The seminal vesicle is a juvenile hormone-responsive tissue in adult male Drosophila melanogaster
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Abstract
Juvenile hormone (JH) is one of the most essential hormones controlling insect metamorphosis and physiology. While it is well known that JH affects many tissues throughout the insects life cycle, the difference in JH responsiveness and the repertoire of JH-inducible genes among different tissues has not been fully investigated. In this study, we monitored JH responsiveness in vivo using transgenic Drosophila melanogaster flies carrying a JH response element-GFP ( JHRE-GFP ) construct. Our data highlight the high responsiveness of the epithelial cells within the seminal vesicle, a component of the male reproductive tract, to JH. Specifically, we observe an elevation in the JHRE-GFP signal within the seminal vesicle epithelium upon JH analog administration, while suppression occurs upon knockdown of genes encoding the intracellular JH receptors, Methoprene-tolerant and germ cell-expressed . Starting from published transcriptomic and proteomics datasets, we next identified Lactate dehydrogenase as a JH-response gene expressed in the seminal vesicle epithelium, suggesting insect seminal vesicles undergo metabolic regulation by JH. Together, this study sheds new light on biology of the insect reproductive regulatory system.
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Manuscript number: RC-2024-02438
Corresponding author(s): Ryusuke, Niwa
1. General Statements [optional]
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Below are quotes from the Reviewers' overall evaluations:
As might be expected based on the authors' skills and expertise, the study is well executed, nicely documented with perfect microscopy images, and well presented. It has been easy to follow. However, suitability for publication depends on where the authors aim to place their paper. Although I like the paper very much, it might seem incomplete for high-end journals.
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Reply to the reviewers
Manuscript number: RC-2024-02438
Corresponding author(s): Ryusuke, Niwa
1. General Statements [optional]
This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.
Below are quotes from the Reviewers' overall evaluations:
As might be expected based on the authors' skills and expertise, the study is well executed, nicely documented with perfect microscopy images, and well presented. It has been easy to follow. However, suitability for publication depends on where the authors aim to place their paper. Although I like the paper very much, it might seem incomplete for high-end journals.
This is a very nice paper and solid piece of work.
Its major strength is the focus on poorly studied the male reproductive organ and identification of Ldh as a novel target of JH activity in the seminal vesicles.
While the developmental roles of insect Juvenile Hormone (JH) are very well studied, its adult functions are largely unknown. Target genes of JH signaling are poorly described. This study adds significant insight into both of these aspects. The study underscores the usefulness of the JHRE-GFP reporter that identifies JH function, and not just JH presence since the reporter is only expressed after JH binding to Met and Gce, a prerequisite for JHRE reporter activation.
The authors have identified the epithelial cells of the ____Drosophila____ seminal vesicle as a JH target tissue. The authors nicely extended this finding by mining already existing expression data to identify a specific JH induced gene in these cells.
This small study reports new but limited results (one tissue of one stage, one hormone) that could be useful for specialists. The work is solid and includes controls and interpretable data.
2. Description of the planned revisions
Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.
1) The study suggests an important role for JH signaling in the SV, likely affecting reproductive capacity of males. The authors depleted the JH receptors through RNAi, achieving a loss in the expression of the WT JHRE-GFP reporter as well as of the authentic target Ldh. Surprisingly, no phenotypic consequences of the double KD of Met and gce are presented. Does that mean that there were none? The authors only discuss a potential impact of Ldh loss for metabolism. Unless I am missing something, the study reports molecular phenotypes that clearly document JH signaling in the SV but no physiological impact of loss of this JH signaling, suggesting that there may be no obvious biological role for JH in this context. I think this is unlikely. Have the authors check fertility of the males, sperm viability and quality, mating competitiveness of the RNAi males? Loss of JH epoxidation (only methyl farnesoate present) made mosquito males less fit and less reproductively competitive relative to epox+ controls (Nouzova et al., 2021, PNAS) -- btw, I think the authors should discuss this paper.
Our response: We will conduct the following experiments to answer these criticisms.
1) We will examine the male fertility by counting the number of offspring from wild-type mothers crossed with males of the seminal vesicle-specific ____Met ____& ____gce____ double RNAi and with males of control RNAi.
2) We will also examine the mating competitiveness of the RNAi males. In more detail, we will cross ____w1118____ (white eye) wild-type background females with (i) a mixed population of males of ____w1118____ wild-type background males and____* w+____ (red eye) control RNAi males, and (ii) a mixed population of males of ____w1118____ wild-type background males and____ w+* Met ____& ____gce____ double RNAi males. We can distinguish between the progenies from RNAi males and those from wild-type males by eye colors.
By conducting plans 1) and 2), we will also indirectly evaluate sperm viability and quality.
In addition, we will also discuss the paper of Nouzova et al. PNAS 2021 in the Discussion section.
2) The authors seem to have made no effort to distinguish between Met and Gce functions. It is always the results from the double knockdown of both paralogs that are presented. Does this mean that single-KD had no effect, thereby indicating entirely redundant functions of both proteins in the studied context? Even if so, it would be of interest to document this redundancy by showing the single-gene KD data. However, I would be surprised if both proteins were equally important in the SV. The authors checked mRNA/protein expression levels. Was any of the two paralogs prevalent in the SV?
Our response: To address this criticism, we will conduct a single transgenic RNAi experiment to knock down either Met or gce separately and assess JHRE-GFP signals in the seminal vesicles.
__ Regarding the expression of Met and gce in the seminal vesicles, a previous study (Baumann et al. Scientific Reports 7: 2132, DOI:10.1038/s41598-017-02264-41) has already reported that GFP signals are observed in the seminal vesicles of ____Met-T2A-GAL4>UAS____-GFP and gce-T2A-GAL4>UAS-GFP animals. These results strongly indicate that both Met and gce are expressed in the seminal vesicles. We will describe and discuss this point in our revised manuscript. In addition, we plan to check and analyze gene expression of Met, gce, and Ldh in the seminal vesicles using a publicly-available single-cell RNA-seq database, such as ____DRscDB (https://www.flyrnai.org/tools/single_cell/web/).__
3) The authors argue for direct regulation of Ldh by Met/Gce (again by which one?). Oddly, the statement in the Results (l.187-188; "suggests ... direct target") is stronger than in the Discussion (l.214, "leaving open the possibility"). The putative JHREs upstream and within the Ldh gene are identified but not tested in a functional study. At least a simple luciferase reporter assay and mutagenesis of the JHREs should be attempted.
Our response: To address this criticism, we plan to conduct a luciferase-based promoter/enhancer analysis in *Drosophila *S2 cultured cells. A similar system was used for a JH-responsiveness of the JHRE promoter in a previous study (Jindra et al. PLoS Genetics 11: e1005394, DOI: __10.1371/journal.pgen.1005394). We will generate plasmid constructs carrying the luciferase coding regions. In these plasmids, the luciferase coding regions will be fused with the upstream region and the first intron region of *Ldh *possessing the intact E-boxes or the mutated E-boxes. Then, we will determine whether the luciferase activity is enhanced by the presence of a JH analog (methoprene) when E-boxes are intact. __
__ For this revision, a new collaborator, Ryosuke Hayashi (a graduate student in the Niwa lab), will participate in this analysis. Thus, he becomes a co-author in the revised manuscript.__
l.232-233. It is not surprising that the JHRR-lacZ reporter shows a different expression pattern relative to JHRE-GFP, as these are really different constructs. The problem is that JH-dependent activation of the JHRR-lacZ transgene has not been tested as thoroughly as that of JHRE-GFP. Is it inducible by added JH or methoprene?
Have the authors examined whether JHRE-lacZ expression increases with Methoprene?
Our response: We have yet to do this analysis. To address this important point from Reviewers #1 and #2, we will examine whether JHRR-lacZ expression is upregulated in the seminal vesicles of virgin males fed methoprene-supplemented food. The lacZ signals will be visualized by immunostaining with an anti-LacZ antibody.
Document testis staining of JHRE-GFP. I think the authors missed a chance by not providing a clear/nice picture of the testis staining. Stainings of testes squashed on a slide is easy and would nicely document in which cells the reporter is activated. Similarly, extracting sperm from the seminal vesicle and examining whether the sperm express JHRE-GFP would be informative.
Our response: As the reviewer suggested, we will assess JHRE-GFP signal in sperm in squashed testis samples.
Did the authors try to analyze the 66 genes identified in seminal vesicle whether they had JHRE elements? This could yield additional significant information about other JH responsive genes in the seminal vesicle.
Our response: We have yet to do this analysis. We will follow the reviewer's suggestion and examine whether the 66 genes identified in the seminal vesicle have JHRE elements.
3a. Doublestaining would further confirm that pd8-Gal4 (crossed to UAS-dsRed) and JHRE-GFP overlap.
3b. Similarly, Doublestaining would further confirm that pd8-Gal4 (crossed to UAS-dsREd) and JHRE-GFP overlap.
Our response: To address this question, we will generate males of Pde8-GAL4; UAS-red fluorescent protein (RedStinger, RFP, or DsRed); JHRE-GFP and observe the overlap between the red fluorescent signals and green fluorescent (JHRE-GFP) signals in the seminal vesicle epithelial cells.
Minor comments:
Fig.1a could be in a supplement.
__Our response: At this point, we are unsure whether to follow this reviewer's suggestion. This is because there are no supplemental figures in the current manuscript, so we hesitate to create a supplemental figure just for this one figure. On the other hand, three reviewers now ask us to perform various additional experiments, thus some of the new data may be shown as supplemental figures. In this case, Fig. 1a can be moved to a supplemental figure, but we would like to wait on this decision. __
3. Description of the revisions that have already been incorporated in the transferred manuscript
Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.
l.25,91,117, and throughout, "JH analog" or "JHA". The authors only use methoprene, so it would be better to specifically talk about methoprene, which is a proven agonist ligand of the JHR proteins (reference 10 and/or Jindra and Bittova, 2020 [Arch Insect Biochem Physiol] for a review). This would land more credibility to using methoprene than just referring to a "JHA".
Our response: According to the reviewer's suggestion, we have replaced "JHA" with "methoprene" as many as possible. In Figures, we used "MTP" instead of "methoprene" due to space limitations.
l.42,44, "paralogs". I believe in this case the authors refer to orthologs of Met in other species. Paralogs result from gene duplications within species, such as Met and gce in cyclorrhaphous flies or Met 1 and 2 in the Lepidoptera. I recommend a recent review on all bHLH-PAS proteins featuring reconstruction of the phylogenetic position of Met/Gce (Tumova et al., 2024 in J Mol Biol).
Our response: As suggested, we have replaced "paralogs" and "paralogous" with "orthologs" and "orthologous," respectively on P3. We have also cited Tumova et al. J. Mol. Biol. 2023 as a new Ref 12.
l.54, "Met and Gce act redundantly to regulate JH-responsive gene expression". Ref 10 should be cited here as it provides functional cell-based and genetic rescue evidence for each paralog.
Our response: We have cited Ref 10 as suggested.
l.66, It would be better to start "In this study" or "Here" to distinguish from the last cited paper.
Our response:____ We created a new paragraph with the sentence "In this study..." at the beginning. We hope we understand the reviewer's suggestion correctly.
l.175, levels were
Our response: We have fixed this error in the transferred manuscript.
l.209, might be evolutionarily among.... conserved ??
Our response: We have fixed this error in the transferred manuscript.
l.226, study has
Our response: We have fixed this error in the transferred manuscript.
l.227-229. The authors are missing a paper by Shin et al., 2012 (PNAS) that shows physical interaction of Met with Cycle and their regulation of circadian gene activity and another paper by Bajgar et al., 2013 (PNAS) which describes photoperid-dependent seasonal regulation of circadian genes by Met, Clk and Cyc.
On the other hand, the cited reference [51] does NOT demonstrate Met:Clk heterodimer since coIP is by no means adequate to address complex stoichiometry. In fact, it is suspicious that Met would heterodimerize and either Cyc or Clk, as they present class II and class I bHLH-PAS proteins.
