Brain and circulating steroids in an electric fish: Relevance for non-breeding aggression

Lucia Zubizarreta; Cecilia Jalabert; Ana C. Silva; Kiran K. Soma; Laura Quintana

doi:10.1371/journal.pone.0289461

Peer Review History

Original SubmissionJuly 18, 2023
14 Aug 2023 Decision Letter - Irfan Ahmad Bhat, Editor PONE-D-23-22559Brain and circulating steroids in an electric fish: relevance for non-breeding aggressionPLOS ONE Dear Dr. Jalabert, 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 Sep 28 2023 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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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: No ****** 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Authors have done a great job of investigating the plasma and brain steroids in electric fish and how they influences nonbreeding aggression. I have few comments Please find them below. 1. Why were the individuals collected only during non-breeding season? Authors should consider breeding aggression based on sexual differences in fishes. 2. Authors should consider this manuscript while writing particular statements https://www.jneurosci.org/content/41/44/9177 3. Authors have quantified only androgens and estrogen, why not progesterone metabolites using LCMS/MS. 4. Authors have used different internal standard “deuterated internal standards (progesterone-d9, cortisol-d4, DHEA-d6, testosterone-d5, E2- 146 d4; C/D/N Isotopes Inc., Pointe-Claire, Canada)”. Can they briefly mention which one was used for which compound and their retention time details? 5. Can you please share in detail the method development for the brain steroids? 6. Authors should combine discussion into a single heading, instead of making different sub heading. 7. Please improve the resolution of figures. Reviewer #2: The manuscript is well written. However, I have some minor queries which should be addressed. Authors should mention how neurosteroids are modulating fish behavior? Is aggression mainly attributed due to steroids only or there are some other factors as well that needs to be mentioned in the manuscript? Statistical analysis should be rewritten, it bit unclear Authors should increase the visibility of figures? ****** 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. 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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.0289461.r001
Revision 1
12 Sep 2023 Author Response Reviewer #1: Authors have done a great job of investigating the plasma and brain steroids in electric fish and how they influence nonbreeding aggression. I have a few comments. Please find them below. 1. Why were the individuals collected only during non-breeding season? Authors should consider breeding aggression based on sexual differences in fishes. Response: Our aim was specifically to study the steroid profiles present in the non-breeding season in a species that shows robust territorial aggression during this season. Non-breeding aggression is a useful behavior to understand neuroendocrine mechanisms that differ from those underlying breeding aggression (which is most commonly studied). In the non-breeding season, circulating sex steroids are often at low concentrations, offering a “clean field” to study sex steroids in the brain. We have added a mention of this in the abstract (lines 38-41). This paradigm has been key in revealing the brain as a source of steroids that regulate aggression not only in fish, but also in birds and mammals. In addition, these models strongly suggest a common role of neuroestrogens in the control of aggression during this season. Interestingly, in Gymnotus omarorum, this sexually monomorphic behavior may stem from sexually dimorphic hormonal actions, as suggested by our results. This is an interesting avenue to research, as it may shed light on how sexually-biased mechanisms may lead to the same outcomes. The study of breeding hormonal profiles and behavior is not part of this study, although it is currently an area of research in our lab and will be the focus of future publications. 2. Authors should consider this manuscript while writing particular statements https://www.jneurosci.org/content/41/44/9177 Response: Thank you for this comment. Wartenberg et al. beautifully show neurosteroidal influences during development that are ultimately related to sexual dimorphism in aromatase-expressing neurons at birth. The spatiotemporal data, obtained in ArIC/eR26-τGFP reporter mice, compares embryos to neonates to shed light on novel mechanisms of estrogenic regulation during development. In our manuscript, we state that the sex differences in hormonal profiles may be due to “...ii) sex differences in brain steroidogenic enzymes that synthesize or metabolize steroids…” (line 406), which encompasses the data in the aforementioned paper, and we have now added it to our cited references. 3. Authors have quantified only androgens and estrogen, why not progesterone metabolites using LCMS/MS. Response: Progesterone was included in our panel of steroids to be quantified by LC-MS/MS (mentioned in our original manuscript, now in lines 198 and 199). However, progesterone was not detectable in either plasma or brain samples in both sexes (stated in lines 290 and 307). We agree with the referee that the role of progestogens in aggression is very important, and it is now discussed in lines 383-389. This is the first time circulating and brain steroids have been measured in this species, and we did not include the quantification of progesterone metabolites in this first study, as acknowledged in the original manuscript (and the current one in lines 387-389). 4. Authors have used different internal standard “deuterated internal standards (progesterone-d9, cortisol-d4, DHEA-d6, testosterone-d5, E2- 146 d4; C/D/N Isotopes Inc., Pointe-Claire, Canada)”. Can they briefly mention which one was used for which compound and their retention time details? Response: Thank you for this observation. In our original manuscript, we stated retention time details for all analytes and internal standards (specified in Table 1). We have now clarified which internal standard was used for each analyte in the methods section (lines 148-150). 5. Can you please share in detail the method development for the brain steroids? We are not sure what the reviewer refers to in this point. We understand that the description of the method in the manuscript was already detailed enough to be replicated: extraction method (lines 141-170), LC-MS/MS method (lines 173-190), and validations (lines 208-218). The changes made addressing the previous point have added important details to our method description. 6. Authors should combine discussion into a single heading, instead of making different sub heading. Response: Thank you. We have carefully considered this suggestion, and we find that the three sub-headings help organize our different lines of discussion. 7. Please improve the resolution of figures. Response: We are grateful for this observation and have corrected the figures accordingly. Reviewer #2: The manuscript is well written. However, I have some minor queries which should be addressed. Authors should mention how neurosteroids are modulating fish behavior? Response: We have now included this point in the final part of the discussion. Is aggression mainly attributed due to steroids only or there are some other factors as well that needs to be mentioned in the manuscript? Response: Thank you for this comment. The neuropeptide arginine vasotocin (AVT) has a rapid status-dependent effect on non-breeding aggressive behavior, and serotonin also modulates aggression. We have added this information to the introduction (lines 94-96). Statistical analysis should be rewritten, it bit unclear. Response: We have revised the Statistics section to make it clearer. Authors should increase the visibility of figures? Response: We are grateful for this observation and have corrected the figures accordingly. Attachments Attachment Submitted filename: Zubizarreta Jalabert et al response to reviewers.docx https://doi.org/10.1371/journal.pone.0289461.r002
25 Sep 2023 Decision Letter - Irfan Ahmad Bhat, Editor Brain and circulating steroids in an electric fish: relevance for non-breeding aggression PONE-D-23-22559R1 Dear Dr. Jalabert, 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, Irfan Ahmad Bhat Academic Editor PLOS ONE Additional Editor Comments (optional):NA 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. Reviewer #1: All comments have been addressed ******** 2. 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. 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 ****** 5. 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 ****** 6. 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: The manuscript should be accepted for publication. I have no further comments for the authors. The manuscript is revised properly. ****** 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. ******** https://doi.org/10.1371/journal.pone.0289461.r003
Formally Accepted
29 Sep 2023 Acceptance Letter - Irfan Ahmad Bhat, Editor PONE-D-23-22559R1 Brain and circulating steroids in an electric fish: relevance for non-breeding aggression Dear Dr. Jalabert: 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. Irfan Ahmad Bhat Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0289461.r004

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