PONE-D-22-20565A novel open-source raspberry Pi-based behavioral testing in zebrafishPLOS ONE

Dear Dr. Wang,

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PLOS ONE

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Reviewer #1: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: Thank you for sharing your manuscript with me. The author designed a low-cost automatic zebrafish behavior detection device and software to measure zebrafish behavior parameters. The results show that the device designed by the author can be used to obtain video data and determine various behavioral parameters to reliably evaluate the behavioral performance of zebrafish. Generally speaking, the theme of the manuscript is very meaningful and necessary. But there are many problems that need to be corrected. These issues are listed below and may help improve the manuscript.

1. Figure 4 is not clear enough. The horizontal and vertical coordinates of each result cannot be seen clearly. Please replace the result map with a higher definition.

2. What is the composition of the system water mentioned in line 91?

3. The zebrafish of one month old is used in the experiment. Can you track and analyze the behavior of small fish several days old after fertilization or adults of three months old through this device and software?

4. Line 163: regions of interest (ROIs) were defined to eliminate the false recognition caused by the mirror effect (fish reflection on the well-wall) of zebrafish in the multi-well plate and the possible influence of the edge of the multi-well plate. The region of interest here refers to which region?

Reviewer #2: This study provides a system package for determining multiple behavioral parameters of zebrafish, a model organism in the behavioral tests. In addition, the authors used an alcohol exposure test to examine the validity of their method. The MS has some novelty, but the following issues must be clarified before being recommended for publication.

（1）Tables: Three-line tables should be used, and footnotes should be used to indicate the meaning of abbreviations.

（2）Statistical analysis method: Since behavioral data usually cannot satisfy normal distribution and Homogeneity, one-way ANOVA and unpaired t-test are not necessarily applicable. At this time, it is best to use the generalized linear model (GzLM) for analysis. See the references:

Persistent impact of amitriptyline on the behavior, brain neurotransmitter, and transcriptional profile of zebrafish (Danio rerio). Aquatic Toxicology, 2022, 245 10.1016/j.aquatox.2022.106129.

Impacts of chronic exposure to sublethal diazepam on behavioral traits of female and male zebrafish (Danio rerio). Ecotoxicology and Environmental Safety, 2021, 208 10.1016/j.ecoenv.2020.111747.

The author should provide some videos to give readers an intuitive impression.

The authors should also discuss and identify the innovations and advantages of their methods. For example, using their systems and commercial equipment to track and analyze the trajectory of the same subjects, compare their results, and then identify the advantages and disadvantages.

Social behavior is the most difficult in fish behavioral analysis. Because when multiple individuals are located in the same container, it is easy to cause exchange between subjects (especially when behavioral trajectories cross each other), and it also involves complex interactions between subjects. I would like to know if there is an excellent way to solve the above problem with this methods.

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Reviewer #1: No

Reviewer #2: No

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A novel open-source raspberry Pi-based behavioral testing in zebrafish

PONE-D-22-20565R1

Dear Dr. Wang,

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,

Hai O. Xu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: