Dear Dr. Hamilton,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, I invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Editor comments: Thank you for this interesting manuscript. As an avid user of noldus programs myself, could the lab please clarify the frame rate used for ethovision (and whether this was changed as the fish got older)? 

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Benjamin Tsang

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Johnson and colleagues present a nice study of zebrafish behavior across time. They examine the variability and repetability of zebrafish behavior across three ages (90, 120, and 150 dpf) in the novel tank test. Overall, the manuscript is well written and the results are clear. My comments are mostly minor to improve data presentation, to point out some more recent citations they may want to incorporate, and to clarify some of the statistics.

Minor points

Line 136. I believe luminance is measured as candela per meter squared (not meter cubed) as it refers to a surface.

Lines 174 and 180. These methods refer to 'period 1' and 'period 2'. However, in the results section and figure (figure 5) the terms 'early' and 'late' are used. I would keep these consistent throughout the paper to make it easier for the reader to understand what 'early' and 'late' refer to when reading the results

Lines 189-190 and figure 5. The author's state that significant differences between groups are when the 95% CrIs do not overlap. However, in figure 5 it appears that several 95% CrIs overlap yet the author's state that there are differences between groups. For example, in figure 5A, the results (lines 257-259) state that there is a difference between males and females. But the 95% CrI's overlap. So this does not appear to be a significant difference. However, I'm not that familiar with Bayesian statistics, and so perhaps I'm misunderstanding something here. If so, I would suggest the author's be a bit clearer about what constitutes a 'significant' or relevant difference in the results section.

Line 331. The author's state that "all n=100 fish were then ranked by time spent...". However, I got the impression earlier in this section (lines 311-314) that only 30 fish were used (10 in each group of HAZ, LAZ, and MAZ). Or was it that only the 'top 10, bottom 10, and middle-most 10' fish were used for the different groups. If so, that wasn't entirely clear from the writing in this section.

It is not clear when different post-hoc tests are used in different figures/results. In the methods section its stated that sometimes a Dunnet (line 156) or Tukey HSD (line 157) or Fisher's LSD (line 165) is used as a post-hoc test. However, it's not clear in the results when the different tests are used. This is important because, for example, the Fisher's LSD does not manage family wise error rates like the Dunnett or Tukey does. This will help readers better interpret which effects are likely to be more robust.

Discussion

Line 353-355. The increase in velocity could also be due to an increase in size. Especially if fish are being housed at the low density indicated here, they are likely to grow quite a bit more between 90 and 120 dpf if they were housed at higher density beforehand. This latter point should also be made.

Finally, I would also kindly point the author's to papers we have recently published that touches on several of the topics discussed in this paper. For example, in Rajput et al, 2022 we explicitly study the consistency of individual differences over time and look at strain and sex differences. We show, for example, that males have higher locomotor activity than females (line 368) and that individual differences in behavior are largely consistent over days and weeks (lines 80, 85, and 92). In another paper (Beigloo et al, 2024), we also show that behavioral type can affect behavioral responses to anxiolytic compounds (escitalopram), an idea suggested in the discussion (lines 414-417). That being said, because these papers are from my own lab, it is completely at the discretion of the author and editor whether it is appropriate to include these citations.

References

Rajput, N., Parikh, K., & Kenney, J. W. (2022). Beyond bold versus shy: Zebrafish exploratory behavior falls into several behavioral clusters and is influenced by strain and sex. Biology Open, 11(8), bio059443.

Beigloo, F., Davidson, C. J., Gjonaj, J., Perrine, S. A., & Kenney, J. W. (2024). Individual differences in the boldness of female zebrafish are associated with alterations in serotonin function. Journal of Experimental Biology, 227(12).

Reviewer #2: This manuscript presents a longitudinal behavioral analysis of adult zebrafish using the Novel Tank Dive Test (NTT), highlighting individual variability and repeatability across development. The study is methodologically ambitious and holds promise to inform future work on zebrafish anxiety-like behavior. However, there are several major concerns regarding the experimental design, interpretation of results, and reproducibility that must be addressed before publication.

Point 1. Habituation and Repeated Testing Effects is a major concern. Although the authors acknowledge the possibility that behavioral changes over time may result from re-exposure rather than developmental progression, they nonetheless interpret their findings as age-related decreases in anxiety-like behavior. This is methodologically problematic.

Repeated testing in the NTT is well known to produce habituation effects, which can mimic anxiolysis. Therefore, it is inappropriate to infer developmental reductions in anxiety without a control group of naïve animals tested only once at each time point. I strongly recommend the authors to either include additional experiments with naïve age-matched groups or substantially reframe their claims.

Point 2. Lack of Arena Randomization and Positional Control is also a problem. There is no description of how the arenas were assigned or randomized across testing days or fish. Since four fish were tested simultaneously, arena position or time-of-day effects could bias results or artificially inflate repeatability. Please, clarify whether randomization procedures were implemented.

Point 3. The manuscript fails to describe how sex was confirmed. This is especially important in adult zebrafish as external sexual dimorphism can be subtle and unreliable, and gonadal dissection or genetic confirmation is often necessary for accurate assignment. Please clarify how sex was identified, whether through dissection, visual dimorphism, or another method. If not confirmed post-mortem, this introduces potential misclassification bias in sex-based analyses.

Point 4. It is stated that 7 females and 9 males were lost during the study, yet no details are provided regarding the causes, timing, or potential impact on longitudinal behavioral profiles. Given the experimental design, if fish were sharing a tank 1:1, how did the authors proceed with the fish that shared the tank with the one that died? Could that affect the behavior of the remaining fish?

Point 5. The division of fish into “low,” “medium,” and “high” anxiety groups based on 150 dpf data is methodologically problematic. Using endpoint data for retrospective grouping can lead to circular reasoning and ignores behavioral plasticity or adaptation over time. For instance, individuals classified as "low anxiety" may simply be more habituated or more adaptable, not inherently less anxious.

Point 6. Two recent studies that directly relate to the present work are not cited but are essential to contextualize the findings. One study (doi: 10.1242/bio.059443) examined individual exploratory behavior in a novel environment over time, revealing consistent inter-individual differences and reinforcing the importance of accounting for stable behavioral phenotypes in repeated-measures designs. Another study (doi: 10.1371/journal.pone.0300227) found that individual variability in novelty response is influenced by strain, sex, and age, which directly informs the present study’s focus on behavioral repeatability and developmental trajectories. Please revise the reference list to include relevant studies and ensure that other foundational papers on sex differences in zebrafish behavior, particularly in the Novel Tank Test, are cited, as some seminal articles appear to be missing.

Point 7. Anxiety-like behavior in zebrafish, particularly in older individuals, may not follow the same trajectory or behavioral signatures as in larvae or juveniles. The Novel Tank Test (NTT) was originally validated for acute anxiogenic responses, and its use as a developmental or trait-based marker of anxiety in adult zebrafish is still under debate. In this context, it is unclear why the authors chose time spent in tank zones (e.g., upper vs. lower) as the primary behavioral feature to stratify individuals into anxiety-level groups, while other relevant parameters such as immobility, freezing-like behavior, or overall activity were not used as equally weighted indicators. Furthermore, while zone-based scoring is conventional, the use of continuous variables such as distance from bottom may offer a more robust and fine-grained analysis of vertical exploration, without the need to define arbitrary cutoffs or classify subjects post hoc. This would also allow for correlational or trajectory-based analyses of anxiety-like behavior over time, strengthening the claim of trait-level consistency. The authors should justify their prioritization of zone occupancy as the main behavioral classifier, clarify whether immobility or other anxiety-related metrics were considered in combination, and consider re-analyzing or presenting vertical position as a continuous measure rather than a categorical one. This may offer greater sensitivity to developmental or individual differences and avoid potential oversimplification of complex behavioral traits.

Point 8. The manuscript does not provide a rationale for the chosen sample size (n = 100; 50 males and 50 females). While this is a commendably large N for a behavioral longitudinal study, no information is provided regarding power analysis, effect size expectations, or justification based on previous studies.

Reviewer #3: This study assessed individuality and variability across adult zebrafish, with observation spanning across 2 months, using the novel tank dive test. It highlights the need to consider age, sex, and individual variation when conducting zebrafish behavioral assays. Overall, I am very enthusiastic about this manuscript.

Here are my comments:

Were experimental fish housed individually?

Just out of curiosity – how did the authors deal with the egg-bound issue of female fish over this 2-month period?

9 fish were dead, and the data were not included. Thus, the “n” in the results should be reflected accordingly, instead of “n = 50” or “n = 100”.

Could the author comment on why the locomotion was increased at 90 dpf, compared to 120 dpf, given that the fish were younger and presumably smaller at 90 dpf?

I am not sure if I understand the interpretation of Fig.4. The error bars are mostly overlapping. Are these reported trends (such as “decline”, “reduction”) significant? If not, how are the conclusions justified?

Fig 5, if variance was measured here, should the unit of the y-axis be “squared”?

A couple of comments on statistical analysis:

Line 189-191: I am not sure why would non-overlapping 95% credible interval would correspond to p<0.006 (credible interval is based on Bayesian, p value is based on frequentist). Please elaborate.

Could the authors please add more details about what the sim function of the arm package and the MCMCglmm package are and why they are used?

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Reviewer #1: Yes:  Justin Kenney

Reviewer #2: No

Reviewer #3: No

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<p>Behavioural Variability and Repeatability in Adult Zebrafish (Danio rerio) Using the Novel Tank Dive Test

PONE-D-25-29898R1

Dear Dr. Hamilton,

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.

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Kind regards,

Benjamin Tsang

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: All of my concerns were adequately addressed.

Reviewer #2: The authors have responded thoroughly to all my concerns, including the addition of new analyses and data (e.g., naïve cohort, power analysis, continuous metrics). I now recommend the manuscript for publication in its current form.

Reviewer #3: (No Response)

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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

Reviewer #3: Yes:  Qian Lin

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