Physio-Transcriptomic Mechanism of Antimony Tin Oxide Nanoparticle-Induced Midgut Toxicity in Bombyx mori

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Fang et al is devoted to the investigation of the effects of different concentrations of ANO nanomaterial (Antimony tin oxide) on the differential expression of genes, weight, antioxidant enzymes’ activities and midgut epithelium structure of Bombyx mori 5-instar larvae.

The results obtained demonstrated that feeding with different concentration ANO resulted in a severe decrease in the weight of B. mori’s larvae in 72 and 96 hours. However, it should be noted that the authors used rather small samples for this experiment (three replicates for each concentration only), which did not allow them to register a significant effect after 48 hours, the tendency towards which is clearly visible in the figure 1.

The authors also showed that treatment of 5-instar larvae with 3.2 mkg/mkL of ATO caused prominent changes in the activities of ROS, superoxide dismutase, catalase and glutathione, in the gene expression (transcriptomic analysis revealed 518 DEGs, 6 of which were additionally validated by qRT-PCR) and midgut tissues (demonstrated with the use of histological and transmission electron microscope analyses).

Overall, it can be concluded that the study presented in the MS by Fang et al is well designed, appears to be well executed and provided a nice set of data on the molecular and physiological mechanisms of ATO toxic effects on insects. I would say it is of interest to a wide range of entomologists.

I don’t see any real problems with this MS, except the fact that the authors didn’t delete the text of the Journal Microsoft Word template in the M&M and Discussion sections (lines 80-99 and 417-420).

Author Response

Comments 1: The manuscript by Fang et al is devoted to the investigation of the effects of different concentrations of ANO nanomaterial (Antimony tin oxide) on the differential expression of genes, weight, antioxidant enzymes’ activities and midgut epithelium structure of Bombyx mori 5-instar larvae. The results obtained demonstrated that feeding with different concentration ANO resulted in a severe decrease in the weight of B. mori’s larvae in 72 and 96 hours. However, it should be noted that the authors used rather small samples for this experiment (three replicates for each concentration only), which did not allow them to register a significant effect after 48 hours, the tendency towards which is clearly visible in the figure 1. The authors also showed that treatment of 5-instar larvae with 3.2 mkg/mkL of ATO caused prominent changes in the activities of ROS, superoxide dismutase, catalase and glutathione, in the gene expression (transcriptomic analysis revealed 518 DEGs, 6 of which were additionally validated by qRT-PCR) and midgut tissues (demonstrated with the use of histological and transmission electron microscope analyses). Overall, it can be concluded that the study presented in the MS by Fang et al is well designed, appears to be well executed and provided a nice set of data on the molecular and physiological mechanisms of ATO toxic effects on insects. I would say it is of interest to a wide range of entomologists. I don’t see any real problems with this MS, except the fact that the authors didn’t delete the text of the Journal Microsoft Word template in the M&M and Discussion sections (lines 80-99 and 417-420). Response 1: We appreciate the reviewer’s careful observation regarding the sample size used in the larval weight analysis. We agree that, with three biological replicates per treatment, the reduction trend at 48 h was not sufficient to reach statistical significance, although an early downward tendency could already be observed in Figure 1. In the revised manuscript, we have taken care to present this result more cautiously and to avoid overinterpretation of the 48 h time point (Lines 472-474, page 17). At the same time, the inhibitory effect of antimony tin oxide nanoparticles on larval growth became more evident at later stages, with statistically significant decreases observed at 72 and 96 h. On this basis, and together with the clear toxic phenotype at 3.2 μg/μL, we selected this concentration for subsequent analyses of oxidative stress, transcriptomic responses, and midgut injury, aiming to characterize the physiological and molecular mechanisms associated with a biologically relevant exposure level (Lines 248-260, page 8). We also appreciate the reviewer for pointing out the leftover journal template text, particularly in the Materials and Methods and Discussion sections (Lines 91-92, page 3 and Lines 447-448, page 17). This was an unintentional editing oversight. We have now removed all irrelevant template content from the revised manuscript, including text from the header, footer, and cover page. Additionally, we have replaced the placeholder email addresses and network connections with the correct information. We have carefully rechecked the full text to avoid similar issues.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

While I was reviewing literature pertaining to this topic, I found that ATO is arsenic trioxide. But in this MS, its mentioned as antimony tin oxide

The dosage concentration (3.2 μg/μL) is way too high in my view. If any LC50 studies have been performed prior to this experiment, the same may be provided. Have the authors measured what’s the amount ingested, accumulated in the midgut and also levels in the hemolymph. Pls provide necessary data.

I couldn’t find the details about characterization of nanoparticle viz., particle size, stability etc. Pls provide the details.

Why were the controls including heat killed nanoparticle control, vehicle control for ultrasonic treatment, particle free tin/antimony control not used in the study?

Pls provide survival curves.

Pls justify why only a single dose of 3.2 μg/μL was chosen for ROS and antioxidant enzyme responses. Its better to test multiple concentrations.

With respect to the RNA-Seq analysis, its better to use FDR < 0.05 as cutoff

A few statements were not supported by appropriate data. If I may say and as the authors may agree, ‘uncoupled oxidative phosphorylation…’ ‘activation of apoptosis’ etc…these statements need additional experiments to come to a reliable conclusion. So, pls change the text accordingly.

Authors have performed ANOVA. Pls mention the post hoc test used.

There are so many sentences repeating in the discussion section. Pls avoid. Strengthen the discussion.

Check for tense and grammatical errors.

Comments on the Quality of English Language

Must be largely improved.

Author Response

Reviewer 2

Comments 1: While I was reviewing literature pertaining to this topic, I found that ATO is arsenic trioxide. But in this MS, its mentioned as antimony tin oxide

Response 1: Thank you for this helpful comment. We agree that the abbreviation “ATO” may be ambiguous, as it is used in the literature to refer to both arsenic trioxide (Vineetha and Raghu, 2019, Cardiovascular toxicology; Yang et al., 2024, Colloids and surfaces. B, Biointerfaces) and antimony tin oxide (Yang et al., 2025, ACS applied materials & interfaces; Jin et al., 2021, Journal of colloid and interface science). To eliminate any possible confusion for readers, we have revised the manuscript and replaced “ATO” with the full name “antimony tin oxide” throughout the text, figures, and figure captions.

 

Comments 2: The dosage concentration (3.2 μg/μL) is way too high in my view. If any LC50 studies have been performed prior to this experiment, the same may be provided.

Response 2: Thank you for raising this point. The concentration of 3.2 μg/μL is indeed relatively high. Although a formal LC50 study was not conducted before this experiment, the choice of this dose was guided by our preliminary dose-screening results and survival observations. Under the present experimental conditions, the Kaplan–Meier survival curves showed that mortality increased with concentration, and the 3.2 μg/μL group exhibited approximately 50% mortality during the observation period (Figure 1C). At the same time, this treatment produced pronounced developmental and tissue damage while retaining sufficient surviving larvae for downstream mechanistic analyses. This rationale has been clarified in the revised manuscript (Lines 256-260; 265-268, page 8).

 

Comments 3: Have the authors measured what’s the amount ingested, accumulated in the midgut and also levels in the hemolymph. Pls provide necessary data.

Response 3: Your question about ingested amount, midgut accumulation, and hemolymph levels is well taken. We acknowledge that quantifying the actual ingested amount of antimony tin oxide, as well as its accumulation in the midgut and its levels in the hemolymph, would provide valuable toxicokinetic information and further strengthen the interpretation of the present study.

At the present stage, however, these data are not available, because our experimental design was based on nominal concentrations of antimony tin oxide applied to mulberry leaves rather than on direct toxicokinetic measurements. To ensure quantitative daily intake of treated leaves, each larva was individually fed. The 5 g portion per larva was completely consumed during each feeding session, guaranteed by preliminary consumption tests. This approach is consistent with established oral exposure methods used in insect toxicology, where precise control of ingested dose is achieved by providing defined quantities of treated diet that are fully consumed (Nagloo et al., 2023, Ecotoxicology and environmental safety). In order to keep the exposure conditions as consistent as possible, mulberry leaves were prepared in the same way for each treatment group and larvae were fed under the same schedule throughout the experiment. Although this design does not allow direct determination of internal dose, it does support comparison of the biological responses observed under controlled nominal exposure conditions (Lines 116-121, page 4).

We appreciate the reviewer’s point and have now explicitly acknowledged this limitation in the revised Discussion (Line 574-579, page 19). We have also revised the text to avoid overinterpretation and to make clear that the present conclusions are based on external exposure-associated responses. Future work will need to combine feeding quantification and elemental analysis of midgut and hemolymph samples to address this issue more directly.

