PONE-D-21-29206First insights into the microbiome of Tunisian Hyalomma ticks gained through next-generation sequencing with a special focus on H. scupensePLOS ONE

Dear Dr. Mohamed Aziz Darghouth,

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CGIAR Research Program on Livestock (CRP Livestock)

Laboratory of epidemiology of enzootic infections in herbivores in Tunisia: application to control (LR16AGR01), Ministry of Higher Education and Scientific Research, Tunisia

PromEssE - ANPR MOBIDOC, Ministry of Higher Education and Scientific Research, Tunisia”

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CGIAR Research Program on Livestock (CRP Livestock)

Laboratory of epidemiology of enzootic infections in herbivores in Tunisia: application to control (LR16AGR01), Ministry of Higher Education and Scientific Research, Tunisia

PromEssE - ANPR MOBIDOC, Ministry of Higher Education and Scientific Research, Tunisia”

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

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: Yes

Reviewer #2: No

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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: 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: For the authors:

Thank you for this research, which deals with very interesting subject with little data on ticks and its symbiotic and commensal/pathogenic bacteria.

I have some comments/remarks to the respected authors. As follow:

I- Generally:

• Please add abbreviation list for all abbreviations you used e.g. rRNA, 16S, 25S, V3-V4, OUT, RPA, NGS.

• 'Candidatus Midichloria mitochondrii' please review this bacterium species scientific name and then after unify witting name and style of this bacterium throughout the manuscript.

II- Other comments: many typo errors, please revise the manuscript well

Abstract:

• P7: next generation

The analysis…

….for 16S RNA of?????

Hyalomma spp. or use H. scupense

Common bacterial genera founf in Hyalomma ticks

Introduction

• P8 : Hyalomma spp. is the most prevalent ticks infesting cattle

Materials and methods

• P10: 'adult' not italic

Cattle breeding barns

16S and 25S rRNA genes (repeat in P13)

'bleach' please specify the material you used

• P11: 'tick's egg' replace by Hyalomma spp. egg

• P11 and P13: in table 2 mentioned that you used a specific primer to amplify Cytochrome b mtDNA gene of Theileria annulata from the collected ticks. Interestingly, I did not any results or discussion on the usage of this primer, why??!!, although in the introduction section and study design, you emphasized cattle theileriosis and ticks role in it also, mentioned that ticks were collected from sites with pervious history of theileriosis.

• P12: 9 instead of nine

Results

• P13: spp. not italic

• P14: However, Francisellacea was the dominant family in the female of H. scupense with 78.8% of…..

'adult' not italic

Fig.2: adult ticks

• P15: Fig 4. Hyalomma scupense

• P16: ' suggesting the potential reproductive role of those symbionts and their correlation with the tick stage. ' please omit or rephrase or remove to discussion section as no suggestions in the results. Results are based on facts not suggestions.

Discussion

• P18: Selmi et al. reported

• P19: 'RA'??!! you mean RPA or another meaning?

Dear respected authors, thank you again.

Regards.

Reviewer #2: Dear Authros, The article is written well but there are some shortcomings that can be incorporated to improve it

1. There are formatting errors (pdf file is attached)

2. There need statistical analysis like regression to find prevalence and correlation with relation to area, tick stage and gender of the tick

3. There is a recent published article related to your article which should be incorporated in discussion

Al-Hosary A, Răileanu C, Tauchmann O, Fischer S, Nijhof AM, Silaghi C. Tick species identification and molecular detection of tick-borne pathogens in blood and ticks collected from cattle in Egypt. Ticks Tick Borne Dis. 2021 May;12(3):101676. doi: 10.1016/j.ttbdis.2021.101676. Epub 2021 Jan 26. PMID: 33540276.

4. There is need to change the figure 3 and it is suggested to keep the colours referring to each organism must be same in the whole figure.

Regards

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Reviewer #1: Yes: Khaled Sultan

Reviewer #2: Yes: Prof Muhammad Imran Rashid

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Attachments

Attachment

Submitted filename: PONE-D-21-29206.docx

Attachment

Submitted filename: Comments annotated_complete.pdf

PONE-D-21-29206R1First insights into the microbiome of Tunisian Hyalomma ticks gained through next-generation sequencing with a special focus on H. scupensePLOS ONE

Dear Dr. Mohamed Aziz Darghouth,

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.

ACADEMIC EDITOR: The authors should be reply to the reviewer comments and highlight the corrections in all the manuscript

Please submit your revised manuscript by Apr 28 2022 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,

Shawky M Aboelhadid, PhD

Academic Editor

PLOS ONE

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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.

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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 #2: All comments have been addressed

**********

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 #2: Yes

**********

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

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 #2: No

**********

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 #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 #2: Even though all the comments have been responded but i can't see the file with the changes in different coloured text

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Reviewer #2: Yes: Muhammad Imran Rashid

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First insights into the microbiome of Tunisian Hyalomma ticks gained through next-generation sequencing with a special focus on H. scupense

PONE-D-21-29206R2

Dear Dr. Mohamed Aziz ,

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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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,

Shawky M Aboelhadid, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

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 #2: All comments have been addressed

**********

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 #2: Yes

**********

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

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 #2: Yes

**********

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 #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 #2: The study was interesting and i congratulate all authors for their efforts. They have incorporated all changes.

Goodluck

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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 #2: Yes: Muhammad Imran Rashid