Quantifying Threespine Stickleback Gasterosteus aculeatus L. (Perciformes: Gasterosteidae) Coloration for Population Analysis: Method Development and Validation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Title

To facilitate the reader on the phylogenetic location of the species, I recommend including the taxonomical indication such as “Gasterosteus aculeatus (Perciformes: Gasterosteidae)”.

 

Abstract and Keywords

No comments on these sections.

 

Introduction

- Lines 31 to 32: “Water pool” may be hard to follow for readers who are not familiar with the terms of aquaculture, so I suggest substituting this term or adding a brief explanation.

-Lines 42 to 43: Care must be taken when mentioning polarization of structural colors in fishes. For example, the reference of Yoshioka et al 2011 does not report polarization in the reflective coloration of neon tetra. For conservative purposes, I would recommend using the term structural coloration instead of polarized coloration.

- Lines 52 to 54: Consider rephrasing since “including, e.g.” in such a long sentence may make the sentence hard for the reader to follow. 

-Line 62: Unless the authors are specifically referring to the seas and not including coastal or oceanic habitats, I suggest rephrasing ”In the seas”.

- Lines 62 to 69: Please provide references for the information stated in the sentences of this paragraph.

- Line 70: Since the geographic sampling area is detailed in the Materials and Methods section, consider not mentioning “which we used as a source of material”.

-Line 73: Using unpublished data should be avoided, so I would recommend being careful when including the reference used in this sentence, unless the information is indispensable for the reader to follow the content of this study.

-Lines 86 to 96: These two paragraphs mentioning the aims and questions include information that overlaps, so please consider rearranging their content into one paragraph in a more straightforward way while avoiding sounding repetitive.

 

Materials and Methods

- Lines 101 to 104: I suggest shortening or splitting this sentence into two, since it becomes hard to follow for the reader.

- Lines 104 to 106: Since this information complements the information mentioned in lines 118 – 120. To avoid repetition and overlapping, I would recommend summing all this content in lines 118 – 120.

- Lines 106 to 108: Unless this content is indispensable to support the methodology detailed in this section, please consider moving this part of the text to the Introduction section. Regarding

- Line 111: Please consider detailing or altering “around the clock light,” for which the meaning may seem ambiguous for the reader.

- Lines 123 to 125: Please mention if the fish collected in 2023 were sampled using the same methods as the ones sampled in 2021; if not, provide the respective information. Also, please mention clearly which fishes are representative of “experimental” and “field data”, making it easier for the reader to follow the Results section.

- Line 146: “were characterized by lack of spawning coloration signs” may sound confusing to the reader, so I recommend rephrasing this part of the sentence.

- Line 139: I recommend using either the terminology “color card” (this sentence) or ”color checker plate” (line 145), for purposes of homogeneity and making it easier for the reader to follow the methodology described.

- Lines 158 to 167: This part of the methodology, while very well detailed, may be overexplained, so please consider summarizing the steps of the described methodology.  

 

Results

General comments:

- When using comparative statements such as “was/were slightly shifted” (lines 278 and 280) and “tended to be less greenish” please add to what these comparisons are respective to, for example “In males from the field conditions, the range was slightly shifted to the greenish compared to the experimental ones”. On the other hand, if these statements are not indicating any comparison but merely the nature of coloration of the SSs (greenish, bluish, yellow), please consider replacing the word “shift” since it may lead the reader to an erroneous interpretation of a comparison statement.

- I would suggest mentioning Figure 6 when referring to statistical significance, since it is represented in that figure.

 

- Line 209: Since the color of squares in Figure 2 was described for SS1-SS8 in line 206, I would suggest indicating the respective information for SS4-SS7.

- Line 229: Please remove “–“ in “ – 20.4.” since the reader may confuse it for a negative value.

- Lines  230 to 232: I would recommend adding and altering the punctuation marks in such a long sentence to make it clearer to the reader.

- Lines 248 to 252 – Please consider rephrasing this long sentence for clearness purposes.

- Line 265: I suggest moving the explanation that coloration in green hues is represented as negative a* values to lines 262 – 263, where the greenish coloration is firstly shown with the mean of negative values for the dorsum of females.

- Line 289: Please consider adding an explanation of the values representing a yellow shift in coloration (such as mentioned earlier for negative values representing a green shift).

- Lines 291 to 293: I suggest detailing which SSs are included when mentioned “across the six different SS” (line 292), as it may not be applied to the SSs for females in the previous sentence (SS1, SS2, SS4, SS6, SS8) since the mean of SS6 for males in experimental condition is not within the mean values mentioned 15.9 to 20.7 (line 293) because it is 7.9 (line 294). Please add a space in “3.6to” (line 293).

- Lines 293 to 294: Please consider adding an explanation of the values representing a blue shift in coloration (such as mentioned earlier for negative values representing a green shift).

- Lines 324 to 325: I recommend rephrasing this sentence since according to the PCA data represented in Figure 7, where overlapping values are observed between males (field and experimental conditions), field condition (males and females), and experimental condition (males and females) which is observed in the quadrant for positive values of both PC1 and PC2.

- Line 367 – Please alter the mentioned values of means in brackets for SS4 and SS6, since these do not match the values shown in Table 1.

- Line 377 – Please close brackets after “SD = 8.31”.

 

Discussion

- Lines 385 to 388: I suggest a structure of enumeration to highlight the items for which the reported method could be effective, for example, “effective for: 1. Describing (…); 2. Capturing…".

- Lines 391 to 392: I would recommend adding a brief explanation of the reasons for coloration alteration in fishes related to the alteration of background, which are described in the following sentences for scorpionfishes, flounders, and sticklebacks.  

- Lines 436 to 438: Please consider adding a couple of references to support the content stated in this sentence, in the case that such a bibliography is available.

- Line 526: Please add an end stop after “yellow”.

- Line 532: To make it easier for the reader to follow the content of this subsection, I would suggest briefly describing the concepts of “patterned and background areas”, what distinguishes them, and how they relate to “pattern-specific variation”, namely in the species for which this study is focused.

 

Conclusion

No comments on this section.

 

References

General comments:

- For homogeneity purposes, please decide to mention either the full names of scientific journals or their abbreviations.

- Please ensure that the references for book chapters are properly structured.

 

Figures and Tables

- Figure 5: Please consider revising the plot representing the field data on cryptic coloration SSs, since the values represented do not agree with the text in the Results section (lines 305 to 309) and the values represented in the respective plot for this data in Figure 6.   

- Table 1: In the legend, consider moving the content of the sentence in lines 352 – 373 to the section of Materials and Methods in the text regarding the pigmentation index, for example,e in lines 173 – 177.

 

Comments on the Quality of English Language

While the content in the Discussion section is well supported and adequately discusses the data in the Results section, it is sometimes hard for the generic reader to follow the information mentioned, and so I would like to suggest rephrasing some parts of the text in a simpler and clearer way. This comment applies particularly to lines 392-395, line 402, lines 413-414, lines 424-427, lines 431-434, lines 452-454, lines 475-477, and line 497.

Author Response

Title

 

To facilitate the reader on the phylogenetic location of the species, I recommend including the taxonomical indication such as “Gasterosteus aculeatus L. (Perciformes: Gasterosteidae)”.

We agreed with the comment, and this is now included in the title. We also extended the latin name to Gasterosteus aculeatus L.

 

Abstract and Keywords

 

No comments on these sections.

 

Introduction

 

Lines 31 to 32: “Water pool” may be hard to follow for readers who are not familiar with the terms of aquaculture, so I suggest substituting this term or adding a brief explanation.

We agreed with the comment and changed the text according to your comment.

 

Lines 42 to 43: Care must be taken when mentioning polarization of structural colors in fishes. For example, the reference of Yoshioka et al 2011 does not report polarization in the reflective coloration of neon tetra. For conservative purposes, I would recommend using the term structural coloration instead of polarized coloration.

We agreed and changed the text according to your comment. .

 

Lines 52 to 54: Consider rephrasing since “including, e.g.” in such a long sentence may make the sentence hard for the reader to follow. 

We simplified the sentence.

 

Line 62: Unless the authors are specifically referring to the seas and not including coastal or oceanic habitats, I suggest rephrasing ”In the seas”.

We agreed and rephrased it as “In the marine environment”.

 

Lines 62 to 69: Please provide references for the information stated in the sentences of this paragraph.

We added new references on this topic.

 

Line 70: Since the geographic sampling area is detailed in the Materials and Methods section, consider not mentioning “which we used as a source of material”.

We agreed with the comment and simplified the sentence.

Line 73: Using unpublished data should be avoided, so I would recommend being careful when including the reference used in this sentence, unless the information is indispensable for the reader to follow the content of this study.

We removed reference to unpublished data and replaced it by some new references.

 

Lines 86 to 96: These two paragraphs mentioning the aims and questions include information that overlaps, so please consider rearranging their content into one paragraph in a more straightforward way while avoiding sounding repetitive.

We combined two paragraphs and rearranged the text to avoid the repetitiveness.

 

Materials and Methods

 

Lines 101 to 104: I suggest shortening or splitting this sentence into two, since it becomes hard to follow for the reader.

We agreed and simplified the sentence.

 

Lines 104 to 106: Since this information complements the information mentioned in lines 118 – 120. To avoid repetition and overlapping, I would recommend summing all this content in lines 118 – 120.

This information was rearranged to the paragraphs describing fish sample size and peculiarities on morphological traits and sex determination.

 

Lines 106 to 108: Unless this content is indispensable to support the methodology detailed in this section, please consider moving this part of the text to the Introduction section.

Introduction is dedicated to only general stickleback biology and in the White Sea and specified on coloration. As size characteristics are used for description of fish samples from the pool and the Seldianaia Inlet, we decided to leave this information here.

 

Line 111: Please consider detailing or altering “around the clock light,” for which the meaning may seem ambiguous for the reader.

We rephrased natural photoperiod conditions as polar-day photoperiod conditions.

 

Lines 123 to 125: Please mention if the fish collected in 2023 were sampled using the same methods as the ones sampled in 2021; if not, provide the respective information. Also, please mention clearly which fishes are representative of “experimental” and “field data”, making it easier for the reader to follow the Results section.

We added information on fishing gear and sampling technique, as well as clarified field and experimental datasets.

 

Line 146: “were characterized by lack of spawning coloration signs” may sound confusing to the reader, so I recommend rephrasing this part of the sentence.

We inserted clarification on the signs of spawning coloration absence.

Line 139: I recommend using either the terminology “color card” (this sentence) or ”color checker plate” (line 145), for purposes of homogeneity and making it easier for the reader to follow the methodology described.

We unified the term within the text.

 

Lines 158 to 167: This part of the methodology, while very well detailed, may be overexplained, so please consider summarizing the steps of the described methodology.  

We have agreed with this comment and summarized the paragraph.

 

Results

General comments:

- When using comparative statements such as “was/were slightly shifted” (lines 278 and 280) and “tended to be less greenish” please add to what these comparisons are respective to, for example “In males from the field conditions, the range was slightly shifted to the greenish compared to the experimental ones”. On the other hand, if these statements are not indicating any comparison but merely the nature of coloration of the SSs (greenish, bluish, yellow), please consider replacing the word “shift” since it may lead the reader to an erroneous interpretation of a comparison statement.

- I would suggest mentioning Figure 6 when referring to statistical significance, since it is represented in that figure.

We agreed with the comments and rephrased the description and reference to the figure where it is needed.

