Comparing Macroscopic and Quantitative Histological Methods to Determine Sexual Maturity in the Female European Plaice, Pleuronectes platessa Linnaeus, 1758

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

REVIEW REPORT Article no. 4048723

BRIEF SUMMARY 

The submitted manuscript in ANIMALS-4048723 entitled “Sexual Maturity Determination for the Female European Plaice, Pleuronectes platessa, Through Quantitative Histology” addresses a significant issue in fisheries reproductive biology and presents a stereology-based quantitative histology approach to classify maturity stages in female Pleuronectes platessa, comparing results with macroscopic staging and estimating maturity ogives (L50). The manuscript is well organized and written, methodologically sound and the dataset is a welcome addition to the field. However, there are some significant matters need to be solved before it is suitable for publication. My comments are outlined below.

 

Positive Aspects

I would particularly like to highlight the following strong aspects of the manuscript:

• The stereological technique is strong, as it is performed well and described clearly.
• The inter-rater reliability after calibration is high.
• The comparison of the assessment of maturity on a macroscopic ns a stereological level is useful in standardization within ICES.
• The classification tree is a useful exploratory technique for use in the field.
• The availability of open data improves transparency and reproducibility of the manuscript.

 

SPECIFIC COMMENTS 

MAJOR COMMENTS

1. Novelty and Overlap with Previous Publications

Page 1, Lines 1–18 (Abstract)

The abstract doesn’t indicate what is new in this work compared to the previously published Sauger et al. (2023) or Zenodo dataset.

Recommendations: State the novelty of the study based on previous literature.

 

2. Redundant description of methods already published

Page 5, Lines 93–105

The general description of sampling retains most of the content from Sauger et al. (2020) and Sauger et al. (2023).

Recommendations: State the novelty of the study with respect to the previous literature.

 

For the above two comments, a substantial portion of the data and methodological basis has previously been published in Sauger et al. (2023) and in the dataset deposited to Zenodo. You do refer to those sources, but the manuscript does not clearly outline what is new in the present work.

Recommendations: Please add a specific explanation, detailing:

• which analyses and results are novel, such as maturity phase classification model, confusion matrix, L50 comparisons, classification tree),
• which components derive directly from previously published material, and
• why the present manuscript constitutes a distinct scientific contribution.

This makes obtaining clarity on the originality of the work absolutely indispensable.

 

3. Cell homogeneity testing limited by sample composition

Page 6, Lines 149–154

Homogeneity test is based on 15 individuals (Line 154), which do not have any late-vitellogenic or hydrated oocytes. This may limit the generality of conclusions across the full reproductive cycle. Also, the dataset used for the homogeneity test contains no vit3, pho, ho stages.

Recommendations: Include a limitation statement explaining that late reproductive stages were not represented. Expand the limitations discussion to mention how homogeneity might differ during peak vitellogenesis or spawning.

 

4. Insufficient pre-calibration missing data limitation

Page 10, Lines 196–198

Text: “the first readings were not recorded…could not be compared.”

Recommendations: Include a clear acknowledgment of this limitation in the Discussion section (Section 4.2).

 

5. Inter-Rater Reliability

In the manuscript, it is stated that the pre-calibration readings values were not documented, making it impossible to assess improvements that might have been realized by implementing the reading technique.

Recommendations: This limitation should be clearly recognized and written about. It would be useful to include any available retrospective comparison, even partial, that has been completed.

 

6. Overlap with the previously published figures/lexicons

Page 11, Figure 2; Page 6, Table 2

The figure 2 and the list of cellular structure (in Table 2) conceptually overlap with Sauger et al. (2023).

Recommendations: The figure should say if it reproduces previous published work or perhaps re-analyses data in a new manner.

 

7. Classification Rules for Maturity Phases

Page 12, Lines 245 – 262

Given the stereology-based maturity classification relies on several decision thresholds (e.g., ≥50% alpha atresia for omitted spawning, presence/absence of certain follicle types).

Recommendations: These thresholds need more detailed biological or statistical justification. If a test on sensitivity to threshold changes was conducted using the model, please describe this. Otherwise, a short justification of the chosen values is needed.

 

8. Interpretation of L50 Differences

Page 16, Lines 391–403

The discussion recognizes discrepancies between the L50 median from macroscopic observations and that of stereological observations, but it is not concerned with implications regarding biological systems or management.

The stereological and macroscopic L50 values are quite different (20.6 cm vs. 28.6 cm). The implementation of such a difference are significant, namely in stock assessment, minimum landing size (MLS), and interpretation of past maturity trends.

Recommendations: Enhancement of interpretation of the L50 results. Additionally, consideration should be given to discussion on:

• Potential biological reasons for earlier maturity under stereology,
• Sampling bias (e.g., attempting to collect small immature fish towards the end of the study),
• Comparison with historical L50 values for Channel Plaice,
• Implications from management to ICES maturity staging practice.
• Sampling bias, comparisons with historical L50 values, implications for management (ICES, MLS 27 cm).

 

MINOR COMMENTS

9. Figure 1 workflow too compressed

Page 4, Figure 1

Much information is visually crowded.

Recommendations: Make Figure 1 (the workflow diagram) clearer by increasing text size, adding distinctions within stages, etc. Also improve resolution.

 

10. Dense formatting in Table 4

Page 9, Table 4

Table 4 is heavily loaded with many details that make the table difficult to read.

Recommendations: To make Table 4 easier to read, consider rearranging the formatting (text alignment/spacing,), possibly even moving it to the Supplement.

 

11. The limitation of oogonia counts might be clarified

Page 10, Lines 204–208

The text briefly mentions underrepresentation because of small size.

Recommendations: Add 1–2 sentences to enhance the accuracy.

 

12. Abbreviation “rap” undefined at first use

Page 13, Line 301

“rap < 0.17” appears without explanation.

Recommendations: Define all abbreviations (for instance, "rap") when they are introduced (for example, earlier in the Methods section).

 

13. Missing sample sizes per maturity phase after stereology

Page 13, Table 6

The Table shows confusion matrix but not a count that is based on a stereologically determined phase.

Recommendations: It is a requirement to state the number of persons who within each stage of maturity after applying the stereological model. Add a summary sentence providing totals for the numbers reported in Class A, B, C, D, E.

 

14. Enhance clarity of transition in Discussion section

Page 14–15, Lines 314–344

In a few sentences, some of the paragraphs tend to detail background information as opposed.

Recommendations: Refine to concentrate on original results.

 

15. Small editorial corrections

Recommendations: Minimal editorial changes for improved methodology section

• Page 5, Line 99: “Nixon D3200”. The manufacturer is Nikon, not Nixon. To cite the use of a Nikon D3200 digital camera in the manuscript, write: “images were captured using a Nikon D3200 digital camera (Nikon Corporation, Tokyo, Japan)”.

 

Literature Review

The literature review for studies similar to the submitted manuscript 4048723 found several papers that are quite close to the subject studied, suggesting that the manuscript may not be entirely novel, depending on its precise goals and methods, as follows:

• Sauger, C, Quinquis, J, Berthelin, C, Lepoittevin, M, Elie, N, Dubroca, L, Kellner, K.A, (2023) Quantitative Histologic Analysis of Oogenesis in the Flatfish Species Pleuronectes platessa as a Tool for Fisheries Management. Animals, 13: 2506. https://doi.org/10.3390/ani13152506
• de Boois, I.J., Wilkes, T., Blom, E., Koelemij, E., Pennock, I., Wiegerinck, H., van Damme, C.J.G. (2022). Gonad development in plaice (Pleuronectes platessa) and sole (Solea solea) in the North Sea: Histological analysis of samples from 2019 and 2020. CVO Report 22.001.
• Sauger, C., Quinquis, J., Kellner, K., Heude-Berthelin, C., Lepoittevin, M., Elie, N. & L. Dubroca (2020). A macroscopic and stereological imaging dataset of Pleuronectes platessa Scientific Data, 7: 165.
• Sauger, C., Quinquis, J., Lepoittevin, M., Heude-Berthelin, C., Kellner, K., Elie N., Dubroca L. (2019). An objective take on defining sexual maturity in the plaice, Pleuronectes platessa (Linné, 1758). 14e colloque de l’AFH "Recherche Halieutique et Développement Durable". 26 au 28 juin 2019 , Université de Caen Normandie.
• Kennedy, J. (2006). Maternal effects and fecundity of place (Pleuronectes platessa) in the Irish Sea.Phd. 168 p.

