Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a high-quality study featuring a rigorous experimental design, robust data, and conclusions of significant scientific importance. The authors successfully identified and functionally characterized two Benzaldehyde-responsive olfactory receptors in Mythimna separata, revealed their distinct ligand selectivities, and explored their ecological and evolutionary implications. This study provides new insights into how insects encode chemical signals with multiple ecological functions, and potential targets for odor-based pest management

 

Some Comments:

 

Q1:

Page 1 line 31-33: The sentence is structurally incomplete as it lacks a main predicate verb. Please add the appropriate linking verb.

 

Q2:

Page 2 line 50: There is a redundant expression in the phrase "rely depend on". Please correct this to either "rely on" or "depend on" to eliminate the duplication.

 

Q3:

Page 3 line 101: The phrase “In H. armigera, functional has analyses have identified...” is grammatically incorrect. Please revise the sentence accordingly.

 

Q4:

Page 4 line141: The phrase “to support normal, development and reproduction” contains an unnecessary comma. Please remove the comma.

 

Q5:

Page 4 line 159: The word “identify” should be changed to “identity” to fit the grammatical context of the sentence.

 

Q6:

Page 4 line 176: The phrase “EAG was amplitudes were corrected...” is grammatically incorrect. Please revise the sentence accordingly.

 

Q7:

Page 4 line 179: Please change “were” to “was” to ensure grammatical agreement.

 

Q8:

Page 4 line 185: The formatting of the IQ-TREE parameters appears to be incomplete. In particular, the quotation marks and closing punctuation around “-m MFP -bb 1000” should be checked and corrected.

 

Q9:

Page 5 line 215: delete “included”.

 

Q10:

The capitalization of section headings in the Materials and Methods and Results sections is inconsistent. Please revise the headings to follow a consistent capitalization style throughout the manuscript.

 

Q11:

In Sections 2.7 and 3.4, both “quantitative real-time PCR (qPCR)” and “qRT-PCR” are used. Please use one terminology consistently throughout the manuscript.

 

Q12:

Concentration units are expressed using both "M" and "mol/L" in different parts of the manuscript. Please use a consistent format throughout.

 

Q13:

Page 9, line 330 and 332: The EC₅₀ values should be presented using standard subscript formatting. Please ensure that “EC₅₀” is consistently formatted throughout the manuscript.

 

Q14:

Please check the punctuation and parentheses in the Figure 2 legend. In particular, the phrase “Msep, M. separata);” contains a mismatched parenthesis and should be corrected.

 

Q15:

Page 11, lines 348–355: The opening paragraph of the Discussion largely repeats the main findings presented in Section 3.5, particularly the different ligand profiles of MsepOR29 and MsepOR42. Please shorten this description and focus more on the significance of the contrasting ligand tuning profiles.

 

Q16:

Page 12, lines 392–407: The Discussion proposes several possible ecological functions of BA-responsive ORs, including host or nectar-resource localization and social communication. Since these functions were not directly examined in the present study, please briefly clarify that these interpretations are speculative and should be considered as potential functions rather than established roles.

 

Author Response

Response to Reviewer 1

Comments and Suggestions for Authors

This is a high-quality study featuring a rigorous experimental design, robust data, and conclusions of significant scientific importance. The authors successfully identified and functionally characterized two Benzaldehyde-responsive olfactory receptors in Mythimna separata, revealed their distinct ligand selectivities, and explored their ecological and evolutionary implications. This study provides new insights into how insects encode chemical signals with multiple ecological functions, and potential targets for odor-based pest management.

Answer: We thank the reviewer for the positive and encouraging evaluation of our study. We also appreciate the constructive comments, which have helped us further improve the clarity and quality of our manuscript.

 

Some Comments:

Q1: Page 1 line 31-33: The sentence is structurally incomplete as it lacks a main predicate verb. Please add the appropriate linking verb.

Answer: We have modified. See line 32-33.

 

Q2: Page 2 line 50: There is a redundant expression in the phrase "rely depend on". Please correct this to either "rely on" or "depend on" to eliminate the duplication.

Answer: We have modified. See line 50.

 

Q3: Page 3 line 101: The phrase “In H. armigera, functional has analyses have identified...” is grammatically incorrect. Please revise the sentence accordingly.

Answer: We have modified. See line 102.

 

Q4: Page 4 line141: The phrase “to support normal, development and reproduction” contains an unnecessary comma. Please remove the comma.

