “affected” or “infected” (because your hypothesis is based on disease transmission) or other term you find more suitable.

 

Ln49 – “Thus, sea cucumbers…”

 

It would be more easy to understand the objectives if these were written by aggregation of similar experiments. Set a common ground for the three experiments (e.g. A. japonicus juveniles at high temperature settings and morbidity and mortality decrease) and then describe the specific goals. It would be easier to read and understand.

 

M&M

 

Section 2.1 – what parameters were measured? There is no indication of nitrogen compounds that are crucial to control for a good physiological condition for the trials. This is particularly relevant during the trials and is not described, even with daily water exchanges. High ammonium or nitrites concentrations would preclude any interpretation from the effect of the treatment in the experiments.

A brief indication of diet composition is also relevant for reproducibility of the study.

 

Section 2.2 – Are the fitness related behaviours a proxy for morbidity? How do you define it?

 

Ln87 – Was it three replicates per condition?

Lines87, 89, 97 – You should replace the word devices. Tank?

 

For this and the other experiments you should mention why density is not a potential confounding effect. Your density appears to be ok, way below 1kg/m2 (half of that, in fact), so it should be mentioned and the choice for that density supported by bibliography.

 

Did the sea cucumbers go through a fasting period before the trial? Were the animals fed during the trial?

 

Describe was used as an “artificial reef”.

 

What are considered healthy animals that, apparently, can be identified immediately?

 

Ln97-100 – Animals of a given condition were TRANSFERRED to different containers/tanks, correct? Please clarify to facilitate interpretation of the text. Also, please state that (or if) the replicates structure was respected and if these were analysed independently. Only at the end of this section we can find a reference to that but it’s not clear.

 

Fig1. There is a legend for “Inside the artificial reef”, but no such image (transparent cylinder with a sea cucumber) is found in the figure.

 

 

Section 2.3 – This section has the exact same detail problems of the previous sections. Again, what are healthy individuals? Were there “healthy individuals inside and outside the plastic cylinders? It’s hard to follow.

 

The analysis and quantification of fitness related behaviours made (feeding, crawling, and adhesion) should be sections that come before the experiments.

 

Section 2.4

Ln137-138 – What do you mean by “Six sea cucumbers were selected from each group and were placed into cubic plastic devices”? From which group, which experiment…?

 

Ln139 – “Sufficient food” is a very vague term for a scientific experiment.

 

 Section 2.5 – Again “Six sea cucumbers were selected from each group and were placed into cubic plastic devices”. The experiments indicate that 18 individuals “…were randomly selected from each of the three cubic plastic devices for the determination of three behaviors…”.

 

 

Ln161 – Please, indicate the precision of the weight measurements.

 

Ln162 – Why is “Adhesion index” represented as AR and not Ai?

 

I see nowhere in the methodology what was considered morbidity. What parameters were taken for that and how was a percentage estimated?

 

Results

 

Legend of Fig2 – “Letters indicate significant differences…” but there are no letters, only “**”. Correct every other figure legend.

 

Fig3 – It is the first time the term “control group” is mentioned. These terms must be defined in the methods. Legend: No letters to indicate differences. There are three levels of “*” that must be explained.

 

Authors state that “Consistently, tentacles activity frequency was not significantly different in sea cucumbers between outside and inside of the artificial reef”. First, how is it consistent? Second, I suspect some problem may have occurred with the data. The mean value and the SD are lower in “outside” then it is in the control group. If there is a difference between “inside” and control, there must be a difference between “inside” and “outside”. Something does not add-up here. We find the same problem for the adhesion analysis but here the differences are even higher! Statistical analyses must be redone.

 

Sections 3.2.2 and 3.2.4 has the same statistical issues as the previous one, seen in Fig5 A and C. One other possible explanation is that the authors indicate SD as a dispersion measure but used, in fact, SE.

 

Discussion

 

English issues and misused terms (e.g. whereas in ln259)

 

Ln 261 – In the discussion should not refer to the control group, but to the condition itself. It facilitates the comprehension in studies with more than one experiment or tested conditions.

 

Considered all the problems mentioned above, the discussion has a serious weakness regarding the interpretation of the results. Statements like “Disease challenge assays were conducted” and subsequent interpretations become unfounded in this context. Mortality and morbidity (which was not defined) are not necessarily a result of a pathogenic disease (which was defined as an objective when the authors aimed to assess disease transmission). Likewise, “pathogenic bacterial communities” was not assessed as a cause of morbidity or death.

 

 

Considering the information available I find it more likely that individuals that were able to seek shelter in the cylinders already had a favourable condition before the beginning of the trials, which made them better fitted to deal with the adverse conditions of the trials.

Author Response

General comments

The manuscript “Artificial reef reduces morbidity and mortality of small sea cucumbers Apostichopus japonicus at high temperature” presents a relevant topic dealing with a particular problem on the viability of early life stages, which is crucial for a successful and optimized rearing of this species. However, I have several major concerns, mainly regarding the methodology that is, currently, not reproducible. Many of the procedures and quantifications are not described and other I am not sure were conducted (please check the comments below). The part of the objectives, that aim to assess disease transmission, are also not addressed in the experiments. The authors mention disease transmission (one of the objectives) and the whole experiment is based on the control of contact between healthy and affected sea cucumbers (Exp II), but the authors never state what is the condition “disease” and how was it determined. Never once the authors addressed and assessed disease transmission, as indicated in the objectives of the study. The manuscript also needs a deep revision in English because many of the ideas and sentences regarding procedures are confusing.

