Rapid Detection of A282S Mutation in the RDL1 Gene of Rice Stem Borer via the Mutation-Specific LAMP Technique

Round 1

Reviewer 1 Report

In this manuscript, authors established a LAMP technique for detecting the A282S mutation in CsRDL1 by the naked eye in short time. It is interesting and innovative work. Meanwhile, the manuscript is clearly organized, and related to a topic of Agronomy. Nevertheless, some minor points should be revised before it is accepted. Therefore, I think it is acceptable after a minor revision.

 

Minor points:

1.     Line 15: Add the full name if the abbreviation appears in the abstract for the first time. It should be “A282S (A2’S)”.

2.    Line 58-59: The Latin name of Plutella xylostella, Heliothis virescens and Nilaparvata lugens appear should be intact.

3.     Line 69, 87, 128, 140, 192 and 242: The unit “℃” are inconsistent. Please check through the full text to avoid similar mistakes.

4.     Line 106-110: Pay attention to the grammar modification in this paper.

5.     Line 141: Please add a space before “min”.

6.     Line 158, 166, 175: Cs in “CsRDL1” should be intact if it is the abbreviation of Chilo suppressalis.

Author Response

Response to reviewer 1

In this manuscript, authors established a LAMP technique for detecting the A282S mutation in CsRDL1 by the naked eye in short time. It is interesting and innovative work. Meanwhile, the manuscript is clearly organized, and related to a topic of Agronomy. Nevertheless, some minor points should be revised before it is accepted. Therefore, I think it is acceptable after a minor revision.

 

Minor points:

1. Line 15: Add the full name if the abbreviation appears in the abstract for the first time. It should be “A282S (A2’S)”.

Response: Thank you for your comments. The sentence “As well knowledge……A2’S mutation of GABA receptor RDL subunit’’ has been revised as “As well knowledge……A282S mutation of GABA receptor RDL subunit’’.

 

2. Line 58-59: The Latin name of Plutella xylostella, Heliothis virescens and Nilaparvata lugens appear should be intact.

Response: Thank you for your comments. The Latin name of Plutella xylostella have been intact as Plutella xylostella (Linnaeus). The Latin name of Heliothis virescens (Fabricius) has been been revised as Heliothis virescens Fabricius. The Latin name of Nilaparvata lugens (Stål) is already intact. In addition, The Latin name of Plutella xylostella, Heliothis virescens and Nilaparvata lugens have been italicised as Plutella xylostella, Heliothis virescens and Nilaparvata lugens respectively. In addition, other Latin names have been corrected throughout the revised manuscript.

 

3. Line 69, 87, 128, 140, 192 and 242: The unit “℃” are inconsistent. Please check through the full text to avoid similar mistakes.

Response: Thanks for your comment. The units including “℃” in line 20, 69, 87, 128, 140, 192 and 242 have been modified consistently in the revised manuscript.

 

4. Line 106-110: Pay attention to the grammar modification in this paper.

Response: Thank you for your comment. The sentence “Note, nucleotides in the box and underlined are the artificially mismatched and mutant nucleotide, respectively……The italic nucleotides are F1c and B1c, and the underlined nucleotides are F2 and B2, respectively’’ has been revised as “Note, nucleotides in the box and underlined are the artificially mismatched and mutated from the the genome sequence of CsRDL1, respectively……F1c and B1c were indicated in italics, F2 and B2 were indicated in bold, respectively’’.

 

5. Line 141: Please add a space before “min”.

Response: Sorry for this mistake. The space before “min” in line 141 has been added in the revised manuscript.

 

6. Line 158, 166, 175: Cs in “CsRDL1” should be intact if it is the abbreviation of Chilo

Response: Thank you for your suggestion. We don't know if you want to describe Cs in “CsRDL1” should be italicized instead of intact, if so, we have corrected it in the revised manuscript.

Author Response File: Author Response.docx

Reviewer 2 Report

Comment: Minor revision

This is an interesting study that has potential application in the identification of mutation using the LAMP technique. However, the strength of the manuscript is greatly affected by grammatical mistakes and readability. The author should revise the manuscript more carefully and scientifically. Some sentences are very difficult to read. Please, revise the manuscript carefully before submission. The overall introduction and discussion need to be improved.

 

Line 14: Please, replace the word “plenty” with another appropriate word.

