Locust cGAS-like Receptors Recognize Derivatives of a Gypsy Retrotransposon to Synergize with RNAi Against Viral Invasion
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsLi et al. investigate the role of a transposon in Locusta migratoria’s immune response against Acrididae reovirus (ARV). They show that infection with ARV increases LmGypsy expression and that LmGypsy knockdown affects virus replication and insect survival. In addition, authors investigate the interaction of LmGypsy with vDNA and viral siRNA production. I generally found the paper interesting, well-written, with clear figures and well-explained methods.
There are a few aspects that would benefit from clarification or revision:
- In the discussion, the authors wrote: ‘A key finding of our work is that ARV infection triggers the selective derepression of LmGypsy, which in turn generates viral DNA (vDNA) via its encoded reverse transcriptase (RT) activity.’
I am not convinced the findings point directly to this. Authors effectively show that LmGypsy expression is affected by AZT treatment, suggesting to me that vDNA plays a role in LmGypsy expression (Figure 1h). Although they show the quantity of vDNA is lower when LmGypsy is knocked down, this effect can also be indirect (Figure 2). I suggest rephrasing this section accordingly and including some discussion about the possible effect of the regulatory functions of the transposon (e.g. could silencing Gypsy element silence a neighbouring gene?).
- I find the results on viral DNA very interesting, especially that the vDNA was produced solely by RdRP and was sufficient to generate viral siRNAs. It would be great to visualise exactly where in the ARV genome these vsiRNAs are produced from. Is it only RdRP in the vDNA-treated group? Does it look the same for the infection group?
- I found Figure 5 rather confusing and difficult to understand regarding the comparison groups. On one hand, authors examine the effect of ARV infection during AZT treatment, showing that viral infection increases cGas expression independently of RT. This shows that cGAS expression is independent of vDNA production. They also show that LmGypsy knockdown reduces expression of some CGAS and Sting. I think these two results should be separated, as the experiments can’t be evaluated together because of different controls. I think this part should be clarified in the results and elsewhere as necessary. For instance, in the results, it is not clear to me how the presented results suggest this :
“Both treatments abolished the ARV-induced upregulation of LmcGAS1, LmcGAS2, LmcGAS4, and LmSting (all p < 0.05; Fig. 5b), whereas LmcGAS3 and LmMab21 expression remained unchanged (all p > 0.05; Fig. 5b).”
Minor comments:
The supplementary table only contains sequences and not the primers, as indicated in the manuscript
Caption Figure 5b says 5 replicates, but it looks like there is more than that. I think this is meant for Figure 5a?
Discussion, second paragraph, first sentence is repeated twice.
Methods, AZT treatment part is written in bold.
Figure 2C: Error bars are not visible for the last bar plot.
Author Response
Dear Reviewer 1,
We would like to thank you for your thoughtful and constructive comments on our manuscript. Your insights have helped us substantially improve the clarity, rigor, and nuance of our work. Below we provide a point-by-point response to all your comments and describe the corresponding revisions made to the manuscript.
Major comments:
Comment 1: In the discussion, the authors wrote: ‘A key finding of our work is that ARV infection triggers the selective derepression of LmGypsy, which in turn generates viral DNA (vDNA) via its encoded reverse transcriptase (RT) activity.’ I am not convinced the findings point directly to this. Authors effectively show that LmGypsy expression is affected by AZT treatment, suggesting to me that vDNA plays a role in LmGypsy expression (Figure 1h). Although they show the quantity of vDNA is lower when LmGypsy is knocked down, this effect can also be indirect (Figure 2). I suggest rephrasing this section accordingly and including some discussion about the possible effect of the regulatory functions of the transposon (e.g. could silencing Gypsy element silence a neighbouring gene?).
Response 1: We appreciate this thoughtful comment. We agree that the original wording implied a direct causal chain that our data do not fully resolve. We have therefore revised the Discussion to better reflect the strength of the evidence and to incorporate the regulatory complexity you highlighted.
