MicroRNA Novel-m0027-3p Negatively Regulates Jhamt Gene and Affects Juvenile Hormone Biosynthesis in Apis mellifera Larvae

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

In the revised manuscript, the authors corrected inappropriate methodological statements. Additional descriptions, such as the JH extraction method, strengthened their claims. However, there are still some overstatements and incorrect explanations throughout the manuscript, that should be corrected before publication.

 

 

1) In the current manuscript, the authors provide the evidences that Novel-m0027-3p downregulates Jhamt expression as well as JH titers during larval development. However, they do not demonstrate a causal relationship between these two effects. Nevertheless, the authors state as if Novel-m0027-3p reduces JH titers by downregulating Jhamt expression (e.g. in the revised title and the last sentence of the Discussion). Because Novel-m0027-3p has 2,109 potential targets (ll. 360-362), the authors did not rule out the possibility that Novel-m0027-3p mimic reduces JH titers through other target genes. In addition, the authors do not demonstrate that reduction of Jhamt expression in the CA after Novel-m0027-3p treatment, due to technical difficulty of dissection. Therefore, the authors cannot make this conclusion without providing additional evidences such as recovery of JH titers by overexpression of Jhamt. I understand the technical difficulty of manipulating gene expression in the honey bee. Therefore, the authors should tone down these statements throughout the manuscript to avoid overstatement.

 

2) The authors also claim that altered JH titers lead to changes in body sizes after Novel-m0027-3p mimic or inhibitor treatments (ll. 383-384 “thereby affecting larval body weight”), although this effect is limited in later sentences (ll. 389-394). The causal relationship between JH titers and body size is not experimentally supported in this study. Application of a JH analog to rescue the reduced body size after mimic treatment is strongly recommended to confirm that Novel-m0027-3p effects body size through reduced JH signaling. Otherwise, the Discussion should be revised to avoid overstatement.

 

3) In the figure 4A and D, JH titers in the control larvae (Mimic-NC and Inhibitor-NC) differ substantially, especially at day-5. The authors need to explain why this inconsistence happened. Because of these large differences between controls, it is unclear whether the observed differences in JH titers are caused by microRNA treatment or simply reflect biological variation among individuals.

 

4) Inconsistent figure formats should be corrected in Figure 4. The authors present individual data points in Figure 4C and F, but not in the other graphs. In addition, it is unclear how Kr-h1 expression levels were normalized across samples. In other figures, the mean expression level of control samples is shown as 1. However, this normalization does not appear to be applied to Figure 4C and F. Figure styles and normalization methods should be standardized throughout the manuscript.

 

5) There are several incorrect or unclear statements that should be corrected:

  - “JH is synthesized through the mevalonate pathway” (ll. 77-78). Although this sentence was just copied from Noriega, 2014, it is inaccurate. Farnesyl-pyrophosphate (FPP) is synthesized via the mevalonate pathway, after which JH is produced through the JH branch. This sentence should be corrected.

  - The sentence in ll. 115-116 is unclear. E75 and Usp are ecdysteroid response genes, not JH response genes. This sentence should be deleted.

  - “while the other target genes are downstream genes of the JH signaling pathway” (ll. 267-268). Although these genes are categorized as hormone pathway-related genes, many of them are not downstream targets of JH signaling. This sentence should be rephrased.

  - The description in ll. 291-292 is grammatically incorrect. This should be corrected. 

  - The description of miR-8 is inaccurate (ll. 375-376). First, miR-8 does not directly target Jhamt, because overexpression of miR-8 upregulates Jhamt expression. Second, the relationship between corpus allatum (CA) cell growth of and Jhamt expression is unclear. This statement should be corrected.

 

 

Other points:

6) “Juvenile hormone and Ecdysteroid” (l. 71) should be written in lowercase, as they are not protein names.

 

7) “frugiperda” (l. 99) should be written as “Spodoptera frugiperda”

 

8) Product information for the microplate reader (l. 229) should be provided.

 

9) Statistical analyses are unclearly described. The authors mention ANOVA in Section 2.9 but apply t-test in several figures (which should be specified as Student’s t-test). The statistical methods should be fully and consistently described. In addition, Figure 1C does not indicate the post-hoc test (Tukey’s HSD test) in the figure caption. This information should be added.

 

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4159525 entitled "MicroRNA Novel-m0027-3p Negatively Regulates Jhamt gene and Affects Juvenile Hormone Biosynthesis in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

 

Reviewer 1

Comments to the Author:

In the revised manuscript, the authors corrected inappropriate methodological statements. Additional descriptions, such as the JH extraction method, strengthened their claims. However, there are still some overstatements and incorrect explanations throughout the manuscript, that should be corrected before publication.

Response: Thank you very much for your comments concerning our manuscript. We have made concerted efforts to rectify certain overstated and incorrect interpretations in the manuscript, implementing numerous revisions in the hope of earning your approval.

Comment 1: In the current manuscript, the authors provide the evidences that Novel-m0027-3p downregulates Jhamt expression as well as JH titers during larval development. However, they do not demonstrate a causal relationship between these two effects. Nevertheless, the authors state as if Novel-m0027-3p reduces JH titers by downregulating Jhamt expression (e.g. in the revised title and the last sentence of the Discussion). Because Novel-m0027-3p has 2,109 potential targets (ll. 360-362), the authors did not rule out the possibility that Novel-m0027-3p mimic reduces JH titers through other target genes. In addition, the authors do not demonstrate that reduction of Jhamt expression in the CA after Novel-m0027-3p treatment, due to technical difficulty of dissection. Therefore, the authors cannot make this conclusion without providing additional evidences such as recovery of JH titers by overexpression of Jhamt. I understand the technical difficulty of manipulating gene expression in the honey bee. Therefore, the authors should tone down these statements throughout the manuscript to avoid overstatement.

Response 1: Accepted. To avoid overstating our findings and implying a causal relationship between these two effects, we have revised the manuscript title as well as the discussion sections.

Revision title: MicroRNA Novel-m0027-3p Negatively Regulates Jhamt gene and Affects Juvenile Hormone Biosynthesis in Apis mellifera Larvae

Revision discussion: In conclusion, this study confirms that novel-m0027-3p negatively regulates the expression of AmJhamt, and affects juvenile hormone biosynthesis in Apis mellifera larvae.

Comment 2: The authors also claim that altered JH titers lead to changes in body sizes after Novel-m0027-3p mimic or inhibitor treatments (ll. 383-384 “thereby affecting larval body weight”), although this effect is limited in later sentences (ll. 389-394). The causal relationship between JH titers and body size is not experimentally supported in this study. Application of a JH analog to rescue the reduced body size after mimic treatment is strongly recommended to confirm that Novel-m0027-3p effects body size through reduced JH signaling. Otherwise, the Discussion should be revised to avoid overstatement.

Response 2: Accepted. We have deleted the description of the body weight in lines 383-384

Comment 3: In the figure 4A and D, JH titers in the control larvae (Mimic-NC and Inhibitor-NC) differ substantially, especially at day-5. The authors need to explain why this inconsistence happened. Because of these large differences between controls, it is unclear whether the observed differences in JH titers are caused by microRNA treatment or simply reflect biological variation among individuals.

Response 3: We understand the reviewer's concern. Indeed, this phenomenon is difficult to explain at the molecular level, and we consider that it may be related to differences in the microenvironment during rearing, such as temperature or humidity, which could lead to minor developmental variations. During the experiment, larvae treated with Mimic-NC and Mimic were reared in the plate, while those treated with Inhibitor-NC and Inhibitor were reared together in another plate. Although differences were observed between the control larvae, the JH trends within each group (control and treatment) were relatively consistent, leading us to conclude that this is not caused by individual variation. We have added a relevant description in the Discussion section, and the revision is as follows:

Revision: Consistent with previous findings, our results demonstrate that upregulating or inhibiting the expression of Novel-m0027-3p can modulate AmJhamt expression and affect JH titers in larvae. This, in turn, suppresses or promotes the expression levels of downstream JH‑responsive genes, AmHex70b and AmKr-h1 (Fig. 4). Of note, the JH titers in the control groups (Mimic-NC and Inhibitor-NC) differed considerably, particularly in 5-day-old larvae. We speculate that this may be attributed to differences in the rearing environment between the two groups of larvae, such as temperature and humidity, leading to minor developmental variations.

Comment 4: Inconsistent figure formats should be corrected in Figure 4. The authors present individual data points in Figure 4C and F, but not in the other graphs. In addition, it is unclear how Kr-h1 expression levels were normalized across samples. In other figures, the mean expression level of control samples is shown as 1. However, this normalization does not appear to be applied to Figure 4C and F. Figure styles and normalization methods should be standardized throughout the manuscript.

Response 4: Accepted. We have revised the formatting of Figure 4C and 4F to ensure consistency with the other figures. The mean expression levels of the control samples were normalized to "1" to maintain uniformity throughout the manuscript. However, due to considerable variation among individual biological replicates in the control group of Figure 4C, the mean values deviated (the control group means for 4, 5, and 6 were 1.073, 1.098, and 1.242, respectively).

Comment 5: There are several incorrect or unclear statements that should be corrected:

  - “JH is synthesized through the mevalonate pathway” (ll. 77-78). Although this sentence was just copied from Noriega, 2014, it is inaccurate. Farnesyl-pyrophosphate (FPP) is synthesized via the mevalonate pathway, after which JH is produced through the JH branch. This sentence should be corrected.

  - The sentence in ll. 115-116 is unclear. E75 and Usp are ecdysteroid response genes, not JH response genes. This sentence should be deleted.

  - “while the other target genes are downstream genes of the JH signaling pathway” (ll. 267-268). Although these genes are categorized as hormone pathway-related genes, many of them are not downstream targets of JH signaling. This sentence should be rephrased.

  - The description in ll. 291-292 is grammatically incorrect. This should be corrected. 

  - The description of miR-8 is inaccurate (ll. 375-376). First, miR-8 does not directly target Jhamt, because overexpression of miR-8 upregulates Jhamt expression. Second, the relationship between corpus allatum (CA) cell growth of and Jhamt expression is unclear. This statement should be corrected.

Response 5: Thank you for your valuable suggestions, which have helped us improve the accuracy and clarity of our manuscript. We have carefully revised the manuscript as per your requirements.

Comment 6: “Juvenile hormone and Ecdysteroid” (l. 71) should be written in lowercase, as they are not protein names.

Response 6: Accepted. We have modified it in the manuscript.

Comment 7: “frugiperda” (l. 99) should be written as “Spodoptera frugiperda”.

Response 7: Accepted. We have modified it in the manuscript.

Comment 8: Product information for the microplate reader (l. 229) should be provided.

Response 8: Accepted. In accordance with the reviewer’s suggestion, we have added the model and manufacturer of the microplate reader to the JH Titer Assay section. The specific revision is as follows:

Revision: Absorbance (optical density, OD value) is measured at a wavelength of 450 nm using a microplate reader (Varioskan LUX, Thermos Fisher, USA).

Comment 9:  Statistical analyses are unclearly described. The authors mention ANOVA in Section 2.9 but apply t-test in several figures (which should be specified as Student’s t-test). The statistical methods should be fully and consistently described. In addition, Figure 1C does not indicate the post-hoc test (Tukey’s HSD test) in the figure caption. This information should be added.

Response 9: Thank you for your valuable suggestions. We have corrected all erroneous statistical methods.

 

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

This study identified a novel miRNA in Apis mellifera, designated novel-m0027-3p. It was shown to directly target and downregulate the expression of Jhamt, a rate-limiting enzyme gene in the juvenile hormone (JH) biosynthesis pathway. This suppression leads to reduced JH titer and decreased expression of downstream signaling genes, ultimately influencing larval development. The revised manuscript presents more complete content, clear logic, and a well-established experimental system. However, several aspects could still be optimized:

1. "Honeybee" is not entirely equivalent to "Apis mellifera". The use of these terms in the paper should be more precise to ensure academic rigor.
2. It is recommended to improve the layout and formatting of figure, such as Figure 1.
3. In the discussion, studies on other insects like Drosophila melanogaster and Spodoptera frugiperda are cited to illustrate that miRNA regulation of Jhamt is a conserved mechanism. However, the connection with Apis mellifera remains somewhat superficial. It is advisable to deepen this analysis, for instance, discuss the specificity of this mechanism in honeybees as social insects.It would enhance the novelty of the research.
4. It is recommended to include a discussion on the limitations of the study. For example, while novel-m0027-3p is predicted to target a large number of genes, this study mainly focused on AmJhamt. The actual regulatory roles of other predicted target genes remain important unresolved questions and could be proposed as future research directions in the discussion.

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4159525 entitled "MicroRNA Novel-m0027-3p Negatively Regulates Jhamt gene and Affects Juvenile Hormone Biosynthesis in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 2

Comments to the Author:

This study identified a novel miRNA in Apis mellifera, designated novel-m0027-3p. It was shown to directly target and downregulate the expression of Jhamt, a rate-limiting enzyme gene in the juvenile hormone (JH) biosynthesis pathway. This suppression leads to reduced JH titer and decreased expression of downstream signaling genes, ultimately influencing larval development. The revised manuscript presents more complete content, clear logic, and a well-established experimental system. However, several aspects could still be optimized:

Response: Thank you very much for your comments concerning our manuscript. The comments are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our research. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.

Comment 1: "Honeybee" is not entirely equivalent to "Apis mellifera". The use of these terms in the paper should be more precise to ensure academic rigor.

Response 1: Accepted. We carefully examined the manuscript and revised the imprecise expressions.

Comment 2: It is recommended to improve the layout and formatting of figure, such as Figure 1.

Response 2: Accepted. We modified the layout of Figures 1 and Figures 4.

Comment 3: In the discussion, studies on other insects like Drosophila melanogaster and Spodoptera frugiperda are cited to illustrate that miRNA regulation of Jhamt is a conserved mechanism. However, the connection with Apis mellifera remains somewhat superficial. It is advisable to deepen this analysis, for instance, discuss the specificity of this mechanism in honeybees as social insects.It would enhance the novelty of the research.

Response 3: Thank you very much for your comments. We agree that deepening the discussion on the specificity of miRNA-mediated Jhamt regulation in honeybees, particularly in the context of their social biology, would significantly enhance the novelty and impact of our study. The revised discussion are as follows:

Revision: In Locusta migratoria, miR‑276 has been shown to negatively regulate Jhamt expression, and knocking down Jhamt leads to premature molting, precocious metamorphosis, and impaired vitellogenesis in nymphs [30]. In Drosophila melanogaster, Overexpression of miR-8 in the corpora allata increased the cell size of the gland and elevated the expression level of Jhamt. [47]. Furthermore, the miR‑9993/miR‑2a‑3p−FPPS/JHAMT−JH pathway mediates larval and pupal development in Spodoptera frugiperda [31]. JH is considered to play a key role in regulating the caste differentiation of social insects such as honeybees [41,42]. The regulatory effect of novel-m0027-3p on AmJhamt may influence JH titers, thereby impacting Apis mellifera caste differentiation. The ability of miRNAs to modulate gene expression subtly and efficiently makes them ideal candidates for regulating the complex developmental pathways that produce distinct phenotypes within a colony.

Comment 4: It is recommended to include a discussion on the limitations of the study. For example, while novel-m0027-3p is predicted to target a large number of genes, this study mainly focused on AmJhamt. The actual regulatory roles of other predicted target genes remain important unresolved questions and could be proposed as future research directions in the discussion.

Response 4: Thank you very much for your comments. We agree that acknowledging the limitations of the study and discussing the unresolved questions regarding other potential target genes will enhance the scientific rigor and transparency of our manuscript. The revised discussion are as follows:

Revision: Our results suggested that novel‑m0027‑3p regulate AmJhamt, thereby affecting JH levels to a certain extent, but its impact on larval body weight is relatively limited. The potential hormone-related target genes bound by novel-m0027-3p are not limited to AmJhamt but may also include key hormonal signaling factors such as AmE75 and AmUsp. Therefore, the alteration in larval body weight induced by novel-m0027-3p may not be mediated solely through AmJhamt. Although novel-m0027-3p is predicted to target a large number of genes, this study primarily focused on AmJhamt. The regulatory roles of other predicted target genes remain important unresolved questions, representing a direction for future research. In conclusion, this study confirms that novel‑m0027‑3p negatively regulates the expression of AmJhamt, and affects juvenile hormone biosynthesis in Apis mellifera larvae.

 

Reviewer 3 Report (Previous Reviewer 4)

Comments and Suggestions for Authors

Accept in present form

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4159525 entitled "MicroRNA Novel-m0027-3p Negatively Regulates Jhamt gene and Affects Juvenile Hormone Biosynthesis in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 3

Comments to the Author:

Accept in present form 

Response: We sincerely thank you and the reviewers for the time and effort dedicated to evaluating the manuscript.

Reviewer 4 Report (Previous Reviewer 5)

Comments and Suggestions for Authors

The study demonstrates that the miRNA novel-m0027-3p negatively regulates the expression of the JHAMT gene in Apis mellifera larvae, then mediating JH biosynthesis. It is a worthy work for understanding of the regulation of insect development. It is a relatively meaningful study. The manuscript is well-written, the data are of high quality, and the conclusions are supported by the results. The revisions have addressed the previous technical concerns. I have no additional comments.

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4159525 entitled "MicroRNA Novel-m0027-3p Negatively Regulates Jhamt gene and Affects Juvenile Hormone Biosynthesis in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 4

Comments to the Author:

The study demonstrates that the miRNA novel-m0027-3p negatively regulates the expression of the JHAMT gene in Apis mellifera larvae, then mediating JH biosynthesis. It is a worthy work for understanding of the regulation of insect development. It is a relatively meaningful study. The manuscript is well-written, the data are of high quality, and the conclusions are supported by the results. The revisions have addressed the previous technical concerns. I have no additional comments.

Response: We sincerely thank you and the reviewers for the time and effort dedicated to evaluating the manuscript.

Reviewer 5 Report (Previous Reviewer 6)

Comments and Suggestions for Authors

The author has addressed most of my concerns through the revisions, and I support the publication of the manuscript in its current form.

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4159525 entitled "MicroRNA Novel-m0027-3p Negatively Regulates Jhamt gene and Affects Juvenile Hormone Biosynthesis in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 5

Comments to the Author:

The author has addressed most of my concerns through the revisions, and I support the publication of the manuscript in its current form.

Response: We sincerely thank you and the reviewers for the time and effort dedicated to evaluating the manuscript.

Round 2

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

In the revised manuscript, the authors have addressed all of my previous comments. The descriptions have been carefully revised to avoid overstatement of their findings. Therefore, the manuscript is now acceptable for publication.

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4159525 entitled "MicroRNA Novel-m0027-3p Negatively Regulates Jhamt gene and Affects Juvenile Hormone Biosynthesis in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

 

Reviewer 1

Comments to the Author:
In the revised manuscript, the authors have addressed all of my previous comments. The descriptions have been carefully revised to avoid overstatement of their findings. Therefore, the manuscript is now acceptable for publication.

Response: We sincerely thank you and the reviewers for the time and effort dedicated to evaluating the manuscript.

 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the current manuscript, Wang and others examined a microRNA, Novel-m0027-3p, that interacts with the mRNA of a Juvenile hormone (JH) synthetic enzyme, jhamt using the honey bee, Apis mellifera. JH is a pleiotropic hormone that regulates multiple biological processes, such as metamorphosis and reproduction in insects. Although several reports have identified multiple microRNAs that regulate jhamt expression, studies in bees remain limited.

The authors previously reported RNA-seq data the larval gut of the honey bee. Based on this dataset, the authors hypothesized that Novel-m0027-3p is a potential negative regulator of jhamt expression. To test this hypothesis, the authors analyzed the function of Novel-m0027-3p using cell culture techniques and in vivo analyses with a microRNA agonist and antagonist.

Considering the importance of JH in insect physiology, and the contribution of pollinators to agriculture, the current study has a potential to attract a wide variety of researchers. Unfortunately, however, the provided data are difficult to interpret properly due to inappropriate methodologies and conclusions, as detailed below.

 

 

Major points:

1) The authors identified Novel-m0027-3p in the gut. Because JH is produced in a specific endocrine gland, corpora allata (CA; Bomtorin et al., PLoS one, 2014), it is unclear whether Novel-m0027-3p inhibits JH production. Although a few studies suggests that jhamt is expressed in the gut to activate local JH signaling (Rahman et al., Sci. Rep., 2017), the CA is the main source of JH during larval development (Jindra et al., Annul. Rev. Entomol., 2013). Therefore, Novel-m0027-3p should be expressed in the CA if it regulates JH production by inhibiting jhamt expression. In the current manuscript, the authors analyzed expression of Novel-m0027-3p and jhamt using cDNA generated from the whole bodies or guts. The authors should analyze the expression of Novel-m0027-3p and jhamt in the CA.

 

2) Although the authors showed that application of Novel-m0027-3p mimic decreases the JH amount in body homogenates (Fig. 4), this experiment was performed using an inappropriate methodology. The authors used the supernatant of larval homogenates dissolved in physiological saline (ll. 192-193). Because JH is an aliphatic compound, it cannot be extracted into the aquatic phase (Noriega et al., Curr. Opin. Insect Sci., 2025). Therefore, the results in Figure 4 likely reflect immuno-crossreactivity of the antibody used in the JH detection kit rather than true JH levels. The authors need to extract JH using an appropriate extraction method.

 

3) The authors analyzed the gene expression of hexamerin (Hex) and ultraspiracle (Usp) as JH response genes (Figure 4). These analyses are inappropriate. Although studies suggest that JH interacts with Usp protein to modulate its activity (Jones et al., FEBS J, 2006), no study demonstrated that JH induces Usp transcription, to the best of my knowledge. Considering that JH antagonizes ecdysteroid signaling, and that ecdysteroids induce Usp expression, it is more likely that JH represses Usp expression. Moreover, it remains unclear whether JH III actively modulates Usp activity in vivo due to its low binding affinity (Jindra et al., Annul. Rev. Entomol., 2013).

Furthermore, the cited article (Martins et al., BMC. Mol. Biol., 2010) did not demonstrate that JH induces Hex expression. This study suggests the presence of potential Usp DNA-binding sites at Hex-encoding loci. Although it is possible that Usp regulates Hex expression, JH is unlikely to be involved in this interaction. The authors need to provide appropriate citations or experimental data demonstrating direct evidence that JH induces transcription of these genes.

Kruppel homolog 1 (Kr-h1) is widely used as a JH response gene (Minakuchi et al., Mech. Dev., 2008), should be analyzed to confirm JH signaling.

 

4) It is unclear whether the observed changes in body size depend on JH signaling following treatment with mimic or inhibitor of Novel-m0027-3p. Rescue experiments such, as treatment of a JH analog (e.g. methoprene), should be conducted to confirm that Novel-m0027-3p regulates larval body size through JH signaling.

 

 

Other points that should be corrected:

 

5) The term “microRNA” should be added to the title (e.g. “The microRNA, Novel-Novel-m0027-3p Negatively Regulates Jhamt Gene to Mediate 2 Juvenile Hormone Biosynthesis”).

 

6) Several articles are cited inappropriately in the introduction. For example, review articles should be cited when providing general background information (e.g. citations 1, 4, and 10). In addition, studies identifying microRNAs that regulate jhamt expression should be added in the introduction (ll. 86-88).

 

7) The roles of JH and 20E are unclear in the current text (ll. 72-73). The authors need to explain these hormones in more detail before describing the roles of known miRNAs in their regulation. For example, the functions of let-7 in hormone production should be fully explained (ll 84-85).

 

8) “Ecdysterone” is not standard nomenclature and should be replaced with “Ecdysteroid” (l. 73).

 

9) Methods for the synthesis of microRNA mimics and inhibitors should be described in detail (ll. 156-158).

 

10) The source of HEK cell culture medium (DMEM, FBS, and penicillin-streptomycin) should be specified.

 

11) In Figure S1, the names of GO terms should be clearly described in the figure or legend.

 

12) In Figure 2C, an appropriate multiple-comparison test should be applied to compare relative activity between cells transfected with jhamt-wt and jhamt-mut constructs.

 

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4091456 entitled "MicroRNA Novel-m0027-3p Regulates Juvenile Hormone Biosynthesis by Targeting Jhamt in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

The revision has met the format specification of your reminder, and the English grammar was edited by a native English speaker from International Science Editing Scientific Services.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

 

Reviewer 1

Comments to the Author:

In the current manuscript, Wang and others examined a microRNA, Novel-m0027-3p, that interacts with the mRNA of a Juvenile hormone (JH) synthetic enzyme, jhamt using the honey bee, Apis mellifera. JH is a pleiotropic hormone that regulates multiple biological processes, such as metamorphosis and reproduction in insects. Although several reports have identified multiple microRNAs that regulate jhamt expression, studies in bees remain limited.

The authors previously reported RNA-seq data the larval gut of the honey bee. Based on this dataset, the authors hypothesized that Novel-m0027-3p is a potential negative regulator of jhamt expression. To test this hypothesis, the authors analyzed the function of Novel-m0027-3p using cell culture techniques and in vivo analyses with a microRNA agonist and antagonist.

Considering the importance of JH in insect physiology, and the contribution of pollinators to agriculture, the current study has a potential to attract a wide variety of researchers. Unfortunately, however, the provided data are difficult to interpret properly due to inappropriate methodologies and conclusions, as detailed below.

Response: Thank you very much for your comments concerning our manuscript. The comments of Reviewers and Associate Editor are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our research. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.

Comment 1: The authors identified Novel-m0027-3p in the gut. Because JH is produced in a specific endocrine gland, corpora allata (CA; Bomtorin et al., PLoS one, 2014), it is unclear whether Novel-m0027-3p inhibits JH production. Although a few studies suggests that jhamt is expressed in the gut to activate local JH signaling (Rahman et al., Sci. Rep., 2017), the CA is the main source of JH during larval development (Jindra et al., Annul. Rev. Entomol., 2013). Therefore, Novel-m0027-3p should be expressed in the CA if it regulates JH production by inhibiting jhamt expression. In the current manuscript, the authors analyzed expression of Novel-m0027-3p and jhamt using cDNA generated from the whole bodies or guts. The authors should analyze the expression of Novel-m0027-3p and jhamt in the CA.

Response 1: Thank you very much for your comments. We analyzed the expression of novel‑m0027‑3p and AmJhamt using cDNA from whole larval bodies and measured JH titers. We fully agree with the reviewer’s suggestion regarding analyzing the expression of novel‑m0027‑3p and AmJhamt in the corpora allata (CA). However, due to the extremely small size of the CA in honeybee larvae, complete dissection or isolation presents a significant technical challenge. Current studies have only succeeded in isolating the corpora cardiaca‑corpora allata (CC‑CA) complex from larvae 7 days after hatching (Bomtorin et al., 2014), and adult studies also rely on the CC‑CA complex (Audsley and Weaver, 2005).

We attempted to isolate the CC‑CA from 4‑ to 6‑day‑old larvae but were unfortunately unsuccessful. On the other hand, AmJhamt expression in the CA has been demonstrated in various insect species. Although novel‑m0027‑3p was initially identified in the gut, as a small nucleic acid molecule, it is capable of crossing cellular barriers, including those of gut and CA cells. Therefore, we believe that examining the expression profiles of novel‑m0027‑3p and AmJhamt in whole larvae does not compromise the interpretation of their regulatory relationship. Instead, whole‑larva expression profiles may provide a more comprehensive overview.

Reference

• Bomtorin AD, Mackert A, Rosa GC, Moda LM, Martins JR, Bitondi MM, Hartfelder K, Simões ZL. Juvenile hormone biosynthesis gene expression in the corpora allata of honey bee (Apis mellifera L.) female castes. PLoS One. 2014, 9(1):e86923. doi: 10.1371/journal.pone.0086923.
• Audsley N, Weaver RJ. Analysis of peptides in the brain and corpora cardiaca-corpora allata of the honey bee, Apis mellifera using MALDI-TOF mass spectrometry. Peptides. 2006, 27(3):512-520. doi: 10.1016/j.peptides.2005.08.022.

Comment 2: Although the authors showed that application of Novel-m0027-3p mimic decreases the JH amount in body homogenates (Fig. 4), this experiment was performed using an inappropriate methodology. The authors used the supernatant of larval homogenates dissolved in physiological saline (ll. 192-193). Because JH is an aliphatic compound, it cannot be extracted into the aquatic phase (Noriega et al., Curr. Opin. Insect Sci., 2025). Therefore, the results in Figure 4 likely reflect immuno-crossreactivity of the antibody used in the JH detection kit rather than true JH levels. The authors need to extract JH using an appropriate extraction method.

Response 2: We apologize for the misunderstanding caused by omissions in the method description. We extracted JH following the protocol established in a previous study (Qiu et al., 2023). Briefly, larval homogenates were mixed with n-hexane and 70% methanol, centrifuged, and the upper n-hexane layer was collected and dried. Subsequently, 100 μL of standard diluent buffer (PBS buffer containing 1% bull serum albumin and 0.05% Tween-20) was added.

We have provided a more detailed description of the JH detection method. The revised version is as follows:

Revision: The 4- to 6-day-old larvae (n = 3 per time point) fed with Mimics, Inhibitors, Mimic‑NC, or Inhibitor‑NC were collected, rinsed three times with phosphate buffered saline (PBS), and surface moisture was gently removed. An enzyme-linked immunosorbent assay (ELISA) kit (ml321040, mlbio, China) was used to detect juvenile hormone (JH) titer following the manufacturer's instructions, with improvements made during JH extraction from larvae (Qiu et al., 2023; Chen et al., 2024). Briefly, each sample was homogenized in 200 μL of physiological saline on ice, followed by the addition of 1000 μL of n-hexane and 500 μL of 70% methanol. The mixture was centrifuged at 12,000 rpm for 10 minutes. The upper n-hexane layer was collected, and the solvent was removed using a freeze-dryer (ALPHAL-2, Christ, Germany). Subsequently, 100 μL of standard diluent buffer (PBS buffer containing 1% Bull Serum Albumin and 0.05% Tween-20) was added. Add 50μL of the diluted standard and 50μL of the sample to the reaction wells respectively, then immediately add 50 μL of horseradish peroxidase (HRP)-labeled antibody to allow the hormone to bind with the antibody, forming an antibody–antigen–enzyme-labeled antibody complex. After thorough washing, 3,3′,5,5′-tetramethylbenzidine (TMB) is added. Due to the catalytic action of HRP, TMB is initially converted into a blue product. Under acidic conditions, it turns into a yellow product. The intensity of the color is positively correlated with the insect hormone level in the sample. Absorbance (optical density, OD value) is measured at a wavelength of 450 nm using a microplate reader. The assay was performed with three biological replicates, each consisting of three technical replicates.

Reference

• Chen JJ, Liu XX, Guo PH, Teets NM, Zhou JC, Chen WB, Luo QZ, Kanjana N, Li YY, Zhang LS. Regulation of forkhead box O transcription factor by insulin signaling pathway controls the reproductive diapause of the lady beetle, Coccinella septempunctata. Int J Biol Macromol. 2024, 258(Pt 1):128104. doi: 10.1016/j.ijbiomac.2023.128104.
• Qiu J, Dai T, Luo C, Cui W, Liu K, Li J, Sima Y, Xu S. Circadian clock regulates developmental time through ecdysone and juvenile hormones in Bombyx mori. Insect Mol Biol. 2023, 32(4):352-362. doi: 10.1111/imb.12835.

Comment 3: The authors analyzed the gene expression of hexamerin (Hex) and ultraspiracle (Usp) as JH response genes (Figure 4). These analyses are inappropriate. Although studies suggest that JH interacts with Usp protein to modulate its activity (Jones et al., FEBS J, 2006), no study demonstrated that JH induces Usp transcription, to the best of my knowledge. Considering that JH antagonizes ecdysteroid signaling, and that ecdysteroids induce Usp expression, it is more likely that JH represses Usp expression. Moreover, it remains unclear whether JH III actively modulates Usp activity in vivo due to its low binding affinity (Jindra et al., Annul. Rev. Entomol., 2013).

Furthermore, the cited article (Martins et al., BMC. Mol. Biol., 2010) did not demonstrate that JH induces Hex expression. This study suggests the presence of potential Usp DNA-binding sites at Hex-encoding loci. Although it is possible that Usp regulates Hex expression, JH is unlikely to be involved in this interaction. The authors need to provide appropriate citations or experimental data demonstrating direct evidence that JH induces transcription of these genes.

Kruppel homolog 1 (Kr-h1) is widely used as a JH response gene (Minakuchi et al., Mech. Dev., 2008), should be analyzed to confirm JH signaling.

Response 3: We fully agree with the reviewer's comment regarding USP. Indeed, it is widely recognized in our knowledge that USP serves as the receptor for ecdysteroids rather than juvenile hormone. Accordingly, we have removed the USP‑related results and supplemented the expression profile of Kr‑h1, the JH receptor.

However, regarding hexamerin, the hexamerin family represents the most important storage proteins in insects, which are secreted by the fat body and released into the hemolymph to support physiological activities (Haunerland, 1996). In addition, proteins containing hexameric domain are also involved in juvenile hormone transport (Braun and Wyatt, 1996; Tawfik et al., 2006; Gilbert et al., 2000). More importantly, multiple lines of evidence support that JH induces Hex expression, as shown in Fig. 3 of the cited article (Martins et al., BMC Mol. Biol., 2010), and Fig. 6 of Zhu et al. (2024), and Zhou et al. (2006). In social insects, JH plays a crucial role in caste differentiation, while hexamerin proteins are also essential for this process.

We have supplemented the introduction to make the evidence more comprehensive. The revised version is as follows:

Revision: Synthesized JH is released into the hemolymph and transported via the hemolymph to various tissues. Among these, Hexamerin (Hex) is synthesized in the fat body and serves as one of the most crucial storage proteins in insects (Haunerland, 1996). It is also involved in the transport of JH (Braun and Wyatt, 1996; Tawfik et al., 2006; Gilbert et al., 2000). Research on the Western honey bee [16], the American cockroach (Zhu et al., 2024), and termites (Zhou et al., 2006) has demonstrated that JH can induce the expression of Hex. However, the main response gene of JH is Kruppel homolog 1 (Kr-h1) (Minakuchi et al., 2008). A direct or indirect regulatory interplay exists between miRNAs and hormonal pathways in insects.

Reference

• Haunerland NH. Insect storage proteins: gene families and receptors. Insect Biochem Mol Biol. 1996 Sep-Oct;26(8-9):755-65. doi: 10.1016/s0965-1748(96)00035-5. PMID: 9014325.
• Braun RP, Wyatt GR. Sequence of the hexameric juvenile hormone-binding protein from the hemolymph of Locusta migratoria. J Biol Chem. 1996 Dec 6;271(49):31756-62. doi: 10.1074/jbc.271.49.31756. PMID: 8940201.
• Tawfik AI, Kellner R, Hoffmann KH, Lorenz MW. Purification, characterisation and titre of the haemolymph juvenile hormone binding proteins from Schistocerca gregaria and Gryllus bimaculatus. J Insect Physiol. 2006 Mar;52(3):255-68. doi: 10.1016/j.jinsphys.2005.11.005. Epub 2005 Dec 27. PMID: 16384579.
• Gilbert LI, Granger NA, Roe RM. The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem Mol Biol. 2000 Aug-Sep;30(8-9):617-44. doi: 10.1016/s0965-1748(00)00034-5. PMID: 10876106.
• Zhou X, Oi FM, Scharf ME. Social exploitation of hexamerin: RNAi reveals a major caste-regulatory factor in termites. Proc Natl Acad Sci U S A. 2006 Mar 21;103(12):4499-504. doi: 10.1073/pnas.0508866103. Epub 2006 Mar 14. PMID: 16537425; PMCID: PMC1450200.
• Zhu S, Chen X, Xia S, Li Q, Ye Z, Zhao S, Liu K, Liu F. Hexamerin and allergen are required for female reproduction in the American cockroach, Periplaneta americana. Insect Sci. 2024 Feb;31(1):186-200. doi: 10.1111/1744-7917.13218. Epub 2023 Jun 16. PMID: 37327125.
• Minakuchi C, Zhou X, Riddiford LM. Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster. Mech Dev. 2008 Jan-Feb;125(1-2):91-105. doi: 10.1016/j.mod.2007.10.002. Epub 2007 Oct 11. PMID: 18036785; PMCID: PMC2276646.

 

 

Comment 4: It is unclear whether the observed changes in body size depend on JH signaling following treatment with mimic or inhibitor of Novel-m0027-3p. Rescue experiments such, as treatment of a JH analog (e.g. methoprene), should be conducted to confirm that Novel-m0027-3p regulates larval body size through JH signaling.

Response 4: Thank you very much for your valuable comment. The aim of measuring larval body weight was to investigate the impact of novel-m0027-3p on JH content, given the established causal relationship between JH levels and larval growth. In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. A limitation is that only body weight data were used to assess the larval phenotypes resulting from the changes in JH content induced by novel‑m0027‑3p. Because other conventional morphological or developmental markers for JH action are either absent or not applicable at this life stage (Larval stage to pupal stage). Therefore, I do not believe it is necessary to further demonstrate the effect of JH on body weight through JH analog treatment.

We supplemented the relevant descriptions in the discussion section:

Revision: This, in turn, suppresses or promotes the expression levels of downstream JH‑responsive genes, AmHex70b and AmUsp (Fig. 4), thereby affecting larval body weight (Fig. 5). In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. These findings align with our previous study showing that Ame‑miR‑2161 affects the larval‑to‑pupal transition in honey bees via the AmJhamt gene [38]. Our results suggested that novel‑m0027‑3p regulate AmJhamt, thereby affecting JH levels to a certain extent, but its impact on larval body weight is relatively limited. The potential hormone-related target genes bound by novel-m0027-3p are not limited to AmJhamt but may also include key hormonal signaling factors such as AmE75 and AmUsp. Therefore, the alteration in larval body weight induced by novel-m0027-3p may not be mediated solely through AmJhamt. In conclusion, this study confirms that novel‑m0027‑3p mediates JH synthesis by negatively regulating the expression of AmJhamt.

• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

Comment 5: The term “microRNA” should be added to the title (e.g. “The microRNA, Novel-Novel-m0027-3p Negatively Regulates Jhamt Gene to Mediate 2 Juvenile Hormone Biosynthesis”).

Response 5: Accepted. We agree and have revised the title to explicitly include “microRNA” and improve specificity: MicroRNA Novel-m0027-3p Regulates Juvenile Hormone Biosynthesis by Targeting Jhamt in Apis mellifera Larvae

Comment 6: Several articles are cited inappropriately in the introduction. For example, review articles should be cited when providing general background information (e.g. citations 1, 4, and 10). In addition, studies identifying microRNAs that regulate jhamt expression should be added in the introduction (ll. 86-88).

Response 6: Thank you very much for your valuable comment. We replaced or deleted inappropriate references ([1]-[4]), and supplemented relevant studies on the regulation of Jhamt expression by miRNA in other insects. The modifications are as follows:

Revision: A direct or indirect regulatory interplay exists between miRNAs and hormonal path-ways in insects. 20-hydroxyecdysone (20E) or JH can induce or suppress the expression of let-7 [Sempere et al., 2002,2003]. Conversely, miRNAs can also indirectly modulate insect hormone production by targeting mRNA [Li et al., 2025]. Studies have shown that the miRNA bantam in Drosophila melanogaster negatively regulates the expression of Jhamt, affecting larval metamorphosis (Qu et al., 2017). miR-276 and miR-182013-5p influence metamorphosis and reproduction in migratory locusts by regulating Jhamt [Song et al., 2024]. Spinetoram and cyantraniliprole disrupt juvenile hormone synthesis by mediating the expression of the farnesyl diphosphate synthase gene (FPPS) and Jhamt through microRNA-9993/microRNA-2a-3p, leading to mortality in frugiperda (Wang et al., 2025). Furthermore, Although the regulation of Jhamt by miRNAs has been reported in several insect species mentioned above, research on this interaction in honeybees remains limited.

Reference

• Naeli P, Winter T, Hackett AP, Alboushi L, Jafarnejad SM. The intricate balance between microRNA-induced mRNA decay and translational repression. FEBS J. 2023 May;290(10):2508-2524. doi: 10.1111/febs.16422. Epub 2022 Mar 11. PMID: 35247033.
• Bartel MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004 Jan 23;116(2):281-97. doi: 10.1016/s0092-8674(04)00045-5. PMID: 14744438.
• Zhang B, Wang Q, Pan X. MicroRNAs and their regulatory roles in animals and plants. J Cell Physiol. 2007 Feb;210(2):279-89. doi: 10.1002/jcp.20869. PMID: 17096367.
• Jindra M, Palli SR, Riddiford LM. The juvenile hormone signaling pathway in insect development. Annu Rev Entomol. 2013;58:181-204. doi: 10.1146/annurev-ento-120811-153700. Epub 2012 Sep 17. PMID: 22994547.
• Wang A, Yang Y, Zhang Y, Xue C, Cheng Y, Zhang Y, Zhang W, Zhao M, Zhang J. Insecticide-induced sublethal effect in the fall armyworm is mediated by miR-9993/miR-2a-3p - FPPS/JHAMT - JH molecular module. Pestic Biochem Physiol. 2025 May;210:106400. doi: 10.1016/j.pestbp.2025.106400. Epub 2025 Apr 1. PMID: 40262877.
• Qu Z, Bendena WG, Nong W, Siggens KW, Noriega FG, Kai ZP, Zang YY, Koon AC, Chan HYE, Chan TF, Chu KH, Lam HM, Akam M, Tobe SS, Lam Hui JH. MicroRNAs regulate the sesquiterpenoid hormonal pathway in Drosophila and other arthropods. Proc Biol Sci. 2017 Dec 20;284(1869):20171827. doi: 10.1098/rspb.2017.1827. PMID: 29237851; PMCID: PMC5745405.

 

Comment 7: The roles of JH and 20E are unclear in the current text (ll. 72-73). The authors need to explain these hormones in more detail before describing the roles of known miRNAs in their regulation. For example, the functions of let-7 in hormone production should be fully explained (ll 84-85).

Response 7: We have supplemented the introduction. The modifications are as follows:

Revision: The timing of molting and metamorphosis in holometabolous insects is primarily regulated by hormones, including Juvenile Hormone (JH) and Ecdysteroid[Hiruma and Kaneko, 2013]. Ecdysteroids are synthesized in the prothoracic glands and secreted into the hemolymph, where they are converted to the active hormone 20-hydroxyecdysone (20E), which triggers larval molting and metamorphosis (Scanlan et al., 2023). It is generally believed that the role of JH is to prevent premature metamorphosis, allowing larvae to undergo multiple rounds of molting until they reach an appropriate size (Jindra et al., 2012). This indicates that the regulation of developmental transitions in insects by ecdysteroids and JH is antagonistic. JH is synthesized through the mevalonate pathway (MVAP). Acetyl-CoA undergoes a series of enzymatic reactions to produce farnesyl pyrophosphate (FPP). FPP is then converted into farnesoic acid by the actions of farnesyl pyrophosphatase and farnesol dehydrogenase. Finally, farnesoic acid is catalyzed by Juvenile hormone acid methyl-transferase (JHAMT) to form JH (Noriega, 2014). JHAMT is the rate-limiting enzyme controlling JH synthesis. The Jhamt gene has been cloned in various insect species such as the Bombyx mori [12], Drosophila melanogaster [13], Tribolium castaneum [14] and Aedes aegypti [15].

Reference

• Hiruma, K.; Kaneko, Y. Hormonal regulation of insect metamorphosis with special reference to juvenile hormone biosynthesis. Curr. Top. Dev. Biol. 2013, 103: 73-100.
• Jindra M, Palli SR, Riddiford LM. The juvenile hormone signaling pathway in insect development. Annu Rev Entomol. 2013, 58:181-204. doi: 10.1146/annurev-ento-120811-153700.
• Scanlan JL, Robin C, Mirth CK. Rethinking the ecdysteroid source during Drosophila pupal-adult development. Insect Biochem Mol Biol. 2023, 152:103891. doi: 10.1016/j.ibmb.2022.103891.
• Noriega, F. G.; Bloch, G.; Moos, M.; Simek, P.; Jindra, M. Approaches to Quantify and Manipulate Insect Hormone Signals. Curr. Opin. Insect Sci. 2025, 72, 101425. https://doi.org/10.1016/j.cois.2025.101425.

Comment 8: “Ecdysterone” is not standard nomenclature and should be replaced with “Ecdysteroid” (l. 73).

Response 8: Accepted. We have modified it in the manuscript.

Comment 9: Methods for the synthesis of microRNA mimics and inhibitors should be described in detail (ll. 156-158).

Response 9: We have supplemented the synthesis methods of microRNA mimics and inhibitors, which are for chemical synthesis. The modifications are as follows:

In the Materials and Methods section

Revision: Based on the sequence of novel-m0027-3p, the mature novel-m0027-3p sequence was simulated as miRNA mimics (double-stranded), while the antisense strand of the novel-m0027-3p sequence was used as miRNA inhibitors (single-stranded). At the same time, their corresponding negative controls (Mimic-NC and Inhibitor-NC) were designed and chemically synthesized by Sangon Biotech (Sangon Biotech, China). All oligonucleotides were fully modified with 2′-O-methyl and phosphonothioates linkages to enhance stability and were HPLC-purified. The sequences are listed in Supplementary Table 2.

Comment 10: The source of HEK cell culture medium (DMEM, FBS, and penicillin-streptomycin) should be specified.

Response 10: Accepted. We have added the manufacturer and catalog information for DMEM, FBS, and penicillin–streptomycin in the cell culture Methods section.

Revision: HEK‑293T cells were maintained in DMEM (6125546, Gibco, USA) supplemented with 10% fetal bovine serum (3168648, Gibco, USA) and 1% penicillin‑streptomycin (60162ES76, Yeasen, China), and cultured at 37 ℃ under 5% CO₂ and 95% relative humidity.

Comment 11: In Figure S1, the names of GO terms should be clearly described in the figure or legend.

Response 11: Accepted. We revised legends of Figure S1 and Figure S2 to display full GO term names, improving readability and interpretability.

Comment 12: In Figure 2C, an appropriate multiple-comparison test should be applied to compare relative activity between cells transfected with jhamt-wt and jhamt-mut constructs.

Response 12: Thank you very much for your valuable comment. We normalized the Mimics-NC treatment to "1" within the jhamt-wt group and the jhamt-mut group, respectively, for intra-group comparisons, so the use of the t-test was appropriate. We have supplemented the explanation in the caption of the Figure 2C.

 

Reviewer 2 Report

Comments and Suggestions for Authors

This study focuses on the molecular regulatory mechanism of honeybee larva development, it identifies and systematically elucidates the crucial role of a novel miRNA, novel-m0027-3p, in regulating honeybee larva development. Also, it confirms that novel-m0027-3p mediates JH synthesis by negatively regulating the expression of AmJhamt.

The paper has a complete structure, clear logic, and provides multi-level experimental evidence.The system of the experimental is complete. The detailed description of the method and the chain of evidence provide convincing experimental evidence for the topic. However, there are still deficiencies in the paper that need to be revised.

Suggestion:

1. Check whether to spell "AmJhamt" or "Amjhamt". For example, different forms of spelling appear in lines 35 and 39. It is recommended to unify the format.
2. The content of "Hex" and "USP" in the third paragraph of the introduction is incongruity with the previous content. It is suggested to add content for transition to make the paragraph more smooth.
3. In line 305 of the discussion section, it is suggested to use the plural form of "insect".
4. What is the purpose of measuring the body weight of larva? If this method is to characterize "development" through changes in body weight, it is simple and quantifiable in operation, but it does have limitations in terms of scientific rigor and depth of biological significance. Can this study comprehensively confirm the effect of AmJhamt on larva development through more indicators or discussion.
5. As the article mentioned, prediction of novel-m0027-3p target genes revealed that it potentially binds to 2,109 mRNAs, including other key hormone signaling components such as E75 and Usp. However, in the overexpression or knockdown experiments, the observed phenotype cannot be excluded as a result of the combined action of multiple targets (including E75, Usp, etc.). Verifying only one target, AmJhamt, although establishing a direct targeting relationship, is not sufficient to prove that it is the sole or primary effector mediating the physiological phenotype. Could the discussion on this part be expanded to enhance the rigor of the conclusion.

 

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4091456 entitled "MicroRNA Novel-m0027-3p Regulates Juvenile Hormone Biosynthesis by Targeting Jhamt in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

The revision has met the format specification of your reminder, and the English grammar was edited by a native English speaker from International Science Editing Scientific Services.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 2

Comments to the Author:

This study focuses on the molecular regulatory mechanism of honeybee larva development, it identifies and systematically elucidates the crucial role of a novel miRNA, novel-m0027-3p, in regulating honeybee larva development. Also, it confirms that novel-m0027-3p mediates JH synthesis by negatively regulating the expression of AmJhamt.

The paper has a complete structure, clear logic, and provides multi-level experimental evidence. The system of the experimental is complete. The detailed description of the method and the chain of evidence provide convincing experimental evidence for the topic. However, there are still deficiencies in the paper that need to be revised.

Response: Thank you very much for your comments concerning our manuscript. The comments of Reviewers and Associate Editor are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our research. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.

Comment 1: Check whether to spell "AmJhamt" or "Amjhamt". For example, different forms of spelling appear in lines 35 and 39. It is recommended to unify the format.

Response 1: We have standardized nomenclature throughout the manuscript (AmJhamt).

Comment 2: The content of "Hex" and "USP" in the third paragraph of the introduction is incongruity with the previous content. It is suggested to add content for transition to make the paragraph more smooth.

Response 2: Accepted. We have added transitional sentences. The modifications are as follows:

Revision: Synthesized JH is released into the hemolymph and transported via the hemolymph to various tissues. Among these, Hexamerin (HEX) is synthesized in the fat body and serves as one of the most crucial storage proteins in insects (Haunerland, 1996). It is also involved in the transport of JH (Braun and Wyatt, 1996; Tawfik et al., 2006; Gilbert et al., 2000). Research on the Western honey bee [16], the American cockroach (Zhu et al., 2024), and termites (Zhou et al., 2006) has demonstrated that JH can induce the expression of Hex. However, the main response gene of JH is Kruppel homolog 1 (Kr-h1) (Minakuchi et al., 2008).

Reference

• Haunerland NH. Insect storage proteins: gene families and receptors. Insect Biochem Mol Biol. 1996 Sep-Oct;26(8-9):755-65. doi: 10.1016/s0965-1748(96)00035-5. PMID: 9014325.
• Braun RP, Wyatt GR. Sequence of the hexameric juvenile hormone-binding protein from the hemolymph of Locusta migratoria. J Biol Chem. 1996 Dec 6;271(49):31756-62. doi: 10.1074/jbc.271.49.31756. PMID: 8940201.
• Tawfik AI, Kellner R, Hoffmann KH, Lorenz MW. Purification, characterisation and titre of the haemolymph juvenile hormone binding proteins from Schistocerca gregaria and Gryllus bimaculatus. J Insect Physiol. 2006 Mar;52(3):255-68. doi: 10.1016/j.jinsphys.2005.11.005. Epub 2005 Dec 27. PMID: 16384579.
• Gilbert LI, Granger NA, Roe RM. The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem Mol Biol. 2000 Aug-Sep;30(8-9):617-44. doi: 10.1016/s0965-1748(00)00034-5. PMID: 10876106.
• Zhou X, Oi FM, Scharf ME. Social exploitation of hexamerin: RNAi reveals a major caste-regulatory factor in termites. Proc Natl Acad Sci U S A. 2006 Mar 21;103(12):4499-504. doi: 10.1073/pnas.0508866103. Epub 2006 Mar 14. PMID: 16537425; PMCID: PMC1450200.
• Zhu S, Chen X, Xia S, Li Q, Ye Z, Zhao S, Liu K, Liu F. Hexamerin and allergen are required for female reproduction in the American cockroach, Periplaneta americana. Insect Sci. 2024 Feb;31(1):186-200. doi: 10.1111/1744-7917.13218. Epub 2023 Jun 16. PMID: 37327125.

Comment 3: In line 305 of the discussion section, it is suggested to use the plural form of "insect".

Response 3: Accepted. Corrected as suggested.

Comment 4: What is the purpose of measuring the body weight of larva? If this method is to characterize "development" through changes in body weight, it is simple and quantifiable in operation, but it does have limitations in terms of scientific rigor and depth of biological significance. Can this study comprehensively confirm the effect of AmJhamt on larva development through more indicators or discussion.

Response 4: Thank you very much for your valuable comment. The aim of measuring larval body weight was to investigate the impact of novel-m0027-3p on JH content, given the established causal relationship between JH levels and larval growth. However, in the specific context of honey bee larvae, body weight remains the sole quantifiable phenotypic indicator that has been established to correlate with JH titer, as other conventional morphological or developmental markers for JH action are either absent or not applicable at this life stage.

In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. On the other hand, current artificial feeding techniques for honeybee larvae struggle to successfully rear them to the pupal stage, while feeding mimics or inhibitors directly on the comb can easily be removed by worker bees during cleaning.

We supplemented the relevant descriptions in the discussion section:

Revision: This, in turn, suppresses or promotes the expression levels of downstream JH‑responsive genes, AmHex70b and AmKr-h1 (Fig. 4), thereby affecting larval body weight (Fig. 5). In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. These findings align with our previous study showing that Ame‑miR‑2161 affects the larval‑to‑pupal transition in honey bees via the AmJhamt gene [38]. Our results suggested that novel‑m0027‑3p regulate AmJhamt, thereby affecting JH levels to a certain extent, but its impact on larval body weight is relatively limited. The potential hormone-related target genes bound by novel-m0027-3p are not limited to AmJhamt but may also include key hormonal signaling factors such as AmE75 and AmUsp. Therefore, the alteration in larval body weight induced by novel-m0027-3p may not be mediated solely through AmJhamt. In conclusion, this study confirms that novel‑m0027‑3p mediates JH synthesis by negatively regulating the expression of AmJhamt.

• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

Comment 5: As the article mentioned, prediction of novel-m0027-3p target genes revealed that it potentially binds to 2,109 mRNAs, including other key hormone signaling components such as E75 and Usp. However, in the overexpression or knockdown experiments, the observed phenotype cannot be excluded as a result of the combined action of multiple targets (including E75, Usp, etc.). Verifying only one target, AmJhamt, although establishing a direct targeting relationship, is not sufficient to prove that it is the sole or primary effector mediating the physiological phenotype. Could the discussion on this part be expanded to enhance the rigor of the conclusion.

Response 5: Accepted. We supplemented the deficiencies of the research in the discussion section. The modifications are as follows:

Revision: This, in turn, suppresses or promotes the expression levels of downstream JH‑responsive genes, AmHex70b and AmUsp (Fig. 4), thereby affecting larval body weight (Fig. 5). In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. These findings align with our previous study showing that Ame‑miR‑2161 affects the larval‑to‑pupal transition in honey bees via the AmJhamt gene [38]. Our results suggested that novel‑m0027‑3p regulate AmJhamt, thereby affecting JH levels to a certain extent, but its impact on larval body weight is relatively limited. The potential hormone-related target genes bound by novel-m0027-3p are not limited to AmJhamt but may also include key hormonal signaling factors such as AmE75 and AmUsp. Therefore, the alteration in larval body weight induced by novel-m0027-3p may not be mediated solely through AmJhamt. In conclusion, this study confirms that novel‑m0027‑3p mediates JH synthesis by negatively regulating the expression of AmJhamt.

• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The aim of this manuscript entitled “Novel-m0027-3p regulates Jhamt gene to mediate Juvenile hormone biosynthesis” is to demonstrate that the novel miRNA m0027-3p regulates the expression of the gene JHAMT, a key enzyme involved in juvenile hormone (JH) biosynthesis in Apis mellifera. There is no doubt that this miRNA is an interesting candidate and that the authors provide evidence for a link between m0027-3p and the juvenile hormone pathway. However, I am not convinced by the demonstration that this effect acts directly on JHAMT ant it is precisely the title of this paper.

A major concern is that the authors focus almost exclusively on the hypothesis of a specific action on JHAMT, whereas their own bioinformatic predictions identify 22 potential mRNA targets associated with six hormonal pathways. It is therefore unclear why JHAMT was selected as the sole functional target. At the very least, other predicted targets should have been examined to assess the specificity of the miRNA’s action, or the rationale for focusing uniquely on JHAMT should be more clearly justified.

In my view, the only experiment that can demonstrate a direct interaction between miRNA m0027-3p and JHAMT is the dual-luciferase reporter assay. However, the observed effects appear relatively weak. Similarly, the physiological consequences of miRNA manipulation would be more convincing if they were assessed using parameters more directly and specifically related to the juvenile hormone pathway.

More generally, the manuscript is written in a very technical manner and appears strongly centered on a specific experimental model or on the establishment of a miRNA action catalogue. The broader biological relevance of the question and the general context of the study are insufficiently highlighted, which limits the overall impact of the work.

Below are more specific comments and questions:

• Fig1 What is the expression level of novel m0027-3p in adult bees? This information seems important to better understand the biological relevance of this miRNA beyond larval stages.
• I do not clearly understand Figure 1. Panel 1A appears to show a conventional RT-PCR with visualization of DNA bands, whereas the quantification shown in panel 1C seems to correspond to quantitative expression data. Is panel 1C based on qPCR? If so, this should be clearly stated in the figure legend. If not, the rationale for this type of quantification should be explained.
• In Figure 2, the inhibition of luciferase activity by the miRNA appears relatively modest. In addition, it is unclear to what extent the lack of effect observed with the mutant JHAMT construct might be due to higher variability in the control condition without miRNA mimics.
• The authors investigate the effect of miRNA m0027-3p on larval body weight. This choice requires stronger justification with respect to what is known about juvenile hormone action on growth or body mass. Why was the larva-to-pupa metamorphic transition, which is more directly linked to JH activity, not examined instead? Extending the analysis to other physiological traits more closely associated with the targeted hormonal pathway would considerably strengthen the conclusions.
• There is no discussion regarding the fact that miRNA m0027-3p was identified in the larval gut, nor about how this localization relates to juvenile hormone signaling. Are there other tissues or developmental stages in which this miRNA might be expressed or synthesized?
• It is unclear why the study focuses so strongly on JHAMT rather than on other predicted targets such as E75 or USP. In particular, it is problematic to analyze the effect of the miRNA on USP if USP itself could be a direct target of m0027-3p, and then use this result to conclude an indirect effect on JHAMT.
• Similarly, changes in juvenile hormone titers could be mediated through effects on E75. While such results may be interesting, they do not demonstrate a direct regulatory effect of miRNA m0027-3p on JHAMT.

In conclusion, while this study addresses an interesting miRNA and provides evidence for its involvement in the juvenile hormone pathway, the data do not convincingly demonstrate a direct and specific regulation of JHAMT. Additional experiments and a broader consideration of alternative targets and physiological outcomes would be necessary to support the main claims of the manuscript.

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4091456 entitled "MicroRNA Novel-m0027-3p Regulates Juvenile Hormone Biosynthesis by Targeting Jhamt in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

The revision has met the format specification of your reminder, and the English grammar was edited by a native English speaker from International Science Editing Scientific Services.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 3

Comments to the Author:

The aim of this manuscript entitled “Novel-m0027-3p regulates Jhamt gene to mediate Juvenile hormone biosynthesis” is to demonstrate that the novel miRNA m0027-3p regulates the expression of the gene JHAMT, a key enzyme involved in juvenile hormone (JH) biosynthesis in Apis mellifera. There is no doubt that this miRNA is an interesting candidate and that the authors provide evidence for a link between m0027-3p and the juvenile hormone pathway. However, I am not convinced by the demonstration that this effect acts directly on JHAMT ant it is precisely the title of this paper.

Comment 1: A major concern is that the authors focus almost exclusively on the hypothesis of a specific action on JHAMT, whereas their own bioinformatic predictions identify 22 potential mRNA targets associated with six hormonal pathways. It is therefore unclear why JHAMT was selected as the sole functional target. At the very least, other predicted targets should have been examined to assess the specificity of the miRNA’s action, or the rationale for focusing uniquely on JHAMT should be more clearly justified.

Response 1: We did not treat AmJhamt as the sole target of novel-m0027-3p. Each miRNA regulates dozens or even hundreds of target genes, and it is not feasible for us to test every one of them. The reason we selected AmJhamt is that Jhamt is the rate-limiting enzyme in juvenile hormone synthesis, acting upstream in the hormone pathway, making it more significant compared to other hormone-related target genes, which are downstream responsive genes. We have added the reasons for selecting the six target genes. Through GO pathway enrichment, we identified two hormone-related pathways (GO:0009755 and GO:0032870), from which we obtained six target genes involved in hormone synthesis or secretion.

We have clarified this point in the introduction and result section, with revisions as follows:

Revision: Based on previously obtained transcriptomic data from the gut of Apis mellifera ligustica worker larvae [28], this study screened and identified the miRNA novel-m0027-3p in the larval gut. The expression profile of novel-m0027-3p was investigated during the larval and pupal stages. We focused on six target genes of novel-m0027-3p associated with insect hormone pathways. Among these, the target gene AmJhamt, as a rate-limiting enzyme in juvenile hormone synthesis, acts upstream in hormone biosynthesis. It is considered more important compared to other downstream hormone-responsive target genes, such as E75 and Usp. Furthermore, the regulatory relationship between novel-m0027-3p and its key target gene AmJhamt was validated, and its functional role in the larval development of A. m. ligustica was investigated. This work holds importance for elucidating how miRNAs modulate hormonal pathways to influence honeybee growth and development.

Revision: KEGG enrichment analysis identified target involvement in 10 metabolic pathways, including Glycolysis/Gluconeogenesis, Biosynthesis of amino acids, Inositol phosphate metabolism, and Citrate cycle (TCA cycle); The five genetic information processing pathways including RNA degradation, Nucleocytoplasmic transport, and RNA polymerase; The four environmental information processing pathways including Phosphatidylinositol signaling system, TGF‑β signaling pathway, FoxO signaling pathway, and Hedgehog signaling pathway; The one cellular processes pathway (Endocytosis); and Two organismal systems pathways (Dorso-ventral axis formation and Cytosolic DNA-sensing pathway) (Fig. S2). We identified two hormone-related pathways (GO:0009755 and GO:0032870) through GO pathway enrichment, from which we obtained a total of six target genes associated with hormone synthesis or secretion. Subsequent research focused on Jhamt (AmJhamt), a key rate-limiting enzyme gene in JH synthesis in the honeybees, while the other target genes are downstream genes of the JH signaling pathway (Fig. 1D).

Comment 2: In my view, the only experiment that can demonstrate a direct interaction between miRNA m0027-3p and JHAMT is the dual-luciferase reporter assay. However, the observed effects appear relatively weak. Similarly, the physiological consequences of miRNA manipulation would be more convincing if they were assessed using parameters more directly and specifically related to the juvenile hormone pathway.

More generally, the manuscript is written in a very technical manner and appears strongly centered on a specific experimental model or on the establishment of a miRNA action catalogue. The broader biological relevance of the question and the general context of the study are insufficiently highlighted, which limits the overall impact of the work.

Response 2: In the validation of the regulatory relationship between miRNA and its target gene, the two most critical experiments are (1) the dual‑luciferase assay, and (2) the overexpression/knockdown of the miRNA to examine changes in target gene expression. These approaches constitute the core evidence for demonstrating genuine binding and regulation between a miRNA and its target gene (Song et al., 2024; Pu et al., 2019). We have robustly confirmed the binding relationship between novel‑m0027‑3p and AmJhamt. Furthermore, we investigated JH hormone levels and the response genes in the JH signaling pathway. A limitation is that only body weight data were used to assess the larval phenotypes resulting from the changes in JH content induced by novel‑m0027‑3p. Because other conventional morphological or developmental markers for JH action are either absent or not applicable at this life stage (Larval stage to pupal stage). In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. On the other hand, current artificial feeding techniques for honeybee larvae struggle to successfully rear them to the pupal stage, while feeding mimics or inhibitors directly on the comb can easily be removed by worker bees during cleaning.

On the other hand, as indicated by the title, this study aims to demonstrate that Novel‑m0027‑3p mediates JH biosynthesis by influencing the expression of the AmJhamt gene. Therefore, the results are more technically oriented rather than delving into deeper issues of insect physiology.

Reference

• Pu M, Chen J, Tao Z, Miao L, Qi X, Wang Y, Ren J. Regulatory network of miRNA on its target: coordination between transcriptional and post-transcriptional regulation of gene expression. Cell Mol Life Sci. 2019, 76(3):441-451. doi: 10.1007/s00018-018-2940-7.
• Song J, Li W, Gao L, Yan Q, Zhang X, Liu M, Zhou S. miR-276 and miR-182013-5p modulate insect metamorphosis and reproduction via dually regulating juvenile hormone acid methyltransferase. Commun Biol. 2024, 7(1):1604. doi: 10.1038/s42003-024-07285-0.
• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

Comment 3: Fig1 What is the expression level of novel m0027-3p in adult bees? This information seems important to better understand the biological relevance of this miRNA beyond larval stages.

Response 3: Thank you very much for your valuable comment. JH plays a major role in larval development, caste differentiation, and metamorphosis in Honeybee. In the adult stage, JH is primarily associated with lifespan (Gong et al., 2023; Rembold et al., 1974). We considered investigating the effects of Novel‑m0027‑3p on adult bees. However, in honeybee adults, due to the social behaviors of worker bees—such as nursing, cleaning, and pollen foraging—it is difficult to control a single experimental variable. Therefore, even if the expression profile of Novel‑m0027‑3p in the adult stage were determined, studying its impact on adult honeybees would be challenging to achieve.

Reference

• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

Comment 4: I do not clearly understand Figure 1. Panel 1A appears to show a conventional RT-PCR with visualization of DNA bands, whereas the quantification shown in panel 1C seems to correspond to quantitative expression data. Is panel 1C based on qPCR? If so, this should be clearly stated in the figure legend. If not, the rationale for this type of quantification should be explained.

Response 4: We have revised the Figure 1 legend to clearly state that panel 1A shows stem-loop RT-PCR band visualization, whereas panel 1C reports stem-loop RT-qPCR quantification. We also clarify the quantification approach and normalization.

Comment 5: In Figure 2, the inhibition of luciferase activity by the miRNA appears relatively modest. In addition, it is unclear to what extent the lack of effect observed with the mutant JHAMT construct might be due to higher variability in the control condition without miRNA mimics.

Response 5: Thank you very much for your valuable comment. We understand your concern. In Fig. 2, we normalized the control groups for Jhamt-wt and Jhamt-mut to "1", and calculated the inhibition rates for the two groups as 19.33% and 8.03%, respectively. Therefore, the high variation in the Jhamt-mut group does not affect the validity of the result.

Comment 6: The authors investigate the effect of miRNA m0027-3p on larval body weight. This choice requires stronger justification with respect to what is known about juvenile hormone action on growth or body mass. Why was the larva-to-pupa metamorphic transition, which is more directly linked to JH activity, not examined instead? Extending the analysis to other physiological traits more closely associated with the targeted hormonal pathway would considerably strengthen the conclusions.

Response 6: In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. On the other hand, current artificial feeding techniques for honeybee larvae struggle to successfully rear them to the pupal stage, while feeding mimics or inhibitors directly on the comb can easily be removed by worker bees during cleaning.

Reference

• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

Comment 7: There is no discussion regarding the fact that miRNA m0027-3p was identified in the larval gut, nor about how this localization relates to juvenile hormone signaling. Are there other tissues or developmental stages in which this miRNA might be expressed or synthesized?

Response 7: Thank you very much for your valuable comment. Novel‑m0027‑3p was initially discovered in the intestine, but it is not specifically expressed there. On the other hand, as a small nucleic acid molecule, miRNA may traverse any type of cell, including brain cells.

Comment 8: It is unclear why the study focuses so strongly on JHAMT rather than on other predicted targets such as E75 or USP. In particular, it is problematic to analyze the effect of the miRNA on USP if USP itself could be a direct target of m0027-3p, and then use this result to conclude an indirect effect on JHAMT.

Comment 9: Similarly, changes in juvenile hormone titers could be mediated through effects on E75. While such results may be interesting, they do not demonstrate a direct regulatory effect of miRNA m0027-3p on JHAMT.

Response 8 and 9: Regardless of whether Novel‑m0027‑3p can directly regulate the expression of the Usp/E75 gene, our dual‑luciferase assay has demonstrated a direct binding relationship between Novel‑m0027‑3p and AmJhamt — a result obtained without the presence of USP or other predicted target genes (HEK 293T cell). On the other hand, even if Novel‑m0027‑3p were able to directly regulate the expression of Usp/E75, its impact on the expression of the upstream JH synthase gene AmJhamt and on JH titers would be limited.

We have clarified this point in the introduction and result section, with revisions as follows:

Revision: Based on previously obtained transcriptomic data from the gut of Apis mellifera ligustica worker larvae [28], this study screened and identified the miRNA novel-m0027-3p in the larval gut. The expression profile of novel-m0027-3p was investigated during the larval and pupal stages. We focused on six target genes of novel-m0027-3p associated with insect hormone pathways. Among these, the target gene AmJhamt, as a rate-limiting enzyme in juvenile hormone synthesis, acts upstream in hormone biosynthesis. It is considered more important compared to other downstream hormone-responsive target genes, such as E75 and Usp. Furthermore, the regulatory relationship between novel-m0027-3p and its key target gene AmJhamt was validated, and its functional role in the larval development of A. m. ligustica was investigated. This work holds importance for elucidating how miRNAs modulate hormonal pathways to influence honeybee growth and development.

Revision: KEGG enrichment analysis identified target involvement in 10 metabolic pathways, including Glycolysis/Gluconeogenesis, Biosynthesis of amino acids, Inositol phosphate metabolism, and Citrate cycle (TCA cycle); The five genetic information processing pathways including RNA degradation, Nucleocytoplasmic transport, and RNA polymerase; The four environmental information processing pathways including Phosphatidylinositol signaling system, TGF‑β signaling pathway, FoxO signaling pathway, and Hedgehog signaling pathway; The one cellular processes pathway (Endocytosis); and Two organismal systems pathways (Dorso-ventral axis formation and Cytosolic DNA-sensing pathway) (Fig. S2). We identified two hormone-related pathways (GO:0009755 and GO:0032870) through GO pathway enrichment, from which we obtained a total of six target genes associated with hormone synthesis or secretion. Subsequent research focused on Jhamt (AmJhamt), a key rate-limiting enzyme gene in JH synthesis in the honeybees, while the other target genes are downstream genes of the JH signaling pathway (Fig. 1D).

Comment 10: In conclusion, while this study addresses an interesting miRNA and provides evidence for its involvement in the juvenile hormone pathway, the data do not convincingly demonstrate a direct and specific regulation of JHAMT. Additional experiments and a broader consideration of alternative targets and physiological outcomes would be necessary to support the main claims of the manuscript.

Response 10: In the validation of the regulatory relationship between miRNA and its target gene, the two most critical experiments are (1) the dual‑luciferase assay, and (2) the overexpression/knockdown of the miRNA to examine changes in target gene expression. These approaches constitute the core evidence for demonstrating genuine binding and regulation between a miRNA and its target gene (Song et al., 2024; Pu et al., 2019). We have robustly confirmed the binding relationship between novel‑m0027‑3p and AmJhamt. Furthermore, we investigated JH hormone levels and the response genes in the JH signaling pathway. A limitation is that only body weight data were used to assess the larval phenotypes resulting from the changes in JH content induced by novel‑m0027‑3p. Because other conventional morphological or developmental markers for JH action are either absent or not applicable at this life stage (Larval stage to pupal stage). In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. On the other hand, current artificial feeding techniques for honeybee larvae struggle to successfully rear them to the pupal stage, while feeding mimics or inhibitors directly on the comb can easily be removed by worker bees during cleaning.

Reference

• Pu M, Chen J, Tao Z, Miao L, Qi X, Wang Y, Ren J. Regulatory network of miRNA on its target: coordination between transcriptional and post-transcriptional regulation of gene expression. Cell Mol Life Sci. 2019, 76(3):441-451. doi: 10.1007/s00018-018-2940-7.
• Song J, Li W, Gao L, Yan Q, Zhang X, Liu M, Zhou S. miR-276 and miR-182013-5p modulate insect metamorphosis and reproduction via dually regulating juvenile hormone acid methyltransferase. Commun Biol. 2024, 7(1):1604. doi: 10.1038/s42003-024-07285-0.
• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

Reviewer 4 Report

Comments and Suggestions for Authors

This study investigates miRNA-mediated regulation in honeybees, revealing that miRNA novel-m0027-3p directly targets Jhamt (a key gene in juvenile hormone (JH) synthesis), thereby regulating JH titers and signaling during larval development to impact honeybee development. It also identifies hormones as a crucial bridge in this regulatory process, providing new insights into how miRNAs modulate hormone pathways to govern honeybee physiology.

This study exhibits certain innovativeness, and the research subject (honeybees) endows it with practical application value. The experimental design of the paper is basically rational, and the results can generally support the conclusions. However, the manuscript requires further revisions prior to publication. Below are my specific suggestions.

1. In the Introduction, the manuscript states that it focuses on the relationship between miRNA novel-m0027-3p and the JH pathway, with the basis derived from Reference 28. However, it fails to clearly explain the specific evidence and the reasoning process behind the hypothesis regarding the interaction between miRNA novel-m0027-3p and the JH pathway. This leaves readers unable to fully grasp the rationale for initiating this study, hindering their smooth comprehension of the manuscript.
2. For the miRNA mimics and inhibitors, the authors only provided their sequences but failed to elaborate on the corresponding design principles. These critical methodological details should be supplemented in the manuscript.
3. Lines 230–232: The rationale for how the authors screened 6 target genes from a total of 2109 predicted candidates, as well as the specific reasons for selecting Jhamt as the sole target for subsequent in-depth investigations, should be clearly elaborated on in the manuscript.
4. The authors are required to design additional experiments to clarify that the altered expression levels of Hex70b and Usp are caused by the changes in the JH signaling pathway. Meanwhile, it is necessary to rule out the possibility that miRNA novel-m0027-3p directly targets Hex70b and Usp (independent of the JH pathway).
5. There is an apparent inconsistency between the statement in the Abstract and the conclusion in the Results section regarding the effect of miRNA novel-m0027-3p on larval body weight. Specifically, the Abstract claims that "These alterations in JH levels further affected larval body weight", whereas the Results section states that "The results indicate that novel-m0027-3p has a limited effect on body weight". In addition, the experimental results of miRNA mimics and inhibitors on larval body weight are logically inconsistent. . If the authors argue that novel-m0027-3p can indeed affect larval body weight, the authors should provide a clearer and more explicit interpretation of this discrepancy.
6. The language of the manuscript needs further polishing.

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4091456 entitled "MicroRNA Novel-m0027-3p Regulates Juvenile Hormone Biosynthesis by Targeting Jhamt in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

The revision has met the format specification of your reminder, and the English grammar was edited by a native English speaker from International Science Editing Scientific Services.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 4

Comments to the Author:

This study investigates miRNA-mediated regulation in honeybees, revealing that miRNA novel-m0027-3p directly targets Jhamt (a key gene in juvenile hormone (JH) synthesis), thereby regulating JH titers and signaling during larval development to impact honeybee development. It also identifies hormones as a crucial bridge in this regulatory process, providing new insights into how miRNAs modulate hormone pathways to govern honeybee physiology.

This study exhibits certain innovativeness, and the research subject (honeybees) endows it with practical application value. The experimental design of the paper is basically rational, and the results can generally support the conclusions. However, the manuscript requires further revisions prior to publication. Below are my specific suggestions.

Response: Thank you very much for your comments concerning our manuscript. The comments of Reviewers and Associate Editor are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our research. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.

Comment 1: In the Introduction, the manuscript states that it focuses on the relationship between miRNA novel-m0027-3p and the JH pathway, with the basis derived from Reference 28. However, it fails to clearly explain the specific evidence and the reasoning process behind the hypothesis regarding the interaction between miRNA novel-m0027-3p and the JH pathway. This leaves readers unable to fully grasp the rationale for initiating this study, hindering their smooth comprehension of the manuscript.

Response 1: The reason we selected AmJhamt is that Jhamt is the rate-limiting enzyme in juvenile hormone synthesis, acting upstream in the hormone pathway, making it more significant compared to other hormone-related target genes, which are downstream responsive genes.

We have clarified this point in the introduction and result section, with revisions as follows:

Revision: Based on previously obtained transcriptomic data from the gut of Apis mellifera ligustica worker larvae [28], this study screened and identified the miRNA novel-m0027-3p in the larval gut. The expression profile of novel-m0027-3p was investigated during the larval and pupal stages. We focused on six target genes of novel-m0027-3p associated with insect hormone pathways. Among these, the target gene AmJhamt, as a rate-limiting enzyme in juvenile hormone synthesis, acts upstream in hormone biosynthesis. It is considered more important compared to other downstream hormone-responsive target genes, such as E75 and Usp. Furthermore, the regulatory relationship between novel-m0027-3p and its key target gene AmJhamt was validated, and its functional role in the larval development of A. m. ligustica was investigated. This work holds importance for elucidating how miRNAs modulate hormonal pathways to influence honeybee growth and development.

Revision: KEGG enrichment analysis identified target involvement in 10 metabolic pathways, including Glycolysis/Gluconeogenesis, Biosynthesis of amino acids, Inositol phosphate metabolism, and Citrate cycle (TCA cycle); The five genetic information processing pathways including RNA degradation, Nucleocytoplasmic transport, and RNA polymerase; The four environmental information processing pathways including Phosphatidylinositol signaling system, TGF‑β signaling pathway, FoxO signaling pathway, and Hedgehog signaling pathway; The one cellular processes pathway (Endocytosis); and Two organismal systems pathways (Dorso-ventral axis formation and Cytosolic DNA-sensing pathway) (Fig. S2). We identified two hormone-related pathways (GO:0009755 and GO:0032870) through GO pathway enrichment, from which we obtained a total of six target genes associated with hormone synthesis or secretion. Subsequent research focused on Jhamt (AmJhamt), a key rate-limiting enzyme gene in JH synthesis in the honeybees, while the other target genes are downstream genes of the JH signaling pathway (Fig. 1D).

Comment 2: For the miRNA mimics and inhibitors, the authors only provided their sequences but failed to elaborate on the corresponding design principles. These critical methodological details should be supplemented in the manuscript.

Response 2: Accepted. We have supplemented the design method for miRNA mimics and inhibitors. The modifications are as follows:

In the Materials and Methods section

Revision: Based on the sequence of novel-m0027-3p, the mature novel-m0027-3p sequence was simulated as miRNA mimics (double-stranded), while the antisense strand of the novel-m0027-3p sequence was used as miRNA inhibitors (single-stranded). At the same time, their corresponding negative controls (Mimic-NC and Inhibitor-NC) were designed and chemically synthesized by Sangon Biotech (Sangon Biotech, China). All oligonucleotides were fully modified with 2′-O-methyl and phosphonothioates linkages to enhance stability and were HPLC-purified. The sequences are listed in Supplementary Table 2.

Comment 3: Lines 230–232: The rationale for how the authors screened 6 target genes from a total of 2109 predicted candidates, as well as the specific reasons for selecting Jhamt as the sole target for subsequent in-depth investigations, should be clearly elaborated on in the manuscript.

Response 3: Accepted. We have added the reasons for selecting the six target genes. Through GO pathway enrichment, we identified two hormone-related pathways (GO:0009755 and GO:0032870), from which we obtained six target genes involved in hormone synthesis or secretion. The reason we selected AmJhamt is that Jhamt is the rate-limiting enzyme in juvenile hormone synthesis, acting upstream in the hormone pathway, making it more significant compared to other hormone-related target genes, which are downstream responsive genes.

We have clarified this point in the introduction and result section, with revisions as follows:

Revision: Based on previously obtained transcriptomic data from the gut of Apis mellifera ligustica worker larvae [28], this study screened and identified the miRNA novel-m0027-3p in the larval gut. The expression profile of novel-m0027-3p was investigated during the larval and pupal stages. We focused on six target genes of novel-m0027-3p associated with insect hormone pathways. Among these, the target gene AmJhamt, as a rate-limiting enzyme in juvenile hormone synthesis, acts upstream in hormone biosynthesis. It is considered more important compared to other downstream hormone-responsive target genes, such as E75 and Usp. Furthermore, the regulatory relationship between novel-m0027-3p and its key target gene AmJhamt was validated, and its functional role in the larval development of A. m. ligustica was investigated. This work holds importance for elucidating how miRNAs modulate hormonal pathways to influence honeybee growth and development.

Revision: KEGG enrichment analysis identified target involvement in 10 metabolic pathways, including Glycolysis/Gluconeogenesis, Biosynthesis of amino acids, Inositol phosphate metabolism, and Citrate cycle (TCA cycle); The five genetic information processing pathways including RNA degradation, Nucleocytoplasmic transport, and RNA polymerase; The four environmental information processing pathways including Phosphatidylinositol signaling system, TGF‑β signaling pathway, FoxO signaling pathway, and Hedgehog signaling pathway; The one cellular processes pathway (Endocytosis); and Two organismal systems pathways (Dorso-ventral axis formation and Cytosolic DNA-sensing pathway) (Fig. S2). We identified two hormone-related pathways (GO:0009755 and GO:0032870) through GO pathway enrichment, from which we obtained a total of six target genes associated with hormone synthesis or secretion. Subsequent research focused on Jhamt (AmJhamt), a key rate-limiting enzyme gene in JH synthesis in the honeybees, while the other target genes are downstream genes of the JH signaling pathway (Fig. 1D).

Comment 4: The authors are required to design additional experiments to clarify that the altered expression levels of Hex70b and Usp are caused by the changes in the JH signaling pathway. Meanwhile, it is necessary to rule out the possibility that miRNA novel-m0027-3p directly targets Hex70b and Usp (independent of the JH pathway).

Response 4: Thank you very much for your valuable comments. We have deleted the results related to the potential target gene Usp, further supplemented the expression data of the JH-responsive gene Kr‑h1, and added descriptions regarding JH and Hex genes in the introduction. The revised version is as follows:

Revision: Synthesized JH is released into the hemolymph and transported via the hemolymph to various tissues. Among these, Hexamerin (Hex) is synthesized in the fat body and serves as one of the most crucial storage proteins in insects (Haunerland, 1996). It is also involved in the transport of JH (Braun and Wyatt, 1996; Tawfik et al., 2006; Gilbert et al., 2000). Research on the Western honey bee [16], the American cockroach (Zhu et al., 2024), and termites (Zhou et al., 2006) has demonstrated that JH can induce the expression of Hex. However, the main response gene of JH is Kruppel homolog 1 (Kr-h1) (Minakuchi et al., 2008). A direct or indirect regulatory interplay exists between miRNAs and hormonal pathways in insects.

Reference

• Haunerland NH. Insect storage proteins: gene families and receptors. Insect Biochem Mol Biol. 1996 Sep-Oct;26(8-9):755-65. doi: 10.1016/s0965-1748(96)00035-5. PMID: 9014325.
• Braun RP, Wyatt GR. Sequence of the hexameric juvenile hormone-binding protein from the hemolymph of Locusta migratoria. J Biol Chem. 1996 Dec 6;271(49):31756-62. doi: 10.1074/jbc.271.49.31756. PMID: 8940201.
• Tawfik AI, Kellner R, Hoffmann KH, Lorenz MW. Purification, characterisation and titre of the haemolymph juvenile hormone binding proteins from Schistocerca gregaria and Gryllus bimaculatus. J Insect Physiol. 2006 Mar;52(3):255-68. doi: 10.1016/j.jinsphys.2005.11.005. Epub 2005 Dec 27. PMID: 16384579.
• Gilbert LI, Granger NA, Roe RM. The juvenile hormones: historical facts and speculations on future research directions. Insect Biochem Mol Biol. 2000 Aug-Sep;30(8-9):617-44. doi: 10.1016/s0965-1748(00)00034-5. PMID: 10876106.
• Zhou X, Oi FM, Scharf ME. Social exploitation of hexamerin: RNAi reveals a major caste-regulatory factor in termites. Proc Natl Acad Sci U S A. 2006 Mar 21;103(12):4499-504. doi: 10.1073/pnas.0508866103. Epub 2006 Mar 14. PMID: 16537425; PMCID: PMC1450200.
• Zhu S, Chen X, Xia S, Li Q, Ye Z, Zhao S, Liu K, Liu F. Hexamerin and allergen are required for female reproduction in the American cockroach, Periplaneta americana. Insect Sci. 2024 Feb;31(1):186-200. doi: 10.1111/1744-7917.13218. Epub 2023 Jun 16. PMID: 37327125.
• Minakuchi C, Zhou X, Riddiford LM. Krüppel homolog 1 (Kr-h1) mediates juvenile hormone action during metamorphosis of Drosophila melanogaster. Mech Dev. 2008 Jan-Feb;125(1-2):91-105. doi: 10.1016/j.mod.2007.10.002. Epub 2007 Oct 11. PMID: 18036785; PMCID: PMC2276646.

Comment 5: There is an apparent inconsistency between the statement in the Abstract and the conclusion in the Results section regarding the effect of miRNA novel-m0027-3p on larval body weight. Specifically, the Abstract claims that "These alterations in JH levels further affected larval body weight", whereas the Results section states that "The results indicate that novel-m0027-3p has a limited effect on body weight". In addition, the experimental results of miRNA mimics and inhibitors on larval body weight are logically inconsistent. If the authors argue that novel-m0027-3p can indeed affect larval body weight, the authors should provide a clearer and more explicit interpretation of this discrepancy.

Response 5: Thank you very much for your valuable comment. We have removed the contradictory descriptions in the abstract, and supplemented the content in the discussion section. After miRNA mimics treatment, the weight of the larvae should be reduced; After treatment with miRNA inhibitors, the body weight of the larvae should increase. Our results suggested that novel‑m0027‑3p can regulate Jhamt, thereby affecting JH levels to a certain extent, but its impact on larval body weight is relatively limited. The modifications are as follows:

Revision: This, in turn, suppresses or promotes the expression levels of downstream JH‑responsive genes, AmHex70b and AmKr-h1 (Fig. 4), thereby affecting larval body weight (Fig. 5). In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. These findings align with our previous study showing that Ame‑miR‑2161 affects the larval‑to‑pupal transition in honey bees via the AmJhamt gene [38]. Our results suggested that novel‑m0027‑3p regulate AmJhamt, thereby affecting JH levels to a certain extent, but its impact on larval body weight is relatively limited. The potential hormone-related target genes bound by novel-m0027-3p are not limited to AmJhamt but may also include key hormonal signaling factors such as AmE75 and AmUsp. Therefore, the alteration in larval body weight induced by novel-m0027-3p may not be mediated solely through AmJhamt. In conclusion, this study confirms that novel‑m0027‑3p mediates JH synthesis by negatively regulating the expression of AmJhamt.

Comment 6: The language of the manuscript needs further polishing.

Response 6: Accepted. We have already had the manuscript professionally polished for language.

Reviewer 5 Report

Comments and Suggestions for Authors

The research explored a new Micro RNA (Novel-m0027-3p) that can negatively regulate JH biosynthesis link between larval body weight and genes expression. The study showed the function of Novel-m0027-3p on honey bee development. But there are a few problems need to be resolved before published.

 

Main points:

1. Title: I think the title is deficient. The title should contain the research subject (including species name, biological type, development stage).
2. Introduction: This part is so insufficiency. The authors mentioned that Novel-m0027-3p can negatively regulates jhamt gene to Mediate JH biosynthesis. But they did not introduce the JH biosynthesis pathway (Hex70b and Usp, et al.).
3. I am very curious why the authors assign the body wight as a parameter but ignore the development time.
4. Discussion: This part is too short! I think the important defect is that the authors did not discuss their results on the JH biosynthesis pathway.

Comments on the Quality of English Language

The english is OK. But the manuscript need to be revised carefully.

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4091456 entitled "MicroRNA Novel-m0027-3p Regulates Juvenile Hormone Biosynthesis by Targeting Jhamt in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

The revision has met the format specification of your reminder, and the English grammar was edited by a native English speaker from International Science Editing Scientific Services.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 5

Comments to the Author:

The research explored a new Micro RNA (Novel-m0027-3p) that can negatively regulate JH biosynthesis link between larval body weight and genes expression. The study showed the function of Novel-m0027-3p on honey bee development. But there are a few problems need to be resolved before published.

Response: Thank you very much for your comments concerning our manuscript. The comments of Reviewers and Associate Editor are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our research. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.

Comment 1: Title: I think the title is deficient. The title should contain the research subject (including species name, biological type, development stage).

Response 1: We agree and have revised the title to include the research subject (species), developmental stage, and miRNA identity: “MicroRNA Novel-m0027-3p Regulates Juvenile Hormone Biosynthesis by targeting Jhamt in Apis mellifera Larvae”.

Comment 2: Introduction: This part is so insufficiency. The authors mentioned that Novel-m0027-3p can negatively regulates jhamt gene to Mediate JH biosynthesis. But they did not introduce the JH biosynthesis pathway (Hex70b and Usp, et al.).

Response 2: Hex70b and Usp are downstream signaling pathway genes of JH. We have added content on the JH synthesis pathway in the Introduction, revised as follows:

Revision: The timing of molting and metamorphosis in holometabolous insects is primarily regulated by hormones, including Juvenile Hormone (JH) and Ecdysteroid[Hiruma and Kaneko, 2013]. Ecdysteroids are synthesized in the prothoracic glands and secreted into the hemolymph, where they are converted to the active hormone 20-hydroxyecdysone (20E), which triggers larval molting and metamorphosis (Scanlan et al., 2023). It is generally believed that the role of JH is to prevent premature metamorphosis, allowing larvae to undergo multiple rounds of molting until they reach an appropriate size (Jindra et al., 2012). This indicates that the regulation of developmental transitions in insects by ecdysteroids and JH is antagonistic. JH is synthesized through the mevalonate pathway (MVAP). Acetyl-CoA undergoes a series of enzymatic reactions to produce farnesyl pyrophosphate (FPP). FPP is then converted into farnesoic acid by the actions of farnesyl pyrophosphatase and farnesol dehydrogenase. Finally, farnesoic acid is catalyzed by Juvenile hormone acid methyl-transferase (JHAMT) to form JH (Noriega, 2014). JHAMT is the rate-limiting enzyme controlling JH synthesis. The Jhamt gene has been cloned in various insect species such as the Bombyx mori [12], Drosophila melanogaster [13], Tribolium castaneum [14] and Aedes aegypti [15].

Synthesized JH is released into the hemolymph and transported via the hemolymph to various tissues. Among these, Hexamerin (HEX) is synthesized in the fat body and serves as one of the most crucial storage proteins in insects (Haunerland, 1996). It is also involved in the transport of JH (Braun and Wyatt, 1996; Tawfik et al., 2006; Gilbert et al., 2000). Research on the Western honey bee [16], the American cockroach (Zhu et al., 2024), and termites (Zhou et al., 2006) has demonstrated that JH can induce the expression of Hex. However, the main response gene of JH is Kruppel homolog 1 (Kr-h1) (Minakuchi et al., 2008).

Reference

• Hiruma, K.; Kaneko, Y. Hormonal regulation of insect metamorphosis with special reference to juvenile hormone biosynthesis. Curr. Top. Dev. Biol. 2013, 103: 73-100.
• Jindra M, Palli SR, Riddiford LM. The juvenile hormone signaling pathway in insect development. Annu Rev Entomol. 2013, 58:181-204. doi: 10.1146/annurev-ento-120811-153700.
• Scanlan JL, Robin C, Mirth CK. Rethinking the ecdysteroid source during Drosophila pupal-adult development. Insect Biochem Mol Biol. 2023, 152:103891. doi: 10.1016/j.ibmb.2022.103891.
• Noriega, F. G.; Bloch, G.; Moos, M.; Simek, P.; Jindra, M. Approaches to Quantify and Manipulate Insect Hormone Signals. Curr. Opin. Insect Sci. 2025, 72, 101425. https://doi.org/10.1016/j.cois.2025.101425.

Comment 3: I am very curious why the authors assign the body wight as a parameter but ignore the development time.

Response 3: In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. On the other hand, current artificial feeding techniques for honeybee larvae struggle to successfully rear them to the pupal stage, while feeding mimics or inhibitors directly on the comb can easily be removed by worker bees during cleaning. Because other conventional morphological or developmental markers for JH action are either absent or not applicable at this life stage (Larval stage to pupal stage). Therefore, the investigation results on the developmental time are very unstable.

Reference

• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

Comment 4: Discussion: This part is too short! I think the important defect is that the authors did not discuss their results on the JH biosynthesis pathway.

Response 4: Jhamt catalyzes the final enzymatic step in JH synthesis; therefore, all experimental results reflect differences after JH synthesis, such as changes in JH titer and downstream responsive genes. Consequently, the discussion section does not emphasize the JH synthesis pathway. A description of the JH synthesis pathway has been added to the introduction. The modifications are as follows:

Revision: The timing of molting and metamorphosis in holometabolous insects is primarily regulated by hormones, including Juvenile Hormone (JH) and Ecdysteroid[Hiruma and Kaneko, 2013]. Ecdysteroids are synthesized in the prothoracic glands and secreted into the hemolymph, where they are converted to the active hormone 20-hydroxyecdysone (20E), which triggers larval molting and metamorphosis (Scanlan et al., 2023). It is generally believed that the role of JH is to prevent premature metamorphosis, allowing larvae to undergo multiple rounds of molting until they reach an appropriate size (Jindra et al., 2012). This indicates that the regulation of developmental transitions in insects by ecdysteroids and JH is antagonistic. JH is synthesized through the mevalonate pathway (MVAP). Acetyl-CoA undergoes a series of enzymatic reactions to produce farnesyl pyrophosphate (FPP). FPP is then converted into farnesoic acid by the actions of farnesyl pyrophosphatase and farnesol dehydrogenase. Finally, farnesoic acid is catalyzed by Juvenile hormone acid methyl-transferase (JHAMT) to form JH (Noriega, 2014). JHAMT is the rate-limiting enzyme controlling JH synthesis. The Jhamt gene has been cloned in various insect species such as the Bombyx mori [12], Drosophila melanogaster [13], Tribolium castaneum [14] and Aedes aegypti [15].

Synthesized JH is released into the hemolymph and transported via the hemolymph to various tissues. Among these, Hexamerin (HEX) is synthesized in the fat body and serves as one of the most crucial storage proteins in insects (Haunerland, 1996). It is also involved in the transport of JH (Braun and Wyatt, 1996; Tawfik et al., 2006; Gilbert et al., 2000). Research on the Western honey bee [16], the American cockroach (Zhu et al., 2024), and termites (Zhou et al., 2006) has demonstrated that JH can induce the expression of Hex. However, the main response gene of JH is Kruppel homolog 1 (Kr-h1) (Minakuchi et al., 2008).

Reference

• Hiruma, K.; Kaneko, Y. Hormonal regulation of insect metamorphosis with special reference to juvenile hormone biosynthesis. Curr. Top. Dev. Biol. 2013, 103: 73-100.
• Jindra M, Palli SR, Riddiford LM. The juvenile hormone signaling pathway in insect development. Annu Rev Entomol. 2013, 58:181-204. doi: 10.1146/annurev-ento-120811-153700.
• Scanlan JL, Robin C, Mirth CK. Rethinking the ecdysteroid source during Drosophila pupal-adult development. Insect Biochem Mol Biol. 2023, 152:103891. doi: 10.1016/j.ibmb.2022.103891.
• Noriega FG. Juvenile Hormone Biosynthesis in Insects: What Is New, What Do We Know, and What Questions Remain? Int Sch Res Notices. 2014, 2014:967361. doi: 10.1155/2014/967361.

Reviewer 6 Report

Comments and Suggestions for Authors

Wang and his colleagues conducted a study on microRNA-mediated regulation of the Jhamt gene in honeybees. The study is meaningful; however, several issues need to be addressed before publication. My comments are as follows:

1. In the Materials and Methods section, the titles of Sections 2.2 and 2.3 are both listed as stem-loop RT-PCR. One of them should be qPCR. Please revise accordingly.
2. In general, quantification of juvenile hormone titers in insects is technically challenging. I checked the ELISA kit used in this study (YJ201349, mlbio, China), but no corresponding product information could be found for this catalog number. If this kit has been used in previous studies, please add the relevant references to support the validity of the juvenile hormone titer measurements.
3. What does “t-text” mean? This error appears in Figures 2, 3, 4, and 5 and should be corrected to t-test. In addition, in Line 110, “bechtop” should be corrected to benchtop.
4. In the Results section (Section 3.1), the authors state that "novel-m0027-3p could be detected across all eight time points examined, spanning the larval, prepupa, and adult stages (Fig. 1A)". However, according to the experimental description in the Materials and Methods (Section 2.2) and the labels in Figure 1A and Figure 1C, the collected samples only include 4-day-old and 6-day-old larvae, 2-day-old prepupae, and pupae at 1, 3, 5, 7, and 9 days of age. The adult stage was not actually involved or tested in this study。
5. The capitalization and abbreviation of the target gene are used inconsistently throughout the manuscript. For instance, "AmJhamt" appears frequently in the Abstract , Introduction , results text , and Discussion. Conversely, "Amjhamt" is used elsewhere in the Abstract , the Materials and Methods , and within the labels and axes of Figure 2 and Figure 3. In a formal scientific publication, gene and protein nomenclature must be maintained with strict consistency across the entire paper.
6. In result 3.3, the author said” The results indicate that novel-m0027-3p has a limited effect on body weight.”. however, in abstract, they claimed “These alterations in JH levels further affected larval body weigh”. Whether body weight is affected should be stated in a unified and clearly defined manner.
7. “Fourth-, fifth-, and sixth-instar larvae (n = 3 per group)”，The sample size is too small, and I am not sure whether this is the reason why no significant change in body weight was observed.

Author Response

Dear Editor,

We would thank you and the reviewers for your positive and constructive comments on the manuscript ID: insects-4091456 entitled "MicroRNA Novel-m0027-3p Regulates Juvenile Hormone Biosynthesis by Targeting Jhamt in Apis mellifera Larvae".

We have studied the reviewer’s comments carefully and have made a revision marked with “track changes” in the original manuscript. We have copied and pasted all reviewers’ comments below and addressed each one individually. Attached please find the revised version and the supplemental data, which we would like to submit for your kind consideration.

The revision has met the format specification of your reminder, and the English grammar was edited by a native English speaker from International Science Editing Scientific Services.

We are looking forward to hearing from you. If you have any queries, please don’t hesitate to contact me at the address below.

 

Thank you and best regards.

 

Yours sincerely,

 

Pro. Dafu Chen and Guojun Xu

 

College of Bee Science and Biomedicine,

Fujian Agriculture and Forestry University

 

E-mail: dfchen826@fafu.edu.cn (D.C.); xuguojun@fafu.edu.cn.

 

Reviewer 6

Comments to the Author:

Wang and his colleagues conducted a study on microRNA-mediated regulation of the Jhamt gene in honeybees. The study is meaningful; however, several issues need to be addressed before publication. My comments are as follows:

Response: Thank you very much for your comments concerning our manuscript. The comments of Reviewers and Associate Editor are all valuable and very helpful for revising and improving our manuscript, as well as the important guiding significance to our research. We have tried our best to improve the manuscript and have made a lot of changes which we hope meet with approval.

Comment 1: In the Materials and Methods section, the titles of Sections 2.2 and 2.3 are both listed as stem-loop RT-PCR. One of them should be qPCR. Please revise accordingly.

Response 1: Thank you very much for your valuable comment. Corrected. One section is now correctly titled stem-loop RT-qPCR, and the descriptions were checked for consistency with the assays performed.

Comment 2: In general, quantification of juvenile hormone titers in insects is technically challenging. I checked the ELISA kit used in this study (YJ201349, mlbio, China), but no corresponding product information could be found for this catalog number. If this kit has been used in previous studies, please add the relevant references to support the validity of the juvenile hormone titer measurements.

Response 2: We apologize for the incorrect catalog number. We have corrected the information for the ELISA kit and provided a more detailed description of the JH detection method. The revised version is as follows:

The 4- to 6-day-old larvae (n = 3 per time point) fed with Mimics, Inhibitors, Mimic‑NC, or Inhibitor‑NC were collected, rinsed three times with phosphate buffered saline (PBS), and surface moisture was gently removed. An enzyme-linked immunosorbent assay (ELISA) kit (ml321040, mlbio, China) was used to detect juvenile hormone (JH) titer following the manufacturer's instructions, with improvements made during JH extraction from larvae (Qiu et al., 2023; Chen et al., 2024). Briefly, each sample was homogenized in 200 μL of physiological saline on ice, followed by the addition of 1000 μL of n-hexane and 500 μL of 70% methanol. The mixture was centrifuged at 12,000 rpm for 10 minutes. The upper n-hexane layer was collected, and the solvent was removed using a freeze-dryer (ALPHAL-2, Christ, Germany). Subsequently, 100 μL of standard diluent buffer (PBS buffer containing 1% Bull Serum Albumin and 0.05% Tween-20) was added. Add 50μL of the diluted standard and 50μL of the sample to the reaction wells respectively, then immediately add 50 μL of horseradish peroxidase (HRP)-labeled antibody to allow the hormone to bind with the antibody, forming an antibody–antigen–enzyme-labeled antibody complex. After thorough washing, 3,3′,5,5′-tetramethylbenzidine (TMB) is added. Due to the catalytic action of HRP, TMB is initially converted into a blue product. Under acidic conditions, it turns into a yellow product. The intensity of the color is positively correlated with the insect hormone level in the sample. Absorbance (optical density, OD value) is measured at a wavelength of 450 nm using a microplate reader. The assay was performed with three biological replicates, each consisting of three technical replicates.

Reference

• Chen JJ, Liu XX, Guo PH, Teets NM, Zhou JC, Chen WB, Luo QZ, Kanjana N, Li YY, Zhang LS. Regulation of forkhead box O transcription factor by insulin signaling pathway controls the reproductive diapause of the lady beetle, Coccinella septempunctata. Int J Biol Macromol. 2024, 258(Pt 1):128104. doi: 10.1016/j.ijbiomac.2023.128104.
• Qiu J, Dai T, Luo C, Cui W, Liu K, Li J, Sima Y, Xu S. Circadian clock regulates developmental time through ecdysone and juvenile hormones in Bombyx mori. Insect Mol Biol. 2023, 32(4):352-362. doi: 10.1111/imb.12835.

Comment 3: What does “t-text” mean? This error appears in Figures 2, 3, 4, and 5 and should be corrected to t-test. In addition, in Line 110, “bechtop” should be corrected to benchtop.

Response 3: Thank you very much for your valuable comment. All occurrences corrected (figures and main text).

Comment 4: In the Results section (Section 3.1), the authors state that "novel-m0027-3p could be detected across all eight time points examined, spanning the larval, prepupa, and adult stages (Fig. 1A)". However, according to the experimental description in the Materials and Methods (Section 2.2) and the labels in Figure 1A and Figure 1C, the collected samples only include 4-day-old and 6-day-old larvae, 2-day-old prepupae, and pupae at 1, 3, 5, 7, and 9 days of age. The adult stage was not actually involved or tested in this study。

Response 4: We apologize for the descriptive error, and have replaced "adult" with "pupal" in the results section.

Comment 5: The capitalization and abbreviation of the target gene are used inconsistently throughout the manuscript. For instance, "AmJhamt" appears frequently in the Abstract , Introduction , results text , and Discussion. Conversely, "Amjhamt" is used elsewhere in the Abstract , the Materials and Methods , and within the labels and axes of Figure 2 and Figure 3. In a formal scientific publication, gene and protein nomenclature must be maintained with strict consistency across the entire paper.

Response 5: Thank you very much for your valuable comment. We have standardized gene nomenclature throughout the Abstract, Methods, Results, Discussion, and figure axes/legends.

Comment 6: In result 3.3, the author said” The results indicate that novel-m0027-3p has a limited effect on body weight.”. however, in abstract, they claimed “These alterations in JH levels further affected larval body weigh”. Whether body weight is affected should be stated in a unified and clearly defined manner.

Response 6: Thank you very much for your valuable comment. We have removed the contradictory descriptions in the abstract, and supplemented the content in the discussion section. Our results suggested that novel‑m0027‑3p can regulate Jhamt, thereby affecting JH levels to a certain extent, but its impact on larval body weight is relatively limited. The modifications are as follows:

Revision: This, in turn, suppresses or promotes the expression levels of downstream JH‑responsive genes, AmHex70b and AmUsp (Fig. 4), thereby affecting larval body weight (Fig. 5). In contrast to the phenotypic responses to JH in other insects, honeybee larvae are highly sensitive to JH between days 4 and 6, which is critical for caste differentiation (Gong et al., 2023; Rembold et al., 1974). The most important phenotypic indicators of caste differentiation are body weight and size. These findings align with our previous study showing that Ame‑miR‑2161 affects the larval‑to‑pupal transition in honey bees via the AmJhamt gene [38]. Our results suggested that novel‑m0027‑3p regulate AmJhamt, thereby affecting JH levels to a certain extent, but its impact on larval body weight is relatively limited. The potential hormone-related target genes bound by novel-m0027-3p are not limited to AmJhamt but may also include key hormonal signaling factors such as AmE75 and AmUsp. Therefore, the alteration in larval body weight induced by novel-m0027-3p may not be mediated solely through AmJhamt. In conclusion, this study confirms that novel‑m0027‑3p mediates JH synthesis by negatively regulating the expression of AmJhamt.

• Gong ZX, Cheng FP, Xu JN, Yan WY, Wang ZL. The Juvenile-Hormone-Responsive Factor AmKr-h1 Regulates Caste Differentiation in Honey Bees. Biomolecules. 2023, 13(11):1657. doi: 10.3390/biom13111657.
• Rembold H, Czoppelt C, Rao PJ. Effect of juvenile hormone treatment on caste differentiation in the honeybee, Apis mellifera. J Insect Physiol. 1974, 20(7):1193-1202. doi: 10.1016/0022-1910(74)90225-x.

Comment 7: “Fourth-, fifth-, and sixth-instar larvae (n = 3 per group)”，The sample size is too small, and I am not sure whether this is the reason why no significant change in body weight was observed.

Response 7: We agree with the reviewer's point that the small sample size may be the reason for the lack of significant differences in body weight. Subsequent experiments will prioritize increasing the sample size.