The Effect of Ultrasound Waves on the Pre-Settlement Behavior of Barnacle Cyprid Larvae

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The topic addressed in this paper is important for searching antifouling agents. This manuscript is prepared well with a good research design. The methods are clearly explained in the methodology section. Few points need to addressed in the final draft

1. Why Tetraclita stalactifera was selected for this study? Is it a common model organism for antifouling studies?

2. What is the reason for keeping the cyprids for three days before the experiments? 

3. If I understand correctly, there are 20 cyprids for each treatment, but performed in single larvae in each trial. However, there is no error bars for showing these values in figures.  

4. The results section needs attention. It is starting with a discussion part. A concise results section followed by a discussion section will add more clarity or both sections may be combined to avoid confusion. 

Author Response

We thank the reviewer for the thorough analysis of our manuscript and the valuable comments provided.

Comment 1. Why Tetraclita stalactifera was selected for this study? Is it a common model organism for antifouling studies?

This barnacle is a common and abundant intertidal species found along the coastlines of the Western Atlantic, especially in tropical and subtropical regions. Although it is not a standard model organism for antifouling tests, previous studies have utilized locally abundant barnacles, as we did in our research.

Comment 2. What is the reason for keeping the cyprids for three days before the experiments? 

Since they are lecithotrophic (i.e., they do not feed), we aimed to ensure that the cyprid larvae were in the final stages of their nutritional reserves, which would prompt them to settle as quickly as possible.

Comment 3. If I understand correctly, there are 20 cyprids for each treatment, but performed in single larvae in each trial. However, there is no error bars for showing these values in figures.  

The reviewer is correct; we used 20 cyprid larvae for each treatment. Error bars were included for quantitative variables (speed and NGDR, shown in Figures 5 and 6, respectively), but not for the semi-categorical variable (time frequency, %; shown in Figures 3 and 4). We believe that adding error bars for the semi-categorical variable would not add significant value, as the statistical results from the G-test are already included.

Comment 4. The results section needs attention. It is starting with a discussion part. A concise results section followed by a discussion section will add more clarity or both sections may be combined to avoid confusion. 

Thank you for pointing this out. We have moved the paragraphs discussing the results to the appropriate section.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors perform a study to correlate the pre-settlement behavior of barnacles and the parameters of ultrasound waves. The research is indeed interesting and the manuscript is clear and well-written. Therefore, acceptance following minor revision is recommended. 

A few minor questions and notes

1) It is not clear in the article how the effects of ultrasound waves on the pre-settlement behavior of the barnacles will affect their fouling activity. In other words, how if one knows the pre-settlement behavior of the foulants, he/she will be able to predict whether the surface will be colonized or no? This is important to be address in the article in order to clarify the purpose of the research. 

2) The Introduction is balanced and presents in detail the state-of-the-art, but it is wondering why the authors do not mention the possibility for "passive" biofouling protection via extremely water-repellent coatings? They have the advantage to not use an additional energy source, can be combined with active sources, such as ultrasound systems or solar irradiation systems and some of these coatings possess very high mechanical durability and anti-fouling performance towards a cocktail of colonizing microorganisms even upon loss of water-repellency. So it will be good to mention that possibility. Literature: https://doi.org/10.1016/j.jfoodeng.2014.01.031 ; https://doi.org/10.1016/j.msec.2016.11.115 ; https://doi.org/10.1016/j.colsurfa.2019.123880 ; https://doi.org/10.1016/j.porgcoat.2023.107719 

3) To make the manuscript more appealing, photographs of the experiments (the water bath with larvae and the ultrasound system) are recommended and mandatory to be included in the main body. 

Author Response

We would like to acknowledge the valuable comments provided by the reviewer.

Comment 1. It is not clear in the article how the effects of ultrasound waves on the pre-settlement behavior of the barnacles will affect their fouling activity. In other words, how if one knows the pre-settlement behavior of the foulants, he/she will be able to predict whether the surface will be colonized or no? This is important to be address in the article in order to clarify the purpose of the research. 

Thank you for your comment. However, we believe this point is clearly addressed at the beginning of the Discussion section. We emphasize that the 10 kPa treatment resulted in more pronounced behavioral changes, suggesting that a lower acoustic pressure is more effective than higher pressures of 15 and 20 kPa. The fact that surface inspection was less frequent for the 10 kPa treatment, resulting in more time spent in the water column, indicates that this acoustic pressure is more effective at repelling cyprid larvae from artificial substrates.

Comment 2. The Introduction is balanced and presents in detail the state-of-the-art, but it is wondering why the authors do not mention the possibility for "passive" biofouling protection via extremely water-repellent coatings? They have the advantage to not use an additional energy source, can be combined with active sources, such as ultrasound systems or solar irradiation systems and some of these coatings possess very high mechanical durability and anti-fouling performance towards a cocktail of colonizing microorganisms even upon loss of water-repellency. So it will be good to mention that possibility.

We appreciate your comment and suggestion. However, we believe that the third paragraph of the Introduction already provides sufficient detail on the widespread use of "passive" water-repellent coatings to prevent biofouling.

Comment 3. To make the manuscript more appealing, photographs of the experiments (the water bath with larvae and the ultrasound system) are recommended and mandatory to be included in the main body. 

Thank you for the suggestion. We have added a photograph of the experimental setup to the original Figure 1.