Untargeted UHPLC-MS Metabolomics Reveals the Metabolic Perturbations of Helicoverpa armigera under the Stress of Novel Insect Growth Regulator ZQ-8

Round 1

Reviewer 1 Report

 

The manuscript focuses on the terpenoid ester compound ZQ-8, which was previously synthesized and found to have a significant impact on the growth and development of H. Armigera. The authors used untargeted UHPLC-MS/MS metabolomics to evaluate the differences in metabolite profiles of the epidermis and viscera of H. armigera treated with ZQ-8, in order to validate the mechanism of ZQ-8 in regulating pest growth and development. The results indicate that decreased glycolysis, increased TCA cycle, and decreased amino acid synthesis were the main causes of growth inhibition and death of H. armigera larvae. The study provides new insights into the underlying metabolic molecular mechanisms of H. armigera under insecticide stress. The manuscript is clear, relevant for the field and presented in a well-structured manner. 15 out of 35 references have been published within the last 5 years. All cited publications are relevant to the field. The manuscript is scientifically sound, experimental design is appropriate and results are clearly presented. Discussion is worthy of attention.  

 

The manuscript deserves to be published after minor adjustments: 

 

1. In the abstract, please describe the ZQ-8 shortly. 

2. Line 153 delete “of”. 

3. Line 158 give explanation for abbreviations x, y, n, N 

4. Please recheck the sentence 157 – 160 for thought clarity. The sentence is too long.  

5. Lines 163, 248, 257, 261 – species Latin name should be in italics.  

6. Line169, I think that you mean R2 instead of R2 

7. Lines 224 – 226 belong to discussion, not results.  

 

Author Response

Dear Reviewer,

 

We are so grateful for the excellent suggestions and the detailed revising from you. We have benefited greatly from the revision. Now we response all the questions point by point and made modification accordingly.

 

Kind regards,

 

4^th 5, 2023

 

Point 1: In the abstract, please describe the ZQ-8 shortly. 

Response 1: Thank you for your kindly suggestions. We had added it.

 

Point 2: Line 153 delete “of”. 

Response 2: Thank you for your kindly suggestions. We had revised it.

 

Point 3: Line 158 give explanation for abbreviations x, y, n, N. 

Response 3: Thank you for your kindly suggestions. We have explained this in the table notes attached to Table S2 of the supporting information. Note: x represents the number of differential metabolites associated with this pathway; y indicates the number of background (all) metabolites associated with the pathway; n represents the number of differential metabolites annotated by KEGG; N represents the number of background (all) metabolites of the KEGG annotation; Enrich Direct means enrichment pointing, Over represents enrichment, that is, x/n>y/N.

 

Point 4: Please recheck the sentence 157 – 160 for thought clarity. The sentence is too long.  

Response 4: Thank you for your kindly suggestions. We had revised it.

 

Point 5: Lines 163, 248, 257, 261 – species Latin name should be in italics.  

Response 5: I am very sorry for the mistake. We had revised it.

 

Point 6: Line169, I think that you mean R² instead of R2. 

Response 6: Thank you for your kindly suggestions. We had revised it.

 

Point 7: Lines 224 – 226 belong to discussion, not results.

Response 7: Thank you for your kindly suggestions. We had put that sentence in the discussion section.

Author Response File: Author Response.pdf

Reviewer 2 Report

Dear authors, 

The topic of this manuscript is well-written and the obtained results provided interesting insight into the metabolism of the Helicoverpa armigera.  However, this paper suffers from a lack of entomological data which are very relevant for the given subject. Therefore, it is difficult to comment on the results if there is not enough information given in the material and method. 

My suggestion is to explain in more detail the whole process of the rearing of insects until L3 and the conditions that were used to rear them. 

It is also not clear how the authors treated larvae. Did you use some trays to do it? How many insects? How many replicates? 

It is necessary to correct that before taking the MS into consideration. 

we don't know if the results are trustworthy when we don't know what the methodology were. Authors we working on insects and they mantioned them only in two sentences in M&M. There is no sence to correct results before we know the M&M. If they accept to add these very relevant data I will review the MS again. 

The manuscript is written in simple and understandable English. 

Author Response

Dear Reviewer,

 

We are so grateful for the excellent suggestions and the detailed revising from you. We have benefited greatly from the revision. Now we response all the questions point by point and made modification accordingly.

 

Kind regards,

 

4^th 5, 2023

 

Point 1: The topic of this manuscript is well-written and the obtained results provided interesting insight into the metabolism of the H. armigera. However, this paper suffers from a lack of entomological data which are very relevant for the given subject. Therefore, it is difficult to comment on the results if there is not enough information given in the material and method.

My suggestion is to explain in more detail the whole process of the rearing of insects until L3 and the conditions that were used to rear them. 

Response 1: Thank you for your kindly suggestions. The H. armigera pupae purchased were soaked in 0.2% sodium hypochlorite solution disinfectant for 5 minutes and then rinsed. The male and female pupae were kept separately in beakers for 5 to 7 days before emergence. The eclosed male is put into the female's beaker for mating, and the appropriate amount of cotton soaked in honey water is added to the beaker. Cover the mouth of the beaker with clean gauze. Adult worms waiting to emerge lay eggs on the gauze. And change the gauze daily. One day before the eggs hatch (blackhead stage), soak the gauze with eggs in 5% formaldehyde solution for 15-25 min. Then wash them with sterile water for 3 times, hang them on sterilized plastic sticks, dry them naturally in a sterile room, put them in a zip-lock bag, and then put them in an incubator at 26℃ and 60% relative humidity. Let them hatch. The H. armigera larvae are placed in a sterilized plastic box with appropriate feed. They were kept in the incubator until L3. (Feeding conditions: temperature 26 ± 1 °C, relative humidity 70 ± 10%, photoperiod 14L:10D).

 

Point 2: It is also not clear how the authors treated larvae. Did you use some trays to do it? How many insects? How many replicates? 

It is necessary to correct that before taking the MS into consideration. 

we don't know if the results are trustworthy when we don't know what the methodology were. Authors we working on insects and they mantioned them only in two sentences in M&M. There is no sence to correct results before we know the M&M. If they accept to add these very relevant data I will review the MS again.

Response 2: Thank you for your kindly suggestions. These problems have been mentioned in the Materials and Methods section of the manuscript. The 3rd instar larvae of H. armigera were treated at 200 mg/L ZQ-8 medicine and 0.01% Triton X-100 aqueous solution (control group) for 5 s, and then transferred to a light incubator and fed with artificial feed for 48 h. The treated H. armigera were placed on ice for 2 min. As their activity waned, pinned the larvae to a wax plate, removed the epidermis with tweezers and scissors, wrapped the remaining organs with aluminum foil, and quickly freezed with liquid nitrogen. The epidermis was quickly rinsed in pre-cooled PBS solution to remove blood and dirt, then absorbed with absorbent paper. Finally, the epidermis was divided into freezer tubes and transferred to the refrigerator at −80 °C for storage. There were 6 replicates per treatment and 30 larvae per replicate.

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript is written as a scientific work because it has all the parts important for this type of work. The novelty of the described work and the well-cited data accompanying the topic are well-underlined. The experiment was organized appropriately; the measurements performed are appropriate, the statistical processing of the results is very detailed. The results are supported by data and provide useful conclusions. The authors present the recent results in this area well, using new references. Given that it is a new insect growth regulator, such research is of great importance. Because of the subject matter, the manuscript is important and should be published, after a minor revision according to the instructions in the indicated places in the manuscript.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer,

 

We are so grateful for the excellent suggestions and the detailed revising from you. We have benefited greatly from the revision. Now we response all the questions point by point and made modification accordingly.

 

Kind regards,

 

4^th 5, 2023

 

The manuscript is written as a scientific work because it has all the parts important for this type of work. The novelty of the described work and the well-cited data accompanying the topic are well-underlined. The experiment was organized appropriately; the measurements performed are appropriate, the statistical processing of the results is very detailed. The results are supported by data and provide useful conclusions. The authors present the recent results in this area well, using new references. Given that it is a new insect growth regulator, such research is of great importance. Because of the subject matter, the manuscript is important and should be published, after a minor revision according to the instructions in the indicated places in the manuscript.

