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Dear Dr. Veenstra:
Thanks for re-submitting your manuscript to PeerJ. I now believe that your manuscript is suitable for publication, as the reviewers (as well as myself) feel you have addressed the major problems. Congratulations! There is one minor item you may choose to address (per reviewer 1, it is entirely up to you); if you entertain this concern, please handle this before sending your manuscript to production.
I look forward to seeing this work in print, and I anticipate it being an important resource for research on neuropeptide signaling in beetles. Thanks again for choosing PeerJ to publish such important work.
Best,
-joe
# PeerJ Staff Note - this decision was reviewed and approved by Jennifer Vonk, a PeerJ Section Editor covering this Section #
no comment
no comment
no comment
I only have one comment for the author (for clarification) which he can choose to address if he wishes to. It is a response to the comment I made earlier (below).
Lines 225-227: These are in vitro studies and should be treated with caution. Has it been shown that CCHa1 can activate CCHa2 receptor in vivo and vice versa? As far as I am aware, only the Drosophila PDF receptor has been shown to be activated by DH31 in vivo. I understand the point that the author is trying to make but perhaps these 2 examples of sNPF/NPF and CCHa-1/2 might not be the best ones.
-------------It is very difficult to to do these things in vivo in insects. The reviewer states that the only example is DH31 acting on the PDF receptor in Drosophila. I described the myosuppressin receptor in Bombyx. Although I did not describe all the details (but they are in the references cited), the FMRFamide neurons innervate directly the prothoracic gland and all evidence suggests that in vivo FMRFamide acts on the myosuppressin receptor. Why else would neurons producing a peptide that is active on the receptor present in the prothoracic gland synapse onto these glands.? Given the very high affinity of this receptor for myosuppressin and the release of myosuppressin into the hemolymph one has to assume that it similarly activates this receptor in vivo. I described this example rather than that of DH31/PDF, because of the sequence similarities between FMRFamide and myosuppressin and because there are several other examples of structurally related peptides that in vitro can activate each others receptors. This suggests that this is likely a very general phenomenon. I disagree with the implicit argument of the reviewer that since it has not been demonstrated in vivo it remains to be seen whether this actually happens. The vertebrate example of CCK using the gastrin receptor in the brain is very telling in my opinion in that it illustrates the consequences of having a sensitive neuropeptide receptor in the periphery (the CCK receptor) that can not be used within the CNS, simply because it is too sensitive. When a neuropeptide is released into the hemolymph its concentrations are very low, but when it is released inside the brain, locally its concentrations will be much higher. It is for this reason that DH31 can activate the PDF receptor in the Drosophila brain and that FMRFamide, when it is released locally within the prothoracic gland in Bombyx mori can still activate the myosuppressin receptor even though its affinity for this receptor is a thousand-fold lower than that of myosuppressin. Neuropeptides are not only used at the periphery, but also within the CNS.
Response: I am not disagreeing about the conclusion of the author based on the findings of Yamanaka et al 2005. 2006. My point was that in vitro assays used to deorphanize GPCRs are usually correct but sometimes they do give a response for other ligands (depending on the type of assay used in deorphanization). Hence Hansen et al 2011 see some response (at very high doses) when CCHamide1 is applied to CCHa2 receptor and CCHamide2 is applied to CCHa1 receptor. However, it is highly unlikely that these high doses will ever be encountered in vivo. That is why I advise the author to treat those deorphanization studies which show that multiple ligands can activate a single receptor with caution.
The few minor errors I pointed out were corrected,
No concerns in original review.
no comment
Excellent summary of beetle nueropeptides that will be useful to the community. I again recommend to accept.
The revised version is much improved and has carefully taken into account the reviewer's comments.
Ok
ok
ok
Dear Dr. Veenstra:
Thanks for submitting your manuscript to PeerJ. I have now received three independent reviews of your work, and as you will see, the reviewers raised some concerns about the research. Despite this, these reviewers are optimistic about your work and the potential impact it will lend to research on neuropeptide signaling in beetles. Thus, I encourage you to revise your manuscript accordingly, taking into account all of the concerns raised by the reviewers.
While the concerns of the reviewers are relatively minor, this is a major revision to ensure that the original reviewers have a chance to evaluate your responses to their concerns.
In your revision, please ensure that all figures (including supplements) are presented clearly.
