PONE-D-22-09391Nutrient Sensing Pathways Regulating Adult Reproductive Diapause in C. elegansPLOS ONE

Dear Dr. Hanover

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Eustice et al present a detailed genetic analysis of the metabolic requirements for a form of caloric restriction in C. elegans called adult reproductive diapause (ARD). From their data, they conclude:

1) Initiation of ARD is regulated by fatty acid metabolism, sirtuins, AMPK, and the OGlcNAc pathway

2) Maintenance of ARD is not influenced by any of the tested metabolic pathways

3) Recovery from ARD required energy sensing, stress response, insulin-like signaling, and TOR pathway function

4) Fatty acid B-oxidation regulates ARD through a ogt-1-nhr-49 pathway

5) Mutants with defects in ARD entry also exhibit changes in the levels of neutral lipids.

Overall, the experiments are extensive and make excellent use of both C. elegans and the ARD pathway to define genetic requirements for this form of caloric restriction. Experiments were performed with a high level of replication and statistical analysis of the data was robust and well done. The discussion is thorough, scholarly, and appropriate. I have some concerns related to the conclusions drawn from the genetic epistasis and the presumed functions of O-GlcNAc pathway and felt that these data could also be interpreted in ways not discussed by the authors.

Major points

1) The authors interpret the ARD entry phenotypes of the nhr-49; ogt-1, nhr-49; oga-1, and acs-2;ogt-1 double mutants in Figure 1 and 4 to mean that these genes function in the same pathway to regulate ARD entry. This is because the double mutants have phenotypes similar to the single mutants. However, these data appear to have a classic ‘floor effect’ problem. For example, the alternative hypothesis is that the genes operate in different pathways. In such a case, the phenotypes should be additive, leading to lower % ARD entry than either of the single mutants. However, the mutants they are analyzing already have extremely low % ARD entry (<10%) and the variability in this rate is fairly large (CV appears to be close to 100% for most). Would it even be possible to detect the alternative hypothesis that the genes function in different pathways? This has significant implications related to the model presented in Figure 6 and in the conclusions made in the discussion.

2) The authors assume that the role of ogt-1 in ARD entry is due to its catalytic addition of O-GlcNAc. However, there is ample evidence that ogt-1 also has non-catalytic roles, particularly in C. elegans. Moreover, the fact that oga-1 mutants exhibit milder phenotypes suggests there may be other aspects to the role of ogt-1 in ARD than O-GlcNAc cycling. The mutant alleles they are using eliminate both catalytic and non-catalytic activities of ogt-1. To better support their conclusion that the role of ogt-1 in ARD relates to the addition of O-GlcNAc, the authors should test catalytically inactive ogt-1 point mutants, which are publicly available.

3) The authors make several qualitative comparisons related to Oil Red O staining that I do not observe. For example, they comment that the ORO staining in ‘frozen’ embryos is stronger in the nhr-49 and ogt-1 single mutants after 30 days of ARD as shown in Figure 5 (I presume they mean compared to N2?). To me, the mutants look either lower than or similar to N2 in these images and no quantification of the embryo ORO levels was performed. What is the evidence for this conclusion? Likewise, Figure S5 lacks an N2 control for comparison. But with the exception of the age-1 mutant, I’m not sure any of the other are obviously ‘stronger’ than the N2 image in Figure S5 without some type of quantification.

4) Minor points

a. I’m not sure I understand the DAPI experiment in Fig S4A. Is this done in live or fixed worms? I think the authors are trying to determine if some of the mutants might have more or less cuticular permeablity to ORO that might explain the differences in staining? But if this is being done in fixed animals (as is the case for ORO), cuticular permeability should not matter since the animal is fully permeabilized.

b. Is the term ‘frozen embryo’ typical for ARD field? I find this to sometimes be confusing, ie is it referring to how the sample was prepared? Would ‘arrested embryo’ serve the same purpose? Seems a little more precise. But I certainly understand sticking with frozen if that is the field standard.

c. Line 176 – I think you mean ‘Hatched L1s’ and not ‘Hatched L4s’?

Reviewer #2: The adult reproductive diapause (ARD) of C. elegans is an intriguing phenomenon from which there is still so much to learn. In this manuscript by Eustine et al, they divide the ARD in three steps: 1) entry, 2) maintenance, and 3) recovery. They explored many pathways that participate in each of these steps. They identified several regulators of ARD initiation like sir-2.1, aak-2, acs-2, ogt-1 and oga-1. They also identified skn-1, rsks-1 and daf-16 as important pathways for ARD recovery. Unexpectedly they did not find any pathway involved in ARD maintenance. They also found that the loss of the O-GlcNAc cycling enzymes converges on the fatty acid metabolic pathways that involved mitochondrial fatty acid B-oxidation. The manuscript contains a fair amount of work that has merits and contributes significantly into the understanding of ARD. However, there are few concerns. The first concern is that the authors did not review carefully all the literature about ARD. The original paper by Angelo and Van Gilst contains two findings that are not reproducible. The first one is that embryos observed during ARD are not arrested. Embryogenesis continues normally during ARD (Seidel and Kimble, 2011). Gametes production does not stop during ARD, in fact oogenesis continues slowly, and that is the main reason there are always embryos in the utero (Carranza-García and Navarro, 2019).

The second one is that Angelo and Van Gilst paper showed that the caspase ced-3 is essential to maintain fertility, however later was shown that ced-3 animals are able to recover their fertility after ARD (Carranza-García and Navarro, 2019).

The insulin pathway was also originally implicated in ARD in 2019. An important contribution to the field was made in 2020 by the group of Antebi showing that the transcription factor HLH-30/TFEB promotes the morphological and physiological remodeling involved in ARD entry, survival and recovery and is not included in the discussion of this manuscript. It would be important to make the appropriate correction throughout the manuscript, and integrate all this literature to strength the paper.

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Reviewer #1: Yes: Todd Lamitina

Reviewer #2: No

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PONE-D-22-09391R1Nutrient Sensing Pathways Regulating Adult Reproductive Diapause in C. elegansPLOS ONE

Dear Dr. Hanover,

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 08/12/2022. 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,

Myon-Hee Lee, Ph.D

Academic Editor

PLOS ONE

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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

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Reviewer #1: Yes

Reviewer #2: Yes

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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

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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: The authors have done an excellent job of responding to my previous comments and suggestions. The lack of an ADR phenotype in the catalytically dead ogt-1 allele is particularly exciting. Outstanding study - congratulations!

Reviewer #2: The authors have responded satisfactorily to all my concerns. The manuscript has improved considerably and will be ready for acceptance after few minor corrections.

1) Authors have changed the term “embryo arrest” in most of the manuscript but there are still a couple of places in which they still use this term. Please change it in lines 307 and 320.

2) Correct the line indent in line 369.

3) Line 505: check partially spelling.

This reviewer suggests outlining the gonad in the ARD worms in Figure 2 because it is not easy to see them.

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Reviewer #2: No

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Nutrient Sensing Pathways Regulating Adult Reproductive Diapause in C. elegans

PONE-D-22-09391R2

Dear Dr. Hanover

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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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,

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Academic Editor

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Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #2: -----------------------------------------------------------------------------------------------------

The authors have responded satisfactorily to my concerns. The paper is ready for publication.

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Reviewer #2: No

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