Point 1: "cor ()" There is something missing.

Response 1: Thank you for your question. This is an R language function name. The correlation between differential metabolites were analyzed by cor () in R language (method = pearson).

 

Point 2: Do not start a sentence with a number.

Response 2: Thank you for your kindly suggestions. We had revised it.

 

Point 3: The scientific name of insect should be italicized.

Response 3: I am very sorry for the mistake. We had revised it.

Author Response File: Author Response.pdf

Reviewer 4 Report

Authors Liu et al. used Untargeted UHPLC-MS Metabolomics method to investigate the mechanisms of action of a terpenoid ester product, ZQ-8 on the cotton bollworm, Helicoverpa armigera, a notorious agricultural pest worldwide.  Authors preformed molecular biology, molecular docking, and protein ligand interaction studies in their previous publications, and they found that ZQ-8 targets on the epidermis of H. armigera, and chitinase 2 and endochitinase. In current study, they use metabolomics strategy to analyze the substance changes in epidermis and viscera tissues of H. armigera 48h after treated with 200mg/L ZQ-8 compared with controls. They found that ZQ-8 mainly inhibits energy metabolism, amino acid biosynthesis as well as chitin synthesis pathways. The metabolomics strategy offers a very promising viewpoint on the readout products during the cellular process affected to ZQ-8 treatment. The design of this study is very logic and scientifically sound. The introduction and discussion are very well written. I only have a few comments associated with M&M and figures.

Materials and Methods

2.3 Sample preparation. Why treated insects with 200 mg/L ZQ-8 solution for 5 s? Any previous studies for this specific dose (LD20 or LD50)? Please clarify it in the context or cite previous studies. I am also curious how authors prepare the epidermis (DB and CB) and viscera (DN and CN) tissues. And what viscera of H. armigera includes? Foregut, midgut, and hindgut?

 

The section titles of 2.7 and 2.5 are same (UHPLC-MS/MS conditions). The title of 2.7 should be changed (Metabolites annotation?).

 

Page 7, line 248, the scientific name of insect should be italicized. Check the MS thoroughly to fix it.

 

Figure 5. the resolution of this figure is very poor. Should be improved significantly.

 

Figure 6. Please illustrate the up or down regulation (in red or blue) by ZQ-8 treatment in the figure legend as well. Similar changes needed for Figures 7&8

Author Response

Dear Reviewer,

 

We are so grateful for the excellent suggestions and the detailed revising from you. We have benefited greatly from the revision. Now we response all the questions point by point and made modification accordingly.

 

Kind regards,

 

4^th 5, 2023

 

Point 1: Materials and Methods

2.3 Sample preparation. Why treated insects with 200 mg/L ZQ-8 solution for 5 s? Any previous studies for this specific dose (LD₂₀ or LD₅₀)? Please clarify it in the context or cite previous studies.

Response 1: Thank you for your kindly suggestions. In our previous study, the concentration of ZQ-8 to determine the growth and development activity of H. armigera was 200 mg/L, and the treatment time was 5 s. We cited the previous study in the materials and methods part of the manuscript.

 

Point 2: How authors prepare the epidermis (DB and CB) and viscera (DN and CN) tissues.

Response 2: Thank you for your kindly suggestions. The preparation of materials for the epidermis (DB and CB) and viscera (DN and CN) tissues of H. armigera is to put the treated H. armigera on ice for 2 min. As their activity waned, pinned the larvae to a wax plate, removed the epidermis with tweezers and scissors, wrapped the remaining organs with aluminum foil, and quickly freezed with liquid nitrogen. The epidermis was quickly rinsed in pre-cooled PBS solution to remove blood and dirt, then absorbed with absorbent paper. Finally, the epidermis was divided into freezer tubes and transferred to the refrigerator at -80 °C for storage. It was described in detail in the Materials and Methods section of the manuscript.

 

Point 3: What viscera of H. armigera includes? Foregut, midgut, and hindgut?

Response 3: Thank you for your kindly suggestions. The viscera parts we treated included the foregut, midgut and hindgut.

 

Point 4: The section titles of 2.7 and 2.5 are same (UHPLC-MS/MS conditions). The title of 2.7 should be changed (Metabolites annotation?).

Response 4: I am very sorry for the mistake. We had revised it to data analysis.

 

Point 5: Page 7, line 248, the scientific name of insect should be italicized. Check the MS thoroughly to fix it.

Response 5: I am very sorry for the mistake. We had revised it.

 

Point 6: Figure 5. the resolution of this figure is very poor. Should be improved significantly.

Response 6: Thank you for your kindly suggestions. We had improved the resolution.

 

Point 7: Figure 6. Please illustrate the up or down regulation (in red or blue) by ZQ-8 treatment in the figure legend as well. Similar changes needed for Figures 7&8.

Response 7: Thank you for your kindly suggestions. We had added it.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Dear authors

Thank you for your responses. The added text explains the procedure applied during the experiment.

Considering the introduction, the authors well presented the significance of the pest insect species, giving the problems that we are facing considering the control of this insect.

Considering the material and method, with the focus on the lack of an entomological part, it could be accepted as given in the replies. However, the English language in responses has to be revised. Please correct the English in the response if you will use this text directly. The required corrections are:

Response 1:

The eclosed males WERE put into the female's beaker...

Cover the mouth of the beaker with clean gauze. ... Should be changed into - The beaker was covered with clean gauze.

Adult worms waiting to emerge lay eggs on the gauze... should be corrected to ... After emergence adults were left to lay eggs on the gauze... (if that was the context).

Please act accordingly in the rest of the text.

Additionally, it is not given what was “appropriate feed”. What was amount of feed?

This complete response 1 should be placed in the section “Test insects”

 

Response 2: Please add the number of larvae per replicate from the response to the main text because you did not give it there.

The sentence given in response 2 is: Each treatment and control group consisted of six replicates (Table S1).

 

The Results are adequately presented and discussed. Conclusions are in accordance with the obtained results. The authors correctly cited articles in the manuscript.

 Two specific comments are:

L88 Please correct “feeding conditions“ to the “rearing conditions“

 

L276 Please replace “body wall“ with  “the rigid cuticle that cannot be expanded”

English is understandable but it requires minor changes. 

Author Response

Dear Reviewer,

 

We are so grateful for the excellent suggestions and the detailed revising from you. We have benefited greatly from the revision. Now we response all the questions point by point and made modification accordingly.

 

Kind regards,

 

6^th 5, 2023

 

Point 1: The eclosed males WERE put into the female's beaker...

Cover the mouth of the beaker with clean gauze. ... Should be changed into - The beaker was covered with clean gauze.

Adult worms waiting to emerge lay eggs on the gauze... should be corrected to ... After emergence adults were left to lay eggs on the gauze... (if that was the context).

Please act accordingly in the rest of the text.

Respond 1: Thank you for your kindly suggestions. We had revised it.

 

Point 2: Additionally, it is not given what was “appropriate feed”. What was amount of feed? This complete response 1 should be placed in the section “Test insects”

Respond 2: Thank you for your kindly suggestions. The amount of feed per box is 70-80 g. This section has been written into the "Testing Insects" section.

 

Point 3: Please add the number of larvae per replicate from the response to the main text because you did not give it there. The sentence given in response 2 is: Each treatment and control group consisted of six replicates (Table S1).

Respond 3: Thank you for your kindly suggestions. We had added it.

 

Point 4: L88 Please correct “feeding conditions“ to the “rearing conditions“

Respond 4: Thank you for your kindly suggestions. We had revised it. 

 

Point 5: L276 Please replace “body wall“ with  “the rigid cuticle that cannot be expanded”

Respond 5: Thank you for your kindly suggestions. We had revised it.

Author Response File: Author Response.pdf