PONE-D-23-27327Neonicotinoids, more than herbicides, land use, and climate, drive recent butterfly declines in the American MidwestPLOS ONE

Dear Dr. Haddad,

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 manuscript does not currently meet the PLoS ONE standards for publication #3 (Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail) or #4 (Conclusions are presented in an appropriate fashion and are supported by the data).   I have provided additional details and discussion of the reports of the reviewers and the manuscript below.

Please submit your revised manuscript by Nov 26 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,

Travis Longcore, Ph.D.

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“Naresh Neupane for extracting climate data; Elise Larsen for aggregating butterfly survey data; Frank Lupi, Doug Landis, Cheryl Schultz, and attendees of the Heartland Environmental and Resource Economics Workshop at the University of Illinois for comments on earlier versions. This research was completed while BVD was funded by an NSF LTER grant at Michigan State University. Funding for the Ries Lab was supported by funding from the USGS- Midwest Climate Adaptation Science Center (G21AC10369). This research would not be possible without the contributions of hundreds of volunteers and the directors of the programs that coordinate their efforts.  We thank Doug Taron of the Illinois BMN (Butterfly Monitoring Network), Jerry Wiedmann of the Ohio BMN, Ronda Spink of the Michigan BMN, and Nathan Brockman of the Iowa BMN, and the hundreds of volunteers who contribute data through these programs. The conclusions and opinions expressed in this article do not necessarily reflect the views or policy of the Washington Department of Fish and Wildlife.”

We note that you have provided funding information that is currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“NSF Long-term Ecological Research Program grant DEB 2224712 (SMS, BVD, NH)

Michigan State University AgBioResearch (SMS)

USDA National Institute of Food and Agriculture (SMS)

Elton R. Smith Endowment for Food and Agricultural Policy (DAH)

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide.

4. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ

5. We note that Figure 1 in your submission contain copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1. You may seek permission from the original copyright holder of Figure(s) [#] to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

6. We note that Figure 2 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1. You may seek permission from the original copyright holder of Figure 2 to publish the content specifically under the CC BY 4.0 license. 

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world-factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Natural Earth (public domain): http://www.naturalearthdata.com/

Additional Editor Comments:

As you may have anticipated, the approach and results presented in this manuscript are polarizing, and led to my expansion of the reviewer number to ensure a fair and thorough review. As you'll see, the reviews range from outright hostility to complete support. I would encourage consideration of criticisms in each of the reviews providing detailed commentary as they get at similar challenges for the reader and the question of whether the conclusions are indeed supported by the data.

There is some question about the methods and exactly how some of the time series contribute to the analysis, especially when there are, for example, only two points in the time series. It would seem that a county with only two surveys would need to be excluded. As it stands, the models are not convincing the reviewers, and will need further explanation (first) and then the question may still remain if they show what they are characterized as showing.

The overall framing and incorporating the breadth of literature in the field on these topics needs to be revisited, especially if the "blame" for monarch decline is going to be shifted now to neonics the literature on actual effects of neonics on monarchs needs to be evaluated as part of describing the purported mechanism that the models are suggesting. That is, drawing conclusions from the model needs to be supplemented by an engagement of the mechanism by which the purported effect is happening and compared with studies that investigate the direct effect of neonics on monarchs.

The manuscript leaves the impression that glyphosate does not impact monarch population trajectories. You discuss that this is probably because the damage has already been done (first paragraph page 8), but this observation needs to take a much bigger role in structuring the narrative overall if this is to be a blame-shifting paper for monarch populations. The argument in the discussion would need to be that "agricultural practices using glyphosate hammered butterfly habitat in agricultural landscapes and got to a point were so much habitat was gone that additional use had no further impact (the earth was already scorched) and now we are seeing that there is a signal that neonics are having a further impact on monarchs on top of the destruction that occurred already." Assuming this is your argument, of course.

As a structural matter, please separate out results and discussion. The ms reads like it started its life at Science or Nature. For PLoS ONE we aren't dealing with those length and style constraints. Should you prepare a revision, it would be appropriate expand the introduction and include a background literature review to address the holes identified by reviewer 1, go into more detail on the methods, report the results separately and without commentary, and then provide a discussion and conclusions that meets PLoS ONE standards ("[A]uthors should avoid overstating their conclusions. Authors may discuss possible implications for their results as long as these are clearly identified as hypotheses instead of conclusions.").

I am requesting a major revision of the manuscript, but please note that does not guarantee that it will be accepted following revision.

An engagement of the comments of each of the reviewers is requested, especially reviewers 2 and 4 relevant to the methods. Reviewer 1 was convinced of the fatal weakness of the manuscript and although pointed out areas where additional context and background information is necessary, did not deeply engage the methods. The "big picture" critiques from reviewer 1 do need to be substantively addressed, however.

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

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: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Partly

**********

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: I Don't Know

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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: General

This manuscript describes an analysis of multiple butterfly monitoring programs in the American Midwest, over a number of years, and attempted to ascertain if any anthropogenic factors such as pesticides, herbicides, landuse, or climatic variables, influence either the diversity of butterfly species or their abundance. They evaluated all monitored species, but highlighted the effects on monarchs, because (I think) of their popularity, and because of the ongoing narrative about their population trends. There were a number of significant trends with some the variables tested, though the authors highlighted only one of these, which was a negative effect of neonicitinoid pesticides on the species richness, and also on the abundance of monarchs.

I have to say that I was initially very intrigued by the idea of this study, but after reading this paper, I am rather shocked at so many instances where the authors appeared to go out of their way to either mischaracterize or purposely ignore the latest science around butterfly and insect populations here in North America, plus some recent research on monarch butterflies (including their response to neonicitinoids), and even some prior work by one of these labs. I find this very troubling, and it does not speak well of the leadership of the senior authors here. Further, the results of these analyses here seem to be counter to much of the established science just mentioned, which calls the data or analyses into question. If this paper is to go forward (either in this journal or after rejection, to another) then the authors need to do a gut-check, and think hard about how this study will affect their reputations.

Specific points

There is so much to point out about this paper that it is hard to know where to start. But for now, let me point out the parts/statements that are mischaracterized. First, the introduction begins with a loaded statement that all insects are declining, which is not entirely true, at least in North America. Indeed, much of this paper is built around this story of insects (butterflies) being in trouble. There have certainly been analyses of long-term data that shows declines of some insects, but then increases in others (Van Klink et al 2020). There has been work showing declines of butterflies in the west (Forister et al 2021) but then a more recent study (Crossley et al 2021) showed this decline was specific to the west, and the butterflies in the east were improving. Another of Forister’s papers showed that some butterfly species are “winning” in the Anthropocene. I note that these studies showing good news were distinctly missing from the text of this paper. Really, the situation is not a straightforward “decline of all insects”, at least in North America.

Along the same vein, the paper has much language devoted to describing the monarch butterfly as being in trouble, which is also a mischaracterization, based on the latest research (which was also left out here). Some of this research even was authored by one of the authors here. Ries et al 2015 described an analysis of long-term monitoring data from another program, which showed no decline of monarch abundance. Similarly, Inamine et al 2016 described a similar analysis of even more data. Crossley et al. 2022 had probably the most comprehensive analysis of monarch data to date, which again, showed no long term decline in the number of summertime monarchs across the continent. Then, the latest study of the monarch genome (Boyle et al 2023) describes how there has been no long-term decline in the effective population size in the last 75 years. Further, it now looks like the population size now is essentially larger than it ever was prior to human clearing of the prairies and eastern forests 200 years ago. So, the story that monarchs are in decline (even though it is popular in the media) is not accurate based on the latest science. I also note that none of the studies I mentioned were brought up in this paper. I find it hard to believe that this was a simple case of carelessness. More likely the authors did not want to include them, which is highly inappropriate.

