Sublethal Broflanilide Exposure Induces Developmental and Reproductive Costs and Early Detoxification Responses in Tuta absoluta

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Manuscript Title:
“Sublethal Broflanilide Exposure Induces Developmental and Reproductive Costs, and Early Detoxification Responses in Tuta absoluta”

Dear Authors,

I have carefully reviewed the manuscript entitled “Sublethal Broflanilide Exposure Induces Developmental and Reproductive Costs, and Early Detoxification Responses in Tuta absoluta.” The study addresses an important topic; however, I would appreciate clarification on the following points:

1. Why was the chi-square test used to analyse continuous variables such as larval period, pupal weight, fecundity, and longevity?

2. Were normality and homogeneity of variance tested prior to statistical analyses?

3. How many biological replicates were used for RNA-seq per treatment group?

4. Were transcriptomic replicates derived from independent biological cohorts?

5. How many individuals were pooled per RNA extraction sample?

6. Why were detoxification-related genes (e.g., cytochrome P450S) not validated by qPCR despite being central to the conclusions?

7. On what basis do the authors conclude that sublethal exposure may drive resistance evolution without measuring enzyme activity, synergist effects, or resistance selection over generations?

8. How do the authors justify interpreting vertebrate-associated KEGG pathways such as “Bile secretion” in an insect model?

9. Have the raw RNA-seq data been deposited in a public repository, and if so, what is the accession number?

10. Were sequencing depth and mapping rates consistent across all biological replicates?

11. What criteria were used to select Vg, VgR, chs1, and chs2 for validation?

12. Can the authors clarify the apparent discrepancy between the number of replicates used in bioassays and transcriptomic analyses?

13. Why is the scientific name Tuta absoluta not consistently italicised throughout the manuscript?

14. Could the Discussion section be condensed to avoid repetition and overinterpretation?

Author Response

Reply to the reviewer 1

Dear reviewer,

Thanks very much for taking your time to review this manuscript. I really appreciate all your comments and suggestions on the manuscript include some details, etc. They were important for improving the whole quality for this manuscript. Please find my itemized responses in below and my revisions/corrections in the re-submitted files (highlighted in yellow). Thanks again!

Dear Authors,

I have carefully reviewed the manuscript entitled “Sublethal Broflanilide Exposure Induces Developmental and Reproductive Costs, and Early Detoxification Responses in Tuta absoluta.” The study addresses an important topic; however, I would appreciate clarification on the following points:

Question 1. Why was the chi-square test used to analyse continuous variables such as larval period, pupal weight, fecundity, and longevity?

Reply to question 1: Thank you for the suggestions about the data analysis. As suggested, the correct calculation method is critical. I have recalculated the data, and the significant results remain consistent with those obtained previously. Accordingly, the description of the methods in the manuscript has been revised. “This experiment was conducted in an incubator at a constant temperature. Data were analyzed with GraphPad Prism 8.0, and statistical significance was determined using the chi-square test, such as, the emergence rate. And the statistical significance was determined using the t-test (p < 0.05), such as, larval period, pupal weight, pupal duration, fecundity, and adult longevity. Web site: http://www.graphpad.com (Accessed November 2025).” Please refer to the revised content highlighted for more detail, line 183-188.

Question 2. Were normality and homogeneity of variance tested prior to statistical analyses?

Reply to question 2: Thank you for the suggestions about the data analysis. It is crucial for obtaining scientifically accurate results. We conducted a Levene test to check the homogeneity of variance in the data. The results showed that the data met the condition of homogeneity of variance, so we subsequently used the chi-square and t-test to analyze the significance between the data.

Question 3. How many biological replicates were used for RNA-seq per treatment group?

Reply to question 3: Thank you for the suggestions about the sample. For each treatment, four biological replicate samples were prepared and each sample was made by mixing 10 larvae that were treated for 72 hours. I have revised the description of material for RNA-seq. Please refer to the highlighted content for more detail. (Line 192-194)

Question 4. Were transcriptomic replicates derived from independent biological cohorts?

Reply to question 4: Thank you for the suggestions about the design of the material. The sequencing samples for transcriptomics were derived from the biological bioassay. After 72 hours of pesticide treatment, the mortality rate was statistically analyzed. Four biological replicate samples were randomly selected for each treatment, each consisting of 40 surviving larval individuals, with each sample prepared by mixing 10 larvae together.

Question 5. How many individuals were pooled per RNA extraction sample?

Reply to question 5: Thank you for the suggestions about the samples. Four biological replicate samples were randomly selected for each treatment, each consisting of 40 surviving larval individuals, with each sample prepared by mixing 10 larvae together. I have revised the description of materials for the transcriptomic sequencing. Please refer to the highlighted content for more detail. (Line 192-194)

Question 6. Why were detoxification-related genes (e.g., cytochrome P450S) not validated by qPCR despite being central to the conclusions?

Reply to question 6: Thank you for the suggestions about the data analysis. As you noted, the analysis of gene expression related to P450 enzymes is critically important. Accordingly, we performed qPCR analysis of two P450 genes, CYP338A1 and CYP6B5, which strengthens the robustness of our conclusions. Compared with the control group, the expression levels of both genes were significantly increased in the LC₁₀ and LC₃₀ treatment groups (t-test, P < 0.05) (Figure S2).

Question 7. On what basis do the authors conclude that sublethal exposure may drive resistance evolution without measuring enzyme activity, synergist effects, or resistance selection over generations?

Reply to question 7: Thank you for the suggestions about the manuscript. In this study, t. absoluta larvae were exposed to sub-lethal concentrations of broflanilide. The results of transcriptome sequencing revealed that the expression of P450 enzyme-related genes up-regulated significantly compared to the control group. Given that P450 enzyme genes are associated with detoxification metabolism, previous research suggests that alterations in their expression may correlate with pest resistance and play a crucial role in balancing survival and the development of resistance (Yuan et al., 2026). Additionally, the differentially expressed genes were significantly enriched in the pathways of Metabolism of xenobiotics by cytochrome P450 and Drug metabolism - cytochrome P450. This finding indicates that the P450 enzyme metabolic pathways related to detoxification actively respond to pesticide stress. Consequently, based on the transcriptome data obtained, this study posits that sublethal pesticide treatment alters the expression patterns of P450 resistance-related genes. As you rightly pointed out, it is important to draw sound conclusions from the study while avoiding overinterpretation. Accordingly, the descriptions suggesting a role in promoting resistance evolution have been removed.

Question 8. How do the authors justify interpreting vertebrate-associated KEGG pathways such as “Bile secretion” in an insect model?

Reply to question 8: Thank you for the suggestions about the result. Although insects do not have a gallbladder or bile system like mammals, the tissues such as the fat body in insects possess metabolic functions similar to those of the liver, and may be involved in the synthesis and secretion of substances similar to bile functions, such as lipid transport proteins, digestive enzymes, or metabolic regulators. These substances may play a role similar to bile in the lipid metabolism, nutrient absorption, or detoxification processes of insects. Additionally, transcriptome enrichment analysis is usually based on known gene function databases (such as KEGG, GO, etc.), and the pathway information in these databases is cross-species integrated. Some genes involved in bile secretion in mammals may have conserved functions in insects, but they act on different physiological processes. For example, genes involved in lipid metabolism or membrane transport may be involved in intestinal lipid absorption or cell secretion processes in insects, but due to the similarity in gene functions, they are mapped to the "bile secretion" pathway. Other studies' enrichment analyses also have related reports, such as "Multi-omics analyses provide molecular insights into host immune responses and metabolic disruption in Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) parasitized by Pyemotes zhonghuajia. Pest Manag Sci, 81: 6303-6315. https://doi.org/10.1002/ps.8969"

Question 9. Have the raw RNA-seq data been deposited in a public repository, and if so, what is the accession number?

Reply to question 9: Thank you for the suggestions about the data. Our data is fully available and shared. Readers can contact the corresponding author to obtain all the raw sequencing data of the transcriptome. Additionally, we are submitting the transcriptome data to NCBI. As the data is under review, we will provide the accession number for the transcriptome data before the manuscript is accepted.

Question 10. Were sequencing depth and mapping rates consistent across all biological replicates?

Reply to question 10: Thank you for the suggestions about the data. For genome sequencing, we usually determine the overall sequencing quality by the sequencing depth. However, for transcriptome sequencing data, we assess the sequencing quality by analyzing the total sequencing data volume, the number of reads, and the alignment rate. The average amount of data obtained from the transcriptome sequencing in this study was 14G for each sample. Other descriptions related to transcriptome analysis are provided in the methods section: "For the LC₁₀ treatment group, the clean reads generated ranged from 55,909,722 to 74,948,656; for the LC₃₀ treatment group, it was 64,484,120 to 79,274,760; and for the control group, it was 67,652,630 to 75,333,706 (Table S1). The corresponding Q30 values were 97.72%–97.91%, 97.79%–97.95%, and 97.80%–97.84%, respectively." Among the four biological replicate samples of CK, the maximum alignment rate was 88.47%, the minimum was 88.02%, and the average was 88.22%. In the four biological replicate samples of LC10, the maximum, minimum, and average values were 87.77%, 86.56%, and 87.07% respectively. In the four biological replicate samples of LC₁₀, the maximum, minimum, and average values were 88.44%, 86.80%, and 87.54% respectively. From the above sequencing data, it can be seen that the alignment rates between the biological replicate samples are high and have stable repeatability.

Question 11. What criteria were used to select Vg, VgR, chs1, and chs2 for validation?

Reply to question 11: Thank you for the suggestions about the design for method. The study performed a statistical analysis of the growth, development, and reproductive capacity of the t. absoluta following treatment with broflanilide. Results indicated that treatment led to a reduction in the epidermis of the t. absoluta larvae and inhibited their growth. The number of eggs laid by individual females was significantly lower compared to the control group. To elucidate this phenomenon at the molecular level, the study employed qPCR to assess the expression levels of several chitinase genes associated with epidermal development, as well as yolk protein genes related to fecundity. The findings revealed a decrease in the expression of both chitin synthase genes and yolk protein genes, thereby clarifying the observed inhibition of larval growth, epidermal shrinkage, and reduced fecundity following pesticide treatment.

Question 12. Can the authors clarify the apparent discrepancy between the number of replicates used in bioassays and transcriptomic analyses?

Reply to question 12: Thank you for the suggestions about the samples. For sublethal effect assessment, 200 second-instar larvae were exposed to LC₁₀ or LC₃₀ concentrations of broflanilide, with acetone as the control. Since the experiment required continuous monitoring of the survival of the larvae and the statistics of the fecundity, 200 larvae were set for each treatment. Considering the experimental requirements and existing similar studies, this number was sufficient for the biological measurement experiment. For the transcriptomic RNA-seq, the transcriptome sequencing samples were set up with three treatments, and each treatment had four biological replicates. Each biological replicate was prepared by mixing 10 larvae together. The number of samples designated for the transcriptome samples is adequate to fulfill the requirements of transcriptome sequencing and aligns with similar studies. While discrepancies exist in the number of samples between the biological assays and the transcriptome sequencing, both experiments can satisfy their respective experimental needs with the available quantities of samples.

Question 13. Why is the scientific name Tuta absoluta not consistently italicised throughout the manuscript?

Reply to question 13: Thank you for the suggestions about the text format. I have changed all the scientific name "Tuta absoluta " in the entire text to be in italics and highlighted in yellow.

Question 14. Could the Discussion section be condensed to avoid repetition and overinterpretation?

Reply to question 14: Thank you for the suggestions about the discussion. I have revised all the discussion contents, eliminated the repetitive parts and removed the overly interpreted descriptions. For more detailed information, please refer to the discussion section of the manuscript.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, I have reviewed the manuscript entitled "Sublethal Broflanilide Exposure Induces Developmental and Reproductive Costs, and Early Detoxification Responses in Tuta absoluta." The results presented are interesting, but many improvements are needed before proceeding with publication.

Abstract Section.

L9. The first mention of an arthropod species must include the order, family, and taxonomic authority.

Before mentioning the objective of the study, provide two lines about the insecticide broflanilide.

L12. Why do you mention "early second-stage larvae of T. absoluta"? Or did you mean newly emerged?

It is recommended to mention only "second-stage larvae of T. absoluta."

L13. There should be a space between "T. absoluta."

L31-32. But how will your results help IPM?

Words found in the keywords should not be repeated in the title.

Introduction Section.

L44. There should be a space between "T. absoluta."

L52. "Botrytis cinerea" is italicized.

L56. Is T. absoluta's resistance to insecticides also related to its intrinsic growth rate?

L67-69. The sentence is confusing; this should be "several tens". Please clarify the sentence.

Before starting with the background of the insecticide broflanilide, you should include a paragraph on T. absoluta.

L121. What do you mean by "growth"?

L125. There should be a space between "T. absoluta".

L127. Change "," to "." and start with "We identified..."

Materials and Methods Section

Subsections (second level) should have continuous numbering.

For example, 2.1. Tuta absoluta strain and insecticides

L141. Separate the subsection "T. absoluta strain and insecticides".

Suggestion

2.1 Tuta absoluta strain

2.2 Insecticides

Bioassay Subsection

How did you perform the mating procedure between female and male T. obsoluta?

How many males and females did you use to achieve successful mating? What were the environmental conditions during mating?

What was the experimental area for the mating trial?

Please be more specific.

L151. How many eggs were there?

L152. How many second-instar larvae were on each leaf?

L156. The number of replicates per concentration is low. Is there any citation that supports the number of replicates?

L158. Just as you mention the make, model, and manufacturer of the stereomicroscope, please do the same for the equipment (growth chamber) you used in the laboratory and controlled the temperature, relative humidity, and photoperiod.

L161-162. What type of camera did you use to capture the photographs of the larvae?

What software did you use to take the measurements of the larvae?

L163. Please cite the Poloplus software.

L164. How did you obtain 200 second-instar larvae of T. absoluta for each treatment? L165. In the introduction, you mentioned that sublethal concentrations do not cause mortality, and here you mention that you recorded mortality in the second larval stage.

L167. What equipment did you use to weigh the pupae?

L168-174. How did you place the females and males together for mating?

L175. What data were analyzed?

L175. Please cite the GraphPad Prism 8.0 software.

L176. Why did you use the chi-square test?

L178. Of the 20 larvae, how many were used for RNA extraction?

L198-214. Please cite all software such as R Project.

Results Section

L233. Correct the insect's scientific name.

 

In Figure 1, the insect's scientific name must be corrected.

L257 and Table 1. Why wasn't an ANOVA or Tukey's test used to analyze the data? Or the Kruskal-Wallis test?

How is the chi-square test performed to group the means?

The chi-square test is a non-parametric test, and Table 1 shows the means ± the standard error.

L272-295. The multivariate methods mentioned were not described in the Materials and Methods section.

The title or description of Figure 2 is too long.

L368. Figure 3 is of poor quality.

L392. The statistical test was not described in the Materials and Methods section.

L413. Helicoverpa armigera should be in italics. Furthermore, when mentioned for the first time, it must be accompanied by the taxonomic authority and Order:Family of the insect species.

L434. Spodoptera frugiperda should be in italics. Furthermore, when mentioned for the first time, it must be accompanied by the taxonomic authority and Order:Family of the insect species.

L4562. Myzus persicae, similar to the above.

In the conclusion:

A paragraph should explain how the results can be applied in practical settings, such as improving pest management strategies, optimizing the use of chemical insecticides, or supporting decision-making in IPM programs. Another paragraph of the requirement should mention future research and the limitations of the study.

Comments on the Quality of English Language

The English could be improved to more clearly express the research. It is recommended that a native English speaker review the document or that an editing service be hired.

Author Response

Reply to the reviewer 2

Dear reviewer,

Thanks very much for taking your time to review this manuscript. I really appreciate all your comments and suggestions on the manuscript include some details, etc. They were important for improving the whole quality for this manuscript. Please find my itemized responses in below and my revisions/corrections in the re-submitted files (highlighted in green). Thanks again!

Comments and Suggestions for Authors

Dear authors, I have reviewed the manuscript entitled "Sublethal Broflanilide Exposure Induces Developmental and Reproductive Costs, and Early Detoxification Responses in Tuta absoluta." The results presented are interesting, but many improvements are needed before proceeding with publication.

Abstract Section.

Question 1. L9. The first mention of an arthropod species must include the order, family, and taxonomic authority.

Reply to question 1: Thank you very much for your suggestion regarding the format of species names. I added the right description content in the abstract and highlighted in green. “The tomato leaf miner, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) poses a significant threat to global tomato production.” Please refer to the highlighted content for more detail, page 1, line 9.

Question 2. Before mentioning the objective of the study, provide two lines about the insecticide broflanilide.

Reply to question 2: Thank you for the suggestions about the abstract. I have added the description of the pesticide and highlighted in green. “Broflanilide, a novel meta‑diamide compound, which can bind specifically to the transmembrane domain of the RDL subunit, causing prolonged opening of the chloride channel, disruption of neurotransmission, and ultimately insect paralysis and death.” Please refer to the revised content for more detail, page 1, line 12-15.

Question 3. L12. Why do you mention "early second-stage larvae of T. absoluta"? Or did you mean newly emerged? It is recommended to mention only "second-stage larvae of T. absoluta."

Reply to question 3: Thank you for the suggestions about the material. Considering the end stage of each larval stage, the pests will stop feeding to prepare for molting and entering the next stage. To prevent the adverse effects of the phenomenon, this study used the larvae that had just reached the second larval stage. But as you mentioned, perhaps there is no need to emphasize the early stage. I have already made the necessary revisions to the content based on your suggestion, changing it to "second-stage larvae of T. absoluta." Please refer to the revised content for more detail, line 16 and line 154-155.

Question 4. L13. There should be a space between "T. absoluta."

Reply to question 4: Thank you for the suggestions about the format. I added the space between "T. absoluta" in the entire manscript and highlighted in green.

Question 5. L31-32. But how will your results help IPM?

Reply to question 5: Thank you for the suggestions. By investigating the physiological, reproductive, and behavioral effects of sublethal pesticide concentrations on pest species, researchers can establish more precise application timing and dosage regimens. For instance, certain pesticides at sublethal levels may not cause immediate mortality but can markedly compromise reproductive output, developmental rate, or mating behavior, thereby suppressing pest populations without necessitating increased chemical inputs. This strategy not only reduces overall pesticide usage but also mitigates environmental contamination and delays the onset of resistance. Moreover, insights into sublethal effects can inform the design of novel pest management tools. As an example, targeting and disrupting the expression of key genes involved in pest reproduction or development offers a pathway to develop environmentally benign control strategies that lessen reliance on conventional pesticides, such as, RNAi.

Question 6. Words found in the keywords should not be repeated in the title.

Reply to question 6: Thank you for the suggestions about the keywords. I have changed the “T. absoluta” to “Tomato leaf miner” and highlighted in green, line 37. I couldn't find an appropriate keyword to replace the name of the “Broflanilide”.

Introduction Section.

Question 7. L44. There should be a space between "T. absoluta."

Reply to question 7: Thank you for the suggestions about the format. I added the space between "T. absoluta" in the entire manscript and highlighted in green.

Question 8. L52. "Botrytis cinerea" is italicized.

Reply to question 8: Thank you for the suggestions about the format. I have changed "Botrytis cinerea" to be in italics and highlighted it in green, line 56.

Question 9. L56. Is T. absoluta's resistance to insecticides also related to its intrinsic growth rate?

Reply to question 9: Thank you for the suggestions about the description. The original sentence was intended to highlight the potential issues associated with chemical pesticides used in pest control, including environmental safety concerns, pesticide residues, and the development of resistance in target pests. It did not address the relationship between growth inhibition in the T. absoluta and pesticide resistance. In our study, we observed significant enrichment of physiological pathways related to detoxification metabolism involving P450 enzymes. Although altered expression of P450 enzyme-related genes has been linked to resistance development in previous studies—for example, "cytochrome P450 genes CYP6CY3 and CYP6CY4 have been shown to confer resistance to flupyradifurone in the green peach aphid, Myzus persicae (Yuan et al., 2026)"—our findings indicate changes in P450 gene expression and enrichment in detoxification pathways. However, these results do not necessarily imply increased resistance in the T. absoluta, as such a conclusion would require multi-generational resistance monitoring data. Accordingly, we have revised the statements concerning resistance development to avoid over-interpretation.

Question 10. L67-69. The sentence is confusing; this should be "several tens". Please clarify the sentence.

Reply to question 10: Thank you for the suggestions about the description.

I have changed the content into “Resistance to the diamide insecticides chlorantraniliprole and flubendiamide was detected in populations from northern China, with resistance ratios reaching up to tens of fold in certain regions.” Please refer to the revised content for detail, line 71-73.

Question 11. Before starting with the background of the insecticide broflanilide, you should include a paragraph on T. absoluta.

Reply to question 11: Thank you for the suggestions about the introduction. Before introducing the pesticide broflanilide used in this study, the background section of this research first provided detailed information about the T. absoluta, including its name, origin, and damage characteristics and so on. Please refer to the revised content for more detail, line 41-60.

Question 12. L121. What do you mean by "growth"?

Reply to question 12: Thank you for the suggestions about the content. The statistics on the life parameters of the T. absoluta, including larval period, pupal weight, pupal duration, emergence rate, fecundity and adult longevity was necessary. The word "growth" is not specific enough, so I have removed this word and replaced it with "life parameters". “A systematic evaluation was conducted to determine the physiological effects, particularly life parameters, of sublethal concentrations (LC₁₀ and LC₃₀) of broflanilide on a susceptible strain of second-instar T. absoluta larvae.” Please refer to the revised content for more detail, line 124-126.

Question 13. L125. There should be a space between "T. absoluta".

Reply to question 13: Thank you for the suggestions about the format. I added the space between "T. absoluta" in the entire manscript and highlighted in green.

Question 14. L127. Change "," to "." and start with "We identified..."

Reply to question 14: Thank you for the suggestions about the manuscript. I have changed the content into “We identified key gene clusters and biological pathways significantly altered by sublethal stress through analysis of differentially expressed genes (DEGs), weighted gene co expression network analysis (WGCNA) and multi-level functional enrichment.” Please refer to the revised content for more detail, line 128-131.

Materials and Methods Section

Question 15. Subsections (second level) should have continuous numbering.

For example, 2.1. Tuta absoluta strain and insecticides

Reply to question 15: Thank you for the suggestions about the format. I have consecutively numbered the subsections and highlighted them in green according to your suggestion. (Line 143-152)

Question 16. L141. Separate the subsection "T. absoluta strain and insecticides". Suggestion 2.1 Tuta absoluta strain 2.2 Insecticides

Reply to question 16: Thank you for the suggestions about the method. I have changed the subsection “T. absoluta strain and insecticides” into “2.1. Tuta absoluta strain  2.2. Insecticides”. (Line 143-152)

Bioassay Subsection

Question 17. How did you perform the mating procedure between female and male T. obsoluta?

Reply to question 17: Thank you for the suggestions about the method.

In the methods section of the biological measurement experiment, I outlined the procedure for conducting the statistical analysis of fecundity. “To evaluate fecundity, male and female adults that emerged within two days were paired and placed in an oviposition setup. A cylindrical floral water tube (1 cm × 4 cm) was fixed to the bottom of a disposable round transparent plastic container (17 cm × 11.5 cm × 9.5 cm). The tube was filled with fresh tap water, and a 7 cm tomato branch with leaves was inserted. A 6 cm × 6 cm piece of tissue paper was placed inside the container; half of the paper was sprayed with 10% sucrose solution and the other half with deionized water to provide nutrition and moisture for the adults (Figure S1B). Data were analyzed with GraphPad Prism 8.0, and statistical significance was determined using the chi-square test and t-test (p < 0.05).”

For the adult mating experiment, the initial step involved distinguishing between female and male pupae. Once the pupae matured into adults, one female and one male adult were placed in a transparent plastic container to facilitate mating. The presence of eggs at a later stage indicated successful mating between the paired adults; conversely, the absence of eggs signified that the paired adults had not mated, and those non-mated pairs were excluded from the egg production count.

Question 18. How many males and females did you use to achieve successful mating? What were the environmental conditions during mating?

Reply to question 18: Thank you for the suggestions about the method. During the experiment, one adult male and one adult female insect were placed together in a container. The likelihood of successful mating was high, provided that the pupae had been accurately sexed and that appropriate oviposition conditions were maintained. Owing to variation in pupal emergence times, the procedure was repeated daily until sufficient data had been collected. For each treatment, a total of 80 male and 80 female adults were used to obtain complete oviposition records. The environmental conditions for the fecundity were the same as those for the biological assay experiment, as described in the biological assay experiment: "All bioassays were conducted under controlled laboratory conditions (Temperature 27 ± 1 °C, 65 ± 5% RH, 16L:8D photoperiod)."

Question 19. What was the experimental area for the mating trial?

Please be more specific.

Reply to question 19: Thank you for the suggestions about the method. The mating experiments were conducted in multiple disposable plastic lunch boxes, which were placed in the incubator. The specific description is as follows: "To assess fecundity, male and female adults that emerged within two days were paired and placed in an oviposition setup. A cylindrical floral water tube (1 cm × 4 cm) was fixed to the bottom of a disposable round transparent plastic container (17 cm × 11.5 cm × 9.5 cm). The tube was filled with fresh tap water, " and a 7 cm tomato branch with leaves was inserted. A 6 cm × 6 cm piece of tissue paper was placed inside the container; half of the paper was sprayed with 10% sucrose solution and the other half with deionized water to provide nutrition and moisture for the adults (Figure S1B).” I have revised the description in the manuscript and highlighted in green. Please refer to the revised content for more detail, line 179-180.

Question 20. L151. How many eggs were there?

Reply to question 20: Thank you for the suggestions about the method. On the leaves of plants that had completed oviposition 12 hours earlier, up to several hundred eggs were observed. The number of eggs far exceeded the quantity of larvae required for the experiment. These eggs were subsequently hatched and used in the biological measurement assays.

Question 21. L152. How many second-instar larvae were on each leaf?

