Environmentally Friendly and Efficient Methods for Mitigating the Density of Ascidian Fouling in Mediterranean Mussel Farming

Round 1

Reviewer 1 Report

The authors report on management practices for eliminating ascidian fouling for mussel aquaculture. The experimental work is performed using appropriate methods. The references are abundant and relevant and conclusions are based on the results obtained in this study. The manuscript itself is clear and easy to follow. The suggestions to possible corrections and improvements of the manuscript are listed as follows.

Line 19 change “(Lamarck 1819)” to “Lamarck, 1819”. Also, add it in line81.

Line 28 “mussel farming” and “Mediterranean” should not be italic. Also “spp.” In line 264.

Line 47 “… very high density or biomass…..a relatively short time…” but how high and how short? Examples are needed here.

Line 48 and others [6,7,8,9,10] should be [6-10].

Line 86 It would be better to place a subtitle before the paragraph.

Line 114 Since high salinity solutions could eliminate ascidians, the salinity of seawater should be marked.

Line 125-128 Also, why did not record the salinity? This parameter is more important.

Line 115 “…in a shaded place…” but in Figure 2, we can see the shadow. Were the socks exposed under sunshine??

Line 135 It would be better to add a mussel sock in each trial to show “4 socks each”

Line 167-171 Parameters values are needed.

Line 230 subtitles are needed for Discussion part.

Line 263 add “(Pallas, 1774)” for Styela clava.

Line 286 change “Styela plicata” to “S. plicata (Lesueur, 1823) ”

Line 480 and 503 Italicize “Perna canaliculus” and “Crassostrea gigas”. Delete “p.” in “p.e016..”

Space characters and commas are needed, for example, “50ppt” and “70ppt” in Abstract, right after “the control” in line 194, Styela clava in line 461, and Perna canaliculus in line 480.

Author Response

The authors report on management practices for eliminating ascidian fouling for mussel aquaculture. The experimental work is performed using appropriate methods. The references are abundant and relevant and conclusions are based on the results obtained in this study. The manuscript itself is clear and easy to follow. The suggestions to possible corrections and improvements of the manuscript are listed as follows.

Response: we would like to thank the reviewer for the positive criticism, as well as for the valuable corrections recommended, which have been all embedded in the revised manuscript.

Line 19 change “(Lamarck 1819)” to “Lamarck, 1819”. Also, add it in line81.

Response: Corrected according to the reviewer’s comment

Line 28 “mussel farming” and “Mediterranean” should not be italic. Also “spp.” In line 264.

Response: Italics were transformed to regular as correctly mentioned by the reviewer

Line 47 “… very high density or biomass…..a relatively short time…” but how high and how short? Examples are needed here.

Response: Following the reviewers comment, the phrase “e.g. Ciona intestinalis and Styela plicata can establish population numbers up to 10⁴ fouling individuals per hectare” was added in the revised manuscript (please see 1^st paragraph of the introduction).

Line 48 and others [6,7,8,9,10] should be [6-10].

Response: corrected according to the reviewer’s comment in the whole manuscript.

Line 86 It would be better to place a subtitle before the paragraph.

Response: We would like to thank the reviewer for mentioning this comment. Accordingly, the subtitle “Study area description” was added in the beginning of the Materials and Methods in the revised manuscript

Line 114 Since high salinity solutions could eliminate ascidians, the salinity of seawater should be marked. 

Response: Sea-water salinity was added (35.3 psu) in the revised manuscript, as recommended by the reviewer (please see section Field experiment of the Materials and Methods in the revised manuscript)

Line 125-128 Also, why did not record the salinity? This parameter is more important.

Response: Indeed, we agree with the reviewer with the comment concerning salinity. We have to state however, that salinity is stable, of approximately 35-36 psu in the study area and hence it was not periodically measured. This statement was added in the revised Materials and methods section, according to the reviewer’s comment

Line 115 “…in a shaded place…” but in Figure 2, we can see the shadow. Were the socks exposed under sunshine??

