PONE-D-20-30716

Effectiveness of orally-delivered double-stranded RNA on gene silencing in the stinkbug Plautia stali

PLOS ONE

Dear Dr. Nishide,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Both reviewers have raised concerns about deficiencies in the Materials and Methods, and these must be thoroughly addressed in your revision. Comments regarding English language quality should be addressed as well. Note that comments made regarding novelty are not relevant to PLOS ONE. 

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

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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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

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: Dear Editor

The manuscript entitled "Effectiveness of orally-delivered double-stranded RNA on gene silencing in the stinkbug Plautia stali” describes a RNAi study by oral-delivered and microinjection dsRNA of several genes in order to evaluate Plautia stali response. This study has interesting data. However, I have several concerns about how the manuscript is written, and lack some comments that I'd like the authors to consider for the possible improvement of the manuscript, outlined below.

Major comments:

1.The English in the present manuscript is not of publication quality and require major improvement.

2. Abstract. Is recommended don't include too many details

3. Description of microinjection experiments should be more detailed in materials and methods, including sample size, treatments, dsRNA concentrations. Most data included in figures references should be included in Materials and Methods section.

4. In methods section, it should include the sample size used to obtain microinjection and feeding assays data. The values only are mentioned in figure references. Only 5 nymphs were used in order to evaluate adult emergence rates, 6-8 nymphs were used in some gene knockdown experiments and some feeding assays. These samples size seem to be too small. Similar published papers used between 20-30 insect in each treatment. Also, dsRNA GFP synthesis (control) is not mentioned in materials and methods.

5. During qPCR experiments analysis only one gene is used as reference gene. How de standard curve was performed? Dilutions? The information about de reference gene is lacked.

6. The exposition time to dsRNA or time of insect feeding with dsRNA is an important factor for successful induction of gene silencing. In this study, the authors performed a feeding assay on first-instar nymphs to second-instar nymphs (using with dsRNA solutions at different concentrations). In other assay, the authors evaluated the dsRNA effect from fifth-instar nymph to adults by insect feeding through green bean pods. Is possible evaluate the continuous feeding with dsRNA from first-instar nymphs to adults?

Reviewer #2: The main objective of this study was to compare the efficiency of RNAi by injection vs feeding in the brown-winged green stinkbug Plautia stali (Hemiptera: Pentatomidae). Authors concluded that P. stali shows high sensitivity to RNAi with injected dsRNA but, unlike the allied pest stinkbugs Halyomorpha halys and Nezara viridula, very low sensitivity to RNAi with orally delivered dsRNA. Overall, this article is well presented and organized. The following suggestion might strengthen the manuscript and make it suitable for publication in PlosOne:

1. It would be important to highlight the novelty of the study in the introduction and conclusions. Are any of the target genes have been tested before by other researches? RNAi background on Plautia stali is missing.

2. Authors should explain why the concentrations of dsRNA tested through injection vs feeding were different.

3. The method section is missing details. A separate section should be created for the feeding assays and injection.

4. It is not clear whether the insects were starving before exposing the insects to diets supplemented with dsRNA.

5. Authors should consider the use of cationic nanoparticles to increase the effectiveness of oral delivery.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: Comments to the authors_PlosOne.docx

Effectiveness of orally-delivered double-stranded RNA on gene silencing in the stinkbug Plautia stali

PONE-D-20-30716R1

Dear Dr. Nishide,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Zach N Adelman

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

While one reviewer requested a few small additional edits to the manuscript, these were based on increasing perceived impact, which cannot be used as a criteria for acceptance. 

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

**********

6. 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: Dear Editor

The authors have made valuable improvements in the manuscript and most of the problems that I pointed were solved. In my opinion, it is now acceptable for publication.

Reviewer #2: Question one from reviewer 2 was partially answered. The authors only added one line and two references. Explaining how this study is different from similar articles showing dsRNA silencing effects in Plautia stali will draw more attention. Also, it would be desirable to add more information about the target genes.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No