PONE-D-23-43144Gene expression signatures of salinity transitions in Limia perugiae (Poeciliidae), with comparisons to other teleostsPLOS 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?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I have thoroughly reviewed the manuscript concerning the gene expression changes in Limia perugiae during transitions between freshwater and saltwater environments. This study employs RNA sequencing to elucidate the genomic responses associated with environmental salinity changes, which is a critical aspect of aquatic biology. Researchers are carefully looking at how gene expression is different in hypersaline and freshwater populations of Limia perugiae. They want to find out how this species can tolerate high salt levels. The manuscript is well-structured, detailing the experimental approach, data analysis, and interpretation of results, reflecting the scientific rigor and depth of the research.

The study's findings, focusing on genes involved in ion transport, chemical homeostasis, and cell signaling, are intriguing and contribute significantly to our understanding of salinity adaptation in fish. Furthermore, the comparative analysis with other teleosts provides valuable insights into the evolutionary aspects of salinity tolerance. These contributions are noteworthy and enrich the field of ichthyology. In the following, several significant points should be revised in this manuscript:

Introduction:

(1) The experimental materials were not adequately introduced in the background. The selection of pearl trevally as a model organism and a concise explanation of its biological attributes should be provided.

(2) The article's structure may use some clarification. To facilitate readers' comprehension of the article's structure and primary arguments, straightforward subheadings such as "research gaps" and "objectives" may be implemented.

(3) Certain assertions may be articulated with greater agility and precision. For example, the first sentence, “Variation in salinity imposes osmoregulatory challenges on aquatic organisms,” could be changed to “Salinity variation imposes osmoregulatory challenges for aquatic organisms.”.

(4) The study's objective and novelty may be expounded upon. However, the study's specific purpose, content, and innovation are insufficiently clear.

Materials and Methods:

(1) The study lacks measurements of background environmental parameters, including salinity levels and physico-chemical metrics, such as pH, at the sites where experimental animals were collected. Quantifying these parameters would provide important context about the habitat conditions.

(2) The description of the RNA extraction and library construction methodology is fairly concise. Elaborating on the experimental procedures and specifics would enhance reproducibility.

(3) Additional details can be provided when outlining the statistical workflow for the differential gene expression analysis, particularly regarding dispersion estimation approaches, design matrix building, etc. Expanding on these analytical steps may improve transparency.

(4) The rationale behind parameter selection for the co-expression network analysis should be explained further, notably the criteria for choosing the soft thresholding power. Describing the decision process more thoroughly would augment the methodology.

(5) In the comparative analysis between species, conveying quality control measures such as homology assessments would strengthen the integrity of the cross-species alignments. Justifying the selection of certain analytical parameters would also enhance robustness.

Results:

(1) The functional enrichment analysis results could benefit from more visual representations, such as bar charts illustrating significantly enriched GO terms, rather than just tables. While informative, tabular-only reporting reduces readability. Utilizing straightforward data visualization techniques can aid in conveying complex results.

(2) The visualization of the 10 shared differential genes from the cross-species comparative analysis (Fig. 4) provides limited information. Incorporating quantitative outcomes for each species, such as comparisons of differential expression directionality and magnitude, would better showcase this data.

(3) The reporting format for the functional enrichment outcomes in the multi-species comparative section is inconsistent with other segments, lacking statistical details like P-values and FDR corrections. Formatting should be unified for clarity.

(4) Certain results around candidate environmental response pathways are repeated in the Discussion. Reducing the reiteration of key data in the Discussion could improve conciseness.

Discussion:

(1) The discussion should directly address how the major findings relate to the original hypotheses and research questions stated in the introduction. Explicitly connecting the conclusions to the study goals would strengthen the framing.

(2) A number of the unique GO terms enriched in the blue module pertain to cellular metabolism and biosynthetic pathways. However, discussions integrating energy metabolism analyses and adaptive capacity were notably absent, despite their relevance. Many transmembrane ion channels critically rely on ATP to function, representing substantial energetic costs. Considering the many complex processes that depend on ion and transporter activity, it would have been helpful to look at metabolic needs and trade-offs along with the processes themselves. This absence of in-depth discussion into the interdependency of bioenergetics and osmoregulation, unfortunately, represents a missed opportunity within the study. Direct analyses quantifying such relationships should be prioritized in this work.

(3) The observed instances of consistent differential expression across a few genes warrant further interpretation. The discussion would benefit from speculating upon why genes integral to these key processes did not demonstrate expected co-occurrence patterns.

(4) When comparing multi-species differences in differential expression, the dialogue on potential explanatory factors like life history and genetic structure distinctions feels disconnected. Weaving this into the overarching narrative flow could provide clearer context.

(5) Considering the genetic relationships between the study populations is advised when evaluating whether sympatric groups exhibit substantial genetic differentiation. Incorporating this aspect lends broader relevance.

(6) Commenting on whether environmental cues elicited plastic or genetically-programmed gene expression changes would add an epigenetic dimension currently lacking.

(7) Elaborating on the implications of the discoveries for the fields of evolutionary biology and aquatic ecology would highlight the potential wider impacts. Discussing how the results advance the current understanding of gene expression in salinity transitions would also be insightful.

(8) While limitations are noted, more fully detailing study constraints and avenues for future research, like repeatability and comparative pursuits, would enhance the scholarship and constructive criticism.

Reviewer #2: I have completed a thorough review of the manuscript titled "Gene expression signatures of salinity transitions in Limia perugiae (Poeciliidae), with comparisons to other teleosts" by Elizabeth J. Wilson et al., submitted for consideration in PLoS One (Manuscript ID: PONE-D-23-43144). Overall, the study provides valuable insights into the gene expression patterns associated with salinity transitions in Limia perugiae. However, I have identified several points that require clarification or further attention:

Title Clarification:

Given the study involves Limia perugiae populations from a hypersaline lagoon and a freshwater stream, rather than transitions between seawater and freshwater, the title may benefit from clarification. I recommend considering a title such as "Gene expression signatures in Limia perugiae populations from hypersaline and freshwater habitats" to accurately reflect the environmental context.

Salinity Percentage in Hypersaline Lagoon:

The authors are encouraged to provide information on the salinity percentage in the hypersaline lagoon (Laguna Oviedo: 17.801° N, 71.363° W). Clarifying the salinity range or percentage in the hypersaline lagoon is crucial for a comprehensive understanding of the study system.

SRA Accessions in Table 1:

In Table 1, the term "Pending" under the SRA Accessions column needs to be addressed. I recommend that the authors provide the specific SRA Accessions or update the information to ensure the reproducibility and accessibility of their RNA-seq data.

Explanation of Abbreviations in Figure 2(A):

In Figure 2(A), clarification is needed for the abbreviations "DC" and "MD" associated with the color bars below the plot. The authors should explicitly state the meanings of these abbreviations, aiding readers in the accurate interpretation of the data presented.

Confirmation of Transcriptomic Data through Real-time qPCR:

The manuscript mentions that confirmation of the transcriptomic data through real-time quantitative PCR (qPCR) is pending. I suggest the authors emphasize the importance of validating their RNA-seq results using qPCR and discuss how this confirmation would strengthen the reliability of their findings.

In addition to these specific points, I commend the authors for their comprehensive analysis of gene expression patterns in Limia perugiae and the comparison with other teleosts. The study contributes significantly to our understanding of salinity transitions in fish. Addressing the mentioned concerns will further enhance the clarity, reproducibility, and overall quality of the manuscript.

Reviewer #3: In this study, the author and the research team devoted extensive effort to the collection of samples across a broad scope. I greatly admire the dedication demonstrated by the author in undertaking this research.

Following the collection of samples from various regions, the authors conducted transcriptome sequencing and extensive comparative analysis, a task of considerable difficulty. The scope of sampling was broad and intricate, and transcriptome sequencing required substantial resources as well as meticulous statistical analysis. This highlights the authors' dedication and contribution to this research.

Furthermore, the research team dedicated significant time to comparing transcriptomic expressions among different fish species and conducted extensive statistical analyses in an attempt to identify whether similar mechanisms exist across different phylogenies in the regulation of salinity. However, the conclusions drawn suggest that teleosts from diverse phylogenies may employ distinct regulatory mechanisms for salinity acclimation. I highly commend the statistical and analytical efforts of the research team. This study provides a valuable direction for future researchers investigating the genetic regulation of euryhaline fish in response to salinity changes.

However, I posit that the author and research team should consider several critical aspects in future studies aimed at examining the regulatory mechanisms and acclimatization of euryhaline fish species across different salinity environments. Firstly, the native habitat of the species, whether seawater or freshwater, is vital, as fish originating from seawater acclimating to freshwater conditions and those from freshwater acclimating to seawater conditions may utilize distinct regulatory mechanisms. Secondly, if the species has been residing in either freshwater or seawater for an extended period, their regulatory mechanisms may have already adapted to those specific environments, rather than being amenable to acclimatization across varying salinity levels.

I express my gratitude towards the authors and the research team for their efforts and contributions to this manuscript and recommend the publication of this original research article.

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Reviewer #1: Yes: Yung-Che Tseng

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: 1130205_Reviewer Comments for Manuscript ID PONE-D-23-43144.docx

Gene expression signatures betweenLimia perugiae (Poeciliidae) populations from freshwater and hypersaline habitats, with comparisons to other teleosts

PONE-D-23-43144R1

Dear Dr. Tobler,

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,

Tzong-Yueh Chen, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

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

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

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

Reviewer #2: Yes

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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.

Reviewer #2: Yes

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

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Based on the revisions and responses provided, I find the manuscript acceptable for publication. The study contributes valuable knowledge to the field of salinity tolerance in teleosts and provides a well-rounded approach to gene expression analysis across environmental gradients. Thank you for your thorough work and for addressing the previous concerns so effectively.

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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 Privacy Policy.

Reviewer #2: No

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