Line 23: Add “the” before the word “LAMP”.

Line 25: Please, replace the word “feasibility and easily” with “feasible and easy”.

Line 26: Add “a” before the word “short time”.

Line 28:  Remove “(words count: 250)”.

Line 50: Add “a” before the “large base”. Replace the word “serious” with “severe”. Please, specify what do you mean by “large base”.

Line 50-52: The sentence “Its…..and nutrients” needs to rewrite. Suggested “Its larvae drill into the rice stem and destroy the host plant's tissue, which leads the rice plant to die due to lack of water and nutrients.”

Line 54: Replace the “to noting” with “noting”. Remove “of” after the word “different”.

Line 55: Replace the “level” with “levels”, and “shows” with “show”.

Line 58: Add “the” before the “RDL”.

Line 62: Replace “insecticides target is given priority” with “insecticide targets prioritizes”.

Line 71-72: Relace “prospect in the field” with “prospects in field”.

Line 80: Add “the” before the “RDL”.

Line 114: Replace the “to noting” with “noting”.

Line 118: Replace the “nucleotide acids” with “nucleotides”.

Line 124: Add “the” before the “LAMP”.

Line 137: Remove the word “both”.

Line 138: Replace the “reaction” with “reactions”.

Line 139: Add “the” before the “LAMP”. Replace the “on” with “at”.

Line 141: Replace the “condition” with “conditions”.

Line 151: Replace the “was” with “were”.

Line 160: Replace the “Design the specific primer for LAMP reaction” with “Designing specific primers for the LAMP reaction”.

Line 162: Replace the “was” with “were”, and “chose” with “chosen”.

Line 168: Remove “from”.

Line 169: add “the” before the word “reaction”.

Line 173: Replace the “screen” with “screening”.

Line 191: Add “the” before the “LAMP”.

Line 211: Replace the “pest” with “pests”.

Line 213-215: Rewrite the sentence “However…….recent years.” for clear understanding.

Line 217: Replace the “level” with “levels”.

Line 223: Replace the “were” with “was”.

Line 224: Add “a” before the word “template”.

Line 225-226: Rewrite the sentence “Meanwhile, the successful ………specificity as well.” Suggested “Meanwhile, the successful mutation-detection of C. suppressalis samples from the field also confirmed its specificity.”

Line 228: Add “the” before the “LAMP”.

Line 229: Remove “is” after the word “technique”.

Line 230: Add “the” before the “LAMP”.

Line 231: Replace the “chose” with “chosen”.

Line 235: Replace “the contamination while open” with “contamination while opening”.”.

Line 240-241: Rewrite the sentence “Both reaction ………….. in field”. Suggested “Both the reaction time and temperature of the LAMP reaction were also optimized, making the LAMP technique more convenient and suitable for usage in the field.”

Line 247: Add “the” before the “LAMP”.

Line 254: Add “the” before the “LAMP”.

Line 258: Add “the” before the “field”.

Line 225-226: Rewrite the sentence “The establishement……………… C. suppressalis.” Suggested “The establishment of the LAMP technique can provide a new means for monitoring the resistance of C. suppressalis to phenylpyrazole insecticides and a reference and basis for managing C. suppressalis.”

Author Response

Dear Editor Darcy Dai,

 

Thank you very much for your email and the reviewers’ comments on our manuscript entitled “Rapid detection of A282S mutation in the RDL1 gene of rice stem borer via the mutation-specific LAMP technique” (Manuscript ID: agronomy-2165886). We have read carefully the manuscript again and made a thorough revision according to the reviewer’s comments, and hope you will find these corrections in red satisfied and accept our manuscript for publication in Agronomy. Enclosed file is a response letter detailing our answers to the reviewers’ comments on the manuscript. We appreciate the reviewers’ comments and the resulting clarity has improved the manuscript.

 

With my best regards,

Chun-Qing Zhao

Response to reviewer 2

Comment: Minor revision

This is an interesting study that has potential application in the identification of mutation using the LAMP technique. However, the strength of the manuscript is greatly affected by grammatical mistakes and readability. The author should revise the manuscript more carefully and scientifically. Some sentences are very difficult to read. Please, revise the manuscript carefully before submission. The overall introduction and discussion need to be improved.

 

1. Line 14: Please, replace the word “plenty” with another appropriate word.

Response: Thank you for your advice. The “plenty” in line 14 has been revised as “various”.

