Review Reports - Characterization of a Stably Maintained, Highly Beta-Cypermethrin-Resistant Strain of the Bird Cherry-Oat Aphid Rhopalosiphum padi (Hemiptera: Aphididae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review of the Manuscript “Characterization of field-evolved, stably heritable beta-cypermethrin resistance in the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae)” by Xinan Li, Peipan Gong, Zilan Zhao, Fengfan Wang, Qiuchi Li, Xiangrui Li, Yunhui Zhang, and Xun Zhu

The manuscript (MS) presents an experimental investigation into a broad range of issues related to the development and maintenance of resistance to a pyrethroid insecticide in an economically important aphid species. The authors' work includes the identification of physiological, biochemical, and genetic mechanisms, as well as assessments of cross-resistance to different compounds and the fitness costs in the insects. The results, based on substantial experimental data, are of considerable interest for the optimization and improvement of chemical plant protection of insect pests.

Despite an overall positive assessment, the MS requires some revision, as in its current form it raises a number of questions.

First, it is advisable to clearly formulate the aim and objectives of the study; this requires editing the final section of the Introduction (lines 99–108).

The MS text needs to be made clearer and more consistent. For instance, many abbreviations (e.g., DMF, Tween 80, EDTA, 1 mM DTT, 1 mM PTU, 1 mM PMSF) are used without definition, whereas others (e.g., CarE, GST, P450) are explained.

Furthermore, the information regarding statistical data processing is rather superficial. It remains unclear what exactly the authors mean by "DPS software, version 7.05" (line 143) – possibly the Development Programming System from General Motors, which is engineering software for programming and testing electronic control units (ECUs) in GM vehicles. In any case, a reference to the software used should be provided, as it is clearly not among the commonly used tools in the field of biological research. In addition, the meaning of the term "Slope" in the same line is unclear. Moreover, in the MS text (e.g., in section 2.4. Synergist bioassays), the formulas for calculating the synergistic ratio and relative synergism ratio should have been given; similarly, in section 2.3 Life-table analysis, the formulas for the intrinsic rate of increase, net reproductive rate, and mean generation time mentioned in Table 2 should have been presented. In general, statistical issues in the Methods section are treated rather cursorily (e.g., the Student’s t‑test, lines 183–185), whereas the use of Fisher’s criterion is only mentioned in the note to Figure 2 in the Results section. This lack of detail in the Methods leaves the reader without an understanding of what the "p values" columns in Tables 1, 3, and 4 represent. Incidentally, even the note to Table 4 (which repeats the text from the Methods) makes it difficult to understand how the SR estimates were calculated. Here, as well as in the Methods, the formula for calculating this estimate should simply have been provided.

In places, the wording needs improvement. For example, given that n=5 (which is quite small), instead of stating "There were no significant differences in CarE activity between the two strains (F = 5.927, P = 0.072)" (lines 250–251), it would be better to write something like "we failed to detect statistically significant differences." Indeed, Figure 2A, which at first glance does not differ markedly from Figures 2B and 2C, leads the reader to suspect that with a slightly larger number of replicates, say 7–8, P = 0.05 might have been reached.

Among minor issues, the Latin name Drosophila melanogaster (line 126) should be italicized. Reference #28 in the References, oddly referred to as a DOI (140.120.197.173/Ecology/Papers/04-1985-Chi-Liu-2011%20ver.pdf), is not a DOI at all. Finally, reference #9 does not provide an actual DOI link.

As for the file Text S1. Supplementary Text "Insecticides, Chemicals, and Synergists", the term "synergist" is mentioned only once (i.e., in the title) and does not appear in the body of the text.

Author Response

First, it is advisable to clearly formulate the aim and objectives of the study; this requires editing the final section of the Introduction (lines 99–108).

Author’s Response: We have revised it in lines 97 to 105.

The aim of this study was to characterize beta-cypermethrin resistance in R. padi and to investigate the potential mechanisms by which this resistance can reach and persist at a high level. The specific objectives were: (1) to quantify the resistance level and its stability across generations; (2) to evaluate the associated fitness cost using the age-stage, two-sex life-table approach; (3) to determine cross-resistance to other insecticides; (4) to identify the roles of detoxifying enzymes and (5) to identify VGSC target-site mutations and function-ally confirm the M918L mutation using transgenic D. melanogaster. These findings will fa-cilitate effective resistance management and the rational use of pyrethroids against R. padi.