The focus here on the Midwest only did not help to boost my view of this paper either, as it can lead to misleading conclusions. As the Crossley et al 2022 study demonstrates, the butterflies in the U.S. are experiencing declines in some regions, increases in others, and are stable in lots more. So, taking a snapshot of one region (even with a boatload of data) is still just one region, and so the conclusions from this analysis would really only be pertinent to here (i.e. probably could not be extrapolated). Moreover, even focusing on the monarchs alone in this region is problematic. The authors state that this is appropriate for monarchs since "95% of monarchs that reach the winter colonies originated in the cornbelt" (citing Wassenaar and Hobson 1998). This is an outdated conclusion, and again, a mischaracterization because the proper (more recent) science was not even mentioned. Wassenaar and Hobson only examined one collection of monarchs from 1997 (an exceptional year). A more thorough study was done by Flockhart et al 2017, in which 40 years of samples were examined. Their results highlighted that the Midwest only produces about 37% of the monarchs that reach Mexico, while 63% come from elsewhere. In fact, their results showed that this varies by year – in some years, most monarchs that reach Mexico come from the northeast, and in others it is the far north. Again, a seeming lack of understanding of the science is the problem here, or perhaps a purposeful effort to neglect the important studies.

I’m going to jump right into the main conclusions next for the sake of my time, since there is just too much to go through line-by-line. The main conclusion of this analysis is that neonicitinoid pesticides are causing declines to butterflies, and to monarchs in particular. This is a result that sounds ridiculous, based on the accumulated laboratory research thus far. The effects of neonics on monarchs specifically have been assessed multiple times, by multiple labs, and each time, there is little to no effect found, at least when standard, field-relevant doses are used. For some reason, monarch butterflies are resistant to nearly every neonicitinoid chemical tested, likely because of their ability to take up the toxins from milkweeds. In a few cases, declines have been found, but only when unrealistic doses are given. Further, multiple labs have examined if the plant concentrations of these chemicals are high enough in the field to cause harm to monarchs, and the answer there is also no. Off the top of my head, I can think of the following studies: Prouty et al 2023, Hall et al 2022, Bargar et al 2020, Olaya-Arenas and Kaplan 2019. None of these studies were mentioned at all. Again, an egregious oversight.

There was another result of these analyses that seemed to be purposely overlooked by the authors, but yet would have tremendous implications if this paper were ever to see the light of day. Apparently, there is a statistically significant, positive effect of glyphosate use on butterfly diversity, and at least a positive trend on monarch abundance (Fig. 4A). This result seems to strain credulity. Not only that, the authors here found NO negative effect of glyphosate use on monarch abundance, even though multiple prior studies (even by these authors) have apparently concluded that glyphosate use in the Midwest was destroying monarch habitat, and leading to their demise.

Yet another important (and credulity-straining) result is that Bt-treated plants are improving the abundance of monarch butterflies, according to these analyses. Yet again, here is a result that blows a giant hole into a suite of prior (older) research that suggested that this crop modification would harm monarchs. This research was from the early 2000s, and some of that hoopla later died down because it turns out the monarchs were never exposed to as much contaminated anthers as people thought. But, this new analysis seems to suggest that Bt-corn is even good for monarch abundance?

So with this new paper, the authors want the reader to believe that glyphosate is NOT harming monarch numbers in the Midwest, and if anything, is helping to improve butterfly diversity there! So is Bt-corn! AND, the authors want people to believe that neonicitinoid pesticides are now to blame for all of the woes of butterflies, and especially monarchs (which all lab studies have shown are resistant to neonics). This is why I think the authors need to do some soul-searching here, and decide if they really want to damage their reputation(s) with this paper. By publishing this, the authors will essentially be undermining much of all of this the prior work, which will call into question either this paper, or, the validity of prior analyses. And I haven’t even begun to discuss what would happen if the media caught wind of these new results. The media hoopla over these “new” findings would cause an upheaval and undermine public faith in scientists.