 

 Line 209: Since the color of squares in Figure 2 was described for SS1-SS8 in line 206, I would suggest indicating the respective information for SS4-SS7.

We have added color identification of SS4-SS7 to the text.

 

Line 229: Please remove “–“ in “ – 20.4.” since the reader may confuse it for a negative value.

Agreed.

 

Lines  230 to 232: I would recommend adding and altering the punctuation marks in such a long sentence to make it clearer to the reader.

We agreed and restructured the text.

 

Lines 248 to 252 – Please consider rephrasing this long sentence for clearness purposes.

We agreed and restructured the text.

 

Line 265: I suggest moving the explanation that coloration in green hues is represented as negative a* values to lines 262 – 263, where the greenish coloration is firstly shown with the mean of negative values for the dorsum of females.

We agreed and rewrote the description a bit.

 

 

Line 289: Please consider adding an explanation of the values representing a yellow shift in coloration (such as mentioned earlier for negative values representing a green shift).

We agreed with the comment and added clarification on b* parameter values.

 

Lines 291 to 293: I suggest detailing which SSs are included when mentioned “across the six different SS” (line 292), as it may not be applied to the SSs for females in the previous sentence (SS1, SS2, SS4, SS6, SS8) since the mean of SS6 for males in experimental condition is not within the mean values mentioned 15.9 to 20.7 (line 293) because it is 7.9 (line 294). Please add a space in “3.6to” (line 293).

We clarified information on the six standards sites and corrected the formatting.

 

Lines 293 to 294: Please consider adding an explanation of the values representing a blue shift in coloration (such as mentioned earlier for negative values representing a green shift).

We clarified our results in this section.

 

Lines 324 to 325: I recommend rephrasing this sentence since according to the PCA data represented in Figure 7, where overlapping values are observed between males (field and experimental conditions), field condition (males and females), and experimental condition (males and females) which is observed in the quadrant for positive values of both PC1 and PC2.

We agreed with the comment and rephrased PCA results description.

 

Line 367 – Please alter the mentioned values of means in brackets for SS4 and SS6, since these do not match the values shown in Table 1.

There were two typos and we corrected them to the right values according to Table 1 “...in SS4 (13.19±5.93), SS6 (13.85±6.51)...”.

 

Line 377 – Please close brackets after “SD = 8.31”.

Corrected.

 

Discussion

 

Lines 385 to 388: I suggest a structure of enumeration to highlight the items for which the reported method could be effective, for example, “effective for: 1. Describing (…); 2. Capturing…".

We agreed and corrected this. 

 

Lines 391 to 392: I would recommend adding a brief explanation of the reasons for coloration alteration in fishes related to the alteration of background, which are described in the following sentences for scorpionfishes, flounders, and sticklebacks.  

It should be noted that one of reviewers recommended deleting the fragment on other fish, except sticklebacks (gobies and others), due to over-explanation, thus we only have added general info on the reasons of color changing in the beginning of the paragraph.

 

Lines 436 to 438: Please consider adding a couple of references to support the content stated in this sentence, in the case that such a bibliography is available.

We have managed to find some references and added them into text.

 

Line 526: Please add an end stop after “yellow”.

Agreed.

 

Line 532: To make it easier for the reader to follow the content of this subsection, I would suggest briefly describing the concepts of “patterned and background areas”, what distinguishes them, and how they relate to “pattern-specific variation”, namely in the species for which this study is focused.

We agreed with the comment and included details on patterned or background areas of a fish body.

 

Conclusion

 

No comments on this section.

 

References

 

General comments:

- For homogeneity purposes, please decide to mention either the full names of scientific journals or their abbreviations.

- Please ensure that the references for book chapters are properly structured.

We agreed with all comments and made some revisions in names of scientific journals and book chapters.

 

Figures and Tables

 

Figure 5: Please consider revising the plot representing the field data on cryptic coloration SSs, since the values represented do not agree with the text in the Results section (lines 305 to 309) and the values represented in the respective plot for this data in Figure 6.  

