Morphological and Molecular Alterations Induced by Lead in Embryos and Larvae of Danio rerio

Round 1

Reviewer 1 Report

This manuscript contains interesting results and is clearly written.  Several minor modifications would help the presentation.
1.  Line 38.  The text states that lead “may” adversely affect humans and wildlife and then proceeds to document that humans are greatly affected.  If the intent is to say that lead may affect wildlife, that should be separated from humans, where “may” is misleading.
2. Line 48.  The text gives a concentration of 566,2, but 566.2 appears to be intended.
3.  Line 88.  The word “Instead” appears incorrect, since it indicates a difference.  However, that sentence simply explains how the previous sentence is developed.
4.  Line 103.  It seems “responses” should be singular , i.e., “response”.
5.  Line 262.  The phrase “proceeding of development” is not clear.  The authors may intend “As development proceeded,…”.
6.  The figures all (and especially 3 and 4) suffer from labels being far too small to read without a magnifying glass.  Even the asterisks are too small.
7.  Line 399.  The authors presumably intend to use a semi-colon between “here” and "still” instead of a comma.
8.  Lines 446-7.  The hypothesis referred to comes out of nowhere and is hard for the reader to accept without further explanation.
9.  Line 448.  The phrase “which plays a pivotal role…” is misplaced.  As written, it modifies evolution instead of beta-catenin.

Author Response

Reviewer #1:

This manuscript contains interesting results and is clearly written.  Several minor modifications would help the presentation.

1. Line 38. The text states that lead “may” adversely affect humans and wildlife and then proceeds to document that humans are greatly affected. If the intent is to say that lead may affect wildlife, that should be separated from humans, where “may” is misleading.

We accepted and delete “may” from the sentence.

2. Line 48. The text gives a concentration of 566,2, but 566.2 appears to be intended.

We accepted and modified as suggested.

3. Line 88. The word “Instead” appears incorrect, since it indicates a difference. However, that sentence simply explains how the previous sentence is developed.

We accepted and modified as suggested.

4. Line 103. It seems “responses” should be singular , i.e., “response”.

We accepted and modified as suggested.

5. Line 262. The phrase “proceeding of development” is not clear. The authors may intend “As development proceeded,…”.

We accepted and modified as suggested.

6. The figures all (and especially 3 and 4) suffer from labels being far too small to read without a magnifying glass. Even the asterisks are too small.

We accepted and modified as suggested.

7. Line 399. The authors presumably intend to use a semi-colon between “here” and "still” instead of a comma.

We accepted and modified as suggested.

8. Lines 446-7. The hypothesis referred to comes out of nowhere and is hard for the reader to accept without further explanation.

We accepted and corrected the sentence as suggested.

9. Line 448. The phrase “which plays a pivotal role…” is misplaced. As written, it modifies evolution instead of beta-catenin.

We accepted and corrected the sentence as suggested.

Reviewer 2 Report

Please refer to the attachment.

Comments for author File: Comments.docx

Author Response

Reviewer #2:

The comments on the MS “Morphological and molecular alterations induced by lead in embryos and larvae of Danio rerio.” The work presents significant progress addressing the influences of lead in zebrafish. The results are substantial together with detailed illustration and discussion provided on the acquired results which further improved the understanding of the physiology of zebrafish and made on step further for practical applications for serving pollution indicator using zebrafish. Still, minor revision needs to be made before the archival publication can be granted.

1. On page 3 line 115-117 the lead concentration was determined for median lethal concentration and considered very low concentration in surface water. Can the author elaborate more on this from the perspective of human health? If this is very low concentration, what is its impact to human as the future application of this design may serve as the pollution indicator.

We accepted an modified accordingly. Please see lines 71-81.

2. As known that the lead pollution can result in negative effects on the human cardiovascular system. However, no cardiac measurement is presented in the current study.

Thank you for this remark. Here we only evaluated morphological alterations during zebrafish development after Pb exposure. Still, we are just working on a new project in which we assessed the cardiotoxic effects induced by Pb (obtained by measuring the heart rate in zebrafish embryos and larvae).

3. For the morphological check, was it done by manual operation or it was done through imaging assessment with automatic imaging recognition to give the score?

To evaluate the severity of morphological defects, the morphological scoring system proposed by Herrmann, 1995 was used. The extent of each malformation was quantified by observing the images acquired for each individual (dorsal and frontal view). We modified the sentence to clarify better the applied method (please see lines 158-164).

4. In Table 1 the tested number for each group should be provided even it was mentioned in the text.

We accepted and modified as suggested.

5. In Fig 5 I guess for one of the future work the authors may want to manage to proceed is to identify the lower limit of the acceptable concentration for zebrafish where no significant difference can be found with respect to the control group. As of now all the tested group showed differences compared to the control groups which do not give the whole picture of the pollution effects.

We agree. We hope to identify the lower limit of the acceptable concentration for zebrafish embryos and larvae in our following research.

Author Response File: Author Response.docx