I look forward to seeing your revision, and thanks again for submitting your work to PeerJ.
Good luck with your revision,
-joe
No issues
Meets all the criteria
No issues
Manuscript: Coleoptera genome and transcriptome sequences reveal numerous differences in neuropeptide signaling between species
General comments:
This manuscript describes the identification of neuropeptides/neurohormones from multiple coleopterans in publicly available genomic and transcriptomic datasets. I commend the author on such a comprehensive analysis which even takes in to account neuropeptides (eg Hansolin) that have been recently discovered. I don’t have any major issues with the manuscript – just some typos, queries and suggestions to improve the figures.
Introduction:
Lines 45-46: Gain and loss of neuropeptides are two phenomena in the broad sense. But what about the modification of existing neuropeptides/receptors? This type of peptide evolution is later described for PDF and NPF.
Line 74: delete “there are”
Line 81: replace “in stead” with “instead”
Material and methods:
Lines 98-99: which precursor is this?
Lines 225-227: These are in vitro studies and should be treated with caution. Has it been shown that CCHa1 can activate CCHa2 receptor in vivo and vice versa? As far as I am aware, only the Drosophila PDF receptor has been shown to be activated by DH31 in vivo. I understand the point that the author is trying to make but perhaps these 2 examples of sNPF/NPF and CCHa-1/2 might not be the best ones.
Results:
Lines 270-275: This part is a bit confusing and can be elaborated.
“none of Leptinotarsa neuropeptide genes predict the same structure as the allatotropin ortholog from Locusta migratoria” – does this mean that Locusta allatotropin ortholog was not identified here?
“while this species has a proctolin gene, it does not predict an [Ala1]-proctolin” – what is the significance of [Ala1]-proctolin?
“Two other peptides that were reportedly isolated...” – what are these peptides?
Line 323: Figure 4 is for Elevenin and Figure 5 is for calcitonin. Swap the two.
Calcitonin B: Any speculation on how such a precursor could evolve? What is the similarity between DH31 and the calcitonin peptide without the disulfide bridge?
Line 392: Replace Figure S7 with Figure S8.
Lines 394-395: How can you be sure that a gene is no longer “functional”? I assume based on the alignment that it is missing the bits encoding the N-terminal fragment and hence doesn’t get translated. Please clarify.
Line 407: Figure 7 is AKH alignment not elevenin.
Line 445: Replace figure S12 (bursicon) with figure S11 (RFLa).
Line 460: Should be Figure S12 and S13. Make sure all the figure references are accurate.
Line 470: Is this calcitonin A?
Line 474: Although this gene is duplicated, the second gene produces a non-amidated peptide and might thus be non-functional. This is mentioned in the figure caption but should be elaborated in text.
Also, as mentioned above, CCHa1 has its own specific receptor and CCHa2 has its own. That is also the conclusion of Hansen et al that deorphanized these receptors. So I don’t see how the duplication of CCHa1 receptors is related to CCHa2.
Line 476: add “are” before “more similar”
Line 567: delete the extra “one to”
Line 584: Figure 12 refers to DH37 and DH47 genes.
DH31 transcripts appear to be quite interesting. Do you predict the other neuropeptides to have a separate receptor? Are there any extra Family B GPCRs in the species which produce these extra peptides? Also, Rhodnius DH31 also has an interesting splicing pattern (see Ons et al 2011). Any similarities between Rhodnius and Coleoptera splicing?
RYamide: Any comments on the conservation of Cys residues in the C-terminus of the precursor? Seems similar to the conservation seen for AKH precursors.
Discussion:
Line 691: add “it” after “possible that”
Line 705: “neuropeptide structures” – are you referring to the primary structure, secondary structure or both? Do you think this is the reason why insulin structures are quite variable?
Line 721: What about the possibility that the evolution of peptides is what facilitates complete metamorphosis? Difficult to tell what is the causative factor.
Line 738: “accidental elimination” – what do you mean by this?
Figures:
For most of the alignments in supplementary files, it is difficult to spot the mature peptide sequences and count the variability when paracopies are present. Perhaps you could underline or bold them to make them standout.