I’m going to assume that this journal will reject the paper, and so these comments are really more for the authors to consider, for gauging whether to resubmit elsewhere. This is probably the harshest review I’ve ever written, but it is because the level of arrogance here is palpable – both for purposely misleading the reader on key issues, and for presenting results that are so at odds with so much recent and established evidence, without even as much as mentioning that evidence. The lack of objectivity here is shocking. Please consider what I said. If I were you, I would table this project. It will be a hot potato if published elsewhere.

Reviewer #2: The paper is very well written. The analysis goals of evaluating different drivers of butterfly abundance are interesting and valuable. The dataset is opportunistic but nonetheless well put together (more on that). It covers a useful number of years and locations (more on that). The analysis is well done but could use a few more components for readers to understand possible confounding (more on that). The conclusions are reasonable and are not overblown given the data and analysis methods (more on that). The tables and figures are great. I only have a few thoughts on how to make the paper more convincing (or not).

First, I like the counterfactual analysis as a way of exploring the implications of the model. Given it’s complete dependence on the model, a reader must be convinced that you got the model right if you want them to trust your conclusions. This is a big task in this context, where you have an observational study, two somewhat-vague community-level response variables, a cobbled together dataset, study counties popping in and out of the dataset, and likely loads of correlation among the independent variables. So, here are some thoughts.

First, related to the community metrics. It would be nice to know which species are driving them. Consider creating a plot or table that gives the median abundance per species, like a rank-abundance bar plot. Readers might like to know if the abundance metric is entirely driven by cabbage whites, for example. Or maybe diversity depends entirely on prairie-dependent species. All this will help a reader understand your results better.

Second, regarding an opportunistic dataset, is there geographic or temporal confounding going on? Can you make something like a map that shows when the different counties enter the dataset? Maybe it is like fig 2D? Or maybe it is a table? With 60 rows and columns summarizing the extent of different data types included? And, by the way, does having a county with 1 year of data really help for this analysis? Or is it more likely to cause unexpected weirdness? Related to this, seems like random effects for counties and years would be better than fixed effects. Maybe using fixed effects is an econometrics thing? Related to spatiotemporal confounding, you probably know that it’s good practice on these types of large-scale and -extent projects to look for spatial and temporal autocorrelation in model residuals. Think Luke Anselin.

Third, I imagine there is plenty of correlation among your independent variables. Maybe it is not enough to cause computational problems (extreme multicollinearity). But it is probably enough to cause interpretation challenges. In this case, it is helpful to run univariate correlations or regression models and note the model coefficients. It sure is convenient, and ultimately more convincing, when the univariate effect estimates are similar to the multivariate effect estimates. When that is not the case, due to independent variable correlations, you have to be extra careful in interpreting model results, right?

Why all this annoying model scrutiny? Because if your model is not a slam dunk then any counterfactual analysis, and subsequent conclusions, and subsequent someday ag or environmental policies, and subsequent economic impacts on real people, are not well founded. You could probably publish this paper without any of this additional junk. But if you do the extra work and still come to the same conclusions, then your conclusions will be that much more powerful and valuable. From a less lofty perspective, the monarch mafia is going to have a hard time believing that monarch decline is more about seed coat treatment than glyphosate application or loss of hedgerows due to corn ethanol. So, you’ll need to convince them your results are not confounded by correlations between independent variables.

Reviewer #3: I enjoyed reading this manuscript; it is rigorous and important piece of work. In particular, the scientific approach of contrasting alternative predictors for a problematic species’ (in this case, declining) population abundance is critical to get past simple correlations. Much past work on the focal species examined here has not taken this approach and provided on parts of the story, some of which suffered from correlations. Finally, the authors present a complete and balanced picture. I particularly appreciated the distinction of insecticides vs. herbicides in this study. Finally, the 17 year scope of the work, the use of multiple abundance databases, and the diversity of butterflies covered makes the study of exceptional importance.

Although I am not familiar with the specifics of counterfactual analysis, I did find the data summary, statistics, and reporting convincing.

There are clear policy implications of this work.

Figures are excellent.

