“Classifying-together” phenomenon in fish (Xenotoca eiseni): Simultaneous exposure to visual stimuli impairs subsequent discrimination learning

Valeria Anna Sovrano; Greta Baratti; Davide Potrich; Tania Rosà; Veronica Mazza

doi:10.1371/journal.pone.0272773

Peer Review History

Original SubmissionMarch 28, 2022
7 Jun 2022 Decision Letter - Livia D'Angelo, Editor PONE-D-22-09159“Classifying-together” phenomenon in fish (Xenotoca eiseni): Simultaneous exposure to visual stimuli impairs subsequent discrimination learningPLOS ONE Dear Dr. Sovrano, 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 Jul 22 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. 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. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Livia D'Angelo 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. 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. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 5. Please include a separate caption for each figure in your manuscript. Please include a separate caption for each figure in your manuscript. 6. 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: No Reviewer #2: No ****** 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 clever study investigates the “classifying-together” or “Bateson effect” in a teleost fish, Xenotoca eiseni. This is a well-written paper with persuasive findings. The multiple experiments and controls are well thought-out to rule out possible alternatives. The findings were novel and suitable for publication in PlosOne, thus I have very limited queries. - The learning criterion was set at least 70% of correct trials. How was it established? - The authors stated that “the exposure-phase lasted at least 50 days”; since the exposure phase in this kind of experiment is a relevant variable, I wonder whether the authors can specify the duration of the exposure. Moreover, I wonder whether a longer duration of the exposure-phase might influence the performance of the fish. - In some conditions, the number of subjects seems small (n=7; e.g. lines 537-540). Can the authors justify their use of parametric statistics? - Specify how the sample size was calculated. Reviewer #2: The authors have conducted an extensive and clever set of experiments to investigate the phenomenon known as the "Bateson-effect" in a behavioral model that has been used in previous cognitive studies, the Redtail splitfin. The work done disentangles some of the uncertainty seen with studies of this phenomenon in other species, and also provides original evidence for this "classifying-together effect" in the teleost species, previously seen before only in higher order species. Also the interesting results found regarding differences in visual discrimination abilities for different shape stimuli presented under incongruence (easier discrimination of cross vs. circle) are discussed in detail leading in to discussion of potential differences seen in this body of work vs. the literature on the "Bateson-effect"/"classifying-together effect" in other species. The analysis and interpretation of the results are sound and thorough. Overall I think this research is insightful and contributes novel evidence in the field which will be of interest to a wide audience, therefore I recommend publication. I would point out however, a few changes that I think need to be made before publication. 1. The main issue I have with the manuscript is that the general quality and clarity of writing needs improvement. Some of the phrasing is odd and can result in some confusion at what the author is trying to say. Also there are some typos (or maybe just odd word choice). 2. I would maybe choose to refer to the stimuli couples as "pairs" or "coupled" rather than 'a couple of' as you use in the manuscript. This is as this is more commonly used when referring to coupled stimuli in literature on visual discrimination. 3. There is a fair amount of methodical information at the end of the introduction which is not necessary to lead into your methods section. You really just want to give an overview of what the set of experiments is investigating rather than a whole lot of detail on the experiments as you are about to give that in the following methods section. 4. In line 128-129 you need a reference for the statement regarding male motivation to join female conspecifics. 5. When describing the stimuli pairs you say that they are balanced for perimeter (disc and amputated disc) and area (disc and cross) however both of these statements are not true. The values are different between the stimuli so you need to correct the statement. 6. The use of 'cozy' to describe the environment throughout is slightly anthropomorphizing, familiar or comfortable may be better. 7. There is a 10-second holding cylinder protocol at the start of the trials however it is not clear what this is for? Is it to view both stimuli? If this is the case can you be sure that the fish will have seen both in such a short period of time? If it is for another reason this needs to be stated. 8. The figures: Figures 6-9 would be better of being just two figure panels of 4 graphs (6a,7a,8a,9a then 6b,7b,8b,9b) to improve data visualization. There also don't seem to be any figure legends in the document I have been given. This makes the figures pretty useless as they should stand on their own without the manuscript text. ****** 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: Yes: Eva Sheardown ******** [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.0272773.r001