FigS2: Indicate the baratin mature peptide sequence
FigS3: Indicate the myosuppressin mature peptide sequence
FigS9: Indicate the sNPF mature peptide sequences
FigS10: Indicate the proctolin mature peptide sequences
FigS11: Indicate the RFLa mature peptide sequences
FigS18: Indicate the mature peptide sequences
FigS20: Indicate the vasopressin mature peptide sequences
FigS22: Indicate the allatotropin mature peptide sequences
FigS26: Indicate the DH47 mature peptide sequences
FigS27: Indicate the DH37 mature peptide sequences
FigS28: Indicate the ETH mature peptide sequences
FigS29: Indicate the RYa mature peptide sequences
FigS30: Indicate the sulfakinin mature peptide sequences. Do this for the remaining alignments where the location and number of peptides are fairly constant.
Figure 4: Pogonus elevenin sequence is missing.
Figure 10: Instead of using red (which is usually used to indicate losses in this manuscript), use green or another color to indicate an increase in paracopies. Also, the Tenebrio gene appears to be translucent (not shaded). Can this be made more pronounced?
Typo in caption: “hat” should be “that”
Figure 12- Typo in caption: “hat” should be “that”
Figure 13- Typo in caption: “hat” should be “that”
This manuscript titled “Coleoptera genome and transcriptome sequences reveal numerous differences in neuropeptide signaling between species” examines the neuropeptide composition of 17 beetle species. Overall this is an in depth and compressive look at neuropeptides in a diverse range of Coleoptera. It provides useful insights into the flexibility and necessity of these peptides in insects and provides some interesting findings on those neuropeptides that are essential versus those that can be lost, the level of conservation in the primary structure of these neuropeptides, and which ones have undergone duplication.
Basic Reporting: I only have few minor concerns on this topic.
The manuscript is well written, provides relevant and up to date references and succinctly covers this topic as much as possible considering the scope of this study. There are a few confusing issues regarding figure labeling. Specifically, on lines 323 and 326 it appears that the figures have been switched. Similarly, I believe that in line 407 the author is actually referring to Figure 4. There are also a few simple typos throughout that need to be identified and corrected.
This work, while important, is largely descriptive and as such does not have a traditional experimental design. As such I have no concerns with this section.
The author has done a good job interpreting the findings and providing possible explanations for the loss, duplication and amino acid changes in the characterized neuropeptides. Obviously at this stage the discussion is largely speculation and additional work will need to be done to verify the biological activity of these peptides. I am not certain that I buy into the idea that loss of a neuropeptide signaling system is typically led by changes to or deletion of the receptor, but it is valid speculation. Importantly, this work lays the groundwork for these future studies.
Overall all this is a very thorough body of work that expands our understanding of neuropeptides, not only in beetles, but in invertebrates in general.
1. Have there been studies that compared neuropeptidomes in other insect orders, or other groups of organisms? I did not find a reference to this in the introduction. In the discussion you say that it is now the first time that neuropeptidomes were compared for species that are not closely related. This seems to imply that such studies exist for more closely related insect species. So, please add the references.
2. line 733: You state that several neuropeptides were also missing in other insect species, but it is not indicated which insect species/orders and the statement does not contain any references.
3. Figures:
- The purple branch in Figure 1 and 2 is not the same. Is there a difference?
- Figure 2: It is not stated what the asterisk stands for.
- Figure 8c: unclear what the dots mean. Not explained what the asterisk indicates.
4. How representative are the Polyphaga for the order of Coleoptera? In this analysis, there is only one species included for which a genome is available, that is not part of this sub-order. Even if this contains most of the current Coleoptera species, you might still miss important information from an evolutionary point of view.
5. There is no comparison at a level lower than infraorder. It is stated the studied species were not closely related (line 661). Is there a reason that closely related species were not included? It seems to be interesting to see whether there are significant neuropeptidome differences between these closely related species. Is it known on what time-scale gain and loss of neuropeptides occurs?
6. Loss of a receptor gene can equally affect neuropeptide signaling as loss of the neuropeptide. Why did you not look at loss of receptors?
7. It is stated that the variability in neuropeptidome composition between insect species from the same insect order may be as large as the one that exists between species from different orders. How is this variability quantified? Was this variability compared or is it a hypothesis? How about variability within an order? Can this statement be generalized from one order to another?
8. Minor comments:
- Conclusion: “insect species from from the same insect order”
- Line 677: “it might be become obsolete"
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