Response: Figures 2C and 2D were obtained after transferring the mussels to a sunshine place in order to be better displayed. This clarification was added in the legend of Figure 2 in the revised manuscript.

Line 135 It would be better to add a mussel sock in each trial to show “4 socks each”

Response: Figure 3 has been modified according to the reviewer’s comment. Particularly a mussel sock was added in each trial to show “4 socks each” (please see Figure 3 in the revised manuscript)

Line 167-171 Parameters values are needed.

Response: Values were added in section 3.1 of the revised manuscript, in accordance to the reviewer’s comment

Line 230 subtitles are needed for Discussion part.

Response: Subtitles were added in the Discussion, as recommended by the reviewer

Line 263 add “(Pallas, 1774)” for Styela clava.

Response: Added, as recommended by the reviewer

Line 286 change “Styela plicata” to “S. plicata (Lesueur, 1823) ”

Response: Corrected according to the reviewer’s comment

Line 480 and 503 Italicize “Perna canaliculus” and “Crassostrea gigas”. Delete “p.” in “p.e016..”

Response: Corrected according to the reviewer’s comment

Space characters and commas are needed, for example, “50ppt” and “70ppt” in Abstract, right after “the control” in line 194, Styela clava in line 461, and Perna canaliculus in line 480.

Response: All the mentioned typo errors were corrected in the revised manuscript following the reviewer’s mention.

Reviewer 2 Report

Dear authors.

The manuscript “Environmental friendly efficient methods for fouling-ascidians mitigation density in Mediterranean Mussel Farming” explores a relevant issue in mussel aquaculture as it is the occurrence of fouling organisms that limit the commercial potential of farms worldwide. The authors properly rise the topic in the Introduction section and develop two central Experiments to test different methodologies to control/eliminate the quantity of ascidians in farmed mussels. Globally I appreciate the manuscript, but I would recommend a revision on some central issues that I detect and pointed in my detailed review (please see bellow). Overall, in my opinion, the manucript deserves to be considered for publication after the addressing of this issues (minor revision). I hope that you might publish this work soon. Good luck!

Yours sincerely

TITLE:

I would put “density” before “mitigation”

ABSTRACT

Lines 20-24 – These sentences are too much detailed for what it is needed in this “Abstract” section.

KEYWORDS

You shall not repeat Keywords that are already placed in the Title. Add some others to catch a wider audience.

INTRODUCTION

Lines 38-44 – It is hard to follow these sentences since it is not clear If you are talking about Ascidians or biofouling in general. Please organize better the text not mixing the facts.

“Previous assessments 42 considering the final market prices have indicated that biofouling could cause economic 43 losses of up to 20% for oyster farming [3] and 30% for scallop aquaculture [4].” Is this only regarding the ascidians biofouling ?

Line 42 – You do not need “as reported by Lane and Willemsen” if you add the reference in the end of the sentence.

Lines 49-51 – Reference [11] is a review article. You shall refer the study where a “total loss of mussel production” occurred.

Line 55 – I would start a new paragraph here since you will star another subject regarding ascidians biology. Anyway, I think that Lines 55-60 are wrongly placed in this part of the Introduction section.

Lines 61-63 – Here you are repeating previous information about the impacts of ascidians in farms. You shall be more effective.

Lines 64 – I guess that the first “first” shall be deleted.

Lines 76-84 – The aims of the study are too vague in this paragraph. You shall be sharper in presenting the study goals.

MATERIAL AND METHODS

Lines 86-87 – The study was conducted in one or more mussel farms? In line 80 you refer “aquaculture farms” but here sounds like it was only one.

Line 91 – Replace by “The water quantity entering the gulf is insufficient…”

Line 94- “affect the area more” do not sound wright.

General comment: In my opinion, give the main goals of the study, I do not see much relevance to describe so exhaustively the study area and the environmental parameters. I rather have information on mussel farm annual production (in tonnes) and on the annual water salinity and temperature in the mussel farm, information that you will need in the discussion to evaluate your results. Readers will seek your study in order to apply ascidian cleaning in elsewhere mussel farms and not in Amvrakikos Gulf so you shall turn your manuscript for a wider audience.

Lines 99-101- I am not seeing the relevance of this information in this section.

Figure 1 – Is this figure showing the mussel farm? It is not clear

Line 108 – “free of ascidians”. Did they have other organisms or they were prepared to be as cleaned as possible from epifaunal organisms?

Line 111 – Subtitle – You shall add to the title of describe it in the first sentence of this paragraph the designation Experiment 1. Otherwise it will only be referred in the Table 1 caption. Please revise this issue also in Experiment 2 (Line 136).

Lines 114 –You shall call the groups A, B, C and D instead of a, b, c and d to ease the reading.

Lines 115 – “…a shaded place for one day…” What kind of shaded day? Always the same shadow spot? I would replace “a day” for “24h” if that was the case. When you say “a day” it looks very informal. In this sentence you write “one day” and in the line 117 you write “1 day”. You shall uniform it.

Line 118 – “(Table 1, Figure 2, Figure 3)”. I understand that these experiments are not easy to described in words, but 1 table and 2 Figures are too much for describe it. I would pass these 3 items to Supplementary material or let Table 1 and transfer Figure 2 and 3 for Supp. Mat.

Line 121 – Mortality and Losses are not the same think here?

Line 122 – “…mussels mortality was recorded…”. Mortality was recorded taking into account human eye observation or other method ? Please clarify.

Ascidians are tunicates, but not all tunicates are ascidians. Did you counted and consider all the tunicates or just the ascidians? Please pay attention to this issue along the manuscript.

Line 124 – “were” instead of “was”

Line 125 – “All the data were quadrupled”. What do you mean, did you collect four replicates of mussel samples in each depth? Please clarify

Here, in this section you shall add information about the importance of quantify and determining the Temperature, Dissolved oxygen and Chl-a. Are they proxys of what? All the readers have to understand it. I would add salinity information in the Experiment 1 since it would be related to Experiment 2.

Figure 2 – I would add best quality photos here since it is hard to visualize the mussels.

Figure 3 – In my opinion this figure brings confusion to your Experiment description and do not clarify it. I would consider to remove it or at most transfer it for Supp. Mat.

Line 137 – Again, “tunicates colonies”. You shall clarify this. Were these mussels collected from the same raft and longlines of the mussels in Experiment 1 ? It is not clear.

Line 142 – I guess that “lit” is not the abbreviation for “liters”

Line 144 – Replace “estimate” by “regist “. “water salinity” instead of “salinity”.

Line 150 – Please see my comment above regarding Experiment 1 and the use of 1 table and 2 figures.

Line 160 – Statistics – I would replace this title for “Data treatment and statistical analysis”. This subsection must to be improved.

“Comparisons among samples were made…” Which samples are you referring? Which statistical factors did you use? Please clarify.

RESULTS

Line 169 – “Dissolved oxygen” instead of “O₂”.

Lines 166-171 - Were you expecting other temperature, dissolved oxygen and Chl-a patterns than these ones? In my opinion there is no need to highlight these results with a paragraph and a graph in the manuscript. Please add a sentence or two in Material and Methods sections referring the common pattern of these parameters during your experiments. As it is, the manuscript is not enriched with such information.

Line 176 – Heavy recruitment of what? Please clarify for a better reading.

Line 184 – I guess that “already” is not the word here.

Figure 7 – Please add to the figure “Number of tunicate colonies” instead of “Number of colonies”. The way you add the Anova results in the figure is wise but the result is very difficult to follow. I would add a table in Supp. Mat with further results of Anova and I would describe the most important ones here in the Result section.

Line 195 – Delete “which were”

I could not find any results or comments about mussel mortality regarding Experiment 1. You only refer it in the Discussion section (Line 271). This manuscript shall be better organized regarding these issues.

Figure 7, 8 and 9 are based in the same graph format. I would be more creative in the presentation of your results. Otherwise the manuscript will not have any style and become less attractive.

Figure 8 A, B and C shall be bigger.

Line 210 – “No mussel mortality”. Did you had zero mussel dead individuals along your experiment?

