Tergal and pleural structures contribute to the formation of ...rsos.royalsocietypublishing.org/content/royopensci/3/8/...of the sternum absent in the beetle Tribolium (ref 24) and

© 2016 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited

Review History

RSOS-160157.R0 (Original submission) Review form: Reviewer 1 (James Marden) Is the manuscript scientifically sound in its present form? No Are the interpretations and conclusions justified by the results? No Is the language acceptable?

Yes

Tergal and pleural structures contribute to the formation of

ectopic prothoracic wings in cockroaches

Moysés Elias-Neto and Xavier Belles

Article citation details R. Soc. open sci. 3: 160347. http://dx.doi.org/10.1098/rsos.160347

Review timeline

Original submission: 3 March 2016 1st revised submission: 18 May 2016 2nd revised submission: 28 June 2016 Final acceptance: 29 June 2016

Note: Reports are unedited and appear as submitted by the referee. The review history appears in chronological order.

Note: This manuscript was transferred from another Royal Society journal without peer review.

on July 10, 2018http://rsos.royalsocietypublishing.org/Downloaded from

http://rsos.royalsocietypublishing.org/

2

Is it clear how to access all supporting data? They have gene expression data that could readily be made available (the raw data, for all three wings) in a single large supplement file. I didn't notice if this was done. If present fine; otherwise make it available. Do you have any ethical concerns with this paper? No Have you any concerns about statistical analyses in this paper?

No Recommendation?

Major revision is needed (please make suggestions in comments) Comments to the Author(s) Jim Marden Dept. of Biology, Penn State University This paper reports the morphology and gene expression phenotypes of the cockroach, Blattella germanica, when the homeotic gene Scr is experimentally repressed during nymphal development. There are three main conclusions: 1. Repression of Scr causes an ectopic wing-like structure on the otherwise wingless (in all extant insects) prothorax (T1). This corroborates a number of previous demonstrations of the same effect in other insect species, including another roach (ref 9). 2. Formation of the ectopic wing on T1 involves not only changes in dorsal morphology but also a reduction in size of a ventral exoskeletal feature, the epimeron. This corroborates previous demonstrations of changes in ventral morphology in other Scr repressed species, including parts of the sternum absent in the beetle Tribolium (ref 24) and a subtle but noted change in ventral features of the bug Oncopeltus (ref 7,8). 3. Repression of Scr causes the T1 segment to transform to a T2 segment. The first two conclusions are well supported by the data and require little further comment, other than to point out that ventral morphology was not examined in the previous study of Scr repression phenotypes in a roach (ref 9), so they have added an important observation. However, the authors should acknowledge that their data in Blatella is otherwise very similar to Periplaneta (and Oncopeltus), reinforcing the view that in hemimetbolous species the T1 wing is mainly a dorsal structure (featuring wing-like lateral margins) and does not develop into a true T2 wing as in the holometabolous insect Tribolium (ref 6, 24). The third conclusion is not supported by the data and this is a major flaw. If the Scr-repressed T1 segment contained flight muscles and had ventral morphology of the T2 segment, this would be prima facie evidence for a transformation to T2 segmental identity. However, they do not present such data. Lacking such a clear effect, they need to discuss how such a conclusion could be made (i.e. construct a falsifiable hypothesis) and then present an objective measure or analysis to determine segmental identity. They present gene expression data and state that the “network” (based on expression changes) in the transformed T1 is more like a T2 than a T3 segment (supp Fig 2). They do not however grapple with the differences in gene expression between the transformed T1 and the T2 segment. Most importantly, they show that nub expression in the transformed T1 is at least four-fold less than in T2, but this key result (which corroborates rather than refutes results and conclusions in ref 8) is buried in a supplemental figure and hidden by



3

their Fig 3e that shows only gene expression relative to the wild type T1 (as fold-change rather than absolute data). Low expression of nub in the wild type T1 creates the impression of very high expression in transformed T1, but that is not the case. Their Fig3e makes a less extreme but also distorted view of what’s going on with Ubx, a gene that is barely expressed at all (essentially “noise”) in T1 and T2. They argue that Scr repression in Oncopeltus (ref 8) also transforms T1 to T2, which is opposite a major conclusion in that paper (based on major differences in global gene expression and additional single and double gene knockout experiments). However, the Blattella results corroborate rather than refute the hypothesis that the T1 ectopic wing in hemimetabolous insects is a unique structure. Ultimately this study uses methods that have been previously applied to similar insects, and they show similar results. Their observation of epimeron reduction in the Scr-repressed roach is a new contribution and strengthens the evidence for both dorsal and ventral contributions to wing formation. Their conclusion that the Scr-repressed T1 transforms to a T2 segment is not rigorously analyzed or supported by the data, and they seek to refute a recent study that their gene expression data corroborates. This aspect of the paper is not high-quality science. Summary: This study presents results that confirm previous observations in other insects, but those corroborations of previous studies are not interpreted as such. Specific remarks: title: “On the origin of insect wings”. There is little here that advances the understanding of insect wing origins, from either a developmental or evolutionary perspective. They need a more narrowly defined title. L 101: Ref 6 should also be cited here. L 103 - 112: This paragraph doesn't belong here. First, the critique is shallow (doesn't address the transcriptome evidence in Ref 8) and is overstated because it ignores the subtle ventral changes near the T1 leg base in Oncopletus Scr-repressed insects. Second, the "at odds with" language ignores the difference between direct and pupal development. Why should a Hemipteran T1 develop under homeotic regulation the same as a holometabolous insect? If the authors want to make these comparisons, they should do so in the conclusions and more carefully discuss and consider the evidence. L 113: The first sentence addresses the "at odds with" bit in the previous paragraph and is unrelated to the remainder of the present paragraph. Start this paragraph with "The present work aims to ….". L 120: "phylogenetically more basal" is not supported by the most recent and complete phylogenetic + fossil analyses (e.g. http://science.sciencemag.org/content/346/6210/763.full). Hemiptera + Thysanoptera appear to have originated 375 Mya, whereas roaches + termites originated ~175 Mya, so the evidence isn't even close to supporting their contention. They would also need a recent reference for "evolutionarily less modified" or delete if this is just an opinion. Bottom line: these are old and obsolete ideas. L 163: The RNAi protocol wouldn't take many words to describe and should be reiterated here. It is central to the study. When in development? Controls? Sample sizes?



4

L 229: This header contains a conclusion rather than a result, and in any case isn’t supported by the data. L 236: “.. the pleural area of T1 did not appear to be significantly modified in Scr-depleted specimens, except for the epimeron, ...”. A highly localized ventral modification on T1 after Scr repression is very similar to the result in ref 8, yet the intro attempts to dismiss that study with a couple of sentences. Why refute things that your results corroborate? Is this an attempt to gain attention or importance, or is it adherence to conventional wisdom? 247: Or did they more closely resemble control T1 than T2? What is the objective basis for comparison here? 257: The wing and the dorsum do not comprise the entire segment. General lack of ventral change contradicts this, and there is no examination of flight muscle presence/absence. The authors need to define what comprises transformation of a body segment. Note that T2-like wing morphology appears to be the default state regardless of segmental identity (supported by extensive evidence that T3 morphology is controlled by Ubx). L. 275: “It is worth noting that our results contrast with the conclusions inferred by Medved et al. [8]”. This is not correct. The results are consistent rather than different. L. 277: “They deduced that the ectopic T1 wing-like structures were formed from a uniquely tergal origin ..” No: that study reported a change on the ventral prothorax in "an area surrounding the leg base" and concluded that the transformed T1 wing is of "primarily dorsal origin". "Uniquely" is not a fair characterization, but more importantly, the results here could be described in the same (primarily dorsal) words as used in Ref 8. As pointed out above, the expression of nub is also very similar between the two studies, both indicating a striking difference from T2. In short, the contrasts being drawn here are not supported by the data. L 287. Ref 8 considered this question (T2 transformation vs. unique structure), particularly with transcriptome data. The present study purports to overturn that conclusion without a more in depth examination of the data or even acknowledgement that the present results are in most ways more similar than different from what was found in ref 8. Fig 3e. Showing fold-change expression relative to a control T1 is very deceptive because low expression in control T1 can create a false impression of very high fold-change difference in transformed T1. Fig S1, which shows absolute expresssion, is much more informative, particularly the low exp of nub in a transformed T1 compared to T2 segment (4-5 times lower than T2). Decision letter (RSOS-160157) 10-May-2016 Dear Dr Bellés: Manuscript ID RSOS-160157 entitled "On the origin of insect wings. Insights from the wingless prothorax of cockroaches" which you submitted to Royal Society Open Science, has been reviewed. The comments from reviewers are included at the bottom of this letter. In view of the criticisms of the reviewers, the manuscript has been rejected in its current form. However, a new manuscript may be submitted which takes into consideration these comments.



