PONE-D-24-37286Transcriptome analyses reveal differences in the response to temperature in Florida and Northern largemouth bass (Micropterus spp.) during early life stagesPLOS ONE

Dear Dr. Bernal,

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“This project was supported by funds from Red Hill Farms. Additional support provided by Agriculture and Food Research Initiative Competitive Grant from the USDA National Institute of Food and Agriculture hatch projects 1013854 to IAEB, ALA-016-1-19075 to AMK, and ALA016-1-19053 to LAR.”

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“We would like to thank the staff of Red Hill Farms and the E.W. Shell Fisheries Center for their support with this research project. Thanks to the staff of the GSAF of UT Austin for their support with sequencing. This project was supported by Agriculture and Food Research Initiative Competitive Grant from the USDA National Institute of Food and Agriculture hatch projects 1013854 to IAEB, ALA-016-1-19075 to AMK, and ALA016-1-19053 to LAR. Support was also provided by the Smithsonian Tropical Research Institute to MAB. We would like to thank the Alabama Supercomputer Authority for their assistance with bioinformatic analyses and data processing.”

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“This project was supported by funds from Red Hill Farms. Additional support provided by Agriculture and Food Research Initiative Competitive Grant from the USDA National Institute of Food and Agriculture hatch projects 1013854 to IAEB, ALA-016-1-19075 to AMK, and ALA016-1-19053 to LAR.”

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: 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

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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: In general, this is an interesting and well-organized manuscript. The authors sequenced and annotated the transcriptome from two different species of largemouth bass larvae in responding to divergent temperatures. The resulting differential expression profiles reveal some genetic aspects that reflect changes of temperature at early development stages of LMBs. Here, I listed some questions relevant to experimental design and data analysis. If the authors can adequately address these questions in the revised manuscript, I will suggest to accepting the revision for publication.

1. According to the preliminary studies (line 81 - 82), 27 oC can be seeing as the optimal growing temperature for LMBs and their range of tolerating is quite large. So, my question is what’s the reason that three experimental temperatures (21, 24 and 27 oC) were selected? Why is that only colder (21 and 24 oC) temperatures were examined as control (21 oC) and intermediate (24 oC) in the current study? Do you have plan to evaluate warmer living conditions (>28 oC) for LMBs?

2. The authors defined critical early life stages as <30-days post hatch (line 98), and therefore, larvae at 8-DPH and 28-DPH were selected to be examined in the present study. Do you have any literature evidence to support this notion that <30-DPH is significant to larval development and/or critical to aquacultural husbandry, which considering as justifying the experimental design?

3. Is there any specific reason for the authors to use Trinity to assembling transcriptomic sequences (line 200)? Since Trinity is known to have problems handling lowly expressed transcripts and high coverage regions, and its normalization process is also questionable (Honaas et al., 2016) and (https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-FAQ).

4. Although the authors summarized the experimental conditions with fish treatments in Table 1, there is no detail information stating how many samples were sequenced and how the technical and biological repeats were generated. Please provide this information for further evaluation of the reliability of experiment. In addition, do you pool tissue? If not, how do you generalize the bias between individuals?

5. In regarding to determining the differential expression genes (DEGs), since there are two variables included in the experimental design (temperatures and age: DPH), I will need the authors to clarify how to normalize the sequencing data into relative abundances of each mRNA in representing relative expression levels of each gene?

6. The qualities of picture and diagram in Figure 2, 3, and 5 are poor. Please revise these figures with better quality.

7. For Figure 5, What does the color bar indicate to? Does the red color indicating the degree of positively correlated and blue color indicating the degree of negatively correlated to certain GO terms? Clarification and a more meaningful figure legend are required.

Reviewer #2: The manuscript (PONE-D-24-37286) presents a technically sound study using transcriptomic data to explore how two lineages of largemouth bass respond to temperature variations during early life stages. The data is well-supported by the methodology, and the conclusions regarding differential gene expression under various temperature conditions are valid based on the presented results. The inclusion of gene ontology (GO) and weighted gene co-expression network analysis (WGCNA) enriches the interpretation of the functional significance of differentially expressed genes (DEGs). However, to ensure the completeness of the content, there are some areas that could be improved:

The introduction provides a solid overview of the importance of temperature regulation in early fish development. However, the link between the specific hypotheses tested in this study and previous research on largemouth bass transcriptomics could be made clearer. I recommend adding more detail on how this study builds upon existing work in this field, specifically emphasizing the novelty of comparing the two largemouth bass lineages (Florida and Northern). This addition would provide a stronger foundation for the reader to understand how the study advances the current knowledge on fish transcriptomics and thermal adaptation.