Our response: In response to both comments from Reviewer #1, ____we have cited these references and rewritten the discussion on P10-11 as below: "An interesting previous study has reported that the seminal vesicle expresses multiple clock genes such as period, Clock (Clk), and timeless, all of which are necessary for generating proper circadian rhythm [52]. In the case of the mosquito Aedes aegypti female, it is reported that JH controls gene expression via a heterodimer of Met and circadian rhythm factor Cycle (CYC) [53]. It was also suggested that Met binds directly to CLK in D. melanogaster [54]. In addition, in the linden bug, Pyrrhocoris apterus, JH alters gene expression via Met, CLK, and CYC in the gut [55]. Considering these previous reports and our results, circadian rhythm factors and JH may cooperate to regulate gene expression in the seminal vesicles."
l.245. It is not "whether", but for sure the existing reporters only reflect limited JHR activity, being based on Kr-h1 JHREs. These reporters likely uncover only a small subset of JH activity in vivo.
Our response: We have rewritten the sentence as follows: "..., more comprehensive JH reporter strains will be needed in D. melanogaster as well as other insects in future studies."
reference 10/11 is duplicated.
Our response: We have fixed this error in the transferred manuscript.
Have the authors done a careful comparison of JHRE-GFP expression and the Met/gce reporter expression described by Baumann et al (Scientific Reports | 7: 2132 | DOI:10.1038/s41598-017-02264-4)? Would be nice to add a few more sentences in the discussion.
Our response: As suggested, we have added some sentences to explain this point on Page 11 as below: "P____revious studies reported that ____Met-T2A-GAL4____ and ____gce-T2A-GAL4____ labeled male accessory glands, ejaculatory duct, and testes as well as seminal vesicles. On the other hand, in our results, JHRE-GFP only labels cells in seminal vesicles and testes [21]. Considering that Met and Gce are expressed in almost all cell types of male reproductive tracts [21], more comprehensive JH reporter strains will be needed in ____D. melanogaster____ as well as other insects in future studies."
- In the discussion:*
6.1 Would have liked to see a more in depth discussion of the role of the seminal vesicle. How could that be supported by JH / metabolic processes? Does it have secretory functions that might be induced by JH? Important functions relative to sperm storage? How could that relate to the finding that JH response is enhanced by mating?
Our response: Unfortunately, the function of the seminal vesicles is largely unknown. However, ____in response to the reviewer's suggestion, we have added some sentences to discuss this point and cited some references describing the seminal vesicles in insects other than the fruit fly, as follows on P9-10: "Furthermore, in some insects other than D. melanogaster, morphological and ultrastructural studies revealed that secretory vesicles were observed in the epithelial cells of the seminal vesicles [37,38,40,44]. JH is known to stimulate secretory activity in the male accessory glands of many insects [45]. Based on the JH response in the seminal vesicles, it is possible that JH signaling affects the secretory activity of the seminal vesicles in D. melanogaster."
The arrow in figure is not defined
Our response: We believe that the reviewer pointed out the arrow in Figure 1e. We have added a sentence to define the arrow in the Figure legend as "The arrow indicates the cell with a GFP signal."
Figure 2b graph labels are flipped
Our response: We have fixed the error.
Line 624: Change "Allow heads" to "Arrowheads"
Our response: We have fixed this error in the transferred manuscript.
Major Comments:
The work uses standard methods and strains. Although the specific findings are new and believable, the authors interpret them beyond what is appropriate. For example, based on increased amounts of a single RNA, they propose that JH regulates metabolism in seminal vesicles and because circadian rhythm genes were known to be expressed in this tissue they propose that JH and circadian systems work together there.
Our response: In response to the reviewer's criticisms, we have discussed our arguments more appropriately in the Discussion. For example, we have mentioned circadian rhythm more carefully on Pages 10-11 as follows: "An interesting previous study has reported that the seminal vesicle expresses multiple clock genes such as period, Clock (Clk), and timeless, all of which are necessary for generating proper circadian rhythm [52]. In case of mosquito Aedes aegypti female, it is reported that JH controls gene expression via a heterodimer of Met and circadian rhythm factor Cycle (CYC) [53]. It was also suggested that Met binds directly to CLK in D. melanogaster [54]. In addition, in the linden bug, Pyrrhocoris apterus, JH alter gene expression via Met, CLK and CYC in the gut [55]. Considering these previous reports and our results, it is possible that circadian rhythm factors and JH cooperatively regulate gene expression in the seminal vesicles."
__ Regarding Ldh, we have added a sentence on Page 10 as "Also, the biological significance of the induction of Ldh expression by JH signaling is not clear."__
4. Description of analyses that authors prefer not to carry out
Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.
l.244, tract
Our response: We have carefully checked out the usage of "tract" and "tracts" not only on Page 11 but also throughout the manuscript. We have decided to use "tracts," but not "tract," throughout the manuscript.