5

Please note that resubmitting your manuscript does not guarantee eventual acceptance, and that your resubmission will be subject to peer review before a decision is made. You will be unable to make your revisions on the originally submitted version of your manuscript. Instead, revise your manuscript and upload the files via your author centre. Once you have revised your manuscript, go to https://mc.manuscriptcentral.com/rsos and login to your Author Center. Click on "Manuscripts with Decisions," and then click on "Create a Resubmission" located next to the manuscript number. Then, follow the steps for resubmitting your manuscript. Your resubmitted manuscript should be submitted by 07-Nov-2016. If you are unable to submit by this date please contact the Editorial Office. We look forward to receiving your resubmission. Sincerely, Matthew Allinson, Editorial Coordinator, Royal Society Open Science on behalf of Kevin Padian, Royal Society Open Science [email protected] Reviewers' Comments to Author: Reviewer: 1 Comments to the Author(s) Jim Marden Dept. of Biology, Penn State University This paper reports the morphology and gene expression phenotypes of the cockroach, Blattella germanica, when the homeotic gene Scr is experimentally repressed during nymphal development. There are three main conclusions: 1. Repression of Scr causes an ectopic wing-like structure on the otherwise wingless (in all extant insects) prothorax (T1). This corroborates a number of previous demonstrations of the same effect in other insect species, including another roach (ref 9). 2. Formation of the ectopic wing on T1 involves not only changes in dorsal morphology but also a reduction in size of a ventral exoskeletal feature, the epimeron. This corroborates previous demonstrations of changes in ventral morphology in other Scr repressed species, including parts of the sternum absent in the beetle Tribolium (ref 24) and a subtle but noted change in ventral features of the bug Oncopeltus (ref 7,8). 3. Repression of Scr causes the T1 segment to transform to a T2 segment. The first two conclusions are well supported by the data and require little further comment, other than to point out that ventral morphology was not examined in the previous study of Scr repression phenotypes in a roach (ref 9), so they have added an important observation. However,



6

the authors should acknowledge that their data in Blatella is otherwise very similar to Periplaneta (and Oncopeltus), reinforcing the view that in hemimetbolous species the T1 wing is mainly a dorsal structure (featuring wing-like lateral margins) and does not develop into a true T2 wing as in the holometabolous insect Tribolium (ref 6, 24). The third conclusion is not supported by the data and this is a major flaw. If the Scr-repressed T1 segment contained flight muscles and had ventral morphology of the T2 segment, this would be prima facie evidence for a transformation to T2 segmental identity. However, they do not present such data. Lacking such a clear effect, they need to discuss how such a conclusion could be made (i.e. construct a falsifiable hypothesis) and then present an objective measure or analysis to determine segmental identity. They present gene expression data and state that the “network” (based on expression changes) in the transformed T1 is more like a T2 than a T3 segment (supp Fig 2). They do not however grapple with the differences in gene expression between the transformed T1 and the T2 segment. Most importantly, they show that nub expression in the transformed T1 is at least four-fold less than in T2, but this key result (which corroborates rather than refutes results and conclusions in ref 8) is buried in a supplemental figure and hidden by their Fig 3e that shows only gene expression relative to the wild type T1 (as fold-change rather than absolute data). Low expression of nub in the wild type T1 creates the impression of very high expression in transformed T1, but that is not the case. Their Fig3e makes a less extreme but also distorted view of what’s going on with Ubx, a gene that is barely expressed at all (essentially “noise”) in T1 and T2. They argue that Scr repression in Oncopeltus (ref 8) also transforms T1 to T2, which is opposite a major conclusion in that paper (based on major differences in global gene expression and additional single and double gene knockout experiments). However, the Blattella results corroborate rather than refute the hypothesis that the T1 ectopic wing in hemimetabolous insects is a unique structure. Ultimately this study uses methods that have been previously applied to similar insects, and they show similar results. Their observation of epimeron reduction in the Scr-repressed roach is a new contribution and strengthens the evidence for both dorsal and ventral contributions to wing formation. Their conclusion that the Scr-repressed T1 transforms to a T2 segment is not rigorously analyzed or supported by the data, and they seek to refute a recent study that their gene expression data corroborates. This aspect of the paper is not high-quality science. Summary: This study presents results that confirm previous observations in other insects, but those corroborations of previous studies are not interpreted as such. Specific remarks: title: “On the origin of insect wings”. There is little here that advances the understanding of insect wing origins, from either a developmental or evolutionary perspective. They need a more narrowly defined title. L 101: Ref 6 should also be cited here. L 103 - 112: This paragraph doesn't belong here. First, the critique is shallow (doesn't address the transcriptome evidence in Ref 8) and is overstated because it ignores the subtle ventral changes near the T1 leg base in Oncopletus Scr-repressed insects. Second, the "at odds with" language ignores the difference between direct and pupal development. Why should a Hemipteran T1 develop under homeotic regulation the same as a holometabolous insect? If the authors want to make these comparisons, they should do so in the conclusions and more carefully discuss and consider the evidence.



