RNA Interference Targeting MaCht-2 Induces Severe Molting Defects and Lethality in Monochamus alternatus

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors The manuscript by Fang et al. aims to establish the role of the MaCht-2 gene as a critical regulator of molting and to evaluate its potential as a target for RNAi-based control of Monochamus alternatus. While the silencing of genes involved in chitin formation in M. alternatus has been previously demonstrated, the exact functional role of MaCht-2 had not yet been assessed in detail. The authors combine phenotypic and molecular monitoring to confirm that this target gene induces lethal and sublethal effects upon dsRNA injection across different instars. Furthermore, electron microscopy results clearly demonstrate structural alterations in the integument of the treated specimens.   My primary concern involves the conclusion—reiterated throughout the discussion—that this gene exhibits stronger and more consistent RNAi-induced effects than other chitin-related genes in this species. To strengthen this claim, it would be more effective to cite the specific parameters used in previous studies and directly compare those outcomes with the current findings. Additionally, the conclusions should explicitly state that the reported behavioral effects (e.g., walking ability) did not derive from a formal experimental design and were not supported by statistical analysis.   Minor comment: Please ensure that all scientific names are italicized throughout the manuscript.  

Author Response

Comments 1: My primary concern involves the conclusion—reiterated throughout the discussion—that this gene exhibits stronger and more consistent RNAi-induced effects than other chitin-related genes in this species. To strengthen this claim, it would be more effective to cite the specific parameters used in previous studies and directly compare those outcomes with the current findings. Additionally, the conclusions should explicitly state that the reported behavioral effects (e.g., walking ability) did not derive from a formal experimental design and were not supported by statistical analysis.

Response 1: Thank you for this insightful comment. We agree that the comparative claims should be better substantiated. As suggested, we have revised the Discussion and Conclusions to include a direct, parameter-based comparison with our previously published data on MaCht-3 and MaIDGF-4 (Fang et al., 2025), obtained under comparable experimental conditions. Specifically, we now highlight that in third-instar larvae, MaCht-2 dsRNA injection induced markedly higher larval mortality (54.55%–84.62%) than that reported for the other two genes, and that severe malformation and locomotor impairment were consistently more frequent. We also now explicitly acknowledge that locomotion was assessed qualitatively and not through a formal quantitative assay. These revisions have been made in the Discussion (Section 4.3, paragraph 3; Section 4.4, paragraph 2) and in the Conclusions.

Revised text in page 13,Section 4.3 (paragraph 3), line 494:
When compared with our previously reported data on MaCht-3 and MaIDGF-4, which were obtained under the same laboratory conditions and with dsRNA prepared by identical methods (Fang et al., 2025), the phenotypic differences are evident. For third-instar larvae, injection of 5 µg dsMaCht-3 produced no larval mortality and limited malformation, whereas the same dose of dsMaCht-2 caused 54.55% larval mortality. Similarly, dsMaIDGF-4 at 5 µg and 10 µg resulted in no larval death in third-instar larvae or produced mortality only inconsistently across doses, in contrast to the clear dose-dependent lethality observed for MaCht-2. Severe malformation rates and the incidence of complete locomotor impairment were also consistently higher after MaCht-2 silencing than after knockdown of the two previously characterized genes. Although a formal statistical meta-analysis across studies is precluded by differences in experimental design and sample sizes, the overall pattern supports the conclusion that MaCht-2 is a comparatively high-impact RNAi target within the chitin metabolic network of M. alternatus.

Revised text in page 14, Section 4.4 (paragraph 2),line 526:
It should be noted that locomotion was assessed by visual observation and descriptive recording, not through a dedicated quantitative behavioral assay. Therefore, the reported impairment of walking ability should be regarded as a qualitative indication of functional compromise rather than a statistically validated behavioral endpoint. Future studies would benefit from incorporating structured locomotor assays to quantify these effects more rigorously.

Revised text in Conclusions:
Compared with previously characterized chitin-related genes in M. alternatus for which comparable injection-based RNAi data are available, MaCht-2 showed higher larval mortality and a greater proportion of severe morphological defects, supporting its potential value for target prioritization in RNAi-based control. The observed locomotion defects were based on qualitative assessment, and formal behavioral quantification is needed to confirm the extent of functional impairment. Further studies should also evaluate alternative dsRNA delivery strategies and assess the practical applicability of MaCht-2 targeting under more realistic conditions.

Comments 2: Minor comment: Please ensure that all scientific names are italicized throughout the manuscript.

Response 2: Thank you for noting this. We have carefully reviewed the entire manuscript and corrected all instances where scientific names were not properly italicized, including the title, Simple Summary, Abstract, main text, tables, figures, and references.

Reviewer 2 Report

Comments and Suggestions for Authors

Fang et al. present a systematic experimental design, sufficient data, and generally standardized writing. However, there are still some issues or areas for improvement in terms of scientific logic, experimental details, accuracy of expression, and formatting. My detailed review comments are as follows:

1. In Figure 4, only dsGFP was used as a negative control, without including an injection of pure water or a non-injected group as a blank control. This makes it impossible to distinguish the effect of the injection procedure itself on development. It is recommended to improve this. Furthermore, gene expression levels were only examined at 12 h, 24 h, and 48 h, yet the phenotypic effects persisted into the adult stage. There is a lack of expression test at later time points (e.g., around pupation or before eclosion). Consequently, it remains unclear whether the observed phenotypes result from sustained gene silencing or from a lagged effect of early transient silencing. It is recommended to add additional monitoring time points for RNAi efficacy.
2. The manuscript does not indicate whether the differences in mortality rates between different dosage groups are statistically significant.
3. "CBM14" is mentioned in Figure 2 but is not explained in the main text.
4. The formatting of the references is inconsistent, with some citations using abbreviated journal titles and others using full titles.

Author Response

Comments 1: In Figure 4, only dsGFP was used as a negative control, without including an injection of pure water or a non-injected group as a blank control. This makes it impossible to distinguish the effect of the injection procedure itself on development. It is recommended to improve this. Furthermore, gene expression levels were only examined at 12 h, 24 h, and 48 h, yet the phenotypic effects persisted into the adult stage. There is a lack of expression test at later time points (e.g., around pupation or before eclosion). Consequently, it remains unclear whether the observed phenotypes result from sustained gene silencing or from a lagged effect of early transient silencing. It is recommended to add additional monitoring time points for RNAi efficacy.

Response 1:  Thank you for these valuable suggestions. Regarding the first concern, we fully acknowledge the importance of distinguishing the effect of the injection procedure from gene-specific silencing. In this study, we used dsGFP as a control following the widely adopted convention in insect RNAi research, including in M. alternatus (Sheng et al., 2023; Ye et al., 2025), where dsGFP-injected insects serve as the standard negative control to account for both injection wounding and dsRNA exposure. All dsGFP-injected individuals in our study developed normally without observable molting defects or mortality, indicating that the injection procedure itself did not cause detectable developmental abnormalities. Nevertheless, we agree that the inclusion of a non-injected or buffer-only group would further strengthen experimental rigor, and we have now added this point as a recommendation for future studies.

Regarding the second concern, we agree that monitoring of expression levels at later developmental time points would provide a more complete picture of silencing persistence. Due to limitations in the number of available insects and the destructive nature of sampling for qPCR (which prevents tracking the same individuals through to phenotypic manifestation), our expression analysis was performed on parallel cohorts at 12, 24, and 48 h post-injection. We have now explicitly acknowledged this limitation and suggested that future studies incorporate later time-point expression monitoring. These revisions have been made in the Discussion (Section 4.2, paragraph 3).

Revised text in page 12,Section 4.2 (paragraph 3),line 429:
It should be noted that the current study did not include a non-injected or buffer-only blank control group, following the common practice of using dsGFP-injected insects as the negative control in M. alternatus RNAi studies. Although all dsGFP-injected individuals developed normally, the inclusion of an additional blank control in future experiments would help to further isolate the potential effects of the injection procedure. In addition, because gene expression was monitored only up to 48 h post-injection, whereas phenotypic defects were observed through later developmental stages, it remains unclear whether the adult-stage phenotypes resulted from sustained transcript suppression or from a cascade triggered by early transient silencing. Future studies with larger sample sizes and extended expression monitoring at later time points, such as during the prepupal or pre-eclosion period, would be valuable to clarify this relationship.

Comments 2:  The manuscript does not indicate whether the differences in mortality rates between different dosage groups are statistically significant.

Response 2:  Thank you for highlighting this important point. The primary objective of our RNAi phenotypic experiments was to describe the range and nature of developmental defects induced by MaCht-2 silencing, and the sample sizes per treatment group (minimum of five individuals) were designed accordingly for qualitative phenotypic characterization. For this reason, formal statistical comparisons of mortality rates among dosage groups were not conducted. We have now explicitly stated this limitation in the Discussion (Section 4.3, paragraph 3). In addition, we have added a note to the Methods (Section 2.5) to clarify the primary purpose of the sample sizes used.

Comments 3:  "CBM14" is mentioned in Figure 2 but is not explained in the main text.

Response 3:  Thank you for pointing out this omission. CBM14 refers to Carbohydrate-Binding Module family 14, a type of chitin-binding domain. These modules were identified alongside ChtBD2 domains in the domain architecture of MaCht-2. We have now added an explanation of CBM14 in the Results section where Figure 2 is described (Section 3.1, paragraph 2).

Revised text in page 6,Section 3.1 (paragraph 2, revised sentence),line 235:
The deduced protein was predicted to comprise 8,274 amino acids, with a theoretical molecular weight of 718,610.43 Da and a predicted isoelectric point of 4.60. Three-dimensional structure prediction further showed that MaCht-2 contained conserved GH18 chitinase-like regions and chitin-binding modules, including ChtBD2 and CBM14 (Carbohydrate-Binding Module family 14), consistent with the structural characteristics of insect Cht2 proteins (Figure 2).

Comments 4:  The formatting of the references is inconsistent, with some citations using abbreviated journal titles and others using full titles.

Response 4:  Thank you for noting this formatting issue. We have carefully reviewed the entire reference list and standardized all journal titles to the abbreviated format according to the Insects journal guidelines. All references now use consistent abbreviated journal names.