Response:

We appreciate your comments, which were very helpful for us to improve the ms. Based on your comments, we carefully revised the whole manuscript, especially the Abstract, Introduction, Materials and methods, and Discussion. We have added some experimental details and have a clear definition of "diseased sea cucumbers". Further, we asked Wei Tang, a professional translator who works at Alibaba, to edit this manuscript to ensure the English quality of the manuscript. We believe the current version of the ms is now ready for publication in Journal of Marine Science and Engineering. Or, we shall be happy to take any further revisions if necessary.

 

General comments 1:

Abstract – needs to be re-written. It should have a sequential structure, with intro, objective, m&m (essencials) results and conclusions. This abstract has a confusing structure and goes back and forth in results and conclusions. Also, as is, the interpretations and conclusions are speculative, but that is mainly related to the issues the study has (see below). Besides, the English needs a deep revision.

Response:

Thank you for the comments. We tried our best to improve the abstract according to your suggestions. Please see lines 9–28.

 

General comments 2:

introduction

Since temperature is the central issue in this article, the introduction must describe why high temperature is a problem for early A. japonicus juveniles and what is considered a high temperature for this species. What are the common constraints and resulting problems? Hatcheries cannot control room/water temperature in the summer? If the authors do not provide more information it feels like a random factor to be analysed.

Response:

Many thanks for the valuable suggestions. 25 °C is the threshold for severe heat stress in sea cucumbers, leading to a significantly increased risk of death and disease from sea cucumbers. It is very expensive to regulate the water temperature in seed production. More detailed information was added in the ms. Please see lines 39–43.

 

Comments 1: Ln42 – replace stresses by stressors.

Response:

The sentence was removed during revision.

 

Comments 2: Throughout the text replace “problematic”, which is a wrong term for this, by something disease related, like “affected” or “infected” (because your hypothesis is based on disease transmission) or other term you find more suitable.

Response:

We replaced “problematic” with “affected”.

 

Comments 3: Ln49 – “Thus, sea cucumbers…”

It would be more easy to understand the objectives if these were written by aggregation of similar experiments. Set a common ground for the three experiments (e.g. A. japonicus juveniles at high temperature settings and morbidity and mortality decrease) and then describe the specific goals. It would be easier to read and understand.

Response:

As suggested by the reviewer, we rewrote this part in the revised ms. Please see lines 78–84.

 

Comments 4: Section 2.1– what parameters were measured? There is no indication of nitrogen compounds that are crucial to control for a good physiological condition for the trials. This is particularly relevant during the trials and is not described, even with daily water exchanges. High ammonium or nitrites concentrations would preclude any interpretation from the effect of the treatment in the experiments.

A brief indication of diet composition is also relevant for reproducibility of the study.

Response:

Thank you for the comments. Dissolved oxygen, pH and salinity were measured. More detailed information can be found in the revised ms. Please see lines 96–98.

We agree on the importance of nitrogen compounds for the experiment. However, due to the experimental conditions, we did not determine the content of nitrogen compounds in aquaculture water. In the future, we will pay more attention to this point.

We supplemented the information on dietary composition, and please see lines 98–100, 112–114, 148–149, 175–176, 185–186.

 

Comments 5: Section 2.2 – Are the fitness related behaviours a proxy for morbidity? How do you define it?

Response:

Clarified. Fitness-related behaviors reflect the state of the sea cucumbers and are not a proxy for morbidity. We identified the sea cucumber without ulcerated skin and has better fitness-related behaviors as an unaffected individual. Morbidity and mortality calculations are added in the revised manuscript, and please see lines 158–167.

 

Comments 6: ln87 – Was it three replicates per condition?

Lines 87, 89, 97 – You should replace the word devices. Tank?

Response:

Corrected. It is six replicates per condition. Please see lines 107–109.

Corrected. We replaced “devices” with “boxes”.

 

Comments 7: For this and the other experiments you should mention why density is not a potential confounding effect. Your density appears to be ok, way below 1kg/m2 (half of that, in fact), so it should be mentioned and the choice for that density supported by bibliography.

Response:

Thank you for the comments. We have added information about culture density in the manuscript. Please see lines 90–92.

As an environmental stress factor, density affects the survival, growth, and behaviors of sea cucumbers. The main aim of this study was to explore the effects of air exposure and disease on sea cucumbers at high temperature, hence it was necessary to exclude density effects. When the density is greater than 525 g/m², the energy intake of sea cucumbers decreases with the increase in density [1]. Thus, the sea cucumber at the present experimental density (400 g/m²) may have more energy to mitigate the effects of heat, air exposure, and disease.

[1] Xia, B., Ren,Y., Wang, Y., Sun, Y., Zhang, Z., 2017. Effects of feeding frequency and density on growth, energy budget and physiological performance of sea cucumber Apostichopus japonicus (Selenka). Aquaculture 466, 26–32. http://dx.doi.org/10.1016/j.aquaculture.2016.09.039

 

Comments 8: Did the sea cucumbers go through a fasting period before the trial? Were the animals fed during the trial? 

Response:

Sea cucumber was not fasted before the trial and were fed ad libitum food every day during the trial.

 

Comments 9: Describe was used as an “artificial reef”.

Response:

We apologize for unclear descriptions. Artificial reef ((length × width × height: 50 × 55 × 60 mm) consists of seven plastic cylinders (diameter × length: 15 × 50 mm), in which only one sea cucumber is accessible. Please see lines 105–107, figure 1A and 1B.

 

Comments 10: What are considered healthy animals that, apparently, can be identified immediately?

Response:

In the revised manuscript, we replaced “healthy” with “without ulcerated skin”. We identified the sea cucumber without ulcerated skin and has good fitness-related behaviors as an unaffected individual, and without ulcerated skin and has poor fitness-related behaviors as an affected individual. The ulcerated skin of the sea cucumber can be recognized immediately. Please see figure 1E.

 

Comments 11: Ln97-100 – Animals of a given condition were TRANSFERRED to different containers/tanks, correct? Please clarify to facilitate interpretation of the text. Also, please state that (or if) the replicates structure was respected and if these were analysed independently. Only at the end of this section we can find a reference to that but it’s not clear.

 Response:

After the experiment, individuals without ulcerated body walls in group O were divided into two groups, among which sea cucumbers in one group were inside the artificial reefs and sea cucumbers in the other group were outside the artificial reefs. Then, the two groups were placed in two different cubic plastic boxes (top length × bottom length × height: 20 × 16.5 × 14 cm). Finally, we obtained three groups of sea cucumbers without ulcerated body walls, which were the ones from group C, the ones inside the artificial reefs in group O, and the ones outside the artificial reefs in group O.  The above-mentioned three groups of sea cucumbers had six replicate sets, respectively. Each of the subsequent behavioral experiments was performed in six replicates and analyzed independently.

 

Comments 12: Fig 1. There is a legend for “Inside the artificial reef”, but no such image (transparent cylinder with a sea cucumber) is found in the figure.

 Response:

We apologize for unclear descriptions. As suggested by the reviewer, we revised the manuscript accordingly. Please see figure 1A.

 

Comments 13: Section 2.3 – This section has the exact same detail problems of the previous sections. Again, what are healthy individuals? Were there “healthy individuals inside and outside the plastic cylinders? It’s hard to follow.

Response:

This is an excellent suggestion. The same details section was deleted in the revised manuscript. In the revised manuscript, we replaced “healthy” with “without ulcerated skin”. We identified the sea cucumber with ulcerated skin as diseased individuals. The ulcerated skin of the sea cucumber can be recognized immediately. After the experiment, sea cucumbers without ulcerated body walls were placed in plastics boxes. We suppose the revised ms is better for understanding.

[1] Deng, H.; He, C.; Zhou, Z.; Liu, C.; Tan, K.; Wang, N.; Jiang, B.; Gao, X.; Liu, W. Isolation and pathogenicity of pathogens from skin ulceration disease and viscera ejection syndrome of the sea cucumber Apostichopus japonicus. Aquaculture, 2009, 287, 18–27.[2] Wang, Y.G.; Rong, X.J.; Zhang, C.Y.; Sun, S.F Main diseases of cultured Apostichopus japonicus: prevention and treatment. Marine Sciences 2005, 9(3), 1–7. (in Chinese with an English abstract)

 

Comments 14: The analysis and quantification of fitness related behaviours made (feeding, crawling, and adhesion) should be sections that come before the experiments.

 Response:

Thank you for this suggestion. All sea cucumbers in the experiment were randomly selected from the same batch of seedlings and they were co-cultured for two weeks to acclimate to the experimental environment. Thus, we suppose that there is no significant behavioral difference between them before the experiment. This is commonly accepted in a number of similar studies on sea cucumbers (for example, Ding et al., 2019; Clement et al., 2020;).

[1] Ding, K., Zhang, L., Zhang, T., Yang, H., Brinkman, R. The effect of melatonin on locomotor behavior and muscle physiology in the sea cucumber Apostichopus japonicus. Front. Physiol., 2019, 10, 221.[2] Clement, J.C., Schagerström, E., Dupont, S., Jutfelt, F., Ramesh, K., 2020. Roll, right, repeat: short-term repeatability in the self-righting behaviour of a cold-water sea cucumber. J Mar. Bio. Assoc. UK, 2020, 100, 115–120.

[2] Clement, J.C., Schagerström, E., Dupont, S., Jutfelt, F., Ramesh, K., 2020. Roll, right, repeat: short-term repeatability in the self-righting behaviour of a cold-water sea cucumber. J Mar. Bio. Assoc. UK 2020, 100, 115–120.

 

Comments 15: Section 2.4

Ln137-138 – What do you mean by “Six sea cucumbers were selected from each group and were placed into cubic plastic devices”? From which group, which experiment…?

 Response:

Crawling, feeding, and adhesion behaviors were measured at the end of air exposure and disease outbreak experiments.

After the experiment, individuals without ulcerated body walls in group O were divided into two groups, among which sea cucumbers in one group were inside the artificial reefs and sea cucumbers in the other group were outside the artificial reefs. Then, the two groups were placed in two different cubic plastic boxes (top length × bottom length × height: 20 × 16.5 × 14 cm). Finally, we obtained three groups of sea cucumbers without ulcerated body walls, which were the ones from group C, the ones inside the artificial reefs in group O, and the ones outside the artificial reefs in group O. The above-mentioned three groups of sea cucumbers had six replicate sets, respectively.

Three sea cucumbers were randomly selected from each group for crawling behavior test, and each group has six parallels. We clarified it in the revised ms. Please see lines 119–129, 173–175.

 

Comments 16: Ln139 – “Sufficient food” is a very vague term for a scientific experiment.

Response:

We replaced “ad libitum” with “sufficient”. Please see lines 99 and 148.

 

Comments 17: Section 2.5 – Again “Six sea cucumbers were selected from each group and were placed into cubic plastic devices”. The experiments indicate that 18 individuals “…were randomly selected from each of the three cubic plastic devices for the determination of three behaviors…”.

Response:

We apologize for unclear descriptions and incorrect writing. After the experiment, individuals without ulcerated body walls in group O were divided into two groups, among which sea cucumbers in one group were inside the artificial reefs and sea cucumbers in the other group were outside the artificial reefs. Then, the two groups were placed in two different cubic plastic boxes (top length × bottom length × height: 20 × 16.5 × 14 cm). Finally, we obtained three groups of sea cucumbers without ulcerated body walls, which were the ones from group C, the ones inside the artificial reefs in group O, and the ones outside the artificial reefs in group O. The above-mentioned three sets of sea cucumbers had six replicates, respectively. Three sea cucumbers were randomly selected from each parallel group for behavior test. 3 individuals × 6 replicates = 18 individuals per group.

