Aging in zebrafish is associated with reduced locomotor activity and strain dependent changes in bottom dwelling and thigmotaxis

Jacob Hudock; Justin W. Kenney

doi:10.1371/journal.pone.0300227

Peer Review History

Original SubmissionNovember 9, 2023
2 Jan 2024 Decision Letter - Manabu Sakakibara, Editor PONE-D-23-37201Aging in zebrafish is associated with reduced locomotor activity and strain-dependent changes in predator avoidance behaviors related to anxiety.PLOS ONE Dear Dr. Kenney, 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. Two experts in the field have carefully reviewed the manuscript entitled, "Aging in zebrafish is associated with reduced locomotor activity and strain-dependent changes in predator avoidance behaviors related to anxiety."　Their comments are appended below. The first reviewer acknowledged the manuscript is fairly well written, however, the second one points out several serious critiques which preclude its acceptance as it stands. This Academic Editor strongly suggests that the authors would rewrite according to the comments and looks forwards to receiving the necessary replies to each concerns and the revised manuscript. Please submit your revised manuscript by Feb 16 2024 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. Please include the following items when submitting your revised manuscript: 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'. A marked-up copy of your manuscript that highlights changes made to the original version. 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Currently, your Funding Statement reads as follows: NIGMS R35GM142566 Please include your amended statements within your cover letter; we will change the online submission form on your behalf. 7. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. [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: This paper presents a highly intriguing study and unveils some fascinating findings. The authors investigated on zebrafish behaviors in the novel tank test, with a specific focus on the impact of age and fish strain. The results revealed that even slight differences in age lead to reduced locomotor activity, while strain-dependent variations were observed in predator avoidance behaviors associated with anxiety. Older TL fish exhibited increased bottom dwelling tendencies, whereas older TU fish displayed decreased thigmotaxis. Overall, this paper was well organized and written. I have only several minor comments as below. 1. I am uncertain about PLOS ONE's specific requirements regarding manuscript line numbers. However, it is common for journals to request authors to include line numbers in their submissions as this can aid the review process. 2. The sentences, such as "The distance from the bottom of the tank is considered a predator avoidance response, which has been extensively investigated in zebrafish (Gerlai, 2010; Kalueff et al., 2013)," and "This behavior has also been interpreted as 'anxiety-like,' where spending more time near the bottom of the tank indicates higher levels of anxiety (Maximino et al., 2010) (page 4, first paragraph in the results section)," were not actually presented as interpretations of the results. Therefore, I would suggest that these sentences be included in the discussion section. 3. “Thigmotaxis (i.e. time spent near the periphery) of a tank is also commonly interpreted as a predator avoidance response in zebrafish, although this interpretation has recently come under question”. What is the problem with use of thigmotaxis as an indicator for predator avoidance response? It is necessary to clarify why this approach is still employed in this study despite its questionable reliability. Reviewer #2: Behavioral changes induced by aging is an interesting topic, and the specific associations remain unexplored. It is essential that the authors systematically assessed a wide range of behavioral changes in zebrafish of different strains, sexes, and ages, by using zebrafish as a model organism. However, in my opinion, this work lacks a deep investigation of the molecular mechanisms involved. Therefore, I suggest that this manuscript needs major revisions before it can be accepted for publication. Specific comments follow: 1. Continuous line numbers in manuscripts are necessary. 2. Abstract. The fact that the zebrafish may be a useful model organism does not seem to be attributable to the fact that, like humans, it also ages, so please rewrite. 3. Please add the information on behavioral changes in zebrafish of different sexes in the Abstract. 4. The significance labeling in all the figures is not explicit enough, please replace the significance labeling with a clearer one. 5. In general, the authors only compare phenotypic behavioral differences between zebrafish of different ages, sexes, and strains, and I'm more interested in the specific mechanisms involved. 6. Neurobehavioral changes are a hot topic today, but the publications cited throughout the manuscript are too old, so please update the citations. 7. Information on the level of dissolved oxygen and hardness in water is necessary. 8. The author mentions that the sex of the fish is finally determined on the basis of the presence or absence of eggs after dissection, which in my opinion is misleading to the reader. Please delete it. 9. The authors are requested to provide a detailed description of how each behavioral indicator is assessed. 10. The authors should provide details on the origin of the water used for the behavioral tests, as changes in water quality can seriously affect the locomotor behavior of zebrafish. 11. Overall, this work systematically evaluates behavioral indicators in zebrafish of different strains, sexes, and ages. However, so far, it seems to me that this work does not highlight the need for zebrafish as a test subject. In other words, zebrafish are widely used by scholars because of their well-defined and highly homologous genome and conserved disease signaling pathways, but this work does not explore in depth the molecular mechanisms involved in the behavioral changes. I think this section will be of interest to future readers. 12. The discussion section lacks depth, it is more of a summary of the published paper, please rewrite. ****** 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: 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. 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Revision 1
12 Feb 2024 Author Response Dear Dr. Manabu Sakakibara, Thank you for taking the time to serve as editor for our recently submitted manuscript, “Aging in zebrafish is associated with reduced locomotor activity and strain-dependent changes in predator avoidance behaviors related to anxiety” at PLoS One. We were pleased to learn that the reviewers found our work “highly intriguing”, “well organized”, and an “interesting topic”. Below, we have described how we addressed reviewer comments. Comments are in bold with our responses in italics. In the modified manuscript file, changes made are in red. I would also like to note that we made a modest change to the title of the manuscript so that it is more descriptive. It is now: “Aging in zebrafish is associated with reduced locomotor activity and strain-dependent changes in bottom dwelling and thigmotaxis.” Reviewer #1 I am uncertain about PLOS ONE's specific requirements regarding manuscript line numbers. However, it is common for journals to request authors to include line numbers in their submissions as this can aid the review process. We apologize for the oversight of not including line numbers. They have now been included. The sentences, such as “The distance from the bottom of the tank is considered a predator avoidance response, which has been extensively investigated in zebrafish (Gerlai, 2010; Kalueff et al., 2013),” and “This behavior has also been interpreted as ‘anxiety-like,’ where spending more time near the bottom of the tank indicates higher levels of anxiety (Maximino et al., 2010) (page 4, first paragraph in the results section),” were not actually presented as interpretations of the results. Therefore, I would suggest that these sentences be included in the discussion section. We discuss the interpretation of our data in the context of anxiety in the second paragraph of the discussion (lines 309-322). “Thigmotaxis (i.e. time spent near the periphery) of a tank is also commonly interpreted as a predator avoidance response in zebrafish, although this interpretation has recently come under question”. What is the problem with use of thigmotaxis as an indicator for predator avoidance response? It is necessary to clarify why this approach is still employed in this study despite its questionable reliability. We expand on this point in more depth in the discussion (lines 323-345). After this sentence, we have now added a parenthetical reference to the discussion (line 127). Briefly, in the third paragraph of the discussion we lay out the challenges with the interpretation of thigmotaxis. The issue is that thigmotaxis does not correlate well with other more well-established markers of predator avoidance or anxiety (e.g., freezing or bottom dwelling). Thigmotaxis also does not change within session as would be expected of an anxiety-like/predator avoidance behavior (and how bottom dwelling often does). At the end of the paragraph, we also discuss a novel interpretation of the thigmotaxis data, i.e., that genetic background, perhaps through the natural history of the strain, may mean that some strains of fish express predator avoidance through thigmotaxis, but others do not (lines 339-345). Reviewer #2 Continuous line numbers in manuscripts are necessary. This has now been included. We apologize for the oversight. Abstract. The fact that the zebrafish may be a useful model organism does not seem to be attributable to the fact that, like humans, it also ages, so please rewrite. The abstract indicates that zebrafish may be a useful model for human aging because they age gradually, not simply because they age. This contrasts with the very slow to negligible aging in other animals of indeterminate growth (e.g. many reptiles and sea urchins). To make this point more prominent, we have added a couple of lines to the beginning of the introduction (lines 57-59). Please add the information on behavioral changes in zebrafish of different sexes in the Abstract. This has now been included in the abstract The significance labeling in all the figures is not explicit enough, please replace the significance labeling with a clearer one. We agree with the reviewer that the significance labeling for the results of the ANOVAs was not particularly clear. To improve the labelling, we now only refer to the ANOVA results in the text, and the figures only include the results of post-hoc tests within sex that we performed after significant ANOVAs or the one-sample t-tests. We have also updated the text accordingly. We have also updated the conclusions and abstract to match the more refined results of the post-hoc tests. We appreciate the suggestion as it has enhanced the readability and interpretation of the results. In general, the authors only compare phenotypic behavioral differences between zebrafish of different ages, sexes, and strains, and I'm more interested in the specific mechanisms involved. Identifying the underlying mechanisms are of interest to us as well. However, we do not have the resources at the moment to do this research. Nonetheless, we believe our behavioral findings are worth reporting as they may spur others to use zebrafish as a model to uncover the molecular mechanisms that underlie the effects of aging on behavior. Neurobehavioral changes are a hot topic today, but the publications cited throughout the manuscript are too old, so please update the citations. We have added some updated citations (lines 314-315, 318, 348, 351). Nonetheless, we kept many of the older citations because we believe it is important to give credit to those who first made a discovery. Information on the level of dissolved oxygen and hardness in water is necessary. This has now been added to the methods section (lines 429-430). The author mentions that the sex of the fish is finally determined on the basis of the presence or absence of eggs after dissection, which in my opinion is misleading to the reader. Please delete it. This line has now been removed. The authors are requested to provide a detailed description of how each behavioral indicator is assessed. This has now been included on lines 457-469 The authors should provide details on the origin of the water used for the behavioral tests, as changes in water quality can seriously affect the locomotor behavior of zebrafish. We have now included this in the methods section (lines 431-434). Briefly, water from the city of Detroit was filtered through a carbon filter and water softener before being reverse osmosis filtered. Sea salts and sodium bicarbonate are then added to the water to maintain the pH and salinity at the desired levels. Overall, this work systematically evaluates behavioral indicators in zebrafish of different strains, sexes, and ages. However, so far, it seems to me that this work does not highlight the need for zebrafish as a test subject. In other words, zebrafish are widely used by scholars because of their well-defined and highly homologous genome and conserved disease signaling pathways, but this work does not explore in depth the molecular mechanisms involved in the behavioral changes. I think this section will be of interest to future readers. The discussion section lacks depth, it is more of a summary of the published paper, please rewrite. In our discussion we try not to speculate too much beyond what our data supports. However, we do offer some novel ideas, such as the discussion on thigmotaxis (lines 323-345) where we suggest that whether thigmotaxis indicates an anxiety-like state in zebrafish may depend on the background strain of the animals. We also suggest that sarcopenia (i.e., muscle loss) may contribute to the changes in locomotor activity we observe (lines 354-362). Nonetheless, we added a paragraph on the potential role that telomere biology may play in the interplay between sex and aging we observed (lines 362-383). Sincerely, Justin W. Kenney & Jacob Hudock https://doi.org/10.1371/journal.pone.0300227.r002
26 Feb 2024 Decision Letter - Manabu Sakakibara, Editor Aging in zebrafish is associated with reduced locomotor activity and strain dependent changes in bottom dwelling and thigmotaxis PONE-D-23-37201R1 Dear Dr. Kenney, 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, Manabu Sakakibara, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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: All of the issues I concerned have been well addressed. The manuscript has been improved a lot, and I have no further suggestion. ****** 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 ******** https://doi.org/10.1371/journal.pone.0300227.r003
Formally Accepted
17 Mar 2024 Acceptance Letter - Manabu Sakakibara, Editor PONE-D-23-37201R1 PLOS ONE Dear Dr. Kenney, 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 If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks 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. 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. Manabu Sakakibara Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0300227.r004

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