Effects of High Temperature on Development, Survival, and Antioxidant Responses of Immature Monolepta hieroglyphica

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a valuable and timely study that contributes to our understanding of the thermal physiology of Monolepta hieroglyphica. The focus on temperature-dependent enzyme activity is relevant to current questions in insect ecophysiology and has clear implications for pest management. The findings are interesting, and I believe the manuscript has good potential for publication. However, several aspects of framing, interpretation, and statistical analysis would benefit from clarification and revision. Specifically, there is a mismatch between what was actually done and how the results are framed, including the title and abstract: the study does not measure a thermal optimum and does not elucidate physiological mechanisms. These claims must be removed or substantially revised.

1. Study focus. There is at least one directly relevant study on temperature-dependent development in M. hieroglyphica that is not cited: Threshold temperature and effective accumulated temperature of Monolepta hieroglyphica. Li Guang-Wei et al., Chinese Bulletin of Entomology 2008 45(4). This omission is serious, as that study examines development across a biologically meaningful temperature range. In contrast, the present manuscript only considers four temperatures (25–34 °C), which largely represent the upper, potentially stressful range. I would also add that, since larvae of M. hieroglyphica live in the soil, they experience lower temperature than the air above. Because of this, the study should be explicitly framed as investigating high-temperature effects. At minimum, the title should reflect this (e.g., “Effects of High Temperature on…”). Also, the current design does not allow estimation of a thermal optimum. With only four temperature treatments and no lower-temperature data, it is not correct to claim that 25–28 °C represents an optimal temperature range. The authors can only state that performance metrics were highest within the tested range. Any broader claim about an optimum is not supported and should be removed. Finally, the developmental times reported here at 28 and 31 °C are longer than those reported in Li et al. This discrepancy may indicate a stronger stress, but it is not discussed and should be addressed.

2. Overall presentation. The manuscript contains a few inaccuracies in how the study is described. Larval body weight and length are said to be measured, but these measurements were taken from adults. The authors repeatedly refer to a “temperature gradient” and “increasing temperature,” which is incorrect - the experiment used four fixed temperature treatments. This should be described as differences among discrete temperature treatments, not a gradient. The stated aim of the study - “to elucidate the physiological mechanisms underlying the adaptability and heat tolerance” - is not supported by the data. The authors measure standard antioxidant enzyme activities, which are widely used indicators of stress. This does not constitute elucidation of mechanisms. The aim should be rewritten to reflect what was actually done.

3. Statistics. I am seriously concerned about the statistical analysis. The authors report three replicates per treatment and ANOVA degrees of freedom of 3 and 8. At the same time, they report extremely large F-values (in some cases >1000) with p-values <0.001. While not mathematically impossible, such results would require almost no within-group variance, which is highly unlikely for biological data. The authors need to clearly explain what constitutes a replicate, how the data were summarized before analysis, and how variance was calculated. Currently, it appears that 30–60 individuals per treatment were averaged into only three values, which were then used in ANOVA. This approach discards most of the data and can produce misleading results. A more appropriate analysis would use linear mixed-effects models, with replicate as a random effect and individual measurements retained. This would allow the full dataset to be used and would provide a more reliable statistical model. At minimum, I would encourage the authors to provide raw data (at the level of individual measurements) in the supplementary material so that the results can be evaluated and reproduced.

I have also a few minor edits that I added as comments to the pdf file.

Overall, this is a solid and potentially useful piece of research, but the manuscript currently overstates its scope and contains avoidable issues in both framing and statistical analysis. With clearer wording, more conservative interpretation, and improved transparency in the analysis, the study would make a meaningful contribution to Insects.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

The manuscript generally reads well but there are quite a few word choices can be improved and grammatical errors that need to be fixed, including but not limited to: "implementation of global warming," "larvaes," "disaster early warning and green pest control strategies," "adult species," "all instar larvae," "has a significantly affected on the body weight," etc.