7

L 113: The first sentence addresses the "at odds with" bit in the previous paragraph and is unrelated to the remainder of the present paragraph. Start this paragraph with "The present work aims to ….". L 120: "phylogenetically more basal" is not supported by the most recent and complete phylogenetic + fossil analyses (e.g. http://science.sciencemag.org/content/346/6210/763.full). Hemiptera + Thysanoptera appear to have originated 375 Mya, whereas roaches + termites originated ~175 Mya, so the evidence isn't even close to supporting their contention. They would also need a recent reference for "evolutionarily less modified" or delete if this is just an opinion. Bottom line: these are old and obsolete ideas. L 163: The RNAi protocol wouldn't take many words to describe and should be reiterated here. It is central to the study. When in development? Controls? Sample sizes? L 229: This header contains a conclusion rather than a result, and in any case isn’t supported by the data. L 236: “.. the pleural area of T1 did not appear to be significantly modified in Scr-depleted specimens, except for the epimeron, ...”. A highly localized ventral modification on T1 after Scr repression is very similar to the result in ref 8, yet the intro attempts to dismiss that study with a couple of sentences. Why refute things that your results corroborate? Is this an attempt to gain attention or importance, or is it adherence to conventional wisdom? 247: Or did they more closely resemble control T1 than T2? What is the objective basis for comparison here? 257: The wing and the dorsum do not comprise the entire segment. General lack of ventral change contradicts this, and there is no examination of flight muscle presence/absence. The authors need to define what comprises transformation of a body segment. Note that T2-like wing morphology appears to be the default state regardless of segmental identity (supported by extensive evidence that T3 morphology is controlled by Ubx). L. 275: “It is worth noting that our results contrast with the conclusions inferred by Medved et al. [8]”. This is not correct. The results are consistent rather than different. L. 277: “They deduced that the ectopic T1 wing-like structures were formed from a uniquely tergal origin ..” No: that study reported a change on the ventral prothorax in "an area surrounding the leg base" and concluded that the transformed T1 wing is of "primarily dorsal origin". "Uniquely" is not a fair characterization, but more importantly, the results here could be described in the same (primarily dorsal) words as used in Ref 8. As pointed out above, the expression of nub is also very similar between the two studies, both indicating a striking difference from T2. In short, the contrasts being drawn here are not supported by the data. L 287. Ref 8 considered this question (T2 transformation vs. unique structure), particularly with transcriptome data. The present study purports to overturn that conclusion without a more in depth examination of the data or even acknowledgement that the present results are in most ways more similar than different from what was found in ref 8. Fig 3e. Showing fold-change expression relative to a control T1 is very deceptive because low expression in control T1 can create a false impression of very high fold-change difference in transformed T1. Fig S1, which shows absolute expresssion, is much more informative,



8

particularly the low exp of nub in a transformed T1 compared to T2 segment (4-5 times lower than T2).

Author's Response to Decision Letter for (RSOS-160347)

See Appendix A.

RSOS-160347.R0 (Revision)

Review form: Reviewer 1 (James Marden)

Is the manuscript scientifically sound in its present form?

Yes

Are the interpretations and conclusions justified by the results?

Yes

Is the language acceptable?

Yes

Is it clear how to access all supporting data? The title page has a so-far blank indication of a link to data deposition. The supplementary material is fine.

Do you have any ethical concerns with this paper?

No

Have you any concerns about statistical analyses in this paper?

No

Recommendation? Accept with minor revision (please list in comments)

Comments to the Author(s) Despite making a few comments that the authors considered overly harsh, my initial review was apparently quite constructive. The revision contains all of the requested changes, so I have little more to say. Well done.

A very few minor things to fix: L. 78 "result rather counterintuitive when trying to image how a leg branch" Wording needs to be fixed. I suggest "result is counterintuitive when trying to imagine how a leg branch"

L. 209 - 213: Use a different word than disc (or disk). So much of the literature on wing development refers to imaginal discs that using this word here has the potential to cause confusion.



9

L. 223: "This kind of unfolded" ????? There is a fragment of a sentence hanging at the end of the paragraph. Fix this. L. 257 "reminds that of T2". Proper wording: "is reminiscent of T2". L. 330 If I provided a constructive non-anonymous review, perhaps I have earned an acknowledgement. Decision letter (RSOS-160347) 23-Jun-2016 Dear Dr Bellés On behalf of the Editor, I am pleased to inform you that your Manuscript RSOS-160347 entitled "Tergal and pleural structures contribute to the formation of ectopic prothoracic wings in cockroaches" has been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee suggestions. Please find the referees' comments at the end of this email. The reviewers and Subject Editor have recommended publication, but also suggest some minor revisions to your manuscript. Therefore, I invite you to respond to the comments and revise your manuscript. • Ethics statement If your study uses humans or animals please include details of the ethical approval received, including the name of the committee that granted approval. For human studies please also detail whether informed consent was obtained. For field studies on animals please include details of all permissions, licences and/or approvals granted to carry out the fieldwork. • Data accessibility It is a condition of publication that all supporting data are made available either as supplementary information or preferably in a suitable permanent repository. The data accessibility section should state where the article's supporting data can be accessed. This section should also include details, where possible of where to access other relevant research materials such as statistical tools, protocols, software etc can be accessed. If the data has been deposited in an external repository this section should list the database, accession number and link to the DOI for all data from the article that has been made publicly available. Data sets that have been deposited in an external repository and have a DOI should also be appropriately cited in the manuscript and included in the reference list. If you wish to submit your supporting data or code to Dryad (http://datadryad.org/), or modify your current submission to dryad, please use the following link: http://datadryad.org/submit?journalID=RSOS&manu=RSOS-160347 • Competing interests Please declare any financial or non-financial competing interests, or state that you have no competing interests. • Authors’ contributions



