Transcriptomic Analysis Reveals the Hepatotoxicity of Perfluorooctanoic Acid in Black-Spotted Frogs (Rana nigromaculata)

Round 1

Reviewer 1 Report

PFOA is of great concern for researchers because of its high concentration and toxic effect in the environment. In this work, the authors investigated the effect of PFOA exposure on gene expression in liver tissue using transcriptomic techniques. This is well designed and very practical, which provide useful information on understanding hepatotoxic mechanism of PFOA in amphibians. The manuscript is well written and structured, the omics methods are of great importance and timely, and the results are analyzed in depth. However, some details should be revised to fulfill the goals of this study. Thus, I recommend accepting this manuscript after a few minor amendments listed below.

1. Line 31: The word “transcriptome sequencing” is repeat with the previous sentence. Please delete it.

2. Line 45-47: Please add some literatures.

3. Line 57-58: This sentence is stated unclear, please rewrite it.

4. Line 81-84: It seems that PFOA seriously threat the frog habitat, but why suddenly mention the liver concentration of PFOA? Does PFOA preferentially accumulated in the frog liver?

5. Line 84: Is the word “high levels of PFOA” appropriate for this study? Because the exposure concentration of PFOA used in this study is close to the environment concentration.

6. Line 99: Why male frog was chosen for this study? Please provide sentences to explain it.  

7. Line 110: Instrument information including country, region and company needs to be provided.

8. Line 126: Is RNA extraction method in qPCR consistent with transcriptome? Please specify.

9. Line 145: Please define the words “filtered sequencing data (clean read)”. It is not very clear.

10. Make sure that symbols, sub- and super-scripts, upper- and lower-case are presented correctly, and that there is correct and consistent use of italics, brackets, and punctuation etc.

Author Response

Please see the attachment, thank you!

Author Response File: Author Response.pdf

Reviewer 2 Report

Major comments:

As this study carried out different expression analysis using transcriptome assembly-based approach, the data analysis pipeline would be different from commonly used methods. So, methodology regarding transcriptome assembly, differential expression analysis, as well as functional enrichment analyses should be specified in details.

Minor comments:

Line 5, name disappears after “and”

Table 1 can put in supplemental materials

Line 98 what software and related versions for differential expression analysis needed be to specified? Throughout the manuscript as well.

Table 2 mentioned clean reads, but in Methods no relevant methodology is mentioned?

Line 120, same above how transcriptome is assembled not mentioned?

Line 126 “unigenes” should be defined?

Line 159, why the 15 DEGs were selected not mentioned?

Line 340, in Table 2 it’s unclear what samples are control and what others are PFOA? Sample ID should be included? why unigene numbers, average length as well as N50 are exactly same across samples?

Line 341, these numbers have been mentioned in text, so it’s not necessary to use this table. Instead authors can include tables showing top enriched GO and KEGG terms will be more understandable.

Figure 1A, it’s strange to have many genes with completely same fold change (two lines of dots vertically at two sides)?

Figure 1B, what does color mean from green to red? If they are fold change, how they were calculated?

Figure 4 why RNAseq did not have standard error bar?

Author Response

Please see the attachment, thank you!

Author Response File: Author Response.pdf