Oxidative Stress Response in the Seaweed Padina pavonica Associated with the Invasive Halimeda incrassata and Penicillus capitatus

Round 1

Reviewer 1 Report

 

 

The topic investigated by the authors is interesting and attempts to answer how allelopathy and competition for resources induce oxidative stress between seaweed populations. However, the first thing the authors need to do is to seek the services of a professional language proofreader to correct their manuscript for language-related errors. 

 

The introduction of the manuscript appears to be disjointed. Try to create a flow between paragraphs – connect the stories.

 

Materials and methods lake information on how you identified the macroalgae. How or where was this done? Any accession numbers? 

Line 126, there is a better name given to the 50 by 50 cm frame used to determine the densities of the P. pavonica and the two invasive species. Provide the correct word. 

 

Did you measure temporal changes in the densities of the three species? This permits a better understanding of the ecological dynamics, not just a single snapshot of the presumed interactive effect on the antioxidant response of the macroalgae. 

 

Did you care to measure the levels of ROS species in the macroalgae? This enables you to correlate or associate changes in antioxidant response with increasing ROS levels. Without this information, most of the discussion on oxidative stress will be speculative.

 

Lines 157: State the factor tested with the one-way ANOVA.

 

Figure 2 is poorly presented. When you say P. pavonica + H. incrassate or P. pavonica + P. capitatus, these imply that the densities you see are the combined densities of both plants. If that is the case, what is the biomass of each species? It should be easily seen and understood if you intend to portray individual species' biomass or density. 

 

Where are the oxidative stress response datasets or results of the invasive plants? Do they suffer stress or not? Your data has not shown this. Please provide these results to give your study credence and make it more comprehensive. 

 

Beyond the analysis of variance, a multivariate analysis will enhance our understanding of your dataset. Note that other environmental factors could be responsible for your observed oxidative response. The physicochemical changes can also induce these responses. Also, physicochemical conditions could interact with the presence of invasive plants or macroalgae to cause oxidative stress in the endemic or native species. Without consideration of the contributory roles of other factors using multivariate analysis, it is difficult to accept the findings of your study. 

Author Response

Reviewer 1

The topic investigated by the authors is interesting and attempts to answer how allelopathy and competition for resources induce oxidative stress between seaweed populations. However, the first thing the authors need to do is to seek the services of a professional language proofreader to correct their manuscript for language-related errors. 

The manuscript has been carefully revised to improve grammar and avoid potential errors, with the help of a native English university colleague.

 

The introduction of the manuscript appears to be disjointed. Try to create a flow between paragraphs – connect the stories.

The paragraphs of the introduction have been modified to try to give greater continuity to the information provided. Additionally, some references have been incorporated to support the new information.

Guzzetti, E., Salabery, E., Ferriol, P., Díaz, J. A., Tejada, S., Faggio, C., & Sureda, A. (2019). Oxidative stress induction by the invasive sponge Paraleucilla magna growing on Peyssonnelia squamaria algae. Marine environmental research, 150, 104763.

Quetglas-Llabrés, M. M., Tejada, S., Capó, X., Langley, E., Sureda, A., & Box, A. (2020). Antioxidant response of the sea urchin Paracentrotus lividus to pollution and the invasive algae Lophocladia lallemandii. Chemosphere, 261, 127773.

 

Materials and methods lack information on how you identified the macroalgae. How or where was this done? Any accession numbers? 

The algae used in the work do not present any difficulty for their identification since they cannot be confused with any other on the coasts of the Balearic Islands. In addition, there are two marine biologists on the team who are experts on the subject and, to confirm, we have reviewed their morphological characteristics following a guide to marine macroalgae and phanerogams from the Western Mediterranean:

Rodríguez-Prieto, C., Ballesteros, E., Boisset, F., & Afonso-Carrillo, J. (2013). Guía de las macroalgas y fanerógamas marinas del Mediterráneo Occidental Omega. SA Barcelona, 656.

More specific information with the new corresponding bibliographic citation has been incorporated in the new version of the manuscript.

 

Line 126, there is a better name given to the 50 by 50 cm frame used to determine the densities of the P. pavonica and the two invasive species. Provide the correct word. 

The correct term is “Quadrat sampling”. In accordance we have corrected the name in the revised version.

 

Did you measure temporal changes in the densities of the three species? This permits a better understanding of the ecological dynamics, not just a single snapshot of the presumed interactive effect on the antioxidant response of the macroalgae. 

This comment is really interesting and it is an option that we want to carry out in the future. In any case, Padina pavonica follows an annual cycle where the thallus detaches every winter and regrows in spring. Something similar occurs with the alien species, since Penicillus capitatus also disappears in winter, while Halimeda incrassata partly disappears and partly remains calcified. In future studies we want to evaluate the dynamics of appearance and disappearance of the three species throughout a year to determine their dynamics and understand their development.

In the revised manuscript, we have more clearly justified why the study was carried out in summer, since that is when the interaction between species is more evident in these islands.

Also, a new reference has been added:

Benita, M.; Dubinsky, Z.; Iluz, D. Padina pavonica: Morphology and Calcification Functions and Mechanism. Am. J. Plant Sci. 2018, 9, 1156–1168.

 

Did you care to measure the levels of ROS species in the macroalgae? This enables you to correlate or associate changes in antioxidant response with increasing ROS levels. Without this information, most of the discussion on oxidative stress will be speculative.

In Figure 4, in addition to MDA levels, ROS production was presented. In this sense, the subsequent discussion was based on these results, so we believe that it is not a speculative text but based on data obtained.

