PONE-D-22-15726Risk factors for tick exposure across an urbanization gradientPLOS ONE

Dear Dr. Janzen,

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.

 As you'll read, reviewers were split with respect to the value of your manuscript.  Given these split perspectives, my editorial decision is to allow you opportunity to potentially address the primary concern of Reviewer 1, which deals with the skewed representation of life stages in your collection and how that limits applicability of your data to any of your findings and general assessment of 'risk'.   Additional concerns:

• Both reviewers were not convinced that 'risk' had actually been assessed. 
• Both also thought that sampling was insufficiently described.
• The Results section could be more meaningfully organized to increase general understanding/relevance.

Note, given the split perspectives of the reviewers and "major revision" designation, the manuscript will go back out for review after a revised version is submitted.   Please submit your revised manuscript by Oct 26 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,

Dr. Janice L. Bossart

Academic Editor

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: 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: Line 101: Note that "mopping" is modified dragging technique, as dragging is much better known.

Line 109: I did not see any details about the frequency of collections from each site per season. How frequently were sites visited per year? The large number of larvae and relatively small number of adults (lines 198 - 199) could indicate undersampling during peak nymph/adult activity.

Lines 250 - 285: A lot of information is provided here that recites what can be read in the tables. Written text here would be best used for highlighting the patterns to help draw the reader's eye to particular areas of the table. There are several ways I can suggest to consider organizing the text: 1) organize by broad general patterns related to land cover (e.g. lines 244 - 245, mixed forest) vs. unusual patterns (e.g. lines 257 - 258, broadleaved with hardwood, and 268 - 269, wetlands) or 2) organize by positive effects vs. negative effects or 3) organize by strength of the association, i.e. coefficients. The text explanation of these results should help aid in understanding or highlighting parts of the tables presented.

Line 324 -326: Vegetation height for adults in particular could be biased by a small collection (n = 13). Much more variation might be seen if more adults were collected.

Line 377: Is this a reference to the wetlands results? Please clarify

Spelling/Grammar/Word usage

Line 322: "tick's" should be changed to "ticks"

Line 330: grass

Line 332: change "is indicating" to indicates

Line 342: represents

Line 372: Revise this sentence, "absolutely most ticks" is awkward

Line 388: measures

Line 406 - 407: Incomplete sentence

Line 412: compared

Final note: Given the author's stated interest in tick-borne disease surveillance, life stages relevant to transmission should be included in larger numbers. Typically, researchers look at density of infected nymphs (DIN) to discuss human risk of exposure to disease. As the authors noted, the clustering of larvae interfered with the analysis, but larvae are also over represented in this study especially given that this is a life stage that generally poses less disease risk to humans and animals.

Limited collection of adults (n = 13) compared to larvae (n = 992) is a concern because some major tick-borne disease, e.g. Borrelia spp. infections, are typically transmitted by adults and nymphs only. Maternal transmission of Borreliae to eggs/larvae does not occur, so unfed larvae are not considered a disease risk for Borrelia. Thus, targeting adult and nymph life stages in collections is imperative to understand the whole system.

Although the authors here are not considering infection in ticks, I recommend looking more closely at literature in which DIN is calculated and used to interpret exposure risk.

In one paper, Jouda et al. 2004, the authors collected and analyzed 108 - 145 nymphs per altitude class, per year, with a total of 1112 nymphs included in the study. Likewise, 9 - 149 adults were included per altitude class, per year, for a total of 507 adults across three years. The authors should seek to collect comparable numbers, particularly when evaluating such a large collection of environmental variables for their effect on densities.

I applaud the authors for the spatial study design and analyses. The limited and skewed tick collections, unfortunately undermine the results overall, and the applicability of this data to understand human exposure risk.

