Characterization and deorphanization of RYamide signaling in Aedes aegypti: A potential regulator of hindgut-associated physiology

Jinghan Tan; Thomas Luong; Jean-Paul V. Paluzzi

doi:10.1371/journal.pone.0342341

Peer Review History

Original SubmissionOctober 14, 2025
30 Nov 2025 Decision Letter - Pedro Oliveira, Editor Dear Dr. Paluzzi, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jan 14 2026 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. 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We look forward to receiving your revised manuscript. Kind regards, Pedro L. Oliveira Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. 3. 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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? -->?> Reviewer #1: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available??> The PLOS Data policy Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English??> Reviewer #1: Yes ****** Reviewer #1: The manuscript aims to deorphanize two putative GPCRs from the mosquito Aedes aegypti. The manuscript is well written, but the introduction could be shortened a little bit. Also in the results section the authors present some information about the literature that should be moved to the discussion (line 438). The possible link to the hindgut physiology is based on the higher staining for the Ryamide in that region. There is no additional evidence to show that the head is the source and the hindgut a target. I would suggest removing that statement from the title because the only evidence of this biological role is a positive immunostaining. More evidence is needed. Is it possible to test the expression of the putative receptors in the different tissues, particularly in the hindgut? That would add more evidence to the hypothesis of the head being the source (RYamide staining) and the hindgut a target by having a high expression of the receptors. The immunofluorescence images are very informative however the contrast is not very good. It is hard to visualize the tissue overall structure. Do the authors have any morphological staining for the general structure of the tissue? If not, it would be good to have an outline of the tissue or a better contrast of the pictures. Minor comments - Please add titles to your graphs. For example, in figure 1 – Ryamide expression – Males. Please add to all graphs. ****** what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] To ensure your figures meet our technical requirements, please review our figure guidelines: https://journals.plos.org/plosone/s/figures You may also use PLOS’s free figure tool, NAAS, to help you prepare publication quality figures: https://journals.plos.org/plosone/s/figures#loc-tools-for-figure-preparation. NAAS will assess whether your figures meet our technical requirements by comparing each figure against our figure specifications. https://doi.org/10.1371/journal.pone.0342341.r001
Revision 1
2 Jan 2026 Author Response We have appended the detailed response to reviewer comments with our revised manuscript below. However, please see the attached detailed reviewer responses that have our comments in blue coloured text. ******************* Response to reviewer comments/suggestions (in blue font): Reviewer #1: The manuscript aims to deorphanize two putative GPCRs from the mosquito Aedes aegypti. The manuscript is well written, but the introduction could be shortened a little bit. Also in the results section the authors present some information about the literature that should be moved to the discussion (line 438). We thank the reviewer for their positive comments. We have revised the introduction by removing some details from the literature making this section more concise. The last few sentences in the results section have also been revised, as requested. The possible link to the hindgut physiology is based on the higher staining for the Ryamide in that region. There is no additional evidence to show that the head is the source and the hindgut a target. I would suggest removing that statement from the title because the only evidence of this biological role is a positive immunostaining. More evidence is needed. We thank the reviewer for this suggestion. As we mentioned in discussion (lines 602–605), based on available RNA-seq datasets (cited in our manuscript and see Supplementary Fig. S3) and a recent preprint study (Greppi et al., 2025), RYaR2 that is now confirmed as an authentic RYamide receptor, is highly enriched in the adult mosquito hindgut. More specifically, RYaR2 is expressed exclusively in rectal pad cells (Greppi et al., 2025), which is an important site of ion transport. These findings strongly support that there is a potential RYamide and RYaR2 signaling pathway associated with the hindgut. Furthermore, we demonstrate that RYamide is secreted by two neurons in the terminal abdominal ganglia, with axonal projections extending toward the rectal pads. This observation further supports a potential role for RYamide in regulating hindgut-related functions, as the terminal abdominal ganglia have been reported to directly innervate ion transporting regions in the insect hindgut (Cook et al., 1991; Van Handek and Klowden, 1996). Is it possible to test the expression of the putative receptors in the different tissues, particularly in the hindgut? That would add more evidence to the hypothesis of the head being the source (RYamide staining) and the hindgut a target by having a high expression of the receptors. We thank the reviewer for this valuable suggestion. The RYaR2 gene is intronless, making it very difficult to accurately assess transcript abundance using qPCR without interference from genomic DNA. We had designed and tried several primer sets but could not reliably quantify transcript expression for this gene (data not shown). Publicly available RNA-seq data indicate that RYaR1 is highly enriched in the head, whereas RYaR2 is expressed almost exclusively in the hindgut of adult mosquitoes (see Supplementary Fig. S3; Hixson et al., 2022). We do have simple RT-PCR (not quantitative) data/results for both receptors but felt the available RNA-seq data (in Fig. S3) is more appropriate. The immunofluorescence images are very informative however the contrast is not very good. It is hard to visualize the tissue overall structure. Do the authors have any morphological staining for the general structure of the tissue? If not, it would be good to have an outline of the tissue or a better contrast of the pictures. We thank the reviewer for this feedback. Images have been revised as requested, with the gut, abdominal ganglion, and brain tissues all outlined (specifically in panels with preparations difficult to visualize like Fig. 4a and all panels in Fig. 5). Minor comments - Please add titles to your graphs. For example, in figure 1 – Ryamide expression – Males. Please add to all graphs. We thank the reviewer for this feedback. All figure captions, including figure title sentences, are provided at the end of the manuscript (line 942-1032). ******************* Attachments Attachment Submitted filename: Tan et al_Responses to reviewers.docx https://doi.org/10.1371/journal.pone.0342341.r002
21 Jan 2026 Decision Letter - Pedro Oliveira, Editor Characterization and deorphanization of RYamide signaling in Aedes aegypti: a potential regulator of hindgut-associated physiology PONE-D-25-55793R1 Dear Dr. Paluzzi, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. For questions related to billing, please contact billing support . If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Pedro L. Oliveira Academic Editor PLOS One Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0342341.r003
Formally Accepted
Acceptance Letter - Pedro Oliveira, Editor PONE-D-25-55793R1 PLOS One Dear Dr. Paluzzi, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS One. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. You will receive an invoice from PLOS for your publication fee after your manuscript has reached the completed accept phase. If you receive an email requesting payment before acceptance or for any other service, this may be a phishing scheme. Learn how to identify phishing emails and protect your accounts at https://explore.plos.org/phishing. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Pedro L. Oliveira Academic Editor PLOS One https://doi.org/10.1371/journal.pone.0342341.r004

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