Reviewer #4: Review of “Neonicotinoids, more than herbicides, land use, and climate, drive recent butterfly declines in the American Midwest”

This is an ambitious study of existing data, attempting to demonstrate a spatio-temporal correlation of butterfly richness, numbers, and specifically monarch numbers with various factors, including different aspects of herbicide and pesticide use. The authors draw a strong conclusion about the negative effects of neonicotinoids based on the analysis. Given the inevitable controversy that monarch research, especially insecticide-related, generates, there must be more clarity in the methods and discussion of uncertainties for this to be an effective contribution.

Extracting trend data from butterfly monitoring programs can be tricky, although strong signals usually come through the noise. Time series have many factors correlated with time itself, and the combination of counterfactual analysis and the negative binomial GLM attempts to disentangle the factors.

But, the following aspects of the analysis need clarification:

A key figure would be the raw time series of species richness, overall abundance, and monarch counts with the same format as in Figure 2 – a mean/median line with the cloud of county level data.

The period 1998-2014 had data from 78 counties (SI table). The abstract says 60 counties. Which 60? (in the S.I Table). Is it the counties with at least 2 years? Is that how many faded lines there are in each graph in Figures 2 and 3? It is not clear how the interrupted time series of county-level counts were handled – 28 counties had only 1-4 years of data, while 13 had complete time series. The inclusion of the “Year” and “County” co-variables in the GLM (maybe?) addressed this - i.e., factors well beyond the study region, like the 2011 drought in Texas and winter storms in Mexico in the “Year” factor, and the samplingincompleteness in the "County" factor, but much more explanation needs to be made, especially the counties that had 1-4 (or 2-4) years data. Could be in the SI.

In trying to interpret the results, is it correct say that the leverage of each factor is dependent on the spatial variability among counties in, say, insecticide treatments, and the sharp increase in neonicotinoids from 2003-2009?

A very recent paper (not available when this MS was written) has some methods for splitting the time series into periods corresponding to changing factors like herbicide use, since the elimination of within field milkweeds was nearly complete by 2006. The discussion on p.14 of glyphosate hints at this break, but more explicit consideration

(Pleasants, J., Thogmartin, W.E., Oberhauser, K.S., Taylor, O.R. and Stenoien, C., 2023. A comparison of summer, fall and winter estimates of monarch population size before and after milkweed eradication from crop fields in North America. Insect Conservation and Diversity.)

The inclusion of the annual weather data by county is an important control. But the lack of post 2014 pesticide data prohibits consideration of the more recent hottest years (as mentioned on p. 14). Given that neonicotinoids leveled off after 2009, could a similar counterfactual analysis of climate impacts since 2014 reveal more about the weather impacts?

And some of the other significant results from the GLM deserve more discussion (like species richness-glyphosate positive)

The proportion of butterflies in Mexico from this region should be reported as a range, rather than a single value (P.13). It does change from year to year, but there is no doubt that this region serves as a major source of migratory monarchs.

The published literature on experimentally determined lethal and sublethal effects of neonicotinoids is confusing; some studies show minimal or no effects at field relevant concentrations, other show important mortality and sublethal effects. While neonicotinoids (and other insecticides) undoubtedly kill their targets, it is important to acknowledge the disparate results.

**********

6. 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.

Reviewere#1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

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

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

Insecticides, more than herbicides, land use, and climate, are associated with declines in butterfly species richness and abundance in the American Midwest

PONE-D-23-27327R1

Dear Dr. Haddad,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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,

Travis Longcore, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

This revision addresses comments from reviewers who ranged from "publish as is" to "burn the manuscript" in detail and does so thoroughly. I have read and in some cases re-read all of the critiques and responses and believe that the manuscript meets the PLoS ONE criteria for publication. Namely that this is 1) original research, 2) reports results not previously published, 3) contains analyses performed to a high technical standard and described in sufficient detail, 4) contains conclusions supported by the data without overstatement (here I appreciate the careful discussion of the limitations inherent in the time period of the data and the effort to preempt misinterpretation by readers), 5) written well in English, and 6) meeting standards of research ethics and integrity. During proofs, please be sure to update the information about data availability with the appropriate repository.

Reviewers' comments: