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Article
Peer-Review Record

Metabolic and Behavioral Impacts of Gustatory Receptor NlGr23 Silencing in the Brown Planthopper

Agronomy 2025, 15(8), 1797; https://doi.org/10.3390/agronomy15081797
by Kui Kang 1, Jie Zhang 1, Renhan Fang 1 and Jun Lü 2,*
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Agronomy 2025, 15(8), 1797; https://doi.org/10.3390/agronomy15081797
Submission received: 17 June 2025 / Revised: 15 July 2025 / Accepted: 23 July 2025 / Published: 25 July 2025

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript by Kang et al. describes how RNAi-mediated targeting of a gustatory receptor, Gr23, results in transcriptomic, proteomic, and metabolic changes in the brown planthopper. The researchers provide evidence that injections of dsRNA targeting Gr23 caused upregulated transcripts and protein abundance associated with glycerophospholipid metabolism, fatty acid synthesis and AMPK signaling. These changes may explain why triacylglycerol levels rose while glycerol and fatty acids decreased, leading to weight gain. The study’s findings drawn interesting connections between chemosensation and metabolic changes, and I think it would be of interest to many researchers interested in the molecular basis of chemoreception and downstream signaling outcomes. While the study has made some good progress in tying different “omics” level data together, there are some issues that I would like the authors to consider before publication:

  1. What was the degree of transcript knockdown of Gr23 following dsRNA injection. Lines 97-99 indicate that qRT-PCR was used to assess knockdown, but the data are not presented.
  2. Do the transcriptomic analyses provide confirmation of the efficacy of RNAi? i.e. are Gr23 transcripts reduced in the transcriptome of dsRNA-treated insects? Similarly, are the Gr23 protein levels reduced in the proteomic analyses?
  3. Why was 48 h post-injection chosen as the time point for assessing changes in the transcriptome? Would more time points provide more information about the short- and long-term impacts of Gr23 knockdown?
  4. When were proteins extracted following dsRNA treatments? What was the level of Gr23 at the time of protein extraction? If Gr23 was not fully depleted, would downstream signaling and metabolism be only partially perturbed? A time course study would also be valuable to explore downstream changes in protein abundances.
  5. The assumption has been made that knockdown of the gustatory receptor resulted in decreased sensitivity to oxalic acid, resulting in increased feeding on rice. This implies that oxalic acid is a modulator of the insect’s feeding behavior. Firstly, what are the negative impacts of ingesting too much oxalate for the insects? Secondly, have you conducted tests that show that increasing oxalic acid in the diet slows the feeding rate?
  6. Are the changes in the transcriptome or changes in lipid homeostasis following Gr23 knockdown due to increased levels of oxalate levels in the gut? i.e. do you see similar changes in gene expression or lipid metabolism when insects are fed diets with increased concentrations of oxalate, compared to those treated with the Gr23 dsRNA? If not, it might be helpful to explain that at this stage, it is not possible to determine if the changes observed in the insects is due to direct disruption of chemosensory signaling of oxalate, or instead, is due to impacts that increased oxalate may have on the insects.
  7. The concluding comments of the abstract and the discussion suggest that this study provides some insights on how gustatory receptors could be a focus for biocontrol of this pest insect, but it is not clear how blocking NlGr23 (lines 300-302) would provide crop protection, when this blocking would result in increased crop damage. What the authors have neglected to address is the survivorship or reproductive potential of insects following the Gr23 knockdown, to support this idea that disrupting Gr23 will help control the insects.

 

Author Response

The manuscript by Kang et al. describes how RNAi-mediated targeting of a gustatory receptor, Gr23, results in transcriptomic, proteomic, and metabolic changes in the brown planthopper. The researchers provide evidence that injections of dsRNA targeting Gr23 caused upregulated transcripts and protein abundance associated with glycerophospholipid metabolism, fatty acid synthesis and AMPK signaling. These changes may explain why triacylglycerol levels rose while glycerol and fatty acids decreased, leading to weight gain. The study’s findings drawn interesting connections between chemosensation and metabolic changes, and I think it would be of interest to many researchers interested in the molecular basis of chemoreception and downstream signaling outcomes. While the study has made some good progress in tying different “omics” level data together, there are some issues that I would like the authors to consider before publication:

  1. What was the degree of transcript knockdown of Gr23 following dsRNA injection. Lines 97-99 indicate that qRT-PCR was used to assess knockdown, but the data are not presented.

Response: Thank you for your comments. We have added the data in Figure S1 and the following description in the main text (Lines 184-186).

qRT-PCR analysis showed NlGr23 transcript knockdown at 24 h, 48 h, and 72 h post-injection. No significant difference in efficiency was found between timepoints (Fig S1).

 

  1. Do the transcriptomic analyses provide confirmation of the efficacy of RNAi? i.e. are Gr23 transcripts reduced in the transcriptome of dsRNA-treated insects? Similarly, are the Gr23 protein levels reduced in the proteomic analyses?

Response: Thank you for your comments. We sincerely appreciate this critical question. While our transcriptomic and proteomic analyses revealed systemic impacts of NlGr23 silencing, neither dataset detected NlGr23 expression in control or dsRNA-treated groups, because of NlGr23 is exclusively expressed in gustatory sensilla of the proboscis ([25], Kang et al. 2023), whereas our omics used whole insects (to capture systemic metabolic effects). Actually, we have confirmed the efficacy of RNAi by used qRT-PCR before RNA-seq (see question 1).

 

Localization of NlGr23a in the distal part of the female labium of the BPH (Kang et al. 2023)

Kang, K.; Zhang, M.; Yue, L.; Chen, W.; Dai, Y.; Lin, K.; Liu, K.; Lv, J.; Guan, Z.; Xiao, S.; et al. Oxalic acid inhibits feeding behavior of the brown planthopper via binding to gustatory receptor Gr23a. Cells 2023, 12:771.

 

  1. Why was 48 h post-injection chosen as the time point for assessing changes in the transcriptome? Would more time points provide more information about the short- and long-term impacts of Gr23 knockdown?

Response: Thank you for your comments. Knockdown efficiency of NlGr23 showed no significant difference across 24 h, 48 h, and 72 h post-injection (Fig S1). Consequently, we selected the intermediate 48 h time point for transcriptomic profiling to ensure consistency with our other assays while capturing the earliest robust transcriptional response.

 

  1. When were proteins extracted following dsRNA treatments? What was the level of Gr23 at the time of protein extraction? If Gr23 was not fully depleted, would downstream signaling and metabolism be only partially perturbed? A time course study would also be valuable to explore downstream changes in protein abundances.

Response: Thank you for your comments. Proteins were extracted at 48 h post-injection per Methods 2.6. qRT-PCR confirmed 72.4% knockdown of NlGr23 transcripts (p < 0.05; Fig S1). Although whole-body samples lacked sufficient target protein for Western blot detection due to NlGr23's exclusive localization in proboscis gustatory sensilla (Kang et al., 2023), three phenotypic outcomes collectively validate functional silencing: (i) 29.7% increased body weight (Fig 1A), (ii) 41.2% elevated honeydew production (Fig 1B), and (iii) 68.83% TAG accumulation (Fig 6A)."

We added the description to the Materials and methods section (Lines 127-128, 149-151).

Lines 127-128: “Total RNA was extracted from whole insects 48 h after dsRNA injection (Section 2.2). Three biological replicates per treatment (5 insects each) were used for RNA-seq.”

Lines 149-151: “For proteomic analysis, whole insects were harvested 48 h post dsRNA injection (Section 2.2) and processed for protein extraction. Each treatment included three biological replicates with 10 insects pooled per replicate.”

Kang, K.; Zhang, M.; Yue, L.; Chen, W.; Dai, Y.; Lin, K.; Liu, K.; Lv, J.; Guan, Z.; Xiao, S.; et al. Oxalic acid inhibits feeding behavior of the brown planthopper via binding to gustatory receptor Gr23a. Cells 2023, 12:771.

 

  1. The assumption has been made that knockdown of the gustatory receptor resulted in decreased sensitivity to oxalic acid, resulting in increased feeding on rice. This implies that oxalic acid is a modulator of the insect’s feeding behavior. Firstly, what are the negative impacts of ingesting too much oxalate for the insects? Secondly, have you conducted tests that show that increasing oxalic acid in the diet slows the feeding rate?

Response: We sincerely thank you for the valuable comments. Our published study (Kang et al., 2023) directly demonstrates that oxalic acid (OA) acts as a potent feeding deterrent in the brown planthopper. Behavioral assays show that OA at 100 μM in artificial diets reduced food acceptance by 41% within 1.5 hours (p < 0.05, Fig 3A), while dual-choice tests revealed strong avoidance (Position Index = -0.52, p < 0.01, Fig 3B). Electropenetrography (EPG) recordings confirmed that OA suppresses feeding duration dose-dependently: at 10 mM, ingestion phases (N4) comprised only 0.56% of total observation time versus 32.7% in controls (p < 0.01, Fig 3C). This aversion is mediated specifically by NlGr23a, as RNAi silencing abolished OA avoidance (Fig 3E-H). High OA concentrations (>5 mM) further induce metabolic stress, consistent with its role as a phytotoxin that disrupts insect physiology. These data collectively validate OA as a biologically significant antifeedant through NlGr23a-dependent sensing.

Kang, K.; Zhang, M.; Yue, L.; Chen, W.; Dai, Y.; Lin, K.; Liu, K.; Lv, J.; Guan, Z.; Xiao, S.; et al. Oxalic acid inhibits feeding behavior of the brown planthopper via binding to gustatory receptor Gr23a. Cells 2023, 12:771.

 

  1. Are the changes in the transcriptome or changes in lipid homeostasis following Gr23 knockdown due to increased levels of oxalate levels in the gut? i.e. do you see similar changes in gene expression or lipid metabolism when insects are fed diets with increased concentrations of oxalate, compared to those treated with the Gr23 dsRNA? If not, it might be helpful to explain that at this stage, it is not possible to determine if the changes observed in the insects is due to direct disruption of chemosensory signaling of oxalate, or instead, is due to impacts that increased oxalate may have on the insects.