 

Comments 4: I couldn’t find the details about characterization of nanoparticle viz., particle size, stability etc. Pls provide the details.  

Response 4: Thank you for this important comment. In the revised manuscript, we have added the available physicochemical information for the commercial antimony tin oxide nanoparticles used in this study. Specifically, the material was purchased from Macklin (A801372), and according to the supplier’s specifications, the primary particle size is approximately 20–80 nm and the purity is >99.5% (Lines 101-107, page 4). Before exposure, the nanoparticles were dispersed in ultrapure water and homogenized by ultrasonication prior to leaf-coating treatment. We also acknowledge that independent characterization of hydrodynamic size distribution, zeta potential, morphology, and colloidal stability under our experimental exposure conditions was not performed. This limitation has now been clarified in the revised manuscript (Lines 583-587, page 20), and we indicate that future studies should combine TEM/DLS/zeta potential analysis with biological endpoint assessment.

 

Comments 5: Why were the controls including heat killed nanoparticle control, vehicle control for ultrasonic treatment, particle free tin/antimony control not used in the study?

Response 5: Thank you for this valuable comment. We acknowledge that incorporating additional controls would enhance the mechanistic understanding of nanoparticle toxicity. In the present study, the control group received mulberry leaves treated with ultrapure water, which underwent the same ultrasonication and coating procedure as the antimony tin oxide suspension, but without nanoparticles. Since ultrapure water was the only dispersion medium used and no surfactants or organic solvents were introduced, this control served as the vehicle control for the ultrasonication-based exposure procedure.

We did not include a heat-treated nanoparticle control, as antimony tin oxide is an inorganic nanomaterial, and heat treatment would likely alter its physicochemical properties-such as aggregation, surface characteristics, and dissolution behavior - rather than produce an inert or biologically inactive control. Additionally, we did not incorporate particle-free tin/antimony controls, as the aim of this study was to evaluate the overall toxicological effects of the commercial antimony tin oxide nanoparticle preparation, rather than to distinguish between nanoparticle-specific effects and ionic contributions. We recognize that the lack of soluble metal controls limits the mechanistic insights of the current study. This has now been clarified in the revised Methods (Lines 107, page 4), and we have added it as a limitation in the Discussion (Lines 586-588, page 20). Future studies should include soluble tin/antimony controls and dissolution analysis to better differentiate between particle-mediated and ion-mediated toxicity.

 

Comments 6: Pls provide survival curves.

Response 6: Thank you for this valuable suggestion. We recognize that survival curves provide a clearer presentation of the temporal pattern of antimony tin oxide toxicity. According to the reviewer’s suggestion, we have now added the larval survival analysis to the revised manuscript. Specifically, Kaplan–Meier survival curves have been included as Figure 1C, and the differences among treatment groups were analyzed using the log-rank test. The corresponding description has been added to the Materials and Methods section (Lines 243-244, page 7) and the Results section (Lines 256-260, page 8).

 

Comments 7: Pls justify why only a single dose of 3.2 μg/μL was chosen for ROS and antioxidant enzyme responses. Its better to test multiple concentrations.

Response 7: Thank you for this valuable suggestion. The concentration of 3.2 μg/μL was chosen on the basis of our toxicity evaluation, which showed that this dose caused approximately 50% mortality in silkworm larvae (Figure 1C). Such an effect level indicates a substantial but not overwhelming toxic challenge, making it suitable for assessing ROS production and antioxidant enzyme responses in surviving individuals. We fully agree that inclusion of multiple concentrations would allow a more complete dose–response analysis. Nevertheless, the present work was designed to investigate the toxic mechanism under a biologically effective exposure condition, and therefore focused on one representative dose. We have now clarified this rationale in the revised manuscript and also acknowledged the lack of a multi-concentration oxidative stress analysis as a limitation of the current study (Lines 579-583, page 19-20).

 

Comments 8: With respect to the RNA-Seq analysis, its better to use FDR < 0.05 as cutoff

Response 8: Thank you for this important suggestion. We agree that controlling for multiple testing is more appropriate for RNA-seq differential expression analysis. Following the reviewer’s recommendation, we re-analyzed the transcriptomic data using an adjusted significance threshold of FDR < 0.05 together with |log2 fold change| > 1, and have revised the Methods, Results, figures, and relevant text accordingly. The number of DEGs and the downstream enrichment results have been updated in the revised manuscript. Our main interpretation has also been adjusted to reflect the more stringent statistical criterion (Lines 188-189, page 5; 283-320, page 10-11; 517-529, page 18).

 

Comments 9: A few statements were not supported by appropriate data. If I may say and as the authors may agree, ‘uncoupled oxidative phosphorylation…’ ‘activation of apoptosis’ etc…these statements need additional experiments to come to a reliable conclusion. So, pls change the text accordingly. Authors have performed ANOVA. Pls mention the post hoc test used.

Response 9: This is an important point. Several statements in the previous version were phrased too conclusively for the level of experimental evidence. In the revised manuscript, expressions such as “uncoupled oxidative phosphorylation” and “activation of apoptosis” have been systematically removed or rephrased throughout the text. The corresponding outcomes are now cautiously described as changes associated with mitochondrial ultrastructural alterations, disrupted lipid metabolism, autophagosome accumulation, and apoptosis-like phenotypes – rather than as definitive proof of specific mechanistic events that would require further functional validation (Lines 545-551, page 18-19).

We appreciate the reviewer’s comment. For comparisons involving only two groups, Student’s t-test was used. For experiments involving more than two groups, one-way ANOVA followed by Dunnett’s multiple-comparison test was applied to compare each treatment group with the control. This clarification has now been incorporated into the revised manuscript (Lines 240-245, page 7).

 

 

 

Comments 10: There are so many sentences repeating in the discussion section. Pls avoid. Strengthen the discussion.

Response 10: Thank you for this valuable comment. We carefully revised the Discussion section to reduce repetitive descriptions and improve its clarity and depth. Specifically, we removed overlapping statements on growth inhibition, oxidative stress, and midgut structural damage; merged several repetitive paragraphs; and reorganized the Discussion to improve the logical flow from physiological toxicity to histological injury and transcriptomic alteration. In addition, we strengthened the Discussion by placing our findings in the broader context of metal oxide nanoparticle toxicity while adopting a more cautious interpretation of the proposed mechanisms. These revisions have substantially improved the conciseness, readability, and scientific rigor of the Discussion section.

 

Comments 11: Check for tense and grammatical errors.

Response 11: Thank you for your careful review. We have thoroughly checked the revised manuscript for tense consistency and grammatical accuracy and have corrected the relevant errors throughout the text.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript entitled “Physio-Transcriptomic Mechanism of Antimony Tin Oxide Nanoparticle-Induced Midgut Toxicity in Bombyx mori” presents a comprehensive and well-designed investigation into the toxicological effects of ATO nanoparticles on silkworm larvae. The study integrates physiological assays, oxidative stress biomarkers, transcriptomic profiling, histopathology, ultrastructural analysis, and bioinformatics approaches to elucidate the underlying mechanisms of midgut toxicity. The work is logically structured, the experimental design is appropriate, and the results are clearly presented with convincing multi-level validation. Overall, the manuscript is scientifically sound, well presented, and provides valuable insights into the environmental risk assessment of nanomaterials in agricultural ecosystems. Only minor revisions are required to further improve clarity and presentation quality.

1. Minor grammatical correction: “resulted in a significantly reduced in larval weight” should be revised to “resulted in a significant reduction in larval weight.”
2. Clarify whether ethical approval was required for silkworm experimentation and provide a statement if applicable.
3. Specify whether RNA-seq raw data have been deposited in a public database (accession number should be included).
4. Indicate the exact statistical post hoc test used after ANOVA.
5. While multiple concentrations were tested for growth analysis, oxidative stress and transcriptomics were performed only at 3.2 μg/μL. A brief justification for selecting this concentration would improve clarity.
6. In Figure 10, ATO is described as arsenic trioxide in the caption; this appears inconsistent with antimony tin oxide and should be corrected.
7. The discussion is strong but could briefly compare ATO toxicity with other metal oxide nanoparticles to place findings in broader nanotoxicology context.

Author Response

The manuscript entitled “Physio-Transcriptomic Mechanism of Antimony Tin Oxide Nanoparticle-Induced Midgut Toxicity in Bombyx mori” presents a comprehensive and well-designed investigation into the toxicological effects of ATO nanoparticles on silkworm larvae. The study integrates physiological assays, oxidative stress biomarkers, transcriptomic profiling, histopathology, ultrastructural analysis, and bioinformatics approaches to elucidate the underlying mechanisms of midgut toxicity. The work is logically structured, the experimental design is appropriate, and the results are clearly presented with convincing multi-level validation. Overall, the manuscript is scientifically sound, well presented, and provides valuable insights into the environmental risk assessment of nanomaterials in agricultural ecosystems. Only minor revisions are required to further improve clarity and presentation quality.

Comments 1: Minor grammatical correction: “resulted in a significantly reduced in larval weight” should be revised to “resulted in a significant reduction in larval weight.”

Response 1: Thank you for your correction, we have made the amendment (Lines 32-33, page 1).

 

Comments 2: Clarify whether ethical approval was required for silkworm experimentation and provide a statement if applicable.

Response 2: Thank you for pointing this out. We agree with this comment. According to the guidelines of our institution and national regulations, ethical approval is not required for experiments involving Bombyx mori, as they are invertebrates and not classified as protected species. Nevertheless, all experiments were conducted in accordance with standard animal care and use practices for insects. Therefore, we have added a statement in the “Institutional Review Board Statement” section as follows: Ethical review and approval were not required for this study involving silkworms, as they are invertebrate animals not subject to ethical oversight under current institutional and national guidelines (Lines 625-627, page 20). 

 

Comments 3: Specify whether RNA-seq raw data have been deposited in a public database (accession number should be included).

Response 3: Agree. We have, accordingly, updated the ‘Data Availability Statement’ to include the BioProject accession number for the raw RNA-seq data. The data have been deposited in the Genome Sequence Archive (https://ngdc.cncb.ac.cn/gsa/search? searchTerm=CRA039701, reference number CRA039701). The accession number and the reviewer link have been added to the revised manuscript to ensure transparency and accessibility upon publication. (Lines 629-631, page 21)

 

Comments 4: Indicate the exact statistical post hoc test used after ANOVA.

Response 4: Thank you for this helpful suggestion. We have clarified the statistical analysis in the revised manuscript. For comparisons involving only two groups, Student’s t-test was used. For experiments involving more than two groups, one-way ANOVA followed by Dunnett’s multiple-comparison test was applied to compare each treatment group with the control. The corresponding description has been added to the revised Materials and Methods section (Lines 240-245, page 7).

 

Comments 5: While multiple concentrations were tested for growth analysis, oxidative stress and transcriptomics were performed only at 3.2 μg/μL. A brief justification for selecting this concentration would improve clarity.

Response 5: Thank you for this helpful comment. We have now clarified the basis for selecting 3.2 μg/μL in the revised manuscript (Lines 135-139, page 4; 256-268, page 8). Although larval growth was assessed across several exposure concentrations to establish the dose-response relationship, the downstream oxidative stress and transcriptomic analyses were conducted at 3.2 μg/μL because this concentration elicited the clearest toxic phenotype. In particular, it caused marked growth suppression and approximately 50% mortality, indicating a robust biological effect, yet still allowed collection of enough surviving larvae for subsequent biochemical and molecular analyses. For this reason, 3.2 μg/μL was used as the representative exposure level for mechanistic investigation.

 

Comments 6: In Figure 10, ATO is described as arsenic trioxide in the caption; this appears inconsistent with antimony tin oxide and should be corrected.

Response 6: Thank you for noting this mistake. The description of antimony tin oxide (ATO) as “arsenic trioxide” was inaccurate. We have corrected the caption to “antimony tin oxide” in the revised version and re-examined the manuscript to avoid similar inconsistencies elsewhere (Lines 553, page 19).

 

Comments 7: The discussion is strong but could briefly compare ATO toxicity with other metal oxide nanoparticles to place findings in broader nanotoxicology context.

Response 7: We appreciate this constructive comment. In the revised Discussion, we have incorporated a short comparison between antimony tin oxide and other metal oxide nanoparticles to broaden the interpretive context of our study. The added text emphasizes that oxidative imbalance, growth impairment, and tissue injury are common toxicological themes reported for several metal oxide nanoparticles in insects, including ZnO and CuO. At the same time, we also point out that the biological outcomes are particle-dependent, as TiO₂ exposure in silkworms has been reported to produce distinct responses under certain exposure conditions. We believe this revision improves the general relevance of the Discussion and clarifies the broader significance of our findings (Lines 481-490, page 17).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed all my comments satisfactorily and made necessary changes to the MS which may now be accepted. 

Line 489: ATO may be changed while proof reading