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Mei Li

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: The authors investigated Cd-induced DAG-PKC/IP3-Ca2+ double signaling pathway and cell injury in gill of freshwater crabs Sinopotamon henanense. The authors systematically designed the work by performing some valuable experimental works accordingly. It is also necessary to evaluate new data critically and not make hasty conclusions that may lead to misinterpretations. However, several important points must be addressed before publication in this journal. Also, the authors need to address all points in the revision stage for a broad range of readers' understanding.

Specific comments:

1. The introduction mentions the toxicity of cadmium and its association with organ injury, but does not specify the exposure levels of Cd in the aquatic environment. It is recommended to add "the current exposure levels of cadmium in the aquatic environment" to highlight the scientific value of this study.

2. The introduction should explain the reason for choosing the freshwater crab Sinopotamon henanense, such as "this species is a regional dominant species, sensitive to heavy metals, and is often used for toxicity assessment in aquatic environments." to enhance the rationality of the experimental design.

3. The exposure concentrations (14.5, 29, 58 mg/L) and duration (4 d) set in the Materials and Methods lack justification. It needs to be clarified whether the concentrations refer to the actual pollution levels in the environment (such as the measured concentration range of cadmium-contaminated water bodies) or preliminary experimental results.

4. In the preparation of samples on page 5, line 88 of the main text, explain why the exposure time of DAG, IP3, PKC, RNA, and histological observation for each indicator detecting Cd in the samples is different, and provide an explanation in the original text to enhance the rationality of the experimental design.

5. Is a sample size of (n=3) reasonable in the statistical analysis on page 7, line 137 of the main text? Please verify.

6. The conclusion in the main text should be cautious. The study measured the destruction of DNA and decrease of ATP as two indicators, but did not detect the genes related to apoptosis and necrosis pathways at the gene or protein level, and the evidence is insufficient.

7. Please note the language modifications, for example, the ℃ for line 117 is not Times New Roman; there are errors in the references in line 252, etc.

8. Please improve the clarity of the images and the aesthetics of the layout. The layout of Figure 2 is not very attractive; please reformat it. Figure 3 only has one image; it is recommended to combine it with other suitable images. In Figure 5A, the arrow marking for GC was not found. Additionally, the markings for Co and GL are not very clear; it is suggested to change the font color for better readability. It is recommended to explain the meaning of the triangle in the B/C/D images and the red arrows in the notes for Figure 5. In Figure 6, the B image does not indicate statistical differences, and the concentration of Cd2+ treatment is also not marked.

Reviewer #2: This study investigates the toxicological effects of cadmium (Cd²⁺) exposure in freshwater crabs (Sinopotamon henanense), with a particular focus on the underlying signaling pathways involved. Crabs were exposed to Cd²⁺ concentrations of 0, 14.5, 29, and 58 mg/L for four days. The results showed that Cd²⁺ exposure activates both the DAG–PKC and IP3–Ca²⁺ signaling pathways in gill tissue, as evidenced by elevated cytoplasmic Ca²⁺ levels and increased PKC activity. Furthermore, Cd²⁺ exposure also upregulated six stress-related genes (CYP450, BAG3, PDI, Hsp21, Hsp27, and Hsp70), suggesting involvement in detoxification and protein folding. Histological analysis revealed gill damage, including filament swelling and epidermal cell death. Additionally, DNA fragmentation and reduced ATP content were observed, potentially contributing to apoptosis and necrosis.

In conclusion, this study provides mechanistic insights into Cd²⁺-induced cellular injury in aquatic invertebrates, highlighting dual-pathway activation, oxidative stress, and disrupted energy metabolism as key contributors. The findings enhance our understanding of cadmium toxicity in freshwater ecosystems and support the study’s relevance to the scope of PLoS ONE. However, the manuscript requires substantial revisions prior to consideration for publication.

Specific comments and suggestions are as follows:

1. The final paragraph of the Introduction should be strengthened by clearly outlining the main objectives and emphasizing its scientific relevance. A more explicit statement of purpose will help readers appreciate the study’s contribution to the field of aquatic toxicology.

2. Important methodological details are missing in the Materials and Methods section. Please specify the number of biological replicates per treatment group, clarify whether n = 3 refers to biological or technical replicates, and provide details on DNA extraction and sample preservation protocols. Additionally, indicate whether normality assumptions (e.g., via Shapiro–Wilk tests) were tested prior to applying one-way ANOVA. The use of standard error (SE) rather than standard deviation (SD) to represent variability should also be justified.

3. The legend for Figure 3 incorrectly includes the description for Figure 4. Please revise to ensure that each figure legend accurately corresponds to its respective figure.

4. The primer sequences used for qPCR were not provide. These should be included in a supplementary table to ensure transparency and allow reproducibility.

5. The rationale for selecting 58 mg/L Cd²⁺ for qPCR and histological analyses is unclear. Please clarify whether this concentration was chosen based on observed toxicity, environmental relevance, or previous literature?

6. At 8 hours, both DAG and IP3 levels declined following initial elevation. This pattern warrants further discussion—could it be reflect negative feedback regulation or cellular adaptation? Similarly, the observed downregulation of several genes (PDI, BAG, Hsp27, and Hsp70) at 48 hours should be discussed to better contextualize these transcriptional responses and their implications for Cd²⁺-induced stress mechanisms.

7. In Section 3.5, the current HE-stained images lack sufficient resolution and clarity. Higher-magnification, better-focused images are recommended to improve visualization of pathological changes in gill tissues.

8. Although the Discussion provides a detailed narrative, it lacks clear structure and thematic focus. Introducing subheadings such as “Oxidative Stress,” “Gene Expression,” and “Histological Damage”would improve readability and organization. A more concise and logically structured synthesis of the proposed toxicological mechanisms would further strengthen this section.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.. Please note that Supporting Information files do not need this step.

Cadmium-induced DAG-PKC/IP3-Ca2+ double signaling pathway and cell injury in gill of freshwater crabs Sinopotamon henanense

PONE-D-25-30435R1

Dear Dr. Jiaxiang Wang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Mei Li

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.-->

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: (No Response)

Reviewer #2: The authors have adequately addressed my comments raised in a previous round of review. I think that this manuscript is now acceptable for publication.

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what does this mean?). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our For information about this choice, including consent withdrawal, please see our Privacy Policy..-->

Reviewer #1: No

Reviewer #2: No

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