How did you obtain these mortality percentages for tunicate colonies? How did you count colonies?

Line 211 – “into” instead of “to”

Lines 217 – Sometimes you use “one day” other times you use “24h”. You shall uniform this along the manuscript.

DISCUSSION

The first paragraph of the Discussion contains valuable information to be add in the Material and Methods section.

Lines 235 – During the previous sections (Material and Methods and Results) you referred the tunicates. Here, you start writing ascidians. As I call to attention above, you shall clarify what did you consider during the Experiments.

Line 235 – Please replace “being increased” by “increase” and “declined” by “declining”

Line 248 – You shall not begin a sentence with a number.

Lines 253-258 – So, you shall refer in the Material and Methods section that mussel washing clean them from ascidians but also from other epifauna species.

Line 258-259 – This is widely documented. Add some references or be less categoric here.

Line 260 – Again the Tunicate vs Ascidian nomenclature. Please revise it along the whole manuscript.

Line 217 – “Minor mussel mortality occurred”. This information is not placed in the Results yet you write in Material and Methods “mussel mortality was recorded “. What do you mean by “minor” here? This information is crucial when evaluation the efficiency of an anti-fouling methodology.

Line 291 – “…and other biofoulings…” Did you collect this information on “other biofoulings”?

Lines 297-298 – Who said that? Is it based on your experience?

Lines 300-302 – Again, you write ascidians and then tunicates turning the text sense very confusing.

Lines 313-315 – 

Line 318 – Please delete “for ascidians metamorphosis inhibition” as it is redundant. “air exposure” instead of “air”

Line 321 – “Currently” instead of “Up to date”

Lines 339-341 - Your results show that a salinity shock and an exposure period might reduce the present of ascidians in mussels. But is this method realistic for most of the mussel farms? That is, do the typical mussel farms have conditions to perform this type of action as a routine? What are the costs? Is this methodology practicable in mussel farms where tonnes of mussels are collected to be sold every day? How big must be the tanks in these cases? Please improve your discussion with the “dark” side of your findings as it is important to be cautious regarding their practicability and applicability.

I would add to the discussion the benefit of this apparent “success” in eliminating most of ascidians and “other biofoulings” in respect to the potential presence, transport and introduction of non-indigenous/invasive species. That is, this type of methodologies that you propose here might also mitigate the spread of such species into elsewhere area. Please see the study of Piló et al 2021 to develop this topic.

Piló, D., Pereira, F., Carvalho, A. N., Vasconcelos, P., Cunha, A. M., & Gaspar, M. B. (2021). Are non-indigenous species hitchhiking offshore farmed mussels? A biogeographic and functional approach. Marine Pollution Bulletin, 171, 112776.

CONCLUSIONS

Line 345 - Please just start with “We proposed…”. I don’t know if “treating” is the best choice here.

Line 347 – I believe that “mussel” after “immersion” shall be deleted.

Line 348 – Please rephrase: “… of ascidians with 0% mortality of the cultured organisms”

Line 358-361- This final sentence shall be deeply rephrased as it is very hard to understand.

REFERENCES

I did not perform a thorough revision on the reference list but I recognize that it contains relevant, updated and diverse information that was used in the text.

The english written is acceptable along the manuscript, yet a revision of a english native speaker (i am not) is desirable before publication, seeking to improve the manuscript style and readability.

Author Response

Dear authors. 

The manuscript “Environmental friendly efficient methods for fouling-ascidians mitigation density in Mediterranean Mussel Farming” explores a relevant issue in mussel aquaculture as it is the occurrence of fouling organisms that limit the commercial potential of farms worldwide. The authors properly rise the topic in the Introduction section and develop two central Experiments to test different methodologies to control/eliminate the quantity of ascidians in farmed mussels. Globally I appreciate the manuscript, but I would recommend a revision on some central issues that I detect and pointed in my detailed review (please see bellow). Overall, in my opinion, the manucript deserves to be considered for publication after the addressing of this issues (minor revision). I hope that you might publish this work soon. Good luck!