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All submissions, other than those with a single author, must include an Authors’ Contributions section which individually lists the specific contribution of each author. The list of Authors should meet all of the following criteria; 1) substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) drafting the article or revising it critically for important intellectual content; and 3) final approval of the version to be published. All contributors who do not meet all of these criteria should be included in the acknowledgements. We suggest the following format: AB carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study and drafted the manuscript; CD carried out the statistical analyses; EF collected field data; GH conceived of the study, designed the study, coordinated the study and helped draft the manuscript. All authors gave final approval for publication. • Acknowledgements Please acknowledge anyone who contributed to the study but did not meet the authorship criteria. • Funding statement Please list the source of funding for each author. Because the schedule for publication is very tight, it is a condition of publication that you submit the revised version of your manuscript within 7 days (i.e. by the 02-Jul-2016). If you do not think you will be able to meet this date please let me know immediately. To revise your manuscript, log into https://mc.manuscriptcentral.com/rsos and enter your Author Centre, where you will find your manuscript title listed under "Manuscripts with Decisions". Under "Actions," click on "Create a Revision." You will be unable to make your revisions on the originally submitted version of the manuscript. Instead, revise your manuscript and upload a new version through your Author Centre. When submitting your revised manuscript, you will be able to respond to the comments made by the referees and upload a file "Response to Referees" in "Section 6 - File Upload". You can use this to document any changes you make to the original manuscript. In order to expedite the processing of the revised manuscript, please be as specific as possible in your response to the referees. When uploading your revised files please make sure that you have: 1) A text file of the manuscript (tex, txt, rtf, docx or doc), references, tables (including captions) and figure captions. Do not upload a PDF as your "Main Document". 2) A separate electronic file of each figure (EPS or print-quality PDF preferred (either format should be produced directly from original creation package), or original software format) 3) Included a 100 word media summary of your paper when requested at submission. Please ensure you have entered correct contact details (email, institution and telephone) in your user account 4) Included the raw data to support the claims made in your paper. You can either include your data as electronic supplementary material or upload to a repository and include the relevant doi within your manuscript



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5) Included your supplementary files in a format you are happy with (no line numbers,vancouver referencing, track changes removed etc) as these files will NOT be edited in production

Once again, thank you for submitting your manuscript to Royal Society Open Science and I look forward to receiving your revision. If you have any questions at all, please do not hesitate to get in touch.

Best wishes

Andrew Dunn Senior Publishing Editor, Royal Society Open Science

on behalf of Kevin Padian Subject Editor, Royal Society Open Science [email protected]

Comments to Author: Reviewer: 1

Comments to the Author(s) Despite making a few comments that the authors considered overly harsh, my initial review was apparently quite constructive. The revision contains all of the requested changes, so I have little more to say. Well done.

A very few minor things to fix: L. 78 "result rather counterintuitive when trying to image how a leg branch" Wording needs to be fixed. I suggest "result is counterintuitive when trying to imagine how a leg branch"

L. 209 - 213: Use a different word than disc (or disk). So much of the literature on wing development refers to imaginal discs that using this word here has the potential to cause confusion.

L. 223: "This kind of unfolded" ????? There is a fragment of a sentence hanging at the end of the paragraph. Fix this.

L. 257 "reminds that of T2". Proper wording: "is reminiscent of T2".

L. 330 If I provided a constructive non-anonymous review, perhaps I have earned an acknowledgement.

Author's Response to Decision Letter for (RSOS-160347)

See Appendix B.



Appendix A Reviewers' Comments to Author:

Reviewer: 1

Comments to the Author(s)

Jim Marden

Dept. of Biology, Penn State University

This paper reports the morphology and gene expression phenotypes of the cockroach,

Blattella germanica, when the homeotic gene Scr is experimentally repressed during

nymphal development. There are three main conclusions:

1. Repression of Scr causes an ectopic wing-like structure on the otherwise wingless

(in all extant insects) prothorax (T1). This corroborates a number of previous

demonstrations of the same effect in other insect species, including another roach (ref

9).

2. Formation of the ectopic wing on T1 involves not only changes in dorsal

morphology but also a reduction in size of a ventral exoskeletal feature, the

epimeron. This corroborates previous demonstrations of changes in ventral

morphology in other Scr repressed species, including parts of the sternum absent in

the beetle Tribolium (ref 24) and a subtle but noted change in ventral features of the

bug Oncopeltus (ref 7,8).

3. Repression of Scr causes the T1 segment to transform to a T2 segment.

The first two conclusions are well supported by the data and require little further

comment, other than to point out that ventral morphology was not examined in the

previous study of Scr repression phenotypes in a roach (ref 9), so they have added an

important observation. However, the authors should acknowledge that their data in

Blatella is otherwise very similar to Periplaneta (and Oncopeltus), reinforcing the

view that in hemimetbolous species the T1 wing is mainly a dorsal structure

(featuring wing-like lateral margins) and does not develop into a true T2 wing as in

the holometabolous insect Tribolium (ref 6, 24).

The third conclusion is not supported by the data and this is a major flaw. If the Scr-

repressed T1 segment contained flight muscles and had ventral morphology of the T2



segment, this would be prima facie evidence for a transformation to T2 segmental

identity. However, they do not present such data. Lacking such a clear effect, they

need to discuss how such a conclusion could be made (i.e. construct a falsifiable

hypothesis) and then present an objective measure or analysis to determine segmental

identity. They present gene expression data and state that the “network” (based on

expression changes) in the transformed T1 is more like a T2 than a T3 segment (supp

Fig 2). They do not however grapple with the differences in gene expression

between the transformed T1 and the T2 segment. Most importantly, they show that

nub expression in the transformed T1 is at least four-fold less than in T2, but this key

result (which corroborates rather than refutes results and conclusions in ref 8) is

buried in a supplemental figure and hidden by their Fig 3e that shows only gene

expression relative to the wild type T1 (as fold-change rather than absolute data).

Low expression of nub in the wild type T1 creates the impression of very high

expression in transformed T1, but that is not the case. Their Fig3e makes a less

extreme but also distorted view of what’s going on with Ubx, a gene that is barely

expressed at all (essentially “noise”) in T1 and T2. They argue that Scr repression in

Oncopeltus (ref 8) also transforms T1 to T2, which is opposite a major conclusion in

that paper (based on major differences in global gene expression and additional

single and double gene knockout experiments). However, the Blattella results

corroborate rather than refute the hypothesis that the T1 ectopic wing in

hemimetabolous insects is a unique structure.

Ultimately this study uses methods that have been previously applied to similar

insects, and they show similar results. Their observation of epimeron reduction in

the Scr-repressed roach is a new contribution and strengthens the evidence for both

dorsal and ventral contributions to wing formation. Their conclusion that the Scr-

repressed T1 transforms to a T2 segment is not rigorously analyzed or supported by

the data, and they seek to refute a recent study that their gene expression data

corroborates. This aspect of the paper is not high-quality science.

Summary: This study presents results that confirm previous observations in other

insects, but those corroborations of previous studies are not interpreted as such.

The first two conclusions are well supported by the data and require little further

comment, other than to point out that ventral morphology was not examined in the

previous study of Scr repression phenotypes in a roach (ref 9), so they have added an

important observation.



REPLY: We appreciate the comments “Their observation of epimeron

reduction in the Scr-repressed roach is a new contribution and strengthens the

evidence for both dorsal and ventral contributions to wing formation”, and, in

the Summary, “The first two conclusions are well supported by the data and

require little further comment, other than to point out that ventral morphology

was not examined in the previous study of Scr repression phenotypes in a roach

(ref 9), so they have added an important observation”.

We have paid special attention to the third point raised by the reviewer, which

led us to fundamentally modify our conclusions in general agreement with the

reviewer comment. More details on the issue are provided in most of the

following replies.

Specific remarks:

title: “On the origin of insect wings”. There is little here that advances the

understanding of insect wing origins, from either a developmental or evolutionary

perspective. They need a more narrowly defined title.

REPLY: We have changed the title. The title proposed in the new version is

“Tergal and pleural structures contribute to the formation of ectopic

prothoracic wings in cockroaches”, which captures what is probably the most

original contribution of the present report.

L 101: Ref 6 should also be cited here.

REPLY: We agree. Ref 6 has been added here.

L 103 - 112: This paragraph doesn't belong here. First, the critique is shallow

(doesn't address the transcriptome evidence in Ref 8) and is overstated because it

ignores the subtle ventral changes near the T1 leg base in Oncopletus Scr-repressed

insects. Second, the "at odds with" language ignores the difference between direct

and pupal development. Why should a Hemipteran T1 develop under homeotic

regulation the same as a holometabolous insect? If the authors want to make these

comparisons, they should do so in the conclusions and more carefully discuss and

consider the evidence.

REPLY: We have rewritten this paragraph is a more descriptive (not

conclusive) way (often using the original wording of Popadić and colleagues,

2015), so fitting better in what should be an “Introduction”. The wording used

is as follows: “A most recent paper by Popadić and colleagues, using the



hemimetabolan species, O. fasciatus (Condylognatha, Hemiptera) as model,

reports that the T1 wing structures formed after Scr depletion in this species are

mainly of dorsal origin, although gene expression differences between the

ectopic T1 wing structure and T2 and T3 wings suggest that ventral structures

also contribute to wing formation (8). These results would also support, thus, a

dual origin for insect wings.”