 

Comments 18: Ln161 – Please, indicate the precision of the weight measurements.

 

Ln162 – Why is “Adhesion index” represented as AR and not Ai?

 

I see nowhere in the methodology what was considered morbidity. What parameters were taken for that and how was a percentage estimated?

Response:

Data are accurate to one decimal place. Please see lines 119–200.

Corrected. Adhesion index represented as A_i in the revised ms. Please see line 200.

Morbidity and mortality calculations are added in the revised manuscript, and please see line 158–167.

 

Comments 19: Legend of Fig2 – “Letters indicate significant differences…” but there are no letters, only “**”. Correct every other figure legend.

Response:

We apologize for the mistake. Relevant content was corrected in the manuscript. Please see lines 220–221, 232–233, 259–260, 273–274.

 

Comments 20: Fig3 – It is the first time the term “control group” is mentioned. These terms must be defined in the methods. Legend: No letters to indicate differences. There are three levels of “*” that must be explained.

Response:

We apologize for the mistake. Relevant content was corrected in the manuscript. Information about the control group was explained in the legend. Please see lines 220–221, 232–233, 259–260, 273–274.

 

Comments 21: Authors state that “Consistently, tentacles activity frequency was not significantly different in sea cucumbers between outside and inside of the artificial reef”. First, how is it consistent? Second, I suspect some problem may have occurred with the data. The mean value and the SD are lower in “outside” then it is in the control group. If there is a difference between “inside” and control, there must be a difference between “inside” and “outside”. Something does not add-up here. We find the same problem for the adhesion analysis but here the differences are even higher! Statistical analyses must be redone.

Sections 3.2.2 and 3.2.4 has the same statistical issues as the previous one, seen in Fig5 A and C. One other possible explanation is that the authors indicate SD as a dispersion measure but used, in fact, SE.

Response:

We apologize for the mistake. Data for this experiment were taken as the mean ± S.E., not the standard deviation. Relevant content is corrected in the manuscript. Please see lines 220–221, 232–233, 259–260, 273–274.

Clarify. We scrutinized the results and found that standard deviation of the data was very large to result in no significant differences in the behavior of sea cucumbers cultured inside and outside the artificial reefs.

 

Comments 22: English issues and misused terms (e.g. whereas in ln259)

 

Ln 261 – In the discussion should not refer to the control group, but to the condition itself. It facilitates the comprehension in studies with more than one experiment or tested conditions.

 

Considered all the problems mentioned above, the discussion has a serious weakness regarding the interpretation of the results. Statements like “Disease challenge assays were conducted” and subsequent interpretations become unfounded in this context. Mortality and morbidity (which was not defined) are not necessarily a result of a pathogenic disease (which was defined as an objective when the authors aimed to assess disease transmission). Likewise, “pathogenic bacterial communities” was not assessed as a cause of morbidity or death.

 

Considering the information available I find it more likely that individuals that were able to seek shelter in the cylinders already had a favourable condition before the beginning of the trials, which made them better fitted to deal with the adverse conditions of the trials.

Response:

We feel great thanks for your professional review work on our article. As you are concerned, there are several problems that need to be addressed. According to your nice suggestions, we have made extensive corrections to our previous draft. We have added the definitions of mortality and morbidity in the Materials and methods. We further discussed the Discussion and revised the interpretation of the results. In addition, we asked Wei Tang, a professional translator who works at Alibaba, to edit this manuscript to ensure the English quality of the manuscript. All sea cucumbers in the experiment were randomly selected from the same batch of seedlings and they were co-cultured for two weeks to acclimate to the experimental environment. Thus, we suppose that there is no significant behavioral difference between them before experiment. A number of similar studies have this concern as well (for example, Ding et al., 2019; Clement et al., 2020;).

[1] Ding, K., Zhang, L., Zhang, T., Yang, H., Brinkman, R. The effect of melatonin on locomotor behavior and muscle physiology in the sea cucumber Apostichopus japonicus. Front. Physiol., 2019, 10, 221.[2] Clement, J.C., Schagerström, E., Dupont, S., Jutfelt, F., Ramesh, K., 2020. Roll, right, repeat: short-term repeatability in the self-righting behaviour of a cold-water sea cucumber. J Mar. Bio. Assoc. UK, 2020, 100, 115–120.

[2] Clement, J.C., Schagerström, E., Dupont, S., Jutfelt, F., Ramesh, K., 2020. Roll, right, repeat: short-term repeatability in the self-righting behaviour of a cold-water sea cucumber. J Mar. Bio. Assoc. UK 2020, 100, 115–120.

Reviewer 3 Report

This paper aims to evaluate effect of artificial reef on morbidity, mortality and behaviors of Apostichopus japonicus treated by high temperature. The study is generally well designed and shown. However, novelty of this study is not clearly explained, especially in relation to a previous paper by the same research team (i.e., Tian et al. 2023 Aquaculture 738755). Tian et al. (2023) has very similar experimental design to the present paper. The study theme is also very similar, namely negative effect of high temperature. Parameters such as crawling, feeding, adhesion behaviors are common to Tian et al. (2023). Tian et al. (2023) already noted that artificial reefs improve behaviors of A. japonicus. Therefore, it is very unclear why the present study is significant, at least in the present state of the paper, which lacks detailed explanation. In Introduction, the authors should explain what has done and what has not been done by Tian et al. (2023), and what can be additionally done by the present study. These should be also clearly noted in Discussion. Were some data used from Tian et al. (2023)? These are important to avoid criticism of plagiarism.

Methodological description lacks some critical information for reproducibility, which will be mentioned later.

Minor comments are shown in the following.

L11 "physical contact": What to what?

L12 Why is it environmentally friendly?

L12-13 Why handling stressess probably worsen the impact of high temperature? Is it shown by previous studies or hypothesis by the present study?

L71 A. japonicus has green and red types, which has been suggested as different species by a taxonomic study (Woo et al. 2017 https://doi.org/10.11646/zootaxa.4350.1.7). However, no other studies support this taxonomy at present. Thus, I suggest to use species name "Apostichopus japonicus" in the present paper but also to clarify which type (i.e., green or red) was used in the experiment.

L76 Why temperature of 25C was used? Is it known temperature of high-temperature mortality etc for this sea cucumber?

L78 Details of seawater used in the experiments should be provided: Filter type, UV-treatment etc.

L80 "detection" should be deleted.

L82 "diet": catalogue number? Also better to note major components (e.g., seaweed, mud).

L83 No information is provided about "artificial reefs". Material, shape, size should be described as well as how they were set in tanks.

Figure 1 Is this figure reproduced from Tian et al. (2023)?

L114 Size description seems different from that shown in Figure 1.

L117 Condition of "diseased" is unclear.

L139 and L149 Please describe the food type.

L149 "crawling": Is it "feeding" correct here?

L164 I suggest to modify the adhesion index, because body weight works to detach sea cucumber from the wall in this experiment. That is, large sea cucumber tends to detach earlier than smaller one due to the heavy body. If heavier and lighter sea cucumbers stay on the wall during the same time, heavier one should have higher adhesion index. Thus, Ai=TW is better. But larger sea cucumber has larger ambulacral area, which increases adhesion, which could be roughly expressed as W^2/3. If the effect of ambulacral area needs to be uniformed, then, the formula may be Ai=T*W^1/3.

Figure 3 and 5 "Adhension" in y-axis should be "Adhesion"

L240-247 This part of text seems redundant and may be more simply described in a sentence.

Author Response

General comments

This paper aims to evaluate effect of artificial reef on morbidity, mortality and behaviors of Apostichopus japonicus treated by high temperature. The study is generally well designed and shown. However, novelty of this study is not clearly explained, especially in relation to a previous paper by the same research team (i.e., Tian et al. 2023 Aquaculture 738755). Tian et al. (2023) has very similar experimental design to the present paper. The study theme is also very similar, namely negative effect of high temperature. Parameters such as crawling, feeding, adhesion behaviors are common to Tian et al. (2023). Tian et al. (2023) already noted that artificial reefs improve behaviors of A. japonicus. Therefore, it is very unclear why the present study is significant, at least in the present state of the paper, which lacks detailed explanation. In Introduction, the authors should explain what has done and what has not been done by Tian et al. (2023), and what can be additionally done by the present study. These should be also clearly noted in Discussion. Were some data used from Tian et al. (2023)? These are important to avoid criticism of plagiarism.

Methodological description lacks some critical information for reproducibility, which will be mentioned later.

Response:

We appreciate your valuable suggestions. Your concerns were revised in the Introduction and Discussion of the revised ms.

The main aims of the present study are to investigate whether artificial reefs improve the survival of sea cucumbers exposed to air exposure and disease outbreak at high temperature. The study of Tian et al., on the other hands was to explore whether artificial reefs improve the digestion and fitness-related behaviors of sea cucumbers in summer. Our experimental data are completely different from Tian et al. The present study found that artificial reef distinguished unaffected individuals from both affected and diseased individuals and in turn reduced the mortality and morbidity of sea cucumbers at high temperature.

Related information was added in the revised ms. Please see lines 72–73, 303–305.

 

Comment 1: L11 "physical contact": What to what?

Response:

When unaffected sea cucumbers (without ulcerated skin and good fitness-related behaviors) entered the artificial reefs, the affected (without ulcerated skin and poor fitness-related behaviors) and diseased (with ulcerated skin) sea cucumbers were unable to make physical contact with each other.

 

Comment 2:  Why is it environmentally friendly?

Response:

Correct. We changed the environmental to cost-effective. Please see line 27.

 

Comment 3: L12-13 Why handling stressess probably worsen the impact of high temperature? Is it shown by previous studies or hypothesis by the present study?

Response:

Yes. Tian et al., (2023) found that handling stress deteriorates the adverse effects on fitness-related behavior of A. japonicus at a high temperature. Related information was added and cited in the revised ms.

[1] Tian, R.; Hu. F.; Wu, G.; Wang, H.; Ding, J.; Chang, Y.; Zhao, C. An effective approach to improving fitness-related behavior and digestive ability of small sea cucumbers Apostichopus japonicus at high temperature: New insights into seed production. Aquaculture 2023, 562, 738755. https://doi.org/10.1016/j.aquaculture.2022.738755

 

Comment 4: L71 A. japonicus has green and red types, which has been suggested as different species by a taxonomic study (Woo et al. 2017 https://doi.org/10.11646/zootaxa.4350.1.7). However, no other studies support this taxonomy at present. Thus, I suggest to use species name "Apostichopus japonicus" in the present paper but also to clarify which type (i.e., green or red) was used in the experiment.

Response:

Clarified. Green Apostichopus japonicus were used in this experiment. Please see line 87.

 

Comment 5: L76 Why temperature of 25C was used? Is it known temperature of high-temperature mortality etc for this sea cucumber?

Response:

It is known that this temperature is regarded as the threshold for severe heat stress at 25 °C (Li et al., 2019). Related information was added and cited in the revised ms. Please see lines 39–43.

[1] Li, C., Feng, H., Xu, D., 2019. Effect of seasonal high temperature on the immune response in Apostichopus japonicus by transcriptome analysis. Fish and Shellfish Immunology, 92, 765–771. https://doi.org/10.1016/j.fsi.2019.07.012

 

Comment 6: L78 Details of seawater used in the experiments should be provided: Filter type, UV-treatment etc.

Response:

Seawater was extracted directly from the ocean and then filtered by sand. Related information was added in the revised ms. Please see lines 95–96, 114.

 

Comment 7: L80 "detection" should be deleted.

Response:

Corrected. Please see lines 97.

 

Comment 8: L82 "diet": catalogue number? Also better to note major components (e.g., seaweed, mud).

Response:

Sea cucumbers were fed a mixture of sea mud and commercial feed (Anyuan Industrial Co., Ltd.) at a 1:4 ratio every day. Related information was added in the revised ms. Please see lines 98–100, 112–114, 148–149, 175–176, 185–186.

 

Comment 9: L83 No information is provided about "artificial reefs". Material, shape, size should be described as well as how they were set in tanks.

Response:

We added the information about artificial reefs in the revised manuscript. Please see lines 105–107.

 

Comment 10: Figure 1 Is this figure reproduced from Tian et al. (2023)?

Response:

We have revised Figure 1, and this was not reproduced from Tian et al., (2023). Please see lines 135.

 

Comment 11: L114 Size description seems different from that shown in Figure 1.

Response:

Corrected. Please see lines 107–108, 120, 124, 144–147.

 

Comment 12: L117 Condition of "diseased" is unclear.

Response:

We apologize for the unclearness and have added information about diseased sea cucumbers in the revised manuscript. Please see lines 117–118.

 

Comment 13: L139 and L149 Please describe the food type.

Response:

We apologize for the unclearness and have added information about food type in the revised manuscript. Please see lines 98–100, 112–114, 148–149, 175–176, 185–186.

 

Comment 14: L149 "crawling": Is it "feeding" correct here?

Response:

We apologize for the mistake and have revised it in the manuscript. Please see lines 186–187.

 

Comment 15: L L164 I suggest to modify the adhesion index, because body weight works to detach sea cucumber from the wall in this experiment. That is, large sea cucumber tends to detach earlier than smaller one due to the heavy body. If heavier and lighter sea cucumbers stay on the wall during the same time, heavier one should have higher adhesion index. Thus, Ai=TW is better. But larger sea cucumber has larger ambulacral area, which increases adhesion, which could be roughly expressed as W^2/3. If the effect of ambulacral area needs to be uniformed, then, the formula may be Ai=T*W^1/3.

Response:

Thank you for this suggestion. To reduce the effect of different body weights on adhesion, we think it is more reasonable to divide the adhesion time by the body weight of sea cucumbers. Because this represents the time of adsorption per weight of sea cucumber. We beg your understanding. We would like to further revise the ms if it is not appropriate.

 

Comment 16: L Figure 3 and 5 "Adhension" in y-axis should be "Adhesion"

Response:

We apologize for the mistake and have revised it in the manuscript. Please see figure 3C and 5C.

 

Comment 17: L L240–247 This part of text seems redundant and may be more simply described in a sentence.

Response:

We think this is an excellent suggestion and have revised it in the manuscript. Please see lines 277–279 of the revised ms.

Reviewer 4 Report

The objective of this study was to detect the effect of artificial reefs on sea cucumbers' morbidity and mortality after exposure to air and high temperature. Although the goal of this study is quite interesting and important for improving sea cucumber culture, neither the experimental setup nor the discussion of the results was described appropriately. The manuscript seems to be sketchy and incomplete.

Specific comments can be found throughout the text.

Comments for author File: Comments.pdf

Author Response

General comments

The objective of this study was to detect the effect of artificial reefs on sea cucumbers' morbidity and mortality after exposure to air and high temperature. Although the goal of this study is quite interesting and important for improving sea cucumber culture, neither the experimental setup nor the discussion of the results was described appropriately. The manuscript seems to be sketchy and incomplete.

Specific comments can be found throughout the text.

Response:

We feel great thanks for your professional review work on our article and apologize for the unclearness. Based on your comments, we carefully revised the whole manuscript. Especially, we clarified the research background in Introduction and added related information of the correlations and measurement method of each experiment in Materials and methods. Further, we asked Wei Tang, a professional translator who works at Alibaba, to edit this manuscript to ensure the English quality of the manuscript. You can find our endeavor through the revised manuscript. We believe the current version of the ms is now ready for publication in Journal of Marine Science and Engineering. Or, we shall be happy to take any further revisions if necessary.

 

Comment 1: I am not sure that I understand what is seed production. Could you please add more info?

Response:

We apologize for the unclearness and have added information about seed production in the revised manuscript. Please see lines 35–39.

 

Comment 2: What is the temperature range for A. japonicus culture? Which temperature is considered "high"?

Response:

We apologize for the unclearness and have added information about high temperature in the revised manuscript. Please see lines 39–43.

 

Comment 3: Please, expand a little bit the Introduction. Why air exposure harm sea cucumbers?

Response:

We apologize for the unclearness and have added information about air exposure in the revised manuscript. Please see lines 47–52.

 

Comment 4: What does this detector measure? Oxygen? Organic carbon? Please, add this info.

Response:

We apologize for the unclearness. Salinity, pH, and dissolved oxygen were measured. We clarified it in the revised ms. Please see lines 96–98.

 

Comment 5: Please, provide the biomass and life stage of the individuals

Response: We apologize for the unclearness. Related information was added in the revised ms. Please see lines 110–111.

 

Comment 6: What do you mean by "diseased"? Please, explain. In addition, were these animals marked? Instead of coping the paragraph in lines 97–107, you should mention something like this: " The dead and healthy individuals were separated and their activity was recorded following the same procedure described above..."

Response:

We identified the sea cucumber without ulcerated skin has better fitness-related behaviors as an unaffected individual, and sea cucumbers with ulcerated skin as a diseased individual. The ulcerated skin of the sea cucumber can be recognized immediately. We agree your suggestion and have revised it in the manuscript. Please see lines 155–157.

 

Comment 7: I can't see in the text how you estimated morbidity... You should explain your input data and also the experimental design. Did you pull the data

Response:

We apologize for the mistake. Relevant content is corrected in the manuscript. Please see lines 206–207.

We explained the experimental design. Please see lines 102–105,140–144. We shall be happy to take any further revisions if necessary.

 

Comment 8: Where are these letters?

Response:

We apologize for the mistake. Relevant content was corrected in the manuscript. Please see lines 220–221, 232–233, 259–260, 273–274.

 

Comment 9: This is strange because the mean of feeding activity outside was lower than in the control...

Response:

We apologize for the mistake. Data for this experiment were taken as the mean ± S.E., not the standard deviation. Relevant content is corrected in the manuscript. Please see lines.

Clarify. We scrutinized the results and found that too large a standard deviation of the data resulted in no significant differences in the behavior of sea cucumbers cultured inside and outside the artificial reefs.

 

Comment 10: Overall, the Discussion needs to be re-written. There is a lot of repetition in it and specific discussion on the results is missing. Nevertheless, there are some parts that are interesting.

Response:

Thank you for the comments. Based on your comments, we carefully revised the whole Discussion. Especially, we clarified the significance of the present findings. Artificial reefs significantly reduced the mortality and morbidity of small A. japonicus exposed to the air and disease conditions at high temperature. Crevice is probably critical for mitigating the deterioration of fitness-related behaviors in sea cucumbers exposed to the air and disease conditions at high temperature. Sea cucumbers with better movement probably proactively enter inside artificial reefs, thereby reducing frequent physical contact between healthy and problematic individuals, and decreasing the likelihood of the disease transmission.

Round 2

Reviewer 1 Report

The improvement of the revised manuscript is quite evident.

Author Response

Thanks for your suggestions, which contributed greatly to our revision of the manuscript. We revised the manuscript slightly based on the suggestions from the second reviewer. We believe the current version of the manuscript is now ready for publication in Journal of Marine Science and Engineering. Or, we shall be happy to take any further revisions if necessary.

Author Response File: Author Response.docx

Reviewer 2 Report

Dear editor and authors, the manuscript is significantly improved and worthy of publication. I just have some minor suggestions to make easier to follow and to be more transparent and correct regarding the results. The results are good and consistent, so you should be transparent and clear to avoid any misjudgement from your pears. Congratulations.

 

 

Abstract:

“Artificial reefs thus provide…” -> “Thus, artificial reefs provide…”

“thereby reducing disease transmission” -> “showing a potential to reduce disease transmission” It’s an hypothesis and not a result.

 

Introduction:

Please clarify “The annual production of sea cucumber was 34 222,707 t in 2022 in China”. Was this in aquaculture, fisheries or both combined?

 

When using “thus” in the middle of a sentence should be between commas.

 

Materials and methods:

Salinity has no units. Please remove ‰.

 

Still need to clarify “Two groups of 16 sea cucumbers (~1 g of wet body weight) without ulcerated body walls were randomly selected and placed in each box with aeration”. Do you mean simply 16 sea cucumbers per box (in each of the 12 boxes – 6 O and 6 C)?

 

“Three diseased sea cucumbers and sixteen sea cucumbers without ulcerated body walls were placed in each box”. Do you mean 19 in total? My point is: this is not easy to follow with this much variation in numbers and experiments. So, whatever you can do to facilitate the reader helps. For example, the figure shows the right number of individuals in Exp.I, but Exp.II has also 16 sea cucumbers. Could you place three sea cucumbers with a different colour representing the affected individuals? It would help a lot.

 

Ln216-217 – move (48.61 ± 1.39%) to after “higher morbidity”

 

Figures 3 and 5 – Do not use the term “control”. Keep the terms used in the manuscript. Gets confusing. You can write, for example, “Group C”; “Group O – Out”; “Group O – In”

 

Regarding your answer to the use of SE: I understand that SD in such cases have negative values because of the high differences in dispersion and between max and min values, but this does not seem transparent. Also, SE is not informative in this case and not correct to use. It is a confidence interval for the mean and you have non-normal distributions. So, the mean should not be the only measure to present. To solve this I suggest using boxplots instead of barplots. Boxplots are, thus, more adequate to your data because it presents the median and not the mean. So, please replace all barplots that represent non-normal data with boxplots.

You have good results that even represent your initial expectations. Be clear and correct in presenting them and avoid criticism.

 

Discussion:

Ln298 – you shouldn’t start a sentence with a number.

 

Ln291 – replace “from” with “in”

 

Ln293 – replace “Further” with “Furthermore”

 

Ln294 – Not sure what you mean by “compounding”. Do you mean synergistic (one increase the negative effect of the other)?

 

Ln297 and Ln325 – State it was “8 times and 7 times HIGHER” of replace with “8 fold and 7 fold”. In line 325 put it in numerical order.

 

Ln311 – Replace “stress” with “stressors”

 

Ln344 – Replace “contact” with “contacting” and should delete “during their escape”

 

This paper does not require a conclusion section but, if the authors want to keep it, they should change it. In its present form is a repetition of the discussion and some results. It’s unfit for this section. It should be something like the last sentences of the discussion and the last of the conclusions. So, I suggest to eliminate the conclusions and move these last (not in red) sentences to the end of the discussion or take the last sentences of the discussion and integrate them in the conclusion section, discarding everything (in red) that is repeating what was said before.

 

Author Response

General comments:

Dear editor and authors, the manuscript is significantly improved and worthy of publication. I just have some minor suggestions to make easier to follow and to be more transparent and correct regarding the results. The results are good and consistent, so you should be transparent and clear to avoid any misjudgement from your pears. Congratulations.

Response:

Thanks for your value suggestions, which contributed greatly to our revision of the manuscript. Based on your comments, we carefully revised the manuscript, especially the Figures, Materials and methods, and Discussion. We believe the current version of the manuscript is now ready for publication in Journal of Marine Science and Engineering. Or, we shall be happy to take any further revisions if necessary.

 

Comment 1: Abstract:

“Artificial reefs thus provide…” -> “Thus, artificial reefs provide…”

“thereby reducing disease transmission” -> “showing a potential to reduce disease transmission” It’s an hypothesis and not a result.

Response:

Corrected. Please see lines 25–27.

 

Comment 2: Introduction:

Please clarify “The annual production of sea cucumber was 34 222,707 t in 2022 in China”. Was this in aquaculture, fisheries or both combined?

When using “thus” in the middle of a sentence should be between commas.

Response:

Clarified. These data are for aquaculture. Please see line 34.

Corrected. Related problems were revised in the manuscript. Please see lines 48, 64.

 

Comment 3: Materials and methods:

Salinity has no units. Please remove ‰.

Still need to clarify “Two groups of 16 sea cucumbers (~1 g of wet body weight) without ulcerated body walls were randomly selected and placed in each box with aeration”. Do you mean simply 16 sea cucumbers per box (in each of the 12 boxes – 6 O and 6 C)?

Response:

Corrected. Please see line 100.

Clarified. There were 12 plastic boxes in the experiment, of which 6 groups with artificial reefs were named group O and 6 groups without artificial reefs were named group C. There were 16 sea cucumbers in each box. Please see lines 114–115.

 

Comment 4:

“Three diseased sea cucumbers and sixteen sea cucumbers without ulcerated body walls were placed in each box”. Do you mean 19 in total? My point is: this is not easy to follow with this much variation in numbers and experiments. So, whatever you can do to facilitate the reader helps. For example, the figure shows the right number of individuals in Exp.I, but Exp.II has also 16 sea cucumbers. Could you place three sea cucumbers with a different colour representing the affected individuals? It would help a lot.

Ln216-217 – move (48.61 ± 1.39%) to after “higher morbidity”

Response:

Thank you for the comments. Please see figure 1C and lines 147–179.

Corrected. Please see line 222.

 

Comment 5:

Figures 3 and 5 – Do not use the term “control”. Keep the terms used in the manuscript. Gets confusing. You can write, for example, “Group C”; “Group O – Out”; “Group O – In”

Regarding your answer to the use of SE: I understand that SD in such cases have negative values because of the high differences in dispersion and between max and min values, but this does not seem transparent. Also, SE is not informative in this case and not correct to use. It is a confidence interval for the mean and you have non-normal distributions. So, the mean should not be the only measure to present. To solve this I suggest using boxplots instead of barplots. Boxplots are, thus, more adequate to your data because it presents the median and not the mean. So, please replace all barplots that represent non-normal data with boxplots.

You have good results that even represent your initial expectations. Be clear and correct in presenting them and avoid criticism.

Response:

Thank you for the comments. Please see figures 3 and 5, and lines 129–130, 159–160, 238–239, 280–281.

Corrected. Please see figures 2–5.

 

Comment 6: Discussion:

Ln298 – you shouldn’t start a sentence with a number

Response:

Corrected. Please see line 296.

 

Comment 7: Ln291 – replace “from” with “in”

Response:

Corrected. Please see line 298.

 

Comment 8: Ln293 – replace “Further” with “Furthermore”

Response:

Corrected. Please see line 300.

 

Comment 9: Ln294 – Not sure what you mean by “compounding”. Do you mean synergistic (one increase the negative effect of the other)?

Response:

Corrected. Please see line 301.

 

Comment 10: Ln297 and Ln325 – State it was “8 times and 7 times HIGHER” of replace with “8 fold and 7 fold”. In line 325 put it in numerical order.

Response:

Corrected. Please see lines 304–305, 332–334.

 

Comment 11: Ln311 – Replace “stress” with “stressors”

Response:

Corrected. Please see lines 318, 324.

 

Comment 12: Ln344 – Replace “contact” with “contacting” and should delete “during their escape”

Response:

Corrected. Please see lines 352–353.

 

Comment 10: This paper does not require a conclusion section but, if the authors want to keep it, they should change it. In its present form is a repetition of the discussion and some results. It’s unfit for this section. It should be something like the last sentences of the discussion and the last of the conclusions. So, I suggest to eliminate the conclusions and move these last (not in red) sentences to the end of the discussion or take the last sentences of the discussion and integrate them in the conclusion section, discarding everything (in red) that is repeating what was said before.

Response:

Corrected. Please see lines 363–383.

Author Response File: Author Response.docx

Reviewer 3 Report

The authors properly revised the manuscript. I consider that this manuscript can be published in this version.

Author Response

General comments: The authors properly revised the manuscript. I consider that this manuscript can be published in this version.

Response:

Thanks for your suggestions, which contributed greatly to our revision of the manuscript. We revised the manuscript slightly based on the suggestions from the second reviewer. We believe the current version of the manuscript is now ready for publication in Journal of Marine Science and Engineering. Or, we shall be happy to take any further revisions if necessary.

Author Response File: Author Response.docx