We would like to thank you for this finding as it is crucial for understanding the whole results patterns. We remastered the picture and added an updated version in the Results section.

 

Table 1: In the legend, consider moving the content of the sentence in lines 352 – 373 to the section of Materials and Methods in the text regarding the pigmentation index, for example,e in lines 173 – 177.

We agreed and moved this description to Materials and Methods.

 

 

While the content in the Discussion section is well supported and adequately discusses the data in the Results section, it is sometimes hard for the generic reader to follow the information mentioned, and so I would like to suggest rephrasing some parts of the text in a simpler and clearer way. This comment applies particularly to lines 392-395, line 402, lines 413-414, lines 424-427, lines 431-434, lines 452-454, lines 475-477, and line 497.

We accepted comments and made corrections to the text where they were appropriate.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript addresses an essentially methodological issue, yet it possesses bioconductual applications in a species of very useful fish as a biological model, thereby endowing the manuscript with scientific significance and rendering it a suitable candidate for publication in Hydrobiology.

The work is well-organized, structured, and clearly articulated; however, I believe it should be condensed to enhance comprehension of the subject matter. Certain paragraphs are excessively lengthy, particularly in the methodology and results sections. All specific comments and suggestions for modifications to the form and substance of the manuscript were incorporated within the text to provide optimal guidance for the authors.

The principal structural findings are summarized below:

1) The initial keyword is present in the title and requires substitution.

2) In Figures 3, 4, and 5, it is advisable to eliminate the minor fluctuating points that denote the value of each individual, as they do not contribute additional information and impair visibility.

3) The initial portion of the conclusion statement requires revision to pertain specifically to the study subject.

Comments for author File: Comments.pdf

Author Response

General comments

 

1) The initial keyword is present in the title and requires substitution.

We agreed and deleted this keyword.

2) In Figures 3, 4, and 5, it is advisable to eliminate the minor fluctuating points that denote the value of each individual, as they do not contribute additional information and impair visibility.

See our comment on figures 3-5 below in the Results section.

3) The initial portion of the conclusion statement requires revision to pertain specifically to the study subject.

We agreed and clarified our statement.

Abstract

 

Line 27: This keyword is already in the title

We agreed and deleted this keyword.

Materials and Methods

 

The English writing is appropriate, however, it would be very useful for the manuscript that the authors economize the writing to maximize the main message. Less is more

We combined and shortened some paragraphs, but at the same time we had to add details on statistics analysis. Anyway, we expected this section to be easier to read after this revision.

 

Results

 

Line 242-243: These data seem to overload the figure, from my point of view they are unnecessary, since they are already included with the violin graph.

We believe that retaining the individual data points is beneficial in our case. According to recommendations by Weissgerber et al. (2015) (Weissgerber TL, Milic NM, Winham SJ, Garovic VD (2015) Beyond Bar and Line Graphs: Time for a New Data Presentation Paradigm. PLoS Biol 13(4): e1002128. doi:10.1371/journal.pbio.1002128), univariate scatterplots are the best choice for showing the distribution of the data in small samples. Overlaying these points on the violin plot allows readers (i) to visualise sample size and spot any potential outliers at a glance, (ii) avoid misinterpreting kernel-smoothed shapes that can appear overly precise with modest n and (iii) to evaluate whether particular sub-groups drive the overall distribution.

 

Line 273-274: Same observation as in the previous figure

See the previous comment on the figure design and conception.

 

Line 301-302: Same observation as in the previous figure

See the previous comment on the figure design and conception.

 

Conclusion

 

Line 551-552: This phrase does not seem to take anywhere

We rewrote this sentence as it was described earlier.