 

2. Line 23: Add “the” before the word “LAMP”.

Response: Thank you for your advice. The sentence “It is the first report of LAMP technique applied……” has been been revised as “It is the first report of the LAMP technique applied……”.

 

3. Line 25: Please, replace the word “feasibility and easily” with “feasible and easy”.

Response: Thank you for your advice. The words “feasibility and easily” in line 25 have been revised as “feasible and easy”.

 

4. Line 26: Add “a” before the word “short time”.

Response: Thank you for your advice. The sentence “According to our results……short time directly……” has been revised as “According to our results……a short time directly……”.

 

5. Line 28:  Remove “(words count: 250)”.

Response: Thank you for your advice. “(words count: 250)” in line 28 has been removed.

 

6. Line 50: Add “a” before the “large base”. Replace the word “serious” with “severe”. Please, specify what do you mean by “large base”.

Response: Thank you for your advice. The sentence “and has the characteristics of large base has been revised as “and has the characteristics of a large base”. And the meaning of “large base” is that C. suppressalis widely distributed in the world, but it’s difficult to prevent, so the rice stem borer C. suppressalis has the characteristics of a large base”.

 

7. Line 50-52: The sentence “Its…..and nutrients” needs to rewrite. Suggested “Its larvae drill into the rice stem and destroy the host plant's tissue, which leads the rice plant to die due to lack of water and nutrients.”

Response: Thank you for your comment, some of your comments have been accepted and revised. The sentence “Its……and nutrients” has been been revised as “Its larvae drill into the rice stem and destroy the rice tissue, which leads the rice to die due to lack of water and nutrients”.

 

8. Line 54: Replace the “to noting” with “noting”. Remove “of” after the word “different”.

Response: Thank you for your advice. The sentence “However, it is worth to noting that C. suppressalis has developed different of resistance level to these insecticides” has been been revised as “However, it is worth noting that C. suppressalis has developed different resistance levels to these insecticides”.

 

9. Line 55: Replace the “level” with “levels”, and “shows” with “show”.

Response: Thank you for your advice. The sentence “However, it is worth…… level to these insecticides……Existing studies shows that……” has been revised as “However, it is worth…… levels to these insecticides……Existing studies show that……”.

 

10. Line 58: Add “the” before the “RDL”.

Response: Thank you for your comment, but we could not entirely agree with that. As described by Liu et al. [1], in the abstract, it is described as “RDL subunits” not “the RDL subunits”, so we think we could not replace it.

 

11. Line 62: Replace “insecticides target is given priority” with “insecticide targets prioritizes”.

Response: Thank you for your comment, but we could not entirely agree with that. As described by Aydin et al. [2], in the abstract, it is described as “be given priority”, so we think we could not replace it.

 

12. Line 71-72: Relace “prospect in the field” with “prospects in field”.

Response: Thank you for your advice. We have removed the word “the” before the word “field” in the revised manuscript.

 

13. Line 80: Add “the” before the “RDL”.

Response: Thank you for your comment, but we could not entirely agree with that. As the same as described in the 10^th problem, in the abstract, it is described as “RDL subunits” not “the RDL subunits”, so we think we could not replace it.

 

14. Line 114: Replace the “to noting” with “noting”.

Response: Thank you for your advice. The sentence “It is worth to noting that the design principle of specific primers follows” has been revised as “It is worth noting that the design principle of specific primers follows”.

 

15. Line 118: Replace the “nucleotide acids” with “nucleotides”.

Response: Thank you for your advice, some of your comments have been accepted and revised.  The sentence “There are 120 ~ 160 bp, 40 ~ 60 bp, and 0 ~ 60 bp nucleotide acids……” has been revised as “120 ~ 160 bp, 40 ~ 60 bp, and 0 ~ 60 bp nucleotides were between the 5' end of F2 and B2……”.

 

16. Line 124: Add “the” before the “LAMP”.

Response: Thank you for your advice. The sentence “The basic component of LAMP reaction system……”has been revised as “The basic component of the LAMP reaction system……”.

 

17. Line 137: Remove the word “both”.

Response: Thank you for your advice. The sentence “both temperature and time of LAMP reaction were optimized” has been revised as “The temperature and time of LAMP reactions were optimized”.

 

18. Line 138: Replace the “reaction” with “reactions”.

Response: Thank you for your advice. The sentence “both temperature and time of LAMP reaction were optimized” has been revised as “temperature and time of LAMP reactions were optimized”.

 

19. Line 139: Add “the” before the “LAMP”. Replace the “on” with “at”.

Response: Thank you for your advice. The sentence “……each component in LAMP reaction system. The LAMP reaction was performed on different temperatures……” has been revised as “……each component in the LAMP reaction system. The LAMP reaction was performed at different temperatures……”.

 

20. Line 141: Replace the “condition” with “conditions”.

Response: Thank you for your advice. The sentence “……60 min to optimize the isothermal condition” has been revised as “……60 min to optimize the isothermal conditions”.

 

21. Line 151: Replace the “was” with “were”.

Response: Thank you for your advice. The sentence “and the nucleotide sequencing results was used to identify the mutation and to determine the accuracy of the LAMP technique” has been revised as “and the nucleotide sequencing results were used to identify the mutation and to determine the accuracy of the LAMP technique”.

 

22. Line 160: Replace the “Design the specific primer for LAMP reaction” with “Designing specific primers for the LAMP reaction”.

Response: Thank you for your comment, some of your comments have been accepted and revised. The sentence “Design the specific primer for LAMP reaction” has been revised as “Design specific primer for LAMP reaction”, which is consistently with the title “2.3 Design of specific primers for LAMP reaction”.

23. Line 162: Replace the “was” with “were”, and “chose” with “chosen”.

Response: Thank you for your advice. The sentence “The specific primers…… CsRDL1-S282 sequence was designed (Fig. 3) and chose by using the plasmid Blunt-CsRDL1-S282” has been revised as “The specific primers…… CsRDL1-S282 sequence were designed (Fig. 3) and chosen by using the plasmid Blunt-CsRDL1-S28”.

 

24. Line 168: Remove “from”.

Response: Thank you for your advice. The sentence “the HNB color becomes from violet to sky blue” has been revised as “the HNB visualization becomes violet to sky blue”.

 

25. Line 169: add “the” before the word “reaction”.

Response: Thank you for your advice. The sentence “the HNB color of the reaction solution with the ddH2O……” has been revised as “the HNB color of reaction solution with the ddH₂O……”.

 

26. Line 173: Replace the “screen” with “screening”.

Response: Thank you for your advice. The sentence “Fig. 4 Screen of specific primers for LAMP reaction’’ has been revised as “Fig. 4 Screening of specific primers for LAMP reaction”.

 

27. Line 191: Add “the” before the “LAMP”.

Response: Thank you for your advice. The sentence “Based on the optimized concentration of each component in LAMP reaction system” has been revised as “Based on the optimized concentration of each component in the LAMP reaction system”.

 

28. Line 211: Replace the “pest” with “pests”.

Response: Thank you for your advice, and we add some other things in the sentence. The sentence “The C. suppressalis is one most serious insect pest damaging the product of rice……” has been revised as “The C. suppressalis is one of the most serious insect pests damaging the product of rice……”.

29. Line 213-215: Rewrite the sentence “However…….recent years.” for clear understanding.

Response: Thank you for your advice. The sentence “However……recent years” has been revised as “However, the insecticide resistance of C. suppressalis to phenylpyrazole insecticides is frequently reported, and it is a has become a serious obstacle for control C. suppressalis in recent years”.

 

30. Line 217: Replace the “level” with “levels”.

Response: Thank you for your advice. The sentence “to serine results in high resistant level……” has been revised as “to serine results in high resistant levels……”.

 

31. Line 223: Replace the “were” with “was”.

Response: Thank you for your advice. The sentence “the specificity of four primers were confirmed……” has been revised as “the specificity of four primers was confirmed……”.

 

32. Line 224: Add “a” before the word “template”.

Response: Thank you for your comment, but we have corrected it in another way. The sentence “the specificity of four primers was confirmed using the plasmid Blunt-CsRDL1-S282, genome-CsRDL1-A282, and ddH₂O as template” has been revised as “the specificity of four primers was confirmed using the plasmid Blunt-CsRDL1-S282, genome-CsRDL1-A282, and ddH₂O as templates”.

 

33. Line 225-226: Rewrite the sentence “Meanwhile, the successful ………specificity as well.” Suggested “Meanwhile, the successful mutation-detection of  suppressalissamples from the field also confirmed its specificity.”

Response: Thank you for your comment, some of your comments have been accepted and revised. The sentence “Meanwhile, the successful ………specificity as well” has been revised as “Meanwhile, the successful mutation-detection of the field strains of C. suppressalis also confirmed its specificity”

 

34. Line 228: Add “the” before the “LAMP”.

Response: Thank you for your advice. The sentence “……HNB of LAMP reaction system were also optimized” has been revised as “……HNB of the LAMP reaction system were also optimized”.

 

35. Line 229: Remove “is” after the word “technique”.

Response: Thank you for your advice. The sentence “which made the LAMP technique is more economical and convenient” has been revised as “which made the LAMP technique more economical and convenient”.

 

36. Line 230: Add “the” before the “LAMP”.

Response: Thank you for your advice. The sentence “visualization is an important advantage of LAMP technique” has been revised as “visualization is an important advantage of the LAMP technique”.

 

37. Line 231: Replace the “chose” with “chosen”.

Response: Thank you for your advice. The sentence “the SYBR Green I was firstly chose as the chromogenic agent” has been revised as “the SYBR Green I was firstly chosen as the chromogenic agent”.

 

38. Line 235: Replace “the contamination while open” with “contamination while opening”.”.

Response: Thank you for your advice. The sentence “…… avoid the contamination while open the EP tube cover after the LAMP reaction” has been revised as “…… avoid the contamination while opening the eppendorf tube cover after the LAMP reaction”.

 

39. Line 240-241: Rewrite the sentence “Both reaction ………in field”. Suggested “Both the reaction time and temperature of the LAMP reaction were also optimized, making the LAMP technique more convenient and suitable for usage in the field.”

Response: Thank you for your advice. The sentence has been rewritten as you suggest above.

 

40. Line 247: Add “the” before the “LAMP”.

Response: Thank you for your advice. The sentence “and the results of LAMP technique were consistent with those of nucleotide sequencing and PCR” has been revised as “and the results of the LAMP technique were consistent with those of nucleotide sequencing and PCR”.

 

41. Line 254: Add “the” before the “LAMP”.

Response: Thank you for your advice. The sentence “this is the first time to detect insecticide resistance using LAMP technique in C. suppressalis” has been revised as “this is the first time to detect insecticide resistance using the LAMP technique in C. suppressalis”.

 

42. Line 258: Add “the” before the “field”.

Response: Response: Thank you for your advice. The sentence “……convenient and suitable for usage in field” has been revised as “……convenient and suitable for usage in the field”.

 

43. Line 225-226: Rewrite the sentence “The establishement…… suppressalis.” Suggested “The establishment of the LAMP technique can provide a new means for monitoring the resistance of C. suppressalisto phenylpyrazole insecticides and a reference and basis for managing C. suppressalis.”

Response: Thank you for your comment, some of your comments have been accepted and revised. The sentence “The establishement……C. suppressalis” has been revised as “The establishment of the LAMP technique can provide a new mean for the monitoring the resistance of C. suppressalis to phenylpyrazole insecticides, and a reference and basis for the management of C. suppressalis”.

Author Response File: Author Response.docx

Reviewer 3 Report

The manuscript " Rapid detection of A282S mutation in the RDL1 gene of rice stem borer via the mutation-specific LAMP technique" constructs a rapid detection technology for RDL1 gene mutations. The establishment of LAMP technique can provide a new mean for the monitoring of resistance of C. suppressalis to phenylpyrazole insecticides, and a reference and basis for the management of C. suppressalis. However, there are many deficiencies in the manuscript. For example, there is no block design in the optimization of LAMP reaction component and optimization of LAMP reaction parameters. In addition, the number of experimental samples used to verify the accuracy of LAMP technology is too small to fully reflect the accuracy of the technology. The specific opinions are as follows:

 

Introduction:

Scientific questions need to be clearly raised.

Line 84. This part does not mention the plastid, which needs to be modified.

Line 106-110. There are two places where underline is used at the same time, which is easy to be confused. It is suggested to modify one of them.

Line 147-148. Why only two larvae are used for experiment in each place? Generally, three repetitions are required. Moreover, from your experimental data, we only used the Chilo suppressalis from three regions for detection. The data volume is too small to fully reflect the accuracy of LAMP technology. More experiments are needed to verify the accuracy of LAMP technology.

Line 177. In the process of the experiment, what method is used for the combination of components? From the result of Figure 5, the author did not carry out the combination design. The results including Figures 6 and 7 also involve the same problem.

Line 205. The number of samples is too small to support this conclusion.

Author Response

Dear Editor Darcy Dai,

 

Thank you very much for your email and the reviewers’ comments on our manuscript entitled “Rapid detection of A282S mutation in the RDL1 gene of rice stem borer via the mutation-specific LAMP technique” (Manuscript ID: agronomy-2165886). We have read carefully the manuscript again and made a thorough revision according to the reviewer’s comments, and hope you will find these corrections in red satisfied and accept our manuscript for publication in Agronomy. Enclosed file is a response letter detailing our answers to the reviewers’ comments on the manuscript. We appreciate the reviewers’ comments and the resulting clarity has improved the manuscript.

 

With my best regards,

Chun-Qing Zhao

 

Response to reviewer 3

The manuscript "Rapid detection of A282S mutation in the RDL1 gene of rice stem borer via the mutation-specific LAMP technique" constructs a rapid detection technology for RDL1 gene mutations. The establishment of LAMP technique can provide a new mean for the monitoring of resistance of C. suppressalis to phenylpyrazole insecticides, and a reference and basis for the management of C. suppressalis. However, there are many deficiencies in the manuscript. For example, there is no block design in the optimization of LAMP reaction component and optimization of LAMP reaction parameters. In addition, the number of experimental samples used to verify the accuracy of LAMP technology is too small to fully reflect the accuracy of the technology. The specific opinions are as follows:

 

Introduction:

Scientific questions need to be clearly raised.

1. Line 84. This part does not mention the plastid, which needs to be modified.

Response: Thank you for your suggestion. This section does not refer to plasmids, so the sentence “2.1 C. suppressalis population and plasmid” has been revised as “2.1 C. suppressalis population”.

 

2. Line 106-110. There are two places where underline is used at the same time, which is easy to be confused. It is suggested to modify one of them.

Response: Thank you for your suggestion. In line 109-110, F2 and B2 were replaced underlined with blod.

 

3. Line 147-148. Why only two larvae are used for experiment in each place? Generally, three repetitions are required. Moreover, from your experimental data, we only used the Chilo suppressalis from three regions for detection. The data volume is too small to fully reflect the accuracy of LAMP technology. More experiments are needed to verify the accuracy of LAMP technology.

Response: Thank you for your suggestion. We agree with your comment, because this was a qualitative experiment and three regions were selected, we used two samples from each region to detect the mutation. In addition, the choice of the regions, according to the studies, the resistance development of C. suppressalis to insecticides acting on the insect GABA receptor is particularly prominent in Jiangsu [3], Jiangxi [4] and Hunan province [5], so the field strains of C. suppressalis from above mentioned three regions were used.

 

4. Line 177. In the process of the experiment, what method is used for the combination of components? From the result of Figure 5, the author did not carry out the combination design. The results including Figures 6 and 7 also involve the same problem.

Response: In part 2.3, We have introduced the basic component of LAMP reaction system, In order to optimize the LAMP reaction system, a serial of concentration for component were respectively set, for example, the concentrations of Bst DNA polymeras were set as 0.08, 0.16, 0.24, 0.32 and 0.4 U/μL, the other components were set as the basic. When we have picked out the optimal concentration for Bst DNA polymeras, we replaced the concentration of this component in the basic reaction system with the optimal concentration, and then optimize the other components.

 

 

5. Line 205. The number of samples is too small to support this conclusion.

Response: Thank you for your suggestion. We agree with your comment, our response is same to the question 3.

 

Author Response File: Author Response.docx

Reviewer 4 Report

In this paper, Song et al. reports the development of a LAMP technique for rapid detection of A282S mutation in RDL GABA receptor subunit in Chilo suppressalis. Since the mutation is associated with resistance to a number of phenylpyrazole insecticides, the results of this study would provide a convenient method for resistance monitoring in the field.

 

Minor issues:

1. Please explain the difference between A282S and A2'S. 

2. Is RDL1 a part of RDL?

3. lines 58 and 59, species names should be italicized.

4. Does CsRDL1 gene undergo alternative splicing? Will alternative splicing affect the efficiency of LAMP?

5. In the references section, too much references are in Chinese. It would be better to use literatures published in international academic journals.

Author Response

Dear Editor Darcy Dai,

 

Thank you very much for your email and the reviewers’ comments on our manuscript entitled “Rapid detection of A282S mutation in the RDL1 gene of rice stem borer via the mutation-specific LAMP technique” (Manuscript ID: agronomy-2165886). We have read carefully the manuscript again and made a thorough revision according to the reviewer’s comments, and hope you will find these corrections in red satisfied and accept our manuscript for publication in Agronomy. Enclosed file is a response letter detailing our answers to the reviewers’ comments on the manuscript. We appreciate the reviewers’ comments and the resulting clarity has improved the manuscript.

 

With my best regards,

Chun-Qing Zhao

 

Response to reviewer 4

In this paper, Song et al. reports the development of a LAMP technique for rapid detection of A282S mutation in RDL GABA receptor subunit in Chilo suppressalis. Since the mutation is associated with resistance to a number of phenylpyrazole insecticides, the results of this study would provide a convenient method for resistance monitoring in the field.

Minor issues:

1. Please explain the difference between A282S and A2'S. 

Response: Thank you for your question. A2'S site mutation is the resistance to dieldrin (RDL) subunit of γ-aminobutyric acid (GABA) receptor, the position of the site is in TM2 second amino acid (2') of conserved hydrophobic transmembrane segments, A282S is the resistance to dieldrin (RDL) subunit of γ-aminobutyric acid (GABA) receptor of Chilo suppressalis.

 

2. Is RDL1 a part of RDL?

Response: Thank you for your question. RDL1 is a replication of RDL gene, in Chilo suppressalis, there are two cDNAs encoding RDL subunits (CsRDL1 and CsRDL2).

 

3. lines 58 and 59, species names should be italicized.

Response: Thank you for your comments. The Latin name of Plutella xylostella, Heliothis virescens and Nilaparvata lugens have been italicised as Plutella xylostella, Heliothis virescens and Nilaparvata lugens, respectively.

 

4. Does CsRDL1 gene undergo alternative splicing? Will alternative splicing affect the efficiency of LAMP?

Response: Thank you for your question. CsRDL1 gene undergo alternative splicing, A282S mutation happens on the 7th exon, but alternative splicing of CsRDL1 occurs in exons 3, 6 and 8, so it won’t affect the efficiency of LAMP.

 

5. In the references section, too much references are in Chinese. It would be better to use literatures published in international academic journals.

Response: Thank you for your comments. The Chinese references “Pest status and loss assessment of crop damage caused by the rice borers Chilo suppressalis and Tryporyza incertulas in China” has been replaced with “Loop-mediated isothermal amplification for the detection of R81T mutation in nAChR with crude genomic DNA extracted from individual Myzus persicae”; the Chinese references “Primary study on resistance of rice stem borer (Chilo suppressalis) to fipronil” has been replaced with “Synergistic and antagonistic effects of piperonyl butoxide in fipronil-susceptible and resistant rice stem borrers, Chilo suppressalis”; the Chinese references “Comparison of methods for testing insecticide resistance in Chilo suppressalis and the resistance monitored” has been replaced with “Identification and characterization of glutathione S-transferases and their potential roles in detoxification of abamectin in the rice stem borer, Chilo suppressalis”; The Chinese references “Research advances in motoring and detecting insect insecticide-resistance” has been replaced with “Rapid detection of the Streptococcus mutans cnm gene by loop-mediated isothermal amplification” The Chinese references “Principle of loop-mediated isothermal amplification and its application in agriculture” has been replaced with “Simultaneous multiple target detection in real-time loop-mediated isothermal amplification”. Finally, only one Chinese reference was used in the revised manuscript.

 

References

 

1. Liu, G.Y.; Zhou, C.W.; Zhang, Z.S.; Wang, C.C.; Luo, X.G.; Ju, X.L.; Zhao, C.Q.; Ozoe, Y. Novel insecticidal 1,6-dihydro-6-iminopyridazine derivatives as competitive antagonists of insect RDL GABA receptors. Pest Manag. Sci. 2022, 78, 2872-2882, https://doi.org/10.1002/ps.6911.
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Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

After the author's revision, the quality of the manuscript has been greatly improved, basically meeting the requirements of publication. It is recommended to accept publication.