Yours sincerely

Response: We feel grateful to the reviewer for the nice, constructive and helpful positive criticism. All suggestions were accordingly incorporated or addressed in the revised manuscript, as provided in detail in the point-to-point response to each comment that follows.

TITLE:

I would put “density” before “mitigation”

Response: We agree with the reviewer’s suggestion and thus we have accordingly modified the title.

ABSTRACT

Lines 20-24 – These sentences are too much detailed for what it is needed in this “Abstract” section.

Response: We agree with the reviewer’s observation and therefore these sentences were replaced by a summarized part, as follows: “Various experiments were applied, including temporally differential washing of mussels, air exposure and immersion in 50 ppt and 70 ppt salinity solutions, as well as the combination of these applications.”

KEYWORDS

You shall not repeat Keywords that are already placed in the Title. Add some others to catch a wider audience.

Response: We are grateful to the reviewer for this suggestion. Three repeated words were replaced by “tunicates”, “bivalves” and “management practices”, in accordance to this comment.

INTRODUCTION

Lines 38-44 – It is hard to follow these sentences since it is not clear If you are talking about Ascidians or biofouling in general. Please organize better the text not mixing the facts.

Response: The sentences referred to biofouling in general. Thus, considering the reviewer’s comment, the text has been accordingly modified for clarity.

“Previous assessments 42 considering the final market prices have indicated that biofouling could cause economic 43 losses of up to 20% for oyster farming [3] and 30% for scallop aquaculture [4].” Is this only regarding the ascidians biofouling ?

Response: No, this refers to biofouling in general, here, as well. Taking into consideration the reviewer’s comment, the first paragraph of the introduction was divided into two in the revised manuscript, with the first presenting a general introduction to biofouling and the second specified to ascidians.

Line 42 – You do not need “as reported by Lane and Willemsen” if you add the reference in the end of the sentence.

Response: Corrected according to the reviewer’s comment

Lines 49-51 – Reference [11] is a review article. You shall refer the study where a “total loss of mussel production” occurred.

Response: The reference was replaced by two other ones, in accordance to the reviewer’s comment.

Line 55 – I would start a new paragraph here since you will star another subject regarding ascidians biology. Anyway, I think that Lines 55-60 are wrongly placed in this part of the Introduction section.

Response: A new paragraph was initiated, in accordance to the reviewer’s comment.

Lines 61-63 – Here you are repeating previous information about the impacts of ascidians in farms. You shall be more effective.

Response: This part was transferred to the second paragraph of the introduction, where fits better to the content, in accordance to the reviewer’s comment.

Lines 64 – I guess that the first “first” shall be deleted.

Response: Corrected according to the reviewer’s comment.

Lines 76-84 – The aims of the study are too vague in this paragraph. You shall be sharper in presenting the study goals.

Response: The paragraph presenting the study aims was modified in an effort to be sharper and more focused, as recommended by the reviewer.

MATERIAL AND METHODS

Lines 86-87 – The study was conducted in one or more mussel farms? In line 80 you refer “aquaculture farms” but here sounds like it was only one. 

Response: The study was conducted on two mussel farms, one long line and one raft. Since, indeed, the way it was written, it was confusing, the part has been rephrased in the revised manuscript.

Line 91 – Replace by “The water quantity entering the gulf is insufficient…”

Response: Corrected according to the reviewer’s comment

Line 94- “affect the area more” do not sound wright. 

Response: This phrase has been deleted and the sentence has been accordingly rephrased (please see the first paragraph of Materials and Methods).

General comment: In my opinion, give the main goals of the study, I do not see much relevance to describe so exhaustively the study area and the environmental parameters. I rather have information on mussel farm annual production (in tonnes) and on the annual water salinity and temperature in the mussel farm, information that you will need in the discussion to evaluate your results. Readers will seek your study in order to apply ascidian cleaning in elsewhere mussel farms and not in Amvrakikos Gulf so you shall turn your manuscript for a wider audience.

Lines 99-101- I am not seeing the relevance of this information in this section

Response: We respect the reviewer’s point of view, but the reason we included all this info is to indicate the importance of Amvrakikos Gulf for mussel aquaculture. Additionally, since this info is important for mussel culture, we would like to ask the editor to keep it here. Of course if the editor prefers so, we can pleasantly delete this part.

Figure 1 – Is this figure showing the mussel farm? It is not clear

Response: Yes, the figure shows the location of the mussel farm. The legend of the figure was accordingly modified for clarity.

Line 108 – “free of ascidians”. Did they have other organisms or they were prepared to be as cleaned as possible from epifaunal organisms?

Response: We thank the reviewer for this comment, indeed, the cleaning concerned all possible epifaunal organisms. Thus, the sentence was modified as follows: … cleaned the maximum possible to be free of ascidians or any other epifaunal organisms,…

Line 111 – Subtitle – You shall add to the title of describe it in the first sentence of this paragraph the designation Experiment 1. Otherwise it will only be referred in the Table 1 caption. Please revise this issue also in Experiment 2 (Line 136).

Response: Both subtitles were modified according to the reviewer’s comment

Lines 114 –You shall call the groups A, B, C and D instead of a, b, c and d to ease the reading.

Response: Corrected according to the reviewer’s comment

Lines 115 – “…a shaded place for one day…” What kind of shaded day? Always the same shadow spot? I would replace “a day” for “24h” if that was the case. When you say “a day” it looks very informal. In this sentence you write “one day” and in the line 117 you write “1 day”. You shall uniform it. 

Response: Both “a day” and “1 day” were replaced by “24h” in accordance to the reviewer’s whereas, details were added for the shaded place.

Line 118 – “(Table 1, Figure 2, Figure 3)”. I understand that these experiments are not easy to described in words, but 1 table and 2 Figures are too much for describe it. I would pass these 3 items to Supplementary material or let Table 1 and transfer Figure 2 and 3 for Supp. Mat.

Response: Probably the comment of the reviewer is reasonable. Nevertheless, since Fishes is an online journal, we believe that it is better to include more Figures to explain the experimental procedures with most possible visualization. Again, we therefore prefer to keep the Figures in the main manuscript and not as supplementary material, but in any case if the editor asks we will willingly transfer them to the supplementary material.

Line 121 – Mortality and Losses are not the same think here?

Response: Losses may also refer to mussel fall to the bottom of the sea. This explanation was added in the revised manuscript, according to the reviewer’s comment.

Line 122 – “…mussels mortality was recorded…”. Mortality was recorded taking into account human eye observation or other method ? Please clarify.

Ascidians are tunicates, but not all tunicates are ascidians. Did you counted and consider all the tunicates or just the ascidians? Please pay attention to this issue along the manuscript.

Response: Mortality was recorded by visual observation, whereas the measurements concerned ascidians. These details were added in this section in accordance to the reviewer’s comment.

Line 124 – “were” instead of “was”

Response: Corrected according to the reviewer’s comment

Line 125 – “All the data were quadrupled”. What do you mean, did you collect four replicates of mussel samples in each depth? Please clarify

Response: Yes. Thus the sentence was modified for clarity as follows: “All data were quadrupled by collecting four mussel samples from each depth”

Here, in this section you shall add information about the importance of quantify and determining the Temperature, Dissolved oxygen and Chl-a. Are they proxys of what? All the readers have to understand it. I would add salinity information in the Experiment 1 since it would be related to Experiment 2.

Response: Information regarding the importance of these abiotic parameters for ascidian population development was added in the revised manuscript, as well as the salinity value, as recommended by the reviewer.

Figure 2 – I would add best quality photos here since it is hard to visualize the mussels.

Response: The reviewer’s comment is reasonable, however unfortunately these are the best quality photos we have. Nevertheless, we believe that the reader can understand the mussels.

Figure 3 – In my opinion this figure brings confusion to your Experiment description and do not clarify it. I would consider to remove it or at most transfer it for Supp. Mat.

Response: The Figure was modified according to a comment of the first reviewer. Again, as stated previously, we would like to ask the editor to keep the Figure here and not in the supplementary material.