Answer: We have deleted. See line 142.

 

Q5: Page 4 line 159: The word “identify” should be changed to “identity” to fit the grammatical context of the sentence.

Answer: We have modified. See line 162.

 

Q6: Page 4 line 176: The phrase “EAG was amplitudes were corrected...” is grammatically incorrect. Please revise the sentence accordingly.

Answer: We have modified. See line 178.

 

Q7: Page 4 line 179: Please change “were” to “was” to ensure grammatical agreement.

Answer: We have modified. See line 181.

 

Q8: Page 4 line 185: The formatting of the IQ-TREE parameters appears to be incomplete. In particular, the quotation marks and closing punctuation around “-m MFP -bb 1000” should be checked and corrected.

Answer: We have modified. See line 187.

 

Q9: Page 5 line 215: delete “included”.

Answer: We have deleted. See line 217.

 

Q10: The capitalization of section headings in the Materials and Methods and Results sections is inconsistent. Please revise the headings to follow a consistent capitalization style throughout the manuscript.

Answer: We have carefully checked the section headings throughout the Materials and Methods and Results sections and have modified their capitalization.

 

Q11: In Sections 2.7 and 3.4, both “quantitative real-time PCR (qPCR)” and “qRT-PCR” are used. Please use one terminology consistently throughout the manuscript.

Answer: We have modified. See line 227 and 310.

 

Q12: Concentration units are expressed using both "M" and "mol/L" in different parts of the manuscript. Please use a consistent format throughout.

Answer: We have modified. See line 151, 154, 230, 346 and 352.

 

Q13: Page 9, line 330 and 332: The EC₅₀ values should be presented using standard subscript formatting. Please ensure that “EC₅₀” is consistently formatted throughout the manuscript.

Answer: We have modified. See line 338 and 340.

 

Q14: Please check the punctuation and parentheses in the Figure 2 legend. In particular, the phrase “Msep, M. separata);” contains a mismatched parenthesis and should be corrected.

Answer: We have modified. See line 293.

 

Q15: Page 11, lines 348–355: The opening paragraph of the Discussion largely repeats the main findings presented in Section 3.5, particularly the different ligand profiles of MsepOR29 and MsepOR42. Please shorten this description and focus more on the significance of the contrasting ligand tuning profiles.

Answer: We have shortened the opening paragraph of the Discussion to reduce repetition of the Results and have emphasized the significance of the contrasting ligand tuning profiles of MsepOR29 and MsepOR42. See line 358-366.

 

Q16: Page 12, lines 392–407: The Discussion proposes several possible ecological functions of BA-responsive ORs, including host or nectar-resource localization and social communication. Since these functions were not directly examined in the present study, please briefly clarify that these interpretations are speculative and should be considered as potential functions rather than established roles.

Answer: Thank you for your suggestion. We have revised the Discussion to clarify that the potential roles of MsepOR29 and MsepOR42 in host and nectar resource localization and social communication remain speculative and require further validation through in vivo physiological and behavioral studies. See line 429-440.

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript systematically investigates the peripheral olfactory mechanism underlying benzaldehyde (BA) perception in Mythimna separata, a major migratory pest of cereal crops in Asia. Building on the authors’ prior work on BA as a male-emitted pheromone antagonist, this study identifies two conventional odorant receptors (MsepOR29 and MsepOR42) with distinct ligand selectivity via phylogenetic screening, molecular cloning, tissue expression profiling, and Xenopus oocyte two-electrode voltage-clamp recording. Overall, the experimental design is reasonably rigorous, and the core conclusions are well supported by the collected data. The findings advance our understanding of olfactory coding in noctuid moths while providing candidate molecular targets for odor-based pest management. This work merits publication in Insects provided the authors adequately address the following concerns and revise the manuscript accordingly.

Specific Comments and Suggestions:

Although heterologous expression in Xenopus oocytes characterized the ligand profiles of MsepOR29 and MsepOR42, functional evidence is restricted to in‑vitro systems. There is a shortage of in‑vivo electrophysiological and behavioral validation based on RNAi or CRISPR‑Cas9 knockout. Moreover, the ecological roles of these two receptors remain elusive: do MsepOR29 and MsepOR42 primarily detect plant‑derived benzaldehyde or benzaldehyde signals emitted by conspecific male moths? Future genetic manipulation combined with physiological and behavioral assays will address this open question. The authors should discuss detailed in the manuscript.

 

Minor Typos:

1. EAG experimental parameters: The study uses 4-day-old virgin adults for EAG assays. Please briefly justify the selection of this age point.
2. Statistical test selection for EAG and qPCR: The Mann–Whitney U test was used for sex comparisons. Please explain why a non-parametric test was selected instead of a Student’s t-test — for example, whether the EAG amplitude data failed normality tests. For qPCR analysis, it should be clearly specified which sample group served as the calibrator for normalization (for example: The relative expression level (fold) was calculated based on the value of female antennae by setting that expression level to value of 1).
3. In Table S1, Benzyl alchol is misspelled. It should be corrected to Benzyl alcohol.
4. In Table S1, tran-2-Hexen-1-ol is misspelled. It should be corrected to “trans-2-Hexen-1-ol”.
5. In Table S1, the CAS number for ethyl benzoate is incorrectly duplicated from methyl benzoate. The correct CAS number for ethyl benzoate is 93-89-0.
6. In Section 3.3: The sentence MsepOR29 was contained a 1248 bp CDS encoding 416 amino acids uses an incorrect passive construction. The verb contain should be in active voice here. Revised: MsepOR29 contained a 1248 bp CDS encoding 416 amino acids.
7. Line 305: The term “gonads” is used here, while “genitalia” is used consistently in figure legends and other sections of the manuscript. Please standardize the terminology to “genitalia” throughout for consistency.
8. Line 258: Please clarify the purpose of the word "Subsection" in the heading 3.1. Subsection BA elicits antennal EAG responses. This label appears redundant.
9. Line 185: The quotation mark formatting is improper.
10. The abbreviation "qPCR" appears at line 225 without its full definition on first mention, while the expanded form "quantitative real-time PCR (qRT-PCR)" is used later at line 302. Please define the abbreviation at its first occurrence and standardize the terminology uniformly across the manuscript.
11. Please carefully re-examine the full text and correct any residual spelling mistakes and other minor errors.
12. The discussion should be expanded to achieve greater depth and broader scope. 

 

Comments on the Quality of English Language

The manuscript is generally well‑written and the scientific logic is sound. However, professional English editing is strongly recommended.

Author Response

Response to Reviewer 2

Comments and Suggestions for Authors

This manuscript systematically investigates the peripheral olfactory mechanism underlying benzaldehyde (BA) perception in Mythimna separata, a major migratory pest of cereal crops in Asia. Building on the authors’ prior work on BA as a male-emitted pheromone antagonist, this study identifies two conventional odorant receptors (MsepOR29 and MsepOR42) with distinct ligand selectivity via phylogenetic screening, molecular cloning, tissue expression profiling, and Xenopus oocyte two-electrode voltage-clamp recording. Overall, the experimental design is reasonably rigorous, and the core conclusions are well supported by the collected data. The findings advance our understanding of olfactory coding in noctuid moths while providing candidate molecular targets for odor-based pest management. This work merits publication in Insects provided the authors adequately address the following concerns and revise the manuscript accordingly.

Answer: We thank the reviewer for the positive assessment of our manuscript and for the constructive comments. We have carefully considered all of the reviewer’s comments point by point and have thoroughly revised the manuscript accordingly. In addition, we commissioned a professional native English-speaking editor to polish the language of the entire manuscript, and the corresponding language editing certificate is provided in the file “Editing Certificate.” Our point-by-point responses to each specific comment are as follows.

 

Specific Comments and Suggestions:

Although heterologous expression in Xenopus oocytes characterized the ligand profiles of MsepOR29 and MsepOR42, functional evidence is restricted to in‑vitro systems. There is a shortage of in‑vivo electrophysiological and behavioral validation based on RNAi or CRISPR‑Cas9 knockout. Moreover, the ecological roles of these two receptors remain elusive: do MsepOR29 and MsepOR42 primarily detect plant‑derived benzaldehyde or benzaldehyde signals emitted by conspecific male moths? Future genetic manipulation combined with physiological and behavioral assays will address this open question. The authors should discuss detailed in the manuscript.

Answer: Thank you for your suggestion. We agree that the ecological roles of MsepOR29 and MsepOR42 require further investigation beyond the heterologous expression system. Based on the current data, it remains unresolved whether these receptors primarily detect plant-derived BA, male-derived BA, or both. We have expanded the Discussion section to address this limitation and discuss the potential ecological roles of BA from both plant-associated and conspecific sources, See line 366-384. We also added future perspectives involving RNAi/CRISPR-Cas9-mediated functional validation combined with electrophysiological and behavioral assays. See line 429-440.

 

Minor Typos:

1. EAG experimental parameters: The study uses 4-day-old virgin adults for EAG assays. Please briefly justify the selection of this age point.

Answer: In electrophysiological studies of moths, adults at a defined reproductive stage are commonly used to assess antennal responses to sex pheromones and host-plant volatiles (Prasuna et al., 1998; Fan et al., 2022). Because adult age and reproductive status can influence olfactory physiological responses, standardizing these factors helps minimize variation among experimental individuals. In M. separata adults generally reach sexual maturity at approximately 4 days post-eclosion (Jiang et al., 2019). Therefore, we selected 4-day-old virgin adults for the EAG assays to ensure a consistent physiological and reproductive state among individuals. The use of virgin adults also minimizes potential effects of mating on olfactory physiology and responsiveness, thereby allowing reliable and comparable evaluation of antennal responses to the tested odorants.

Prasuna, A.L.; Jyothi, K.N.; Sighamony, S.; Prasad, A.R.; Yadav, J.S. Antennal olfactory receptivity of Achaea janata Linn. (Lepidoptera: Noctuidae) to certain general plant volatiles—electrophysiological recording. J. Entomol. Res. 1998, 22, 1–9.

Fan, X.B.; Mo, B.T.; Li, G.C.; Huang, L.Q.; Guo, H.; Gong, X.L.; Wang, C.Z. Mutagenesis of the odorant receptor co-receptor (Orco) reveals severe olfactory defects in the crop pest moth Helicoverpa armigera. BMC Biol. 2022, 20, 214.

Chang, H.; Liu, Y.; Ai, D.; Jiang, X.; Dong, S.; Wang, G. A pheromone antagonist regulates optimal mating time in the moth Helicoverpa armigera. Curr. Biol. 2017, 27, 1610–1615.e3.

Jiang, N.-J.; Tang, R.; Wu, H.; Xu, M.; Ning, C.; Huang, L.-Q.; Wang, C.-Z. Dissecting sex pheromone communication of Mythimna separata (Walker) in North China from receptor molecules and antennal lobes to behavior. Insect Biochem. Mol. Biol. 2019, 111, 103176.

2. Statistical test selection for EAG and qPCR: The Mann–Whitney U test was used for sex comparisons. Please explain why a non-parametric test was selected instead of a Student’s t-test — for example, whether the EAG amplitude data failed normality tests.

Answer: Thanks for this helpful comment. We agree that the choice of statistical test should consider the distribution and sample size of the data. In this study, the EAG and qPCR analyses included 11 and 3 biological replicates per sex, respectively. Given the relatively small sample sizes, particularly the qPCR data with only 3 biological replicates, it is difficult to reliably determine from such limited data whether the underlying population distribution is normal. It is important to note that we did not exclude Student’s t-test solely on the basis of a predefined sample-size threshold (e.g., n < 30), as there is no universal minimum sample-size requirement for its application. However, small-sample data contain limited distributional information and are more sensitive to departures from normality (Canavos, 1988; Wang and Riffel, 2011; Kim and Park, 2019); therefore, when the normality assumption cannot be adequately verified, directly applying Student’s t-test may not be prudent. Accordingly, we used the Mann–Whitney U test for comparisons between sexes. This rank-based non-parametric test does not require data to be normally distributed and is therefore considered appropriate for the present data. Notably, the Mann–Whitney U test has been used in insect physiology and olfactory electrophysiology studies to compare differences between groups, including physiological responses such as EAG and molecular expression data (Chen et al., 2026; Ebrahim et al., 2021; Sun et al., 2025; Caragata et ai., 2017).

Canavos, G.C. The sensitivity of the one-sample and two-sample Student t statistics. Comput. Stat. Data Anal. 1988, 6, 39–46.

Wang, M.; Riffel, M. Making the right conclusions based on wrong results and small sample sizes: Interpretation of statistical tests in ecotoxicology. Ecotoxicol. Environ. Saf. 2011, 74, 684–692.

Kim, T.K.; Park, J.H. More about the basic assumptions of t-test: Normality and sample size. Korean J. Anesthesiol. 2019, 72, 331–335.

Chen, R.; Hu, W.; Hu, Q.; Yan, J.; Yin, J.; Khamis, F.M.; Gao, Y.; Leal, W.S. Semiochemicals and odorant receptors underlying potato cultivar susceptibility and resistance to potato tuber moth. Proc. Natl. Acad. Sci. USA 2026, 123, e2537754123.

Ebrahim, S.A.M.; Talross, G.J.S.; Carlson, J.R. Sight of parasitoid wasps accelerates sexual behavior and upregulates a micropeptide gene in Drosophila. Nat. Commun. 2021, 12, 2453.

Sun, D.; Zhang, Y.; Cao, S.; Wang, X.; Cao, Q.; Zhang, S.; Liu, Y. A compound produced by Helicoverpa armigera male genitalia activates a conserved pheromone receptor. J. Integr. Agric. 2025, 24, 1892–1904.

Caragata, E.P.; Pais, F.S.; Baton, L.A.; Silva, J.B.; Sorgine, M.H.; Moreira, L.A. The transcriptome of the mosquito Aedes fluviatilis (Diptera: Culicidae), and transcriptional changes associated with its native Wolbachia infection. BMC Genomics 2017, 18, 6.

3. For qPCR analysis, it should be clearly specified which sample group served as the calibrator for normalization (for example: The relative expression level (fold) was calculated based on the value of female antennae by setting that expression level to value of 1).

Answer: We apologize for not describing this procedure clearly in the manuscript. We have revised the Materials and Methods section to clearly describe the qPCR normalization strategy. ΔCt was calculated as Ct (MsepORx) − Ct (MsepRPS3), and ΔΔCt was calculated as ΔCt (each sample) − mean ΔCt (minimum). The sample with the minimum ΔCt within each biological replicate was used as the calibrator, and the mean ΔCt of the calibrators from three biological replicates was used as the reference for ΔΔCt calculation. Relative transcript abundance was then calculated using the 2^−ΔΔCt method. These details have been added to the revised Materials and Methods section. The detailed description has been added in line 233-237.

In addition, we apologize for the inaccurate description of the statistical analysis in the manuscript. The statistical analysis was performed using the Mann–Whitney U test, rather than a Student’s t-test, for the comparison between sexes. We have corrected this description in the revised manuscript (Line 239).

4. In Table S1, Benzyl alchol is misspelled. It should be corrected to Benzyl alcohol.

Answer: We have modified. See Table S1.

5. In Table S1, tran-2-Hexen-1-ol is misspelled. It should be corrected to “trans-2-Hexen-1-ol”. In Table S1, the CAS number for ethyl benzoate is incorrectly duplicated from methyl benzoate. The correct CAS number for ethyl benzoate is 93-89-0.

Answer: We have modified. See Table S1.

6. In Section 3.3: The sentence MsepOR29 was contained a 1248 bp CDS encoding 416 amino acids uses an incorrect passive construction. The verb contain should be in active voice here. Revised: MsepOR29 contained a 1248 bp CDS encoding 416 amino acids.

Answer: We have modified. See line 296.

7. Line 305: The term “gonads” is used here, while “genitalia” is used consistently in figure legends and other sections of the manuscript. Please standardize the terminology to “genitalia” throughout for consistency.

Answer: We have modified. See line 313.

8. Line 258: Please clarify the purpose of the word "Subsection" in the heading 3.1. Subsection BA elicits antennal EAG responses. This label appears redundant.

Answer: We have deleted the redundant word “Subsection”. See line 265.

9. Line 185: The quotation mark formatting is improper.

Answer: We have modified. See line 187.

10. The abbreviation "qPCR" appears at line 225 without its full definition on first mention, while the expanded form "quantitative real-time PCR (qRT-PCR)" is used later at line 302. Please define the abbreviation at its first occurrence and standardize the terminology uniformly across the manuscript.

Answer: We have modified. See line 227 and 310.

11. Please carefully re-examine the full text and correct any residual spelling mistakes and other minor errors.

Answer: We have carefully re-examined the entire manuscript and corrected the remaining spelling mistakes and other minor errors.

12. The discussion should be expanded to achieve greater depth and broader scope.

Answer: We have expanded the Discussion to address the potential ecological roles of BA from both plant-associated and conspecific sources, as well as the limitations and future directions of the present study. See line 367-385.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have fully addressed all my comments and concerns. Therefore, I recommend that this manuscript be accepted for publication.