Biocontrol Mechanisms of a Chinese Heterorhabditis indica Strain Against Tuta absoluta: Virulence Assay and Time-Course Transcriptomics of Host Immune Responses

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

1) Overall assessment of the manuscript

The study evaluates the biocontrol potential of a native Heterorhabditis indica strain (CQ7-2) against Tuta absoluta larvae (LC₅₀ reported as 1.35 IJs/larva) and characterizes the host immune-related transcriptional response across infection time points via RNA-seq, followed by targeted interpretation of altered immune pathways (IMD/Toll/JAK-STAT/JNK; melanization/encapsulation).

A structured time-course transcriptomic dataset for T. absoluta challenged with a Heterorhabditis EPN is potentially valuable for both insect immunology and biocontrol, particularly because T. absoluta is a globally important invasive pest and omics studies on its interaction with EPNs are limited.

Overall scientific quality. The experimental concept is promising and the dataset could be publishable; however, key design limitations and reporting gaps currently weaken causal claims (especially “immunosuppression strategy”) and reduce reproducibility.

 

2) Strengths of the work

1. Clear applied relevance: addresses urgent need for non-chemical control options for T. absoluta and evaluates a locally sourced EPN.
2. Time-course transcriptomics: multiple post-infection time points (6/12/18 hpi) provide dynamics rather than a single snapshot.
3. Use of reference genome: reads mapped to a defined T. absoluta assembly and differential expression was pursued with a standard framework.
4. RT-qPCR validation: validation of selected DEGs is included
5. Data availability: raw RNA-seq deposited (GSA BioProject PRJCA054879; CRA036293).

3) Critical points — MAJOR COMMENTS

Major 1 — Causal attribution to “immunosuppression” is not experimentally supported

Issue. The manuscript repeatedly interprets early/downregulated immune transcripts as evidence that the nematode “employed an immunosuppressive strategy” and “targeted critical nodes” of the immune network.

Why this is a problem. From whole-larva RNA-seq alone, reduced expression of immune genes can reflect many non-exclusive causes: developmental stage effects, systemic morbidity, metabolic collapse, altered tissue composition, or transcript dilution due to extensive non-immune transcriptional shifts—especially at 18 hpi when larvae are near death.

 

What to do.

• Temper the language throughout Results/Discussion/Conclusions to reflect association rather than demonstrated mechanism.
• Add functional/phenotypic immune readouts (even a minimal set) aligned with the transcript claims: hemocyte counts/viability, phenoloxidase activity, melanization capacity, encapsulation assay, AMP activity proxy, or bacterial load kinetics.

Major 2 — Bioassay design does not support the reported LC₅₀ as presented

Issue. The dose series used is 5–80 IJs/larva, yet LC₅₀ is reported as 1.35 IJs/larva.

Why this is a problem. Reporting an LC₅₀ below the lowest tested dose implies extrapolation well outside the observed range. This can be valid but requires rigorous presentation (model fit, confidence intervals, goodness-of-fit, and ideally inclusion of lower doses).

What to do.

• Include doses below 5 IJs/larva (e.g., 0.5, 1, 2, 3) or explicitly justify extrapolation with complete probit outputs.
• Report 95% CI for LC₅₀ and LT₅₀, slope ± SE, chi-square, df, and p-value for fit.
• Clarify whether mortality was corrected (e.g., Abbott) and whether deaths were confirmed as EPN-associated (cadaver dissection, IJ emergence, or bacterial signs).

 

Major 3 — RNA-seq replication, experimental unit, and batch structure are insufficiently transparent

Issue. RNA samples are pools of ten larvae per sample, but the manuscript does not unambiguously state the number of independent biological replicates per time point used for RNA-seq (distinct from RT-qPCR replication).

Why this is a problem. edgeR inference depends critically on replicate structure and dispersion estimation; pooling can obscure biological variability and risks pseudoreplication if not carefully defined.

What to do.

• Provide a table with n biological replicates per condition, whether each replicate is an independent cohort, and how pooling was performed.
• Clarify randomization, whether larvae came from different cages/plants, and whether sequencing lanes/batches were balanced across conditions.

 

Provide MDS/PCA and sample-to-sample correlation plots to demonstrate replicate coherence and absence of confounding.

Major 4 — Overinterpretation of late-stage “immune activation” at 18 hpi

Issue. The manuscript emphasizes a strong immune upregulation at 18 hpi but also describes systemic collapse and inevitable mortality.

Why this is a problem. At near-terminal stages, transcriptional bursts may reflect generalized stress, tissue breakdown, microbiome translocation, or dysregulated signaling rather than a coordinated protective response.

What to do.

• Reframe 18 hpi as “late/terminal response,” and avoid implying efficacy.

 

4) Specific points — MINOR COMMENTS

1. Typographical/grammatical issues: a few minor typos (e.g., “p-valuie”, spacing like “Cyclin-D2and”).
2. Methods clarity: specify whether larvae were surface-sterilized prior to RNA extraction; whole-body RNA can be influenced by cuticle/plant residues.
3. Data sharing: in addition to raw reads, provide processed count matrices, sample metadata, and analysis scripts to improve reproducibility.

5) Final recommendation

Minor revision

Author Response

Summary

Thank you very much for your thorough and insightful review of our manuscript. Your professional and detailed comments have been invaluable in helping us improve the quality and clarity of our work. We sincerely appreciate the time and expertise you have dedicated to this evaluation.

We have carefully considered all of your suggestions and have revised the manuscript accordingly. Below, we provide a point-by-point response to each of your comments, detailing the specific changes made. All modifications in the manuscript text have been highlighted for your convenience.

We believe that addressing your concerns has strengthened the paper, and we are grateful for your constructive input.

Point-by-point response to Comments and Suggestions

Comments 1:

Major 1 — Causal attribution to “immunosuppression” is not experimentally supported

Issue. The manuscript repeatedly interprets early/downregulated immune transcripts as evidence that the nematode “employed an immunosuppressive strategy” and “targeted critical nodes” of the immune network.

Why this is a problem. From whole-larva RNA-seq alone, reduced expression of immune genes can reflect many non-exclusive causes: developmental stage effects, systemic morbidity, metabolic collapse, altered tissue composition, or transcript dilution due to extensive non-immune transcriptional shifts—especially at 18 hpi when larvae are near death.

Response 1：

We sincerely thank you for raising this critical point, which gets to the heart of our study's interpretative limitations. We fully agree that attributing the observed downregulation of immune transcripts solely to a pathogen-driven "immunosuppressive strategy" is an overinterpretation based on our transcriptomic data alone. 

In direct response, we have conducted a thorough revision of the manuscript to temper the language and remove unwarranted causal claims. Specifically, we have replaced phrases like "employed an immunosuppressive strategy" and "targeted critical nodes" with more precise and cautious formulations that describe the observed transcriptional patterns as being consistent withimmunosuppression or immune modulation, while explicitly acknowledging the correlative nature of the data and the need for future functional validation.

These revisions have been implemented throughout the text, including in the Abstract, Results, and Discussion sections. Key locations include lines 25-27, 43-45, 48-51, 52-54,659-661, 664-666, 701-704, 746-750, 781-786, and 791-797. We believe these changes significantly improve the scientific accuracy and balance of the manuscript.

Comments 2:

Major 2 — Bioassay design does not support the reported LC₅₀ as presented

Issue. The dose series used is 5–80 IJs/larva, yet LC₅₀ is reported as 1.35 IJs/larva.

Why this is a problem. Reporting an LC₅₀ below the lowest tested dose implies extrapolation well outside the observed range. This can be valid but requires rigorous presentation (model fit, confidence intervals, goodness-of-fit, and ideally inclusion of lower doses).

Response 2：

We sincerely thank you for this crucial statistical critique, which rightly questions the extrapolation of the LC₅₀ value. We fully acknowledge that reporting an LC₅₀ (1.35 IJs/larva) below the lowest tested dose (5 IJs/larva) requires clear justification. o address this, we have supplemented the manuscript with the following points, which support the reliability of this extrapolated estimate:

1. Model Fit:​ The probit model demonstrated a good fit to the observed data, as indicated by a non-significant goodness-of-fit test (χ² = 2.196, df = 3, p = 0.533) and a well-defined dose-response slope (1.113 ± 0.303 SE). These statistical parameters are now reported in the manuscript (Lines 326-331).
   
2. High Mortality at the Lowest Tested Dose:​ The substantial mortality (75.6%) observed at the lowest concentration (5 IJs/larva) already far exceeds the 50% threshold, providing empirical confidence that the LC₅₀ lies within a plausible extrapolated range below this dose.
3. Technical Consideration for Dose Range:​ The decision not to test doses below 5 IJs/larva was primarily methodological. At very low concentrations, the accurate dispensing of IJs becomes technically challenging due to factors such as adhesion to pipette tips, which can introduce significant variability in the actual number of IJs inoculated. The chosen range was therefore optimized to ensure dosing accuracy and reproducibility.
   

We have revised the relevant section to incorporate these clarifications and justifications, ensuring that the extrapolation is presented with appropriate transparency and statistical support.

Comments 3:

Major 3 — RNA-seq replication, experimental unit, and batch structure are insufficiently transparent

Issue. RNA samples are pools of ten larvae per sample, but the manuscript does not unambiguously state the number of independent biological replicates per time point used for RNA-seq (distinct from RT-qPCR replication).

Why this is a problem. edgeR inference depends critically on replicate structure and dispersion estimation; pooling can obscure biological variability and risks pseudoreplication if not carefully defined.

Response 3：

We sincerely thank you for highlighting this essential point regarding experimental design transparency, which is crucial for the statistical validity of our RNA-seq analysis. We agree that the replicate structure must be clearly defined to support edgeR's inference.

To address this, we have made the following clarifications and additions to the manuscript:

1. Explicit Replication Details:​ We have now unambiguously stated the number of independent biological replicates​ in both the bioassay and RNA isolation sections. Specifically, the details regarding the setup of biological replicates have been added to Lines 193 and 219-223.

2. Supplementary Quality Controls:​ To directly address concerns about biological variability and potential batch effects obscured by pooling, we have generated and included two new supplementary figures:

   • Fig S2:​ Shows the inter-sample distances, allowing assessment of overall reproducibility.

   • Fig S3:​ Presents the PCA plot, demonstrating that the primary source of variation is driven by treatment and time point.

These revisions provide full transparency regarding our experimental unit and replicate structure, substantiate the robustness of our differential expression analysis, and mitigate the risk of pseudoreplication. We believe these additions significantly strengthen the methodological rigor of the study.

Comments 4:

Major 4 — Overinterpretation of late-stage “immune activation” at 18 hpi

Issue. The manuscript emphasizes a strong immune upregulation at 18 hpi but also describes systemic collapse and inevitable mortality.

Why this is a problem. At near-terminal stages, transcriptional bursts may reflect generalized stress, tissue breakdown, microbiome translocation, or dysregulated signaling rather than a coordinated protective response.

Response 4：

We sincerely thank you for this critical insight, which refines the interpretation of our late-stage data. We fully agree that the transcriptional upregulation observed at 18 hpi, coinciding with systemic morbidity and impending mortality, cannot be unequivocally interpreted as a coordinated, protective immune response. As you astutely notes, it could equally reflect generalized stress, tissue breakdown, or dysregulated signaling.

To address this, we have substantially revised the relevant sections of the manuscript to remove any definitive claims of a "coordinated protective response." We now frame the observed upregulation as a complex terminal transcriptional event occurring in the context of systemic collapse. Specific changes include the deletion of the phrasing in lines 374-375 and the careful re-wording of interpretations in lines 452-458, 662-665, 674-676, and 784-789. This ensures our discussion aligns with the correlative nature of the data and acknowledges the alternative explanations you have highlighted.

Comments 5:

Specific points — MINOR COMMENTS

1. Typographical/grammatical issues: a few minor typos (e.g., “p-valuie”, spacing like “Cyclin-D2and”).
2. Methods clarity: specify whether larvae were surface-sterilized prior to RNA extraction; whole-body RNA can be influenced by cuticle/plant residues.
3. Data sharing: in addition to raw reads, provide processed count matrices, sample metadata, and analysis scripts to improve reproducibility.

Response 5：

1. All noted typographical errors, including “p-valuie” (line 279) and missing spaces (e.g., “Cyclin-D2and” at line 763), have been corrected.
2. We have added a statement in the RNA isolation section (Lines 220-223) specifying that larvae were surface-sterilized before homogenization to minimize potential contamination from cuticular residues or external material, thereby ensuring the quality of the whole-body RNA.
3. In addition to the raw sequencing reads deposited in the public repository, we now provide the processed gene expression count matrix and associated sample metadata as Supplementary Files​ with the manuscript.
   

We would like to express our sincere gratitude for your comprehensive and insightful review of our manuscript. Your expert critique and constructive suggestions have allowed us to better recognize the limitations of our work and have substantially enhanced the scientific quality of the paper. We have carefully addressed each of your concerns, and we greatly appreciate the time and scholarly acumen you invested in this process. Thank you again for your invaluable feedback.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

The study is timely and potentially useful, combining replicated virulence assays of Heterorhabditis indica against Tuta absoluta with a time-course transcriptomic analysis. However, several statements,especially in the Discussion/Conclusions, overstate mechanistic certainty based on RNA-seq alone. Please reframe causal language (“immune evasion strategy/critical nodes”) as transcriptomic evidence “consistent with” immunosuppression unless functionally validated. Also, clarify and proofread the RNA-seq statistical criteria (explicit FDR and any log2FC cutoffs), and acknowledge the limitation of pooling 10 larvae/sample. Streamlining repetitive GO/KEGG narration would improve readability.

Lines 13–25:
The Simple Summary is clear but slightly promotional (“provides a scientific basis”). Consider rephrasing to emphasize what was demonstrated (virulence + time-course immune suppression) rather than claiming direct applicability.

Lines 26–43:
The Abstract is generally strong, but several claims (“sophisticated immunosuppressive strategy”, “critical nodes”) sound mechanistic while evidence is transcriptomic correlation. Consider softening to “consistent with immunosuppression” unless functional validation is provided.

Lines 48–62:
The invasion background is detailed and engaging, but the paragraph is very dense with statistics and geographical spread. Consider condensing and moving some numbers (e.g., economic losses) to a single sentence to improve flow.

Lines 67–77:
The insecticide resistance overview is useful, but it reads as a broad “catalog” of resistance cases. It would help to briefly state the key knowledge gap that this manuscript addresses (why EPNs and what is unknown about host immune responses to them) earlier in the Introduction.

Lines 72–77:
You cite “up to 28-fold resistance” to Bt (Btk). Please ensure this is properly supported and framed (strain, geography, assay type). Otherwise, readers may challenge the generality of the claim.

Lines 78–95:
The biological control paragraph is informative, but some numbers (40–70% suppression; 87–95% reduction) need clearer context (study conditions, application method, and whether measured as infestation reduction vs mortality). This will prevent overinterpretation.

Lines 96–109:
The “three barriers” framing is a nice structure, but the first barrier (“commercial strains biased toward soil pests”) is stated strongly. Consider citing broader comparative reviews or meta-analyses on foliar/endophytic targeting limitations of EPNs.

Lines 110–121:
The study rationale is clear, but novelty is oversold (“first transcriptomic analysis”). Please verify whether previous transcriptomic/immune gene expression studies exist for T. absoluta under EPN/symbiont bacterial challenge, and rephrase if needed.

Lines 123–136:
Rearing details are adequate. However, since the colony has been maintained for >5 generations, please comment on whether this could reduce genetic variation and affect immune responses compared with field populations.

Lines 144–167:
Bioassay design is clearly described and replication is strong. Consider clarifying whether larvae were randomly assigned to treatments and whether the observer was blinded to reduce bias in survival scoring.

Lines 153–162:
The replicate structure is good, but pooling technical replicates after a log-rank homogeneity test may still mask batch effects. Please report replicate-level variation (e.g., LC50 confidence intervals per biological replicate) or justify pooling more explicitly.

Lines 168–174:
RNA samples are pooled (10 larvae/sample). Pooling increases practicality but reduces the ability to quantify individual variability. This limitation should be acknowledged in the Discussion.

Lines 204–228:
The RNA-seq pipeline is detailed, but there are typos/unclear thresholds (“applying a significance threshold of and …”; “p-valuie”). Please correct and explicitly state the FDR cutoff (e.g., FDR < 0.05) and the log2FC threshold used for DEGs.

Lines 252–257:
Data access is provided, which is good. Consider adding a brief note on what is deposited (raw fastq, count matrices, metadata) to make reuse easier.

Lines 266–276:
The Results description of cadaver color change implies a mechanistic link to symbiotic bacteria. If this is inferential (not directly tested), consider stating it as “consistent with” rather than causal.

Lines 277–280 (Figure 1):

Figure 1A (Kaplan-Meier survival):please report n per treatment directly in the caption or panel. Figure 1B images are useful, but a brief note on what the darkening indicates (inference vs. evidence) would prevent overinterpretation.

Lines 281–309:
The Results are readable, but the narrative includes interpretive phrasing (“systemic physiological collapse”). This belongs more in Discussion unless you provide direct physiological measures beyond transcriptome patterns.

Lines 314–332:
GO analysis is presented with many numbers in-line (gene counts, enrichment scores). Consider summarizing key patterns (direction + category) and moving detailed counts to a supplementary table for readability.

Lines 345–377:
The GO narrative is thorough but repetitive across time points. You can reduce text by focusing on the transition from early cuticle/metabolic effects to later immune pathway enrichment (e.g., Toll-related terms).

Lines 371–383:
Statements like “infection … strongly downregulated metabolic processes” are plausible but can be overgeneralized. Consider emphasizing that these are transcript-level changes and may reflect host shutdown, pathogen manipulation, or both.

Lines 384–390 (Figure 3):

Figure 3 is information-rich but visually crowded-many GO terms are angled and hard to read. Consider reducing to fewer top terms or moving full lists to Supplementary and keeping only the strongest signals in the main figure.

Lines 503–510 (Figure 5):

Panel A (Mfuzz clusters) is visually appealing but dense; individual trajectories are hard to distinguish. Consider showing cluster centroids more prominently or adding the number of genes per cluster on the panel. Panel C heatmaps are readable, but label size (gene IDs) may be small in print-please confirm legibility at final PDF scale.

Lines 547–583:
The immune gene interpretation is interesting but reads as if the mechanism is resolved (“critical immune evasion strategy”). Without functional assays (RNAi, enzyme activity, melanization/encapsulation quantification), please tone down certainty or propose these as mechanistic hypotheses for follow-up.

Lines 572–582:
The phrasing “terminal immune response coinciding with systemic collapse” is strong. Consider rephrasing to a more cautious interpretation tied to the observed time-course and mortality pattern.

Lines 584–592 (Figure 6):

Figure 6 heatmaps are strong and align well with the immune-pathway narrative. However, because values are Z-score normalized, please remind readers in the caption that colors reflect relative expression patterns (not absolute expression), to avoid misinterpretation of magnitude across genes.

Lines 680–692:
The Conclusions are well written but overstate mechanistic certainty (“precise interference … mechanistic explanation”). Reframe as “transcriptomic evidence consistent with suppression of…” and highlight the need for functional validation (which you already mention) as a key limitation.

Lines 730–770:
The reference list actually includes several recent sources (2021–2024) on management and resistance, which is good. Still, some classic invasion/control citations are old (expected), but ensure that all major claims in the Introduction are anchored in the most recent syntheses where available (e.g., integrated management updates, current resistance monitoring).

 

Kind regards, R

Comments on the Quality of English Language

The English could be improved to more clearly express the research, particularly in the Methods/Statistics. For example, the RNA-seq differential expression section contains typos and incomplete/unclear phrasing (e.g., “applying a significance threshold of and …”, “p-valuie”), and the reporting of statistical terms is occasionally inconsistent or ambiguous (e.g., using “R” terminology in contexts where PERMANOVA is implied). In addition, several sentences in the Discussion use overly strong mechanistic wording (“immune evasion strategy”, “systemic collapse”) that reads as causal rather than transcriptome-based inference; rephrasing with more cautious language (“consistent with/suggestive of”) would improve precision. Overall, careful proofreading (ideally by a fluent English speaker) and tightening long sentences would substantially improve readability.

Author Response

Summary

Thank you very much for your thorough and insightful review of our manuscript. Your professional and detailed comments have been invaluable in helping us improve the quality and clarity of our work. We sincerely appreciate the time and expertise you have dedicated to this evaluation.

We have carefully considered all of your suggestions and have revised the manuscript accordingly. Below, we provide a point-by-point response to each of your comments, detailing the specific changes made. All modifications in the manuscript text have been highlighted for your convenience.

We believe that addressing your concerns has strengthened the paper, and we are grateful for your constructive input.

Point-by-point response to Comments and Suggestions

Comments 1:  

Lines 13–25:
The Simple Summary is clear but slightly promotional (“provides a scientific basis”). Consider rephrasing to emphasize what was demonstrated (virulence + time-course immune suppression) rather than claiming direct applicability.

Response 1：

Thank you for this objective and valuable suggestion. We agree that the original wording was overly promotional and lacked a precise description of our core findings. Following your advice, we have revised the Simple Summary as follows:

1. We removed a redundant promotional sentence (original lines 18–19).

2. We deleted the subjective adverb “highly.”

3. We rephrased the concluding statement to focus squarely on what was demonstrated in the study, namely the virulence of the nematode and the time-course suppression of key host immune pathways.

The revised text (now lines 13–25 in the marked-up manuscript) now more accurately and objectively summarizes our work. We believe these changes have significantly improved the tone and clarity of this section.

Comments 2:

Lines 26–43:
The Abstract is generally strong, but several claims (“sophisticated immunosuppressive strategy”, “critical nodes”) sound mechanistic while evidence is transcriptomic correlation. Consider softening to “consistent with immunosuppression” unless functional validation is provided.

Response 2：

We thank the reviewer for this critical and appropriate suggestion. We fully agree that, in the absence of direct functional validation, our language should more accurately reflect the correlative nature of the transcriptomic evidence. As suggested, we have softened the relevant statements throughout the Abstract (and the main text) to avoid overstating mechanistic conclusions.