Author’s Response: We thank the reviewer for pointing this out. We have now carefully checked the entire manuscript and revised it so that all abbreviations are defined at their first occurrence in the main text, and are used consistently thereafter. Specifically, we have added definitions for DMF (N,N-dimethylformamide), Tween 80 (polyoxyethylene sorbitan monooleate), EDTA (ethylenediaminetetraacetic acid), DTT (dithiothreitol), PTU (1-phenyl-2-thiourea), PMSF (phenylmethylsulfonyl fluoride), BSA (bovine serum albumin), CDNB (1-chloro-2,4-dinitrobenzene), α-NA (α-naphthyl acetate), PBO (piperonyl butoxide), TPP (triphenyl phosphate), DEM (diethyl maleate), APRP (adult pre-reproductive period), TPRP (total pre-reproductive period), r (intrinsic rate of increase), R0 (net reproductive rate), T (mean generation time), and VGSC (voltage-gated sodium channel) in the text.

Author’s Response:

Regarding “DPS software, version 7.05,” we clarify that DPS stands for Data Processing System, a statistical software package developed by Tang and Zhang specifically for entomological and biological research. It is not the General Motors Development Programming System. We have revised and added the appropriate reference in line 139: Tang Q.Y. and Zhang C.X. (2012). Insect Science, DOI: 10.1111/j.1744-7917.2012.01519. x

In addition, the meaning of the term "Slope" in the same line is unclear.

Author’s Response: We have revised it. The term “Slope” refers to the slope of the probit regression line, which reflects the heterogeneity of the test population’s response to the insecticide. We have now defined this explicitly in line 137.

Moreover, in the MS text (e.g., in section 2.4. Synergist bioassays), the formulas for calculating the synergistic ratio and relative synergism ratio should have been given; similarly, in section 2.3 Life-table analysis, the formulas for the intrinsic rate of increase, net reproductive rate, and mean generation time mentioned in Table 2 should have been presented.

Author’s Response: We have revised it. We have added the missing formulas. In Section 2.4, we now provide: The synergistic ratio (SR) was calculated as LC₅₀ (insecticide alone) / LC₅₀ (insecticide + synergist), and the relative synergism ratio (RSR) was calculated as the SR of the Beta-R strain with the synergist divided by the SR of the SS strain with the synergist. In Section 2.3, we have added the standard life-table formulas: R₀ = ∑lₓmₓ; r is solved from the Euler-Lotka equation ∑e⁻ʳ⁽ˣ⁺¹⁾lₓmₓ = 1; and T = ln(R₀)/r.

In general, statistical issues in the Methods section are treated rather cursorily (e.g., the Student’s t‑test, lines 183–185), whereas the use of Fisher’s criterion is only mentioned in the note to Figure 2 in the Results section.

Author’s Response: We have revised it in line 186-189 and line 269-272.

line 186-189.

The significance of the differences in enzyme activities between the SS and Beta-R strains was determined by Student's t test using GraphPad Prism 8.0.1 (GraphPad Software Inc., La Jolla, CA, USA), with the threshold of significance set at P < 0.05. All data are presented as means ± standard errors of five replicates (n = 5).

line 269-272.

Figure 2. Specific activities of carboxylesterase (CarE, A), glutathione-S-transferases (GSTs, B), and cytochrome P450 monooxygenases (P450s, C) in Rhopalosiphum padi beta-cypermethrin-susceptible (SS) and beta-cypermethrin-resistant (Beta-R) strains after their 62nd generations. Data are presented as means ± SE (n = 5). *Significant difference according to Student's t test (P < 0.05).

This lack of detail in the Methods leaves the reader without an understanding of what the "p values" columns in Tables 1, 3, and 4 represent.

Author’s Response: We checked all the data in manuscript and recalculated the P value. Additionally, we revised lines 139 as well as Tables 1, 3 and 4.

P < 0.05 indicates a significant linear relationship between the logarithm of insecticide dose and the probability of death.

Incidentally, even the note to Table 4 (which repeats the text from the Methods) makes it difficult to understand how the SR estimates were calculated. Here, as well as in the Methods, the formula for calculating this estimate should simply have been provided.

Author’s Response: We have revised it in table 4. SR = LC₅₀ (insecticide alone) / LC₅₀ (insecticide + synergist)

Author’s Response: We have revised it in line 260-261.

Among minor issues, the Latin name Drosophila melanogaster (line 126) should be italicized.

Author’s Response: We have revised it in line 119.

Reference #28 in the References, oddly referred to as a DOI (140.120.197.173/Ecology/Papers/04-1985-Chi-Liu-2011%20ver.pdf), is not a DOI at all.

Author’s Response: Thank you for pointing this out. We apologize for the confusion caused by the incorrect use of the term "DOI" for Reference #29. You are correct that the string "140.120.197.173/Ecology/Papers/04-1985-Chi-Liu-2011%20ver.pdf" is not a DOI, but rather a URL link to a PDF version of the paper.

Reference #28 (Chi, H.; Liu, H. Two new methods for the study of insect population ecology. Bull. Inst. Zool. Acad. Sin. 1985, *24*, 225–240) is the foundational paper on the age-stage, two-sex life table theory. It was published in an early issue of the journal, at a time when the journal had not yet adopted the DOI registration system. Consequently, no official DOI has been assigned to this article, and it cannot be retrieved via DOI in major academic databases such as Scopus, Web of Science, or PubMed. We have therefore replaced the incorrect "DOI" label with the correct format. The reference now reads as follows:

Chi, H.; Liu, H. Two new methods for the study of insect population ecology. Bull. Inst. Zool. Acad. Sin. 1985, *24*, 225–240.

Finally, reference #9 does not provide an actual DOI link.

Author’s Response: we have provided an actual DOI link of reference #9

As for the file Text S1. Supplementary Text "Insecticides, Chemicals, and Synergists", the term "synergist" is mentioned only once (i.e., in the title) and does not appear in the body of the text.

Author’s Response: We have revised it in line 126-127.

Reviewer 2 Report

Comments and Suggestions for Authors

General Assessment

This study reports a striking level of beta-cypermethrin resistance (4,617-fold) in a laboratory-selected strain of Rhopalosiphum padi derived from a field population, and attempts to link this phenotype to a homozygous M918L (super-kdr) mutation and upregulated detoxifying enzymes (P450s and GSTs). The experimental data are extensive, including life-table analyses, synergism assays, enzyme activity measurements, and transgenic Drosophila functional validation.

However, several fundamental conceptual and interpretational flaws undermine the scientific validity of the authors' conclusions. These issues relate primarily to: (i) the misuse of clonal selection as "evolutionary adaptation" in a parthenogenetic organism; (ii) the overstatement of the M918L mutation's contribution to resistance based on weakly supportive transgenic data; (iii) the erroneous interpretation of fitness cost from life-table parameters; and (iv) selective presentation of cross-resistance data. The manuscript requires major revisionto correct these conceptual errors and re-interpret the findings within the appropriate biological framework.

Major Scientific Concerns

Concern 1: Misrepresentation of "Selection" and "Evolution" in a Parthenogenetic Clone

The authors established the Beta-R strain by selecting a single isofemale line (i.e., a single clone) that exhibited the lowest mortality in initial bioassays, and then subjected this single clone to 22 generations of intermittent insecticide exposure. Because R. padi reproduces via apomictic parthenogenesis, all individuals within this isofemale line are genetically identical to the founder female collected from the field.

Consequently, the high-level resistance (4,617-fold) and the homozygous M918L mutation detected in the Beta-R strain are pre-existing characteristics of that particular field-collected clone, not the products of the 22-generation selection regime. The authors' use of the terms "evolution," "development," and "stably inherited resistance" throughout the Abstract and Discussion is scientifically misleading. In a clonal organism, propagation of a pre-existing resistant clone does not constitute evolutionary adaptation.

Required revision: The entire manuscript must be re-interpreted as a characterization of a naturally occurring high-resistance clone, rather than a study of resistance evolution under selection. All instances of "evolved," "developed," or "selected for homozygosity" must be revised to accurately reflect that the authors selected a pre-existing resistant clone from a mixed field population. The authors should also provide genotyping data (or at least a clear statement) confirming that the founder clone of the Beta-R strain was already homozygous for M918L at generation 0.

Concern 2: Overstatement of the M918L Mutation's Contribution to Resistance

The transgenic Drosophila melanogaster functional assay (Figure 3C) shows that the M918L mutation confers only marginal protection: mortality reduction of 3.65%, 18.96%, and 26.80% at beta-cypermethrin concentrations of 0.2, 1, and 5 mg/L, respectively, compared to wild-type flies.

This modest effect is disproportionately weak relative to the 4,617-fold resistance observed in the aphid clone. The data strongly suggest that M918L is a minor contributor to the resistance phenotype, while the upregulated P450/GST activities (and possibly other undiscovered mechanisms) are the primary drivers. The authors' conclusion that "a single homozygous M918L point mutation may be critical for the high resistance" is not supported by the transgenic evidence.

Required revision: The authors must either: (i) provide quantitative in silico evidence (e.g., molecular docking) demonstrating that M918L drastically reduces beta-cypermethrin binding affinity, or (ii) substantially downgrade their claims about the role of M918L and acknowledge that the resistance phenotype is predominantly driven by the upregulated P450/GSTs, with M918L playing a secondary role.

The Abstract, Results, and Discussion must be revised to reflect this corrected interpretation.

Concern 3: Misinterpretation of Fitness Cost from Life-Table Data

The authors conclude that the Beta-R strain has a "low fitness cost" based solely on the relative fitness ratio r_f=0.92 (r_"Beta-R" /r_"SS" ). This interpretation is statistically and ecologically flawed.

Key facts from Table 2:

Intrinsic rate of increase (r): SS = 0.3884 vs. Beta-R = 0.3594

Difference: highly significant (P<0.0001)

Pre-adult duration: SS = 7.16 days vs. Beta-R = 7.80 days (+0.64 days, P<0.0001)

The intrinsic rate of increase (r) is the definitive composite metric of fitness, integrating survival, development, and reproduction. A highly significant reduction in r (0.3884 → 0.3594) represents a substantial demographic cost. Simple exponential projection (e.g., over 30 days, starting with 100 individuals) yields a >2-fold difference in population size between SS and Beta-R strains. This is not "low" by any ecologically meaningful standard.

Required revision: The authors must revise their Abstract, Discussion, and Conclusions to accurately state that beta-cypermethrin resistance carries a significant fitness penalty, which has critical implications for resistance management (e.g., potential for resistance dilution in the absence of selection pressure). The label "low fitness cost" must be removed entirely.

Concern 4: Selective Presentation of Cross-Resistance Data

The Abstract prominently features the positive cross-resistance to lambda-cyhalothrin (869-fold) and bifenthrin (363-fold) but entirely omits the negative cross-resistance to omethoate (0.61-fold) and chlorpyrifos (0.72-fold) reported in Table 3.

Since the negative cross-resistance data provide the only directly actionable chemical solution for managing this resistance in the field, its omission from the Abstract biases the reader toward a pessimistic view and undermines balanced scientific reporting. The negative cross-resistance is arguably the most practical finding for pest management practitioners.

Required revision: The Abstract must include a concise statement on the negative cross-resistance to organophosphates. If space constraints are cited, the authors should replace less critical statements (e.g., "stably inherited") with the cross-resistance data to ensure balanced presentation.

Minor Revisions

Data presentation consistency (Table 2): The decimal places for the parameters are inconsistent. For example, "1.47 ± 0.04" and "2.00 ± 0.01" are acceptable, but some values (e.g., total longevity: 21.34 ± 0.67 vs. 23.28 ± 0.68) require uniform presentation. The authors should standardize the number of decimal places for all comparable parameters across the table to improve readability and scientific rigor.

Statistical annotation in Figure 2: The authors used Student's t-tests for the enzyme activity comparisons (CarE, GST, P450). The exact P-values (or at least significance thresholds: P < 0.05, < 0.01, or < 0.001) should be indicated directly on the figure panels (e.g., as asterisks above the bars), rather than only described in the legend or text.

Supplementary raw data deposition: The authors should provide the complete raw data for: Life-table observations (individual daily survival and fecundity records), and Insecticide bioassay mortality counts at all concentrations. This will enhance data transparency and reproducibility. The authors may consider uploading these datasets as supplementary spreadsheets (e.g., Excel or CSV files) alongside the manuscript.

Author Response

Major Scientific Concerns

Concern 1: Misrepresentation of "Selection" and "Evolution" in a Parthenogenetic Clone

Author's response: The entire manuscript has been reinterpreted as a description of a naturally occurring highly resistant clone, rather than a study of the evolution of resistance under selective conditions. All references to "evolution", "development", or "selecting for homology" have been corrected. Unfortunately, these generations were not systematically preserved for VGSC cloning and sequencing, as the main focus at that stage was to monitor the gradual increase in resistance levels through biological assays.

Concern 2: Overstatement of the M918L Mutation's Contribution to Resistance

The Abstract, Results, and Discussion must be revised to reflect this corrected interpretation.

Author's response: We have revised the claim that M918L plays a significant role in resistance, and have acknowledged that the resistance phenotype is mainly related to P450/GSTs, while the role of M918L is only a minor factor. We have made revisions in the abstract, results, and discussion sections to reflect this revised interpretation.

Concern 3: Misinterpretation of Fitness Cost from Life-Table Data

The authors conclude that the Beta-R strain has a "low fitness cost" based solely on the relative fitness ratio r_f=0.92 (r_"Beta-R" /r_"SS"). This interpretation is statistically and ecologically flawed.

Key facts from Table 2:

Intrinsic rate of increase (r): SS = 0.3884 vs. Beta-R = 0.3594

Difference: highly significant (P<0.0001)

Pre-adult duration: SS = 7.16 days vs. Beta-R = 7.80 days (+0.64 days, P<0.0001)

Author's response: We have revised the abstract, discussion, and conclusion sections to accurately state that resistance to beta-cypermethrin incurs significant fitness costs, which is of crucial importance for resistance management. The phrase "low fitness cost" has also been deleted.

Concern 4: Selective Presentation of Cross-Resistance Data

Author's response: In the abstract, we have described the cross-resistance to organophosphorus insecticides. Owing to space limitations, we have replaced non-essential statements (e.g., "stable inheritance") with cross-resistance data to ensure a balanced presentation of the content.

Minor Revisions

Author’s Response: We have revised it in table 2.

Author’s Response: We have revised it in Figure 2.

Author’s Response: we have already submitted all the original data in the supplementary materials, including the life table observation data and the biological mortality counts of the insecticides at various concentrations.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript (MS) “Characterization of a stably maintained, highly beta-cypermethrin-resistant strain of the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae)” (previously “Characterization of field-evolved, stably heritable beta-cypermethrin resistance in the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae)”) by Xinan Li, Peipan Gong, Zilan Zhao, Fengfan Wang, Qiuchi Li, Xiangrui Li, Yunhui Zhang, and Xun Zhu has been substantially revised in accordance with the reviewers’ comments. However, in its present form, the MS raises a few minor issues that need to be corrected.

Line 3: In the title of the MS, there is an extra hyphen in the word “Rhopalosiphum.”

Line 4: The colon is missing here: “Hemiptera Aphididae”

Lines 49–50: In the Keyword list the words “beta-cypermethrin” and “Rhopalosiphum padi” duplicate words already used in the title of the MS. Instead, other terms should be used, for example, pyrethroid and insecticide resistance.

Lines 308–309: The sentence needs editing: “the non-inclusion of 1 in the 95% CI indicates a significant difference in LC50 values of the insecticides between the two strains” (“non-inclusion” instead of “not inclusion”).

Author Response

The manuscript (MS) “Characterization of a stably maintained, highly beta-cypermethrin-resistant strain of the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae)” (previously “Characterization of field-evolved, stably heritable beta-cypermethrin resistance in the bird cherry-oat aphid Rhopalosiphum padi (Hemiptera: Aphididae)”) by Xinan Li, Peipan Gong, Zilan Zhao, Fengfan Wang, Qiuchi Li, Xiangrui Li, Yunhui Zhang, and Xun Zhu has been substantially revised in accordance with the reviewers’ comments. However, in its present form, the MS raises a few minor issues that need to be corrected.

Line 3: In the title of the MS, there is an extra hyphen in the word “Rhopalosiphum.”

Author’s Response: We have revised it in lines 3.

Line 4: The colon is missing here: “Hemiptera Aphididae”

Author’s Response: We have revised it in lines 4.

Author’s Response: We have revised it in Keywords.

Keywords: Pyrethroids; Insecticide resistance; Detoxifying enzymes; Fitness cost; Volt-age-gated sodium channel

Author’s Response: We have revised it in lines 255, 278, and 308.

Reviewer 2 Report

Comments and Suggestions for Authors

The revised manuscript has adequately addressed the major concerns raised in the first round of review. The authors have provided a more complete and coherent dataset on resistance stability, fitness cost, cross-resistance spectrum, and resistance mechanisms. In particular, the finding that high-level beta-cypermethrin resistance remains stable after 40 generations without insecticide exposure is of considerable value for understanding the persistence of resistance in Rhopalosiphum padi. The revised version shows clear improvements in methodological description, data presentation, and depth of discussion, and the conclusions are well supported by the data. The language has also been polished and is now generally clear and readable.I consider the manuscript to have reached the standard required for publication and recommend it for acceptance.

Author Response

The revised manuscript has adequately addressed the major concerns raised in the first round of review. The authors have provided a more complete and coherent dataset on resistance stability, fitness cost, cross-resistance spectrum, and resistance mechanisms. In particular, the finding that high-level beta-cypermethrin resistance remains stable after 40 generations without insecticide exposure is of considerable value for understanding the persistence of resistance in Rhopalosiphum padi. The revised version shows clear improvements in methodological description, data presentation, and depth of discussion, and the conclusions are well supported by the data. The language has also been polished and is now generally clear and readable.I consider the manuscript to have reached the standard required for publication and recommend it for acceptance.

Author’s Response: Thank you very much for your positive assessment and for recommending the manuscript for acceptance. We sincerely appreciate the time and effort you have devoted to reviewing our work, and we are grateful for your constructive comments in the first round of review, which have substantially improved the quality of the manuscript. Thank you again for your valuable guidance and for your recommendation.