Prey Identification of the Little Tern, Sternula albifrons (Pallas, 1764), by Applying DNA Barcoding to Fecal Materials

Round 1

Reviewer 1 Report

Comments of Reviewer: Manuscripts ID sustainability-1870503

Entitled: "Prey identification of the little tern (Sternula albifrons) by applying DNA barcoding to fecal material" by  Hyunbin et al.

The manuscript in all is well written, coherent and understandable in every way. The identification of prey animals and food web structures are always a hard task in ecological researches. As the authors suggested, only observation based studies lack a lot of information about the prey number and population size. With these types of resaerches we get more detail/information about the structure of aquatic/wetland food web structures. Overall, it gives us new and usefull inforamtion, and new approaches to understand the processes.

Minor problems:

At first mentioning a species, please indicate the descriptor and date too.

line 45: please be more specific about the food web. It can be terrestiral, aquatic or wetland, but in this case I suggest the use of aquatic/wetland habitat. Please change it through the whole manuscript.

line 64-66: The two following sentences are not logically connected. The authors start to wrote down some information about the feeding of little tern, but the nex sentence is about the worldwide spread of the species. Please give more information about the little tern (habitat preference, feeding strategy, mating, fecal production, etc...), and re-phrase this paragraph.

line 72: in natural habitat...

line 86: indicate descriptor and date here too

Figure 1: please indicate the colletion points too, it can be crucial to know from where the samples are collected. Please indicate the number of collected samples in 2.2.

Through the manuscript, the use of little tern is not universal. Sometimes the authors starting it with capital sometimes not, please make it uniformal.

Questions:

1. What is the retention time of the consumed food in case of the little tern? It can be useful to know it, becasue sometimes the less chitinized species may be totally absorbed.

2. Do the sampling sites are scattered from each other? It is also quite informative to know that the land use may have any effect on the feeding strategy.

3. I have a little concern about the number of collected samples. I think it is exiguous. How the authors be 100 % sure, that they covered the whole spectrum of the prey species?

Author Response

Reviewer 1.

The manuscript in all is well written, coherent and understandable in every way. The identification of prey animals and food web structures are always a hard task in ecological researches. As the authors suggested, only observation based studies lack a lot of information about the prey number and population size. With these types of resaerches we get more detail/information about the structure of aquatic/wetland food web structures. Overall, it gives us new and usefull inforamtion, and new approaches to understand the processes.

• All of the authors appreciate Reviewer 1's positive and valuable comments. We had discussions for the improvement of the manuscript in accordance with the suggestions and did our best to accept all of the comments. We marked the changes and revision results in RED.

Minor problems:

At first mentioning a species, please indicate the descriptor and date too.

• We added the descriptor and date at the title, abstract, and the first mention in the main text (Lines 2-3, 16, 63-64)

line 45: please be more specific about the food web. It can be terrestiral, aquatic or wetland, but in this case I suggest the use of aquatic/wetland habitat. Please change it through the whole manuscript.

• We specified food web types and changed them throughout the manuscript (Lines 45, 48).

line 64-66: The two following sentences are not logically connected. The authors start to wrote down some information about the feeding of little tern, but the nex sentence is about the worldwide spread of the species. Please give more information about the little tern (habitat preference, feeding strategy, mating, fecal production, etc...), and re-phrase this paragraph.

• We agree with this comment. We re-phrased the paragraph according to the comment. Please see the Lines 63~67.

line 72: in natural habitat...

• We changed it at Line 72.

line 86: indicate descriptor and date here too

• We indicated the descriptor and date.

Figure 1: please indicate the colletion points too, it can be crucial to know from where the samples are collected. Please indicate the number of collected samples in 2.2.

• Thank you for your detailed comment. We changed figure 1 according to the comment.

Through the manuscript, the use of little tern is not universal. Sometimes the authors starting it with capital sometimes not, please make it uniformal.

• We changed it throughout the manuscript (Lines 255, 262).

Questions:

1. What is the retention time of the consumed food in case of the little tern? It can be useful to know it, becasue sometimes the less chitinized species may be totally absorbed.

• As per the comment, we agree that the retention time of the consumed food for little tern affects the digestion of prey items. We, therefore, discussed it on Lines 212~214.

2. Do the sampling sites are scattered from each other? It is also quite informative to know that the land use may have any effect on the feeding strategy.

à We carried out a scattered sampling to gather fecal materials in Doyodeung Island. However, we did not analyze the feeding strategy at this moment due to the lack of samples. We will provide new insight into feeding strategies for little terns in further research.

3. I have a little concern about the number of collected samples. I think it is  How the authors be 100 % sure, that they covered the whole spectrum of the prey species?

We agree with your concern about the number of collected samples. Figure 2B also showed insufficient samples, and we did not discuss the population level of little tern. We left it as a limitation in our research, which should be addressed in further study.

Reviewer 2 Report

I have found this manuscript very fascinating, and it is written well and described comprehensively. This study provided essential information on the feeding preferences of little tern based on DNA sequence data. I eagerly await follow-up studies to aid the ecological community's understanding of the estuarine ecosystems. I think it is enough quality and attractive to the readers of Sustainability. I hope suggestions and minor comments will help improve the manuscript before it can be published d at the journal. Comments are listed below.

Suggestion: 

1) As I know, contamination is a crucial issue in the metagenome study field. Please explain a method for reducing contamination, such as cross-contamination of each sample in the experiment design.

2) The statistical analysis looks poorly explained and is poor, which might affect the conclusions. It is essential to avoid that to enforce the credibility of a paper with important data. Therefore, you should explain it much more clearly and provide more detailed information (i. e., which kind of data did you use, transformed data or not?)

Minor comments:

1) Line 121 - 135: please provide detailed information, such as nationality, Cat. No. and so on for kits and devices.

2) Line 147: [a 2% -> a 2%

3) Line 182, 183: Figure 2a, 2b --> please capitalize "a" and "b"

4) Figure 1: please delete "A," "B".

Author Response

Reviewer 2.

I have found this manuscript very fascinating, and it is written well and described comprehensively. This study provided essential information on the feeding preferences of little tern based on DNA sequence data. I eagerly await follow-up studies to aid the ecological community's understanding of the estuarine ecosystems. I think it is enough quality and attractive to the readers of Sustainability. I hope suggestions and minor comments will help improve the manuscript before it can be published d at the journal. Comments are listed below.

• The authors appreciate every positive and valuable comment to improve the manuscript. We did our best to revise the manuscript according to your comments. Changes are marked in RED.

Suggestion: 

• As I know, contamination is a crucial issue in the metagenome study field. Please explain a method for reducing contamination, such as cross-contamination of each sample in the experiment design.

à We agree with your concern about contamination. Therefore, we described a method for reducing contamination, such as cross-contamination. Please see the Lines 121-123.

• The statistical analysis looks poorly explained and is poor, which might affect the conclusions. It is essential to avoid that to enforce the credibility of a paper with important data. Therefore, you should explain it much more clearly and provide more detailed information (i. e., which kind of data did you use, transformed data or not?)

à Thank you for your comment. We added detailed information about statistical analysis (Line 156)

Minor comments:

1) Line 121 - 135: please provide detailed information, such as nationality, Cat. No. and so on for kits and devices.:  We accepted the comment and added the requested information.

2) Line 147: [a 2% -> a 2%: We deleted it.

3) Line 182, 183: Figure 2a, 2b --> please capitalize "a" and "b": We modified the letters.

4) Figure 1: please delete "A," "B".: We accepted the comment.