© 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.
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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
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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?
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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.
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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,
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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.
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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,
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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.
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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.
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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
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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.
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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
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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.
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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
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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).
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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.
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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.
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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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