PONE-D-24-01103Sprayable RNAi for silencing of important genes to manage red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)PLOS ONE

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This project was financially supported King Abdulaziz City for Science and Technology (KACST), Saudi Arabia through the Project No. 11BIO1803-06

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Reviewers' comments:

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The manuscript presents new information relevant to the management of red palm weevil. However, it was difficult to interpret the text occasionally. The reviewer has recommendations at the following pages and figures.

Page 20 “Pupal duration recorded was lower in SCP and actin treatment were 12.20±0.20 and 12.00±0.32 days, while it was significantly higher in the control (14.00±0.32).” This sentence is difficult to understand and needs to be rewritten.

Page 23 Please define the meaning of “reduction in transcript levels” Do you mean transcription rate? And what are “pronounced metamorphosis effects”? Are these measured by differences in mortality rates? What is an “efficient effect” This entire section should be rewritten more clearly.

Figure 1 “vector as were named as” possibly should be “vector and were designated as”

Figures 3 -5 What do the letters a and b indicate?

Figure 4 what does (A) and (B) indicate that is not also designated by the color difference?

Table 1 seems to be missing.

Reviewer #2: RNA interference (RNAi) is a powerful tool for knocking down gene function in pest control. In the present study, the authors targeted to silence of several important genes in red palm weevil and obtained promising results. The topic covered in the article is interesting and falls within the scope of the Journal.

The manuscript is well-written and organized.

The experimental design looks good. The study looks promising and adequately replicated

The findings are robust and statistically ok.

Please find the attached manuscript with my comments. I provided several suggestions.

Reviewer #3: I noticed that the article entitled Sprayable RNAi for silencing of important genes to manage red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae) was submitted. However, the author's experimental content limitation, which did not fully resolve my concerns. Furthermore, through further reading, I have developed some new insights. Therefore, I recommend rejection for publication in its current form due to the following major concerns:

1.Why choose 3 rd and 5 th instar ? This issue has not been clearly analyzed in either the Materials and Methods or the results sections.

2.The manuscript presents some initial attempts to explore the research topic, which is commendable. However, these efforts appear to be in the early stages of exploration and lack depth. The experimental contents are too lilmited, only survival of instar, weight change in instar, pupal duration and adult emergence. The experiment should be added, such as the effect of egg hatchability, adult mating ability and offspring.

Reviewer #4: The paper titled "Sprayable RNAi for silencing of important genes to manage red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)" addresses a significant issue related to the sustainable control of the Red Palm Weevil. This insect pest poses a considerable economic threat due to the damage it causes to decorative palms and, more critically, the threat it poses to date palms, particularly in countries where the economy is heavily dependent on them. The authors propose the utilization of dsRNAi in the diet of RPW larvae, which holds promise as a future biopesticide. Several recent studies have also explored similar approaches involving RNAi targeting selected genes such as the olfactory co-receptor, cuticle proteins, and reproductive-related genes. Encouraging and advancing research in this direction will accelerate the development of an effective biopesticide capable of effectively managing the pest population. However, reviewing the results was somewhat challenging as I couldn't locate any of the four tables mentioned in the paper, unless I overlooked them. Before receiving clarification on the tables, I would like to express some comments:

-In section 3.2, regarding the effects of dsRNAs on survival, I would appreciate more details about this experiment. I would prefer a standard analysis such as the Kaplan-Meier method rather than comparing means, as I find it insufficiently informative.

-Regarding the weight gain becarefull to the Y-axis of the Figure 3, please try to harmonize the police and the linear scale. There are 3-7 instar larvea in the RPW, the larval stage can last until 2 months , so maybe if you made a follow-up of more than 6 days the results will be more pronounced.

-Are the selected genes capable of giving direct effects on the weight gain ? I think that they are more involved in some physiological and morphological traits that will not necessarily be reflected by the larvae weight.

- The figures for pupal duration require revision, as there is a title "Treatment" that should be removed. Upon reviewing the figures, it is apparent that pupal duration is reduced. Does this imply that emergence occurred earlier? If so, it suggests that the dsRNA treatment shortened pupal duration, resulting in the emergence of adults in a shorter time frame !! However, the data on emergence indicate a significant and promising effect across almost all genes.

A figure illustrating the relative expression of the genes is absent. Such a figure is crucial for comprehending the underexpression of the genes. Additionally, statistical analysis for this section is lacking. While I recognize that this section primarily aims to confirm the reduction of gene expression, it should be presented in a more comprehensive manner to adequately convey the findings.

Please ensure that the acknowledgment section is corrected and that any typos present in the manuscript are addressed.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Submitted filename: PONE-D-24-01103_R1.pdf

Sprayable RNAi for silencing of important genes to manage red palm weevil, Rhynchophorus ferrugineus (Coleoptera: Curculionidae)

PONE-D-24-01103R1

Dear Dr. Muhammad Naeem Sattar,

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Kind regards,

Nafiu Bala Sanda, PhD

Academic Editor

PLOS ONE

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