The key finding now reads: “A key finding of our work is that ARV infection triggers the selective de-repression of LmGypsy, and that its activity is essential for the accumulation of ARV-derived vDNA.” We have also explicitly acknowledged that our experiments do not formally distinguish direct from indirect mechanisms and have discussed the possibility that the effect of AZT treatment points to a more complex regulatory interplay. Most importantly, following your suggestion, we have added a discussion of the potential regulatory functions of the retrotransposon: “Moreover, because long terminal repeat retrotransposons (LTR) frequently harbor enhancer and promoter activities capable of modulating neighboring genes48-50, silencing LmGypsy may indirectly affect host factors involved in antiviral defense or nucleic acid metabolism.”
We believe these revisions appropriately temper the central claim and address the concern that knockdown of LmGypsy could exert indirect effects through neighboring gene regulation. We thank you for this constructive guidance, which has substantially improved the nuance of our interpretation.
Comment 2: I find the results on viral DNA very interesting, especially that the vDNA was produced solely by RdRP and was sufficient to generate viral siRNAs. It would be great to visualise exactly where in the ARV genome these vsiRNAs are produced from. Is it only RdRP in the vDNA-treated group? Does it look the same for the infection group?
Response 2: We sincerely appreciate your encouraging comment and your interest in our vDNA results. The suggestion to visualize the distribution of vsiRNAs across the ARV genome and to compare the vDNA-treated and infection groups is an excellent one.
Following the reviewer’s suggestion, we attempted to map vsiRNA reads across the ARV genome in both the vDNA-treated and ARV-infected groups. However, although vsiRNAs could be reliably detected, the overall mapped read coverage was relatively limited and unevenly distributed, which prevented robust visualization of reproducible genome-wide vsiRNA profiles suitable for publication-quality presentation.
Importantly, our current data consistently demonstrate that ARV-derived vDNA is sufficient to induce virus-specific siRNA production and activate RNAi-mediated antiviral immunity. We agree that determining whether vsiRNAs are preferentially enriched within the RdRP region, and whether similar patterns occur between the vDNA-treated and infection groups, represents an important question for future investigation.
Comment 3: I found Figure 5 rather confusing and difficult to understand regarding the comparison groups. On one hand, authors examine the effect of ARV infection during AZT treatment, showing that viral infection increases cGas expression independently of RT. This shows that cGAS expression is independent of vDNA production. They also show that LmGypsy knockdown reduces expression of some CGAS and Sting. I think these two results should be separated, as the experiments can’t be evaluated together because of different controls. I think this part should be clarified in the results and elsewhere as necessary. For instance, in the results, it is not clear to me how the presented results suggest this : “Both treatments abolished the ARV-induced upregulation of LmcGAS1, LmcGAS2, LmcGAS4, and LmSting (all p < 0.05; Fig. 5b), whereas LmcGAS3 and LmMab21 expression remained unchanged (all p > 0.05; Fig. 5b).”
Response 3: Thank you for this helpful comment. We agree that the original presentation of Figure 5 was insufficiently clear regarding the comparison groups and the logical relationship between the different experiments. We have now reorganized this section to separate the functional characterization of the cGAS-like pathway from the connection to LmGypsy.
Specifically, the revised Figure 5 first demonstrates that ARV infection induces LmcGAS1–4 and LmSting expression (Fig. 5a) and then confirms their antiviral roles through RNAi silencing (Fig. 5b). In a separate panel (Fig. 5c), we address whether LmGypsy depletion affects the virus-induced expression of these genes. We have removed the sentence that referred to “both treatments” and no longer conflate the AZT and LmGypsy knockdown data in a single interpretation. The text now clearly presents each comparison group independently, and we hope this makes the logic much easier to follow.
Minor comments:
Comment 1: The supplementary table only contains sequences and not the primers, as indicated in the manuscript
Response 1: Thank you for pointing this out. The omission was due to an earlier upload error. We have now uploaded a corrected version of the supplementary table that includes the complete primer sequences.