Reviewer #2: The overall value of presented study is high and the outcome may have additional value in tick-borne diseases ecoepidemiology and use as starting point in landscape planning and urban planning related studies, risk measurements for public health. The strength of the manuscript and presented study is solid basis of up-to date literature, with substantial representation of papers describing local environment characteristics that allowed for good study design. Manuscript is not free from minor redaction issues and few major gaps, yet this should be easily improved by the Authors.

Title: please specify which ticks! Also, In Reviewer's opinion it's abour risk factors, not risks in it's current state.

Line no. – comment

9-10 – broad thesis, please provide examples of epidemiological studies

9-17 – Phrases like: „. Greenspaces of all kinds are crucial for terrestrial biodiversity” or „unwanted insects” or „are not only annoying” sounds maybe catchy for lay-man, but are biased, empty and not suitable for scientific journal.

Thus I strongly advise to review and adjust the language used.

15 „arachnoids” (arachnids).

17 – what inf. Dis.? This statement lacks of examples of vector borne diseases of epidemiological importance for precision.

19 – how long, what is long in tick lifecycle?

24 – rephrase (ticks surely are not hoping for anything)

34-38 – in reviewer’s opinion it is important to underline that these descriptions are for I. ricinus, because other ticks of medical importance, Dermacentor reticulats, have different (almost opposite) patterns of occurence, described i.a. in vast environmental studies performed by Mierzejewska et al.

40 – like above, specify which ticks. It is worth to specify in discussion as well.

43-46 – please ellaborate importance of nymphs, within introduction, before the aims of the project. As for now such insert is confusing.

56 – latin radius, please try rephrase using a singular nom. for clarity throughout the ms.

Fig 1. Please state if inventory plot 2x2 is an example photography or satellite view. It is unclear (not easily distinguishable what is presented in the circle picture). Maybe arrows with description would be helpful.

80 – please provide reference to urbanization index

98 – is this the description of the snowball technique? Could you please confirm if that means that the compass arrow pointed both distance between 0-36 m as 1degree to 10 cm, as well as the direction? This randomization technique seems interesting and I feel it was not covered enough.

101-106 – please state if were collected ticks cathegorized at site or later?

107-112. In line 88 you stated that there were 35 Sites monitored by checking five 2x2 inventory plots. In lines 107-112 you stated that sampling were performed in 8h period of the day for ALL 35 sites at the same day, is that correct? Or do you mean that in each Site sampling was performed within same day? How many timeas each Site was sampled? Please clarify sampling as this is not clear for now, consider adding some suppl. materiały e.g tables.

299 – this is probably Fig 3 description?

General comment:

Aim of the study was to investigate the risk of tick encounters (not done?)

The tool was to identify the potential risk factor (success).

Table 4? I think this is actually risk factors, not risks. How were those risks valued? Is it solely on the strength of effects? Or is there any formula used?

303: risk factors rather than risk, suggestion: it is worth considering explaining in methods that you intended to evaluate risk on the basis of tick presence/abundance in reference to any detected risk factors. For now ms is lacking risk measurement description, it is only resulting from discussion part and it's virtually about risk factors.

406-407 - for rephrasing, seem like different kind of journalism

It seems logical that risk is affected by positive or negative effects, however risk itself was not described in my opinion. E.g., what is the matching value for statement that "there is still a considerable risk for tick encounters in highly urbanized areas.". What risk level is considerable and what is not?

Hopefully these comments will be of use for imprevements of the manuscript.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-22-15726R1Factors responsible for Ixodes ricinus presence and abundance across an urbanization gradient

PLOS ONE

Dear Dr. Janzen,

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.

Between the original and revised version, your manuscript has now been assessed by three external reviewers.  All feel the main focus, an investigation of ticks in urban areas and the risks they pose to humans and pets, has value.  That said, reviewers have also identified major limitations of the study, e.g. experimental design, sampling effort, and the fact so few adults were collected.  These limitations have not been adequately addressed or embraced in the revised version with respect to data interpretation and conclusions that can be drawn.  It is also unclear how exactly sampling was conducted across sites (and time) and whether the approach was adequate for assessing tick activity over the period of the study.  

Given the mixed reviews (from minor revision to reject outright), I'm willing to let the manuscript progress through another round of revision and review.  Please take reviewer concerns fully to heart, otherwise similar concerns will likely continue to be raised by the reviewer community.  Alternatively, you may decide you would prefer submitting your manuscript to another, more focused, journal.

Please submit your revised manuscript by Jan 21 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,

Janice L. Bossart

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: (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: Partly

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

Reviewer #3: I Don't Know

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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: No

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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

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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 a useful investigation into what local site and landscape level factors influence the abundance of Ixodes scapularis in an urban landscape. Importantly, this study highlights the fact that ticks can occur in urbanized areas, and thus may pose a risk to human and companion animal health. Nevertheless, there are several major and minor shortcomings of this study.

The major issues include:

The use of the term “rural-urban gradient”. This term does not accurately reflect the landscape variables tested in this study. Non-urban (aka. artificial surface) doesn’t necessarily mean rural.

This study is better suited to just look at nymphs and larvae. The limited collection of adult ticks makes it difficult to make any conclusions about the adult life stage.

The outcome of objective 2 (effect of land cover at each sampling site) is not clear. The methods state all sampling sites were in greenspaces, but do not provide landcover information.

It seems that there was no repeat sampling of sites over time. This is a major (though not fatal) flaw in study design. To convince readers of the relevance of this data set, greater effort is needed to explain how time was factored into the modeling framework. Furthermore, the distance sampled per site (40m2 if I did my calculations correctly) falls well below sampling efforts in most studies (~1000m2 per site).

There is no explanation in the methods and results on how the response variable (abundance) was adjusted to account for the variation in sampling effort across sites. For example, density of ticks should be calculated per site and then this metric would be comparable across sites with different plot numbers.

Reanalysis and interpretation of the results is warranted prior to publication.

Unable to find data available on the stated website. Please provide direct link.

The minor issues are:

Line 21: describe what “40% urbanization” is in the context of this study.

Line 72: effect of local factors on what? effect of land cover on what? Results and discussion talk about presence/absence of ticks, but this not stated in any objectives.

Line 79: remove “large” – this is a vague qualifier

Line 84: the area of a circle with a 100m radii would be 0.0314km2

Line 107: At what time of year did sampling occur in 2017? How many times were sites visited/resampled? What were the intervals of collection? How does this relate to known activity time of life stages in the region?

Line 127: At what time of year did sampling occur in 2019? How many times were sites visited/resampled? What were the intervals of collection? How does this relate to known activity time of life stages in the region?

Line 128: Were any of the sites sampled in 2017 resampled in 2019? It is unclear.

Line 129: Okay. So, this statement makes it sound like each site was only sampled once over the entire duration of the study. That means a site consists of sampling from a single time point and 5 or 10 4m2 plots. This totals, at most, 40m2 of sampling per data point. While there is no authority over what the minimum sampling effort for ticks should be, this seems very low and may explain why so few adults were collected over the course of the study.

Line 135: It is unclear what local landcover the sampling occurred in. It is just referred to as greenspace here, but was it forest and if so what type? This is central to the second objective of the study.

Line 138: What is the snowball technique? Is that what is described in lines 138-144?

Line 154: How do you “cover” the activity patterns of ticks by only sampling each site a single time and having those single sampling occasions span from May to October?

Line 160: what is meant by the “height of the field layer”? what distance around the plot was used for stem density measurements? Was vegetation height/tree cover also measure in both 2017 and 2019, or only 2019?

Line 179: include list of the eight forest subcategories here

Line 182: This size of the forest fragments that sampling sites were in would be an important variable to include in this analysis.

Line 184: Justification for why 1000m buffers were used for urbanization and landscape composition models is needed. One could perform an analysis to determine the appropriate ‘scale of effect’.

Line 185: Was SHDI and contagion measured using the landcover classification with or without the forest subcategories?