 

Lines 157: State the factor tested with the one-way ANOVA.

We thank the reviewer for this point, the factor tested has been included in the statistical analysis section. 

 

Figure 2 is poorly presented. When you say P. pavonica + H. incrassata or P. pavonica + P. capitatus, these imply that the densities you see are the combined densities of both plants. If that is the case, what is the biomass of each species? It should be easily seen and understood if you intend to portray individual species' biomass or density. 

In accordance with the reviewer's comment, we have modified the figure to make it more understandable for the reader. In addition, we have explained more clearly the different results of the densities in the Results section.

 

Where are the oxidative stress response datasets or results of the invasive plants? Do they suffer stress or not? Your data has not shown this. Please provide these results to give your study credence and make it more comprehensive. 

The reviewer's comment is correct, and in fact, we considered making these determinations at the time. However, at the time of sampling it was impossible for us to find an area in which the alien species were isolated in order to have a control value. All the specimens were found within the Padina pavonica meadow, which means that we cannot determine the existence of oxidative stress as we do not have a control point.

 

Beyond the analysis of variance, a multivariate analysis will enhance our understanding of your dataset. Note that other environmental factors could be responsible for your observed oxidative response. The physicochemical changes can also induce these responses. Also, physicochemical conditions could interact with the presence of invasive plants or macroalgae to cause oxidative stress in the endemic or native species. Without consideration of the contributory roles of other factors using multivariate analysis, it is difficult to accept the findings of your study. 

Takin into account the way of sampling timing, in the present work, a multivariate analysis is not necessary since the environmental conditions of the three study areas were exactly the same and the samplings were carried out on the same day. The next step in our research will be to carry out an annual monitoring, since we are going to analyze the influence of variations in the environmental parameters of the three zones during time.

Reviewer 2 Report

Are there other macroalgae species (except Penicillus capitatus and Halimeda incrassata) or microalgae in the selected areas for analysis.

In the article I would like to see a photo where photos are presented with the number of Individuals in the analysis zone.

 

In the future, it would be interesting to see if Padina pavonica and invasive Halimeda incrassata and Penicillus capitatus occur together. And how they affect each other.

It is also possible to see how the composition of the soil affects.

Author Response

Reviewer 2

Are there other macroalgae species (except Penicillus capitatus and Halimeda Incrassata) or microalgae in the selected areas for analysis.

In the study areas there is an almost exclusive predominance of the species selected for the study. During the sampling we have been able to observe some isolated specimens of Dasycladus vermicularis and Halimeda tuna.

In the revised version of the manuscript, we have introduced this information to better characterize the studied area.

 

In the article I would like to see a photo where photos are presented with the number of Individuals in the analysis zone.

We appreciate the reviewer's comment; however, we do not have the photographs requested. During the sampling we only took some representative photographs to show the alien algae. Now in winter, we cannot do them because they are annual species in our waters and right now they are not present.

 

In the future, it would be interesting to see if Padina pavonica and invasive Halimeda incrassata and Penicillus capitatus occur together. And how they affect each other.

The reviewer's comment is very interesting and, in fact, we currently have a project requested to carry out a long-term study with monthly follow-ups and to be able to see how alien species are occupying new spaces, and if algae appear where they coexist together.

We have added this idea as a proposal for the future in the final section of conclusions of the new version of the manuscript.

 

It is also possible to see how the composition of the soil affects.

In the modified version of the manuscript, a granulometric analysis of the sediments of the three studied areas has been incorporated. No differences have been observed between the different zones, showing the three zones a similar composition. This additional information has also been incorporated in the text in the different sections.

 

 

Round 2

Reviewer 1 Report

I have two additional points for the authors to consider: 

1. The manuscript has not been correctly revised for language-related errors. Please allow proper language revision because it will substantially improve the quality of your write-up.

2. Your reason for not doing a multivariate analysis is not correct. You do not need to perform year-round sampling to confirm the association between the response parameters and physicochemical conditions tested -- though you stated they were the same, they were not. The values were not significantly different from each other. And by the way, your argument would make more sense if you had done a multivariate analysis and seen that it was irrelevant. Outrightly dismissing it is not scientifically correct. Note that you can apply multivariate analysis as long as you replicate your observations, samplings, and analysis. 

P.S.: comments are not made to ridicule your work but to improve the quality of the final published paper. 

 

Author Response

Response to the reviewer

First, we would like to thank the reviewer who has dedicated a valuable time to read the manuscript and provide his comments.

1. The manuscript has not been correctly revised for language-related errors. Please allow proper language revision because it will substantially improve the quality of your write-up.

The manuscript has been revised in detail to improve the language and avoid grammatical and typographical errors.

2. Your reason for not doing a multivariate analysis is not correct. You do not need to perform year-round sampling to confirm the association between the response parameters and physicochemical conditions tested -- though you stated they were the same, they were not. The values were not significantly different from each other. And by the way, your argument would make more sense if you had done a multivariate analysis and seen that it was irrelevant. Outrightly dismissing it is not scientifically correct. Note that you can apply multivariate analysis as long as you replicate your observations, samplings, and analysis. 

According to the comment, a multivariate analysis (MANOVA test) has been carried out to determine if there is any possible relationship between the biomarkers analyzed and the physicochemical conditions measured. The results of the analysis do not show any effect of these conditions. This information has been incorporated into the new version of the manuscript explaining the analysis carried out and the results obtained.