Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

It is recommended that at the end of the introduction you clearly state the objective of this work, as this is not entirely clear to the reader.

The methodology seems adequate for the study conducted; it only needs clarification regarding the type of aphids used in the bioassays: those fed on soybeans or on ragweed?

 

It is also suggested that they review their similarity report, which is 28% and should be at least 15%.

They should also explain why they selected that age for the aphids in the bioassays and why they chose those insecticides for the tests.

Author Response

Reviewer 1

It is recommended that at the end of the introduction you clearly state the objective of this work, as this is not entirely clear to the reader.

Response from author:

Thanks for your kindly comments. We had added this section. Please check Lines 85-86.

 

The methodology seems adequate for the study conducted; it only needs clarification regarding the type of aphids used in the bioassays: those fed on soybeans or on ragweed?

Response from author:

Thanks for your kindly comments. In this study, both experimental populations were selected for bioassays. We had revised this section. Please check Lines 156-157.

 

It is also suggested that they review their similarity report, which is 28% and should be at least 15%.

Response from author:

Thanks for your kindly comments. The higher repeat rate mainly in Methods and Materials section. We have revised our manuscript according to the iThenticate report. Please check.

 

They should also explain why they selected that age for the aphids in the bioassays and why they chose those insecticides for the tests.

Response from author:

Thanks for your kindly comments.

（1）Due to the unstable physiological state, small size, and other characteristics of aphids during the nymph stage, it can lead to inaccurate biological measurement results. Additionally, older adult aphids may accumulate more nutrients or detoxifying enzymes through feeding, leading to an increase in their tolerance to pesticides. If it is a mother aphid producing offsprings, the synthesis of yolk protein in the body will further alter the metabolic profile, affecting the authenticity of the results. Therefore, as in most studies, one-day-old adult aphids were used in bioassays.

（2）Because those insecticides are the most commonly used ones for controlling A .glycines. This reason has been added in the Materials and Methods section. Please check Lines 150-151.

Reviewer 2 Report

Comments and Suggestions for Authors

 

Short summary:

The authors hypothesize that A. glycines growing on A. artemisiifolia causes the aphids to develop counter-adaptations through coevolution. They further claim that this adaptation has increased tolerane to plant secondary metabolites, which might potentially culminate to enhanced resistane to insecticides.

They have reared the aphid population on G. max plants and A. artemissifolia plants.

They have performed differential gene expression analysis and functional annotation to demonstrate that certain genes are differentially expressed among the aphids reared on A. artemissifolia plants versus those reared on G. max. They have confirmed these results with qRT-PCR.

They have performed bioassays using three insecticides: imidacloprid, thiamethoxam, and lambda-cyhalothrin. They have shown that A. glycines reared of A. artemissifolia showed higher insecticide resistance compared to conspecifics reared on G. max.

 

Major concerns:

The assumption that just because A. glycines infest A. artemissifolia plants, they must have developed enhanced insecticide resistance is overreaching. Unless it could be proven that A. glycines do not feed on any plants other than G. max and A. artemissifolia, such a claim would be unwarranted.

Line 84: “provide a foundation for optimizing insecticide use in integrated pest management.” The authors should elaborate on how their work will help optimize insecticide use.

Line 299: “critical insights for developing more proactive and sustainable pest management strategies.” This line appears like an overestimation of the results. The authors are recommended to elaborate on what proactive pest management strategies could be applied based on their study.

Minor suggestions:

Line 56: herbivore resistance to herbivores

Line 95:If possible, the exact number of generations for which the insects were reared should be included.

Line 178: What was the P-value for the 4250 differentially expressed genes.

Figure 1: the top 20 DEG involved in different GO classification is not adding any specific information. For example, significant padj value for a category like “catalytic activity” is fairly vague. Consider not including this. Alternatively, the authors may try to attempt to include only the statistically significant genes or show some significant biological connection between GO classification and KEGG classification.

Figure 2: For simplicity, it might be better to only include the five UGT family genes that show a statistically significant difference between aphids reared on G. max versus A. artemissifolia.

The authors should include future directions, for example potentially perform knock-out experiments to show how insecticide resistance is lost after knocking out specific UGT family genes.

 

 

Author Response

Reviewer 2

Short summary:

The authors hypothesize that A. glycines growing on A. artemisiifolia causes the aphids to develop counter-adaptations through coevolution. They further claim that this adaptation has increased tolerane to plant secondary metabolites, which might potentially culminate to enhanced resistane to insecticides.

They have reared the aphid population on G. max plants and A. artemissifolia plants.

