PONE-D-22-12066Bombyx mori Vps13d is a key gene affecting the silk yieldPLOS ONE

Dear Dr. Li,

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As you can see from the attached comments, both reviewers find the manuscript interesting and novel. However, they recommend to resolve some critical issues (which they highlights in their comments) before acceptance fo publication. I suggest to revise carefully the manuscript according to the reviewers' suggestions. Please, note that the rest of this email is automatically generated.

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Academic Editor

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: The silk yield is an important indicator to measure the quality of silkworm varieties. This paper determined that the difference in BmVps13d gene expression between JS and L10 may be related to the difference in promoter sequence, and verified that BmVps13d gene has an important effect on silk yield of silkworm using the CRISPR/Cas9 system. The findings are interesting and meaningful. It is acceptable after some revision.

1. Why were the sequence characterizations of Vps13d proteins compared in B. mori, D. melanogaster, and H. sapiens in Figure 1?

2. Line 178-180, “The results showed that the expression of BmVps13d were significantly higher in the midgut and silk gland of JS than in that of L10 at the third day of fifth instar larval stage (Fig 2)”. However, Figure 2 does not reflect the expression of BmVps13d significantly higher in the silk gland of JS than in that of L10. It is recommended to provide original data or change the plotting method.

3. Line 252-257, “The body weights of the ΔBmVps13d mutants showed a 24.7% decline than that of Nistari at the third day of fifth instar larval stage (Fig 7 A). ΔBmVps13d females showed 27.3%, 24.5%, and 24.8% declines in the weights of cocoons, pupae, and cocoon shells respectively; ΔBmVps13d males showed 11.9%, 4.3%, and 5.4% declines in the weights of cocoons, pupae, and cocoon shells respectively; 256 females and males of ΔBmVps13d showed 2.4% and 2.9% declines in the cocoon layer ratio respectively (Fig 7 B).” It is known that the body weight of the larvae directly affects the weight of cocoons, pupae, and cocoon shells. Knockout of BmVps13d resulted in reduced larval weight, which directly affected cocoons, pupae, and cocoon shells. Therefore, a direct relationship between the BmVps13d gene and cocoons, pupae, and cocoon shells cannot be demonstrated. Therefore, the conclusion needs to be revised.

4. It is recommended that authors analyze the expression of silk protein genes in Nistari and ΔBmVps13d mutants.

Reviewer #2: 1. The promoter sequence in Figure 4 is the same as in Figure 3?

2. Page 6: lines 114-115: the role of pRL-TK vector in dual luciferase assay should be demonstrated.

3. Two sgRNA sites of BmVps13d were designed. Why was only one detected in Figure 6?

4. Page 8: lines 158-160: Why were ΔBmVps13d mutants and Nistari weighed on the third day after cocooning?

5. How about the effect of BmVps13d on the expression of silk protein genes? As an important information, the author should detect it.

6. There are some writing errors in the manuscript, please proofread the full text carefully.

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Reviewer #1: No

Reviewer #2: No

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Bombyx mori Vps13d is a key gene affecting silk yield

PONE-D-22-12066R1

Dear Dr. Li,

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,

René Massimiliano Marsano, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments: