The Endogenous Metabolic Response of Tribolium castaneum under a High Concentration of CO2
Round 1
Reviewer 1 Report
The manuscript "Study on the endogenous metabolic response in Tribolium castaneum under high concentration of CO2.The idea is good and the manuscript is well written and presented well, there are some minor mistakes that are mentioned in the annotated file for correction. This investigation will surely help to assist policy makers to make decisions for the management of stored grain insect pests which have become a threat to the stored products industry worldwide.
Comments for author File: 
Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 2 Report
This article's subject is quite interesting and timely, it could add valuable information on the effects of CO2 on T. castaneum control strategies. However, there are flaws in terms of the writing that do not allow the acceptance without major revisions, as this impairs article understanding by the readers. Also, the methodology could be improved, as there are some parts that are not fully explained.
For example, in the discussion, between lines 254 and 264 (the beginning) a resume on results about Drosophila spp. flies are done, and this is not linked to this study, so we don't understand what's the advantage of that text for the article. The results of the flies could be compared with the ones obtained in this study, but very carefully, as those organisms are different from the red flour beetle.
The results are interesting, but the discussion should be improved with more references to studies involving CO2 treatments and metabolic responses of insects, as it lacks references and comparisons, which would certainly shed a light on these results interpretation and elevate the quality of this article. The abstract should be improved accordingly with the discussion, stating clearly the advances achieved by this work.
For example, studies like these ones could eventually be consulted and added to the discussion: https://doi.org/10.1649/0010-065X-74.1.127; https://link.springer.com/article/10.1007/s11356-017-8860-5; https://doi.org/10.1016/j.jspr.2014.06.004; https://www.sciencedirect.com/science/article/abs/pii/S0022474X21001338; https://www.sciencedirect.com/science/article/abs/pii/S0022191013001807, among many others that might be selected.
Comments for author File: Comments.pdf
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
The paper is original and deals with important topics, which are selected physiological, biochemical and toxicological aspects of controlled atmospheres (CO2-based) to control the warehouse pest Tribolium castaneum. The data and results are interesting and useful and therefore I recommend them for publication. The problem with the evaluated paper is that it is written in a way that is largely incomprehensible to the reader. For example, the abstract, introduction and the beginning of the "Material and Methodology" section should explain why and for what purpose molecular analyses were used. Although they are an important part of the work there is no mention of them in these sections. This makes the paper difficult to read, especially for readers who are not narrow specialists in the subject. There are a number of incorrect statements in the abstract introduction and discussion and irrelevant citations/references are used. English and incorrect terminology (e.g. „virulence” of CO2) MUST BE revised prior re-submission of the corrected MS.
Title
Delete “study of” ….. Here I suggest to consider more applied and general title since Agronomy is not specialised entomological journal
Abstracts
---It is not written properly. It is not clear, incorrect terminology is used and several sentences are vague- general thus say almost nothing
---Be more specific about using vague statements “low or high” concentrations of CO2 trough the MS.: Define ranges of CO2 concentrations that are considered low or high.
Line 13 - virulence
Pathogens are virulent …. But not CO2.
Line 16- 17“At the same time, biochemical and physiological indexes of different treatment groups were examined”
----This sentence has no content (says nothing). Be more concrete or delete it.
Line -15-16. adaptation indexes under different treatments, including mortality, pupation rate, eclosion rate and body weight
----First to hear that “mortality, pupation rate, eclosion rate and body weight” may be considered as a sort of “adaptation indexes”. Please explain and provide some references fro that…. To me is the wrongly used terminology
Line 1ř counted various adaptation indexes
Better use “estimated” than “counted”
Line 24 adversities
Do you intended “stress factors”?
Introduction
Line 30 Red flour beetles, Tribolium castaneum (Herbst), belonging to Coleoptera, is a
-Change Red flour beetle….. is a
Line 33- 34 - T. castaneum feeds on the germ and endosperm of grain a ….
- Not correct statement it is secondary pest so does not eat endosperme (just the germ) and dust
Line “Once infestation levels are high, its glands secrete a fluid that produces a musty odor in the 35 flour and this secretion also contains the carcinogen benzoquinone, making the grain unfit 36 for consumption [4-5…
- References 4 and 5 are irrelevant to the claim. The should be replaced; e.g. medical aspects of SSP and Tribolium are covered in the recent review published in Annual review of Entomology https://doi.org/10.1146/annurev-ento-020117-043218))
Line 37-38. Pest control strategies for grain storage rely heavily on fumigation, insecticide spraying,…
-Not only chemical but also physical methods are used, particularly low storage temperatures are used to prevent development of stored product pests (e.g. see the review recently published in MDPI Insects -journal - https://doi.org/10.3390/insects10050149)
Materials and methods
Line 85 T. castaneum used in this experiment were
-was
Line 89 Uniform number of 8th instar larvae:
----What do mean by “uniform number of larvae”?
Line 99-100. The survival of the test insects was recorded at 24 h intervals until all test insects died 99 or became adults. recorded pupation rate and eclosion rate if pests pupated.
---What do mean by “recorded pupation rate and eclosion rate if pests pupated” ?
Line 100 -101- Materials of 100 assay were collected at 48 h after TD treatment (according to the mortality statistics of air
I do not understand the sentence.
Line 106 If the test worms
Do not use term worm!!! … larvae
RESULTS
Line 189 - phenotypes?
May be “morphological features”?
Figure 1 - captions.. x-axes o the graphs
What do you mean by “ Treat time” ? It is not clear. Is it “Time after treatment” or “Exposure time”?
Figure 3. The changes of three kinds of carbohydrates of T. castaneum treated with different concentrations of high carbon dioxide.
Check the meaning : the sentence says that carbohydrates were treated by CO2
4. Discussion
Line 250 Insects growing underwater,
Insects develop - they do not grow.
Line 254 or 271 -Drosophila
Latin names are written in Italics
268 - 269 … the pupae were unable to pupate successfully
- Incorrect entomological terminology: larvae pupate, but pupae do not pupate
Author Response
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Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
This version is much better, congratulations to the authors.
Just to clarify one of the questions left open in the responses: The assumptions of the ANOVA are tests made to the data (before the anova) to determine if the data is normally distributed (for example the Shapiro-Wilk test) and if the variances are homogenous (for example, the Levene's test), if your data does not meet these two assumptions then you should eventually use a non-parametric test to investigate your null hypothesis.