Reply to question 21: Thank you for the suggestions about the method. I have revised the description of the bioassay. In each petri dish, five second-instar larvae and three fresh leaves were placed. Three replicate experiments were conducted for each concentration, and 20 larvae were used in each replicate experiment. An acetone solution served as the control. Please refer to the revised content for more detail, line 160-164.

Question 22. L156. The number of replicates per concentration is low. Is there any citation that supports the number of replicates?

Reply to question 22: Thank you for the suggestions about the method.

We conducted across different concentration gradients in the biological assays, three replicates were performed for each concentration, in accordance with established protocols (Lautaro et al., 2024) (“Pervasive sublethal effects of agrochemicals on in-sects at environmentally relevant concentrations”)

Question 23. L158. Just as you mention the make, model, and manufacturer of the stereomicroscope, please do the same for the equipment (growth chamber) you used in the laboratory and controlled the temperature, relative humidity, and photoperiod.

Reply to question 23: Thank you for the suggestions about the equipment. I have revised the description in the manuscript, “All bioassays were conducted under thermostatic incubator (Temperature, 27 ± 1 °C; 65 ± 5% RH; 16L:8D photoperiod). Please refer to the revised content for more detail, line 163-164.”

Question 24. L161-162. What type of camera did you use to capture the photographs of the larvae?

Reply to question 24: Thank you for the suggestions about the method. I have revised the description in the manuscript, “The Olympus SZX7 stereomicroscope, equipped with binocular eyepieces and a dedicated camera adapter, is compatible with Olympus DP series digital cameras and other microscope cameras, thereby facilitating high-resolution image acquisition and research documentation.”

Question 25. What software did you use to take the measurements of the larvae?

Reply to question 25: Thank you for the suggestions about the method. Digital images of the larvae were acquired using Olympus cellSens software integrated with a fluorescence stereomicroscope (Olympus SZX7, Japan). This software functions as the primary platform for image acquisition and control, enabling real-time preview, image capture, multidimensional experiments, and basic measurement and analysis. Please refer to the revised content for more detail, line 167-168.”

Question 26. L163. Please cite the Poloplus software.

Reply to question 26: Thank you for the suggestions about the method. I have added the description in the manuscript, “Poloplus software (v2.0, LeOra, USA).” Please refer to the revised content for more detail, line 170.”

Question 27. L164. How did you obtain 200 second-instar larvae of T. absoluta for each treatment? L165. In the introduction, you mentioned that sublethal concentrations do not cause mortality, and here you mention that you recorded mortality in the second larval stage.

Reply to question 27: Thank you for the suggestions about the content. Following oviposition of the eggs, the hatched larvae burrowed into the leaf tissue. To extract them, the surrounding leaf area was first gently pricked with a dissecting needle, followed by lightly probing near the larval body. This stimulation typically induced the larva to emerge from the leaf, after which it could be collected using a soft brush.

In the introduction, sublethal concentrations were initially defined as “doses that do not cause direct mortality but are sufficient to impair normal physiological, behavioral, developmental, or reproductive functions (Shi et al., 2023).” However, it is important to clarify that LC₃₀ specifically refers to the concentration estimated to cause 30% mortality in the test population. As such, the original phrasing may conflate two distinct toxicological endpoints. To enhance precision, the description in the introduction has been revised as follows: “Sublethal concentrations are sufficient to impair normal physiological, behavioral, developmental, or reproductive functions (Shi et al., 2023).” Please refer to the revised content for more detail, line 94-95.” In the bioassay experiments, larval mortality was recorded at 72h post-treatment to assess survival and developmental progression. These data were subsequently used to calculate larval period, providing a measure of sublethal effects on development.

Question 28. L167. What equipment did you use to weigh the pupae?

Reply to question 28: Thank you for the suggestions about the method. The pupal weight was measured using a balance (Mettler Toledo, Switzerland) with a precision of 0.0001 g. I have revised the description in the manuscript, please refer to the revised content for more detail, line 175-176.

Question 29. L168-174. How did you place the females and males together for mating?

Reply to question 29: Thank you for the suggestions about the method. To assess the reproductive capacity of individual females, adult mating experiments were conducted. For this purpose, pupae were first sexed accurately. Upon emergence, one two-day-old female and one two-day-old male were placed together in a disposable transparent plastic container lined with tomato leaves, allowing them to mate and oviposit freely. The presence of eggs on the tomato leaves after 2–3 days was considered indicative of successful mating; pairs that failed to produce eggs within this period were excluded from subsequent fecundity analyses. The experimental setup was illustrated in Figure S1B.

Question 30. L175. What data were analyzed?

Reply to question 30: Thank you for the suggestions about the method. Data were analyzed with GraphPad Prism 8.0, and statistical significance was determined using the chi-square test, such as, the emergence rate. And the statistical significance was determined using the t-test (p < 0.05), such as, larval period, pupal weight, pupal duration, fecundity, and adult longevity. Web site: http://www.graphpad.com (Accessed November 2025). Please refer to the revised content for more detail, line 183-188.

Question 31. L175. Please cite the GraphPad Prism 8.0 software.

Reply to question 31: Thank you for the suggestions about the method. Following the reference formatting guidelines provided on the official website, I have included the URL and accessed time in the manuscript. “Web site: http://www.graphpad.com (Accessed November 2025).” Please refer to the revised content for more detail, line 187.”

Question 32. L176. Why did you use the chi-square test?

Reply to question 32: Thank you for the suggestions about the method. As suggested, the correct calculation method is critical. I have recalculated the data, and the significant results remain consistent with those obtained previously. Accordingly, the description of the methods in the manuscript has been revised. “This experiment was conducted in an incubator at a constant temperature. Data were analyzed with GraphPad Prism 8.0, and statistical significance was determined using the chi-square test, such as, the emergence rate. And the statistical significance was determined using the t-test (p < 0.05), such as, larval period, pupal weight, pupal duration, fecundity, and adult longevity. Web site: http://www.graphpad.com (Accessed November 2025).” Please refer to the revised content for more detail, line 183-188.

Question 33. L178. Of the 20 larvae, how many were used for RNA extraction?

Reply to question 33: Thank you for the suggestions about the method. I have revised the description. “For each treatment, four biological replicate samples were prepared and each sample was made by mixing 10 larvae that were treated for 72 hours.” Please refer to the revised content for more detail, line 192-194.

Question 34. L198-214. Please cite all software such as R Project.

Reply to question 34: Thank you for the suggestions about the references. I have added the citation for all software in the method and references, line 213-229.

Results Section

Question 35. L233. Correct the insect's scientific name.

 Reply to question 35: Thank you for the suggestions about the format. I have changed the t.absoluta into “T.absoluta”, line 250.

Question 36. In Figure 1, the insect's scientific name must be corrected.

Reply to question 36: Thank you for the suggestions about the insect's scientific name. For each individual section, the full name should be written for the first mention, and the abbreviation should be used thereafter. I have revised the scientific name in Figure 1, line 261-264.

Question 37. L257 and Table 1. Why wasn't an ANOVA or Tukey's test used to analyze the data? Or the Kruskal-Wallis test?

Reply to question 37: Thank you for the suggestions about the method.

New analytical methods were incorporated, and the data were re-analyzed using one-way ANOVA followed by t-tests. The Methods section has been revised accordingly to reflect these updates. “Data were analyzed with GraphPad Prism 8.0, and statistical significance was determined using the chi-square test, such as, the emergence rate. And the statistical significance was determined using the t-test (p < 0.05), such as, larval period, pupal weight, pupal duration, fecundity, and adult longevity. Web site: http://www.graphpad.com (Accessed November 2025).”

Question 38. How is the chi-square test performed to group the means?

The chi-square test is a non-parametric test, and Table 1 shows the means ± the standard error.

Reply to question 38: Thank you for the suggestions about the method. Just as mentioned in Question 32, the correct calculation method is critical. I have recalculated the data, and the significant results remain consistent with those obtained previously. Accordingly, the description of the methods in the manuscript has been revised. “This experiment was conducted in an incubator at a constant temperature. Data were analyzed with GraphPad Prism 8.0, and statistical significance was determined using the chi-square test, such as, the emergence rate. And the statistical significance was determined using the t-test (p < 0.05), such as, larval period, pupal weight, pupal duration, fecundity, and adult longevity. Web site: http://www.graphpad.com (Accessed November 2025).” Please refer to the revised content for more detail, line 183-188.

Question 39. L272-295. The multivariate methods mentioned were not described in the Materials and Methods section.

Reply to question 39: Thank you for the suggestions about the method. Hierarchical clustering analysis was incorporated into the transcriptome data analysis workflow. As described in the Methods section, such as, “To examine sample relationships, distances between samples were calculated with the “vegan” R package, followed by hierarchical clustering using “hclust” to visualize overall expression profile similarities.” (lines 216–218, highlighted in green).

Question 40. The title or description of Figure 2 is too long.

Reply to question 40: Thank you for the suggestions about the figure.

The figure caption has been condensed to include only the essential information. The revised version is as follows: “Figure 2. Overview of transcriptome analyses. (A) Hierarchical clustering dendrogram: branch lengths represent sample distances; colors indicate experimental groups. (B) PCA plot: points are colored/shaped by group; axes scales are relative. (C) Co-expression Venn diagram: colors denote samples; numbers show counts of uniquely or commonly expressed genes; overlaps represent genes expressed in multiple samples. (D) Venn diagram of differentially expressed genes (DEGs) across comparisons: colors denote comparison groups; numbers indicate shared or unique DEGs; overlaps represent DEGs common to multiple comparisons. (E) Bar chart of DEG counts per comparison: up-regulated (red) and down-regulated (blue). (F–H) Line graphs depicting expression trends of sub-modules across groups (standardized expression levels). (I) WGCNA module clustering tree: colors represent co-expression modules; vertical distances reflect gene expression correlations. (J) Heatmap of module–group correlations: rows: modules; columns: groups; color intensity indicates correlation strength; numbers show correlation coefficients and P‑values.” (Line 315-326 highlighted in green).

Question 41. L368. Figure 3 is of poor quality.

Reply to question 41: Thank you for the suggestions about the figure. The image quality has been enhanced, and the revised version has been inserted above Figure 3 in the main text of the manuscript.

Question 42. L392. The statistical test was not described in the Materials and Methods section.

Reply to question 42: Thank you for the suggestions about the method. The revised version is as follows: “Statistical significance was determined and figures were generated using GraphPad Prism 8.0 and the significance was determined using the t-test (p < 0.05).” (Line 247-248，highlighted in green)

Question 43. L413. Helicoverpa armigera should be in italics. Furthermore, when mentioned for the first time, it must be accompanied by the taxonomic authority and Order: Family of the insect species.

Reply to question 43: Thank you for the suggestions about the insect's scientific name. The revised scientific name is as follows: “Helicoverpa armigera (Hübner, 1808) (Lepidoptera: Noctuidae)” (Line 411, highlighted in green).

Question 44. L434. Spodoptera frugiperda should be in italics. Furthermore, when mentioned for the first time, it must be accompanied by the taxonomic authority and Order: Family of the insect species.

Reply to question 44: Thank you for the suggestions about the insect's scientific name. The revised scientific name is as follows: “Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae)” (Line 432, highlighted in green).

Question 45. L4562. Myzus persicae, similar to the above.

Reply to question 45: Thank you for the suggestions about the scientific name. The revised scientific name is as follows:“Myzus persicae (Sulzer， 1776) (Hemiptera: Aphididae)” (Line 450, highlighted in green).

In the conclusion:

Question 46. A paragraph should explain how the results can be applied in practical settings, such as improving pest management strategies, optimizing the use of chemical insecticides, or supporting decision-making in IPM programs. Another paragraph of the requirement should mention future research and the limitations of the study.

Reply to question 46: Thank you for the suggestions about the discussion. I have reorganized and revised the discussion section in accordance with your suggestions. Specifically, I have addressed the implications of our findings for field application as follows: "Integrated pest management strategies should seek to minimize sublethal exposure. Sublethal concentrations not only cause physiological suppression but also act as key selection pressures that prime adaptive defense mechanisms, particularly detoxification metabolism. Therefore, in field applications, it is essential to ensure precise and effective dosing, aiming for complete mortality to reduce the risk of resistance driven by sublethal residues. This requires establishing locally calibrated recommended doses based on sensitivity monitoring and optimizing application techniques to ensure thorough coverage of pest habitats." (Line 461-468, highlighted in green).

Furthermore, I have expanded the discussion to highlight future research directions, incorporating suggestions for integrating chemical and biological control methods: "In addition, the chitin synthesis (CHS) and vitellogenin-related (Vg) pathways—which are suppressed under sublethal stress—represent promising targets for novel interventions. Developing RNA interference (RNAi)-based biopesticides that target these pathways could complement chemical modes of action. Emerging dsRNA delivery platforms using engineered microorganisms show promise for field implementation. Such a 'chemical-biological' integrated approach would not only enhance current control efficacy but also delay resistance evolution by attenuating population resilience." (Line 474-478, highlighted in green).

Comments on the Quality of English Language

Question 47. The English could be improved to more clearly express the research. It is recommended that a native English speaker review the document or that an editing service be hired.

