Review Reports - Unlocking the Power of Metaproteomics: A Novel Tool for Enhancing Shorebird Conservation and Blue-Economy Management

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents an interesting and potentially valuable application of metaproteomics to investigate the diet of waders in coastal wetlands. The study provides substantial data and demonstrates the potential of protein-based approaches to complement conventional dietary analysis. However, the manuscript would benefit from improved structure, clearer presentation, and more cautious interpretation of some findings.

My specific comments are as follows:

The manuscript combines features of a critical review with those of an original research article. The Introduction includes extensive methodological background, while parts of the Results and Discussion overlap. The authors should streamline the Introduction and more clearly distinguish their experimental findings, comparisons with previous studies, and critical evaluation of the methodology.
The advantages of metaproteomics over DNA metabarcoding, stable isotope analysis, and conventional dietary methods are sometimes overstated. The authors should acknowledge more explicitly that these methods provide complementary information and that metaproteomics also has limitations, particularly regarding database completeness and taxonomic resolution.
Several taxa are identified using related organisms as reference proteins because species-specific proteomes are unavailable. The authors should clarify the limitations of these assignments and avoid interpreting all detected proteins as unequivocal evidence of prey consumption, particularly for potential parasites.
The rationale for performing post hoc Dunn tests even when the overall Kruskal–Wallis test is not significant should be clarified. The authors should also ensure that conclusions regarding interspecific and seasonal differences are based on appropriately adjusted p-values.
Some interpretations of dietary preferences, seasonal prey availability, and ecological adaptations appear more speculative than the data directly support. The authors should moderate these statements, and the Conclusions should focus more closely on the findings demonstrated by the present study.
The manuscript requires careful English-language editing. The manuscript contains numerous grammatical errors, inconsistent terminology, typographical mistakes, and formatting problems in headings, figure captions, tables, and the concluding statements. Scientific names and taxonomic ranks should also be checked for consistency throughout the text.

Author Response

We are grateful to the reviewer for the positive evaluation of our study and the constructive feedback, which has contributed to improving the structure, clarity, and scientific interpretation of the manuscript.

My specific comments are as follows:

The manuscript combines features of a critical review with those of an original research article. The Introduction includes extensive methodological background, while parts of the Results and Discussion overlap. The authors should streamline the Introduction and more clearly distinguish their experimental findings, comparisons with previous studies, and critical evaluation of the methodology.

We thank the reviewer for this constructive comment. We agree that certain sections of the manuscript, particularly the Introduction, contain an extensive methodological overview. We also agree that the distinction between the presentation of experimental results and their subsequent discussion, should be improved. So, following reviewer recommendation we have simplified the introduction and also we have clarified the results and Discussion section.

The advantages of metaproteomics over DNA metabarcoding, stable isotope analysis, and conventional dietary methods are sometimes overstated. The authors should acknowledge more explicitly that these methods provide complementary information and that metaproteomics also has limitations, particularly regarding database completeness and taxonomic resolution.

We thank the reviewer for this valuable comment. We would like to emphasize that this study represents a pilot investigation aimed at exploring the potential of metaproteomics as a tool for dietary analysis in shorebirds. We agree that metaproteomics, DNA metabarcoding, stable isotope analysis, and conventional dietary methods provide complementary information, and that further research is needed to fully assess the capabilities of metaproteomic approaches. Nevertheless, our results highlight several promising advantages, including the identification of taxonomic patterns consistent with those previously documented using conventional methods, the detection of invertebrate groups that may be overlooked in morphological analyses due to differential digestibility, and the potential to reduce the time required for taxonomic identification compared with the traditional examination of dietary remains. We also acknowledge that the completeness and taxonomic coverage of protein reference databases can influence the detection of organisms and the taxonomic resolution achieved. We have therefore revised the Discussion to clarify these considerations while maintaining the focus on the promising applications of metaproteomics as a complementary approach to dietary studies of shorebirds (line 734-755).

Several taxa are identified using related organisms as reference proteins because species-specific proteomes are unavailable. The authors should clarify the limitations of these assignments and avoid interpreting all detected proteins as unequivocal evidence of prey consumption, particularly for potential parasites.

We thank the reviewer for this observation. We agree that the availability and taxonomic coverage of reference proteomes are important factors influencing protein identification in metaproteomic studies. As this is a pilot study aimed at exploring the potential of metaproteomics for shorebird dietary analysis, our objective was to characterize the taxonomic groups represented in the fecal protein profiles rather than to establish unequivocal species-level identifications or confirm the consumption of every detected organism. When species-specific reference proteomes are unavailable, protein matches to related organisms has been widely used and can provide useful taxonomic information, although the resulting assignments must be interpreted at an appropriate taxonomic level. We have clarified this limitation in the manuscript and emphasized that the detection of proteins assigned to a particular taxon does not necessarily demonstrate direct prey consumption (line 573-575 and 753-755).

The rationale for performing post hoc Dunn tests even when the overall Kruskal–Wallis test is not significant should be clarified. The authors should also ensure that conclusions regarding interspecific and seasonal differences are based on appropriately adjusted p-values.

We thank the reviewer for this comment. We would like to clarify that Dunn’s post hoc tests were performed only for taxonomic classes or orders showing significant overall differences in the Kruskal–Wallis test. There was an error in the Statistical Analysis section, which has now been corrected (line 322-332). Specifically, the original text referred to the use of Dunn’s test without p-value adjustment, rather than to its application following non-significant Kruskal–Wallis results. To summarize, in addition to Dunn’s post hoc tests with Benjamini–Hochberg-adjusted p-values, we also performed the tests without p-value adjustment to explore potential differences between wader groups that might be difficult to detect due to heterogeneity among replicates. This distinction has now been clarified in the Materials and Methods section.

Regarding the interpretation of statistical differences, only comparisons with Benjamini–Hochberg-adjusted p-values below 0.05 were considered statistically significant. Results based exclusively on unadjusted p-values were interpreted as exploratory findings rather than confirmed statistical differences. We have clarified this distinction in the Results, Discussion, and Conclusions sections of the revised manuscript to ensure that the interpretation of interspecific and seasonal differences is consistent with the statistical evidence (lines:499-503; 517-518; 526; 532-534; 539-541; 629-630; 631-633; 672-683).

Some interpretations of dietary preferences, seasonal prey availability, and ecological adaptations appear more speculative than the data directly support. The authors should moderate these statements, and the Conclusions should focus more closely on the findings demonstrated by the present study.

Following the reviewer’s suggestion, we have revised the Discussion to moderate speculative ecological interpretations and distinguish them from our findings. The Conclusions have also been revised to focus on the main results and the potential of metaproteomics as an alternative to traditional dietary analyses.

The manuscript requires careful English-language editing. The manuscript contains numerous grammatical errors, inconsistent terminology, typographical mistakes, and formatting problems in headings, figure captions, tables, and the concluding statements. Scientific names and taxonomic ranks should also be checked for consistency throughout the text.

We have carefully revised the manuscript to correct language, terminology, formatting, and typographical errors, ensuring consistency in scientific names and taxonomic ranks throughout. We have standardized the manuscript to American English (e.g., “faeces” to “feces” and “analyse” to “analyze”) and consistently adopted the term “shorebirds” instead of “waders”. In addition, we have clarified the terminology used to describe taxonomic results by using expressions such as “relative protein abundance” or “relative abundance of proteins assigned to [taxon]”, to avoid implying that protein-based measurements directly represent organism abundance.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Referee Report

On the paper “ UNLOCKING THE POWER OF METAPROTEOMICS: A NOVEL TOOL FOR ENHANCING WADER CONSERVATION AND BLUE-ECONOMY MANAGEMENT “ (ijms-4615726) by the authors Escobar-Niño, Almudena 1; Calcis-Marzán, María Victoria 1; Forján-Miguéns, Andrea 3; Punta-Sánchez, Irene 2; Coste, Olivier 1; Pérez-Hurtado, Alejandro 3; Fernández-Acero and Francisco-Javier 1 submitted to the International Journal of Molecular Sciences

This is interesting paper. It reports the investigation of the metaproteomics for the first time to analyze the feces of waders in the salt flats and saltmarshes of Cádiz Bay, aiming to determine their diet with unprecedented resolution. Conventional methods, based on direct observation or fecal inspection, are labor-intensive and risk losing critical dietary information. By contrast, metaproteomics provides rapid, high-resolution insights, offering a robust alternative for ecological monitoring. Obtained results demonstrate the technique’s potential to transform natural area management, optimize conservation strategies for avian species, and support the sustainable development of the blue economy. This study highlights metaproteomics as an innovative, cross-sectoral approach capable of advancing both fundamental ecological research and practical conservation outcomes. The obtained results are reliable without any doubts. However, I have some addition only after implementation of which the paper can be recommended to publication:

The authors should provide in the 1. Introduction concrete examples of materials promising for energy storage:

(1). S.H. Jabarov, Z.E. Hakhverdiyeva, Y.I. Aliyev, A.Y. Meshalkin, S.V. Trukhanov, A.V. Trukhanov, E.M. Huseynov, DSC spectroscopic investigation of Nd_0.5Ca_0.5MnO₃, Pr_0.5Ca_0.5MnO₃ and Nd_0.5Sr_0.5MnO₃ compounds, Surf. Engin. Appl. Electrochem., 62, 3–6 (2026) 241–246. https://doi.org/10.3103/S106837552670016X.

The authors should provide in the 1. Introduction concrete examples of modeling and solving optimization problems:

(2). S.H. Jabarov, Z.E. Hakhverdiyeva, S.F. Samadov, A.A. Sidorin, N.V.M. Trung, A.S. Abiyev, Y.I. Aliyev, N.V. Tiep, S.V. Trukhanov, A.V. Trukhanov, A-site cation substitution effects on lattice distortion and open-volume defects in Nd–Ca/Sr and Pr–Ca perovskite manganites, Micro and Nanostructures 214 (2026) 208659. https://doi.org/10.1016/j.micrna.2026.208659.

The proposed 2 papers should be inserted in References.

The paper should be sent to me for the second analysis after the major revisions.

Comments for author File: Comments.pdf

Author Response

On the paper “UNLOCKING THE POWER OF METAPROTEOMICS: A NOVEL TOOL FOR ENHANCING WADER CONSERVATION AND BLUE-ECONOMY MANAGEMENT “ (ijms-4615726) by the authors Escobar-Niño, Almudena 1; Calcis-Marzán, María Victoria 1; Forján-Miguéns, Andrea 3; Punta-Sánchez, Irene 2; Coste, Olivier 1; Pérez-Hurtado, Alejandro 3; Fernández-Acero and Francisco-Javier 1 submitted to the International Journal of Molecular Sciences

We sincerely thank the reviewer for the positive evaluation of our manuscript and for this valuable suggestion.

The authors should provide in the 1. Introduction concrete examples of materials promising for energy storage: (1). S.H. Jabarov, Z.E. Hakhverdiyeva, Y.I. Aliyev, A.Y. Meshalkin, S.V. Trukhanov, A.V. Trukhanov, E.M. Huseynov, DSC spectroscopic investigation of Nd₅Ca_0.5MnO₃, Pr_0.5Ca_0.5MnO₃ and Nd_0.5Sr_0.5MnO₃ compounds, Surf. Engin. Appl. Electrochem., 62, 3–6 (2026) 241–246. https://doi.org/10.3103/S106837552670016X.

Following the reviewer's recommendation, we have incorporated the suggested reference into the Introduction (line 90-100), within the context of recent research on spectroscopic characterization for investigating functional materials and their potential technological applications.

The authors should provide in the 1. Introduction concrete examples of modeling and solving optimization problems:

In accordance with the reviewer's suggestion, we have included the proposed reference in the Introduction (line 90-100), as an example of research addressing the characterization and optimization of material properties. We briefly discuss the relevance of advanced analytical and computational approaches for investigating complex systems.

The proposed 2 papers should be inserted in References. .

We thank the reviewer for the recommendation. Both suggested publications have been incorporated into the reference list and cited in the Introduction of the revised manuscript. The corresponding text has been adjusted to integrate these references while preserving the main focus of the study on the application of metaproteomics to shorebird dietary analysis and its potential implications for coastal wetland conservation.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Referee Report

On the paper “ UNLOCKING THE POWER OF METAPROTEOMICS: A NOVEL TOOL FOR ENHANCING WADER CONSERVATION AND BLUE-ECONOMY MANAGEMENT “ (ijms-4615726-v2) by the authors Escobar-Niño, Almudena 1; Calcis-Marzán, María Victoria 1; Forján-Miguéns, Andrea 3; Punta-Sánchez, Irene 2; Coste, Olivier 1; Pérez-Hurtado, Alejandro 3; Fernández-Acero and Francisco-Javier 1 submitted to the International Journal of Molecular Sciences

This paper has been well corrected and it can be recommended.

Comments for author File: Comments.doc