Reviewer 3 Report

Comments and Suggestions for Authors

I think this is an interesting manuscript, but it still needs some improvements in clarity and content for easy of reading and interpretation 

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript is too long and can easily be reduced with no loss of relevant information

Author Response

General comments

 

I think this is an interesting manuscript, but it still needs some improvements in clarity and content for easy of reading and interpretation 

The manuscript is too long and can easily be reduced with no loss of relevant information

We tried to simplify and shorten information without significant loss of information in different sections and paragraphs.

 

Title

 

Delete “... for population analysis”

We put “population analysis” in the title not to expand, but narrow the scope of our work, make it more focused exactly on this direction to avoid too broad application of our results, because our methodology is directed to large sample size and, potentially, assume analysis of coloration data together with other population characteristics. We paid special attention to this in the further text.

 

 

Introduction

 

Line 25: this is too much given the approach and evidence provided here

We have removed the last phrase “and other species”.

 

Line 56-61: this appears to be lost here; probably better to be included below after the characterization of stickleback populations in the white sea... this way method description will flow better

We agreed and moved the paragraph below in the text to save the logic in description.

 

Lines 62-69: This paragraph must be improved; it appears "too humanized" and requires bibliographic support

We added new references on this topic.

 

Line 74-77: unclear. This is likely a consequence of different hormonal levels manifested in variable individual nuptial coloration intensity

We rewrote the sentence for a better understanding.

 

Line 86: an improved ... universal is too much given the limited conditions tested and/or analysed

We decided to write just a technique without clarifications to delete any misunderstanding, excessive detail or a deeper meaning.

 

Materials and Methods

 

Line 104-108: Instead of this general characterization, relevant data is the size and age structure of the 1100 individuals placed in the experimental pool

We updated the description in the paragraph.

In this section we described general info of this species that inhabits White Sea. A sample with more than 1,000 individuals is a good model of natural population, so we expected the same parameters for the whole fish group (1,100 individuals) in the water pool as in the wild population.

On the other hand, we added the data on mean size in both samples (51 experimental and 60 wild fish), which was similar to the generalized information for the sticklebacks from the White Sea.

Also, we did not plan to connect our results with stickleback population structure, so we did not study the age structure of sampled individuals thoroughly.

 

Line 117: I miss an ethical statement, and think this must be included in the manuscript

We mentioned the ethical statement in the specials section at the end of the article.

 

Line 118-122: again I miss a description of the size and age of individuals used for the analysis

See the previous comment for lines 104-108.

 

Line 125-126: this appears strange given the previous information on 2:1 sex ratio; again some information on the size and age of individuals, is missing and will be important

We added that in this case we simply balanced the sex ratio.

 

Line 194: Tests on scores must be indicated here

We added information on testing scores in PCA.

 

Results

 

Line 224: Simplify results structure

We agreed with this proposal and changed the numeration in the Results section.

 

Line 328-330: only loading over 0.30 should be listed and interpreted

also it is not clear how this relates to individual variables

Clarification is required

We altered this fragment a lot and included only loadings higher than 0.30.

 

Line 336: if that is case, scores along this PC should also be presented in the main manuscript

We described scores for PC3 in the paragraph with t-tests and added them in the text.

 

Line 337-343: As indicated above clarification is required regarding individual variables used for analysis and parameters

See the previous comment.

 

Line 348-349: clarify this

We rewrote the explanation of 24 variables.

 

Discussion

 

This section is too long, and should be easily reduced without loss of relevant information

We truly appreciate your proposal, but suppose that any subsection is important in Discussion. All of them give a better understanding of the technique’s implementation,

advantages, disadvantages and limitations and help to compare the results with other fish coloration studies.

 

Line 492: Reverse: Males also show red coloration in SS4

Agreed.

 

Line 508-509: support references are required here

We included some references to this fragment.

 

Conclusion

 

Line 556: given there is some overlap with small samples sizes, I have concerns about what will happen with larger sample sizes

We rephrase the paragraph for a better understanding.

 

Line 563: This should not be an independent paragraph

Agreed.

 

Line 566: This has not been tested and cannot be assumed here

We agreed and deleted this fragment.