Comment 2: Caption Figure 5b says 5 replicates, but it looks like there is more than that. I think this is meant for Figure 5a?
Response 2: Thank you for catching this discrepancy and for raising this important point. It prompted us to clarify a detail that was missing from the original figure legend.
You are right that Figure 5b displays more than five data points. The five biological replicates are genuine, but each replicate consisted of 15 nymphs. Because individual locust nymphs are relatively large, we pooled five nymphs per sample for RNA extraction and qPCR to ensure sufficient and uniform material. This pooling yielded three parallel measurements within each biological replicate, giving a total of 15 data points across the five replicates.
We have now revised the figure legend to make this explicit. It reads: “Data are shown as mean ± SEM from five biological replicates; each replicate is represented by three measurements obtained from pooled samples of five nymphs.” We hope this clarification resolves the confusion, and we appreciate your careful reading of the figure.
Comment 3: Discussion, second paragraph, first sentence is repeated twice.
Response 3: We have removed the duplicated sentence in the second paragraph of the Discussion section.
Comment 4: Methods, AZT treatment part is written in bold.
Response 4: The unintended bold formatting in the “AZT treatment” subsection of the Methods has been corrected.
Comment 5: Figure 2C: Error bars are not visible for the last bar plot.
Response 5: The figure has been adjusted so that the error bars are now clearly visible.
We are grateful for the time and expertise you have dedicated to reviewing our manuscript. We believe the revisions have significantly strengthened the clarity and rigor of our work, and we hope the revised manuscript is now acceptable for publication in your journal.
Sincerely,
Yao Xu and co-authors,
Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China.
14 May 2026
Reviewer 2 Report
Comments and Suggestions for AuthorsThe manuscript titled “Locust cGAS-like receptors recognize derivatives of a Gypsy retrotransposon to synergize with RNAi against viral invasion” investigates the antiviral mechanism in Locusta migratoria that Acrididae reovirus (ARV) infection triggers the selective de-repression of the Gypsy retrotransposon LmGypsy.
However, the following aspects should be considered to improve the quality of the manuscript.
1. Simple summary is missing.
2. “reovirus” is not necessary to be italicized.
3. The full names of abbreviations should be clearly explained upon their first appearance, either in abstract or main text. For instance, LINE-1, cGAS–STING, etc..
4. A recent review article regarding RNAi and cGAS-STING pathways (doi /10.3389/finsc.2025.1749008) could be included in the introduction.
5. Primers information for qPCR detection and dsRNA synthesis could not be found in supplementary documents. Instead, it is suggested to include as a regular table in M&M.
6. In the discussion on the evolutionary perspective, elaborate more on how the specific activation of LmGypsy rather than multiple TEs might be related to the host's genomic stability and evolutionary advantages.
7. Include more detailed comparisons with similar mechanisms in other organisms or discuss potential evolutionary pressures that have shaped this fine-tuning of TE activation.
8. This manuscript delves into the antiviral mechanisms, particularly focusing on RNAi and the cGAS-STING pathways. I am curious about how other antiviral immune pathways, such as the Toll and IMD pathways, might be associated with LmGypsy activity.
Author Response
Dear Reviewer 2,
We sincerely thank the reviewer for the careful evaluation of our manuscript entitled “Locust cGAS-like receptors recognize derivatives of a Gypsy retrotransposon to synergize with RNAi against viral invasion”. We greatly appreciate the reviewer’s insightful comments and constructive suggestions, which have substantially improved the clarity, rigor, and overall quality of the manuscript.
In response to the reviewer’s comments, we have carefully revised the manuscript throughout. The major revisions include the addition of a Simple Summary section, clarification of abbreviation definitions, refinement of virus nomenclature formatting, incorporation of additional literature discussing RNAi and cGAS-STING pathways, inclusion of primer information in the main manuscript, and expansion of the evolutionary discussion regarding selective TE activation and host–TE coevolution.
Detailed point-by-point responses are provided below.
Comment 1: Simple summary is missing.
