Feeding dsSerpin1 Enhanced Metarhizium anisopliae Insecticidal Efficiency against Locusta migratoria manilensis

Round 1

Reviewer 1 Report

This is an interesting research dealing with a very concerning (and difficult to control) pest. The experimental design is correct and although results are not as good as authors may have expected, they are very useful in the RNAi technology and in developing more sustainable pest control strategies. Here are some comments that might help to improve the manuscript.

First of all, the English needs a deep revision, it is mostly uncorrectly written and some sentences are hard to understand (e.g. sentence in lines 67-70 is confusing: if Serpin1 inhibited infection, how mortality can be increased?). There are sentences incomplete, with no verb (e.g., sentence in lines 62-63).

Please avoid redundance, some information is presented twice (e.g., lines 106 and 109, or lines 112 and 119, or lines 136 and 139).

I am not sure to understand the experimental design in section 2.4. Since you say that “the locust treatments’ protocol was similar as 2.3”, I suppose you are using insects different than those from the bioassay in the 2.3 section. Is that correct? And regarding the “10 treated individuals taken at 6h, 18h, 24h, 48h and 72h after treatments”, it is unclear if you have a total of 10 individuals from each different feeding treatments (and then you are taking them proportionately in every time, i.e., two locusts at 6h, two more at 18h, etc.) or if you have 10 individuals from each different feeding treatment AND for every time (i.e., 10 locusts at 6h, 10 more at 18h, etc.).

In results, there is no need to include in the text all results showed in the figures, that is what the figures are for. You just need to highlight the most important information in those results. Also, there is no point to insist in pointing differences when they are not statistically significant, as in sentence in lines 162-165 (in the parentheses in line 164, if you want to include p it should be p>0.05), or as in the last sentence of that paragraph, since mortality did not significantly increased with higher amount of dsSerpin1 in almost none of the days, especially not in the last three days.

Legend in figure 2 is incorrect: it is the relative expression of Serpin1 in L. m. manilensis.

First paragraph in discussion has no relation with your results. Here you should be discussing why no significant differences after 7 days were found in the treatments. If you are trying to say that the feeding method (versus microinjection) might be the reason, it only may make sense if you were providing evidence of other experiments having better results with microinjections.

Your results do not agree your statement in lines 212-215: it increased significantly the mortality but to a certain point (only for the first 7 days), so I think it would be interesting to discuss why the inhibition of Serpin1 expression is not translated in a enhanced effect of M. anisopliae.

Author Response

First of all, the English needs a deep revision, it is mostly uncorrectly written and some sentences are hard to understand (e.g. sentence in lines 67-70 is confusing: if Serpin1 inhibited infection, how mortality can be increased?). There are sentences incomplete, with no verb (e.g., sentence in lines 62-63).

Response: It is a good suggestion. We have added specific data on how mortality increases in the sentences in lines 67-70. Revised the sentences in lines 62-63 and added verbs.

Please avoid redundance, some information is presented twice (e.g., lines 106 and 109, or lines 112 and 119, or lines 136 and 139).

Response: We have deleted duplicate information.

I am not sure to understand the experimental design in section 2.4. Since you say that “the locust treatments’ protocol was similar as 2.3”, I suppose you are using insects different than those from the bioassay in the 2.3 section. Is that correct? And regarding the “10 treated individuals taken at 6h, 18h, 24h, 48h and 72h after treatments”, it is unclear if you have a total of 10 individuals from each different feeding treatments (and then you are taking them proportionately in every time, i.e., two locusts at 6h, two more at 18h, etc.) or if you have 10 individuals from each different feeding treatment AND for every time (i.e., 10 locusts at 6h, 10 more at 18h, etc.).

Response: The artificial feeding scheme of locusts was consistent with that in 2.3. I took locusts with the same feeding scheme and measured the expression of serpin1 gene at 6h, 18h, 24h, 48h and 72h respectively. And 10 individuals from each different feeding treatment and for every time were taken at 6h, 18h, 24h, 48h and 72h respectively.

In results, there is no need to include in the text all results showed in the figures, that is what the figures are for. You just need to highlight the most important information in those results. Also, there is no point to insist in pointing differences when they are not statistically significant, as in sentence in lines 162-165 (in the parentheses in line 164, if you want to include p it should be p>0.05), or as in the last sentence of that paragraph, since mortality did not significantly increased with higher amount of dsSerpin1 in almost none of the days, especially not in the last three days.

Response: We accept your suggestion, we have deleted unnecessary and modified error messages.

Legend in figure 2 is incorrect: it is the relative expression of Serpin1 in L. m. manilensis.

Response:We accept your suggestion, we have modified Legend in figure 2.

First paragraph in discussion has no relation with your results. Here you should be discussing why no significant differences after 7 days were found in the treatments. If you are trying to say that the feeding method (versus microinjection) might be the reason, it only may make sense if you were providing evidence of other experiments having better results with microinjections.

Response: We made some changes in the first paragraph of the discussion. We provide evidence that other experiments have better results in microinjection.

Your results do not agree your statement in lines 212-215: it increased significantly the mortality but to a certain point (only for the first 7 days), so I think it would be interesting to discuss why the inhibition of Serpin1 expression is not translated in a enhanced effect of M. anisopliae.

Response: Thank you sir. We accept your suggestion. We revised the statement in lines 212-215.

Reviewer 2 Report

Manuscript with title "Feeding dsSerpin1 enhanced Metarhizium anisopliae insecti- 2cidal efficiency against Locusta migratoria manilensis"

It’s subject is new. 
1. Every time you want to say a novelty of research, you should not point to other results. For example: How- 71 ever, no extensive and comprehensive studies on the role of Serpin1 has been carried out 72 by RNAi technology on L. m. manilensis [24].
2. The phrase “significance level of p <0.05” replaced by “significance (p <0.05)”
3. In Fig. 1, the mean comparison revealed by small letter on every column. These comparison are not correct, you must do it for all of treatments, not separately.
4. In Fig. 2, Please check the mean comparison again.
5. I did not find "conclusion" in this reports,  is it according to Journal format.

Author Response

1. Every time you want to say a novelty of research, you should not point to other results. For example: How- 71 ever, no extensive and comprehensive studies on the role of Serpin1 has been carried out 72 by RNAi technology on L. m. manilensis [24].

Response : Thank you sir. We accept your suggestion, we changed to elaborate on the topic of serpin1.
2. The phrase “significance level of p <0.05” replaced by “significance (p <0.05)”

Response : Thank you sir. We accept your suggestion, we changed “significance level of p <0.05” to “significance (p <0.05)”

3. In Fig. 1, the mean comparison revealed by small letter on every column. These comparison are not correct, you must do it for all of treatments, not separately.

Response : Thank you sir. This analysis aimed to analyzed the difference daily. This is important information to reduce the pest damage. Thus we did the comparison by day with all treatments.

4. In Fig. 2, Please check the mean comparison again.

Response : Thank you sir. We accept your suggestion. We checked the average again.
5. I did not find "conclusion" in this reports,  is it according to Journal format.

Response : Thank you sir. We accept your suggestion. We have added the conclusion.

 

Reviewer 3 Report

Dear Authors,

I have reviewed the manuscript "Feeding dsSerpin1 enhanced Metarhizium anisopliae insecticidal efficiency against Locusta migratoria manilensis". I find that your manuscript is an interesting scientific work, but an array of corrections is necessary:

• English language is the most problematic feature of your paper and needs extensive editing
• the aim of the present work needs to be more clearly defined, especially in relation to the work you published last year in MDPI Insects;
• methodology needs clarifications
• results need rearrangements and clearer presentation
• discussion might benefit from further additions, especially ones focusing on your own work and its implications, such as injection vs feeding comparisons, dosage optimization perspectives, and perspectives for implementations in practice as biological control
• a short conclusion section would be beneficial for the article

Please read my detailed comments as given in the attached file.

Reviewer

 

Comments for author File: Comments.pdf

Author Response

• English language is the most problematic feature of your paperand needs extensive editing

Response : Thank you sir. We accept your suggestion. We will polish the pronunciation of the article. Two native English speakers read the manuscript.

• the aim of the present workneeds to be more clearly defined, especially in relation to the work you published last year in MDPI Insects;

Response : Thank you sir. We accept your suggestion, We have added your suggestions on how to increase the mortality rate in MDPI's article.

• methodologyneeds clarifications

Response : Thank you sir. We accept your suggestion. We changed the specific details in the methodology to clarify the sampling quantity. There are 10 locusts in each different feeding treatment (i.e. 10 locusts in 6 hours, 10 locusts in 18 hours, etc.).

• resultsneed rearrangements and clearer presentation

Response : Thank you sir. We accept your suggestion. We deleted the analysis without significance In the results.

• discussionmight benefit from further additions, especially ones focusing on your own work and its implications, such as injection vs feeding comparisons, dosage optimization perspectives, and perspectives for implementations in practice as biological control

Response: Thank you sir. We accept your suggestion. In the first paragraph of the discussion, we compared injection with feeding and provided evidence that other experiments had better results in microinjection. And the actual implementation of biological prevention and control

• a short conclusionsection would be beneficial for the article

Response : Thank you sir. We accept your suggestion. We have added the conclusion.

Please read my detailed comments as given in the attached file.

Response: Thanks a lot.

Author Response File: Author Response.docx

Reviewer 4 Report

This manuscript describes the impact of dsRNA of an immune gene on locust mortality, gene expression levels, and the ability of the insects to generate a behaviorally induced fever.

I found this manuscript difficult to read because the text was poorly written. There were many grammatical and wording errors that at times made it difficult to understand. I would ask the authors to please have someone with a stronger command of written professional English to edit this paper before re-submission.

One major concern is that it is not clear how the authors produced a temperature gradient so that the insects could select an area with increased temperatures. Or was some other method used to elevate the insect's temperature?  In other words, they described how the change in the insect's external temperature was measured, but not how the fever was generated.

Lines 34-35: the first time an insect’s scientific name is mentioned, it should be accompanied by the Order and Family, such as "(Order: Family)".

Lines 148-149: How was the “corrected mortality” calculated?  Did they use Abbott’s formula?  If not, then how did they make this correction?  Also, please provide a reference for this calculation.

I may have other suggestions once the text has been corrected. 

Author Response

I found this manuscript difficult to read because the text was poorly written. There were many grammatical and wording errors that at times made it difficult to understand. I would ask the authors to please have someone with a stronger command of written professional English to edit this paper before re-submission.

Response : Thank you sir. We accept your suggestion. We invite two native English people polished this paper.

One major concern is that it is not clear how the authors produced a temperature gradient so that the insects could select an area with increased temperatures. Or was some other method used to elevate the insect's temperature?  In other words, they described how the change in the insect's external temperature was measured, but not how the fever was generated.

Response : Thank you sir. We accept your suggestion. We use infrared thermal imager to measure the surface temperature of insects. This is due to the application of Metarhizium anisopliae. Insects produce immune response and warm body temperature, which is the result of self-regulation of temperature. We present the changes of body temperature of insects when they encounter fungi and RNAi treatment.

Lines 34-35: the first time an insect’s scientific name is mentioned, it should be accompanied by the Order and Family, such as "(Order: Family)".

Response : Thank you sir. We accept your suggestion. We add the insect’s the Order and Family.

Lines 148-149: How was the “corrected mortality” calculated?  Did they use Abbott’s formula?  If not, then how did they make this correction?  Also, please provide a reference for this calculation.

Response : Thank you sir. We added a formula to calculate the corrected mortality. Corrected mortality = (number of dead insects in the treatment group - number of dead insects in the control group) / (1 - number of dead insects in the control) * 100%.

Round 2

Reviewer 1 Report

After revising the new version of the manuscript, I am totally satisfied with it, it has significantly improved and I do recommend its publication. There are just a few minor mistakes that need to be fixed:

Line 74: “L. m. manilensis” should be in italics.

Lines 98 and 160 and 179: “M. anisopliae” should not be in italics.

Line 152: please change the period after the parenthese for a comma or just start in caps.

Lines 186 and 187: “treatment” and “only”, respectively, should not be in italics.

Line 277: “dsSerpin1” should be in italics, and please remove “s” in “down-regulates”.

Line 279: “dsSerpin1” should be in italics.

Author Response

After revising the new version of the manuscript, I am totally satisfied with it, it has significantly improved and I do recommend its publication. There are just a few minor mistakes that need to be fixed:

Line 74: “L. m. manilensis” should be in italics.

Response : Thank you sir. We accept your suggestion. We have corrected it.

Lines 98 and 160 and 179: “M. anisopliae” should not be in italics.

Response : Thank you sir. We accept your suggestion. We have corrected it.

Line 152: please change the period after the parenthese for a comma or just start in caps.

Response : Thank you sir. We accept your suggestion. We have corrected it.

Lines 186 and 187: “treatment” and “only”, respectively, should not be in italics.

Response : Thank you sir. We accept your suggestion. We have corrected it.

Line 277: “dsSerpin1” should be in italics, and please remove “s” in “down-regulates”.

Response : Thank you sir. We accept your suggestion. We have corrected it.

Line 279: “dsSerpin1” should be in italics.

Response : Thank you sir. We accept your suggestion. We have corrected it.

Reviewer 3 Report

Dear Authors,

I was asked for another round of review of your manuscript "Feeding dsSerpin1 enhanced Metarhizium anisopliae insecticidal efficiency against Locusta migratoria manilensis". I am pleased to see that your manuscript has significantly improved since I first reviewed it, especially with respect to the quality of presentation and discussion. I will recommend it for publication in MDPI Agriculture, following another round of minor revisions for the final correction of the few remaining imperfections. Please read my detailed comments as given below:

• line 18-20: this sentence is still nonsensical because it suggests that the gene LmSerpin1 is performing the injection of its own dsRNA into the locusts. Please correct to: "Previous studies have shown that LmSerpin1 gene upregulates the immune responses of Locusta migratoria manilensis, and that the infection of locusts with Metarhizium anisopliae can be enhanced through injection of the interfering dsSerpin1"
• line 25-26: "1g/g diet" means that the entire diet consisted of 100% anisopliae. Please correct to whatever you actually meant to say.
• line 44: anisopliae is not a fungicide. A fungicide is a substance that kills fungi, whereas M. anisopliae is a fungus intended to kill insects. Hence, you have to correct "fungicide" to "insecticide" or "biological control agent".
• line 65-68: The aim of your current work is now better defined than before but is still unnecessarily spanned over two sentences, which blurs the actual difference from the aim of your previous work. Thus, the first sentence can be dropped, and instead you should just merge its beginning with the second sentence, to put a clearer accent on the feeding approach. ("This study was intended to estimate the efficiency of a field-applicable feeding approach for administering different doses of dsSerpin1 together with anisopliae to L. m. manilensis.")
• line 96: "dsSerpin1" should be a start of a new sentence, and "respectively" should be deleted.
• line 113: In the entire section 2.4. the information is missing, as to, which gene (Serpin1) was the subject of the qRT-PCR analysis. I suggest that you add "Quantitative Real-Time PCR Analysis of Serpin1 Expression" into the section title.
• line 152: delete the full stop "." after the brackets
• line 162-163: If it was significantly higher, then p<0.05 and not p>0.05. Anyway you should delete p>0.05 from the brackets.
• line 187-188: Day 5 is ok, but the graph says that at day 6 there was no significant difference between control ("b") and 10 μg ("ab"), whereas in the text you say that the difference is significant. Please correct either in the text or in the graph.
• line 207: What is "pf dsRNA"? Maybe it was meant to be "of dsRNA"?
• line 221: Other aspects OF innate immunity
• line 223: ...is limited due to THE lower insecticidal efficiency OF anisopliae
• line 225: please add "average" ("decreased the average time required")
• line 227: please add "only" ("treated only with Metarhizium")
• line 238: μg
• line 283: please add "previously" ("previously proved to be useful")
• line 284: please correct "These experiments" to "The experiments in this study".

Author Response

Thank you so much for your very useful suggestions and comments. With your efforts, our manuscript has been improved.

• line 18-20: this sentence is still nonsensical because it suggests that the gene LmSerpin1 is performing the injection of its own dsRNA into the locusts. Please correct to: "Previous studies have shown that LmSerpin1 gene upregulates the immune responses of Locusta migratoria manilensis, and that the infection of locusts with Metarhizium anisopliae can be enhanced through injection of the interfering dsSerpin1"

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 25-26: "1g/g diet" means that the entire diet consisted of 100% anisopliae. Please correct to whatever you actually meant to say.

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 44: anisopliae is not a fungicide. A fungicide is a substance that kills fungi, whereas M. anisopliae is a fungus intended to kill insects. Hence, you have to correct "fungicide" to "insecticide" or "biological control agent".

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 65-68: The aim of your current work is now better defined than before but is still unnecessarily spanned over two sentences, which blurs the actual difference from the aim of your previous work. Thus, the first sentence can be dropped, and instead you should just merge its beginning with the second sentence, to put a clearer accent on the feeding approach. ("This study was intended to estimate the efficiency of a field-applicable feeding approach for administering different doses of dsSerpin1 together with anisopliae to L. m. manilensis.")

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 96: "dsSerpin1" should be a start of a new sentence, and "respectively" should be deleted.

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 113: In the entire section 2.4. the information is missing, as to, which gene (Serpin1) was the subject of the qRT-PCR analysis. I suggest that you add "Quantitative Real-Time PCR Analysis of Serpin1 Expression" into the section title.

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 152: delete the full stop "." after the brackets

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 162-163: If it was significantly higher, then p<0.05 and not p>0.05. Anyway you should delete p>0.05 from the brackets.

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 187-188: Day 5 is ok, but the graph says that at day 6 there was no significant difference between control ("b") and 10 μg ("ab"), whereas in the text you say that the difference is significant. Please correct either in the text or in the graph.

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 207: What is "pf dsRNA"? Maybe it was meant to be "of dsRNA"?

Response : Thank you sir. We accept your suggestion. We have corrected it.

• line 221: Other aspects OF innate immunity

Response : We have corrected it.

• line 223: ...is limited due to THE lower insecticidal efficiency OF anisopliae

Response : We have corrected.

• line 225: please add "average" ("decreased the average time required")

Response : It was added in.

• line 227: please add "only" ("treated only with Metarhizium")

Response : It was added in.

• line 238: μg

Response : It was corrected.

• line 283: please add "previously" ("previously proved to be useful")

Response : It was added in.

• line 284: please correct "These experiments" to "The experiments in this study".

Response : It was corrected.

 

Reviewer 4 Report

The authors did not adequately address my concern as to how the insects generated an increased body temperature.  This is considered a "behavioral fever" meaning the insects need to do something to increase their body temperature, usually move to places with a higher temperature.  This is often accomplished by generating a temperature gradient (even something as simple as a light bulb that produces heat), allowing the insects to seek out higher temperatures. The authors even cite papers where these methods are described but they do not have a section in their methods describing how this was performed. Also, the changes in temperature were minimal (no higher than 29oC). One example of a behavioral fever generated in a related species is in:  Clancy, L.M., Jones, R., Cooper, A.L. et al. Dose-dependent behavioural fever responses in desert locusts challenged with the entomopathogenic fungus Metarhizium acridum. Sci Rep 8, 14222 (2018). https://doi.org/10.1038/s41598-018-32524-w.  In this paper they describe a fevers of 40-45oC (much higher than reported in the current study).  The authors should address this concern.

Also, although the English text was greatly improved, there are still some errors.  I suggest they go through the paper one more time and correct these areas. I have uploaded the pdf if some areas highlighted.

Comments for author File: Comments.pdf

Author Response

The authors did not adequately address my concern as to how the insects generated an increased body temperature.  This is considered a "behavioral fever" meaning the insects need to do something to increase their body temperature, usually move to places with a higher temperature.  This is often accomplished by generating a temperature gradient (even something as simple as a light bulb that produces heat), allowing the insects to seek out higher temperatures. The authors even cite papers where these methods are described but they do not have a section in their methods describing how this was performed. Also, the changes in temperature were minimal (no higher than 29oC). One example of a behavioral fever generated in a related species is in:  Clancy, L.M., Jones, R., Cooper, A.L. et al. Dose-dependent behavioural fever responses in desert locusts challenged with the entomopathogenic fungus Metarhizium acridum. Sci Rep 8, 14222 (2018). https://doi.org/10.1038/s41598-018-32524-w.  In this paper they describe a fevers of 40-45oC (much higher than reported in the current study).  The authors should address this concern.

Response: We read this paper before. The aim of this reference is different comparing to us. Clancy did the body fever is under the bulb/ light condition. Actually, there are similar studies on locust without Metarhizium treatment.

While, our experiment is doing the influence of dsRNA to enhance the mortality. The body fever is side product. In this study, Metarhizium anisopliae as a fungal infection is a positive control, and Clancy's experiment focused on the effect of Metarhizium anisopliae dose.

In terms of the body temperature, the ambient temperature has a big influence on locust body temperature. The two studies used different ambient temperature. Second, the temperature is different among of different species, and different development stages. According to our previously study, 3^rd instar hoppers has lower body temperature comparing 4/5 instars and adults. In practical, 3^rd instar is recommended as the control stage in the field in China.

Also, although the English text was greatly improved, there are still some errors.  I suggest they go through the paper one more time and correct these areas. I have uploaded the pdf if some areas highlighted.

Response: The manuscript has been checked again.  The highlights have been solved.

 The number of alive, dead and lost locusts were daily counted and recorded for 10 days at the same time.

Response: The major reason of lost locusts is the once the locust died, the alive one may eat the dead one as we found the incomplete dead individuals, thus we will record the lost numbers. During the experiment, lost rate is under 10%. Otherwise, we need to restart the test.

Author Response File: Author Response.docx