Line 137 – Again, “tunicates colonies”. You shall clarify this. Were these mussels collected from the same raft and longlines of the mussels in Experiment 1 ? It is not clear.

Response: We thank the reviewer for raising this uncertainty. Indeed, they were collected from the same farm. This was added for clarification in the revised manuscript

Line 142 – I guess that “lit” is not the abbreviation for “liters”

Response: Indeed, and this has been corrected according to the reviewer’s comment

Line 144 – Replace “estimate” by “regist “. “water salinity” instead of “salinity”.

Response: Corrected according to the reviewer’s comment

Line 150 – Please see my comment above regarding Experiment 1 and the use of 1 table and 2 figures.

Response: Again, we would like to ask the editor to keep the figures and the Table in the main text. However in the opposite case we will of course transfer it to the supplementary material.

Line 160 – Statistics – I would replace this title for “Data treatment and statistical analysis”. This subsection must to be improved. 

Response: Modified, in accordance to the reviewer’s comment

“Comparisons among samples were made…” Which samples are you referring? Which statistical factors did you use? Please clarify.

Response: “Samples and factors” were replaced with “recorded values of mortality and ascidian densities”, in accordance to the reviewer’s comment

RESULTS

Line 169 – “Dissolved oxygen” instead of “O₂”. 

Response: Corrected according to the reviewer’s comment

Lines 166-171 - Were you expecting other temperature, dissolved oxygen and Chl-a patterns than these ones? In my opinion there is no need to highlight these results with a paragraph and a graph in the manuscript. Please add a sentence or two in Material and Methods sections referring the common pattern of these parameters during your experiments. As it is, the manuscript is not enriched with such information.

Response: We consider these values important and therefore we would prefer to keep them in the manuscript if the editor agrees.

Line 176 – Heavy recruitment of what? Please clarify for a better reading.

Response: Heavy recruitment of ascidians. This addition was incorporated in the revised manuscript.

Line 184 – I guess that “already” is not the word here.

Response: The word “already” in accordance to the reviewer’s comment

Figure 7 – Please add to the figure “Number of tunicate colonies” instead of “Number of colonies”. The way you add the Anova results in the figure is wise but the result is very difficult to follow. I would add a table in Supp. Mat with further results of Anova and I would describe the most important ones here in the Result section.

Response: The Figure 7 was accordingly modified, based on the reviewer’s comment

Line 195 – Delete “which were”

Response: Corrected according to the reviewer’s comment

I could not find any results or comments about mussel mortality regarding Experiment 1. You only refer it in the Discussion section (Line 271). This manuscript shall be better organized regarding these issues.

Response: The reviewer is correct. The results were added in the section 3.1 in accordance to the reviewer’s comment.

Figure 7, 8 and 9 are based in the same graph format. I would be more creative in the presentation of your results. Otherwise the manuscript will not have any style and become less attractive.

Figure 8 A, B and C shall be bigger.

Response: All figures quality was improved. We trust this version will satisfy the reviewer’s and editor’s expectations.

Line 210 – “No mussel mortality”. Did you had zero mussel dead individuals along your experiment?

How did you obtain these mortality percentages for tunicate colonies? How did you count colonies? 

Response: Mortalities were measured visually with an approximation based on visual observations, as has been added in the Materials and Methods of the revised manuscript.

Line 211 – “into” instead of “to”

Lines 217 – Sometimes you use “one day” other times you use “24h”. You shall uniform this along the manuscript.

Response: Both issues have been corrected in the revised manuscript

DISCUSSION

The first paragraph of the Discussion contains valuable information to be add in the Material and Methods section.

Response: We thank the reviewer for this suggestion. The paragraph has been moved to the Materials and Methods section, in accordance to the reviewer’s comment.

Lines 235 – During the previous sections (Material and Methods and Results) you referred the tunicates. Here, you start writing ascidians. As I call to attention above, you shall clarify what did you consider during the Experiments.

Response: All experiments investigated ascidians, which has been modified in the whole manuscript, in accordance to previous comments of the reviewer.

