A Nemertean-Derived Peptide Toxin Exhibits Oral Insecticidal Activity Against Spodoptera litura

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents an interesting and potentially valuable study on the identification of peptide toxins with oral insecticidal activity against Spodoptera litura. The comparative screening of seven recombinant insecticidal candidates, followed by oral evaluation and subsequent whole-plant testing of the lead candidate, provides a logical experimental progression from candidate selection to a more application-oriented assessment. In particular, the observation that injection potency does not necessarily translate into oral efficacy is relevant for the development of peptide-based foliar bioinsecticides. The extension of the study to foliar application also adds a useful biotechnological and applied dimension. Nevertheless, I believe that several aspects require clarification and, in particular, that the molecular characterization of the recombinant products should be strengthened before firm conclusions can be drawn regarding their comparative biological activities.

Oral insecticidal activity of recombinant nemertide α-1 has already been demonstrated against Mamestra brassicae, Myzus persicae, and Acyrthosiphon pisum, as the authors themselves acknowledge. Therefore, the present study does not establish oral activity of A1 for the first time. Rather, its novelty lies in demonstrating oral efficacy against S. litura and in evaluating the efficacy of foliar-application A1. The manuscript should consistently distinguish these novel contributions. Several molecules included in the screening panel (nemertide α-1, the PI1a/GNA fusion protein, and the R9Q variant of U1-AGTX-Ta1b) were already described and biologically characterized, including in oral insecticidal assays. It is therefore unclear whether any of the seven peptide sequences/variants tested here were newly designed in the present study or whether the novelty resides primarily in their recombinant production and comparative evaluation against S. litura. The authors should clearly distinguish previously reported peptide sequences and engineered variants from any constructs that are genuinely novel in the present study.

Overall, the molecular characterization of the recombinant candidates appears insufficient to unequivocally establish the identity. Direct mass-spectrometric characterization would substantially strengthen the study. Intact-mass spectrometry would be particularly valuable to determine whether the purified products have the expected molecular masses, while LC-MS/MS could provide sequence-level confirmation of their identity. The authors correctly state that recombinant products initially characterized by SDS-PAGE and Western blotting should be further verified by LC-MS/MS to establish sequence identity and molecular integrity (Lines 408–410). I agree with this assessment, particularly given the substantial discrepancies between the theoretical and apparent molecular masses reported for these small peptides. Direct mass-spectrometric characterization would substantially strengthen the study.

2.1. Recombinant Production of Seven Insecticidal Candidates

Lines 153–161; Figure 1A–B
The assignment of the purified products relies primarily on SDS-PAGE migration and anti-His Western blotting. While anti-His immunoreactivity confirms the presence of C-terminally His-tagged species, it does not by itself establish the sequence identity or molecular integrity of the recombinant peptides.

Lines 163–167; Figure 1A–B
All six peptide toxins migrate at apparent molecular masses substantially higher than their calculated values. For example, A1 has a theoretical molecular mass of approximately 4.1 kDa, including the C-terminal 6×His tag, whereas its principal band migrates at approximately 10 kDa. Although anomalous SDS-PAGE migration of small disulfide-rich peptides is plausible, this substantial discrepancy further highlights the need for direct molecular confirmation of the recombinant products.

Lines 168–173; Figure 1A–B
The PIGA preparation raises an additional concern because several electrophoretically distinct species are detected by the anti-His antibody. The authors appropriately describe this preparation as heterogeneous; however, it remains unclear which band corresponds to intact PI1a/GNA and what the additional His-positive species represent. This is particularly relevant for interpretation of the subsequent bioassays, since the lack of activity of PIGA could reflect the biological properties of the intact fusion protein, but could also be influenced by degradation, incomplete processing, or other heterogeneous recombinant products.

4.4. High-Cell-Density Fermentation of A1

The reported A1 titer appears to be an indirect estimate based on total protein determination combined with SDS-PAGE densitometry rather than a direct quantitative measurement of A1. The methodology and limitations associated with this estimate should therefore be more clearly described, particularly because the reported titer is subsequently used to support the potential scalability of recombinant A1 production.

Lines 628–632
The authors state that total extracellular protein was quantified using the BCA assay and that the relative abundance of A1 was estimated by SDS-PAGE densitometry using purified A1 as a reference. Please provide further details on how these two measurements were combined to calculate the A1 titer and clarify the uncertainty associated with this indirect quantitative approach.

4.5. Chromatographic Purification of Recombinant Products

The purification and quantification of the recombinant preparations require further clarification. Importantly, the A1 preparation used in the initial leaf-disc feeding assay and the preparation used in the subsequent whole-plant foliar assay were obtained using different purification workflows. The fermentation-derived A1 used for the whole-plant assay underwent an additional cation-exchange polishing step. Therefore, the two A1 preparations are not directly equivalent in terms of purification procedure and potentially in terms of purity/composition. The authors should clearly acknowledge this difference and provide purity and molecular characterization data for both preparations to establish their comparability.

Lines 662–664
The authors state that the material intended for the initial injection and leaf-disc feeding assays was desalted, concentrated using 3-kDa MWCO devices, and lyophilized. Please clarify how the concentration of each recombinant candidate was determined after these steps and before preparation of the bioassay solutions. In particular, it should be specified whether the reported concentrations represent total protein or quantitatively determined target peptide.

Lines 674–675
The authors state that protein concentration was measured for the final A1 preparation and that electrophoretic purity was assessed by SDS-PAGE. Please specify the method used to determine the final protein concentration and report, if available, a quantitative estimate of the purity of the final A1 preparation.

Discussion

Lines 410–416: The justification provided for not performing additional mass-spectrometric identification is not fully convincing. Previous validation of a similar secretory expression and nickel-affinity purification workflow for related peptide toxins does not establish the identity or molecular integrity of the recombinant products generated in the present study. Likewise, anti-His immunoreactivity and correspondence between biological activity and previously reported properties cannot substitute for direct molecular identification. This limitation is particularly important because the biological activity, potency comparisons, and subsequent selection of A1 as the lead candidate all depend on the identity and integrity of these preparations.

Lines 417–424: This issue is particularly relevant for the three preparations that showed no detectable injection mortality (C1, S1, and PIGA), despite previously reported biological activities. The authors themselves acknowledge that differences between the recombinant and previously tested products could contribute to these results. Without direct molecular characterization, however, it is difficult to distinguish genuine species- or stage-specific lack of activity from differences in the recombinant preparations. The negative result obtained with the heterogeneous PIGA preparation should therefore be interpreted with particular caution.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study “A Nemertean-derived Peptide Toxin Exhibits Oral Insecticidal Activity against Spodoptera litura” presents a promising and innovative idea, demonstrating coherence among the hypothesis, experimental design, results, and conclusions. However, there are some weaknesses that need to be reviewed.

In the introduction, the authors report, on page 2, the need for control agents with new modes of action. On page 11, they report that A1 exhibits a different intoxication phenotype. I would like them to explain this, given that the reported possible mode of action for A1 is inducing convulsions and tremors, which is similar to that of other chemical agents.

Regarding the experimental design, I see an important problem: the oral screening does not include all seven candidates. The authors generated seven candidates, but only those that showed detectable toxicity via injection underwent oral testing. Consequently, candidates A1, D1, L1, and T1 were orally analyzed, while the others (C1, S1, and PIGA) were not tested orally; I consider this contradictory, given that the authors state that activity via injection does not predict oral activity, although they use injection-based results to select the candidates for oral evaluation. I recommend that the remaining candidates, C1, S1, and PIGA, be tested orally.

Another important point to consider is the concerns about the concentration of A1. To achieve about 92% of mortality, 8 µL/µL of A1 is required, which is equivalent to 8 mg/mL, which is high from the perspective of bioinsecticide development. What is the economic viability? The authors themselves note that they did not assess this; however, in my view, this issue should be evaluated, as a biologically active candidate does not necessarily translate into a viable agricultural insecticide.

The study shows mortality after feeding on pepper leaves treated with A1, demonstrating insecticidal activity associated with oral exposure; however, it does not demonstrate gastrointestinal stability, passage through the intestine, absorption, and arrival at the target. The authors acknowledge that they did not perform stability or transport experiments. The authors suggest that the greater oral stability may be due to its origin in the mucus of L. longissimus. Although the hypothesis is interesting, it is speculative. Investigating the stability and resistance of A1 in the intestinal tract is essential to provide higher robustness to the findings.

The authors were quite cautious and acknowledge that the mechanism of action of A1 is not well established. Based on previous studies, they infer a possible action on NaV receptors. However, if the study aims to propose A1 as an insecticide candidate, its mode of action should be investigated, perhaps through electrophysiological assays of A1 on S. litura NaV channels and an evaluation of the dose-response relationship. Furthermore, assessing the toxicity of A1 to non-target insects, such as bees and wasps, is essential, given that A1 is a candidate insecticide for agricultural use and this is a very important concern actually.

Furthermore, what is the residual effect of A1? Given its agricultural application, it is important to demonstrate this effect to determine how long A1 remains active on the leaves. Assays demonstrating stability regarding rainfall, UV light, temperature, humidity, and leaf-surface microbiota, as well as the leaf half-life, would be essential. A comparison with commercial insecticides is also necessary to assess the relative activity of A1.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have adequately addressed the previous comments. The revised manuscript now clearly defines the novelty of the study, appropriately acknowledges the limitations associated with the absence of direct mass-spectrometric characterization of the recombinant products, and provides a more cautious interpretation of the negative bioactivity results. The purification and protein quantification procedures have also been clarified. I consider that the manuscript has been substantially improved and that my major concerns have been satisfactorily addressed. I therefore recommend acceptance in its present form.

Reviewer 2 Report

Comments and Suggestions for Authors

Considering the changes made to the manuscript and the points clarified in the cover letter, I recommend the manuscript for publication in its current form.