Reply to question 47: I sincerely appreciate your valuable suggestions regarding my English expressions. In accordance with your advice, I have carefully revised and improved the manuscript to ensure accurate presentation of the research content while enhancing its readability.

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript analyzes the effects of sublethal concentrations of broflanilide on the development, reproduction, and transcriptomic response of Tuta absoluta. The authors used an approach combining toxicological bioassay with transcriptome analysis and validation of selected gene expression using qPCR. The topic of this work is timely and important from a plant protection perspective, as Tuta absoluta is one of the most important tomato pests worldwide, and understanding the molecular mechanisms of its response to insecticides can contribute to better population resistance management for this species.

 

The insect husbandry methodology and experimental conditions are generally described correctly, but require clarification in several areas. The authors used a susceptible strain of T. absoluta maintained under laboratory conditions for over 30 generations without exposure to insecticides. This research material allows for the assessment of the organism's baseline sensitivity to the test compound. However, the selection of only one laboratory population should be more clearly justified, as field populations may exhibit significantly greater variability in their response to broflanilide. The bioassay description uses the leaf-dip bioassay method, which is standardly used in studies on insecticide toxicity to fluke larvae. The range of tested concentrations and the number of replicates are provided, but the description of the statistical analysis is insufficient. In particular, it is unclear how the LC₁₀ and LC₃₀ values ​​were determined, and whether probit analysis, which is standard in this type of research, was used. Furthermore, using the chi-square test to analyze most biological parameters (e.g., pupal weight or developmental length) seems methodologically inadequate, as these data are continuous and are typically analyzed using parametric tests or linear models.

 

Bioassay results indicate that sublethal concentrations of broflanilide significantly affect larval development and reproductive parameters of T. absoluta. The authors demonstrated an extension of the larval and pupal periods, a reduction in pupal mass, a decrease in survival, and a significant reduction in female fecundity. These results are consistent with the observed inhibition of larval growth and morphological changes, such as reduced body length and darkened cuticle. However, the interpretation of these observations is rather descriptive and should be supplemented with broader reference to previous studies on the sublethal effects of broflanilide or other insecticides acting on the GABAergic system in insects.

 

The transcriptomic analysis is one of the strongest parts of the study. The authors obtained high-quality sequence data, as evidenced by high Q30 values ​​and adequate sequencing depth. Data quality analyses, including PCA and hierarchical clustering, indicate good biological reproducibility of the samples. A significant number of differentially expressed genes were identified between the control and broflanilide-treated groups. Gene co-expression network analysis (WGCNA) additionally allowed for the identification of gene modules associated with the response to chemical stress. However, the description of the bioinformatics methods is in places too brief and does not include all the parameters used in the analysis, which hinders the full reproducibility of the results.

 

The results of functional analyses (GO, KEGG, and GSEA) indicate that sublethal exposure to broflanilide primarily affects metabolic processes and xenobiotic detoxification mechanisms. The enrichment of pathways related to cytochrome P450 metabolism appears particularly significant, consistent with commonly observed mechanisms of insect response to insecticide stress. The authors also indicate changes in metabolic pathways related to fatty acid and amino acid metabolism, as well as immunological processes (Toll and Imd pathways). However, the interpretation of these results requires further investigation, particularly in the context of the potential significance of these changes for the development of insecticide resistance.

 

QPCR validation of the transcriptomic results was performed for genes related to cuticle development (chs1 and chs2) and reproduction (Vg and VgR). The obtained results indicate a significant reduction in the expression levels of these genes in the broflanilide-treated groups, which provides a potential molecular explanation for the observed developmental inhibition and decreased fecundity of the insects. This approach is methodologically sound, but the number of genes analyzed is relatively small. Expanding the validation to include a larger number of genes related to detoxification (e.g., P450s, GSTs, esterases) could strengthen the conclusions drawn from this study.

 

Figures and Tables are clear. References include 26 references.

Comments for author File: Comments.pdf

Author Response

Reply to the reviewer 3

Dear reviewer,

Thanks very much for taking your time to review this manuscript. I really appreciate all your comments and suggestions on the manuscript include some details, etc. They were important for improving the whole quality for this manuscript. Please find my itemized responses in below and my revisions/corrections in the re-submitted files (highlighted in blue). Thanks again!

Comments and Suggestions for Authors

Question 1. This manuscript analyzes the effects of sublethal concentrations of broflanilide on the development, reproduction, and transcriptomic response of Tuta absoluta. The authors used an approach combining toxicological bioassay with transcriptome analysis and validation of selected gene expression using qPCR. The topic of this work is timely and important from a plant protection perspective, as Tuta absoluta is one of the most important tomato pests worldwide, and understanding the molecular mechanisms of its response to insecticides can contribute to better population resistance management for this species.

 Reply to question 1: We would like to express our sincere gratitude to the reviewer for taking the time to evaluate our manuscript and for providing such insightful and encouraging comments. We are pleased that the reviewer recognizes the timeliness and importance of our work on Tuta absoluta, a globally significant pest of tomato crops. The reviewer's positive assessment of our integrated approach—combining toxicological bioassays with transcriptomic analysis and qPCR validation—is particularly gratifying, as our goal was to provide a comprehensive understanding of the sublethal effects of broflanilide at both the organismal and molecular levels.

We agree wholeheartedly with the reviewer's comment that elucidating the molecular mechanisms of insecticide response is crucial for improving resistance management strategies. Your affirmation reinforces the potential impact of our findings. We have carefully considered all your suggestions and believe that incorporating them will substantially strengthen the manuscript. The point-by-point responses to your specific comments are provided below.

Question 2. The insect husbandry methodology and experimental conditions are generally described correctly, but require clarification in several areas. The authors used a susceptible strain of T. absoluta maintained under laboratory conditions for over 30 generations without exposure to insecticides. This research material allows for the assessment of the organism's baseline sensitivity to the test compound. However, the selection of only one laboratory population should be more clearly justified, as field populations may exhibit significantly greater variability in their response to broflanilide. The bioassay description uses the leaf-dip bioassay method, which is standardly used in studies on insecticide toxicity to fluke larvae. The range of tested concentrations and the number of replicates are provided, but the description of the statistical analysis is insufficient. In particular, it is unclear how the LC₁₀ and LC₃₀ values ​​were determined, and whether probit analysis, which is standard in this type of research, was used. Furthermore, using the chi-square test to analyze most biological parameters (e.g., pupal weight or developmental length) seems methodologically inadequate, as these data are continuous and are typically analyzed using parametric tests or linear models.

 Reply to question 2: Thank you for the suggestions about the manuscript. We sincerely appreciate your valuable suggestions, which are instrumental in enhancing the overall rigor and organization of our study. In the present study, the sublethal effects were assessed using a susceptible strain that had been established through multiple generations of laboratory selection. Due to practical constraints, we were unable to include additional field populations for broader biological assays at this stage. Future work will therefore aim to incorporate multiple field populations to enable more comprehensive and in-depth analyses.

In the bioassay experiments, broflanilide was diluted into five concentration gradients, with 20 larvae exposed to each concentration and three biological replicates per treatment. Mortality was recorded after 72 hours. Bioassay data were statistically analyzed using the poloplus software, which automatically calculated and generated the sublethal concentrations corresponding to LC₁₀ and LC₃₀.

As suggested, the correct calculation method is critical. I have recalculated the data, and the significant results remain consistent with those obtained previously. Accordingly, the description of the methods in the manuscript has been revised. “This experiment was conducted in an incubator at a constant temperature. Data were analyzed with GraphPad Prism 8.0, and statistical significance was determined using the chi-square test, such as, the emergence rate. And the statistical significance was determined using the t-test (p < 0.05), such as, larval period, pupal weight, pupal duration, fecundity, and adult longevity. Web site: http://www.graphpad.com (Accessed November 2025).” Please refer to the revised content for more detail. (Line 183-188).

Question 3. Bioassay results indicate that sublethal concentrations of broflanilide significantly affect larval development and reproductive parameters of T. absoluta. The authors demonstrated an extension of the larval and pupal periods, a reduction in pupal mass, a decrease in survival, and a significant reduction in female fecundity. These results are consistent with the observed inhibition of larval growth and morphological changes, such as reduced body length and darkened cuticle. However, the interpretation of these observations is rather descriptive and should be supplemented with broader reference to previous studies on the sublethal effects of broflanilide or other insecticides acting on the GABAergic system in insects.

We thank the reviewer for this insightful comment. We agree that a broader comparison with previous studies on GABAergic insecticides will strengthen our interpretation, and we have revised the Discussion accordingly.

First, we have established a clearer mechanistic link between phenotype and molecular data. The prolonged larval duration and cuticular abnormalities are now explicitly connected to downregulation of chitin synthase genes (chs1/chs2), confirmed by qPCR. As chitin is essential for cuticle integrity, its impaired synthesis explains the observed developmental delays (Zhou et al., 2024). Additionally, KEGG analysis revealed disruption of upstream endocrine pathways (steroid hormone biosynthesis, insulin signaling), suggesting that sublethal broflanilide exposure perturbs hormonal regulation of molting, thereby affecting development (Zhang et al., 2021). Second, we have contextualized our findings within the broader literature on GABAergic insecticides. The reproductive decline in T. absoluta, linked to reduced Vg/VgR expression and metabolic trade-offs (catabolic pathway activation vs. protein synthesis suppression), aligns with reports in Spodoptera frugiperda and Helicoverpa armigera under insecticide stress (Lv et al., 2025; Li et al., 2024). Similar resource reallocation strategies have been documented for other GABA antagonists (e.g., fipronil), indicating a conserved adaptive response across species. Third, we have integrated the observed upregulation of detoxification genes (P450s) and immune pathways (Toll/Imd) into the emerging "detox-immunity" coordination framework (Chen et al., 2025), supported by recent findings in Myzus persicae (Yuan et al., 2026). These revisions (pages 12–14, lines 452–589) transform the discussion from descriptive to mechanistically grounded. We thank the reviewer for guiding this improvement.

Question 4. The transcriptomic analysis is one of the strongest parts of the study. The authors obtained high-quality sequence data, as evidenced by high Q30 values ​​and adequate sequencing depth. Data quality analyses, including PCA and hierarchical clustering, indicate good biological reproducibility of the samples. A significant number of differentially expressed genes were identified between the control and broflanilide-treated groups. Gene co-expression network analysis (WGCNA) additionally allowed for the identification of gene modules associated with the response to chemical stress. However, the description of the bioinformatics methods is in places too brief and does not include all the parameters used in the analysis, which hinders the full reproducibility of the results.

Reply to question 4: Thank you for the suggestions about the method. Your suggestion regarding the Methods section is highly valuable, as it contributes to ensuring the full reproducibility of the study. To provide greater detail on the parameters used in the analysis, I have incorporated eight additional references into the bioinformatics subsection of the transcriptomics methodology. I believe this enhancement improves the transparency of the data analysis, (Line 213-229).

Question 5. The results of functional analyses (GO, KEGG, and GSEA) indicate that sublethal exposure to broflanilide primarily affects metabolic processes and xenobiotic detoxification mechanisms. The enrichment of pathways related to cytochrome P450 metabolism appears particularly significant, consistent with commonly observed mechanisms of insect response to insecticide stress. The authors also indicate changes in metabolic pathways related to fatty acid and amino acid metabolism, as well as immunological processes (Toll and Imd pathways). However, the interpretation of these results requires further investigation, particularly in the context of the potential significance of these changes for the development of insecticide resistance.

 Reply to question 5: Thank you for the suggestions about the discussion. Your comments on the Discussion section are invaluable. A balanced and comprehensive discussion is essential for a study, but overinterpretation must be avoided. For instance, while the enrichment of detoxification pathways related to cytochrome P450 was statistically significant, establishing a definitive link between this enrichment and the development of insecticide resistance would require further empirical support. Therefore, this portion of the discussion has been removed. In summary, following your suggestions, I have carefully revised the Discussion to ensure it remains rigorous and avoids unwarranted extrapolation.

Question 6. QPCR validation of the transcriptomic results was performed for genes related to cuticle development (chs1 and chs2) and reproduction (Vg and VgR). The obtained results indicate a significant reduction in the expression levels of these genes in the broflanilide-treated groups, which provides a potential molecular explanation for the observed developmental inhibition and decreased fecundity of the insects. This approach is methodologically sound, but the number of genes analyzed is relatively small. Expanding the validation to include a larger number of genes related to detoxification (e.g., P450s, GSTs, esterases) could strengthen the conclusions drawn from this study.

Reply to question 6: Thank you for the suggestions about the data. As you noted, the analysis of gene expression related to P450 enzymes is critically important. Accordingly, we performed qPCR analysis of two P450 genes, CYP338A1 and CYP6B5, which strengthens the robustness of our conclusions. Compared with the control group, the expression levels of both genes were significantly increased in the LC₁₀ and LC₃₀ treatment groups (t-test, P < 0.05) (Figure S2).

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors replied to most of the questions.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, I have reviewed the revised version of the manuscript. The new version is enhanced by the suggestions and/or observations made previously. Furthermore, in the point-by-point response section, the authors accurately addressed each question raised about the manuscript. I can only congratulate you on your new contributions to the knowledge of the sublethal effects of the insecticide broflanilide on Tuta absoluta.