Gene Silencing of laccase 1 Induced by Double-Stranded RNA in Callosobruchus maculatus (Fabricius 1775) (Coleoptera: Chrysomelidae) Suggests RNAi as a Potential New Biotechnological Tool for Bruchid’s Control
Perumal Vivekanandhan
Round 1
Reviewer 1 Report
Reviewer
Comments to the authors
The manuscript title is “First report of gene silencing induced by double-stranded RNA in Callosobruchus maculatus (Fabricius 1775) (Coleoptera: Chrysomelidae): insight of a new biotechnological tool for bruchids’ control” by Arnaud Segers et al., In this study, (i) the identification of all core mechanism proteins involved in the RNAi pathway (i.e. Dicer-2, Argonaute-2, and R2D2) in the genome of C. maculatus, (ii) the ORF identification of the Lac1 sequence in C. maculatus, and (iii) the evaluation of gene expression level after micro-injection of dsRNA to adults using Real-Time quantitative PCR analyses are evaluated (RT-qPCR). This is the first report of RNAi in a bruchid species, providing new insight into a potential method of control for these global pests. I saw merit in this research after carefully reviewing this manuscript, but it contains some grammatical and typo errors. As a result, I propose major revision.
Major Points
1. The manuscript title hard to follow so please revise it.
2. Line 17: typo errors
3. The abstract looks like text book I suggest the author please clearly write objectives, methodology and results. The current form not in scientific standard.
4. Line 45: Thanks?
5. Line 58: Insect means all the insects? Or insect pest?
6. Line 60: what is nt?
7. Line 71: cite more recent references
8. Cite reference
9. Line 97: cite following recent references it will support this statement.
a. Naqqash, M.N., Gökçe, A., Bakhsh, A. and Salim, M., 2016. Insecticide resistance and its molecular basis in urban insect pests. Parasitology research, 115(4), pp.1363-1373.
b. Karaağaç, S.U., 2012. Insecticide resistance. IntechOpen.
c. Vivekanandhan, P., Thendralmanikandan, A., Kweka, E.J. and Mahande, A.M., 2021. Resistance to temephos in Anopheles stephensi larvae is associated with increased cytochrome P450 and α-esterase genes overexpression. International Journal of Tropical Insect Science, 41(4), pp.2543-2548.
10. Line 100: cite recent and relevant references it will support your statement;
a. Ojebode, M.E., Olaiya, C.O., Adegbite, A.E., Karigidi, K.O. and Ale, T.O., 2016. Efficacy of some plant extracts as storage protectants against Callosobruchus maculatus. J. Biotechnol. Biomater, 6, p.217.
b. Vivekanandhan, P., Senthil-Nathan, S. and Shivakumar, M.S., 2018. Larvicidal, pupicidal and adult smoke toxic effects of Acanthospermum hispidum (DC) leaf crude extracts against mosquito vectors. Physiological and Molecular Plant Pathology, 101, pp.156-162.
c. Nattudurai, G., Baskar, K., Paulraj, M.G., Islam, V.I.H., Ignacimuthu, S. and Duraipandiyan, V., 2017. Toxic effect of Atalantia monophylla essential oil on Callosobruchus maculatus and Sitophilus oryzae. Environmental Science and Pollution Research, 24(2), pp.1619-1629.
d. Vivekanandhan, P., Usha-Raja-Nanthini, A., Valli, G. and Subramanian Shivakumar, M., 2020. Comparative efficacy of Eucalyptus globulus (Labill) hydrodistilled essential oil and temephos as mosquito larvicide. Natural product research, 34(18), pp.2626-2629.
e. Idoko, J.E. and Ileke, K.D., 2020. Comparative evaluation of insecticidal properties of essential oils of some selected botanicals as bio-pesticides against Cowpea bruchid, Callosobruchus maculatus (Fabricius)[Coleoptera: Chrysomelidae]. Bulletin of the National Research Centre, 44(1), pp.1-7.
f. Pratheeba, T., Vivekanandhan, P., Faeza, A.N. and Natarajan, D., 2019. Chemical constituents and larvicidal efficacy of Naringi crenulata (Rutaceae) plant extracts and bioassay guided fractions against Culex quinquefasciatus mosquito (Diptera: Culicidae). Biocatalysis and Agricultural Biotechnology, 19, p.101137.
g. Vivekanandhan, P., Venkatesan, R., Ramkumar, G., Karthi, S., Senthil-Nathan, S. and Shivakumar, M.S., 2018. Comparative analysis of major mosquito vectors response to seed-derived essential oil and seed pod-derived extract from Acacia nilotica. International Journal of Environmental Research and Public Health, 15(2), p.388.
11. Line 104: Zhang et al., (2018) typo error
12. The entire introduction needs to reframe like following order otherwise reader will confused
i. Background of the study
ii. Insect pest and their problems
iii. Chemical and plant extract mediated control methods and their problems
iv. Alternative techniques
v. Talk about your areas
vi. Finally, objectives
13. Line 123: typo error Zhao et al. 2015; line 126: El sayadi et al. 2016
- In the materials and methods section, please clearly mention the source of insect pests and maintain details.
- Methodological components such as text books are currently not in scientific standards, so they must be carefully revised.
- The figure 3 is not clear; please provide quality images.
- There are some technical and typographical errors in the results section; please double-check them.
- The discussion section is not scientifically correct; please discuss with more relevant ligatures.
- The conclusion section needs to be expanded because it is currently incomplete.
Author Response
Dear Editors, dear Reviewers,
First of all, we would like to thank you for your proofreading and for all your constructive remarks and suggestions which have reoriented the writing of this manuscript towards the most relevant elements with regard to the problematic addressed.
As you will notice in this new manuscript, answers provided to reviewers’ suggestions have induced the rewriting of the manuscript (especially the introduction and the discussion) by fitting better sentences structure, restructuring the development of the information, and a revision of English has been performed according to the received corrections. New informative elements and references have also been included for a more comprehensive reading. All changes made to the original text have been highlighted in blue using the “Track Changes” function in the new manuscript as requested by Editors.
We hope that all your remarks could correctly be responded to, and that your expectations have been met in this new manuscript. We remain available to answer your suggestions regarding to these modifications. The application of each remark is point to point explained here below.
Thank you for considering it for review. We appreciate your time and look forward to hearing from you at your earliest convenience.
On behalf of all authors,
Arnaud Segers
Author Response File: 
Author Response.docx
Reviewer 2 Report
This paper demonstrated the operability of RNA interference technology in Callosobruchus maculatus and firstly reported the successful interference of gene in C. maculatus. The experimental method of this study is detailed and the data is sufficient. Although the interference of laccase 1 gene did not affect the survival of C. maculatus, this study supports that RNAi can be used as a potential method to control the bruchid in the future. However, there are still many problems with the article, requiring major revisions before publication.
Comments:
1. Keywords: “RNAi” and “RNA interference” repeats, suggested to delete one; suggest to replace “Bruchidae” and “Bruchinae” with “Callosobruchus maculatus”; suggest to “laccase”
2. L87: “report” change to “reported”
3. L97: “NTO”-suggest to define this abbreviation
4. L104-“Zhang et al., (2018)”- add the reference number “[36]”, and unify the citation format for references below
5. Table1,2,3,4: modify all tables in the text in three-line format
6. L184: “Lac1 .” change to “Lac1.”
7. L246-252: This description of “reference genes” should appear in the Introduction, not Materials and Methods
8. L278: “ddH20” change to “ddH2O”
9. Figure 3: enlarge font and bold research targets “Cmac-Ago-2” and “Cmac-Dcr-2”. Why not phylogenetic analysis of ' R2D2'?
10. Figure 4: “R2D2”, “Dicer-2”, and “Argonaute-2” in the figure legend whether need be italic, please unify
11. Table 4: Why compared the Lac1 dsRNA treatment group with the control group instead of the gfp dsRNA group
12. Figure 8: Please redraw the figure and add the horizontal and vertical titles and units
13. Figure 9: Add the horizontal and vertical coordinate units of the chart. “The LT50s were not reached in all treatments”-Why was the survival only counted to the seventh day? Since LT50 was used as the standard, the number of days should not be limited.
14. L442: “treatment (blue)” change to “treatment (blue).”
15. Appendix A: Whether all three treatments (Lac1 dsRNA treatment, gfp dsRNA group, Elution buffer control) were injected with 400 ml volume. L267-269-“different dsRNA exposures (no dsRNA, 100 ng, 200 ng, 400 ng of microinjected Lac1 dsRNA; and 100 ng, 200 ng, 400 ng of microinjected gfp dsRNA)”: Is this a pre-experiment? Where are the results?
16. References: Latin names of species and genera should be italicized. For example, L620-“Agrilus Planipennis”.
Author Response
Dear Editors, dear Reviewers,
First of all, we would like to thank you for your proofreading and for all your constructive remarks and suggestions which have reoriented the writing of this manuscript towards the most relevant elements with regard to the problematic addressed.
As you will notice in this new manuscript, answers provided to reviewers’ suggestions have induced the rewriting of the manuscript (especially the introduction and the discussion) by fitting better sentences structure, restructuring the development of the information, and a revision of English has been performed according to the received corrections. New informative elements and references have also been included for a more comprehensive reading. All changes made to the original text have been highlighted in blue using the “Track Changes” function in the new manuscript as requested by Editors.
We hope that all your remarks could correctly be responded to, and that your expectations have been met in this new manuscript. We remain available to answer your suggestions regarding to these modifications. The application of each remark is point to point explained here below.
Thank you for considering it for review. We appreciate your time and look forward to hearing from you at your earliest convenience.
On behalf of all authors,
Arnaud Segers
Author Response File: Author Response.docx
Round 2
Reviewer 1 Report
The author implements all of the reviewer's suggestions. As a result, the current form of the manuscript is publishable.
Reviewer 2 Report
accept
