PFOS Induces Lipometabolism Change, Immune Defense, and Endocrine Disorders in Black-Spotted Frogs: Application of Transcriptome Profiling
Round 1
Reviewer 1 Report
Manuscript entitled “PFOS induces lipometabolism change, immune defense, and endocrine disorders in black-spotted frogs: Application of transcriptome profiling” by Shi et al., investigated the toxicity of the PFOS on frog using transcriptome and found that PFOS induces lipometabolism change, immune defense, and endocrine disorders in black-spotted frogs, which could eventually adversely affect the population of amphibian. Therefore, this work has significance value in PFOS adverse effects and ecological risks. This topic is of interest to the readers of Diversity. The experiments are elaborately designed and the conclusion was well supported by the data. In addition, the manuscript was well organized and written. Although I do have some suggestions for improving (listed below), it should not compromise the significance of this study. Therefore, I recommend acceptance after minor revision.
Specific comments:
Citiation: Please check it. It seems not right.
Line 35-36: Please add a reference.
Line 64: it should be “> 99.9%
How often did you change the water during the exposure period?
Line 97: Please add more information about R software.
Line 101: please add the full name of KEGG
Define and explain all acronyms and abbreviations on first mention in the text.
On first mention of a species in the text give both the common (trivial) and formal name, and make sure that the presentation is correct and consistent.
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
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Author Response File: 
Author Response.docx
Reviewer 2 Report
PFOS have been a topic of worldwide concern because of their high concentration and potential ecological risk in the environment. In this work, the authors investigated RNA-seq of frogs exposed to PFOS at environmentally relevant doses, in order to investigate the the underlying mechanism of PFOS in frogs. This is well designed and of great practical significance, which provide useful information on understanding mechanism of PFOS in frogs. The manuscript is well written and structured, the research methods are timely and innovative, and the results are analyzed in depth. Thus, I recommend accepting this manuscript after a few minor amendments listed below.
Line 35-37: Please add some literature.
Line 43: What’s the meaning of “MAPK”? Please add its full name.
Line 70: “Several mg/L”? please check this important information
Line 167: Please add full name of PPAR and PFAS. Please check it throughout the manuscript.
Line 169-170: Please add more latest relevant literature.
Figures: please improve the quality of these figures
Author Response
Please see the attachment
Author Response File: Author Response.docx
Reviewer 3 Report
The authors have examined how exposure to PFOS affects gene expression in a frog species.
I think the manuscript can be greatly improved with more detail in the methods, specifically regarding animal husbandry and experimental design. In addition, the discussion currently reads as a recap of the results instead of a discussion of the implications of the results.
Section 2.2
In line 67 it is not clear to me what is being stated. Did previous research demonstrate that male frogs accumulate more PFAS than females? If so, please cite the study, or if was from these authors, please include the data. Was it the same species? Was it PFAS in general or the PFOS from this study.
Line 68. Where were the frogs obtained from? Are these collected from the wild or purchased from a laboratory supplier? How old are they?
Line 73. What size tank are they in? is this a large tank with a small amount of water on the bottom or a small tank with deep water? This greatly impacts the route of exposure and needs to be elaborated on. If it’s a small tank, then this density seems extremely high. What are the habits of this species? Are they only aquatic during the breeding season or are they usually in aquatic habitats—this speaks to what the normal exposure would be in the environment. I think more information could be provided about the study species, either in the introduction or the methods.
What was the husbandry over the 21 days? Were the animals fed? Was the water changed? When the water was changed was the PFOS renewed? This section is lacking in detail.
Section 2.3
How many samples were used? There should have been 100 animals per treatment but 20 per replicate. Was a total of 200 animals used? Were the RNA samples kept separate or pooled by replicate. Later in section 2.5 the n=5 (which is the appropriate level of analysis) but how were the 20 within a tank treated—were means and variance calculated per replicate, was only 1 animal from each tank examined or were samples pooled? More detail on sample size and statistical analysis is needed here and in 2.5
Discussion:
It seems that the authors are building a body of research regarding the effects of PFOS on amphibians, which is greatly needed. In the discussion I think it would be beneficial to the readers to address what the impacts of their findings could be and how those relate to other studies and other contaminants. Right now each subsection basically reiterates the list of functions could be impacted based on altered patterns of gene expression. But what happens to a frog with altered lipid metabolism? Are there studies that have examined the fitness impacts of this? Do females produce smaller clutches? Does either sex reproduce less frequently or die earlier? We may not know these things and if not it would be great to at least discuss what these implications could be. Same for immune defense—are there studies that that see a connection between these immunological pathways and susceptibility to pathogens? Is this the direction research needs to move? The authors have done a lot of work to examine these pathways but just knowing that they are disrupted doesn’t directly lead to impacts in survival and reproduction. I think especially for a special issue connecting pollutants to biodiversity and solutions some of these connections either need to be made (if the data exist) or more effort should be put on tying how these endpoints measured could affect the whole organism, population, etc. In ecotoxicology there is a push toward using adverse outcome pathways to make these connections from molecular initiating events to population, community, and even ecosystem level effects. The authors mention molecular initiating events so it would be great to discuss AOPs and what we know that would allow us to make these connections.
Specific comments:
Line 27: I think it is important for “size” to be “sizes” here to emphasize that multiple populations of many species are having their size reduced
Line 31: Similarly, here I think it is important for “population” to be “species”
Line 43: please spell out the MAPK pathway the first time
Line 52: suggest changing “performing” to “evaluating”
Line 75: I think “pitching” should be “pithing”
Table: the header should explain the column meanings and they are cramped so I cannot tell where some columns end and others begin.
Figures: at least in my downloaded version the resolution is very low and the figures are blurry.
Author Response
Please see the attachment
Author Response File: Author Response.docx
Round 2
Reviewer 3 Report
The authors have addressed most of the comments sufficiently. I appreciate that they took the idea of discussing their work in terms of an adverse outcome pathway. However, developing an AOP is more than a sentence. There are papers written providing guidelines for constructing and presenting AOPs. I would suggest either following those carefully or stating that their data could be used toward development of an AOP with the molecular initiating event and several key events.
Author Response
Please see the attachment.
Author Response File: Author Response.docx
