Diet Induced Variation in Gut Microbiota Is Linked to the Growth Performance of an Agricultural Pest Chilo suppressalis
, Mahmoud Magdy 3, Jingnan Chen 2, Weihua Ma 1,* and Letian Xu 2,*
Round 1
Reviewer 1 Report
The manuscript entitled «Diet induced variation in gut microbiota is linked to the growth performance of an agricultural pest Chilo suppressalis» is of undoubted scientific interest and deserves publication in a journal of this type. I believe that the current version of the manuscript should be slightly improved to ensure greater durability of the results achieved. In general, the manuscript is well written and the procedures are well described, but in the section "methods" there are some points that need to be improved (for more details, see below).
I have only a few questions for the methods section.
1. What age were the larvae at the time of harvesting from the fields. How long were the larvae kept in the lab before the experiment started?
I'm confused.
First, the larvae were kept in the laboratory on an artificial diet and then changed the diet to different plants, right? Or were the larvae immediately fed of laboratory of corn, rice, oats and an artificial diet? It's about plants, right? If so, which crops were used: lab-grown or wild? Add information, please.
2. In addition, the abbreviations of the variants LACO, LARE, etc. should be given in section 2.1. where the authors give the design of the experiment.
Author Response
The manuscript entitled «Diet induced variation in gut microbiota is linked to the growth performance of an agricultural pest Chilo suppressalis» is of undoubted scientific interest and deserves publication in a journal of this type. I believe that the current version of the manuscript should be slightly improved to ensure greater durability of the results achieved. In general, the manuscript is well written and the procedures are well described, but in the section "methods" there are some points that need to be improved (for more details, see below).
Response: Thanks for your positive comments and suggestions. And by following your suggestion, we have carefully revised the manuscript to address the issue in methods. Please see the point-to-point response attached below.
I have only a few questions for the methods section.
Comment 1. What age were the larvae at the time of harvesting from the fields. How long were the larvae kept in the lab before the experiment started?
I'm confused.
First, the larvae were kept in the laboratory on an artificial diet and then changed the diet to different plants, right? Or were the larvae immediately fed of laboratory of corn, rice, oats and an artificial diet? It's about plants, right? If so, which crops were used: lab-grown or wild? Add information, please.
Response: We are sorry for the misunderstanding. The 3rd-4th instar larvae were collected from the fields. The larvae were kept in the lab on an artificial diet until the next generation emerged, and the newly laid eggs were collected for the experiment. We fed the newly emerged larvae with different diets immediately. The healthy and fresh plant materials were collected from our experimental fields. The details have been added in P2 lines 83-94.
Comment 2. In addition, the abbreviations of the variants LACO, LARE, etc. should be given in section 2.1. where the authors give the design of the experiment.
Response: Thanks for your suggestion. We have added the abbreviations in the design of the experiment (P2 lines 89-90).
Author Response File: 
Author Response.docx
Reviewer 2 Report
The reviewed manuscript touches on a very interesting topic, the effect of the diet of a pest of rice crops and water oats (Chilo suppressalis) on the formation of the intestinal microbiota which may further affect its functioning. Using amplicon-based next-generation sequencing (amplicon-based NGS), the Authors examined the gut bacterial composition of C. suppressalis larvae reared on rice (Oryza sativa), water oats (Zizania latifolia), corn (Zea mays), and an artificial diet. In addition, the Authors studied the size of the larvae and their foraging rate on different types of diet. The study showed high bacterial diversity, especially between larvae fed an artificial diet (lowest bacterial diversity among all samples) and larvae fed corn (most diverse microbial community). The entire layout of the work is correct and properly prepared. All chapters are adequately and comprehensively described. The hypothesis established by the authors has been confirmed and properly confirmed. I recommend approval of the manuscript in its current form.
Author Response
We really appreciate your positive comments.
Reviewer 3 Report
Dear Authors,
I attached my reviewer's opinion.
Best wishes
Comments for author File: 
Comments.pdf
Author Response
Reviewer’s opinion about MS entitled “Diet induced variation in gut microbiota is linked to growth
studies. growth performance of an agricultural pest Chillo suppressalis”.
General comments
This study extends our knowledge of microbial-insect interactions of Chillo suppressalis larvae in response to dietary variation. The results obtained may serve as an important biological contribution to the host plant choice selection features of this polyphagous pest.
Response: We really appreciate your positive comments. By following your suggestion, we have carefully revised the manuscript. And the specific responses are listed point-to-point below.
Specific remarks
Comment 1: First of all, it should be noted that the order of citation of the manuscript is contrary to the requirements of Agronomy MDPI. The form of citations in the text and the order of the cited bibliographical sources are not alphabetical but chronological.
Response: Thanks for your reminding. The references have been reorganized in accordance with the requirements of Agronomy MDPI.
Comment 2: In the first section of the Introduction, I am less understanding of the highlighting of the Chinese example. Is that the only place where this pest is found? Somehow this idea should be redirected or this section needs to be deleted or rephrased.
Response: Thanks for your concern. By following your suggestion, we rephrased the section as follows: Rice (Oryza sativa) has been considered the most popular staple food, which feeds more than three billion people around the world. Rice was usually planted in a mosaic fashion or under a crop rotation system with 'Jiaobai', which is also called water-oat (Zizania latifolia) (P1 lines 34-36).
Comment 3: In the methodology, the display of the photoperiod is wrong, as not only the light period should be described, but also the dark period L/D=16/8.
Response: Thanks for your reminding, and the sentence has been revised as follows: … on an artificial diet in a climatic chamber with 28 ± 1 °C, a 16: 8 h light: dark photoperiod and 80% relative humidity (P2 lines 84-85).
Comment 4: The literature supporting the abiotic needs for insect raising should be added. What
equipment provided the required abiotic elements during the experiment. Perhaps a climatic chamber, or thermostat, if so the parameters of this should also be presented.
Response: Yes, the insect was raised in a climatic chamber, which has been added in the revised manuscript (P2 line 84).
Comment 5: The exact origin and health status of the plant and insect samples should be mentioned.
Response: Thanks for your suggestion and we are sorry for missing the information. The details of the origin and health status have been added as follows:
Rice shoots, corn, and water-oat pulps were collected from our experimental field at Huazhong Agricultural University, Wuhan, Hubei Province, China. These healthy and fresh plant materials were washed with distilled water three times and dried in the air before larvae feeding (P2 lines 90-93).
The information on insect samples and status was seen in lines 86 to 89: “A total number of 600 healthy larvae were used with an arrangement of 150 larvae for each treatment were reared on four different types of diet: corn, rice, water oat and artificial diet for 20 days till reaching the fourth instar.”
Comment 6: For statistical analysis, the name of the analysis program and its origin are missing.
Response: The details have been added as follows: SPSS (Version 27.0.1), Vegan package in R (Version3.3.1) and PICRUSt2 tool (https://github.com/picrust/picrust2) in line 140, 145 and 146, respectively.
Comment 7: In the labelling of the results, it is necessary to indicate the degrees of freedom (df) and the exact p-values for the statistical values.
Response: Done.
Comment 8: The results are merged with the tables. It is difficult to separate the text from the figure and table captions. Line insertion is required.
Response: Done.
Comment 9: There is a lot of space between lines 189-190 of the manuscript. This section needs to be restructured.
Response: Done.
Comment 10: The evaluation of chapter (3.3) should start with a text, not a diagram.
Response: Done.
Comment 11: Intermediate words in chapter titles should begin in lower case.
Response: Done.
Comment 12: This sentence should be deleted from the discussion "Thus, this study tried to clarify how gut microbiota and its potential function of an important pest C. suppressalis responses to the change of dietary regimes, which was further linked to the pest performance on each diet". No need to repeat the previous data.
Response: Done.
Comment 13: There should be no figure or table reference in the discussion
Response: Done.
Comment 14: The discussion at the end of the paper should also include a description of the advantages, disadvantages, limitations and not least the practical, economic benefits and applicability of the study. It should also present the possibilities for further development of the experiment and the way forward
Response: Thanks for your suggestion. The description has been added as follows: (P9 lines 322-326, 329-330, 336-340 and lines 345-346).
Comment 15: The author's contribution section has not been prepared according to the requirements of Agronomy MDPI.
Response: Thanks for pointing the issue out. The section has been revised as follows: Conceptualization, P.Z., B.M., W.M. and L.X.; Methodology and Validation, P.Z., B.M., M.M. and J.C.; Software, P.Z. B.M. and J.C.; Formal Analysis, P.Z. and B.M.; Data Curation, P.Z., B.M., W.M. and L.X.; Writing – Original Draft Preparation, P.Z., B.M., and L.X.; Writing – Review & Editing, W.M. and L.X.; Visualization, P.Z.; Supervision, W.M. and L.X.; Project Administration, W.M. and L.X.; Funding Acquisition, L.X.. All authors contributed to the manuscript and approved the final version.
Comment 16: The reference form is not following the requirements of Agronomy MDPI.
Response: The references have been reorganized in accordance with the requirements of Agronomy MDPI
Comment 17: I think the manuscript is a well-written and very useful work. The statistical description is well supported and analysed using appropriate methods. The publication of its results could be of great importance for the development of insect olfactory and host plant choice studies. I propose to publish the manuscript after correcting the errors.
Response: We really appreciate your positive comments. By following your suggestion, we have carefully revised the manuscript.
Author Response File: Author Response.docx