Specifically, phrases such as “sophisticated immunosuppressive strategy” and “critical nodes” have been revised. These changes ensure our claims are aligned with the evidence provided by the transcriptomic data. The revised text can be found in lines 33–55 of the updated manuscript.

Comments 3:  

Lines 48–62:
The invasion background is detailed and engaging, but the paragraph is very dense with statistics and geographical spread. Consider condensing and moving some numbers (e.g., economic losses) to a single sentence to improve flow.

Response 3：

We appreciate this suggestion to improve the narrative flow. The detailed statistics and geographical spread have been streamlined into a single, concise sentence that retains the key contextual information without disrupting the flow of the introduction (see lines 70–76). 

Comments 4:  

Lines 67–77:
The insecticide resistance overview is useful, but it reads as a broad “catalog” of resistance cases. It would help to briefly state the key knowledge gap that this manuscript addresses (why EPNs and what is unknown about host immune responses to them) earlier in the Introduction.

Response 4：

Thank you. We have revised this section to reduce the catalog-like listing and instead provide a more concise synthesis (see lines 82–93).

Comments 5:  

Lines 72–77:
You cite “up to 28-fold resistance” to Bt (Btk). Please ensure this is properly supported and framed (strain, geography, assay type). Otherwise, readers may challenge the generality of the claim.

Response 5：

Thank you for highlighting this important point. We acknowledge the citation was imprecise. We have revised the statement to accurately reflect the specific conditions and findings reported in the original reference (see lines 93–96). To achieve a more seamless transition to the rationale for biocontrol, we have added a new sentence citing a recent field study on the efficacy of native Moroccan EPNs against T. absoluta(see lines 98–103).

Comments 6:  

Lines 78–95:
The biological control paragraph is informative, but some numbers (40–70% suppression; 87–95% reduction) need clearer context (study conditions, application method, and whether measured as infestation reduction vs mortality). This will prevent overinterpretation.

Response 6：

Thank you for this suggestion. We agree that additional context was needed. We have revised the paragraph to specify the study conditions, application methods, and efficacy metrics associated with the cited efficacy, as reported in the respective references (see lines 105-110).

Comments 7:  

Lines 96–109:
The “three barriers” framing is a nice structure, but the first barrier (“commercial strains biased toward soil pests”) is stated strongly. Consider citing broader comparative reviews or meta-analyses on foliar/endophytic targeting limitations of EPNs. 

Response 7：

Thank you for this suggestion. We have revised this statement to avoid an overly strong assertion. The text now clarifies that while commercial EPN products are registered for many pests, our survey of current product labels and literature indicates none are specifically promoted for T. absoluta control (see lines 126-132). This observation is based on our review of publicly available product information.

Comments 8: 

Lines 110–121:
The study rationale is clear, but novelty is oversold (“first transcriptomic analysis”). Please verify whether previous transcriptomic/immune gene expression studies exist for T. absoluta under EPN/symbiont bacterial challenge, and rephrase if needed.

Response 8：

We thank the reviewer for urging caution. To our knowledge, while transcriptomic studies on T. absoluta exist for other contexts (e.g., antennal function or response to entomopathogenic fungi), no prior work has investigated its transcriptional response to entomopathogenic nematode (EPN) infection.

Comments 9:

Lines 123–136:
Rearing details are adequate. However, since the colony has been maintained for >5 generations, please comment on whether this could reduce genetic variation and affect immune responses compared with field populations.

Response 9：

We thank the reviewer for raising this valid point. We acknowledge that long-term laboratory rearing can potentially reduce genetic variation. However, the T. absoluta colony used in this study was established from a field collection in 2025, and the experiment was conducted after approximately five generations of laboratory adaptation. This relatively short timeframe is unlikely to have led to a significant loss of genetic diversity relevant to baseline immune function. While traits like insecticide resistance may attenuate without selection pressure, we have no reason to believe that the fundamental immune pathways investigated here would be substantially altered.

Comments 10:

Lines 144–167:
Bioassay design is clearly described and replication is strong. Consider clarifying whether larvae were randomly assigned to treatments and whether the observer was blinded to reduce bias in survival scoring.

Response 10：

Thank you for this suggestion. We have revised the Methods section to explicitly state that larvae of the appropriate instar were randomly assigned​ to each treatment group. Furthermore, we have added more detailed description of the experimental procedure. The relevant text has been updated in lines 193–197.

Comments 11:

Lines 153–162:
The replicate structure is good, but pooling technical replicates after a log-rank homogeneity test may still mask batch effects. Please report replicate-level variation (e.g., LC50 confidence intervals per biological replicate) or justify pooling more explicitly.

Response 11：

Thanks for this insightful statistical point. We agree that transparency regarding replicate-level variation is important. Our decision to pool homogenous technical replicates was primarily statistical: with only 15 larvae per technical replicate, small sample sizes are susceptible to random sampling error, potentially masking the true concentration-response relationship and increasing the risk of false-negative results. Pooling three homogeneous replicates increased the effective sample size to 45 larvae per concentration per biological replicate, yielding more robust and stable estimates of mortality for probit analysis.

To address this concern directly, we have now included a new Supplementary Table S1​ that presents the replicate-level data, allowing readers to assess the variation across biological replicates. 

Comments 12:

Lines 168–174:
RNA samples are pooled (10 larvae/sample). Pooling increases practicality but reduces the ability to quantify individual variability. This limitation should be acknowledged in the Discussion.

Response 12：

Thank you for this important point. We fully agree and acknowledge that pooling RNA samples, while practical for obtaining sufficient material, limits our ability to assess inter-individual transcriptional variation. This limitation has been explicitly stated in the Discussion section (see lines 701-702).

Comments 13:

Lines 204–228:
The RNA-seq pipeline is detailed, but there are typos/unclear thresholds (“applying a significance threshold of and …”; “p-valuie”). Please correct and explicitly state the FDR cutoff (e.g., FDR < 0.05) and the log2FC threshold used for DEGs.

Response 13：

Thank you for your careful reading. We have corrected the typo “p-valuie” to “p-value” and completed the phrase regarding the significance threshold. The text now explicitly states “applying a significance threshold of p-value ≤ 0.05 and |log₂FC| ≥ 1” for defining differentially expressed genes (DEGs), where |log₂FC| ≥ 1 corresponds to the previously mentioned “minimum fold change (FC) of 2”. These edits have been made in the specified lines to ensure clarity and accuracy (see lines 274-278).

Comments 14:

Lines 252–257:
Data access is provided, which is good. Consider adding a brief note on what is deposited (raw fastq, count matrices, metadata) to make reuse easier.

Response 14：

Thank you for this suggestion to improve data accessibility. We have updated the data availability statement accordingly. The raw sequencing reads (FASTQ files) have been deposited. Additionally, the processed count matrices are provided as a Supplementary File for convenient reuse.

Comments 15:

Lines 266–276:
The Results description of cadaver color change implies a mechanistic link to symbiotic bacteria. If this is inferential (not directly tested), consider stating it as “consistent with” rather than causal.

Response 15：

Thank you for this important reminder. We agree and have revised the description accordingly. The text now avoids implying a direct causal link and instead presents the observed color change as a phenomenon “consistent with” the activity of the symbiotic bacteria (see lines 334-336).

Comments 16:

Lines 277–280 (Figure 1):

Figure 1A (Kaplan-Meier survival):please report n per treatment directly in the caption or panel. Figure 1B images are useful, but a brief note on what the darkening indicates (inference vs. evidence) would prevent overinterpretation.

Response 16：

Thank you for these suggestions. We have updated the figure caption accordingly. The caption now explicitly states the number of larvae (n) used in each treatment group for Figure 1A. For Figure 1B, we have added a note clarifying that the color change of the cadavers is an observation consistent with melanization to avoid overinterpretation.   Comments 17:

Lines 281–309:
The Results are readable, but the narrative includes interpretive phrasing (“systemic physiological collapse”). This belongs more in Discussion unless you provide direct physiological measures beyond transcriptome patterns.

Response 17：

We agree that the phrase “systemic physiological collapse” is interpretive and extends beyond the transcriptomic evidence presented. Following your advice, we have removed this phrasing from the Results section (see lines 371-372).

Comments 18:

Lines 314–332:
GO analysis is presented with many numbers in-line (gene counts, enrichment scores). Consider summarizing key patterns (direction + category) and moving detailed counts to a supplementary table for readability.

Response 18：

Thank you for this suggestion to improve readability. We have revised this section accordingly. The detailed statistical data (e.g., specific gene counts and enrichment scores) have been moved. In the main text (lines 377–391), we now present a concise summary focusing on the key biological patterns and directions of enrichment observed.

Comments 19:

Lines 345–377:
The GO narrative is thorough but repetitive across time points. You can reduce text by focusing on the transition from early cuticle/metabolic effects to later immune pathway enrichment (e.g., Toll-related terms).

Response 19：

Thank you for this suggestion to improve the narrative flow. We have substantially rewritten this section to reduce redundancy and to explicitly highlight the dynamic transition​ in biological processes over time. The revised text (lines 415–455) now focuses more clearly on this temporal progression, particularly regarding immune-related functions.

Comments 20:

Lines 371–383:
Statements like “infection … strongly downregulated metabolic processes” are plausible but can be overgeneralized. Consider emphasizing that these are transcript-level changes and may reflect host shutdown, pathogen manipulation, or both.

Response 20：

Thank you for this important nuance. We have deleted the statements in this paragraph to avoid overgeneralization (see lines 455-456).

Comments 21:

Lines 384–390 (Figure 3):

Figure 3 is information-rich but visually crowded-many GO terms are angled and hard to read. Consider reducing to fewer top terms or moving full lists to Supplementary and keeping only the strongest signals in the main figure.

Response 21：

Thank you for this feedback on the figure’s readability. We aimed to present a comprehensive overview of the significantly enriched GO terms, as the infection elicited broad and substantial transcriptional changes. To balance specificity with informativeness, we have already selected terms with high enrichment scores and used mid-level (e.g., GO level 2–3) descriptors. We acknowledge that the number of terms can make the figure appear dense. While we believe maintaining a broad overview in the main figure is valuable for readers, we will carefully re-examine the layout and labeling to improve clarity. We will also ensure the figure is provided at high resolution to facilitate legibility.

Comments 22:

Lines 503–510 (Figure 5):

Panel A (Mfuzz clusters) is visually appealing but dense; individual trajectories are hard to distinguish. Consider showing cluster centroids more prominently or adding the number of genes per cluster on the panel. Panel C heatmaps are readable, but label size (gene IDs) may be small in print-please confirm legibility at final PDF scale.

Response 22：

 Thank you for these constructive suggestions to improve figure clarity. In response:

1. For Panel A, we have now added the number of genes per cluster​ directly onto the figure to provide immediate context on the scale of each expression pattern.
2. Regarding Panel C, we will ensure all text elements remain fully legible at the final publication scale in the PDF.

Comments 23:

Lines 547–583:
The immune gene interpretation is interesting but reads as if the mechanism is resolved (“critical immune evasion strategy”). Without functional assays (RNAi, enzyme activity, melanization/encapsulation quantification), please tone down certainty or propose these as mechanistic hypotheses for follow-up.

Response 23：  

We agree that definitive mechanistic conclusions require functional validation. We have revised this section to tone down the language, replacing overly certain claims with phrasing that presents the observed patterns (see revised text in lines 658–661, 665–666, and 671–673).

Comments 24:

Lines 572–582:
The phrasing “terminal immune response coinciding with systemic collapse” is strong. Consider rephrasing to a more cautious interpretation tied to the observed time-course and mortality pattern.

Response 24：  

Thank you for this correction. We agree that the original phrasing was overly conclusive. We have revised the text to present a more cautious interpretation (see lines 665–666 and 671–673). This revision addresses a pattern of overstatement that we have worked to correct throughout the manuscript in response to your feedback.

Comments 25:

Lines 680–692:
The Conclusions are well written but overstate mechanistic certainty (“precise interference … mechanistic explanation”). Reframe as “transcriptomic evidence consistent with suppression of…” and highlight the need for functional validation (which you already mention) as a key limitation.

Response 25：

 Thank you for this final and crucial suggestion. We have revised the Conclusions section accordingly. As shown in the manuscript (Lines 789–809), we have reframed the language, replacing phrases such as “precise interference … mechanistic explanation” with formulations like “transcriptomic evidence consistent with suppression of…”, thereby more accurately reflecting the correlative nature of our evidence. Furthermore, we have more clearly emphasized the need for functional validation as a key limitation and a central direction for future work.

Additional clarifications

Once again, we would like to express our sincere gratitude for your time, expertise, and the thoroughly constructive review. Your insightful comments have been invaluable in strengthening the scientific rigor, clarity, and overall quality of our manuscript. We greatly appreciate your contribution to our work.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

Thank you for your carefully addressed all major and minor comments raised during the previous review.
The revised manuscript shows substantial improvement in methodological clarity, statistical transparency, data interpretation, and overall coherence between results, discussion, and conclusions.
The transcriptomic analyses are now better contextualized biologically, figures and tables are clearer and internally consistent, and the discussion is more balanced and appropriately framed.

I have no further major concerns and consider the manuscript suitable for publication in its present form.

Kind regards, 

R