Line 235 – Please replace “being increased” by “increase” and “declined” by “declining”

Response: Corrected in accordance to the reviewer’s comment

Line 248 – You shall not begin a sentence with a number.

Response: Corrected in accordance to the reviewer’s comment

Lines 253-258 – So, you shall refer in the Material and Methods section that mussel washing clean them from ascidians but also from other epifauna species.

Response: Added, based on a previous comment of the reviewer (please see revised materials and Methods)

Line 258-259 – This is widely documented. Add some references or be less categoric here.

Response: The sentence has been deleted according to the reviewer’s comment.

Line 260 – Again the Tunicate vs Ascidian nomenclature. Please revise it along the whole manuscript.

Response: The term “Ascidian” has been only kept in the whole document, in accordance to the reviewer’s comment.

Line 217 – “Minor mussel mortality occurred”. This information is not placed in the Results yet you write in Material and Methods “mussel mortality was recorded “. What do you mean by “minor” here? This information is crucial when evaluation the efficiency of an anti-fouling methodology.

Response: In accordance to the reviewer’s comment, this information was added in the section 3.1 in the revised manuscript.

Line 291 – “…and other biofoulings…” Did you collect this information on “other biofoulings”?

Response: The sentence was modified and the parts regarding “other biofoulings” was deleted for clarity.

Lines 297-298 – Who said that? Is it based on your experience?

Response: The statement was indeed very naïve and thus it was deleted from the revised manuscript.

Lines 300-302 – Again, you write ascidians and then tunicates turning the text sense very confusing.

Response: As mentioned in a previous response, the term ascidians was only kept in the whole manuscript.

Lines 313-315 –  

Line 318 – Please delete “for ascidians metamorphosis inhibition” as it is redundant. “air exposure” instead of “air”

Response: Corrected according to the reviewer’s comment

Line 321 – “Currently” instead of “Up to date”

Response: Corrected according to the reviewer’s comment

Lines 339-341 - Your results show that a salinity shock and an exposure period might reduce the present of ascidians in mussels. But is this method realistic for most of the mussel farms? That is, do the typical mussel farms have conditions to perform this type of action as a routine? What are the costs? Is this methodology practicable in mussel farms where tonnes of mussels are collected to be sold every day? How big must be the tanks in these cases? Please improve your discussion with the “dark” side of your findings as it is important to be cautious regarding their practicability and applicability.

Response: A detailed summarizing part was added in the end of the Discussion, presenting the limitations of the proposed methods as well.

I would add to the discussion the benefit of this apparent “success” in eliminating most of ascidians and “other biofoulings” in respect to the potential presence, transport and introduction of non-indigenous/invasive species. That is, this type of methodologies that you propose here might also mitigate the spread of such species into elsewhere area. Please see the study of Piló et al 2021 to develop this topic.

Piló, D., Pereira, F., Carvalho, A. N., Vasconcelos, P., Cunha, A. M., & Gaspar, M. B. (2021). Are non-indigenous species hitchhiking offshore farmed mussels? A biogeographic and functional approach. Marine Pollution Bulletin, 171, 112776.

Response: We thank the reviewer for the interesting suggestion, which has been included in the end of the discussion in the revised manuscript together with the extra reference.

CONCLUSIONS

Line 345 - Please just start with “We proposed…”. I don’t know if “treating” is the best choice here.

Response: Corrected according to the reviewer’s comment

Line 347 – I believe that “mussel” after “immersion” shall be deleted.

Response: Corrected according to the reviewer’s comment

Line 348 – Please rephrase: “… of ascidians with 0% mortality of the cultured organisms”

Response: The phrase was accordingly modified in accordance to the reviewer’s comment

Line 358-361- This final sentence shall be deeply rephrased as it is very hard to understand.

Response: The sentence was deeply simplified based on the comment of the reviewer, to whom we are again grateful for all the constructive improvements suggested.

REFERENCES

I did not perform a thorough revision on the reference list but I recognize that it contains relevant, updated and diverse information that was used in the text.