L 113: The first sentence addresses the "at odds with" bit in the previous paragraph

and is unrelated to the remainder of the present paragraph. Start this paragraph with

"The present work aims to ….".

REPLY: We agree. Modified accordingly.

L 120: "phylogenetically more basal" is not supported by the most recent and

complete phylogenetic + fossil analyses

(e.g. http://science.sciencemag.org/content/346/6210/763.full). Hemiptera +

Thysanoptera appear to have originated 375 Mya, whereas roaches + termites

originated ~175 Mya, so the evidence isn't even close to supporting their contention.

They would also need a recent reference for "evolutionarily less modified" or delete

if this is just an opinion. Bottom line: these are old and obsolete ideas.

REPLY: We agree. We have deleted the wording mentioned, and replaced them

by the more aseptic wording “B. germanica is a hemimetabolan species like O.

fasciatus, but belongs to the subclass Polynopteran, the sister group of

Condylognatha + Psocodea + Endopterygota (=Holometabola)” adding the

Misof reference.

L 163: The RNAi protocol wouldn't take many words to describe and should be

reiterated here. It is central to the study. When in development? Controls? Sample

sizes?

REPLY: We agree. We have expanded the description of the RNAi

experiments, providing the more important details (stage, controls, doses,

sample sizes).

L 229: This header contains a conclusion rather than a result, and in any case isn’t

supported by the data.


http://science.sciencemag.org/content/346/6210/763.full


REPLY: We have reconsidered the conclusion of the T1 to T2 transformation at

the light of the comments of the reviewer. In this context, we have removed this

heading. In the new version, the section “Results” is not subdivided into

subsections.

L 236: “.. the pleural area of T1 did not appear to be significantly modified in Scr-

depleted specimens, except for the epimeron, ...”. A highly localized ventral

modification on T1 after Scr repression is very similar to the result in ref 8, yet the

intro attempts to dismiss that study with a couple of sentences. Why refute things

that your results corroborate? Is this an attempt to gain attention or importance, or is

it adherence to conventional wisdom?

REPLY: All right. In order to clarify everything, we would like to state that our

conjectures might be more or less robustly based on fully justified facts, but

they are honest, and have nothing to do with an attempt to gain attention or

importance, or to adhere to conventional wisdom.

247: Or did they more closely resemble control T1 than T2? What is the objective

basis for comparison here?

REPLY: We have deleted the statement “The general expression trends in T1

Scr-depleted samples (and specifically those of ap and Ubx) resembled those of

T2 more than those of T3.” In this context, Figure 3e, that shows fold-change

expression relative to a control T1 has been eliminated. Instead, we used the

former Figure S1 (absolute expression) as new Figure 4 (see also our reply to the

last point, about Fig. 3e). Figure S2 is retained as Figure S1.

257: The wing and the dorsum do not comprise the entire segment. General lack of

ventral change contradicts this, and there is no examination of flight muscle

presence/absence. The authors need to define what comprises transformation of a

body segment. Note that T2-like wing morphology appears to be the default state

regardless of segmental identity (supported by extensive evidence that T3

morphology is controlled by Ubx).

REPLY: We agree, the wing and the dorsum do not comprise the entire

segment, and we did not examine flight muscles. We have suppressed the entire

sentence, from “Their coriaceous… to… T. castaneum (6)”, which was too

categorical when stating that there was a transformation T1 to T2. We deal with

the issue of T1 to T2 transformation later in the Discussion, with new, much



more prudent texts.

L. 275: “It is worth noting that our results contrast with the conclusions inferred by

Medved et al. [8]”. This is not correct. The results are consistent rather than

different.

REPLY: These dramatically contrasting and rather categorical expressions

have been eliminated in the new version. We agree that our results are more

similar than opposite to those obtained in O. fasciatus, and this is reflected in the

new version of the manuscript.

L. 277: “They deduced that the ectopic T1 wing-like structures were formed from a

uniquely tergal origin ..” No: that study reported a change on the ventral prothorax

in "an area surrounding the leg base" and concluded that the transformed T1 wing is

of "primarily dorsal origin". "Uniquely" is not a fair characterization, but more

importantly, the results here could be described in the same (primarily dorsal) words

as used in Ref 8. As pointed out above, the expression of nub is also very similar

between the two studies, both indicating a striking difference from T2. In short, the

contrasts being drawn here are not supported by the data.

REPLY: Again, these contrasting expressions have been eliminated in the new

version (see previous Reply)

L 287. Ref 8 considered this question (T2 transformation vs. unique structure),

particularly with transcriptome data. The present study purports to overturn that

conclusion without a more in depth examination of the data or even

acknowledgement that the present results are in most ways more similar than

different from what was found in ref 8.