Given the existence of at least three recent and relevant studies on exactly P. platessa + quantitative histological maturity staging, the submitted manuscript is probably not to be the first to apply these methods to plaice.

Additionally, there is a long history of macroscopic maturity staging work in plaice (such as Rijnsdorp, 1989) though this relies on gross (macroscopic) gonad examination, not histology.

• Rijnsdorp A.D. (1989). Maturation of male and female North Sea plaice (Pleuronectes platessa). J. Cons. Explor. Mer., 46: 35-51.

It is well documented in the literature that histology, including stereology / quantitative histology, has been used to make determinations of sexual maturity in the plaice.

Ways of adding novelty include the following.

• The male gonad histology and maturity is documented, however, the 2023 study seems to focus on the female oogenesis. Including data on the male maturity histology and spermatogenesis would be, in itself, a novelty.
• Right now, with the changing climate and fishing regimes, it is especially relevant to provide a longer temporal coverage. Also focusing on inter-annual variation and the effects of changing the environment.
• The use of automated image analysis is also a good way of detecting observer bias in histological slides, which is also a novelty.

The most recent and comprehensive work to date is the one by Sauger et al., 2023, and it is in this work that the histology-stereology maturity of the female plaice is documented in detail. However, there are still some gaps that remain, for example, male maturity, seasonality, ecological / environmental linkage and the use of modern image analysis, which could result in some valuable and novel contributions.

 

Conclusion

In conclusion, the manuscript provides a potential useful contribution but requires major revisions concerning a clearer definition of novelty, improvement in the description of the justification of the methods adopted, as well as interpretational thoughts on major findings. I recommend resubmission after substantial revision.

 

Sincerely,

Overall Recommendation

Reconsider after Major Revisions

Author Response

The authors would like to first and foremost thank all reviewers for the time and attention they have given this article. All comments have helped with clarifying specific points raised in this study, as well as improved the overall quality of the manuscript. The recommendations were also of great help and have truly enhanced and eased the correcting process.

 

Please find below the replies to the Reviewers’ comments. All changes are highlighted in yellow in the PDF version of the manuscript.

 

Reviewer 1 :

 

Comments 1 : The abstract doesn’t indicate what is new in this work compared to the previously published Sauger et al. (2023) or Zenodo dataset. Recommendations: State the novelty of the study based on previous literature.

Response 1 : The previous work focused mainly on the description of the cellular types found within the ovaries, this paper goes more in depth with the methods used. To underline this fact, the first and third sentence of the Abstract was changed.

 

Comments 2 : The general description of sampling retains most of the content from Sauger et al. (2020) and Sauger et al. (2023). For the above two comments, a substantial portion of the data and methodological basis has previously been published in Sauger et al. (2023) and in the dataset deposited to Zenodo. You do refer to those sources, but the manuscript does not clearly outline what is new in the present work. Recommendations: State the novelty of the study with respect to the previous literature. Please add a specific explanation, detailing:

• which analyses and results are novel, such as maturity phase classification model, confusion matrix, L50 comparisons, classification tree),
• which components derive directly from previously published material, and
• why the present manuscript constitutes a distinct scientific contribution.

This makes obtaining clarity on the originality of the work absolutely indispensable.

Response 2 : We agreed that it was important that the novelty of the paper, compared to the first ones (Sauger et al. 2020 and Sauger et al. 2023), be underlined and it was an oversight on our part that it wasn’t clearly written. To address this, a paragraph was added at the end of the Introduction (lines 78-88), and the following paragraph was modified (lines 80-107).

 

Comment 3 : Homogeneity test is based on 15 individuals (Line 154), which do not have any late-vitellogenic or hydrated oocytes. This may limit the generality of conclusions across the full reproductive cycle. Also, the dataset used for the homogeneity test contains no vit3, pho, ho stages. Recommendations: Include a limitation statement explaining that late reproductive stages were not represented. Expand the limitations discussion to mention how homogeneity might differ during peak vitellogenesis or spawning.

Response 3 : The statement was added in Section 4.1, end of paragraph 2 (lines 359-362)

 

Comment 4 : Text: “the first readings were not recorded…could not be compared. Recommendations: Include a clear acknowledgment of this limitation in the Discussion section (Section 4.2).

Response 4 : This bias was acknowledged in section 4.2, paragraph 3, line 419.

 

Comment 5 : In the manuscript, it is stated that the pre-calibration readings values were not documented, making it impossible to assess improvements that might have been realized by implementing the reading technique. Recommendations: This limitation should be clearly recognized and written about. It would be useful to include any available retrospective comparison, even partial, that has been completed.

Response 5 : As answered in response 4, this bias was acknowledged in section 4.2, paragraph 3, lines 419-424.

 

Comment 6 : The figure 2 and the list of cellular structure (in Table 2) conceptually overlap with Sauger et al. (2023). Recommendations: The figure should say if it reproduces previous published work or perhaps re-analyses data in a new manner.

Response 6 : Your comment is absolutely true and it should have been done. This was an oversight on our part. For Table 2, a sentence was added to underline the origin of the terms used in section 2.4, paragraph 3, lines 147-148. For Figure 2, though only the data paper (Sauger et al. 2020) suggests the use of a PCA to help with the visual interpretation, the figure itself and the data was never published in a peer reviewed journal. Thus we found redundant to underline the fact that this was presented here for the first time.

 

Comment 7 : Page 12, Lines 245 – 262. Given the stereology-based maturity classification relies on several decision thresholds (e.g., ≥50% alpha atresia for omitted spawning, presence/absence of certain follicle types). Recommendations: These thresholds need more detailed biological or statistical justification. If a test on sensitivity to threshold changes was conducted using the model, please describe this. Otherwise, a short justification of the chosen values is needed.

Response 7 : During this study, we relied on pre-existing literature for the ‘rules’ used to set up the classification (Brown-Peterson et al. 2011 and the ICES’ WKMATCH and WKASMSF). It was never our intention to question these ‘thresholds’. For the 50% atresia threshold, we added that this was per the definition of the WKMATCH (section 3.3, page 11, line 258). For the presence or absence of certain cellular types, as stated just before and in manuscript (section 2.6, 1rst paragraph, lines 186-190), these are also based on literature. We set up the 2 tables (3 and 4) to summarize the information for our own convenience and to help the reader (and ourselves) to better understand and assimilate what cellular types are present during which sexual maturity phase.  No tests on the sensitivity of these ‘thresholds’ were performed because this wasn’t the aim of the study.

 

Comment 8 : Page 16, Lines 391–403

The discussion recognizes discrepancies between the L50 median from macroscopic observations and that of stereological observations, but it is not concerned with implications regarding biological systems or management.

The stereological and macroscopic L50 values are quite different (20.6 cm vs. 28.6 cm). The implementation of such a difference are significant, namely in stock assessment, minimum landing size (MLS), and interpretation of past maturity trends.

Recommendations: Enhancement of interpretation of the L50 results. Additionally, consideration should be given to discussion on:

• Potential biological reasons for earlier maturity under stereology,
• Sampling bias (e.g., attempting to collect small immature fish towards the end of the study),
• Comparison with historical L50 values for Channel Plaice,
• Implications from management to ICES maturity staging practice.
• Sampling bias, comparisons with historical L50 values, implications for management (ICES, MLS 27 cm).

Response 8 : “but it is not concerned with implications regarding biological systems or management” This was indeed a conversation we had amongst authors during the writing of this article. Did we want to orientate towards the methods used, or did we want to take on the fishery/ stock assessment aspect? The biological aspect? We chose to stay with just what these result differences could mean in stock assessment (section 4.3). Implications on the differences these results could mean for stock assessment have been added in Section 4.3 (lines 440-446).

“Potential biological reasons for earlier maturity under stereology” & “Comparison with historical L50 values for Channel Plaice “ : We acknowledge in the manuscript that size at first maturity has been getting smaller over the years for the plaice, but we by no means have data to infirm of confirm the different hypotheses suggested in the literature.

“Sampling bias (e.g., attempting to collect small immature fish towards the end of the study)” : A sentence as added (Section 4.1, end of paragraph 1, lines 352-356) to underline this potential bias.

 

Comments 9 : Figure 1. Much information is visually crowded. Recommendations: Make Figure 1 (the workflow diagram) clearer by increasing text size, adding distinctions within stages, etc. Also improve resolution.

Response 9 : Figure 1 has been reworked using the suggested recommendations. Thank you.

 

Comment 10 : Table 4 is heavily loaded with many details that make the table difficult to read. Recommendations: To make Table 4 easier to read, consider rearranging the formatting (text alignment/spacing,), possibly even moving it to the Supplement.

Response 10 : We are fully aware of the complexity of this table. It holds a lot of information and has been worked on over the last 3 years to make it as clear as possible, but it is still heavily loaded.  It has been moved to the ‘Supplementary materials’ to not obstruct the flow of the reading, and if the reader is curious, he can go read it at a later date.

 

Comment 11 : Page 10, Lines 204–208. The text briefly mentions underrepresentation because of small size. Recommendations: Add 1–2 sentences to enhance the accuracy.

Response 11 : A sentence was added in section 4.1, end of paragraph 1 (lines 350-352).

 

Comment 12 : Page 13, Line 301“rap < 0.17” appears without explanation. Recommendations: Define all abbreviations (for instance, "rap") when they are introduced (for example, earlier in the Methods section).

Response 12 : The manuscript was checked to make sure that all abbreviations are defined when first introduced. ‘reap’ was defined as ‘ratio between the ventral ovary’s length and the fish’s total length’ at first introduction (page 13, line 326). We decided to put it in bold to make it stand out so as to visually see it’s an abbreviation and help with the flow of the reading.

 

Comment 13 : Page 13, Table 6. The Table shows confusion matrix but not a count that is based on a stereologically determined phase. Recommendations: It is a requirement to state the number of persons who within each stage of maturity after applying the stereological model. Add a summary sentence providing totals for the numbers reported in Class A, B, C, D, E.

Response 13 : A sentence was added to clearly state the data found in the confusion matrix. Section 3.4, paragraph 1,  2^nd sentence (Lines 312-313).

 

Comment 14 : Page 14–15, Lines 314–344. In a few sentences, some of the paragraphs tend to detail background information as opposed. Recommendations: Refine to concentrate on original results.

Response 14 : The discussion was thoroughly examined and a few changes were made, to try to refine the dialogue and concentrate on the original results.

Section 4.1, paragraph 1, last sentence (lines ) “was the limited access to immature individuals, which led to a size targeted sampling (August 2019) for small sized plaice to ensure that immature individuals would also be sampled, and a maturity ogive could be calculated.” Was changed to “was the limited access to immature individuals, which was necessary to calculate a maturity ogive.”.

The second paragraph was reviewed multiple times after requests from another reviewer, and was extended since it was judged to be lacking in detail.

For the following paragraph, we decided to leave as is since it calls back to terms and definitions that the reader may not be perfectly familiar with, and to ease the information since, after this long read, going back to search to the definitions of these terms can be discouraging.

 

Comment 15 : Recommendations: Minimal editorial changes for improved methodology section. Page 5, Line 99: “Nixon D3200”. The manufacturer is Nikon, not Nixon. To cite the use of a Nikon D3200 digital camera in the manuscript, write: “images were captured using a Nikon D3200 digital camera (Nikon Corporation, Tokyo, Japan)”.

Response 15 : Changed as per suggested.

Reviewer 2 Report

Comments and Suggestions for Authors

The present manuscript evaluates and compares macroscopic evaluation method and quantitative histology to determine sexual maturity in female Pleuronectes platessa. Differences in L50 estimates were identified between the two approaches, which may have important implications, for example for the determination of minimum landing size. The study also indicates spatial heterogeneity within the gonad, which could influence sampling strategies and collection protocols. Overall, the manuscript presents interesting data and is generally well written, but would benefit from improvements in presentation and discussion. Below, I provide comments and questions aimed at clarifying specific points and improving the overall quality of the manuscript.

Comment 1: The current title does not fully reflect the main objective of the study, which involves the comparison between two methods for determining sexual maturity in female plaice. The authors may consider revising the title to better capture this comparative approach, for example: “Comparison between macroscopic assessment and quantitative histology to determine sexual maturity for the female European plaice, Pleuronectes platessa Linnaeus, 1758” or “Comparison of macroscopic and quantitative histological methods to determine sexual maturity in female European plaice, Pleuronectes platessa Linnaeus, 1758”  and so.

Comment 2: (Line 78) Please, include the reference for the lexicon previously published by the authors [40].

Comment 3: Figure 1 is blurry and lacks sufficient resolution. The figure should be redesigned or exported at a higher dpi. In addition, if my understating is correct, “2 sections” (median cross) were used for maturity determination, whereas “6 sections” were used to assess cellular homogeneity. If so, this distinction should be clearly indicated in the figure to avoid confusion.

Comment 4: The manuscript does not report the number of points used in the grid-based counting procedure. The number of points per grid and the total number of points counted per slide should be clearly stated, as this information is essential for evaluating the robustness and reproducibility of the quantitative histological analyses. In addition, when a grid point fell on an empty area of the histological section, was it considered an artefact and excluded from the counts? The manuscript should clarify how empty areas were treated in the grid-based counting procedures. Even though this issued is addressed in the Results section (lines 272-285), it would be appropriate to include the number of counted points and how artefacts were treated in the M&M section as well.  

Comment 5: (Lines 170-173) Given the spatial heterogeneity detected by the cellular homogeneity analysis, could the use of a single median ovarian section to determine maturity phase introduce bias? This potential limitation should be discussed and justified.

Comment 6: Regarding inter- and intra-gonadal homogeneity, the statistical analyses applied do not allow the differences between the different portions within the ovary and between the dorsal and ventral ovaries to be identified, since these factors were combined into a single varaible position. It is only possible to affirm that there is a difference within the ovaries, and that the differences between individuals (i.e., slide) are greater than the differences in position. This could be considered a limitation of the results, since it is not possible to determine whether the differences are due to the longitudinal portion or to the difference between the ventral and dorsal gonads. As these differences exist, the authors could consider a new statistical analysis in which it would be possible to identify where the differences lie. These results could help to guide better collection in future studies.

Comment 7: (Lines 333-335) The discussion at this point could be further developed to address the inter- and intra-gonadal homogeneity observed in this study.

Comment 8: I believe that these results (i.e. those regarding gonad homogeneity) should be discussed in the 'Discussion' section, particularly with regard to the implications of inter- and intra-gonadal homogeneity. The authors report that both position and slide have significant effects on their models. This finding has direct methodological implications and may potentially compromise the robustness of the sampling protocol if ovarian spatial heterogeneity is not adequately considered. In light of these results, a more in-depth discussion on how ovarian heterogeneity may affect histological assessments in this species would be beneficial. Specifically, it would be important to address whether sampling a single ovarian region is sufficient or whether a standardised sampling strategy, such as systematically collecting and analysing the anterior, median and posterior portions of each ovary, would be more appropriate. Providing guidance on which ovarian portions should be sampled or recommending the inclusion of multiple regions per ovary would make the proposed protocol more practical.

Comment 9: (Figure 2) I do not see a clear benefit of the PCA presented in Figure 2. The multivariate ordination does not reveal distinct clustering among positions or individuals, and the extensive overlap among groups makes the biological interpretation difficult. Moreover, PCA is an exploratory tool and does not “confirms these results” as said in line 209. The authors should either clarify the specific insight gained from the PCA or consider removing this analysis.

Comment 10: The confusion matrix reveals a low level of agreement between the two approaches, mainly regarding the weakness of macroscopic method in correctly identifying immature and developing stages. This has important implication and should be discussed in greater depth. Such misclassification directly affects maturity ogive construction and L50 estimation, as it may lead to an underestimation of the proportion of mature females and consequently bias management-related metrics. These results warrant a more explicit and critical discussion about the limitations of macroscopic staging.

Comment 11: (Line 349) “at least”.

Author Response

The authors would like to first and foremost thank all reviewers for the time and attention they have given this article. All comments have helped with clarifying specific points raised in this study, as well as improved the overall quality of the manuscript.

 

Please find below the replies to the Reviewers’ comments. All changes are highlighted in yellow in the PDF version of the manuscript.

Reviewer 2 :

 

Comment 1: The current title does not fully reflect the main objective of the study, which involves the comparison between two methods for determining sexual maturity in female plaice. The authors may consider revising the title to better capture this comparative approach, for example: “Comparison between macroscopic assessment and quantitative histology to determine sexual maturity for the female European plaice, Pleuronectes platessa Linnaeus, 1758” or “Comparison of macroscopic and quantitative histological methods to determine sexual maturity in female European plaice, Pleuronectes platessa Linnaeus, 1758”  and so.

Response 1 : This article has undergone major revisions, as it is now, the title should indeed be changed so as to better reflect the main objective of the study. Thank you for underlining this fact. The title has been changed to : “Comparing macroscopic and quantitative histological methods to determine sexual maturity in the female Erupean plaice, Pleuronectes platessa Linnaeus, 1758”

 

Comment 2: (Line 78) Please, include the reference for the lexicon previously published by the authors [40].

Response 2 : Added

 

Comment 3: Figure 1 is blurry and lacks sufficient resolution. The figure should be redesigned or exported at a higher dpi. In addition, if my understating is correct, “2 sections” (median cross) were used for maturity determination, whereas “6 sections” were used to assess cellular homogeneity. If so, this distinction should be clearly indicated in the figure to avoid confusion.

Response 3 : Figure one has been completely reworked and the distinction between the use for the 2 or 6 cross sections has been added. Thank you for the suggestion.

 

Comment 4: The manuscript does not report the number of points used in the grid-based counting procedure. The number of points per grid and the total number of points counted per slide should be clearly stated, as this information is essential for evaluating the robustness and reproducibility of the quantitative histological analyses. In addition, when a grid point fell on an empty area of the histological section, was it considered an artefact and excluded from the counts? The manuscript should clarify how empty areas were treated in the grid-based counting procedures. Even though this issued is addressed in the Results section (lines 272-285), it would be appropriate to include the number of counted points and how artefacts were treated in the M&M section as well.  

Response 4 : A sentence was added in section 2.4 (M&M) at the end of the first paragraph to give details on the number of points used in the counting grid. For the issue concerning how points that fell on an ‘empty area’ were treated, a paragraph was created for the ‘Calibration’ part of the study in section 2.4 (M&M), and a sentence was added in this last paragraph to clarify the situation. During this study, we realized that different readers will make various different choices when they are not in front of a ‘perfect grid point’ placement (a point that is not perfectly on an oocyte for example). Enumerating them all in this paper would take an entire section on its own, if not be its own paper: ‘Guidelines to stereology with the point grid method’. So we decided to strongly suggest that the readers go read the ‘Reading protocol’ document that is in free access if ever they are curious about the details. We are aware that this choice can be argued and contested, but we would rather have more concise paper focused on the results obtained from the methods rather than a paper with a 5 page section with the reading rules.

 

Comment 5: (Lines 170-173) Given the spatial heterogeneity detected by the cellular homogeneity analysis, could the use of a single median ovarian section to determine maturity phase introduce bias? This potential limitation should be discussed and justified.

Response 5 : Yes it could, it’s the reason we checked for cellular homogeneity. To see if the location of the section could be a bias. The results of the study suggest that there is cellular homogeneity for the gonads for individuals at the beginning of the spawning season. However, individuals in other maturity phase have not been tested, and this is indeed a very important point that has not been discussed. We rectified this by adding a few sentences in section 4.1 (Discussion), end of paragraph 2 (lines 344-351).

 

Comment 6: Regarding inter- and intra-gonadal homogeneity, the statistical analyses applied do not allow the differences between the different portions within the ovary and between the dorsal and ventral ovaries to be identified, since these factors were combined into a single varaible position. It is only possible to affirm that there is a difference within the ovaries, and that the differences between individuals (i.e., slide) are greater than the differences in position. This could be considered a limitation of the results, since it is not possible to determine whether the differences are due to the longitudinal portion or to the difference between the ventral and dorsal gonads. As these differences exist, the authors could consider a new statistical analysis in which it would be possible to identify where the differences lie. These results could help to guide better collection in future studies.

Response 6 : Your statement is true, but we only wanted to see if there was a difference or not. To quantify this difference and pin-point it, plots were made (since the number of each structure was quantified). They were just not presented in this study, but are fully available (scripts on demand and data on Zenodo).

 

Comment 7: (Lines 333-335) The discussion at this point could be further developed to address the inter- and intra-gonadal homogeneity observed in this study.

Response 7 : If the changes and explanations of Response 5 and 6 do not cover this, could you please be more specific on what other points are to be detailed?

 

Comment 8: I believe that these results (i.e. those regarding gonad homogeneity) should be discussed in the 'Discussion' section, particularly with regard to the implications of inter- and intra-gonadal homogeneity. The authors report that both position and slide have significant effects on their models. This finding has direct methodological implications and may potentially compromise the robustness of the sampling protocol if ovarian spatial heterogeneity is not adequately considered. In light of these results, a more in-depth discussion on how ovarian heterogeneity may affect histological assessments in this species would be beneficial. Specifically, it would be important to address whether sampling a single ovarian region is sufficient or whether a standardised sampling strategy, such as systematically collecting and analysing the anterior, median and posterior portions of each ovary, would be more appropriate. Providing guidance on which ovarian portions should be sampled or recommending the inclusion of multiple regions per ovary would make the proposed protocol more practical.

Response 8 : See Response 5. Since no cellular difference was seen inter and intra gonads for individuals sampled at the beginning of the spawning season (presenting already ‘advanced’ oocytes (cortical alveoli and vitellogenic oocytes), it was inferred that whatever the section position used, the results would be the same (the maturity phase found would be the same and similar cellular types are found on all slides). This is probably why we didn’t detail this more, but it was an oversight and we hope the details provided is enough to clarify the points you have raised.

 

Comment 9: (Figure 2) I do not see a clear benefit of the PCA presented in Figure 2. The multivariate ordination does not reveal distinct clustering among positions or individuals, and the extensive overlap among groups makes the biological interpretation difficult. Moreover, PCA is an exploratory tool and does not “confirms these results” as said in line 209. The authors should either clarify the specific insight gained from the PCA or consider removing this analysis.

Response 9 : We were not aiming for distinct clusterings, but for overlapping ellipses to visually show the similarity between the data extracted from the histological slide readings. However, it is true that the formulation of “confirms these results” was a little strong. The sentence was rectified (section 3.2, 1rst paragraph, last sentence, lines 216-217).  

 

Comment 10: The confusion matrix reveals a low level of agreement between the two approaches, mainly regarding the weakness of macroscopic method in correctly identifying immature and developing stages. This has important implication and should be discussed in greater depth. Such misclassification directly affects maturity ogive construction and L50 estimation, as it may lead to an underestimation of the proportion of mature females and consequently bias management-related metrics. These results warrant a more explicit and critical discussion about the limitations of macroscopic staging.

Response 10 : This was already stated in section 4.3, but we added a few more sentences to underline this critical flaw with the macroscopic method for this species.

 

Comment 11: (Line 349) “at least”.

Response 11 : Line 350, ‘with’ before ‘at least’, was removed.

Reviewer 3 Report

Comments and Suggestions for Authors

The text is understandable, well-structured, and consistent. However, the use of the variable "Month" in the decision tree is unclear. It should be explained in the Materials and Methods section because it appears in lines 304-306 without any reference indicating what it refers to as months 6, 11, and 12, and is then used in the decision tree without any reference or justification. I recommend restructuring Table 3 for better presentation, including checking the wording and spelling in the title.

Author Response

The authors would like to first and foremost thank all reviewers for the time and attention they have given this article. All comments have helped with clarifying specific points raised in this study, as well as improved the overall quality of the manuscript.

 

Please find below the replies to the Reviewers’ comments. All changes are highlighted in yellow in the PDF version of the manuscript.

 

Reviewer 3 :

 

Comments 1 : the use of the variable "Month" in the decision tree is unclear. It should be explained in the Materials and Methods section because it appears in lines 304-306 without any reference indicating what it refers to as months 6, 11, and 12, and is then used in the decision tree without any reference or justification

Response 1 : In section 3.4, second paragraph (lines 304 to 306), the name of the month was added. Moreover, in the M&M (section 2.6, second paragraph, lines 180-181), the numerical equivalence of the months was highlighted  so as to not cause any confusion when it is later used in the results section.

 

Comments 2 : I recommend restructuring Table 3 for better presentation, including checking the wording and spelling in the title.

Response 2 : Table 3 was widened to allow for better reading, and a spelling mistake was rectified.  For the title, a grammatical mistake was rectified, and the title is now a single sentence (turned a period into a coma).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

At this point, the authors have adequately addressed the previous comments, improving the clarity of the methods, and strengthening results and discussion. The study offers a clear contribution to both reproductive biology and fisheries management, and therefore I consider the manuscript suitable for publication in its present form.