6.2 What do epithelial cells of spermatheca do?
Our response: We agree with the reviewer that this is a very interesting question. However, please note that this paper focuses on males, and females are beyond our current scope. We plan to examine JHRE-GFP signals in the spermatheca in a different project. We do appreciate the reviewer's kind understanding.
6.3 How do the authors envision that JH enters the epithelial cells?
__Our response:____ We don't have any hypotheses on this point. Transporters may exist to achieve intracellular permeability of JH, but we do not think this point has been discussed in current insect physiology. Furthermore, since this issue is related to all JH-responsive cells, not just seminal vesicle epithelial cells, we do not feel the need to discuss it in this paper. __
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Referee #3
Evidence, reproducibility and clarity
Summary:
Using two existing reporters, the authors showed that cells in Drosophila seminal vesicles are responsive to JH. They believe, but do not show, that these are epithelial cells. JH response of those cell is shown to depend on the known JH receptors and to increase after mating, when JH titers are known to rise. RT-qPCRs show that Ldh expression increases in response to JH.
Major Comments:
The work uses standard methods and strains. Although the specific findings are new and believable, the authors interpret them beyond what is appropriate. For example, based on increased amounts of a single RNA, they propose that JH …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Summary:
Using two existing reporters, the authors showed that cells in Drosophila seminal vesicles are responsive to JH. They believe, but do not show, that these are epithelial cells. JH response of those cell is shown to depend on the known JH receptors and to increase after mating, when JH titers are known to rise. RT-qPCRs show that Ldh expression increases in response to JH.
Major Comments:
The work uses standard methods and strains. Although the specific findings are new and believable, the authors interpret them beyond what is appropriate. For example, based on increased amounts of a single RNA, they propose that JH regulates metabolism in seminal vesicles and because circadian rhythm genes were known to be expressed in this tissue they propose that JH and circadian systems work together there.
Minor comments:
Fig.1a could be in a supplement.
Significance
General Assessment, advance, and audience:
This small study reports new but limited results (one tissue of one stage, one hormone) that could be useful for specialists. The work is solid and includes controls and interpretable data.
-
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Referee #2
Evidence, reproducibility and clarity
Summary:
The authors identify the epithelial layer of the Drosophila seminal vesicle as a target of Juvenile Hormone (JH) signaling as evidenced by the transcription of two different reporters that are induced by the JH receptors Met and gce via previously identified JH response elements (JHRE). In agreement with this model, the JHRE-GFP reporter is not activated in Met/gce double RNAi knockdowns. Likewise, knockdown of JHAMT, a JH biosynthetic enzyme, reduces reporter expression. That this response is mediated by Juvenile Hormone (JH) is further supported by the finding that application of Methoprene, a JH analogue, through feeding …
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Referee #2
Evidence, reproducibility and clarity
Summary:
The authors identify the epithelial layer of the Drosophila seminal vesicle as a target of Juvenile Hormone (JH) signaling as evidenced by the transcription of two different reporters that are induced by the JH receptors Met and gce via previously identified JH response elements (JHRE). In agreement with this model, the JHRE-GFP reporter is not activated in Met/gce double RNAi knockdowns. Likewise, knockdown of JHAMT, a JH biosynthetic enzyme, reduces reporter expression. That this response is mediated by Juvenile Hormone (JH) is further supported by the finding that application of Methoprene, a JH analogue, through feeding of intact animals or by adding to cultured seminal vesicles, increases reporter expression. Mating, which has previously shown to increase JH levels, similarly increases reporter expression. By mining available RNA and protein data the authors identify Lactate dehydrogenase as a gene that is specifically expressed in the seminal vehicle under JH control. These findings suggest that metabolic processes in the seminal vesicle are regulated by JH and may be important for the function of this organ.
Major comments:
The claims and the conclusions are supported by the data
The data and the methods presented in such a way that they can be reproduced
The experiments adequately replicated and statistical analysis adequate
Optional suggestions for experiments that would enhance the current set of data and are not very time-intensive:
Document testis staining of JHRE-GFP. I think the authors missed a chance by not providing a clear/nice picture of the testis staining. Stainings of testes squashed on a slide is easy and would nicely document in which cells the reporter is activated. Similarly, extracting sperm from the seminal vesicle and examining whether the sperm express JHRE-GFP would be informative.
Did the authors try to analyze the 66 genes identified in seminal vesicle whether they had JHRE elements? This could yield additional significant information about other JH responsive genes in the seminal vesicle.
a) Doublestaining would further confirm that pd8-Gal4 (crossed to UAS-dsRed) and JHRE-GFP overlap.
b) Similarly, Doublestaining would further confirm that pd8-Gal4 (crossed to UAS-dsREd) and JHRE-GFP overlap.
Have the authors examined whether JHRE-lacZ expression increases with Methoprene?
Have the authors done a careful comparison of JHRE-GFP expression and the Met/gce reporter expression described by Baumann et al (Scientific Reports | 7: 2132 | DOI:10.1038/s41598-017-02264-4)? Would be nice to add a few more sentences in the discussion.
In the discussion:
a) Would have liked to see a more in depth discussion of the role of the seminal vesicle. How could that be supported by JH / metabolic processes? Does it have secretory functions that might be induced by JH? Important functions relative to sperm storage? How could that relate to the finding that JH response is enhanced by mating?
b) What do epithelial cells of spermatheca do?
c) How do the authors envision that JH enters the epithelial cells?
Minor comments:
Prior studies are referenced appropriately
The text and figures clear and accurate
Suggestions that would help the authors improve the presentation of their data and conclusions:
The arrow in figure is not defined
Figure 2b graph labels are flipped
Line 624: Change "Allow heads" to "Arrow heads"
Significance
General assessment / Advance:
While the developmental roles of insect Juvenile Hormone (JH) are very well studied, its adult functions are largely unknown. Target genes of JH signaling are poorly described. This study adds significant insight into both of these aspects. The study underscores the usefulness of the JHRE-GFP reporter that identifies JH function, and not just JH presence since the reporter is only expressed after JH binding to Met and gce, a prerequisite for JHRE reporter activation. The authors have identified the epithelial cells of the Drosophila seminal vesicle as a JH target tissue. The authors nicely extended this finding by mining already existing expression data to identify a specific JH induced gene in these cells.
Audience: Audience interested in the role of insect hormones in general or putative reproductive function (basic research and applied (insect control) will be interested in the finding and the approaches taken by the author.
Reviewer field of expertise: Drosophila sex-specific gene expression and function, molecular genetic approaches
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Referee #1
Evidence, reproducibility and clarity
This is an interesting and straightforward study that utilizes a recently developed in vivo sensor of juvenile hormone (JH) signaling in Drosophila. The authors focus on one understudied aspect of insect reproduction, the adult male seminal vesicle (SV), as a target of JH action. Using simple genetics and gaining from previous RNA-seq and proteomics data, the authors identify lactate dehydrogenase (Ldh) as a prime candidate gene positively regulated by JH in the SV. This regulation is potentially important for the SV physiology (metabolism?) and male reproduction, although this has not been addressed (see below).
As might be …
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Referee #1
Evidence, reproducibility and clarity
This is an interesting and straightforward study that utilizes a recently developed in vivo sensor of juvenile hormone (JH) signaling in Drosophila. The authors focus on one understudied aspect of insect reproduction, the adult male seminal vesicle (SV), as a target of JH action. Using simple genetics and gaining from previous RNA-seq and proteomics data, the authors identify lactate dehydrogenase (Ldh) as a prime candidate gene positively regulated by JH in the SV. This regulation is potentially important for the SV physiology (metabolism?) and male reproduction, although this has not been addressed (see below).
As might be expected based on the authors' skills and expertise, the study is well executed, nicely documented with perfect microscopy images, and well presented. It has been easy to follow. However, suitability for publication depends on where the authors aim to place their paper. Although I like the paper very much, it might seem incomplete for high-end journals.
Major comments:
- The study suggests an important role for JH signaling in the SV, likely affecting reproductive capacity of males. The authors depleted the JH receptors through RNAi, achieving a loss in the expression of the WT JHRE-GFP reporter as well as of the authentic target Ldh. Surprisingly, no phenotypic consequences of the double KD of Met and gce are presented. Does that mean that there were none? The authors only discus a potential impact of Ldh loss for metabolism.
Unless I am missing something, the study reports molecular phenotypes that clearly document JH signaling in the SV but no physiological impact of loss of this JH signaling, suggesting that there may be no obvious biological role for JH in this context. I think this is unlikely. Have the authors check fertility of the males, sperm viability and quality, mating competitiveness of the RNAi males? Loss of JH epoxidation (only methyl farnesoate present) made mosquito males less fit and less reproductively competitive relative to epox+ controls (Nouzova et al., 2021, PNAS) -- btw, I think the authors should discuss this paper.
The authors seem to have made no effort to distinguish between Met and Gce functions. It is always the results from the double knockdown of both paralogs that are presented. Does this mean that single-KD had no effect, thereby indicating entirely redundant functions of both proteins in the studied context? Even if so, it would be of interest to document this redundancy by showing the single-gene KD data. However, I would be surprised if both proteins were equally important in the SV. The authors checked mRNA/protein expression levels. Was any of the two paralogs prevalent in the SV?
The authors argue for direct regulation of Ldh by Met/Gce (again by which one?). Oddly, the statement in the Results (l.187-188; "suggests ... direct target") is stronger than in the Discussion (l.214, "leaving open the possibility"). The putative JHREs upstream and within the Ldh gene are identified but not tested in a functional study. At least a simple luciferase reporter assay and mutagenesis of the JHREs should be attempted.
Minor comments and suggestions (in the order of appearance):
l.25,91,117, and throughout, "JH analog" or "JHA". The authors only use methoprene, so it would be better to specifically talk about methoprene, which is a proven agonist ligand of the JHR proteins (reference 10 and/or Jindra and Bittova, 2020 [Arch Insect Biochem Physiol] for a review). This would land more credibility to using methoprene than just referring to a "JHA".
l.42,44, "paralogs". I believe in this case the authors refer to orthologs of Met in other species. Paralogs result from gene duplications within species, such as Met and gce in cyclorrhaphous flies or Met 1 and 2 in the Lepidoptera. I recommend a recent review on all bHLH-PAS proteins featuring reconstruction of the phylogenetic position of Met/Gce (Tumova et al., 2024 in J Mol Biol).
l.54, "Met and Gce act redundantly to regulate JH-responsive gene expression". Ref 10 should be cited here as it provides functional cell-based and genetic rescue evidence for each paralog.
l.66, It would be better to start "In this study" or "Here" to distinguish from the last cited paper.
l.175, levels were
l.209, might be evolutionarily among.... conserved ??
l.226, study has
l.227-229. The authors are missing a paper by Shin et al., 2012 (PNAS) that shows physical interaction of Met with Cycle and their regulation of circadian gene activity and another paper by Bajgar et al., 2013 (PNAS) which describes photoperid-dependent seasonal regulation of circadian genes by Met, Clk and Cyc.
On the other hand, the cited reference [51] does NOT demonstrate Met:Clk heterodimer since coIP is by no means adequate to address complex stoichiometry. In fact, it is suspicious that Met would heterodimerize and either Cyc or Clk, as they present class II and class I bHLH-PAS proteins.
l.232-233. It is not surprising that the JHRR-lacZ reporter shows a different expression pattern relative to JHRE-GFP, as these are really different constructs. The problem is that JH-dependent activation of the JHRR-lacZ transgene has not been tested as thoroughly as that of JHRE-GFP. Is it inducible by added JH or methoprene?
l.244, tract
l.245. It is not "whether", but for sure the existing reporters only reflect limited JHR activity, being based on Kr-h1 JHREs. These reporters likely uncover only a small subset of JH activity in vivo.
reference 10/11 is duplicated.
Significance
This is a very nice paper and solid piece of work.
Its major strength is the focus on poorly studied the male reproductive organ and identification of Ldh as a novel target of JH activity in the seminal vesicles.
The weakness is the limitation to molecular phenotypes without showing physiological relevance of JHR signaling in the seminal vesicles for male reproductive fitness. Evidence for the Ldh gene being directly regulated by the JHR is indirect.
These limitations will likely reduce the impact of this work although otherwise it would be of great interest to the larger community of developmental biologists and insect endocrinologists.
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