Line 193: do you mean “To analyze the effect of possible risk factors….”?

Line 210: It is unclear how spatial autocorrelation is accounted for within the models. Please explain and/or show results of tests for spatial autocorrelation.

Line 216: What aspect/measure of each land cover type is being used? Proportion?

Line 216: How was this variation in sampling effort (5 vs 10 plots) accounted for in your response variable of abundance?

Line 219: Including the proportion of all landcover types in a single model is likely not appropriate as explanatory variables are very likely to be highly correlated given that the data is proportional. While the authors state that they tested for collinearity, these results are not reported, and the full models probably violated this rule. Please show VIF results and/or reduce variables included in full model.

Line 239: Need justification for why counts of nymphs and adults were combined. Because few adults were collected, it seems reasonable to just run a model for larvae and a model for nymphs.

Line 277: Could the fewer ticks in taller vegetation be a detection issue?

Table 1: Why so little consistency in significant landscape variables across scales for larval presence/absence and abundance?

Line 449: It is confusing to have a discussion of tick abundance under a section title tick presence and absence.

Figure 3: Add an inset map for spatial context and explain the difference between the white and black in the background.

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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 #3: No

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PONE-D-22-15726R2Factors responsible for Ixodes ricinus presence and abundance across a natural-urban gradientPLOS ONE

Dear Dr. Janzen,

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.

I feel you've done a generally good job of addressing the major concerns of previous reviewers.  The exclusion of adults from the analyses and re-focus on nymphs and larvae was necessary given so few adults were collected.  The design, sampling, and analysis are now better described and adequately justified.  However, points where clarity is lacking remain and the text in general needs tightened up.  Also, I think a focused paragraph needs to be added to the discussion that explicitly addresses how the lack of data for adults factors into our understanding of environment-tick-human encounters learned from this study.  For example, would you expect the same patterns or no and why or why not?  Are there any caveats to the conclusions given the lack of data on adults?  The Results section currently contains a fair amount of Materials and Methods.  Below I list a few specific examples, but this section needs to be gone through with close scrutiny such that the Results is only results.  In many of these cases, the text is redundant with what was already written in the Materials and Methods section.  Finally, clarity would be greatly improved if the Results could be restructured in some more meaningful way.  It currently reads like a laundry list of results, which makes it hard for readers to identify key, interesting patterns.  I'm not convinced every significant results needs to be mentioned.  Why not just make a general statement about factors influencing abundance/presence, cite out to the Tables, and then in the text only highlight those results that are particularly meaningful or revealing?  

• As far as I can tell, 10 plots/site were sampled in 2017 and 5 plots/site were sampled in 2019.  If I have that right, then for clarity simply just state that rather than using verbiage like '5 or 10 were sampled'.
• What factors are local site/scale factors?  In the M&Ms (236-237) you list vegetation height, tree stem density, and temperature.  But then in Table 4, you indicate coniferous forests. Could Tables 1&2 be divided into local scale factors vs all others? Do these Tables only include factors that are significant? I'm assuming so, so please explicitly state that in the caption. 
• Numbering and discussion order of Tables & Figures should correspond. Currently, you cite out to Table 4 before Table 3. Either change the order of their discussion or the numbers of the tables.
• In the text Time is identified as a factor but the Tables don't list Time as a factor, they list Month.  Choose either Time or Month.
• Regularity of sampling is still unclear.  Did you sample weekly? bi-weekly? daily? 3 days in a row? some other way?  Please clarify.
• Line 70: N. Europe is a misnomer given you only looked at one spot in N. Europe, i.e. Stockholm County
• Line 84: Delete the period after 'sites'
• Line 105: Change 'picture' to 'figure'
• There are many spots where verb agreement is incorrect, e.g. Line 105: 'show' should be 'shows', line 135 'were' should be 'was', line 137 'allow' should be 'allows', line 222 'account' should be 'accounts', line 291 'was' should be 'were', line 316 'represents' should be 'represent', etc. Given how many I uncovered just quickly reading through certain sections, I'm guessing there are others. Please carefully scrutinize all text to ensure verb usage is correct.
• Line 130: Insert 'them' after 'excluded'
• Line 222: delete either 'these' or 'the'
• Lines 232-238: This is a really long sentence.  Rather than having 'fixed factors' at the end, how about flipping the text around to something like, Fixed factors included...  Then list the factors.
• Line 239: Add 'a' before 'random'
• Line 256: Replace 'but not for adult ticks' with 'and excluded adult ticks from the analyses'.
• Line 273: Replace existing with, "Adult ticks were excluded from all statistical analyses". (I was back and forth on whether this should only be here or only in M&Ms or in both, and decided it seemed best in both locations)
• Lines 282-284: Delete and add as footnote to the Tables.
• Line 285: 'provide' should be 'provided'
• Lines 290-292: If these were the only local factors (as indicated in lines 236-237), then how about just simply state something like, All local site factors, i.e. veg height, tree stem density, and temp, all had a significant effect on...
• Examples of M&Ms included in the Results: Lines 273-274, 280-281, 292-295, 314-316, etc. Please carefully scrutinize the Results section and ensure that M&Ms are in the M&Ms section where they belong.
• Line 275-277: Since 273-274 will be removed since it's not results, simply reword to something like, Highly correlated variables removed from the analyses of landscape configuration...include...  etc.
• Line 318: 'lager' should be 'larger'.

Please submit your revised manuscript by Mar 31 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:

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• 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,

Janice L. Bossart

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.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[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.

PONE-D-22-15726R3Factors responsible for Ixodes ricinus presence and abundance across a natural-urban gradientPLOS ONE

Dear Dr. Janzen,

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.

 The changes you have made have definitely improved manuscript organization and clarity.  Unfortunately, as I read through I'm continuing to find additional errors.  PLOS ONE does not rely on copy editors.  The onerous for ensuring the manuscript is as error free as possible lies with the authors.  Below I've listed errors I noted just reading through the yellow highlighted text.  Given the number found, I'm not at all confident that aren't many additional errors throughout the manuscript.  Since it is easy to miss errors when one is very familiar with a manuscript, please have someone who is unfamiliar with your manuscript read through it slowly and closely to ensure your submission is as error free as possible. Line 84-85.  Delete the 2nd 'in 2019'Line 199. Add a comma after 'attributes'Line 278. Remove 'that'Line 284. 'Curve linear' should be 'curvilinear'Line 307. Capitalize 'spruce' for consistencyLine 403. 'dryer' should be 'drier'Line 406. 'steam' should be 'stem'Line 465. Add a comma after 'stage'Line 467. 'build' should be 'built'Table 2 Heading. 'Ricinus' should be lower caseTable 4 Heading. Should be 'Landscape features include...' Please italicize all scientific names.  The vast majority of these are Ixodes ricinus, but there are a few others. Please submit your revised manuscript by May 18 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,

Janice L. Bossart

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.

[Note: HTML markup is below. Please do not edit.]

[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.

Factors responsible for Ixodes ricinus presence and abundance across a natural-urban gradient

PONE-D-22-15726R4

Dear Dr. Janzen,

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. Congratulations!

Typically, changes between revisions should only include those requested by external reviewers and the editors.  Your Revision 4 text differs substantially from that of Revision 3.  My suggestion to have someone unfamiliar with the manuscript read through was only meant with respect to finding spelling/grammatical errors, not to result in substantial modifications to the text and its structure/organization.  Typically significant modifications require additional review.  That said, in reading through the many text modifications this most recent revision incorporated, I recognize they only deal with wording and flow have made the manuscript easier to read and follow.

I note that scientific names are still not capitalized within the reference section.  Please italicize all scientific names prior to submitting the final version for publication.

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,

Dr. Janice L. Bossart

Academic Editor

PLOS ONE

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