PONE-D-23-14450Interaction of insecticidal proteins from Bacillus thuringiensis and Pseudomonas spp. for boll weevil resistance managementPLOS ONE

Dear Dr. Barbosa Rodrigues,

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.

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We look forward to receiving your revised manuscript.

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Md. Asaduzzaman Shishir, PhD

Academic Editor

PLOS ONE

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Reviewers' comments:

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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: Partly

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

Reviewer #1: I Don't Know

Reviewer #2: No

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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: No

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: Yes

Reviewer #2: No

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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: There is a discussion but no conlusion. Otherwise the manuscript is sound. A conclusion with future direction(s) must be included in the manuscript before publication. There is few minor gramtical mistakes. Those also should be addressed.

Reviewer #2: The manuscript titled "Interaction of insecticidal proteins from Bacillus thuringiensis and Pseudomonas spp. for boll weevil resistance management" describes the expression of toxins from B. thuringiensis and Pseudomonas spp., their isolated and combined activities against the cotton boll weevil (Anthonomus grandis). Despite the significance of finding new molecules to control this important pest, I have several concerns about the manuscript, and unfortunately, it cannot be accepted in its present form.

Major Issues:

1. In the abstract and introduction, the authors have raised several confusions regarding the boll weevil. Firstly, A. grandis is the most important cotton pest in the Americas, not worldwide. Additionally, there are no commercially available transgenic crops or pesticides based on Bt Cry toxins specifically targeting the boll weevil, so the issue of its resistance to Bt toxins does not exist. These statements need to be corrected.

2. The introduction is overly focused on Brazil, while it should be noted that A. grandis is an important pest from the US to Argentina.

3. In lines 79-80, the authors state that "Upon ingestion of these proteins, the high intestinal pH of susceptible larvae activates a protoxin that binds to specific receptors…". This statement is true only for lepidopteran insects; for other insects, such as coleopterans, the mechanism is slightly different. This distinction should be clarified in the text.

4. The statistical analysis of Table 1 data is misinterpreted. The authors claim certain statistical differences that are not valid. For example, at LC50, Cry1Ia10 overlaps with all the other proteins, and at LC90, only Cry8 and PIP-47 show significant differences. These errors should be corrected, along with the sentences referring to these results throughout the manuscript.

5. The discussion section needs to be reformulated as it contains excessive repetition.

6. Additionally, I believe the text would benefit from an additional round of language corrections.

Minor Issues:

Line 34: Change "Coleoptera" to "coleopteran."

Line 37: Specify which Cry8 variant was used: Cry8Aa, B, or C?

Line 83: Change "action" to "activity."

Line 101: Change "methods" to "agents."

Line 195: Change "Assay of interactions between proteins against Anthonomus grandis" to "Assay of different protein combinations against Anthonomus grandis."

Line 268: In Table 1, change "IC" to "CI."

Lines 310 and 314: Change "FS" to "SF."

Lines 327-330: This sentence should be moved to the beginning of the paragraph.

Lines 343-344: Were all these toxins expressed, purified, and tested against coleopteran insects? Or were strains that harbor these protein genes found to be toxic to coleopterans?

Line 345: Change "Coleoptera" to "coleopteran."

Lines 353-354: Correct the decimal numbers; some use periods, while others use commas as separators.

Lines 371-375: Please verify if the bioassay conditions were the same for the compared experiments.

Lines 377-378: I fail to see how this sentence contributes to the discussion. The cited reference pertains to a different toxin expressed in a different system, making it inappropriate for dose comparison.

Lines 386-387: This sentence lacks meaning.

Lines 396-399: Were the tested larvae in the same instar or size in these experiments? Larval size can influence LC values, so it is important to consider this when making comparisons.

Line 425: Cry1Ia is highly toxic to A. grandis compared to which other Cry protein? For comparison, in Lepidoptera, the LC50 values are typically in ng, indicating a much lower dosage is required to kill the insect larvae.

Line 431: The authors could further explore the competition of toxins for binding sites in the discussion.

Line 442: Put "Lymantria dispar" in italics.

Line 460: What is "Howardi" referring to?

In the citations and references, please avoid the use of Master's and PhD theses. Instead, search for the related published articles that have undergone multiple rounds of peer review.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-23-14450R1Interaction of insecticidal proteins from Pseudomonas spp. and Bacillus thuringiensis for boll weevil managementPLOS ONE

Dear Dr. Barbosa Rodrigues,

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.

Please submit your revised manuscript by Nov 04 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Md. Asaduzzaman Shishir, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments (if provided):

The manuscript was improved significantly after the revisions. A few more adjustments are required. Please follow the reviewers instructions.

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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

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

Reviewer #1: I Don't Know

Reviewer #2: Yes

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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: Yes

**********

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: (No Response)

Reviewer #2: All of the aforementioned concerns were effectively resolved. Only one last thing, the letter denoting statistical difference for Cry3Aa in table 1 should be revised from 'a' to 'ab'.

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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

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Interaction of insecticidal proteins from Pseudomonas spp. and Bacillus thuringiensis for boll weevil management

PONE-D-23-14450R2

Dear Dr. Barbosa Rodrigues,

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.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Md. Asaduzzaman Shishir, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: