Isotopic Turnover and Fractionation of δ15N and δ13C in Captive Pseudopleuronectes americanus (Walbaum)
Round 1
Reviewer 1 Report
Dear Authors,
Stable isotope studies in fish species provide a wide range of information, from seasonal feeding and migration pathway patterns to intra- or inter-species resource partitioning and trophic position, so the content of the manuscript holds significant value, there are considerable inconsistencies, particularly in terms of technical aspects and references. The references have not been prepared according to the journal's guidelines. There is a need for reformatting of fonts, especially within tables and graphs, as some appear to have been directly copied from Excel. The article must be revised to adhere to the journal's writing standards. Upon making these necessary corrections, I believe the manuscript could be considered for acceptance.
Best regards.
Comments for author File: 
Comments.pdf
Author Response
Thank you so much for the feedback and comments. Please see the attachment for response to the editor and reviewers.
Author Response File: Author Response.pdf
Reviewer 2 Report
REVIEW: fishes-2594770
This is an interesting paper looking at the turnover and fractionation of carbon and nitrogen stable isotopes in four tissues of winter flounder. The results provide novel information about changes and differences in isotope values over time and among tissue types. I found the manuscript well-written and the results worth publishing. However, the study focuses on only one consumer species (winter flounder), lacks clear study hypotheses and proper statistical testing, and is also based on very low sample sizes (i.e., n = 2 per treatment). Thus, I am not fully confident about the reliability of the findings nor about the general attractiveness of the paper for a broader readership. Below I first elaborate the main concerns and then provide some minor editorial suggestions for further revision of the manuscript.
MAJOR COMMENTS:
Firstly, the authors should aim to come up with clear study hypotheses that can be tested with proper statistical analyses. As the manuscript stands now, it remains somewhat unclear what the authors expect to find and there are also no statistical testing done to support the findings. Although the authors refer to statistical testing (e.g. in line 243->), no statistical analyses have been conducted and thus many of the findings seem somewhat speculative as the result might arise from pure chance. My gut feeling is that the authors have done no statistical testing because of the insufficient sample size (n = 2 fish per treatment). This fact is the main “stumbling block” of the study, as it severely decreases the reliability of the overall study findings.
Secondly, the authors have decided to conduct the study during the winter season with minimal overall growth of fish. I find it hard to come up with any good reasoning for this, because the isotopic turnover and fractionation might be quite different during the summer growing season, which is the time when the winter flounder likely have the most important role also in the food web of the study area. Hence, if the study aims to provide detailed information about isotopic dynamics to support other food web studies in the region, it feels a bit awkward to conduct the study during a period when the consumers (winter flounder) are expected to have little impact on other guilds in the food web.
MINOR COMMENTS:
L9-22: The Abstract reads very well!
L77-83: Please aim to come up with clear, testable study hypotheses. This would help the reader to get an idea of what you are predicting (changes over time & differences among tissue types).
L99 & L127: Only two individuals (n=2) were sacrificed for each treatment. This is insufficient sample size for most statistical tests and the results can be affected more by chance than real isotopic changes/differences.
L156: This paragraph “Statistical Analysis” does not describe any real statistical analyses that would have confirmed the patterns in isotopic changes / differences.
L209-210 and elsewhere: Please include the per mil symbol “‰” in all cases in the body text where you report delta-values, and also in the axis titles (e.g. in Fig. 3) and all figure and table captions. Also provide the sample sizes in tables and figures when appropriate.
Table 4: Please elaborate what “m”, “t50” and “t95” stand for and also use superscript for numbers in the delta-values.
L256, L266 and elsewhere: The wordings like “slightly higher” and “was significant” are somewhat vague and not convincing, because no proper statistical testing has been conducted.
L266: I believe “contribute” should be replaced with “contribution”?
L287: What does “rom” stand for?
L327: Replace “where” with “were”.
L369: Check the incorrect author name “Geroux”.
L372: I believe the Latin names should be provided for all species when first mentioned (here for arctic cod)?
L381: Replace “effect” with the verb “affect”.
L403: The abbreviation “SIA” is only used once. Thus, replace “SIA” with “stable isotope analyses”.
L442-444: Use consistent number of decimals.
L453: I believe “is” should be replaced with “in”?
Author Response
Thank you so much for the feedback and comments. Please see the attachment for response to the editor and reviewers.
Author Response File: Author Response.pdf
Reviewer 3 Report
L32: is the order of citation correct?
L38: please cite at least 1 more recent publication
L87: Watanabe et al., 2006 2005, delete the S and Y as first initial.
L110 and 143: Please explain the reason behind the use of digestive issues for isotope analysis and isotope turnover indicator.
L153-154: Personally I don’t think there is a need to list the equation anymore after 30 plus years of stable isotope study and publication.
Table 1. please state the unit for each measurement
This table showed in some individuals, after 105 to 147 days of experiment, weight gain was very minimal even lost weight while some were double. Which caused the difference and how it impacted the results and interpretation?
Table 2. I am confused. The caption said values shown for initial and final, but there are two groups of data each for each diet I don’t see initial or final. Same as Table 3. For both tables, same subtitle should be listed once only.
Figure 5 and 6: it appears shorter sampling frequency (more samples) is needed to better show when the issue reached isotope equilibrium.
Table 5: machine analytical accuracy is single digit please do not report isotope data with 2 significant digits.
4. Discussion:
The author stated Winter flounder in this study experienced minimal growth, preventing the calculation of an exponential growth constant (k) as there was little to no change in the average total lengths and weights of fish sampled over time. Values for k were therefore estimated from a diet switch in a model marine species, juvenile Atlantic croaker Micropogonias undulatus 314 (Mohan et al. 2016) to model tissue turnover under high, low, and no growth scenarios. Work done by previous researcher including this reviewer showed significant weight gain by the end of experiment. The results from this study probably showed data for flounder under environmental stress which led to poor growth. New issue with new isotope signature was incorporated to replace old issue and isotope value prior to experiment.
Line 411, 463, 464: please added unit for isotope values
Line 434: large isotope enrichment for C-13 (>2ppt) needs more explanation. Is this due to the loss of light isotope during growth stress? Does the diet contain more lipid than the fish? For example.
Are the authors able to calculate the weighted average isotope enrichment using the isotope values of mass of different tissues?
The authors should also discuss results for other similar experiments with low growth if available.
very good
Author Response
Thank you so much for the feedback and comments. Please see the attachment for response to the editor and reviewers.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
The authors have done a good job in responding to the reviewer comments and revising the manuscript. The available data and study setting does not allow overcoming my main concern related to the low sample sizes (n=2 per treatment), so it is up to the Editor to decide whether this is something that prevents the manuscript to be acceptable for publishing in Fishes. Otherwise I think the manuscript is acceptable in its present form.
Reviewer 3 Report
no issue remains, I recommed for publication.