Revision 1
25 Jul 2022 Author Response Reviewer #1: This clever study investigates the “classifying-together” or “Bateson effect” in a teleost fish, Xenotoca eiseni. This is a well-written paper with persuasive findings. The multiple experiments and controls are well thought-out to rule out possible alternatives. The findings were novel and suitable for publication in PlosOne, thus I have very limited queries. We were happy to read that our MS received positive feedback from the Reviewer. We thank the Reviewer for the useful comments and suggestions, which improved the quality of our work. - The learning criterion was set at least 70% of correct trials. How was it established? The ≥ 70% criterion is based: (1) on the choice already made in similar discriminative learning experiments, to maintain procedural continuity and have the opportunity to draw comparisons (for example, see the most recent publication in this same Journal: Sovrano et al., 2022, “Visual discrimination and amodal completion in zebrafish”, PLoS One, 17(3): e0264127 https://doi.org/10.1371/journal. pone.0264127); (2) on the rationale that, having to choose between two options, the chance level is 50%, so 70% for one of the two options represents a consistent deviation from random behavioral choices. Moreover, the probability of performing 70% of correct choices maintained for two consecutive days was statistically different from a chance level: binomial test for first choices p < 0.03; binomial test for total choices p< 0.001. The joint use of these two dependent variables (first choices and total choices ≥70%) might be sensitive to different aspects of the animals’ performance (e.g. the total choices could be more sensitive to show within-session learning events.) We now specify how the learning criterion was established in the text. - The authors stated that “the exposure-phase lasted at least 50 days”; since the exposure phase in this kind of experiment is a relevant variable, I wonder whether the authors can specify the duration of the exposure. Moreover, I wonder whether a longer duration of the exposure-phase might influence the performance of the fish. All the fish were hosted in the rearing/exposure tank with the stimuli and other conspecifics for 50 days. Then, the fish continued to be exposed to those stimuli even during the rest time between each daily session of the discriminative learning task; in this case, the fish were kept separated in individual spaces, so that we could recognize each fish and continue its personal training. We have hopefully improved the description of this section by better differentiating the exposure types (in group and individually) during the learning-phase, and by deleting the word "almost" to avoid confusion in the reader. - In some conditions, the number of subjects seems small (n=7; e.g. lines 537-540). Can the authors justify their use of parametric statistics? We considered 7 animals for each experimental condition in both discriminative learning experiments and in the baseline condition (not only for the baseline, lines 537-540). We have already indicated this in the Procedure section. The sample size satisfies the ethical principle of “Reduction”, according to which it is acceptable to use the smallest number of animals leading to statistically valid results. Since the number of subjects used for each experimental condition has been extensively described in the Procedure, we have now deleted the single two values in the Results, to match the format of all the sub-paragraphs and to avoid confusion or doubts in the reader. We used parametric statistics when the dependent variables could assume values from zero to infinite, such as the number of overall choices in each corridor, the number of trials to learn the discrimination, the time duration (in seconds) of each trial and their total amount. All the range values of these variables can be represented on a scale of greater complexity (such as the ratio scale, which also assumes an absolute zero). - Specify how the sample size was calculated. The sample size used here, in each experimental condition (N=7), was similar to the one used in studies with the same kind of tasks and power analyses already available in literature among fish species (for an example see [18-25]). The sample size was even approved by the Italian official bodies responsible for animal welfare, i.e., the Ethics Committee of the University of Trento and the National Ministry of Health, and it cannot exceed that declared in the approved official documents due to strict ethical reasons. We have now mentioned this crucial aspect in the text, and we thank the Reviewer for raising this issue. Reviewer #2: The authors have conducted an extensive and clever set of experiments to investigate the phenomenon known as the "Bateson-effect" in a behavioral model that has been used in previous cognitive studies, the Redtail splitfin. The work done disentangles some of the uncertainty seen with studies of this phenomenon in other species, and also provides original evidence for this "classifying-together effect" in the teleost species, previously seen before only in higher order species. Also the interesting results found regarding differences in visual discrimination abilities for different shape stimuli presented under incongruence (easier discrimination of cross vs. circle) are discussed in detail leading in to discussion of potential differences seen in this body of work vs. the literature on the "Bateson-effect"/"classifying-together effect" in other species. The analysis and interpretation of the results are sound and thorough. Overall I think this research is insightful and contributes novel evidence in the field which will be of interest to a wide audience, therefore I recommend publication. I would point out however, a few changes that I think need to be made before publication. We are grateful to Eva Sheardown for her positive feedback. Thank you for the useful comments and suggestions, which improved the quality of our work. 1. The main issue I have with the manuscript is that the general quality and clarity of writing needs improvement. Some of the phrasing is odd and can result in some confusion at what the author is trying to say. Also there are some typos (or maybe just odd word choice). A native English speaker has read and carefully revised the manuscript to improve the general quality and clarity of writing. 2. I would maybe choose to refer to the stimuli couples as "pairs" or "coupled" rather than 'a couple of' as you use in the manuscript. This is as this is more commonly used when referring to coupled stimuli in literature on visual discrimination. Done, thank you for the suggestion. 3. There is a fair amount of methodical information at the end of the introduction which is not necessary to lead into your methods section. You really just want to give an overview of what the set of experiments is investigating rather than a whole lot of detail on the experiments as you are about to give that in the following methods section. Following this suggestion, we deleted the methodological information at the end of the Introduction section. We left the description of the purpose of our experiments, briefly presenting the experimental conditions and formulating possible expectations, to progressively introduce the reader to the subsequent sections. 4. In line 128-129 you need a reference for the statement regarding male motivation to join female conspecifics. We added the references as requested (lines 138-140). 5. When describing the stimuli pairs you say that they are balanced for perimeter (disc and amputated disc) and area (disc and cross) however both of these statements are not true. The values are different between the stimuli so you need to correct the statement. Thanks for noticing the mistake. By making a copy-and-paste, we had left the area of the amputated disc, instead of correctly reporting the area of the cross. We have now reported the correct values. As regards to the perimeters, however, we have specified that we made them as balanced as possible (actually, with a slight difference between them). 6. The use of 'cozy' to describe the environment throughout is slightly anthropomorphizing, familiar or comfortable may be better. We replaced the word “cozy” with “comfortable”. 7. There is a 10-second holding cylinder protocol at the start of the trials however it is not clear what this is for? Is it to view both stimuli? If this is the case can you be sure that the fish will have seen both in such a short period of time? If it is for another reason this needs to be stated. The 10-second period is an acclimatization time after the manipulation, necessary before starting each trial, to reduce the normal physiological activation (e.g., after having caught the fish in the comfortable external area and placed it at the center of the experimental arena). The possibility to visually scan both stimuli occurred as soon as the fish was free to move within the arena (i.e., after lifting the transparent cylinder). We provided this piece of information in the text (lines 231-235) 8. The figures: Figures 6-9 would be better of being just two figure panels of 4 graphs (6a,7a,8a,9a then 6b,7b,8b,9b) to improve data visualization. There also don't seem to be any figure legends in the document I have been given. This makes the figures pretty useless as they should stand on their own without the manuscript text. We accepted this suggestion in the overall presentation of the figures. We added the captions (which were missing in the previous version of the MS), adapting them to the new version of the figures. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0272773.r002
27 Jul 2022 Decision Letter - Livia D'Angelo, Editor “Classifying-together” phenomenon in fish (Xenotoca eiseni): Simultaneous exposure to visual stimuli impairs subsequent discrimination learning PONE-D-22-09159R1 Dear Dr. Sovrano, 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, Livia D'Angelo Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0272773.r003
Formally Accepted
16 Aug 2022 Acceptance Letter - Livia D'Angelo, Editor PONE-D-22-09159R1 “Classifying-together” phenomenon in fish (Xenotoca eiseni): Simultaneous exposure to visual stimuli impairs subsequent discrimination learning Dear Dr. Sovrano: 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. Livia D'Angelo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0272773.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .