Candidate Pheromone Receptors of the Red-Belted Clearwing Moth Synanthedon myopaeformis Bind Pear Ester and Other Semiochemicals

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

-Rewrite and re-select keywords.

-The citation of reference [3] should be placed at the end of the sentence.

-“Pheromone Receptors” changed to “pheromone rceptors”

-The introduction should include more research progress on OR recognition of pheromones and less on the application of experimental methods, such as transgenic Drosophila meanogaster.

-The references in the method section range from [36] to [69], are there too many cited references?

-Re-do Fig. 2 to make it more intuitive and clear.

-The content in Table 1 should be distinguished whether it belongs to the method section or the result section.

-The results section should directly describe the results obtained from the experiment, and should not include any references.

-Results rewritten, describing the main trial results, removing irrelevant information.

-What is the behavioral regulation effect of the semiochemicals screened by OR on the target pests in the discussion.

-The discussion should be analyzed based on the key results of the study, the discussion section is lengthy, appropriately reduce.

-“In this study we have identifed, isolated and functionally characterised some key odorant receptors of the red belted clearwing moth S. myophaeformis, conducting their heterologous expression in ab3A neurons of transgenic D. melagogaster that we approached by SSR and GC-SSR. Up to our knowledge, this is the frst contribution to the chemosensory deorphanization of this pest insect's odorant receptors that adds to the ongoing knowledge on its ecology. Since the last decade, semiochemical based control strategies applied for feld trapping of S. myophaeformis demonstrates parallelism between this moth and C. pomonella: both insects are atracted to pear ester, that together with other apple emited ligands, was identifed among the main activators interfering with the behavior of S. myophaeformis [34].” This part is suggested to be deleted.

Author Response

Comment 1: Rewrite and re-select keywords.

Response 1: We are convinced that the keywords, as they are indicated and ordered, represent correctly the content of our study. We profited from the reviewer’s suggestion to integrate “OR” to the keyword “Odorant Receptors”.

 

Comment 2: The citation of reference [3] should be placed at the end of the sentence.

Response 2: Done

 

Comment 3: “Pheromone Receptors” changed to “pheromone receptors”

Response 3: Done, along the whole manuscript.

 

Comment 4: The introduction should include more research progress on OR recognition of pheromones and less on the application of experimental methods, such as transgenic Drosophila melanogaster.

Response 4: We acknowledge the reviewer’s comment, however key results from this manuscript depended on the use of SSR and GC-SSR methods. Experiments have been conducted in comparative view with what has been documented from C. pomonella, selecting key OR candidates (OR3s and OR22s), which were screened to a few ligands based on the emission of apple and on previous knowledge on codling moth’s orthologues. To justify our choice of introducing the application of experimental methods based on transgenic Drosophila, rather than going into a wider description of OR-sensing of pheromones in insects, we have integrated a few texts among lines 95-99.

 

Comment 5: The references in the method section range from [36] to [69], are there too many cited references?

Response 5: We acknowledge, as indicated by the Reviewer, that 34 references cited in the Material and Methods section may represent a high number of citations. However, we are still convinced that this number represents the minimal number of required citations to describe the use of a wide variety of methodologies, that in this manuscript spread from RNA-sequencing to phylogenetic analysis, cloning, electrophysiology and structural analysis.

 

Comment 6: Re-do Fig. 2 to make it more intuitive and clear.

Response 6: We have now provided an updated version of Figure 2, that we think is more intuitive and clearer. We have edited Fig. 2 caption accordingly (lines 392-397).

 

Comment 7: The content in Table 1 should be distinguished whether it belongs to the method section or the result section.

Response 7: As from the reviewer’s request, we have integrated a short clarification into the Table’s caption (lines 249-250), with the intent to distinguish contents of Table 1 between methods and results.

 

Comment 8: The results section should directly describe the results obtained from the experiment, and should not include any references.

Response 8: We agree with the reviewer that references in the results may be superfluous. In accordance, we removed referencing to [29] (Cattaneo et al. 2025 - https://doi.org/10.1007/s10886-025-01579-1) and referencing to [72] (Butterwick et al. 2018 - https://doi.org/10.1038/s41586-018-0420-8 - which is now [108]), that are already cited elsewhere.

However, we decided to keep citation for the following [23] (Dobritsa et al. 2003 - https://doi.org/10.1016/s0896-6273(03)00094-1) and [65] (Cattaneo et al. 2022 - https://doi.org/10.1007/s10340-022-01585-2), motivating evidence reported from previous studies. Indeed, these studies discussed the absence of ab3A-spiking in lines of D. melanogaster lacking the OR22a gene, where recovery of ab3A-spiking upon replacing a heterologous OR represents evidence of the functional expression of the latter, which is of importance before attempting deorphanization to candidate ligands. We have also considered the importance of keeping these citations in this section of the manuscript, since this will help us in responding also to another Reviewer’s request.

In addition, we decided to keep also the following citations at lines 458-460: [71] (Bobkov et al. 2021 - https://doi.org/10.1038/s41598-021-83024-3) and [72] (Miller and Tu 2008 - https://doi.org/10.1673/031.008.5301); in support to our results, we have now stressed in the text of the Results the importance of the conservation of a tryptophan residue that we have found in SmyoORs within a documented ICL-3 hotspots of binding energy that it is shared among insect ORs, including ORs of the codling moth (Bobkov et al. [71]).

 

Comment 9: Results rewritten, describing the main trial results, removing irrelevant information.

Response 9: Done, as indicated in response to the previous comment. We went through the whole result session and we did not find information, which can be considered as irrelevant.

 

Comment 10: What is the behavioral regulation effect of the semiochemicals screened by OR on the target pests in the discussion.

Response 10: Apart from the reported data on pear ester, that was demonstrated as a key attractant from both C. pomonella and S. myophaeformis (Tóth et al. [34]) the semiochemicals that we have screened in this study and that we have found active on the ORs of S. myophaeformis have not been tested behaviorally on this species before, and our evidence is merely electrophysiological on SmyoOR-channels expressed ex vivo. Thus, despite that we agree with the reviewer that this information would add to our manuscript, at the current stage, it is not possible to state and discuss behavioral regulation effects from the semiochemicals active on the ORs of this moth. In our manuscript we could only hypothesize effects or motivate evidence of SmyoOR-binding of various compounds based on previous data from the codling moth. We listed some key examples along the discussion section, that are summarized below.

Between lines 581-587, we have indicated the possibility of SmyoOR22.4-sensing of the same pheromones emitted by C. pomonella that may be related with trophic interactions, since both species share the same plant host; between lines 598-602 we motivated SmyoOR3.4- and OR22.4-binding to the (Z)-4-undecenal aldehydes suggesting possible structural similarities to other aldehyde ligands, given the wide emission of the latter from various plants and animal sources; between lines 611-618 we discussed similarities with aldehyde binding between SmyoOR3.4 and the codling moth CpomOR22, consistent with the idea that various insects’ chemosensory receptor channels can be characterized by a somewhat common pharmacology; between lines 622-629, upon demonstrating the overall binding of SmyoORs to various non-host ligands, we justified consistency with evidence from the insects’ chemosensory combinatorial coding of OR-sensors, which lay at the foundation of insects’ olfactory systems, for which while some ORs may respond to unique ligands, others may respond to only few ligands, or may have a wider spectrum of activation among multiple variegate ligands.

 

Comment 11: The discussion should be analyzed based on the key results of the study, the discussion section is lengthy, appropriately reduce.

Response 11: We went along the whole Discussion session, and we did not find information, which can be considered to be irrelevant.

 

Comment 12: “In this study we have identifed, isolated and functionally characterized some key odorant receptors of the red belted clearwing moth S. myophaeformis, conducting their heterologous expression in ab3A neurons of transgenic D. melagogaster that we approached by SSR and GC-SSR. Up to our knowledge, this is the first contribution to the chemosensory deorphanization of this pest insect's odorant receptors that adds to the ongoing knowledge on its ecology. Since the last decade, semiochemical based control strategies applied for field trapping of S. myophaeformis demonstrates parallelism between this moth and C. pomonella: both insects are attracted to pear ester, that together with other apple emited ligands, was identified among the main activators interfering with the behavior of S. myophaeformis [34].” This part is suggested to be deleted.

Response 12: As to the reviewer’s request, we removed this section, and we have rearranged the description to provide a better flow of the discussion.

Reviewer 2 Report

Comments and Suggestions for Authors

Authors Cattaneo and Walker III presented a study on the functions of ORs in the red-belted clearwing moth Synanthedon myophaeformis. Authors first performed transcriptome analysis and identified 65 candidate ORs, from which at least 62 ORs are specifically expressed in the male antennae. Then authors conducted phylogenic analysis and focus on the pheromone receptor clade, OR3 and OR22 candidate homologues, and chose 8 OR candidates with relatively high expression in male S. myophaeformis antennae for further functional characterization studies. Through transgenic overexpression of 8 candidate ORs in Drosophila followed by SSR to screen the active ORs toward 27 odorant compounds. Based on SSR data, authors focused on the OR3.4 and OR22.4, and found that OR3.4 response to three semiochemicals in a dose-dependent manner, OR3.4 may play a key role in detecting these semiochemicals. In addition, authors performed GC-SSR which provides supplementary evidence to support their SSR results. The overall design of the research is impressive. However, the authors could enhance the clarity and impact of their manuscript by providing additional details or reorganizing some of the data. Below are some suggestions to further improve the quality of the manuscript.

Page 5, line 19 “To drive expression of SmyoOR3s in in the A neuron of ab3 basiconic sensilla…” delete the redundant “in”

Page 5, M&M section for “Cloning of Olfactory Receptors for expression in the Drosophila empty neuron system”--- the authors described the methods for cloning and transgenic expression of SmyoORs in the Drosophila 'A' neuron, however, the successful expression of these eight ORs in the empty neuron system should be confirmed, for example by qRT-PCR or visualization methods such as in situ hybridization.

Page 5, SSR: While the authors performed odor-evoked SSR responses to confirm functional expression of one SmyoOR3.4, similar confirmation is lacking for the other seven receptors. To support the claim that all eight SmyoORs were successfully expressed in the Drosophila empty neuron system, additional functional or molecular validation (e.g., qRT-PCR or in situ hybridization) is needed (as descripted above).

Page 10, Figure 1: The resolution of this figure should be enhanced for better clarity. Additionally, where can the sequences for the other ORs used to construct this tree be found? Please provide this information with the supplementary data.

Page 11, Figure 2 presents the relative expression levels of 65 ORs in male antennae (based on Supplementary File 5); however, expression level alone does not indicate receptor function in pheromone or other semiochemical binding. Since this figure does not directly support functional conclusions and the selection of the 8 ORs appears to be based on phylogenetic relationships rather than expression alone, I recommend moving this figure from the main text.

Figure 3A: The absence of spiking for SmyoOR3.2 and SmyoOR22.2 could be due to either unsuccessful transgenic expression or a lack of responsiveness to the tested compounds. Since the authors did not confirm the expression independently, it remains unclear which is the case. Additional validation would help clarify whether these ORs are truly non-functional or simply not expressed. The quality of this figure needs to be enhanced.

Figure 5B. This figure shows preliminary data from their experiment, it would be helpful to improve the clarity and labeling of this figure properly.

Figure 6, C: This is the 3D structure of CpomOR3. However, it is unclear how this structure specifically contributes to the current study on S. myophaeformis, given that the authors could have used the amino acid sequence of S. myophaeformis OR3.4 to generate a 3D structure using AlphaFold. Could the authors clarify the relevance of including CpomOR3’s structure in this context? In addition, authors presented two figures that appear to be related to the 3D structures, but they are not clearly labeled or differentiated. N-terminal and C-terminals should be labeled in these figures as well.

Comments on the Quality of English Language

The English writing is generally clear, but the manuscript would benefit from minor revisions. I have highlighted a few small errors for correction.

Author Response

Authors Cattaneo and Walker III presented a study on the functions of ORs in the red-belted clearwing moth Synanthedon myophaeformis. Authors first performed transcriptome analysis and identified 65 candidate ORs, from which at least 62 ORs are specifically expressed in the male antennae. Then authors conducted phylogenetic analysis and focused on the pheromone receptor clade, OR3 and OR22 candidate homologues, and chose 8 OR candidates with relatively high expression in male S. myophaeformis antennae for further functional characterization studies. Through transgenic overexpression of 8 candidate ORs in Drosophila followed by SSR to screen the active ORs toward 27 odorant compounds. Based on SSR data, authors focused on the OR3.4 and OR22.4, and found that OR3.4 response to three semiochemicals in a dose-dependent manner, OR3.4 may play a key role in detecting these semiochemicals. In addition, authors performed GC-SSR which provides supplementary evidence to support their SSR results. The overall design of the research is impressive. However, the authors could enhance the clarity and impact of their manuscript by providing additional details or reorganizing some of the data. Below are some suggestions to further improve the quality of the manuscript.

