Dear Dr. Zou,

Thank you very much for submitting your manuscript "Ecdysone signaling mediates the trade-off between immunity and reproduction via suppression of amyloids in the mosquito Aedes aegypti" for consideration at PLOS Pathogens. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

The reviewers made several suggestions for improving the manuscript. However, the performance of any new experimental work is at the discretion of the authors, and is not required for the revision, which can be accomplished by revision of the text.

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Kenneth D Vernick

Associate Editor

PLOS Pathogens

Kirk Deitsch

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Performance of any new experimental work is at the discretion of the authors, and is not required for the revision, which can be accomplished by revision of the text.

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: Immunity and reproduction are two important processes in insects that compete for limited resources. A delicate balance between immunity and reproduction is needed for survival and propagation. While some studies have indicated that insect hormones are implicated in the trade-off, the actual hormoneregulated effector molecules have not been identified. Wang et al. reported in this study that Pirk-like, a negative regulator of the IMD pathway, is upregulated by 20-hydroxyecdysone (20E) after a blood meal in the yellow fever mosquito Aedes aegypti to attenuate mosquito innate immunity in favor of egg maturation. The authors provide clear evidence showing that the 20E receptor complex directly binds to the promoter of the Pirk-like gene to increase its expression after a blood meal. Most importantly, Pirklike was able to directly interact with PGRP-LC, PGRP-LE and IMD. Knockout of Pirk-like enhanced mosquito survival after bacterial infection and this enhancement was nullified by the treatment of Thioflavin T, an inhibitor of amyloid formation, suggesting that Pirk-like represses the formation of functional IMD amyloid fibril which is required for the signal transduction of IMD pathway. This study addresses a fundamental question in mosquito biology and provides a new target that may be exploited for the control of mosquito-borne diseases. The results are supported by substantial evidence and statistical analysis. The manuscript is well written and is comprehensible to the general reader. 

Reviewer #2: This article revealed that insect hormone 20E and its receptor EcR coordinate the activity of the immune pathway, IMD after blood meals. It highlights the role of 20E on maintaining a delicate balance between reproduction and immunity. It demonstrated that the expression of Pirk-like was mainly determined by 20E during vitellogenesis. Pirk-like terminated amyloid formation, which is mediated by PGRP-LC, PGRP-LE and IMD. And it suppressed the downstream IMD signaling and helped female mosquito fertility. Knockout of Pirk-like in mosquitoes led to more resistance to bacterial infection, along with impaired reproduction. A novel aspect is the demonstration that 20E could modulate functional amyloid formation in mosquitoes. The findings presented in this work revealed a mechanism by which the anautogenous mosquitoes inhibit immune activation during blood meals. 

Reviewer #3: The manuscript by Wang et al. is a very interesting study addressing the trade-off balance between immunity and reproduction in adult female mosquito Aedes aegypti. The authors addresses this topic by analyzing the role of the main gonadotrophic hormone, 20-hydroxyecdysone (20E) and its receptor EcR in the regulation of the immune deficiency (IMD) pathway and the oocyte development during the reproductive phase of the mosquito, that is the post blood meal phase. By using a combination of RNAi, CRISPR-Cas9, and several genomic and molecular biology techniques, the authors clearly show that 20E and EcR are required to downregulate the IMD pathway by directly inducing the expression of Pirk-like, a negative regulator of the IMD pathway. In addition, the authors reveal that this effect is mediated by the Pirk-like-mediated impairment of the formation of amyloid aggregates. In summary, the authors clearly show that the dual role of 20E and EcR in inducing proper oogenesis and repressing innate immunity at the same time is critical for mosquito fertility.

The study is thorough, very well executed and the conclusions are properly supported by the data presented. It is relevant to note that the information reported is very interesting contribution to the understanding of the molecular basis of the trade-off between immunity and reproduction in adult insects, which in the case of mosquitoes is of paramount importance given its relevance in the transmission of deadly pathogens. In summary, the work is scientifically very sound and worth it for publication.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: I have several comments that may help the authors to improve this manuscript.

Specific points:

1. In addition to the IMD pathway, Pirk-like also downregulates genes related to ribosome, lysosome, synaptic vesicle cycle, and amino sugar and nucleotide sugar metabolism. What could be the potential mechanism? Will these downregulations affect innate immunity and egg maturation?

2. Lines 55-57. Only the first gonadotrophic cycle consists of a post-eclosion phase (PE) and a post blood meal phase (PBM).

3. Lines 175-176. Pirk-like was induced by 20E in EcR- and USP-overexpressed Ae. aegypti (Aag2) cells.

4. Line 188. The assay coupled with ChIP seems to be quantitative PCR or real-time PCR, not reverse-transcription PCR.

5. Lines 196-197. The extra band could come from (or result from) endogenous EcR and USP. It is not interfered with by endogenous proteins.

6. Lines 222-223. The sequence similarity, not variance, suggested that amyloidal aggregates could be formed in Ae. aegypti.

7. Fig. 6. The Pirk-like-/- mosquitoes induced much higher expression of PGRP-LC and Rel2 after infection with E. cloacae after a blood meal. Will Pirk-like affect immune responses in the PE phase?

8. Line 312. Vg proteins are deposited in maturing oocytes.

9. Fig. 7A. The effect of Pirk-like knockout on ovary development should also be examined before a blood meal.

10. Fig. 7D. This experiment should also include infection with E. cloacae as shown in Fig. 7B and C.

11. Line 355. It is 20E, not EcR, that blocks certain physiological processes.

12. Line 819-821. Was the differential expression compared with the levels of iEGFP-PBS?

Reviewer #2: The manuscript provides a set of solid data and the experiments are presented in a logical sequence, with the following shortcomings that remain to be addressed.

Specific comments:

1. In Abstract, Line 27-20. The two sentences are confusing. “Drosophila melanogaster Pirk, which a negative regulator of the IMD pathway. CRISPR/Cas9 knockout of Pirk-like has shown that it represses the IMD pathway.”

2. Figure 5, amyloid formation in the fat body of iEcR mosquitoes should be detected to further support the conclusion that amyloid was closely associated with 20E.

3. Line 41. Replace “20-hydroxyecdysone” by “20-hydroxyecdysone (20E)”.

4. Line 92. “Aedes. Aegypti” should be “Aedes aegypti”.

5. Line 279. “than did WT_Ec mosquitoes or normal controls” is incorrect.

6. Line 293. “is likely a direct Rel2 transcriptional target” should be “is likely a direct transcriptional target of Rel2”.

7. Line 415. “LB medium no antibiotics” should be “LB medium without antibiotics”.

Reviewer #3: The only concern regarding the manuscript that I believe would improve the manuscript refers to better show the trade off between immunity and reproduction that derives from the experimental approach followed by the authors, that is, the elimination of EcR during the first gonadotrophic cycle of Aedes. It would be interesting that the authors show the effect of eliminating EcR on reproductive parameters and on the response to Enterobacter cloacae infection. That is, as they do with the Pirk-like mutant, it would be interesting to measure ovarian growth, number of eggs deposited and vitellogenin production in EcR-depleted females with and without Enterobacter infection compared to wild type females in order to check the effect of the absence of EcR on the control of both processes.

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Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: PPATHOGENS-D-22-00692 Comments.pdf

Dear Dr. Zou,

We are pleased to inform you that your manuscript 'Ecdysone signaling mediates the trade-off between immunity and reproduction via suppression of amyloids in the mosquito Aedes aegypti' has been provisionally accepted for publication in PLOS Pathogens.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

Kenneth D Vernick

Associate Editor

PLOS Pathogens

Kirk Deitsch

Section Editor

PLOS Pathogens

Kasturi Haldar

Editor-in-Chief

PLOS Pathogens

​orcid.org/0000-0001-5065-158X

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

***********************************************************

Reviewer Comments (if any, and for reference):