They have performed differential gene expression analysis and functional annotation to demonstrate that certain genes are differentially expressed among the aphids reared on A. artemissifolia plants versus those reared on G. max. They have confirmed these results with qRT-PCR.

They have performed bioassays using three insecticides: imidacloprid, thiamethoxam, and lambda-cyhalothrin. They have shown that A. glycines reared of A. artemissifolia showed higher insecticide resistance compared to conspecifics reared on G. max.

Major concerns:

The assumption that just because A. glycines infest A. artemissifolia plants, they must have developed enhanced insecticide resistance is overreaching. Unless it could be proven that A. glycines do not feed on any plants other than G. max and A. artemissifolia, such a claim would be unwarranted.

Response from author:

Thanks for your kindly comments. We agree with your opinion. A. artemisiifolia contains various bioactive secondary metabolites, which collectively constitute a potent chemical challenge to herbivorous insects. It has been reported that chemical substances within A. artemisiifolia show inhibitory activity against many insects, including melon aphid and other Lepidoptera (Han 2021; Zhang et al. 2010). One of the main ways to adapt to new hosts is to enhance the activity of detoxification enzymes. This mechanism is precisely the one that enhances insecticide resistance. Therefore, as we mentioned in Introduction, adaptation to A. artemissifolia likely activates detoxification pathways, potentially driving enhanced insecticide resistance.

 

Line 84: “provide a foundation for optimizing insecticide use in integrated pest management.” The authors should elaborate on how their work will help optimize insecticide use.

Response from author:

Thanks for your comments. This section has been revised. Please check Lines 89-90.

 

Line 299: “critical insights for developing more proactive and sustainable pest management strategies.” This line appears like an overestimation of the results. The authors are recommended to elaborate on what proactive pest management strategies could be applied based on their study.

Response from author:

We have revised this section. Please check Lines 314-316.

 

Minor suggestions:

Line 56: herbivore resistance to herbivores

Response from author:

We have revised this section. Please check Line 57.

 

Line 95:If possible, the exact number of generations for which the insects were reared should be included.

Response from author:

The aphid clone on G. max plants (Ag-G) and A. artemisiifolia plants (Ag-A) was set in 2022, and they have been continuously raised up to now. Generally, one generation takes five days. Therefore, the exact number of generations is not clear, but it's at least ten generations. We have revised this section. Please check Line 99.

 

Line 178: What was the P-value for the 4250 differentially expressed genes.

Figure 1: the top 20 DEG involved in different GO classification is not adding any specific information. For example, significant padj value for a category like “catalytic activity” is fairly vague. Consider not including this. Alternatively, the authors may try to attempt to include only the statistically significant genes or show some significant biological connection between GO classification and KEGG classification.

Figure 2: For simplicity, it might be better to only include the five UGT family genes that show a statistically significant difference between aphids reared on G. max versus A. artemissifolia.

Response from author:

We had added the statistically significant genes in the supplementary file, please check. And the figure 2 had been revised. Please check Line 215.

 

The authors should include future directions, for example potentially perform knock-out experiments to show how insecticide resistance is lost after knocking out specific UGT family genes.

Response from author:

Thanks for your kindly comments, we have added future directions in Conclusions section. Please see Lines 310-312.

Reviewer 3 Report

Comments and Suggestions for Authors

After reading the article, I consider the topic to be current and under-explored, with a clear and conceptually well-founded hypothesis supported by recent literature. The literature review is comprehensive, citing recent works on GTUs, detoxification, host adaptation, and insecticide resistance. The authors articulate well the literature on plant–insect coevolution, xenobiotic detoxification pathways, and metabolic resistance. In order to make the article more fluid for the reader, I offer my considerations.

Replace the keywords Aphis glycines and Ambrosia artemisiifolia, as they appear in the title.

The manuscript, in its current form, needs substantial revisions before it can be considered for publication. The main points that compromise the clarity and soundness of the conclusions are as follows: one severe criticism I have is in relation to the English language. There are several language issues that require careful revision, such as: typos or grammar errors: “educes crop yields,” probably “reduces crop yields.” “thereby for and maintaining individuals with high basal detoxification enzyme activity,” truncated sentence (“thereby favoring and maintaining...,” for example). “A. glycines successfully colonize this invasive plant,” agreement (“colonizes”). Terms changed in the text, as in results. “Bifenthrin” is mentioned, but everywhere else the insecticide is “lambda-cyhalothrin” (Table 4). Please clarify if I have misunderstood something. Also, some sentences are too long, with several ideas strung together, which hinders fluidity. I recommend paying attention to correcting typos, subject-verb agreement, avoiding very long sentences and redundancies, and standardizing the nomenclature of insecticides and species.

In the methodology, essential information is missing about the number of generations maintained in each host; the independence of biological replicates; the composition of the samples used in RNA-seq; the controls and design of the bioassays (including the solvent, experimental independence, and formal statistical tests for LC₅₀ comparison); the discussion of the range of ICs and the limitations of toxicity data. The manuscript does not specify how many generations were maintained or whether the three replicates are truly independent (three separate cages) or subsamples from the same colony, posing a risk of pseudoreplication.

The authors validated 17 UGTs, but did not include genes from other pathways (P450s, GSTs, ABC transporters). The contributions went beyond the empirical support of the results.

Comments on the Quality of English Language

The manuscript, in its current form, requires substantial revisions before it can be considered for publication in English, as it contains serious flaws in its use of language.

Author Response

Reviewer 3

After reading the article, I consider the topic to be current and under-explored, with a clear and conceptually well-founded hypothesis supported by recent literature. The literature review is comprehensive, citing recent works on GTUs, detoxification, host adaptation, and insecticide resistance. The authors articulate well the literature on plant–insect coevolution, xenobiotic detoxification pathways, and metabolic resistance. In order to make the article more fluid for the reader, I offer my considerations.

 

Replace the keywords Aphis glycines and Ambrosia artemisiifolia, as they appear in the title.

Response from author:

We have revised this section, “Aphis glycines and Ambrosia artemisiifolia” have been replaced as “aphid and invasive plants”. Please check Line 31.

 

The manuscript, in its current form, needs substantial revisions before it can be considered for publication. The main points that compromise the clarity and soundness of the conclusions are as follows: one severe criticism I have is in relation to the English language. There are several language issues that require careful revision, such as: typos or grammar errors: “educes crop yields,” probably “reduces crop yields.” “thereby for and maintaining individuals with high basal detoxification enzyme activity,” truncated sentence (“thereby favoring and maintaining...,” for example). “A. glycines successfully colonize this invasive plant,” agreement (“colonizes”). Terms changed in the text, as in results. “Bifenthrin” is mentioned, but everywhere else the insecticide is “lambda-cyhalothrin” (Table 4). Please clarify if I have misunderstood something. Also, some sentences are too long, with several ideas strung together, which hinders fluidity. I recommend paying attention to correcting typos, subject-verb agreement, avoiding very long sentences and redundancies, and standardizing the nomenclature of insecticides and species.

Response from author:

Thanks for your kindly comments. We have carefully revised through manuscript.

 

In the methodology, essential information is missing about the number of generations maintained in each host; the independence of biological replicates; the composition of the samples used in RNA-seq; the controls and design of the bioassays (including the solvent, experimental independence, and formal statistical tests for LC₅₀ comparison); the discussion of the range of ICs and the limitations of toxicity data. The manuscript does not specify how many generations were maintained or whether the three replicates are truly independent (three separate cages) or subsamples from the same colony, posing a risk of pseudoreplication.

Response from author:

Thanks for your kindly comments.

• The aphid clone on maxplants (Ag-G) and A. artemisiifolia plants (Ag-A) was set in 2022, and they have been continuously raised up to now. Generally, one generation takes five days. Therefore, the exact number of generations is not clear, but it's at least ten generations. We have revised this section. Please check Line 99.
• Total RNA was extracted from the following groups: aphids reared on maxplants and A. artemisiifolia plants from separate cages. The extraction was performed on the one-day-old apterous adult A. glycines using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Please check Lines 104-107.
• The composition of the samples used in RNA-seq was the total RNA. Please check Line 109.
• Regarding the section on "the controls and design of the bioassays", we believe that we have clearly stated it in the Materials and methods. Like others and the articles we have published before, everyone operates and writes in this way.
• In this study, the LC₅₀value within the range of ICs indicates the accuracy of the experimental results. In general, this makes no sense for discussion. We haven't seen any related discussions in other research articles either. Therefore, we did not discuss the range of ICs and the limitations of toxicity data.

 

The authors validated 17 UGTs, but did not include genes from other pathways (P450s, GSTs, ABC transporters). The contributions went beyond the empirical support of the results.

Response from author:

Thanks for your comments, as you suggest, many pathways would contribute to detoxification in insects. In this study, differential gene analysis revealed that UDP-glycosyltransferase (UGT) genes were the most prominently enriched functional category among detoxification-related metabolism genes. Additionally, it is confirmed that various roles of UGT would contribute to host adaptation and insecticide resistance. Associated information has been discussed in Discussion section. Therefore, our study concerned UGT.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors The authors were careful to clarify my doubts and make adjustments to the manuscript.
I am satisfied. Comments on the Quality of English Language I consider it important to review the English text before publication.