PONE-D-22-09155Detection of bifenthrin, bifenazate, etoxazole resistance in Tetranychus urticae collected from peppermint fields and hop yards using targeted sequencing and TaqMan approaches in pooled DNA samplesPLOS ONE

Dear Dr. Justin Clements,

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Reviewer #1: Yes

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

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Reviewer #6: Yes

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5. Review Comments to the Author

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Reviewer #1: Abstract

The abstract need to rewritten with key information.

Rewrite the statement “The results suggest the TaqMan approach accurately genotypes T. urticae populations collected from agricultural fields” for better understanding.

Introduction

The molecular mechanism for acaricide resistance may be improved from following latest references

1. https://doi.org/10.1016/j.pestbp.2021.104985

2. https://doi.org/10.11158/saa.26.8.10

3. https://doi.org/10.1186/s13071-020-04227-7

Materials & Method

1. Provide the reference for CethylTrimethyl Ammonium Bromide (CTAB) method and company details for Zymo Research Quick-DNA Tissue/132 Insect Microperp kit.

2. Line no 135, specify type and wavelength of UV light

3. DNA extraction procedure may be shortened by providing suitable reference and the modification made in protocol. Similarly, the kit method shortened by citing manufacturers’ instruction.

4. Line no 198 replicate or replication

Result & Discussion

1. Authors should explain the reasons for the samples did not amplify in Table 3 in results.

Reviewer #2: Dear Dr. Islam Hamim

Manuscript #: PONE-D-22-09155

Title: Detection of bifenthrin, bifenazate, etoxazole resistance in Tetranychus urticae collected from peppermint fields and hop yards using targeted sequencing and TaqMan approaches in pooled DNA samples

Whether the use of using two different molecular approaches to determine the resistance genotypes present in T. urticae from collected field are the main question in this study. For this aim, Authors examined the use of a TaqMan qPCR-based approach to determine acaricide resistance genotypes in field-collected populations of T. urticae from peppermint fields and hop yards in the Pacific Northwest of the United States and confirmed the results with a multiplex targeted sequencing. The results shown the TaqMan approach accurately genotypes T. urticae populations collected from agricultural fields. However, as the researchers of this method stated, it needs to be further developed for the correct determination of resistance in T. urticae. Therefore, this manuscript contains content of applicable value. This manuscript is appropriate to be published in the PLOS ONE after some corrections that I have mentioned below:

1. In "Running Title", acaricide should be used instead of miticide.

2. The abstract should be reviewed and some conclusions should be given.

3. Line 122 “dry” should be removed.

4. Information about the laboratory population of T. urticae should be given in the material and method.

5. In DNA isolation from Tetranychus urticae section, the mite stage used in DNA isolation should be specified.

6. In the determination of the bifenazate L50 value, 4 doses were used, except for the control. At least 5 doses were required to determine reliable LC50 (Yu,2015, Simon, J. Y. (2015). The toxicology and biochemistry of insecticides. CRC press.).

7. In the medium lethal dose assay section, how bifenazate was applied to mites should be written clearly.

8. Line 351, mint should be written instead of mites.

Reviewer #3: Detection of bifenthrin, bifenazate, etoxazole resistance in Tetranychus urticae collected from peppermint fields and hop yards using targeted sequencing and TaqMan approaches in pooled DNA samples

Authors Shumate et al. presented a study to detect SNPs that are associated with acaracide resistance in Tetranychus urticae by using targeted sequencing and TaqMan approaches. Their results showed that both diagnostic methods exhibited consistent results, but TaqMan approach may have potential to be used in the field since this method is relatively easier and taking less time compared to targeted sequencing. However, when authors interpreted the results of phenotypes and genotypes, more discussion is needed. G126S is a mutation associated with low level of bifenezate resistance (several citations needed here) but recent study (reference 24) suggested that G126S may not link to bifenezate resistance. Besides target site insensitivity (mutations on the target proteins that have been functional studied), other mechanisms such as enhanced metabolic detoxification (P450s, esterases, GSTs) or sequestrations (ABC transporters) may also play roles in acaricide resistance. Authors only cited 24 papers in their manuscript. More previous studies associated with this topic should be discussed when authors interpreted their results and pointed out future directions.

1. Change to a more specific title: Using targeted sequencing and TaqMan approaches to detect acaricide resistance associated SNPs in Tetranychus urticae collected from peppermint fields and hop yards

2. Material and Method

Page 11, line 115: detailed information of these 13 field-collected populations (location-latitude and longitude, collection time) and the lab susceptible population should be listed in the main text or in a supplementary table.

3. Material and Method

Medium lethal dose assay: not very clear. Some details missed, such as which method was used for pesticide treatment? Any machine used for bioassay? Please cite some previous studies/publications or provide more details here.

4. Table 1 and Table 2 are same as the Table 3. Suggested to delete Table 1&2 and only leave the Table 3

5. Table 1 and Table 3: Population 1&2 in I1017F (Tagman) shows “Resistance”, however, other populations showed “wildtype” or “heterozygous”. Suggest changing “Resistance” to “heterozygous” or “homozygous”

6. Table 4. The bioassay data only exhibited resistance to bifenezate but did not include other two acaricides. Since the purpose of this experiment is to show if phenotype and genotype matches. G126S is not a good predictor for bifenezate resistance, then other two bioassay data should be more important. Authors should add data or at least discuss other possible reasons (previous bioassay data, other resistance mechanisms besides target site insensitivity, etc).

Reviewer #4: Review report PONE-D-22-09155: Detection of bifenthrin, bifenazate, etoxazole resistance in Tetranychus urticae collected from peppermint fields and hop yards using targeted sequencing and TaqMan approaches in pooled DNA samples

Tetranychus urticae is an important pest and recorded on more than 1,000 different plant species infested worldwide. Although several methods have been used frequently, farmers mostly rely on chemical pesticides against T. urticae. But the acaricide resistance can develop through multiple mechanisms, including enhanced metabolic breakdown of acaricides, target site insensitivity, and behavioral resistance. The authors examined the use of a TaqMan qPCR-based approach to determine acaricide resistance genotypes in field-collected populations of T. urticae from peppermint fields and hop yards in the Pacific Northwest of the United States and confirmed the results with a multiplex targeted sequencing. Although the MS is well written, the following points need to be addressed.

L 26-27: Multiple acaricides are currently registered for control including bifenazate, bifenthrin, and extoxazole > Multiple acaricides including bifenazate, bifenthrin, and extoxazole are currently registered for control T. urticae.

L 29-30: Not clear. What is integrated pest management tools?

L 31-34: The authors mentioned “The detection of these mutations through TaqMan qPCR has been suggested as a practical, quick, and reliable tool to inform agricultural producers of acaricide resistance phenotypes present within their fields and have potential utility for making appropriate acaricide application and integrated pest management decisions”—Here is the major concern that the authors emphasis the TaqMan qPCR throughout the MS to determine acaricide resistance genotypes. This is not a new method to determine to detect different chemical resistance status of spider mites. It would be better to compare with other method and confirm the accuracy to determine resistance genotypes against specific chemicals.

L 71-72: Any reference(s).

L 77-79: Pls rephrase

L 197: Why the author performs the median lethal dose assay only for bifenazate?

L 277-280: Add reference(s)

L 281-284: If the method is already well-known molecular tool for growers to detect different chemical resistance status of T. urticae, what is the novelty of present research. (Pls check reference 19, 20)

None of the scientific name is Italic in reference section.

Reviewer #5: Clements et al. used TaqMan qPCR/Multiplex targeted sequencing to determine acaricide resistance genotypes in field populations of Tetranychus urticae in the Northwest of the USA. Their results indicate that TaqMan approach accurately genotypes T. urticae populations but according to the authors interpretation of results is challenging.

The difficult interpretation of the results might partly be caused by the fact that G126S in cytb (used by the the authors for bifenazate resistance screening) is no longer considered as a bifenazate resistance mutation (Xue et al. 2021, PMS). The authors do acknowledge Xue et al. 2021 but only in the penultimate paragraph of their manuscript. I think the authors should be more upfront about this, and change abstract/introduction accordingly (i.e. that their results confirm findings of Xue et al. 2021). They could change title to "Detection of acaricide resistance in Tetranychus urticae collected from ....", mention in the abstract that "G126S is not a good bifenzate resistance indicator" ; also rephrase line 88: "This mutation has been shown to confer resistance" and line 107: "G126S is a well-defined marker of resistance", and change line 211 to "TaqMan quantitive PCR detection of target-site mutations". Wihthout these changes, I believe the manuscript will contribute to the misinterpretation of the role of the G126S in bifenazate resistance and cannot be accepted for publication.

Other comments:

line 90: I1017F in Chs1 was also shown to confer resistance against hexythiazox and clofentezine (10.1016/j.ibmb.2014.05.004). Authors should include this reference in the introduction

DNA extraction methods: The authors do mention two extraction methods (Zymo kit vs CTAB) but do not compare results between the two methods (e.g. 260/280 values or yield?). Author should include this comparison in the main text of the manuscript.

Table 3 shows significant overlap with Table 1/2. Authors should combine these three tables into one table.

line 279: I1017F mutation has also been screened in T. urticae populations using resitriction digest/CT method (j.pestbp.2017.04.003); authors should integrate this reference in the Discussion or Introduction

line 321: other VGSC mutations have also been implicated in bifenthrin resistance (L1024V) and non target site

mediated resistance mechanisms are also involved in bifenthrin resistance (see Riga et al. 2014, 10.1038/s41598-017-09054-y), which cannot be detected by the Taqman assay developed in this study. Author should integrate this reference in the Discussion section.

Reviewer #6: The manuscript “Detection of bifenthrin, bifenazate, etoxazole resistance in Tetranychus urticae collected from peppermint fields and hop yards using targeted sequencing and

TaqMan approaches in pooled DNA samples” by Shumate et al., describes the analysis of 13 T. urticae populations for the bifenthrin, bifenazate, etoxazole resistance. They used two methods to assess target site mutations that were reported to be associated with resistance to these pesticides, TaqMan and Multiplex targeted-sequencing (not for bifenazate), and have determined the actual resistance status of collected field mite populations. They checked the correlation/predictability between these parameters and found that target SNPs were congruently detected with either of the sequencing methods, but that genotyping information did not correlate with the actual resistance status of tested populations, indicating that tested polymorphisms are not tightly correlated with the resistance in T. urticae. Authors confirm the utility of TaqMan to detect mutations in T. urticae and further demonstrate that it can be used for field-collected populations.

The information presented is useful and is adding to several attempts to develop diagnostic tools to predict pesticide resistance in mite populations based on genotype information. The main challenges there is not necessarily detection of the allelic frequency, but their correlation with the resistance phenotype. Next, what are cut offs for defining a population as resistant vs susceptible based on allele frequency, even if it is 100% predictive of the resistance. Manuscript would benefit from additional data, explanations and some corrections.

a) Line 221: Why did authors choose bifenazate (G126S), see Xue W, Wybouw N, Van Leeuwen T. The G126S substitution in mitochondrially encoded cytochrome b does not confer bifenazate resistance in the spider mite Tetranychus urticae. Experimental and Applied Acarology. 2021 Dec;85(2):161-72. At least they should detect S141F SNP as well, as per DOI:10.1038/s41598-017-09054-y.

Likewise, what was the rationale to detect VGSC (F1538I) when it was reported to be a poor predictor of mite resistance to bifenthrin?

chs (I1017F) should be tightly correlated to etoxazole resistance.

b) Line 250: “The TaqMan assay predicted population 2 (collected from hops in Washington) as a completely resistant population when, in fact, there was a high proportion of wildtype alleles (17.45%). As such, the population should be considered heterozygous.” Authors refer to Etoxazole data? They should clearly state it. In addition, they should define criteria for calling populations heterozygous/homozygous, and in particular resistant vs sensitive. Population 2 should give rise to 68% of mites homozygous to I1017F SNP and thus predicted to be resistant. Is this population resistant or susceptible according to authors? What should be cut offs and how are they defined?

c) Line 259:

Median lethal dose assay was performed only for bifenazate resistance. As per Xue W, Wybouw N, Van Leeuwen T. The G126S substitution in mitochondrially encoded cytochrome b does not confer bifenazate resistance in the spider mite Tetranychus urticae. Experimental and Applied Acarology. 2021 Dec;85(2):161-72., this is the least predictable association. Authors concur in line 269, but they should have known it ahead of the time.

Authors should add etoxazole bioassays.

d) Line 284-297: “Our findings confirm the utility of TaqMan to detect mutations in T. urticae and further demonstrates that it can be used for field-collected populations.” “However,

how a grower would interpret the results in the context of pest management decisions needs to be

further addressed.” Authors have to define or at least propose how TaqMan data can be used for diagnostic decision.

e) Line 321: “A pyrethroid (bifenthrin) has never been registered for use on mint in the Pacific Northwest. The TaqMan qPCR and targeted-sequencing data for F1538I (Bifenthrin) suggested that most mint field populations of mites have some proportion of a resistance phenotype for Bifenthrin.”

Careful!!! There was no resistance bioassay performed. According to DOI:10.1038/s41598-017-09054-y, this SNP is not very predictive of the resistance state.

f) Line 339: “To confirm the results of the genotypic assays, we performed an LD50 assessment on bifenazate resistance. We decided to confirm resistance to one of the chemicals we explored, bifenazate, which can be predicted with the mutation G126S in the cytochrome b gene.” This contradicts data presented and data in DOI:10.1038/s41598-017-09054-y and Xue et al., 2021.

Corrections:

Grammar in sentences in lines: 86-88, 99-100

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Reviewer #1: Yes: Devendra Jain

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

Reviewer #6: Yes: Vojislava Grbic

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PONE-D-22-09155R1Using targeted sequencing and TaqMan approaches to detect acaricide resistance associated SNPs in Tetranychus urticae collected from peppermint fields and hop yardsPLOS ONE

Dear Dr. Justin Clements,

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. Authors need to address comments from Reviewer 4 and reviewer 6.

Please submit your revised manuscript by Dec 2, 2022. 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,

Islam Hamim, 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.

Additional Editor Comments:

Authors need to address comments from Reviewer 4 and reviewer 6.

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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

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

Reviewer #5: All comments have been addressed

Reviewer #6: (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 #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

Reviewer #5: Yes

Reviewer #6: Partly

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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: N/A

Reviewer #5: N/A

Reviewer #6: 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 #2: (No Response)

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: 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 #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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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: Dear Dr. Islam Hamim

The authors have made the requested changes in MS and added new information. This manuscript is appropriate to be published in the PLOS ONE.

Reviewer #3: (No Response)

Reviewer #4: Review report PONE-D-22-09155R1: Detection of bifenthrin, bifenazate, etoxazole resistance in Tetranychus urticae collected from peppermint fields and hop yards using targeted sequencing and TaqMan approaches in pooled DNA samples

The authors examined the use of a TaqMan qPCR-based approach to determine acaricide resistance genotypes in field-collected populations of T. urticae from peppermint fields and hop yards in the Pacific Northwest of the United States and confirmed the results with a multiplex targeted sequencing. Although the MS is improved much, the following point need to be addressed.

The authors changed the title as ‘Using targeted sequencing and TaqMan approaches to detect acaricide resistance associated SNPs in Tetranychus urticae collected from peppermint fields and hop yards’

I appreciate their decision and would suggest specifying the acaricide name(s) in the title rather acaricide. Still, I am not convinced that without examining the median lethal dose assay of bifenthrin and etoxazole to detect the acaricide resistance associated SNPs. If this is not possible to perform these studies in this stage, that should be addressed in the discussion.

Reviewer #5: (No Response)

Reviewer #6: I find that revisions did not address the main problem of this manuscript. Yes, authors demonstrated that TaqMan protocol can identify SNPs in mite populations, BUT, authors do not state clearly that we do not have genetic markers for mite pesticide resistance (except etoxazole based on the prior research). Instead, the revised manuscript states in the abstract, line 33: “Within this investigation we examined the use of a TaqMan

34 qPCR-based approach to determine acaricide resistance genotypes in field-collected populations of T.

35 urticae from peppermint fields and hop yards in the Pacific Northwest of the United States and

36 confirmed the results with a multiplex targeted sequencing.” This is MISLEADING! Authors detected SNPs (genotypes), but not acaricide resistance.

Furthermore, in Introduction authors state, Line 81: ” SNPs in multiple, different

82 genes have become associated with the development of resistance to different acaricide chemistries

83 including bifenthrin, bifenazate, and etoxazole (17-20). Historically these mutations have been detected

84 through DNA sequencing that can be analyzed for the presence of the polymorphism. However, multiple

85 investigations have recently suggested using these polymorphisms as a diagnostic tool to inform

86 agricultural growers of the acaricide resistance status of individual field populations of T. urticae. This

87 would allow the grower to make an informed decision into which acaricide would adequately control

88 their local T. urticae populations and may prevent further resistance development (21,22). Three of the

89 most common polymorphisms studied are a mutation that results in the amino acid change. The first

90 results in a change of a glycine to a serine in the cytochrome b gene (G126S) in the mitochondrial

91 respiratory chain. This mutation has been shown to confer resistance to bifenazate, a carboxylic ester, in

92 T. urticae (23). The effects of the G126S mutation have recently been called into question and new

93 research suggests that this mutation cannot be used to predict acaricide resistance (24). Another

mutation at amino acid 1538 in the voltage gate channel gene results in a 94 phenylalanine changing to an

95 isoleucine. This mutation confers resistance to bifenthrin, a pyrethroid (16) and has also been shown to

96 confer resistance to the growth inhibitors, hexythiazox and clofentezine (25). Finally, a mutation in the

97 chitin synthase 1 gene that changes an isoleucine to a phenylamine has been shown to result in

98 resistance to etoxazole, a narrow spectrum systemic acaricide (20). These mutations have been

99 characterized by sequencing both susceptible and resistant populations of T. urticae and have provided

100 in-depth information on the development of resistance to acaricides in T. urticae (16-20, 23). A

101 restriction digest has also been used to screen mite populations for the I1017F mutation(26).”

In line 93, authors provide information that G126S substitution is not predictive of mite resistance to Bifenazate. But they do not provide such disclaimer for F1538I (Bifenthrin)!!

I understand that this research has been done while some of the relationships between SNAs and resistance status were not available. However, the paper is written NOW and has to position facts correctly.

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Reviewer #2: Yes: Recep AY

Reviewer #3: No

Reviewer #4: Yes: Mohammad Shaef Ullah

Reviewer #5: No

Reviewer #6: Yes: Vojislava Grbic

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Attachments

Attachment

Submitted filename: Review report PONE-D-22-09155_R1.docx

PONE-D-22-09155R2Using targeted sequencing and TaqMan approaches to detect acaricide (bifenthrin, bifenazate, and etoxazole) resistance associated SNPs in Tetranychus urticae collected from peppermint fields and hop yardsPLOS ONE

Dear Dr. Justin Clements,

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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Academic Editor

PLOS ONE

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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 #3: All comments have been addressed

Reviewer #5: All comments have been addressed

Reviewer #6: (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 #3: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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

Reviewer #3: Yes

Reviewer #5: N/A

Reviewer #6: 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 #3: Yes

Reviewer #5: Yes

Reviewer #6: (No Response)

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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 #3: Yes

Reviewer #5: Yes

Reviewer #6: Yes

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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 #3: This is the second revision of the original paper PONE-D-22-09155. Authors did a great job in the revision. They answered all questions raised by reviewers. It is ready to be published at this stage.

Reviewer #5: All my comments have been addressed. The manuscript can be considered for publication in PLOS One.

Reviewer #6: The effort to modify the manuscript is appreciated. However, authors have to go over manuscript and systematically change parts that refer to analysed SNPs as a tool for detection of pesticide resistance in mite populations. Authors state:” In the present study, we set out to investigate whether we could use a pooled TaqMan qPCR approach to monitor resistant populations of T. urticae. We chose to examine a limited set of three well defined markers G126S (Cytochrome b), F1538I (Voltage gated sodium channel), and I1017F (Chitin synthase 1) using a pooled TaqMan approach.” This is a good rationale for the study and is independent of utility of these SNPs for detection of mite pesticide resistance. Reading the manuscript, authors are continuously trying to justify the choice of SNPs and to link it to the resistance. Sorry for the insistence, but one should clearly state the purpose of the work and should critically state the utility of analysed SNPs as being diagnostic for mite pesticide resistance. Already in the field, there is confusion of whether they are usable or not. Authors themselves have chosen these SNPs based on initial publications that suggested that they can be diagnostic. This manuscript should not continue to propagate this misconception. For example running title: Detection of acaricide resistance in Tetranychus urticae using molecular based approaches” is not appropriate and should be changed. Also, authors introduced modifications as:” This mutation has been suggested to confer resistance to bifenthrin (please add REF), a pyrethroid (16) and has also been shown to confer resistance to the growth inhibitors, hexythiazox and clofentezine (25). However, some studies bring into question the accuracy of F1538I to predict bifenthrin resistance (26).” Can authors make a single statement in the introduction that will critically establish the utility of reported SNPs as diagnostic? The fact that most of them are not, is not diminishing their work.

Please have a careful reading of the manuscript and eliminate some grammar mistakes.

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Reviewer #3: No

Reviewer #5: No

Reviewer #6: Yes: Vojislava Grbic

**********

[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.

Using targeted sequencing and TaqMan approaches to detect acaricide (bifenthrin, bifenazate, and etoxazole) resistance associated SNPs in Tetranychus urticae collected from peppermint fields and hop yards

PONE-D-22-09155R3

Dear Dr. Justin Clements

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,

Islam Hamim, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Accepted.

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 #3: (No Response)

Reviewer #6: All comments have been addressed

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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 #3: (No Response)

Reviewer #6: Yes

**********

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

Reviewer #3: (No Response)

Reviewer #6: 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 #3: (No Response)

Reviewer #6: 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 #3: (No Response)

Reviewer #6: 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 #3: Authors have adequately addressed my comments raised in a previous round of review. This manuscript is now acceptable for publication.

Reviewer #6: Thank you for making changes in the manuscript. Can you please make a change in the title in line 256: Targeted-sequencing and detection of bifenthrin (F1538I,) and etoxazole (I1017F) resistance. Please change resistance to polymorphisms.

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Reviewer #3: No

Reviewer #6: Yes: Vojislava Grbic

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