Author Response

Response: Thank you very much for your comments. We have revised the manuscript as suggested

Comments and Suggestions for Authors

This is a valuable and timely study that contributes to our understanding of the thermal physiology of Monolepta hieroglyphica. The focus on temperature-dependent enzyme activity is relevant to current questions in insect ecophysiology and has clear implications for pest management. The findings are interesting, and I believe the manuscript has good potential for publication. However, several aspects of framing, interpretation, and statistical analysis would benefit from clarification and revision. Specifically, there is a mismatch between what was actually done and how the results are framed, including the title and abstract: the study does not measure a thermal optimum and does not elucidate physiological mechanisms. These claims must be removed or substantially revised.

Q1: Study focus. There is at least one directly relevant study on temperature-dependent development in M. hieroglyphica that is not cited: Threshold temperature and effective accumulated temperature of Monolepta hieroglyphica. Li Guang-Wei et al., Chinese Bulletin of Entomology 2008 45(4). This omission is serious, as that study examines development across a biologically meaningful temperature range. In contrast, the present manuscript only considers four temperatures (25–34 °C), which largely represent the upper, potentially stressful range. I would also add that, since larvae of M. hieroglyphica live in the soil, they experience lower temperature than the air above. Because of this, the study should be explicitly framed as investigating high-temperature effects. At minimum, the title should reflect this (e.g., “Effects of High Temperature on…”). Also, the current design does not allow estimation of a thermal optimum. With only four temperature treatments and no lower-temperature data, it is not correct to claim that 25–28 °C represents an optimal temperature range. The authors can only state that performance metrics were highest within the tested range. Any broader claim about an optimum is not supported and should be removed. Finally, the developmental times reported here at 28 and 31 °C are longer than those reported in Li et al. This discrepancy may indicate a stronger stress, but it is not discussed and should be addressed..

Response: Thanks. We have revised the manuscript as suggested. (Lines 2-3; Lines 298-301)

Lines 2-3: Effects of High Temperature on Development, Survival, and Antioxidant Responses of Immature Monolepta hieroglyphica.

Lines 298-301: However, the developmental duration of  M. hieroglyphica larval measured in this study was longer than that reported by Li et al. [38]. We speculated that conspecific populations from different geographical regions or different years may vary in their responses to high temperatures.

Q2: Overall presentation. The manuscript contains a few inaccuracies in how the study is described. Larval body weight and length are said to be measured, but these measurements were taken from adults. The authors repeatedly refer to a “temperature gradient” and “increasing temperature,” which is incorrect - the experiment used four fixed temperature treatments. This should be described as differences among discrete temperature treatments, not a gradient. The stated aim of the study - “to elucidate the physiological mechanisms underlying the adaptability and heat tolerance” - is not supported by the data. The authors measure standard antioxidant enzyme activities, which are widely used indicators of stress. This does not constitute elucidation of mechanisms. The aim should be rewritten to reflect what was actually done.

Response: Thank you very much for your comments. We only monitored the feeding capacity of female adults, and more systematic studies will be conducted to increase monitoring of male adults.

 

Q3: tatistics. I am seriously concerned about the statistical analysis. The authors report three replicates per treatment and ANOVA degrees of freedom of 3 and 8. At the same time, they report extremely large F-values (in some cases >1000) with p-values <0.001. While not mathematically impossible, such results would require almost no within-group variance, which is highly unlikely for biological data. The authors need to clearly explain what constitutes a replicate, how the data were summarized before analysis, and how variance was calculated. Currently, it appears that 30–60 individuals per treatment were averaged into only three values, which were then used in ANOVA. This approach discards most of the data and can produce misleading results. A more appropriate analysis would use linear mixed-effects models, with replicate as a random effect and individual measurements retained. This would allow the full dataset to be used and would provide a more reliable statistical model. At minimum, I would encourage the authors to provide raw data (at the level of individual measurements) in the supplementary material so that the results can be evaluated and reproduced.

Response: Thank you very much for your comments. Following the relevant revision suggestions, indices such as body weight and body length of the tested insects were statistically analyzed using a linear mixed model. The covariance parameter test showed P > 0.05, indicating no significant individual differences among the tested insects, which confirmed that the linear mixed model was not appropriate for the data in this study. Differences in the survival curves of the tested insects under different temperature treatments were analyzed using the Kaplan–Meier logrank test. We have revised the manuscript as suggested and added the raw data at the level of individual measurements.