 

Comment 1: Page 5, line 19 “To drive expression of SmyoOR3s in in the A neuron of ab3 basiconic sensilla…” delete the redundant “in”

Response 1: Done.

 

Comment 2: Page 5, M&M section for “Cloning of Olfactory Receptors for expression in the Drosophila empty neuron system”--- the authors described the methods for cloning and transgenic expression of SmyoORs in the Drosophila 'A' neuron, however, the successful expression of these eight ORs in the empty neuron system should be confirmed, for example by qRT-PCR or visualization methods such as in situ hybridization.

Response 2: We agree with the reviewer, that additional PCR analysis would prove the expression of the heterologous ORs in Drosophila, as it would also be the case with in situ hybridizations. However, this study resulted in line with many others where we have used the same technique up to now and where demonstrating re-establishment of ab3A spiking from empty neuron flies, upon crossing with lines carrying the OR-transgene, was sufficient to demonstrate the expression of the latter. To stress this evidence further, we have integrated a few texts in lines 404-405 where we cited references [23;65] where this scenario was widely discussed. Additionally, both me and the senior author moved from the original institution where SSR studies have been conducted (SLU-Alnarp) and we are not currently provided with the technological facilities to undertake similar experiments with transgenic Drosophila. However, lines expressing SmyoORs have been deposited to the Bloomington Drosophila Stock Center (BDSC #98395 to #98401): in the frame of future projects, these flies can be purchased to validate, if needed, the expression of these transgenes.

 

Comment 3: Page 5, SSR: While the authors performed odor-evoked SSR responses to confirm functional expression of one SmyoOR3.4, similar confirmation is lacking for the other seven receptors. To support the claim that all eight SmyoORs were successfully expressed in the Drosophila empty neuron system, additional functional or molecular validation (e.g., qRT-PCR or in situ hybridization) is needed (as descripted above).

Response 3: We acknowledge the reviewer for this comment, which helped us realizing that we dragged a typing error to the submitted version of the manuscript. Indeed, in the previous Single sensillum recordings (SSR) method section we mentioned about SmyoOR3-only, while the same method has been applied to all SmyoORs, including OR3s and OR22s. We have now corrected this error, see from line 218.

 Except for SmyoOR3.2 and SmyoOR22.2 subunits, which did not show evident ab3A spiking, confirmation of OR-expression based on ligand activation is evident for most of the receptors, by both re-establishing ab3A spiking and resulting in one or more active ligands.

 

Comment 4: Page 10, Figure 1: The resolution of this figure should be enhanced for better clarity.

Response 4: For better clarity, we have increased the size of the figure in the word file of the manuscript, which respected requests advanced also from Reviewer 3.

Comment 5: Additionally, where can the sequences for the other ORs used to construct this tree be found? Please provide this information with the supplementary data.

Response 5: The sequences of the ORs from other species may be found from the references cited in the Materials

and Methods section (lines 170-172), citations 50-53. For convenience, we have added a new supplementary data file with all the sequences in FASTA format (now Supplementary Data File 2).

 

Comment 6: Page 11, Figure 2 presents the relative expression levels of 65 ORs in male antennae (based on Supplementary File 5); however, expression level alone does not indicate receptor function in pheromone or other semiochemical binding. Since this figure does not directly support functional conclusions and the selection of the 8 ORs appears to be based on phylogenetic relationships rather than expression alone, I recommend moving this figure from the main text.

Response 6: We agree with the assessment that expression level alone does not indicate receptor function for any kind of ORs. It was not our intention to presume this. It was only our intention to display that several ORs among the OR3 and OR22 subfamilies were among the most highly expressed ORs in the male antennae. This is not indicative of binding to either pheromones or kairomones, it only highlights the relative abundance of the ORs as a correlate of relative potential inputs to the brain. So, we make no claims about what highly expressed ORs will bind based on high expression levels, but there is evidence to support functional significance for highly expressed ORs.

For example: in the course of our investigations on C. pomonella ORs, a similar scenario appeared when we identified CpomOR3 detecting pear ester (Bengtsson et al. [28]) with sensitivity and specificity, which was first identified as a highly attractive candidate kairomone with pheromone-like potency on both males and females. In fact, CpomOR3 was nested in the canonical PR clade and our transcriptome data suggested that this subunit was the most highly expressed OR in female antennae and the third highest in male antennae (Walker et al. [27]).

