PONE-D-19-18603

Modulations of microbehaviour by associative memory strength in Drosophila larvae

PLOS ONE

Dear Dr Schleyer

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.

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First, let me apologise for the little delay in this decision, as i have been on my annual family vacation.

As you see from the attached comments both reviewers and myself agree that the manuscript has a lot of merit, but as detailed by Reviewer #1, there are some largely technical issues to be resolved that will enhance the presentation and comprehension of the manuscript.  Please thoroughly address all comments and suggestions paying particular attention to the comments related to the statistics.

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We would appreciate receiving your revised manuscript by Sep 20 2019 11:59PM. When you are 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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We look forward to receiving your revised manuscript.

Kind regards,

Efthimios M. C. Skoulakis, PhD

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?

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

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

Reviewer #1: No

Reviewer #2: Yes

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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

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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: Yes

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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: General comments

This manuscript 'Modulations of microbehaviour by associative memory strength in Drosophila larvae' shows the associative learning of chemical cues in Drosophila larva for searching foods. This is a nice work, which clarify the usage of environmental cues by learning occurrence with model behavior; microbehaviors. The results and discussions are written well and they are supported well by results. While, the writing of materials and methods including statistics need to be improved. For now, I'm afraid that the lack of information in materials and methods can bring misunderstandings to readers and the statistics seem to contain some mistakes. I recommend authors to ask their natives colleagues to edit their English.

Here are numbers of suggestion, which might improve the manuscript.

Through manuscripts,

*Please correct the p value. It is impossible to get 'p=0'. In addition, suggest to provide x2 value instead of H value in KW test.

In introduction

Page 1 line 36-37, suggest to erase the words 'that can guide this search' and add ' to guide this search' after 'crucial'.

Page 1 line 44, suggest to remove ',' before 'and'

Page 1 line 45-47, suggest to rewrite the sentences 'From the …[8, 10-13]'. Currently it is difficult to understand this part.

Page 2 line 59, suggest to replace the words 'reward concentration' to 'reward quantity'

Then suggest to replace the words 'odour dilution' to 'odour concentration'.

Page 2 line 58-60, please provide the outline of the experiments. The short explanation about n-amyl acetate is important even it is a common chemical used in Drosophila learning: reason of selection, type of odour. This information can be provided in materials and methods section in the paragraph 'general'.

In Materials and Methods

*The repetition number is unclear through the section. How many petri dish was used (which contained 15 individuals)?

Page 2 line 72-73 and 74-75, suggest to remove 'as mentioned in the figure legends'.

Page 2-3 line 82-85, suggest to rewrite the sentences from 'for the typical… (AM+)' since the explanation about '+' next to AM/EM is not clear. Or add the sentence, for example: the presence of fructose is showed by '+'.

Page 3 line 87-92, suggest to provide more details about the unpaired test.

Additionally, suggest to add simple explanation about 'conditioning' (or around line 137-139).

Page 3 line 106, suggest to add the word '(Pref)'after 'a preference score'.

Page 3 line 117 suggest to provide the explanation about 'Stowards' and 'Saway'.

Page 4 line 128, suggest to provide the explanation of 'abs'.

Page 5 line 151, suggest to provide the number of petri dishes used for experiments to each calculations (maybe better to add the info. near each part).

Only figure legends contain the detailed data but it would be better to comment it shortly also in Materials and Methods section.

In Results & Discussion

*Suggest to add some discussions about the Drosophila associative learning on feeding of adult flies.

*Spearman r value under 0.5 does not regard as 'clear' correlation. It is taken as 'slight' correlation in general. It would be better to provide some information about the standard correlation value in insect learning in comparison with adult flies or other insect larva.

Page 5 line 165, suggest to make 'Discussion' to plural.

Page 6 line 199-201, it seems some sentences are missing. The place of quotations seems strange.

Page 7 line 301, suggest to remove 'clearly'.

In figure legends

In Fig. 1, please correct the p value. It is impossible to get 'p=0'.

Please provide x2 value not H value for KW test.

Page 6 line 242-243, suggest to replace '(G-J)' to 'G-J'.

In Fig. 2 please correct the p value.

Suggest to add N value for all parameter, not only (A) but also (B) and (C).

In Fig. 3 please correct the p value.

Reviewer #2: The ability of an animal to form memories is typically tested by assessing changes in the behavioral response after a specific training regime. In the past years the larva of the fruit fly has been consolidated as an intriguing model to study associative learning, in particular since the entire mushroom body circuit has been mapped using ssTEM reconstruction. However, to assess the ability to form memories most experimental analyses are based on simple choice assays and do not explore how the animal adapts its behavior.

In the current manuscript Thane and colleagues use high-resolution analysis of larval locomotion and navigation to understand how different strength of memories are encoded. The approach follows a previous publication the Schleyer lab (Paisios et al., 2017), but rather focusses on the stimulus strength.

Overall the manuscript is well prepared and the data well displayed. I do not see any major issues to be addressed. The findings reported in the current manuscript is certainly relevant for the field, in particular for scientists interested in how naïve or sensory experience alters navigation the manuscript will be of great interest.

My only minor comment is that currently the only data displayed in the figures (relevant for navigation) are the correlation plots and not the statistical analyses of the actual navigational decisions. I feel it would be valuable to also include these analyses in the manuscript on parameters of navigational performance.

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Reviewer #1: No

Reviewer #2: Yes: Simon Sprecher

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Modulations of microbehaviour by associative memory strength in Drosophila larvae

PONE-D-19-18603R1

Dear Dr. Schleyer,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Efthimios M. C. Skoulakis, PhD

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

Reviewer #2: All comments have been addressed

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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

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: 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

Reviewer #2: 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

Reviewer #2: 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: General comments

This manuscript 'Modulations of microbehaviour by associative memory strength in Drosophila larvae' shows the associative learning of chemical cues in Drosophila larva for searching foods. Thank authors for their corporations. I have only minor comments.

Trough manuscript, please unify the number figures after the decimal point.

Maybe 0.001?

Ex) line 257, suggest to change 'p < 0.0001' to 'p < 0.001', Line 266, 'p = 0.0004' to 'p < 0.001' and so on.

Line 67 and Line 186, suggest to change 'Drosophila melanogaster' to 'D. melanogaster'.

Line 175, suggest to remove the sentences after 'H'; '(sometimes reported as the x2-value)'.

Line 260, no df information in the result here.

Line 326, suggest to remove the underline from the word 'differences'.

Reviewer #2: The authors have addressed all points that were raised adequately. I feel the manuscript is an interesting addition in the field, it nicely highlights the quantitative features that may be extracted from "complex" behaviours in the fruit fly larva. In particularly interesting to changing external conditions or - as done here- during learning.

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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

Reviewer #2: Yes: Simon Sprecher