Electrophysiological responses to conspecific odorants in Xenopus laevis show potential for chemical signaling

Heather J. Rhodes; Melanie Amo

doi:10.1371/journal.pone.0273035

Peer Review History

Original SubmissionDecember 9, 2021
15 Mar 2022 Decision Letter - Michael Klymkowsky, Editor PONE-D-21-38952Do Xenopus laevis communicate through chemical signaling? The nose knows.PLOS ONE Dear Dr. Rhodes, Thank you for submitting your manuscript to PLOS ONE (and sorry for the delay). 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 consider and respond to the reviewers' specific comments. Please submit your revised manuscript by Apr 29 2022 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. 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The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: “We thank Heather Eisthen for assistance and training with EOG techniques as well as helpful comments on a draft of this manuscript, Katie Darrah for her work piloting EOG recordings, and members of the Grass Lab 2017 for advice and support. We also acknowledge and thank the following funding sources: The Grass Foundation, Denison University, the R.C. Good Faculty Fellowship, and the Helen L. Yeakel Summer Research Fund.” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “Funding was provided by The Grass Foundation https://grassfoundation.org/ (HJR), and Denison University https://denison.edu/, including the R.C. Good Faculty Fellowship at Denison University (HJR), and the Helen L. Yeakel Summer Research Fund at Denison University (MA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. 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? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: In their manuscript entitled "Do Xenopus laevis communicate through chemical signaling? The nose knows," Heather J. Rhodes and Melanie Amo developed an in situ electroolfactogram preparation and recorded olfactory responses in the adult water nose of Xenopus. The study is very well conducted and straightforward. The logic of the experiments is fine, the methodology is sound, the statistical analysis is well done, and the authors provide convincing evidence for all their claims. I enjoyed reading the manuscript. The results are extremely interesting for individuals working in the fields of chemical senses and (amphibian) olfaction in particular. To the best of my knowledge, this study is the first study that shows odorant responses recorded from the intact adult water nose of Xenopus laevis. The obtained results are, without a doubt, a significant advance in the relevant fields. I find myself in the unusual position of not having major complaints. I only have a few very minor suggestions and points that the authors could consider taking into consideration. Specific suggestions I'm not too fond of questions as manuscript titles. Why don't you shortly state what you did in the title? You could consider adding a figure that shows details about the in situ electroolfactogram preparation. This figure could also show the anatomy of the tripartite olfactory organ of adult Xenopus and give information about how you positioned the electrodes etc. Such a figure would be beneficial for many readers. Lines 4-5: Here, you state that the adult water nose is well placed to receive information about conspecifics. Why do you think this is the case? Line 27: What do you mean by "chemicals originating in the mouth"? Line 32: I do not like the term "fishlike" class I olfactory receptors. I think there are more elegant and up-to-date ways to name/ describe these olfactory receptors. Lines 83-89: What do you mean by "early animals"? Were the 27 male frogs already sexually mature? Also, you should add some more information about the male and female frogs from which you collected the stimuli (size, sexual maturity, etc.). Line 99: ml/m? Do you mean ml/min? Lines 114-121: You could be more precise when explaining how you placed the electrodes. But see also my suggestion to add a figure that better describes the recording of olfactory responses. Lines 124-129: You could explain why L-methionine and L-alanine are suitable control stimuli for the adult water nose. Line 228: Experiment 8? Why do you give this information? Is the number of the experiment important? The discussion section is relatively short. Consider discussing some points in more detail. You could, for instance, speculate what morphological olfactory receptor neuron types and what olfactory receptor families could be responsible for the recorded odorant responses. In the discussion section, you could compare the detection thresholds of the responses to amino acids obtained in your work with detection thresholds obtained in other studies using other methods. There is a paper (Breunig et al., 2010) where thresholds to amino acids, including L-methionine, of single Xenopus receptor neurons have been determined using the calcium imaging technique. Line 323-325: Here, a reference for your statement is missing. Reviewer #2: The authors describe olfactory responses in the water nose of Xenopus laevis to several bodily secretions of males and females using the electroolfactogram technique. Technically this appears to be a solid and carefully designed and performed study. The reported responses are an important step in understanding intraspecies olfactory communication in amphibians. However, the presentation of results and conclusions could be improved in several ways. Sometimes information is missing, misleading or incorrect. A detailed list follows: Abstract, line 7 Please include information whether the explants are from males or females (this information is given for the source of the odors). Line 13 Imprecise, this manuscript is not 'adding new layers' to understanding of 'vocal communication'. Introduction, Line 26 Please explain the term 'choana' and explain the access of liquids to the VNO better. Line 29 would be clearer to write 'through which water is actively circulated' Line 61 Mention here which of the noses (water nose, air nose) are examined. Line 62 Were female noses also examined? Methods, line 92, 'double pithing the frog' Please explain the procedure, as it is not generally known. Also 'euthanizing' means to kill, but since the heart rate is retained, this seems to be the wrong expression. 'Paralysed' might be a more correct term? Also please mention whether anesthesis is necessary/maintained during recording. You should also mention that the frog was killed after the experiment (I assume it was). Line 95 'ice was placed over the frogs body' Is that an approved method? Line 119 'with expected timing' Please specify the timing you expect here. Line 135-135 Please mention the age range of animals pooled. Are there particular (hormonal) states to be considered? Line 138 Which plastic? Different kinds might emit different contaminants. Line 144-145 This is a good control, but not optimal as one could argue that the skin secretions might dissolve plastic components that pure saline could not. Maybe include a caveat here. Line 155-157 Maybe rephrase for clarity in this way: After injection, the stimulus reached the olfactory epithelium after several seconds and somewhat diluted. Line 159 Volume of injected NaCl? Line 162-163 The sentence in parentheses should be a separate sentence and should be stated more directly: why you think the concentration at the olfactory epithelium could vary, and which direction do you expect it to vary. Line 165-166 Give the volume of stimuli. Line 176-178 Please mention whether results thus obtained are similar or different to those going from lowest to highest concentration. Line 202-203 and 205-206 Please clarify: How can you subtract a signal outside of the recorded time range? Results, line 245 This information (estimate of 5 fold dilution) should also be given in the respective Methods section. There the authors write only 'and some dilution of stimuli'. Line 252 'may not be distinguishable'? Should read 'are not distinguishable' according to the criteria set by the authors. Line 287-292 Please state here whether EOG responses to control stimuli reached significance according to your 2 sigma criteria. Discussion, line 300-301 'estimated to be 2 µM' seems a rather strong statement. Please weaken according to the actual accuracy, which which you can estimate the dilution factor. Line 302 What about female frogs? If not tested that should be stated explicitly here and before in Methods and Results. Line 324 'held in amplexus' is not a generally known term. Please explain. Line 330-331 One hopes. But is it really practical to use this EOG assay for some kind of biochemical fractionation? If the authors plan such studies that could be hinted at here. Figures 3-5 Better to label these curves animal1, animal2 or individual1 etc, not 'exp1'. Also, this description could go inside the boxes, there is lots of empty space in the upper right corner. Figure 3 Assuming that your 'exp1-6' are numbered chronologically there seems to be a very clear trend of decreasing z score with time (no such trend for the other curves). If true, please mention and explain (potential) causes for this trend. Is there a possibility it relates to season? Figures 3-5 The x-axis lettering should mention the stimulus directly, including whether it is from male or female animals. Do not just write 'stimulus' there. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Ivan Manzini Reviewer #2: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0273035.r001
Revision 1
25 May 2022 Author Response Please see Response to Reviewers.docx. (the text of which is copied below, but lacks formatting here.) Dear Editors and Reviewers, Thank you for your thoughtful feedback on our manuscript, PONE-D-21-38952. We used your feedback to revise and improve our work and hope that our revisions are to your satisfaction. We feel the manuscript is stronger for this process. Below, we offer a point-by-point response to all feedback. Journal and reviewer comments are in italics and our responses are in purple text. Please note that when we refer to line numbers in our responses, we give line numbers from the Track Changes version of the manuscript. We look forward to your response, Heather J. Rhodes and Melanie Amo 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 We have double checked style requirements and believe all is in order. 2. Thank you for stating the following in the Acknowledgments Section of your manuscript: “We thank Heather Eisthen for assistance and training with EOG techniques as well as helpful comments on a draft of this manuscript, Katie Darrah for her work piloting EOG recordings, and members of the Grass Lab 2017 for advice and support. We also acknowledge and thank the following funding sources: The Grass Foundation, Denison University, the R.C. Good Faculty Fellowship, and the Helen L. Yeakel Summer Research Fund.” We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: “Funding was provided by The Grass Foundation https://grassfoundation.org/ (HJR), and Denison University https://denison.edu/, including the R.C. Good Faculty Fellowship at Denison University (HJR), and the Helen L. Yeakel Summer Research Fund at Denison University (MA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.” Please include your amended statements within your cover letter; we will change the online submission form on your behalf. We removed funding sources from the acknowledgement section. All funding sources are represented in the finding statement. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. No changes are needed. I am prepared to provide access upon acceptance. 4. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. 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. Reference list is complete, correct, and to the best of our knowledge no sources have been retracted. Reviewers' comments: Reviewer #1: In their manuscript entitled "Do Xenopus laevis communicate through chemical signaling? The nose knows," Heather J. Rhodes and Melanie Amo developed an in situ electroolfactogram preparation and recorded olfactory responses in the adult water nose of Xenopus. The study is very well conducted and straightforward. The logic of the experiments is fine, the methodology is sound, the statistical analysis is well done, and the authors provide convincing evidence for all their claims. I enjoyed reading the manuscript. The results are extremely interesting for individuals working in the fields of chemical senses and (amphibian) olfaction in particular. To the best of my knowledge, this study is the first study that shows odorant responses recorded from the intact adult water nose of Xenopus laevis. The obtained results are, without a doubt, a significant advance in the relevant fields. I find myself in the unusual position of not having major complaints. I only have a few very minor suggestions and points that the authors could consider taking into consideration. Thank you. We are gratified that you found the study to be, for the most part, strong, valuable, and clearly reported. Specific suggestions I'm not too fond of questions as manuscript titles. Why don't you shortly state what you did in the title? I understand; question titles usually annoy me too. We altered the title to be a statement rather than a question. You could consider adding a figure that shows details about the in situ electroolfactogram preparation. This figure could also show the anatomy of the tripartite olfactory organ of adult Xenopus and give information about how you positioned the electrodes etc. Such a figure would be beneficial for many readers. We agree that such a figure would be helpful. Unfortunately, we do not have any high-quality photographs of our preparation. We developed schematics which we added as Fig 1. We hope these are useful. Lines 4-5: Here, you state that the adult water nose is well placed to receive information about conspecifics. Why do you think this is the case? We were alluding to the fact that olfaction is a good candidate for social communication. Chemical communication is common among aquatic animals. Xenopus secrete chemicals into the water through skin secretions and cloacal fluids and they sample the chemical composition of the water through the water nose. In case the specific words “well placed” were problematic or confusing, we have adjusted the wording of this sentence. We elaborate on these ideas in the third paragraph of the introduction (apx. Lines 52-62). Line 27: What do you mean by "chemicals originating in the mouth"? The source of stimuli to the adult Xenopus VNO is unclear, to the best of our knowledge. In some frogs, it seems that water passing through the nares may pass over the VNO, but given that Xenopus does not allow water to enter the principal cavity when submerged, instead directing water into the medial cavity, it seems unlikely that water from the nares would reach the VNO. Thus the choana or the nasolacrimal ducts seem likely pathways for stimuli to reach the VNO. We tried to clarify the language in the paper. Line 32: I do not like the term "fishlike" class I olfactory receptors. I think there are more elegant and up-to-date ways to name/ describe these olfactory receptors. We revised this sentence to provide more specific and complete information. Lines 83-89: What do you mean by "early animals"? Were the 27 male frogs already sexually mature? Also, you should add some more information about the male and female frogs from which you collected the stimuli (size, sexual maturity, etc.). Please see the second paragraph in the methods which begins by stating that all animals were sexually mature adults. That includes all males used for physiology, as well as males and females used for stimulus acquisition. We did not obtain specific weights and lengths of animals used for stimulus acquisition, so we can provide no further information. “Early animals” simply meant those used for early experiments as we developed our procedures. We have clarified the language. Line 99: ml/m? Do you mean ml/min? Yes – thanks for catching that! Lines 114-121: You could be more precise when explaining how you placed the electrodes. But see also my suggestion to add a figure that better describes the recording of olfactory responses. A bit of additional information was added, as well as the new Fig 1. We hope this provides the information you’re looking for. Lines 124-129: You could explain why L-methionine and L-alanine are suitable control stimuli for the adult water nose. We added information and citations to support the use of amino acids as positive controls. They have been recognized as odorants Xenopus can detect in the past. Additionally, they effectively drive odorant responses in a wide range of animals, including other frogs, salamanders, fish… even aquatic invertebrates. Line 228: Experiment 8? Why do you give this information? Is the number of the experiment important? This information was provided so that readers can see how the sample data in figure 3 (renumbered from Fig 2) fit with the summary data graphed in figures 4 and 5.(renumbered from Figs 3 and 4). Experiments on individual frogs are numbered so that a reader can examine how a single animal responds to multiple stimuli. We have moved the reference to “experiment 8” out of the figure title and to the end of the legend in the hopes that it will be less distracting to readers. The discussion section is relatively short. Consider discussing some points in more detail. You could, for instance, speculate what morphological olfactory receptor neuron types and what olfactory receptor families could be responsible for the recorded odorant responses. We have modestly expanded the discussion in our revisions, but we don’t feel a compelling need to speculate on receptor types, as we’re not sure we have new insights to offer. The candidate list for molecules in the skin secretions and cloacal fluids that could be triggering these responses is still long and open ended. We appreciate the suggestion, but think this is a topic best left for future work. In the discussion section, you could compare the detection thresholds of the responses to amino acids obtained in your work with detection thresholds obtained in other studies using other methods. There is a paper (Breunig et al., 2010) where thresholds to amino acids, including L-methionine, of single Xenopus receptor neurons have been determined using the calcium imaging technique. Thanks for pointing this paper out! We have included it in the discussion. Line 323-325: Here, a reference for your statement is missing. We added citations that further support this idea. Reviewer #2: The authors describe olfactory responses in the water nose of Xenopus laevis to several bodily secretions of males and females using the electroolfactogram technique. Technically this appears to be a solid and carefully designed and performed study. The reported responses are an important step in understanding intraspecies olfactory communication in amphibians. However, the presentation of results and conclusions could be improved in several ways. Sometimes information is missing, misleading or incorrect. A detailed list follows: Thank you. We are sorry if points were unclear but stand by our work as correct; we will attempt to clear up any misunderstandings. Abstract, line 7 Please include information whether the explants are from males or females (this information is given for the source of the odors). There are no “explants.” The preparation is in situ in that the olfactory epithelium is intact and in place, but the frog has been euthanized, so it is not in vivo. All EOG recordings were made from adult males, as is specified on lines 7-8. Language in the methods has been revised and a new Fig 1 has been added to clarify the nature of the preparation; we also state clearly that no females were used for electrophysiology. Line 13 Imprecise, this manuscript is not 'adding new layers' to understanding of 'vocal communication'. The intention was not to suggest that this paper alone adds to our understanding of vocal communication, but that continued research (as described in the previous sentence, lines 10-12) will. We have clarified the language. Introduction, Line 26 Please explain the term 'choana' and explain the access of liquids to the VNO better. The choana is the opening that connects the principal cavity of the nose with the oral cavity. A parenthetical has been added to better explain this specialized term, as well as a new Fig 1 has been added to provide a visual reference. The choana is part of the path air follows to the lungs. This opening may also provide a path for non-volatile chemicals to reach the VNO, which sits in the bottom of the principal cavity, adjacent to the choana. It is also possible that the nasolacrimal duct provides a path for fluid and stimuli to reach the VNO. In some frogs, it seems that water passing through the nares may pass over the VNO, but given that Xenopus does not allow water to enter the principal cavity when submerged, instead directing water into the medial cavity, it seems unlikely that water from the nares would reach the VNO. This anatomy is complex, we know. We hop the new figure will help clarify. Line 29 would be clearer to write 'through which water is actively circulated' Change made – thank you. Line 61 Mention here which of the noses (water nose, air nose) are examined. Done. It was the water nose – thank you. Line 62 Were female noses also examined? No. This was restated in the methods to clarify. Methods, line 92, 'double pithing the frog' Please explain the procedure, as it is not generally known. Also 'euthanizing' means to kill, but since the heart rate is retained, this seems to be the wrong expression. 'Paralysed' might be a more correct term? Also please mention whether anesthesis is necessary/maintained during recording. You should also mention that the frog was killed after the experiment (I assume it was). Pithing is an accepted form of euthanasia for many animals, including anuran amphibians. Double pithing a frog involves the destruction of all central nervous system structures with a probe inserted through the foramen magnum into the skull, and then into the spinal cord. The probe is moved in a circular motion to destroy all brain and spinal cord structures and nerve connections. We have added a citation from the American Veterinary Medical Association to support this form of euthanasia as well as a short explanation of pithing in the subsequent sentence. We also attempted to clarify the final sentence in the paragraph. We are happy to provide further information, although it feels like it may take the paper off track to explain amphibian biology at length. Once the frog is pithed, it is brain dead and the animal no longer breathes. But the tissues of these animals are fairly hypoxia tolerant and some carbon dioxide can be exchanged through the skin, preventing hypercapnia. The myogenic heart may continue to beat for a couple of hours after brain death. It also continues to beat for hours after decapitation and can even continue to beat for a considerable time if the heart is fully removed from the body. Thus, the heart is not a good indicator of death in the frog. Brain death, from the total destruction of the central nervous system, is a better criterion. After pithing, the frog is dead, even if particular cells and tissues remain functional. Line 95 'ice was placed over the frogs body' Is that an approved method? All procedures were approved by IACUC as written. Ice is generally not allowed in the place of sedation or anesthesia, but here anesthesia was achieved by an injectable sodium channel blocker (MS222) first. Ice was utilized to decrease metabolic rate in the tissues with the intent of extending the time over which the preparation would remain viable. Line 119 'with expected timing' Please specify the timing you expect here. A clarification was added. Line 135-135 Please mention the age range of animals pooled. Are there particular (hormonal) states to be considered? They were all adults, sexually mature, and were not part of any current breeding programs, but beyond that we do not have any specific information. We added a sentence to clarify what we do and don’t know. Line 138 Which plastic? Different kinds might emit different contaminants. Sandwich bags are made of low-density polyethylene. We have added this information to the paper. Line 144-145 This is a good control, but not optimal as one could argue that the skin secretions might dissolve plastic components that pure saline could not. Maybe include a caveat here. This is possible, but it seems far more likely that the many water-soluble compounds in the skin secretions are what is being detected by the animal. We think this was an appropriate control, clearly explained. We are satisfied that a reader, such as yourself, may wonder about the possibility of dissolved plastics, but we don’t think a caveat is needed. Line 155-157 Maybe rephrase for clarity in this way: After injection, the stimulus reached the olfactory epithelium after several seconds and somewhat diluted. Thanks for the suggestion – it was implemented. Line 159 Volume of injected NaCl? 50µl – the same as a stimulus. This was added to the text. Line 162-163 The sentence in parentheses should be a separate sentence and should be stated more directly: why you think the concentration at the olfactory epithelium could vary, and which direction do you expect it to vary. We really can’t say. We were collecting drops of water from the perfusion system, then testing that later on an osmometer. That could have acted like a smoothing function on the concentration over time, removing peaks or valleys. In the preparation, perfusate is allowed to flow freely out of the nasal cavity, but there could be some slight pooling or mixing of liquid on the epithelium that could further alter concentration on the surface over time. The EOG responses themselves also reflect relatively slow and long-lasting receptor potentials, suggesting integration over time in the olfactory epithelium. Thus, even if the concentration delivered is higher or lower for short periods of time (in ways we were unable to measure) the olfactory epithelium itself could act as a smoothing function. Our approach was to get a sense of the timing and dilution and report that transparently to the best of our ability. We have changed these sentences to ensure the information is clear without offering supposition. Line 165-166 Give the volume of stimuli. This is reported in the first line of the paragraph that starts “To deliver a stimulus…” which is now line 175. It was also added on line 181 for further clarity. Line 176-178 Please mention whether results thus obtained are similar or different to those going from lowest to highest concentration. This has been added to the manuscript. We saw no differences. Line 202-203 and 205-206 Please clarify: How can you subtract a signal outside of the recorded time range? Recording was continuous for the entire length of a stimulus block (so a single recording file might be 40+ minutes long). Note on line 127-128 it says that data was “continuously recorded.” Results, line 245 This information (estimate of 5 fold dilution) should also be given in the respective Methods section. There the authors write only 'and some dilution of stimuli'. The 5-fold (or 1:5) dilution is described in the methods section. See specifically lines 182-190. Line 252 'may not be distinguishable'? Should read 'are not distinguishable' according to the criteria set by the authors. We do not claim to know whether stimuli are or are not detectable by the animals. Behavioral experiments would be needed to determine that. We carefully stated at the end of the methods (now lines 247-249) that “z ≥ 2 was set to represent responses that are likely detectable as different from control, since such responses would be greater in amplitude than 97.7% of control responses.” Saying stimuli beyond that threshold are “likely detectable” does not mean stimuli below that threshold are not detectable. Thus, the criteria we set are consistent with the language in the figure legend: “light gray horizontal band from z = -2 to z = 2 indicates responses that may not be distinguishable from saline control.” Line 287-292 Please state here whether EOG responses to control stimuli reached significance according to your 2 sigma criteria. It was the standard deviation of these control stimuli that was used in the z-score calculation. This was stated on lines 240-246 of the methods, but we clarified in the results on lines 270-272. Discussion, line 300-301 'estimated to be 2 µM' seems a rather strong statement. Please weaken according to the actual accuracy, which which you can estimate the dilution factor. This statement has been modified accordingly. Line 302 What about female frogs? If not tested that should be stated explicitly here and before in Methods and Results. A statement was added to the methods to make it clear that only males were used for EOG experiments. In both the introduction (lines 38-61) and the discussion (lines 345 to the end) we provide a description of the various vocal and clasping behaviors that males engage in, and use differentially in the presence of other males or females. Thus, we had reason to look at male EOG responses because we are interested in what sensory cues males use to select appropriate behaviors. Line 324 'held in amplexus' is not a generally known term. Please explain. This is the reproductive clasping position used by anuran amphibians. In the case of Xenopus, the male’s head is on the female’s back, with his forelimbs clasped around her abdomen. In this position, the female lays eggs and the male releases sperm for external fertilization. Males also hold other males in amplexus, likely as an alternative reproductive tactic to gain proximity to a female and engage in sperm competition. We added a brief explanation of the term and citations to support. Line 330-331 One hopes. But is it really practical to use this EOG assay for some kind of biochemical fractionation? If the authors plan such studies that could be hinted at here. It has indeed been done in other species and we hope it will be in Xenopus, by us or others. I have elaborated on these ideas and added citations. Figures 3-5 Better to label these curves animal1, animal2 or individual1 etc, not 'exp1'. Also, this description could go inside the boxes, there is lots of empty space in the upper right corner. We have changed the labels to “Frog#” instead of “exp#” and moved the keys into the space of the graphs. Figure 3 Assuming that your 'exp1-6' are numbered chronologically there seems to be a very clear trend of decreasing z score with time (no such trend for the other curves). If true, please mention and explain (potential) causes for this trend. Is there a possibility it relates to season? We think this was just due to random chance. As you note, there is not a relationship between z-scores and experiment order for other stimuli. All of the data reported here was collected within a one-month time period mid-July to mid-August and animals were housed under tightly controlled conditions, making seasonal change unlikely. Figures 3-5 The x-axis lettering should mention the stimulus directly, including whether it is from male or female animals. Do not just write 'stimulus' there. We have revised the titles and labels on the x-axes of all graphs. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0273035.r002
18 Jul 2022 Decision Letter - Michael Klymkowsky, Editor PONE-D-21-38952R1Electrophysiological responses to conspecific odorants in Xenopus laevis show potential for chemical signaling.PLOS ONE Dear Dr. Rhodes, 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 address review #2's minor comments and return the revised manuscript, no further review will be necessary Please submit your revised manuscript by Sep 01 2022 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. 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If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. 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. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. 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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: The manuscript has much improved in the revision, in particular the readability for a larger audience. The addition of a schematic figure is welcome. Minor points: Figure 1: the opening of the nose (naris) is a white oval in panel A, but a black dot in panel B. Better use the white oval also in panel B, this has the advantage of not having to distinguish between small and large black dots. Figure 1: The drawing looks like the VNO is accessed from the MC. Can you make the drawing so that VNO looks connected to PC, not to MC? Line 36 „ciliated and microvillous receptor neurons expressing class I OR1, OR2, and V1R receptors“ I think you mean class I and class II ORs, not ' class I OR1, OR2'. Line 135-136, „a characteristic negative deflection lasting 2-3 seconds with expected timing based on the perfusion and stimulus deliver system described below)“ Unclear. Better say: „a characteristic negative deflection lasting 2-3 seconds, which is the expected timing based on the perfusion and stimulus deliver system described below)“ ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0273035.r003
Revision 2
31 Jul 2022 Author Response Minor modifications were made to figure one in accordance with the suggestions of reviewer 2. Reviewer 2 also asked for two minor modifications to the text; in both cases the reviewer's suggestions would have changed the meaning of the text and made it inaccurate. Instead, we attempted to clarify the text in light of the reviewer's misunderstanding. We hope the editor is happy with these modifications. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0273035.r004
2 Aug 2022 Decision Letter - Michael Klymkowsky, Editor Electrophysiological responses to conspecific odorants in Xenopus laevis show potential for chemical signaling. PONE-D-21-38952R2 Dear Dr. Rhodes, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Michael Klymkowsky, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0273035.r005
Formally Accepted
26 Aug 2022 Acceptance Letter - Michael Klymkowsky, Editor PONE-D-21-38952R2 Electrophysiological responses to conspecific odorants in Xenopus laevis show potential for chemical signaling. Dear Dr. Rhodes: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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. If we can help with anything else, please email us at plosone@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. Michael Klymkowsky Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0273035.r006

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