The description of the experimental design could benefit from further elaboration on the triplicate data used for each treatment group. The manuscript currently lacks sufficient detail on the replication strategy for the two largemouth bass lineages (Florida and Northern) across the three temperature conditions. Please clarify how the triplicates were incorporated into the experimental setup and any statistical methods used to account for this in the analysis. This is essential for reproducibility and understanding the robustness of the data.

Page 15: In Table 2, which presents the contig statistics and BUSCO results for the Florida and Northern largemouth bass transcriptomes, there is a notable difference in the "Fragmented BUSCOS" category. Specifically, the Florida largemouth bass transcriptome has 113 fragmented BUSCOs (3.1%) compared to 83 fragmented BUSCOs (2.3%) in the Northern transcriptome. This discrepancy warrants further discussion. Could this difference be due to variations in sequencing quality or depth between the two lineages, or might it reflect underlying biological factors such as genetic divergence between the populations? I recommend addressing this point in the manuscript, perhaps by providing additional details on the assembly process or discussing potential biological explanations. Clarifying this difference would enhance the readers' understanding of the assemblies' quality and their implications for downstream analyses.

Page 17: Figure 4. There appears to be an inconsistency between Figure 4 and its caption. The caption refers to comparisons across temperature treatments at different developmental stages for both Florida (A) and Northern (B) largemouth bass, but it also implies the existence of panels (C) and (D), which are either missing or mislabelled. To ensure clarity and accurate representation of the data, I suggest revising the figure to include the missing panels or adjusting the caption to match the figure as currently presented. Consistent labeling of figures and captions is essential for clear communication of the results.

The presentation style of Table 3 resembles more of a diagram than a traditional table, which may lead to confusion. For better clarity and consistency with the rest of the manuscript, I recommend reformatting Table 3 into a standard table layout with clear rows and columns. This will make the data more accessible and easier to interpret for the reader, thereby improving the overall presentation of the results.

Figure 5 is well-structured, but there are areas where the caption and its reference in the manuscript can be improved. Specifically:

1) Please use consistent terminology for "Weighted Gene Network Correlation Analysis" (WGCNA). The abbreviation WGCA is also mentioned elsewhere—ensure uniformity throughout the manuscript.

2) Clarify how correlation strength and statistical significance are represented in the figure. The description of the correlation coefficients and p-values could be more transparent for readers unfamiliar with the methodology.

3) Ensure that the manuscript provides a thorough biological interpretation of the correlations shown in Figure 5, particularly highlighting key findings and their implications for understanding gene expression in response to temperature across developmental stages.

The discussion section interprets the findings effectively within the broader context of thermal tolerance in fish. However, it would benefit from a deeper exploration of how these transcriptomic changes might affect long-term survival, growth, and reproduction in the wild. Additionally, there are opportunities to discuss the potential applications of these findings in management, conservation, and aquaculture. For instance, how might selective breeding programs leverage these results to improve thermal tolerance in largemouth bass populations? Expanding on these points could provide greater relevance to the study's broader ecological and practical implications.

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Reviewer #1: No

Reviewer #2: No

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Transcriptome analyses reveal differences in the response to temperature in Florida and Northern Largemouth Bass (Micropterus spp.) during early life stages

PONE-D-24-37286R1

Dear Dr. Bernal,

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 #1: All comments have been addressed

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 #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

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 #1: Yes

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 #1: Yes

Reviewer #2: Yes

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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 #1: Since the authors adequately addressed my questions and revised the text and figures accordingly, I will recommend to accept the manuscript for publication.

Reviewer #2: The authors have addressed the reviews individually and provided valuable insights into the field. The manuscript is now suitable for publication in its current form.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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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 #1: No

Reviewer #2: No

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