REPLY: Again, we agree that our results are more similar than opposite to

those obtained in O. fasciatus and this has been clearly reflected in the new

version of the manuscript, where we dealt on the issue “T2 transformation vs.

unique structure” following a much more prudent approach, in the penultimate

paragraph of the Discussion.

Fig 3e. Showing fold-change expression relative to a control T1 is very deceptive

because low expression in control T1 can create a false impression of very high fold-

change difference in transformed T1. Fig S1, which shows absolute expresssion, is

much more informative, particularly the low exp of nub in a transformed T1

compared to T2 segment (4-5 times lower than T2).



REPLY: Figure 3e has been eliminated, and we have used instead the former

Figure S1 (absolute expression) as new Figure 4 (see also our reply to the

reviewer comment on l. 247, above.

AUTHORS SUMMARY OF THE REPLY

The most important criticism was against our previous emphasis sharply

contrasting our results with those obtained in Oncopeltus. The reviewer

suggested that our results where more similar than different from what is

described in Oncopeltus, and we agreed after careful consideration and detailed

re-reading of Oncopeltus papers not only the most recent one (Mevdev et al,

2015), but also a previous one from the same group (Chesebro et al., 2009),

which is also very useful. This new view has been followed all along the new

version of the manuscript that we resubmit now.

Another point raised by the reviewer refers to the issue of the homeotic

transformation of T1 into T2. We proposed that in Blattella Scr depletion

triggered such a homeotic transformation, and the reviewer did not agree with

that conclusion. We also agree in this point, as the changes triggered by Scr

depletion mainly affected the dorsal structures (although, importantly, it also

affected the pleural epimeron). Thus, in the new manuscript we do not claim

that the treatment produced a homeotic transformation of T1 into T2, but only

the changes described.

A third important point is the consideration of the T1 ectopic wings of

Oncopeltus as unique structures (Mevdev et al, 2015) (and whether those of

Blattella would deserve the same consideration). We have not entered in detail

in this issue, as we consider that it is still enigmatic and because our

experiments do not add any new light to it. We have only mentioned the

possibility that the differences between the homeotic transformation observed in

Drosophila and Tribolium and the changes observed in Oncopeltus and Blattella

might be due to the different modes of metamorphosis, holometabolan in the

first case and hemimetabolan in the second.

Finally, we have changed the title of the manuscript, also following a suggestion

of the reviewer. The title proposed in the new version is “Tergal and pleural

structures contribute to the formation of ectopic prothoracic wings in

cockroaches”, which captures an important contribution of the present report,

as recognized by the reviewer.



All changes and additions have been highlighted in yellow in the new version of

the manuscript in order to be easily recognizable.

We would like to thank the reviewer for the extensive and constructive work

done on our manuscript.



Dear Editor, Please find you enclosed the last version of our manuscript RSOS-160347, entitled "Tergal and pleural structures contribute to the formation of ectopic prothoracic wings in cockroaches", which was been accepted for publication in Royal Society Open Science subject to minor revision in accordance with the referee and editorial suggestions. The referee’s suggestions were as follows (our reply, included): Comments to the Author(s) Despite making a few comments that the authors considered overly harsh, my initial review was apparently quite constructive. The revision contains all of the requested changes, so I have little more to say. Well done.

A very few minor things to fix: L. 78 "result rather counterintuitive when trying to image how a leg branch" Wording needs to be fixed. I suggest "result is counterintuitive when trying to imagine how a leg branch" REPLY: corrected in the new version.

L. 209 - 213: Use a different word than disc (or disk). So much of the literature on wing development refers to imaginal discs that using this word here has the potential to cause confusion. REPLY: corrected in the new version.

L. 223: "This kind of unfolded" ????? There is a fragment of a sentence hanging at the end of the paragraph. Fix this. REPLY: corrected in the new version (this incomplete sentence has been deleted).

L. 257 "reminds that of T2". Proper wording: "is reminiscent of T2". REPLY: corrected in the new version.

L. 330 If I provided a constructive non-anonymous review, perhaps I have earned an acknowledgement. REPLY: We agree, we have included the reviewer in the acknowledgements. The editorial indications were to include or complete the sections: “Ethics statement”, “Data accessibility”, “Competing interests”, “Authors’ contributions”, “Acknowledgements” and “Funding statement”, which has been done, accordingly. We are very happy with this version of the manuscript, and we hope that it will fulfill the last requirements of the journal. Otherwise, do not hesitate to contact us again. Yours sincerely, Xavier Belles, on behalf of all authors

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Tergal and pleural structures contribute to the formation of ...rsos.royalsocietypublishing.org/content/royopensci/3/8/...of the sternum absent in the beetle Tribolium (ref 24) and