Supplemental Figure S1: Weight and body length of M. hieroglyphica at different temperatures.

Q4: I have also a few minor edits that I added as comments to the pdf file..

Response: Thank you very much for your thorough review and valuable comments on the PDF file. I have carefully considered all your minor edits and revised the manuscript accordingly. Show a portion of it. For more details, please refer to the revised version.

Lines 52-53: Over the past 20 years, the combined effects of global warming and agricultural practices—such as conservation tillage,....

Lines 65-66: The temperature tolerance range of different insect larvae are different.

Lines 349: In response to high-temperature stress, larvae of M. hieroglyphica display distinct...

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript, “Coupled Effects of Temperature on Development, Survival, and Antioxidant Capacity of Immature Monolepta hieroglyphica Motschulsky” by Shi et al. reports on a leaf beetle pest of a variety of crops.

Overall, the paper is generally well-written (English editing needed) and the experiments are designed and replicated in such a way to generate valuable information. Ideally, the authors would have also established a lower temperature to show that 25-27 C is optimal (what if 20 C is optimal and rates are declining above that temperature?).  A closely- related species Serangium japonicum has an ‘optimal’ range of 20–32 C.  All the data in the author’s results support that 28 C is actually optimal. 25 is worse at survival, pupation, eclosion, and similar for adult size.

The authors used a single growth chamber set at each temperature and not to repeat the trials. This leads to potential bias and chamber effects and is generally discouraged. Best practice is to randomize the growth chambers and replicate three times to determine if temperature effects are consistent. Given time and funding limitations, this best practice sometimes cannot be done… but the authors should acknowledge the limitations of their design in the discussion,

For section 2.3, add details about corn roots- were roots imbedded in the agar, were they replaced during feeding?  150 larvae were tested per temp (50 per treatment replicated 3 times). Were these all run at the same time or was this repeated through time? If all were run at the same time, this is another pseudoreplication problem.

For the statistical tests, were data checked for normality prior to running ANOVA? This is important (data may require transformation or tests of median differences may be needed, especially for longevity).

In results, authors need to reword so that the significant results are reported correctly.  For example, developmental duration for first instars was significantly longer at 25 C and similar for other temperatures (Table 1). Based on duration and mortality, 28 C seems to be optimal (20% mortality and 25 days versus 48% mortality and 27 days at 25 C). It would be helpful to construct a life table with these data and show overall % survival and development time versus temperature.

Feeding rate and pupation also supports 28 C as the optimal temp.

In all figures with error rates, specify Mean + 1 S.E. or S.D. (specify what the error lines signify).

Comments on the Quality of English Language

Minor editing needed throughout (missing 'ands' run-on sentences). 

Author Response

Open Review

(x) I would not like to sign my review report

( ) I would like to sign my review report

Quality of English Language

(x) The English could be improved to more clearly express the research.

( ) The English is fine and does not require any improvement..

	Yes	Can be improved	Must be improved	Not applicable
Does the introduction provide sufficient background and include all relevant references?	( )	(x)	( )	( )
Is the research design appropriate?	( )	( )	(x)	( )
Are the methods adequately described?	( )	( )	(x)	( )
Are the results clearly presented?	( )	( )	(x)	( )
Are the conclusions supported by the results?	( )	( )	(x)	( )
Are all figures and tables clear and well-presented?	( )	( )	(x)	( )

Response: Thank you very much for your comments. The manuscript has been edited by native English speakers.

Comments and Suggestions for Authors

The manuscript, “Coupled Effects of Temperature on Development, Survival, and Antioxidant Capacity of Immature Monolepta hieroglyphica Motschulsky” by Shi et al. reports on a leaf beetle pest of a variety of crops.

Q1: Overall, the paper is generally well-written (English editing needed) and the experiments are designed and replicated in such a way to generate valuable information. Ideally, the authors would have also established a lower temperature to show that 25-27 C is optimal (what if 20 C is optimal and rates are declining above that temperature?).  A closely- related species Serangium japonicum has an ‘optimal’ range of 20–32 C.  All the data in the author’s results support that 28 C is actually optimal. 25 is worse at survival, pupation, eclosion, and similar for adult size.

Response: Thank you very much for your comments. The manuscript has been edited by native English speakers, and have revised the manuscript as suggested (Lines 15-16; Lines 31-32; Lines 369-370);

In the preliminary experiments, we set five temperature levels (22, 25, 28, 31, 34). We found that the ecological parameters of the double-spotted leaf beetle were not very satisfactory at 22℃ (low temperature might affect the metabolic activities of the insects). However, our main focus was on the effects of high temperature stress on this insect, so the formal experiment excluded one of the low-temperature treatments (22℃). In the later stage, when we conduct further research on the impact of low temperatures on this insect, we will set up a temperature gradient below 25℃.

Lines 15-16: This study identified 28 ℃ as the optimal temperature for the growth and development of M. hieroglyphica larvae.

Lines 31-32: The highest pupation rate, emergence rate, and biometric indices were observed at 28 ℃, 

Lines 369-370: the highest values for these parameters occurred at 28 ℃,

Q2: The authors used a single growth chamber set at each temperature and not to repeat the trials. This leads to potential bias and chamber effects and is generally discouraged. Best practice is to randomize the growth chambers and replicate three times to determine if temperature effects are consistent. Given time and funding limitations, this best practice sometimes cannot be done… but the authors should acknowledge the limitations of their design in the discussion.

Response: Thank you very much for your comments. All subsequent experiments were conducted in laboratory climate chambers, each monitored using HOBO devices to ensure a constant temperature. (Lines 113-114).

Q3: For section 2.3, add details about corn roots- were roots imbedded in the agar, were they replaced during feeding? 150 larvae were tested per temp (50 per treatment replicated 3 times). Were these all run at the same time or was this repeated through time? If all were run at the same time, this is another pseudoreplication problem.

Response: Thank you very much for your comments. We have revised the manuscript as suggested.

Lines 119-128: Newly hatched (< 12 h) M. hieroglyphica larvae were removed from the breeding population and placed in Petri dishes (5 cm diameter, 1 cm height). The bottom of each dish was covered with 1% hydrogel, followed by a layer of filter paper that completely covered the agar medium. Corn roots were spread flat on the filter paper to allow free feeding by the larvae, and the medium was replaced every 24 h. One larva was placed per dish, and 150 larvae were randomly selected per group. These leaf beetle larvae were placed in climate chambers at different temperatures (25, 28, 31, or 34 ℃, 70 ± 5% RH, and 16:8 h (L/D) photoperiod), respectively. Survival and molting of the larvae were recorded daily, and observations continued until all larvae either reached the pupal stage or died.

Q4: For the statistical tests, were data checked for normality prior to running ANOVA? This is important (data may require transformation or tests of median differences may be needed, especially for longevity).

Response: Thank you very much for your comments. We have revised and added the manuscript as suggested. (Lines185-187)

Lines184-186: All data were first checked for normality and homogeneity of variance and were transformed when they did not fit a normal distribution.

Q5: In results, authors need to reword so that the significant results are reported correctly.  For example, developmental duration for first instars was significantly longer at 25 C and similar for other temperatures (Table 1). Based on duration and mortality, 28 C seems to be optimal (20% mortality and 25 days versus 48% mortality and 27 days at 25 C). It would be helpful to construct a life table with these data and show overall % survival and development time versus temperature

Response: Thank you very much for your comments. We have revised the manuscript as suggested.（Lines 194-203）

Lines 194-203: Different temperatures significantly affected the developmental duration and mortality of M. hieroglyphica larvae (Table 1). As the temperature increased, the developmental duration of larvae at each instar generally decreased. However, the duration of the 3rd-instar larvae was significantly prolonged at 34 ℃ (Tukey’s test: F3,8=10.99, P<0.001). At 28°C, the mortality rate of larvae at all instars reached its minimum values, whereas at 34°C, the mortality rate peaked across all instars, with the highest mortality observed in 3rd-instar larvae (66.36 ± 6.36%) (Tukey’s test: first, F3,8=352.79, P<0.001; second, F3,8=25.92, P<0.001; third, F3,8=92.10, P<0.001). Larvae at each instar exhibited the shorter developmental duration and the lowest mortality at 28 ℃, indicating that 28 ℃ is the optimal temperature for the larval stage of M. hieroglyphica.

Q6: Feeding rate and pupation also supports 28 C as the optimal temp.

Response: Yes, our research shows that 28℃ is the optimal temperature for M. hieroglyphica larvae. (Lines 279-280; Lines 222-223; Lines 235-236; ......)

Lines 279-280: Larvae at each instar exhibited the shorter developmental duration and the lowest mortality at 28 ℃, indicating that 28 ℃ is the optimal temperature for the larval stage of M. hieroglyphica.

Lines 222-223: Specifically, larval food intake increased significantly with rising temperature from 25℃ to 28 ℃, peaking at 28 ℃

Lines 235-236: At 28 ℃, M. hieroglyphica exhibited optimal pupation and emergence rates of 87.04% and 81.25%, respectively.

.....

Q7: In all figures with error rates, specify Mean + 1 S.E. or S.D. (specify what the error lines signify).

Response: Thank you very much for your comments. We have revised the manuscript as suggested. （Lines 206-208；Lines 231-233; Lines 242-244; Lines 265-267; Lines 280-283）

Lines 206-208: Note: The results were represented as mean ± SE. Means followed by the same letter within the same column are not significantly different (ANOVA: Tukey’s post hoc test, P < 0.05) between treatments.

Lines 231-233: Figure 2. Daily food consumption of third instar larvae of M. hieroglyphic at different temperatures. The results were represented as mean ± SE. Different letters above the bars indicate statistically significant differences among temperatures (ANOVA: Tukey’s post hoc test, p < 0.05).

Lines 242-244: Figure 3. Pupation rate (A) and emergence rate (B) of M. hieroglyphica at different temperatures. The results were represented as mean ± SE. Different letters above the bars indicate statistically significant differences among temperatures (ANOVA: Tukey’s post hoc test, P < 0.05).

Lines 265-267: Figure 4. Weight (A) and body length (B) of M. hieroglyphica at different temperatures. The results were represented as mean ± SE. Different letters above the bars indicate statistically significant differences among temperatures (ANOVA: Tukey’s post hoc test, P < 0.05).

Lines 280-283: Figure 5. Effects of temperature stress on the detoxification enzyme activity levels of larvae M. hieroglyphica. (A): CAT activity; (B): GST activity; (C): SOD activity; (D): POD activity. The results were represented as mean ± SE. Different letters above the bars indicate statistically significant differences at P < 0.05 (ANOVA followed by a Tukey’s post hoc test).

Q8: Comments on the Quality of English Language Minor editing needed throughout (missing 'ands' run-on sentences).

Response: Thank you very much for your comments. We have revised the manuscript as suggested.

Reviewer 3 Report

Comments and Suggestions for Authors

The study addresses a relevant and timely topic by evaluating the effects of temperature on key biological and physiological traits of Monolepta hieroglyphica, providing valuable insights into its thermal responses and potential implications under climate change scenarios. The integration of life-history parameters with antioxidant responses represents a meaningful contribution to understanding the species’ adaptability and supports the development of temperature-based pest management strategies. However, despite its strengths, some aspects related to methodological clarity, data interpretation, and scientific writing require further clarification and correction before the manuscript can be considered for publication. Please find my comments below.

Current title

The term “coupled effects” is vague and unclear. The phrase “Immature Monolepta…” is not properly structured.

Suggested improvement: Effects of temperature on development, survival, and antioxidant responses of immature Monolepta hieroglyphica or  Thermal effects on development, survival, and antioxidant enzyme activity in immature Monolepta hieroglyphica

Abstract

Several sentences are not written in standard scientific English.

High-temperatures (31 ℃ and 34 ℃)... Should be: “High temperatures” (no hyphen)

Phrase like: The best pupation rate, emergence rate, biometric indices occurred…(Missing verg agreement, my suggestion is were observed or occurred within).

Introduction

The phrase: explosive polyphagous insect pest. “Explosive” is not appropriate in scientific English. Suggested alternatives: highly destructive  or outbreak-prone.

The phrase: to elucidate the physiological mechanisms…, this is too strong fiven the methods used. The authors should change this statement. A suggestion could be: to evaluate the effects of temperature on…..

Materials and methods

In several sections, it is unclear: what constitutes a replicate, what is the experimental unit. Example:

50 larvae were randomly selected per group… three replicate groups. Does this mean n = 150 per treatment? This is not clearly stated.

Use of root mass as a proxy for consumption. The authors should check this methodology because it could be affected by evaporation/desiccation.

There are lack of detail regarding water loss control and environmental conditions. Gravimetric measurements of feeding may introduce bias unless properly controlled.

About antioxidant assays. There are insufficient methodological details. For example:

Missing information on; enzyme activity units and normalization (e.g., per protein content). There are nsufficient methodological details to ensure reproducibility.

Results

The section contains numerous grammatical errors and non-standard scientific phrasing. Example:

Temperature has a significantly affected on the body weight, Different temperature treatments have a significant impact , M. hieroglyphica species

These should be revised to standard scientific English, e.g.:

Temperature significantly affected body weight, temperature significantly influenced…, M. hieroglyphica.

Overinterpretation in Results

The phrase: We speculate that the extremely high temperature (34 ℃) caused abnormal molting and developmental arrest… This is interpretation, not a result. The authors should move such statements to the Discussion and keep Results strictly descriptive.

Inconsistent formatting:

P<0.001 vs p < 0.001

Extremely high F-values: F = 1927.71,  F = 6020.29. These are unusually high and may raise reviewer concerns. Such large F-values require confirmation of variance structure and data scaling

Discussion

Several mechanistic explanations are speculative and not supported by the data presented. Please, use cautious language: may be associated with…  is consistent with… , has been reported in other species…

The Discussion should explicitly address how the study fills the identified knowledge gaps. Include statements like: This study provides new evidence on…, Our findings extend previous work by…

 

 

 

 

 

 

Author Response

Comments and Suggestions for Authors

The study addresses a relevant and timely topic by evaluating the effects of temperature on key biological and physiological traits of Monolepta hieroglyphica, providing valuable insights into its thermal responses and potential implications under climate change scenarios. The integration of life-history parameters with antioxidant responses represents a meaningful contribution to understanding the species’ adaptability and supports the development of temperature-based pest management strategies. However, despite its strengths, some aspects related to methodological clarity, data interpretation, and scientific writing require further clarification and correction before the manuscript can be considered for publication. Please find my comments below..

Q1: Current title-The term “coupled effects” is vague and unclear. The phrase “Immature Monolepta…” is not properly structured. Suggested improvement: Effects of temperature on development, survival, and antioxidant responses of immature Monolepta hieroglyphica or  Thermal effects on development, survival, and antioxidant enzyme activity in immature Monolepta hieroglyphica.

Response: Thank you very much for your comments. We have revised the title of manuscript as suggested (Lines 2-3)

Lines 2-3: Effects of Temperature on Development, Survival, and Antioxidant Responses of Immature Monolepta hieroglyphica.

Abstract

Q2-Q3: Several sentences are not written in standard scientific English. High-temperatures (31 ℃ and 34 ℃)... Should be: “High temperatures” (no hyphen);

Phrase like: The best pupation rate, emergence rate, biometric indices occurred…(Missing verg agreement, my suggestion is were observed or occurred within).

Response: Thank you very much for your comments. We have revised the abstract of manuscript as suggested (Lines 30-31; Lines 31-33).

Lines 30-31: High temperatures (31 ℃ and 34 ℃) had adverse effects on the developmental duration, survival rates, and feeding efficiency.

Lines 31-33: The best pupation rate, emergence rate, and biometric indices were observed to be within the temperature range of 25-28 ℃, and continued to decline as the temperature increased.

Introduction

Q4-Q5: The phrase: explosive polyphagous insect pest. “Explosive” is not appropriate in scientific English. Suggested alternatives: highly destructive  or outbreak-prone;

The phrase: to elucidate the physiological mechanisms…, this is too strong fiven the methods used. The authors should change this statement. A suggestion could be: to evaluate the effects of temperature on….

Response: Thank you very much for your comments. We have revised the introduction of manuscript as suggested (Lines59-60; Lines 94-96).

Lines 59-60: It exhibits an outbreak-prone pattern featuring "earlier onset, increased population peaks, and expanded distribution scope".

Lines 94-96: The aim was to evaluate the effects of temperature on underlying the adaptability and heat tolerance of its larvae and pupae under varying temperatures. .

Materials and methods

Q6-Q8: In several sections, it is unclear: what constitutes a replicate, what is the experimental unit. Example: 50 larvae were randomly selected per group… three replicate groups. Does this mean n = 150 per treatment? This is not clearly stated;

Use of root mass as a proxy for consumption. The authors should check this methodology because it could be affected by evaporation/desiccation. There are lack of detail regarding water loss control and environmental conditions. Gravimetric measurements of feeding may introduce bias unless properly controlled;

About antioxidant assays. There are insufficient methodological details. For example: Missing information on; enzyme activity units and normalization (e.g., per protein content). There are nsufficient methodological details to ensure reproducibility....

Response: Thank you very much for your comments. We have revised the materials and methods of manuscript as suggested (Lines59-60; Lines 94-96; Lines 99-101).

Lines 99-101: Key characteristics were used to determine the species of M. hieroglyphica, for example, a near-circular pale spot was found on each elytra base, etc [31,32].

Lines 112-116: All subsequent experimental assays were performed in the lab and carried out at climatic chambers with the temperatures of 25 ℃, 28 ℃, 31 ℃, and 34 ℃, which mirror the prevailing temperatures during the corn and other crops growing season under the background of greenhouse effect in north China. Combined with the pre-experimental results, the above four temperatures (25 ℃, 28 ℃, 31 ℃, and 34 ℃) were determined.

Lines 99-101: Key characteristics were used to determine the species of M. hieroglyphica, for example, a near-circular pale spot was found on each elytra base, etc [31,32].

Results

Q9-Q12: The section contains numerous grammatical errors and non-standard scientific phrasing. Example: Temperature has a significantly affected on the body weight, Different temperature treatments have a significant impact , M. hieroglyphica species.

These should be revised to standard scientific English, e.g.: Temperature significantly affected body weight, temperature significantly influenced…, M. hieroglyphica..

Overinterpretation in Results: The phrase: We speculate that the extremely high temperature (34 ℃) caused abnormal molting and developmental arrest… This is interpretation, not a result. The authors should move such statements to the Discussion and keep Results strictly descriptive..

Inconsistent formatting: P<0.001 vs p < 0.001; Extremely high F-values: F = 1927.71,  F = 6020.29. These are unusually high and may raise reviewer concerns. Such large F-values require confirmation of variance structure and data scaling.

Response:  Thank you very much for your comments. We have revised the results of manuscript as suggested (Lines59-60; Lines 94-96; Lines 99-101).

Lines 219-222: The standard curves of antioxidant enzyme activity levels detection were shown in Supplemental Figure S1. The R2 values of all antioxidant enzymes are greater than 0.99, indicating that this experiment is real and stable. And confirm the validity of the enzyme activity experiment results.

Discussion

Q13-Q14: Several mechanistic explanations are speculative and not supported by the data presented. Please, use cautious language: may be associated with…  is consistent with… , has been reported in other species…;

 The Discussion should explicitly address how the study fills the identified knowledge gaps. Include statements like: This study provides new evidence on…, Our findings extend previous work by….

Response: Thank you very much for your comments. We have revised the discussion of manuscript as suggested (Lines59-60; Lines 94-96; Lines 99-101).

Lines59-60: We speculate that the occurrence of developmental delays and increased mortality in the larvae might be related to their prolonged exposure to high temperatures,

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Many thanks to the authors for addressing each comment and concern point by point. The manuscript, in its current form, has the quality required to be published in this journal.