 

According to requests from other reviewers, we have revised this figure for clarity and have decided to keep it among the main figures.

 

Comment 7: Figure 3A: The absence of spiking for SmyoOR3.2 and SmyoOR22.2 could be due to either unsuccessful transgenic expression or a lack of responsiveness to the tested compounds.

Response 7: Based on this comment, we want to stress the evidence that the absence of background levels of ab3A spiking, before testing any ligand demonstrates, a priori, an unsuccessful transgenic expression of the heterologous ORs. We mentioned this evidence between lines 404 and 405, which are supported since long-dated findings (Dobritsa et al. [23]). Indeed, lack of responsiveness to the tested compounds can be demonstrated only upon accessing spiking ab3A neurons.

 

Comment 8: Since the authors did not confirm the expression independently, it remains unclear which is the case. Additional validation would help clarify whether these ORs are truly non-functional or simply not expressed.

Response 8: Acknowledging the reviewer’s point, we have added additional text between lines 539 and 547, stressing the impossibility of proving if these receptors would be functional or not, given the fact that, based on our results, they appeared to be simply not expressed. In this text, we open to future projects attempting expression of SmyoOR3.2 and OR22.2 from updated fly-lines that we have recently deposited at the Bloomington Drosophila Stock Center upon crossing with CRISPR-gene edited mutants replacing their OR22a with Gal4 (Chahda et al. [75] - https://doi.org/10.1371/journal.pgen.1008005). Note: this reference was integrated into the manuscript and cited, accordingly, at line 544.

 

Comment 9: The quality of this figure needs to be enhanced.

Response 9: We have improved the quality of Figure 3A and uploaded an updated version of the figure.

 

Comment 10: Figure 5B. This figure shows preliminary data from their experiment, it would be helpful to improve the clarity and labeling of this figure properly.

Response 10: We have improved the quality of Figure 5 and uploaded an updated version of the figure.

 

Comment 11: Figure 6, C: This is the 3D structure of CpomOR3. However, it is unclear how this structure specifically contributes to the current study on S. myophaeformis, given that the authors could have used the amino acid sequence of S. myophaeformis OR3.4 to generate a 3D structure using AlphaFold.

Response 11: Unfortunately, AlphaFold 3D structure for SmyoOR3.4 is not deposited. For this reason, we chose CpomOR3. We have stressed this aspect further in the method section at lines 338-339.

 

Comment 12: Could the authors clarify the relevance of including CpomOR3’s structure in this context? In addition, authors presented two figures that appear to be related to the 3D structures, but they are not clearly labeled or differentiated. N-terminal and C-terminals should be labeled in these figures as well.

Response 12: We have edited the figure caption accordingly and integrated indications from the first and the last amino acid of the polypeptide to clarify contents of Figure 6C.

Reviewer 3 Report

Comments and Suggestions for Authors

The author has conducted a detailed study on the candidate pheromone receptors of the red-belted clearwing moth Synanthedon myophaeformis that bind pear ester and other semiochemicals, which is of great value and significance. However, there are a few issues:

Line 67 “use of transgenic Drosophila meanogaster represented a” should be corrected to Drosophila melanogaster.

Lines 156-164 The sentence “Read mapping of individual sample reads to the de novo transcriptome and subsequent expression level abundance estimations were carried out, as described [46] with the Trinity Perl script ‘align_and_estimate_abundance.pl’ in the release version of Trinity 2.8.4, using RSEM v.1.2.12 [38], Bowtie v.0.12.6 [47] and samtools v.0.1.19 [48]. The CD-HIT-EST-modified Trinity.fasta file was used as reference transcripts input and the trimmed fastq male adult antennal reads described above were used as mapping input” is repetitive with the earlier mention of Trinity. I suggest the author consolidate this into one section to avoid redundancy.

RNA Sequencing and Transcriptomics Analysis: This section is repetitive and overly lengthy. De novo transcriptome assembly is a common process; I suggest simplifying and consolidating the description to avoid unnecessary repetition.

Lines 239-251 In the sentence "To confirm expression of SmyoOR3-transgenes, basic spiking of ab3-neurons were compared with the same parental…..", the paragraph formatting is problematic.

Line 352 "In total, 237,934 transcripts (>201 nt)" - The abbreviation "nt" should be spelled out for clarity, especially when it is first introduced.

Figure 1 is too small and almost unreadable. It should be enlarged for clarity.

Line 385 The species name Bombyx mori should be italicized in the figure legend.

Figure 2 is hard to interpret and needs to be improved. Additionally, a color bar should be provided indicating log2(FPKM+1). There is also a contradiction between the figure's depiction (log2(FPKM)) and the figure legend's description.

While the discussion is well-written, it lacks in-depth discussion regarding the application and ecological significance of these findings. I suggest that the author appropriately expand on these aspects to deepen the theme and relevance of the work.

According to OA requirements, transcriptome data should be provided with a public data access ID once the manuscript is accepted.

Author Response

The author has conducted a detailed study on the candidate pheromone receptors of the red-belted clearwing moth Synanthedon myopaeformis that bind pear ester and other semiochemicals, which is of great value and significance. However, there are a few issues:

 

Comment 1: Line 67 “use of transgenic Drosophila meanogaster represented a” should be corrected to Drosophila melanogaster.
Response 1: Done.

Comment 2: Lines 156-164 The sentence “Read mapping of individual sample reads to the de novo transcriptome and subsequent expression level abundance estimations were carried out, as described [46] with the Trinity Perl script ‘align_and_estimate_abundance.pl’ in the release version of Trinity 2.8.4, using RSEM v.1.2.12 [38], Bowtie v.0.12.6 [47] and samtools v.0.1.19 [48]. The CD-HIT-EST-modified Trinity.fasta file was used as reference transcripts input and the trimmed fastq male adult antennal reads described above were used as mapping input” is repetitive with the earlier mention of Trinity. I suggest the author consolidate this into one section to avoid redundancy.

Response 2: The use of RSEM with the earlier mention of Trinity (current version lines 141-151) is not repetitive to what is written here because in the first instance RSEM was used for filtering out low abundance reads, and in this instance, it was used for abundance estimations, so these were two distinct processes. Nonetheless, as noted in the response directly below this, substantial reductions in text have been made across this entire section (current version lines 152-165).

 

Comment 3: RNA Sequencing and Transcriptomics Analysis: This section is repetitive and overly lengthy. De novo transcriptome assembly is a common process; I suggest simplifying and consolidating the description to avoid unnecessary repetition.

Response 3: We thank the reviewer for bringing attention to redundancies in this section. Substantial amounts of text have been consolidated to shorten the length of this section.

Comment 4: Lines 239-251 In the sentence "To confirm expression of SmyoOR3-transgenes, basic spiking of ab3-neurons were compared with the same parental…..", the paragraph formatting is problematic.

Response 4: Solved

Comment 5: Line 352 "In total, 237,934 transcripts (>201 nt)" - The abbreviation "nt" should be spelled out for clarity, especially when it is first introduced.

Response 5: Done

Comment 6: Figure 1 is too small and almost unreadable. It should be enlarged for clarity.

Response 6: Done

Comment 7: Line 385 The species name Bombyx mori should be italicized in the figure legend.

Response 7: Done

Comment 8: Figure 2 is hard to interpret and needs to be improved. Additionally, a color bar should be provided indicating log2(FPKM+1). There is also a contradiction between the figure's depiction (log2(FPKM)) and the figure legend's description.

Response 8: Our apologies for the confusion. The figure has been revised. After thoroughly examining our data for this figure, we confirm that the The log2 FPKM+1 metric was not used in this report. The “plus one” adjustment was a carry-over from previous studies where FPKM values for some transcripts were zero, however, that did not happen in this study. So we have deleted the mention in the figure legend of FPKM+1. Our apologies. There should no longer be any contradiction between the figure legend and the figure. Also, labeling of the data headers in the figure and description in the figure legend has been revised for greater clarity.

Comment 9: While the discussion is well-written, it lacks in-depth discussion regarding the application and ecological significance of these findings. I suggest that the author appropriately expand on these aspects to deepen the theme and relevance of the work.

Response 9: We thank the reviewer for suggesting this implementation. We have added additional text in the conclusion section between lines 687 and 694.

Comment 10: According to OA requirements, transcriptome data should be provided with a public data access ID once the manuscript is accepted.

Response 10: Both authors have moved on from the institute where the research was conducted. The transcriptome raw sequencing data currently exists on an external hard drive at the previous institution. We have asked for this hard drive to be shipped to the current address of the senior author so that the files may be appropriately deposited to an online public database. We are still waiting for this shipment and can assure that this will be handled as soon as possible. This point has been clarified also directly with the editorial office of the journal.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

 Accept.