Response: We sincerely thank you for the valuable comments and suggestions, which have significantly enhanced the rigor and clarity of our manuscript. The current data cannot distinguish whether the transcriptomic/proteomic changes result directly from NlGr23 silencing or indirectly from increased oxalate consumption. Future controlled feeding studies with defined oxalate diets will address this limitation.

We added this limitation in the Discussion section (Lines 306-308).

Lines 321-323: “This study shows NlGr23 silencing causes transcriptomic/proteomic changes in BPH, but we cannot determine if these result directly from receptor loss or indirectly from increased oxalate intake.”

 

  1. The concluding comments of the abstract and the discussion suggest that this study provides some insights on how gustatory receptors could be a focus for biocontrol of this pest insect, but it is not clear how blocking NlGr23 (lines 300-302) would provide crop protection, when this blocking would result in increased crop damage. What the authors have neglected to address is the survivorship or reproductive potential of insects following the Gr23 knockdown, to support this idea that disrupting Gr23 will help control the insects.

Response: Thank you for your suggestion. We revised the manuscript accordingly to avoid these misconceptions (Lines 22-24).

Lines 22-24: “This study establishes NlGr23 as a key regulator linking chemosensation to metabolic reprogramming, providing new insights into gustatory-receptor-mediated energy homeostasis in the BPH.”

Reviewer 2 Report

Comments and Suggestions for Authors

Review:

 

Title: Metabolic and Behavioral Impacts of Gustatory Receptor NlGr23Silencing in the Brown Planthopper

 

 

This study establishes NlGr23 as a key regulator linking chemosensation to metabolic reprogramming and providing insights for developing gustatory-receptor-targeted strategies for the biocontrol of the BPH. The results of multiomics profiling revealed disrupted lipid homeostasis, identifying 187 differentially expressed genes and 150 differentially expressed proteins. These genes were enriched in pathways including glycerophospholipid metabolism, fatty acid biosynthesis, and AMPK signaling. The results of biochemical assays showed that NlGr23 silencing elevated triacylglycerol levels by 68.83%, and reduced glycerol and free fatty acid levels, suggesting impaired lipolysis

 

The paper is well written, and presents the important results in the topic of insects, especially Hemipteran endosymbionts.

The Introduction is well presenting the most important and relevant timely references, I do not find problems with this section.

The methods part I also well presented, here, I definitively had to mention the substantial work of the authors, and the meta-analyses methods used.

Please mention here the sample size, how many insects individuals were used for analyses?

Please also do this at the other assessment.

Please add a bit more details about data analyses. How data were i.e. analyses by distributions of the normality?

 

I have no specific other problems with the results and discussions, I do appreciated the work behind this manuscript. Altogether I consider that this paper is valuable, minor revision in data presentation has to be made.

 

Author Response

Title: Metabolic and Behavioral Impacts of Gustatory Receptor NlGr23Silencing in the Brown Planthopper

This study establishes NlGr23 as a key regulator linking chemosensation to metabolic reprogramming and providing insights for developing gustatory-receptor-targeted strategies for the biocontrol of the BPH. The results of multiomics profiling revealed disrupted lipid homeostasis, identifying 187 differentially expressed genes and 150 differentially expressed proteins. These genes were enriched in pathways including glycerophospholipid metabolism, fatty acid biosynthesis, and AMPK signaling. The results of biochemical assays showed that NlGr23 silencing elevated triacylglycerol levels by 68.83%, and reduced glycerol and free fatty acid levels, suggesting impaired lipolysis

The paper is well written, and presents the important results in the topic of insects, especially Hemipteran endosymbionts.

The Introduction is well presenting the most important and relevant timely references, I do not find problems with this section.

The methods part I also well presented, here, I definitively had to mention the substantial work of the authors, and the meta-analyses methods used.

Response: Thank you for your comments.

 

Please mention here the sample size, how many insects individuals were used for analyses?

Response: Thank you for your suggestion. We added the description to the Materials and methods section (Lines 127-128, 149-151)

Lines 127-128: “Total RNA was extracted from whole insects 48 h after dsRNA injection (Section 2.2). Three biological replicates per treatment (5 insects each) were used for RNA-seq.”

Lines 149-151: “For proteomic analysis, whole insects were harvested 48 h post dsRNA injection (Section 2.2) and processed for protein extraction. Each treatment included three biological replicates with 10 insects pooled per replicate.”

 

Please also do this at the other assessment.

Please add a bit more details about data analyses. How data were i.e. analyses by distributions of the normality?

Response: Thank you for your suggestion. We added the description to the Materials and methods section (Lines 179-180).

Lines 179-180: “Normality was confirmed with the Shapiro-Wilk test (p > 0.05). Two-group comparisons employed Student's t-test.”

 

I have no specific other problems with the results and discussions, I do appreciated the work behind this manuscript. Altogether I consider that this paper is valuable, minor revision in data presentation has to be made.

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