Response 1: Thank you for this helpful suggestion. We have now added a concise “Simple Summary” section to the revised manuscript to improve accessibility for readers outside the immediate field and to highlight the major biological significance of our findings.
Comment 2: “reovirus” is not necessary to be italicized.
Response 2: Thank you for this careful observation. We agree that the term “reovirus” should not be italicized when referring to the virus group or family in a general sense, and we have corrected these instances throughout the revised manuscript.
However, in the case of Acrididae reovirus (ARV), the italicization was intentionally retained because it refers to a specific viral species rather than the reovirus group in general. We have carefully checked the manuscript to ensure that italics are now used consistently and only for formal virus species names.
Comment 3: The full names of abbreviations should be clearly explained upon their first appearance, either in abstract or main text. For instance, LINE-1, cGAS–STING, etc..
Response 3: We agree completely. We have carefully reviewed the entire manuscript (including the Abstract, main text, and figure legends) and have ensured that every abbreviation is spelled out at its first occurrence. Specific examples:
“LINE‑1” is now introduced as “long interspersed nuclear element‑1 (LINE‑1)”
“cGAS–STING” is introduced as “cyclic GMP‑AMP synthase (cGAS) – stimulator of interferon genes (STING)”
Other abbreviations (e.g., ARV, dsRNA, RT, RdRP, vDNA, vsiRNA) have been similarly defined at first use. We have also checked that no abbreviation appears without prior definition.
Comment 4: A recent review article regarding RNAi and cGAS-STING pathways (doi /10.3389/finsc.2025.1749008) could be included in the introduction.
Response 4: We thank the reviewer for this valuable recommendation. The suggested review article has now been incorporated into the Introduction to strengthen the discussion of the interplay between RNAi and cGAS-like antiviral pathways in insects.
Comment 5: Primers information for qPCR detection and dsRNA synthesis could not be found in supplementary documents. Instead, it is suggested to include as a regular table in M&M.
Response 5: Thank you for this suggestion. We agree that providing primer information in the main manuscript improves clarity and accessibility. We have now added a dedicated table containing all primer sequences used for qPCR detection and dsRNA synthesis in the Materials and Methods section of the revised manuscript.
Comment 6: In the discussion on the evolutionary perspective, elaborate more on how the specific activation of LmGypsy rather than multiple TEs might be related to the host's genomic stability and evolutionary advantages.
Response 6: We thank the reviewer for this insightful suggestion. In the revised manuscript, we have substantially expanded the Discussion to address the evolutionary implications of selective LmGypsy activation during ARV infection. We now discuss how the host preferentially induces a single retrotransposon family, rather than triggering broad derepression of multiple TEs. This selectivity may reflect an adaptive strategy. It allows the host to amplify antiviral immunity while safeguarding genome integrity. We also address the risks associated with uncontrolled TE mobilization, including insertional mutagenesis and excessive immune stimulation. We propose that selective activation of LmGypsy minimizes these harmful effects, yet still harnesses the immunological benefits of TE-derived nucleic acids. In addition, we broaden the discussion of host–TE coevolution. We suggest that LmGypsy may represent a partially domesticated, immune-responsive retrotransposon. It could have been co-opted as a regulated amplifier of antiviral defense in L. migratoria. These additions have been incorporated into the evolutionary perspective section of the Discussion.
Comment 7: Include more detailed comparisons with similar mechanisms in other organisms or discuss potential evolutionary pressures that have shaped this fine-tuning of TE activation.
Response 7: We thank the reviewer for this insightful suggestion. In the revised manuscript, we have substantially expanded the Discussion to provide a more detailed comparison of TE activation patterns across organisms and to discuss the evolutionary pressures that likely shape the fine‑tuned regulation of TE activation observed in our study.
Specifically, we now contrast the broad derepression of multiple retroelement families (e.g., LINE‑1 and SINEs) frequently reported in mammals during viral infection with the more selective, context‑dependent TE activation documented in insects such as Drosophila, mosquitoes, and silkworms. Our finding that ARV infection preferentially induces a specific LTR retrotransposon, LmGypsy, rather than causing widespread TE activation, fits this insect pattern. We then discuss the potential evolutionary trade‑off underlying such selectivity: restricted activation of a limited TE subset may provide sufficient immunostimulatory nucleic acids to amplify antiviral signaling through conserved pathways (e.g., cGAS/cGLR) while minimizing the risks of insertional mutagenesis, genome instability, and excessive immune activation. We further propose that recurrent viral infections may have promoted the evolutionary retention of immunostimulatory TE families, and that hosts have co‑evolved regulatory mechanisms to constrain their mobilization, with LmGypsy potentially representing a retrotransposon that has become functionally integrated into the antiviral defense of L. migratoria.
We believe these additions address the reviewer’s concern and significantly strengthen the evolutionary framing of our study.
Comment 8: This manuscript delves into the antiviral mechanisms, particularly focusing on RNAi and the cGAS-STING pathways. I am curious about how other antiviral immune pathways, such as the Toll and IMD pathways, might be associated with LmGypsy activity.
Response 8: We thank the reviewer for this thoughtful question. In the initial phase of this study, we indeed explored the potential contribution of several canonical insect immune pathways by knocking down key signaling components, including Myd88 (Toll pathway) and Domeless (JAK/STAT pathway). As reported in our results (please refer to Fig. 3c; data for Myd88 and Domeless), silencing these genes did not lead to a detectable change in ARV replication, suggesting that the Toll and JAK/STAT pathways are not major determinants of the outcome of ARV infection in locusts. It should be noted, however, that these experiments measured viral load rather than LmGypsy expression or retrotransposon activity per se; therefore, we cannot formally exclude the possibility that the Toll and/or JAK/STAT pathways modulate LmGypsy independently of their effects on overall viral replication.
We agree that additional immune pathways, particularly the IMD pathway, may also contribute to antiviral responses or interact with TE-mediated immune regulation. However, the potential involvement of the IMD pathway in LmGypsy-dependent antiviral immunity was not directly investigated in the present study and will require further functional analyses in future work.
We sincerely thank the reviewer again for the careful evaluation and constructive comments, which have substantially improved the manuscript.
Sincerely,
Yao Xu and co-authors,
Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China.
14 May 2026
Reviewer 3 Report
Comments and Suggestions for AuthorsThis manuscript showed the interplay between transposable elements (TEs) and innate immunity in the migratory locust (Locusta migratoria). The authors demonstrate that the LmGypsy retrotransposon serves a dual role in antiviral defense against the Locust Reovirus (ARV) through two distinct pathways: the amplification of the RNAi response via virus-derived DNA (vDNA) and the activation of a cGAS-STING-like signaling pathway.
This work is significant as it extends our understanding of "genomic parasites" being co-opted for host defense in non-model hemimetabolous insects. The experimental design is robust, and the mechanistic insights into the "double-layered" defense strategy are compelling.
The study identifies four cGAS-like receptors, but the specific functional division between them remains unclear, particularly regarding LmcGAS3, which showed a different expression pattern. A more detailed discussion or preliminary data on the tissue-specific expression or ligand preference of these different receptors would add significant value to the paper.
The authors noted that vDNA can persist for the duration of the host's life. This raises an intriguing question: is there evidence of this vDNA or the associated vsiRNAs being transmitted to the germline? A brief discussion on the potential for TE-mediated transgenerational antiviral immunity would be a forward-looking addition to the Discussion section.
Author Response
Dear Reviewer 3,
We sincerely thank the reviewer for the careful evaluation of our manuscript entitled “Locust cGAS-like receptors recognize derivatives of a Gypsy retrotransposon to synergize with RNAi against viral invasion”. We greatly appreciate the reviewer’s insightful comments and constructive suggestions, which have significantly improved the quality, depth, and clarity of the manuscript.
In response to the reviewer’s comments, we performed additional experiments and substantially revised the manuscript. Major revisions include new tissue-specific expression analyses of LmcGAS receptors following ARV infection, incorporation of new experimental evidence regarding the absence of germline transmission of ARV-derived vDNA, and expansion of the Discussion to better address functional specialization of cGAS-like receptors and the implications of persistent vDNA for antiviral immune memory.
Detailed point-by-point responses are provided below.
Comment 1: The study identifies four cGAS-like receptors, but the specific functional division between them remains unclear, particularly regarding LmcGAS3, which showed a different expression pattern. A more detailed discussion or preliminary data on the tissue-specific expression or ligand preference of these different receptors would add significant value to the paper.
Response 1: Thank you for this insightful suggestion. To further investigate the potential functional divergence among the four LmcGAS receptors, we performed additional tissue-specific expression analyses following ARV infection. Eight tissues were dissected at 3 dpi, and the expression patterns of LmcGAS1–4 were examined by qPCR. The results revealed clear spatial differences among the receptors. LmcGAS1 was predominantly expressed in the midgut, whereas LmcGAS2 showed highest expression in the fat body and midgut. LmcGAS3 displayed a distinct pattern, with strongest expression in the fat body and relatively high expression in the malpighian tubule. In contrast, LmcGAS4 was highly enriched in the midgut and also showed elevated expression in the hemolymph. These findings have now been incorporated into the Results section and discussed in the revised Discussion as additional evidence supporting functional specialization among LmcGAS receptors.
Comment 2: The authors noted that vDNA can persist for the duration of the host's life. This raises an intriguing question: is there evidence of this vDNA or the associated vsiRNAs being transmitted to the germline? A brief discussion on the potential for TE-mediated transgenerational antiviral immunity would be a forward-looking addition to the Discussion section.
Response 2: We sincerely thank the reviewer for this insightful and forward-looking suggestion. The possibility that virus-derived DNA (vDNA) or associated vsiRNAs might access the germline and contribute to transgenerational antiviral immunity is indeed a fascinating question that deserves careful consideration. In direct response to this comment, we have now included new experimental data in the revised manuscript. Specifically, we examined vDNA accumulation in both reproductive tissues (ovaries and testes) and eggs laid by ARV-infected adults after mating. Using qPCR, we were unable to detect ARV-derived vDNA in any of these samples. These negative findings have been added to the Results section and are clearly stated as follows: “To further assess whether ARV-derived vDNA could access the germline, we examined vDNA accumulation in reproductive tissues and offspring derived from ARV-infected locusts. No detectable vDNA signal was observed in either ovaries or testes following ARV infection. Consistently, vDNA was also undetectable in eggs produced by infected adults after mating. These findings suggest that ARV-derived vDNA is unlikely to be transmitted through the germline in L. migratoria under our experimental conditions.”
Accordingly, we revised the Discussion to avoid implying potential germline transmission of vDNA in locusts. Rather than speculating on heritable antiviral responses, we now emphasize that persistent vDNA is more likely associated with sustained antiviral defense or prolonged immune priming within infected individuals during the host’s lifetime. The revised text in the Discussion now reads: “In Drosophila melanogaster, virus-derived DNA has been reported to participate in forms of immune memory and, in some cases, has been associated with heritable antiviral responses. However, in our study, we did not detect ARV-derived vDNA in either ovaries, testes, or eggs produced by infected adults, arguing against efficient germline transmission in L. migratoria under the conditions tested. These findings suggest that the persistence of vDNA in locusts is more likely linked to somatic antiviral defense rather than transgenerational inheritance.”
We sincerely thank the reviewer again for the constructive comments and valuable suggestions, which substantially strengthened both the mechanistic depth and evolutionary perspective of this study.
Sincerely,
Yao Xu and co-authors,
Jiangsu Key Laboratory of Sericultural and Animal Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang 212100, China.
14 May 2026
Round 2
Reviewer 2 Report
Comments and Suggestions for AuthorsMy previous concerns have been address accordingly. I have no other comments.
