Lentinan Impairs the Early Development of Zebrafish Embryos, Possibly by Disrupting Glucose and Lipid Metabolism
Round 1
Reviewer 1 Report
Dear. Authors
Thank you for your very interesting report. But I've wondered a bit, so I'll show you below.
１．"2.4 Pathological alteration analysis"
The last three lines say "After being acclimated for 5 min under lighting condition, the swimming ability in the light/dark cycle (5 min/5 min) was recorded for 25 min using Zebrabox" , but why did you choose this time interval?
2. " Biochemical indexes determination"
Please indicate the methodology a little more clearly.
3. Although "n = X" is written in each figure, please write "each group n = X".
Also, since the statistics are done with "Graph Pad", please convert the graph to "Scatter plot" once and show it.
5. Please indicate the age of the zebrafish larvae used.
For example, "6–7 days post-fertilization"
6. The discussion should explain in more detail the effects of LNT on humans. It should then be compared with what happened in the zebrafish experiment.
Author Response
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Author Response File: 
Author Response.docx
Reviewer 2 Report
These results of this study provide evidence for toxicity effects of lentinan to embryo stages and provide an insight into the potential toxicity mechanisms on embryonic development.
The present topic is interesting. This manuscript can be accepted after revision.
Some comments
- The title is not really fit with the content. The first, what are high concentrations? 100, 300, 900 μg/ml? how about dose higher LC50?
Second, in this study, LNT was exposured to zebrafish embryos,s not zebrafish larvae so the defects induced by LNT relate to its effect on zebrafish embryos.
Third, are you sure that development toxicity of LNT only relate to disrupting the glucose and lipid metabolism?
2/ The manuscript used zebrafish larvae. Therefore the manuscript should provide ethical guidelines which the manuscript followed or supply the name of the ethics committee or institutional review board (Approval number/ID).
3/ Explain more about the reason for select LNT at concentrations of 30, 100, 300, 900 mg/ml (1/36, 1/12, 1/4, 3/4 LC50, respectively) for further evaluations. Can these doses induce mortality of zebrafish larvae at 120 hpf or not?
4/ Method for morphological observation should be more clearly shown. Were Zebrafish larvae anesthetized or not when measure body length, swim bladder or record heart beat?. Please confirm.
Which software support for measure the body length, swim bladder size, eye area and yolk sac area?
Explain the formula for calculating relative swim bladder area, relative eye area, relative yolk sac area?
The hatching rate did not show in the method but present in the result. Consider putting it in the method.
- For Biochemical indexes determination: As described in the manuscript, the number of biological replicates is n=4. How is about of number of technical replicates for each well? Could you explain for number of technical replicates for each test?
- Table S 1 is sloppy. Should be name of the gene, official gene symbol, unified the Gene bank accession No. Name of the primers used in the work are incorrect. For example “FABP6” is name of protein not gene. Similar to other genes. Is Actinb2 correct? Please confirm.
- Please explain more clearly how concentration-response curves for mortality were created and how LC50 was derived?
- Check unit of LNT in Table 1, “μg/ml” not “mg/ml”
Please check the unit of glucose, TG, T-CHO is “mmol/g protein” or “mmol/mg protein”. Please confirm.
- Line 239 “the adverse effects” is incorrect. It should be changed to “toxicity”. the adverse effects are what we observed at treatment dose but not toxicity dose.
- In the manuscript, LNT at 900 μg/ml increased significantly the yolk sac area compare to the control. That means LNT induced yolk sac edema. Yolk sac edema relate to delay nutrition absorption. Nutrition (fat protein, water) were retained in yolk. Please explain why TG and T-CHO decreased in group exposure with LNT 900 μg/ml.
- Add more discussion to link the deformations such as short body length, slow
blood flow velocity, small eyes, and swim bladder and yolk sac edema, inhibition in the locomotor activity with the decrease in the glucose, triglyceride and cholesterol levels. - Consider conclusion: the test used in the manuscript is Zebrafish Embryo Acute Toxicity Test Method (ZFET), the test for checking toxicity in zebrafish embryos but not in zebrafish larvae. So the conclusion should rewrite for zebrafish embryos. The conclusion was too short and did not describe your main points and explain their significance.
Author Response
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Author Response File: Author Response.docx
Reviewer 3 Report
An interesting publication with a very well-developed methodology.Results presented in an orderly and logical manner.
Only the biochemical test results should be plotted like all the others (graph).
I found unequivocal errors but marked them in the attached file.
As for the supplementary data, the sequence of primers should also include
the numbers of these genes (NCBI), as well as the lengths of the amplification
products, the site of primer annealing and their melting point.
Please take this data into account.
Comments for author File: 
Comments.pdf
Author Response
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Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
Dear author
Thank you for your prompt reply. Also, I think that comments are treated in good faith. The article is much easier to understand.
Fixed "Point 2" to make it easier to understand.
“2.6 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis” also needs to be explained in a little more detail. Did you homogenize this with 60 μl PBS?
I confirmed the "scatter plot" pointed out in "Point 3". Trust the data. Please return to the original graph. I think the original data is easy to read as an article.
Author Response
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Author Response File: Author Response.docx
