Abstract
Pest-resistant plants usually utilize secondary metabolites to cope with insect infestation. Betulin, a key bioactive compound in aphid-resistant wild peach, possesses promising applications in crop protection. Here, betulin, in both greenhouse and field experiments, displayed excellent control efficacy against Myzus persicae. RNA-seq, qRT‒PCR, and western blotting revealed that betulin significantly inhibited the expression of MpGABR (encoding a GABAA receptor). Besides, RNAi-mediated silencing of MpGABR markedly increased aphid sensitivity to betulin. Furthermore, MST and voltage-clamp assays indicated that betulin bound to MpGABR (Kd = 2.24 µM) and acted as an inhibitor of MpGABR. Molecular docking, mutagenesis and genome editing suggested that THR228 is a critical and highly conserved site in MpGABR that betulin binds to specifically, causing aphid death. Overall, the activity of betulin depends on specific targeting and inhibition of MpGABR. Elucidating the mechanism of action of this peach-derived insecticide may offer a sustainable green strategy for aphid control.
1. Introduction
Over more than 400 million years of coevolution with herbivores, plants have developed a variety of morphological,1 biochemical,2 and molecular3 defense strategies against insect herbivore attack. Plant secondary metabolites are highly valuable natural compounds, offering abundant and potent biochemical defense driven by plant-insect coevolution.4 Interestingly, wild relatives of crops with insect resistance usually accumulate large amounts of biologically active compounds.5, 6 Accordingly, investigating the bioactivities and potential mechanisms of action of compounds derived from wild resistant germplasms may provide effective and sustainable strategies for pest control in crop protection.
Myzus persicae (Sülzer) is one of the most destructive sap-feeding pests worldwide. It is able to settle on more than 400 plant species globally, displaying extraordinary polyphagia and a wide range of hazards.7 In addition to ingesting phloem saps and secreting honeydew, M. persicae can transmit various plant viruses,8 resulting in severe losses in crop yields. To date, the principal control strategy for M. persicae has relied on synthetic insecticides, including pyrethroids, organophosphorus compounds, carbamates and neonicotinoids.9–11 However, long-term irrational application of these chemical insecticides has led to environmental pollution and promoted the resistance of aphids to insecticides.11–13 Therefore, it is necessary to develop novel green insecticides as potential alternatives to synthetic insecticides. Fortunately, plant secondary metabolites are a “treasure trove of active compounds” that serve as a rich resource for the development of promising green insecticides.
Our previous study revealed that Prunus davidiana, a close wild relative of cultivated peach, displays strong resistance to M. persicae through the accumulation of high levels of betulin.3, 5 In addition, betulin, a lupane-type triterpene, possesses strong insecticidal activity and is a promising substance for the development of novel aphid-control insecticides. A number of studies have reported that betulin and its derivatives exhibit a wide range of pharmacological activities,14 which have been used to treat various diseases, such as cancer,15 glioblastoma,16 inflammation17 and hyperlipidemia.18 However, the insecticidal mechanism of betulin against aphids remains unclear.
Gamma-aminobutyric acid (GABA) receptors have been confirmed to be targets of terpenoids that impair neuronal function in insect herbivores.19 In mammals, there are two types of GABA receptors: ionotropic (GABAA) and metabotropic (GABAB) receptors. GABAA receptors (GABRs) are heteropentameric ligand-gated ion channels in the central nervous system that conduct chloride and bicarbonate ions and are the target of numerous drugs for the treatment of neuropsychiatric disorders.20 GABRs are composed of 5 different types of subunits, each with 4 helical transmembrane domains (TM1-TM4), of which TM2 is located at the center of the pentamer and forms an ion channel.21 However, the structure and assembly of insect GABRs have not been fully elucidated. The first GABA receptor subunit identified in insects is encoded by Rdl, which confers resistance to dieldrin in Drosophila melanogaster.22 Although a variety of terpenoids play roles as positive allosteric modulators or noncompetitive antagonists at GABRs,19 whether betulin targets the GABR in M. persicae needs to be further investigated.
In this study, betulin was confirmed to have excellent control efficacy against M. persicae in both greenhouse and field experiments. RNA-seq, qRT‒PCR, and western blotting assays revealed that betulin significantly inhibited the expression of MpGABR in aphids. In addition, RNAi-mediated silencing of MpGABR markedly increased aphid sensitivity to betulin. Furthermore, microscale thermophoresis (MST) and voltage-clamp assays indicated that betulin can bind to MpGABR (Kd= 2.24 µM) and act as an inhibitor (EC50 = 20.66 µM) of MpGABR. Molecular docking analysis suggested that betulin bound to MpGABR via the THR228 amino acid residue. This site is highly conserved in the family Hemiptera and may be a critical specific binding site for betulin in MpGABR in aphids. Mutagenesis and genome editing experiments revealed that betulin bound specifically to this amino acid residue in aphids but not in Drosophila, resulting in aphid death. This study elucidated the insecticidal mechanism of betulin involving the targeting of MpGABR, providing a sustainable green strategy for aphid control.
2. Materials and methods
2.1 Test insects
M. persicae populations were collected from tobacco (Nicotiana tabacum L.) leaves. Wild-type (WT) flies (D. melanogaster) were purchased from Fungene Biotechnology Co., Ltd., Jiangsu, China. The test insects were cultured in a greenhouse (25 °C, 70% relative humidity and 14 h light/10 h dark cycle) in Beibei, Chongqing, China.
2.2 Bioassay
The toxicity of betulin against M. persicae was measured using a slip-dip bioassay as previously described.5 Thirty apterous adult M. persicae individuals were gently transferred on tobacco (N. tabacum) leaves using a fine brush. The tobacco leaves were subsequently immersed in 0.1% (v/v) Tween-80 and 3% (v/v) acetone solution containing betulin or pymetrozine (as a positive control) at 6 doses (0, 0.0625, 0.125, 0.25, 0.5, 1, and 2 mg· mL-1) for 5 seconds. After drying, the leaves with M. persicae were placed on wet filter paper and cultured on a water-soaked sponge for 48 h. Three replicates were tested for each treatment. Furthermore, the LC30, LC50 and LC70 values of betulin were calculated using log-probit analysis via IBM SPSS Statistics (v.22.0, Chicago, IL, USA).2
2.3 Control effect of betulin against M. persicae
The control effects of betulin against M. persicae in the greenhouse and field were evaluated by a spraying-based method as described previously.23 The commercial insecticide pymetrozine and an aqueous solution containing 3% (v/v) acetone and 0.1% (v/v) Tween-80 were employed as positive and negative controls, respectively. Sprays of 0.1641 mg·mL−1 (LC50) betulin and 1.0612 mg·mL−1 (LC50) pymetrozine (Table S1) were prepared in aqueous solutions including 3% (v/v) acetone and 0.1% (v/v) Tween-80, respectively. Before the control effect assays, each tobacco (N. tabacum L.) seedling was pre-inoculated with 60 apterous adults of M. persicae. In the greenhouse trial, each treatment included 8 replications, and each replication consisted of 3 tobacco plants growing under controlled conditions as described above. The field trial (Beibei District, Chongqing, China.) was conducted under natural conditions with an average temperature of 20 °C. Each treatment had 8 replications, covering an area of 20 m2, with 3 protected rows per replication. Furthermore, each tobacco plant was sprayed with 20 mL of the corresponding solution with an electric sprayer. Mortality was recorded after 1, 5, 9, and 14 days of treatment. The control efficacy of betulin against M. persicae was calculated according to the methods of Zhou.23
2.4 RNA-seq assay
After exposure to 0.1641 mg· mL-1 (48 h LC50) betulin for 48 h, 100 wingless M. persicae adults were sampled, and total RNA was extracted via TRIzol reagent (Tiangen, Beijing, China) to construct mRNA sequencing libraries. RNA-seq analysis was performed on the Ion Proton BGISEQ-500 platform (Beijing Genomic Institute, BGI, China). After the raw reads were filtered, the clean reads were aligned to the M. persicae reference genome, which was downloaded from the National Center for Biotechnology Information database, using HISAT2 (v2.1.0).24 The expression levels of genes in each sample were normalized to the fragments per kilobase million mapped reads (FPKM) value. The differentially expressed genes (DEGs) between groups were identified using the fold change factor (|Log2(FC)| > 1). Functional enrichment analysis of DEGs was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.
2.5 Validation by quantitative real-time PCR (qRT‒PCR)
The gene-specific primers used for qRT‒PCR, which were designed with Primer 5.0, are listed in Table S2. According to the manufacturer’s specifications for the Bio-Rad iQ™ SYBR® Green Supermix Kit, qRT‒PCR was performed with the following conditions: 60 °C for 15 s, followed by 48 cycles of 60 °C for 30 s, and 72 °C for 30 s. The ribosomal protein S18 gene (RPS18) was used as the reference gene to normalize the expression levels. The relative expression levels of the target genes were calculated using the 2-ΔΔCt approach.2 Three biological replicates were used for each assay.
2.6 Cloning, bioinformatics and phylogenetic analysis
The cDNA of M. persicae was synthesized from the extracted total RNA using the PrimeScript® 1st Strand cDNA Synthesis Kit (TaKaRa, Japan). The coding sequences of MpGABR (encoding GABAA receptor), MpGABRAP (encoding GABAA receptor-associated protein) and MpGABRB (encoding GABAA receptor β subunit) were cloned from M. persicae cDNA using the corresponding specific primers (Table S2) and then inserted separately into the pGEM-T Easy vector. The recombinant plasmids were subsequently transferred into Escherichia coli DH5α competent cells for sequencing. BioXM (v2.7.1) was used to analyze the sequence information of these three genes, including the length, molecular weight, and isoelectric point of the deduced amino acid sequence. MEGA7 was used for multiple amino acid sequence alignment (ClustalW) and phylogenetic analysis (neighbor-joining method with 1000 replicates) of MpGABR.
2.5 In vitro RNAi assay
To knock down the target genes (MpGABR, MpGABRAP, and MpGABRB), MpGABR-, MpGABRAP-, and MpGABRB-dsRNA were artificially synthesized using the dsRNA Synthesis Kit (Thermo Scientific, Vilnius, Lithuania, EU). In accordance with a previously described method,25 fresh tobacco leaves were cut into 3.0 cm-diameter discs and placed in an oven at 50 °C for 3 min. Subsequently, the dried leaf discs were exposed to 10 μL of diethyl pyrocarbonate-treated nuclease-free water (DEPC-water), dsGFP (green fluorescent protein, 1000 ng· μL-1, negative control), and MpGABR-, MpGABRAP-, or MpGABRB-dsRNA (1000 ng/·μL-1) at 25 °C for 5 h. After the leaf discs had absorbed the solution, they were placed on wet filter paper on a water-soaked sponge. Subsequently, 30 wingless adult M. persicae, starved for 24 h, were fed on the treated leaf discs for 48 h. The surviving aphids were collected after exposure for qRT‒PCR.
2.6 Prokaryotic expression of MpGABR and western blotting
To produce the MpGABR protein in vitro, an Escherichia coli expression system was constructed.26 Briefly, the coding sequence of MpGABR was inserted into the expression vector PET-B2M and transformed into BL21 (DE3) competent cells. To induce the expression of the MpGABR protein, 2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was used, and then, the transformed BL21 cells were incubated at 180 rpm and 30 °C for 24 h. Finally, the protein was extracted and purified using Solarbio GST-tag Purification Resin for subsequent assays.
The purified MpGABR protein resolved on a 15% SDS‒PAGE (sodium dodecyl sulfate‒ polyacrylamide gel electrophoresis) gel and then transferred to a polyvinylidene fluoride (PVDF) membrane at 20 V for 1 h. Moreover, 1 × TBST (Tris-Borate-Sodium Tween-20) containing 5% fat-free milk powder was used to block the PVDF membrane. Then, the PVDF membrane was incubated with an antibody against MpGABR (polyclonal rabbit antibody, provided by GeneCreate Biotechnology) at 25 °C for 1 h. Subsequently, the membrane was further incubated with the corresponding horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody at 25 °C for 1 h and then washed three times with TBST. Finally, the bands on the membrane were visualized using a Chemi Doc MP Imaging System (Bio-Rad) after incubation with a chemiluminescence reagent (ECL, Bio-Rad).
2.7 Molecular docking
In accordance with a previously reported method,23 the protein structure and function of MpGABR were predicted via the I-TASSER server (Iterative Threading ASSEmbly Refinement). AutoDockTools software (v1.5.7) was used to conduct molecular docking. Furthermore, PyMOL (v3.1) and Discovery Studio (v4.5) were used to evaluate the molecular docking model.
2.8 Site-directed mutagenesis
The cDNA of MpGABR was inserted into the PET-B2M vector and used as the template for site-directed mutagenesis. In accordance with the manufacturer’s specifications, the Fast MultiSite Mutagenesis Kit (TransGen, Beijing, China) was used to generate the multisite mutations in MpGABR.27 Then, the MpGABR mutants, with the mutations R224A (ARG224 replaced by ALA), A226T (ALA226 replaced by THR), F227Y (PHE227 replaced by TYR), and T228R (THR228 replaced by ARG), were amplified, and the amplicons were used to replace the BamHI-SmaI fragment in the recombinant MpGABR plasmid.
2.9 Method for Drosophila genome editing
To generate Drosophila mutants bearing the DmGABRR122T mutation, homologous to THR228 in MpGABR, the Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR/Cas9) genomic editing strategy was used. First, CRISPR Optimal Target Finder (http://targetfinder.flycrispr.neuro.brown.edu/) was used to generate short guide RNA (sgRNA) targets without off-target hits. Then, the Precision gRNA Synthesis Kit (Thermo Scientific) was used to synthesize the sgRNA. The 228 bp homologous arm in R122T, for homology-directed repair, was subsequently employed to synthetize single-stranded DNA (ssDNA) bearing the desired mutation (DmGABRR122T). Subsequently, 1 nL of a mixture containing 160 ng· μL-1 ssDNA, 300 ng· μL-1 both sgRNAs, or 90 ng· μL-1 Cas9 protein was injected into Drosophila embryos via the Drummond Nanoject III (Broomall, PA, USA) system. CRISPR/Cas9-induced mutations were verified by amplifying and sequencing gDNA flanking the genome editing target site. Finally, according to the traditional topical application and bioassay approach described previously,23 the acute insect toxicity of betulin in genome-modified or nonmodified (WT) 3-day-old adult flies was determined. The oligonucleotide sequences used for PCR, the sgRNA synthesis template and the fragment of donor DNA used for homology-directed repair (HDR) are listed in Table S3.
2.10 Microscale thermophoresis (MST) assay
The binding affinity of MpGABR for betulin was measured via a NanoTemper Technologies Monolith Kit (MO, Munich, Germany) according to the manufacturer’s specifications. Briefly, MpGABR was fluorescently labeled via a protein labeling kit (RED-NHS 2nd generation, MonolithTM) and then mixed with sixteen concentrations (from 5 nM to 60 mM) of betulin. The mixtures were subsequently loaded into NanoTemper capillaries for MST measurements with 40% LED and 60% MST power at 25 °C. MO Control NanoTemper Technologies GmbH software (v2.3) and MO Affinity Analysis software (v2.3, NanoTemper Technologies GmbH) were used to acquire and analyze the recorded data, respectively. The dissociation constant (Kd), indicating the binding affinity, was calculated using data from three replicates for each experiment.
2.11 Electrophysiological recording
In accordance with previously described methods,28 oocytes isolated from mature healthy Xenopus females were digested at 25 °C for 1 h to remove the follicular layer. Individual oocytes were selected to express MpGABR and incubated at 18 °C with 1× Ringer’s solution (96 mM NaCl, 5 mM MgCl2, 5 mM HEPES, 2 mM KCl, and 0.8 mM CaCl2) supplemented with 550 mg·mL-1 sodium pyruvate, 100 mg·mL-1 streptomycin, 50 mg·mL-1 tetracycline, and 5% dialyzed horse serum. After 3 days of incubation, the whole-cell currents were recorded via a two-electrode voltage-clamp. The data were collected and analyzed using Digidata 1440A and pCLAMP 10.2 software (Axon Instruments Inc., Union City, CA, USA), respectively.29
2.12 Statistical analysis
Statistical analysis was performed using SPSS (v22). Statistical significance was determined using one-way analysis of variance (ANOVA) for differences among groups. The replicate numbers are indicated by the black dots in the figures.
3. Results
3.1 Control efficacy of betulin against aphids
Bioassays revealed that the LC50 values of betulin and pymetrozine against M. persicae at 48 h were 0.1641 and 1.0612 mg·mL-1, respectively (Table S1). To assess the control efficacy of betulin, tobacco plants infected with M. persicae were exposed to betulin (0.1641 mg·mL-1) and pymetrozine (1.0612 mg·mL-1, as a positive control) in both the greenhouse and field. In the greenhouse test (Fig. 1A-D), after 14 days of treatment, M. persicae in the CK group (negative control) reproduced rapidly, and tobacco leaves turned yellow and wrinkled, whereas tobacco leaves in the betulin and pymetrozine treatment groups were barely affected by the aphids (Fig. 1A). The control efficacy of betulin against M. persicae was significantly greater than that of pymetrozine after 1 day of treatment, while there was no significant difference between the control efficacies of betulin and pymetrozine after 5, 9, and 14 days of treatment (Fig. 1E). After 14 days of treatment, the control efficacies of betulin and pymetrozine reached 91.54% and 95.15%, respectively. Throughout the field test (Fig. 1F), no difference in the control efficacy was observed between betulin and pymetrozine, with values of 87.79% and 90.14%, respectively, after 14 days of treatment (Fig. 1G). These results suggest that betulin has immense potential for development as a commercial aphid insecticide like pymetrozine.

The aphicidal activity of plant-derived derivative betulin against M. persicae.
(A-G) Representative images of the control effects of betulin (A, 0.1641 mg·mL-1, 48 h LC50 value of betulin), pymetrozine (B, 1.0612 mg·mL-1, a positive control), and CK (C, a negative control) against M. persicae in greenhouse (A-E) and field (F, G) tests after treatment for 14 d. The data are shown as the mean ± SD from twelve independent experiments. **p < 0.01. ns, not significant.
3.2 Expression patterns of genes in aphids after treatment with betulin
To investigate the expression patterns of genes in M. persicae after exposure to betulin, RNA-seq was performed on M. persicae with 0.1641 mg·mL-1 betulin treatment for 48 h and without (CK) (Fig. 2A). Principal coordinate analysis (PCA) revealed that the expressed genes were strongly clustered in the CK group and betulin group (Fig. 2B). Compared with those in the CK group, there were 130 up- and 41 downregulated significant differentially expressed genes (DEGs) in M. persicae after betulin treatment (Fig. 2C and D, Table S4). The validation by qRT‒PCR revealed that the relative expression trends of the fifteen DEGs determined via qRT‒PCR were similar to those detected via RNA‒seq, supporting the reliability of the RNA‒ seq data (Fig. 2E). To further analyze the functions of the DEGs, GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses were performed. GO term enrichment demonstrated that the DEGs were enriched mainly in the GABAA receptor activity term (Fig. 2F). Interestingly, KEGG enrichment also found that the DEGs were enriched in the GABAergic signaling related pathway and GABAergic synapse terms (Fig. 2G). Additionally, the expression of DEGs related to GABR, including MpGABR (encoding GABAA receptor), MpGABRAP (encoding GABAA receptor-associated protein) and MpGABRB (encoding GABAA receptor β subunit), in the betulin group was significantly lower than that in the CK group (Fig. 2H). In particular, the log2(fold change) value of MpGABR was the lowest, at -3.8 (Table S4). These results suggested that MpGABR may be a candidate target for betulin against M. persicae.

RNA-Seq analysis revealed candidate targets for betulin against M. persicae.
(A) Diagram of the candidate targets for betulin against aphids as determined by RNA-Seq. Water containing 0.1% (v/v) Tween-80 and 3% (v/v) acetone was used as the control treatment (CK). (B) Principal coordinates analysis (PCA) analysis differentially expressed genes in RNA-Seq. (C-D) Distribution (C) and heatmap (D) of the significantly differentially expressed genes (DEGs) in M. persicae. (E) qPCR validation of 15 DEGs identified by RNA-Seq. (F-G) Top 10 enriched GO (F) and KEGG (G) pathways of the DEGs. The “rich ratio” was defined as the ratio of the number of DEGs enriched in the pathway to the total number of genes enriched in the same pathway. (H) Heatmap of the expression level of the GABR genes identified by RNA-Seq. MpGABR, encoding GABAA receptor; MpGABRAP, encoding GABAA receptor associated protein; MpGABRB, encoding GABAA receptor β subunit.
3.3 MpGABR expression was inhibited by betulin
Analysis of the sequence information revealed that the MpGABR, MpGABRAP and MpGABRB proteins have 694, 118, and 250 amino acids, respectively, with calculated molecular weights of 77.20, 14.08, and 28.01 kDa and isoelectric points of 9.85, 9.63, and 7.20, respectively (Table S5). Additionally, structure prediction indicated that MpGABR, MpGABRAP and MpGABRB contain 4, 0, and 1 transmembrane helical domain, respectively (Fig. 3A), implying that only MpGABR has a complete transmembrane structure.

MpGABR expression in aphids was significantly inhibited by treatment with betulin.
(A) Schematic drawing of MpGABR, MpGABRAP and MpGABRB. TM, transmembrane helices. (B-D) qPCR expression analysis of MpGABR (B), MpGABRAP (C) and MpGABRB (D) transcripts in M. persicae exposed to LC30, LC50, and LC70 of betulin for 48 h. The bars represent the average (± SD). Different letters above the error bars indicate significant difference (ANOVA, Tukey’s test, P < 0.05). (E-G) qPCR expression analysis of MpGABR (E), MpGABRAP (F) and MpGABRB (G) after RNAi at 48 h post-dsRNA feeding relative to the expression levels after DEPC-water treatment. (H) Schematic drawing of the RNAi assay in M. persicae. (I) Mortality of aphids exposed to the LC50 of betulin for 48 h after RNAi. An asterisk (*) on the error bar indicates a significant difference between the treatment and group CK according to t tests, ***P < 0.001, ****P < 0.0001. ns, not significant.
To further verify the inhibitory effect of betulin on GABR-related gene expression, the relative expression of the three GABR-related genes was detected in M. persicae exposed to the LC30, LC50, and LC70 of betulin for 48 h. The relative expression of all three genes decreased gradually as the concentration of betulin increased (Fig. 3B-D). After exposure to the LC30 of betulin for 48 h, the relative expression of MpGABR, MpGABRAP and MpGABRB decreased by 82.91%, 10.53%, and 11.49%, respectively. These results indicated that MpGABR was most sensitive to betulin.
Furthermore, after these three genes were silenced via RNAi (Fig. 3H), the expression levels of MpGABR, MpGABRAP and MpGABRB in M. persicae decreased by 64.57%, 63.28%, and 67.33%, respectively, compared with those in the control group (DEPC-treated water) (Fig. 3E-G). After exposure to the LC50 of betulin for 48 h, the mortality of M. persicae with MpGABR silenced markedly increased, by 30.44%, compared with that in the control group, whereas the mortality of M. persicae in the MpGABRAP- and MpGABRB-silenced groups did not significantly differ from that in the control group (Fig. 3I). Additionally, western blotting analysis revealed that MpGABR protein expression decreased by 69.91% after RNAi (Fig. S1A, B). These results further implied that MpGABR might be a target for betulin against M. persicae.
3.5. Phylogenetic analysis of GABRs in insects
Furthermore, phylogenetic analysis of GABRs in insects was performed using the amino acid sequences of 71 GARB proteins from Hemiptera, Diptera, Lepidoptera, Thysanoptera, Hymenoptera, and Coleoptera. MpGABR (GenBank number: XM022318019.1) was found to be genetically closely related to CAI6365831.1 from Macrosiphum euphorbiae (Fig. 4 and Table S6), indicating that MpGABR may be functionally analogous to CAI6365831.1.

Phylogenetic analysis of GABR in insects.
Maximum likelihood based phylogenetic tree of GABR from six orders: Hemiptera, Diptera, Lepidoptera, Thysanoptera, Hymenoptera, and Coleoptera. Protein sequence alignment was performed using MUSCLE in MEGA X. The sequences used for constructing the tree were listed in Table S6.
3.6. Responses of MpGABR to betulin
To investigate the effect of betulin on the MpGABR protein, M. persicae was exposed to the LC30, LC50, and LC70 of betulin for 48 h. As the concentration of betulin increased, the MpGABR protein content in M. persicae gradually decreased (Fig. 5A and B). Compared with that in the CK group, the MpGABR protein content in M. persicae exposed to the LC30, LC50, and LC70 of betulin decreased by 33.68%, 44.89%, and 76.89%, respectively.

The interaction of betulin with MpGABR protein.
(A, B) Western blotting analysis of MpGABR protein after betulin treatment for 48 h at three different concentrations (LC30, LC50 and LC70). The bars represent the average (± SD). Different letters above the error bars indicate significant difference (ANOVA, Tukey’s test, P < 0.05). (C) Expression of recombinant wild type-aphid MpGABR (1, WT) protein by Escherichia coli. (D) Quantification of the binding affinity of betulin with wild type-aphid MpGABR using MST. Current responses (E) and inhibition activity (F) of MpGABR induced by different concentration of betulin in the presence of GABA. 0 µM MpGABR indicates the presence of only GABA.
Moreover, the WT (wild type) MpGABR was expressed in Escherichia coli (Fig. 5C). The binding affinity of betulin with WT MpGABR was further measured using MST. The Kd value was 2.24 µM (Fig. 5D), suggesting that betulin could bind to MpGABR. Additionally, voltage-clamp-based electrophysiological recording demonstrated that MpGABR strongly responded to betulin (Fig. 5E). In the presence of only GABA, a fast inward current was generated. As the concentration of added betulin increased, the current and channel activity of MpGABR decreased correspondingly, with an EC50 value of 20.66 µM (Fig. 5E and F). These results indicated that betulin acted as an inhibitor of MpGABR.
3.5 Key sites for the binding of betulin to MpGABR
To further study the binding mode of betulin in the active pocket of MpGABR, molecular docking was performed to explore the structure–function relationships between betulin and MpGABR. The docking results revealed that the predicted binding energy between betulin and MpGABR was −6.38 kcal·mol−1 (Table S7), verifying that betulin could act as a specific ligand for MpGABR. The three-dimensional binding pattern of betulin with MpGABR indicated that the four key amino acid residues (ARG224, ALA226, PHE227 and THR228) interacted with betulin in the MpGABR binding pocket (Fig. 6A). Among the four key amino acid residues, ALA226 and THR228 interacted with betulin via hydrogen bonding in the MpGABR binding pocket (Table S7). Betulin generated nonconventional H-bonds (C…H) with ALA226 (3.31 Å) and conventional H-bonds (C-O H) with THR228 (2.16 Å). Furthermore, sequence alignment of the key amino acid residues of GABR in different species from Hemiptera, Diptera, Lepidoptera, Hymenoptera, Thysanoptera, and Coleoptera indicated that only THR228 was conserved in Hemiptera (Fig. 6B), implying that THR228 may be an essential specific site for the action of betulin on MpGABR in aphids. Mutants of aphid MpGABR were subsequently constructed, including R224A (ARG224 replaced by ALA), A226T (ALA226 replaced by THR), F227Y (PHE227 replaced by TYR, similar to the site in Drosophila), and T228R (THR228 replaced by ARG, similar to the site in Drosophila) (Fig. 6C). The binding affinity of betulin with these mutants was measured using MST. The results revealed that the Kd values of R224A, A226T, F227Y, T228R, and WT were 2.31, 2.27, 2.25, 5321, and 2.24 μM, respectively (Fig. 6D and E, Table S8). This result suggested that the mutation at the THR228 site, with the highest Kdvalue, markedly reduced the binding ability of betulin to MpGABR, implying that the THR228 site may be an essential specific site for the binding of betulin to MpGABR.

Molecular docking, binding site and inhibitory effect of betulin on MpGABR.
(A) Best conformations of betulin docked to the binding pocket of MpGABR in aphids. An enlarged view of the betulin binding sites in MpGABR is indicated by a dashed frame. Potential Pi–alkyl (green), Alkyl (yellow), and hydrogen bond (red) interactions are indicated by dashed lines. (B) Sequence alignment of the key amino acids bound to betulin. The conserved residues among the different species in GABR are shown in orange, cyan, and light green. (C) Expression of the recombinant mutation type-aphid MpGABR by Escherichia coli. Lane 1: R244A, Lane 2: A226T, Lane 3: F227Y, Lane 4: T228R. (D, E) Quantification of the binding affinity of betulin with wild-type and mutant type-aphid MpGABR using MST. The bars represent the average (± SD) values. * indicates a significant difference (Student’s t test, ****P < 0.0001).
3.6 Betulin binds specifically to MpGABR via THR228
To further prove that THR228 is the specific binding site for betulin in MpGABR, the WT and mutant (R122T, equivalent to THR228 in MpGABR) Drosophila DmGABR proteins were expressed in E. coli and extracted (Fig. 7A and D). The binding affinities of betulin and pymetrozine with DmGABRWT and DmGABRR122T were evaluated using MST. As shown in Fig. 7B, betulin was able to bind to DmGABRR122T (Kd= 342.4 µM) but not to DmGABRWT. However, pymetrozine exhibited no binding affinity for DmGABRR122T or DmGABRWT (Fig. 7C). Moreover, to generate Drosophila mutant individuals carrying DmGABRR122T, the CRISPR/Cas9 genomic editing strategy was used. After exposure to different concentrations of betulin, the mortality rate of DmGABRR122T Drosophila was significantly greater than that of DmGABRWT Drosophila (Fig. 7E). In addition, the LD50 value of betulin against DmGABRR122T Drosophila at 72 h was 27.19 μg·fly−1, whereas the LD50 value of betulin against DmGABRWT Drosophila could not be calculated because the lethal concentration exceeded 1000 μg·fly−1 (Table S9). Additionally, there was no significant difference in mortality between DmGABRWT and DmGABRR122T Drosophila after exposure to pymetrozine (Fig. 7F). The LD50 values of pymetrozine against DmGABRWT and DmGABRR122T Drosophila at 72 h were 0.23 and 0.24 μg·fly−1, respectively (Table S9). Moreover, THR228 is located at the neurotransmitter-gated ion-channel ligand-binding domain in MpGABR (Fig. S2), implying that betulin may play a role as a competitive antagonist of MpGABR. Taken together, these findings suggested that betulin bound specifically to MpGABR via THR228, resulting in the death of M. persicae.

Gene editing in Drosophila validated the species-specific binding site of betulin.
(A) Expression of the recombinant wild type (WT) and mutation type (R122T) of DmGABR (Drosophila melanogaster) by E. coli. Lane 1: wild type, Lane 2: R122T. (B, C) Quantification of the binding affinity of betulin (B) and pymetrozine (C) with WT and R122T of DmGABR using MST. (D) Sanger sequencing of the DmGABR gene in flies. Direct sequencing chromatograms of PCR products amplified from a fragment of gDNA flanking the WT (a), and introduced R122T (b) mutant flies. (E-F) Toxicity curves of betulin (E), and pymetrozine (F) against WT, and the DmGABRR122T of the Cas9 Drosophila.
4. Discussion
Although chemical pesticides are effective in pest control, the long-term unreasonable application of these substances has led the emergence of resistant pests, environmental deterioration, and deleterious effects on nontarget organisms, including humans, raising widespread and intense concerns.30 These challenges have made it urgent for the development of alternative strategies for pest control. Recently, the utilization of plant secondary metabolites as insecticides has become increasingly popular as an eco-friendly and biocontrol approach.31–33 Our previous study revealed that P. davidiana, a wild relative of cultivated peach, strongly resists M. persicae by accumulating high contents of betulin.3, 5 In addition, betulin, a lupane-type triterpene, possesses potent insecticidal activity and is a promising substance for the development of novel insecticides for aphid control. In this study, the insecticidal effect of betulin was further evaluated by comparing the control efficacy of betulin with that of pymetrozine against aphids in greenhouses and fields (Fig. 1A-G). These results indicate that betulin has a similar control effect to pyrhidone and has immense potential for development as a plant-derived insecticide.
Terpenes are a diverse group of plant secondary metabolites that can increase the resistance of plants to insect herbivores through direct 34 and indirect 35 defense mechanisms. In direct defense against herbivores, triterpenes play important roles in diverse biological activities, including antiparasitic, insecticidal, and antifeedant activities.36, 37 Azadirachtin, a tetracyclic triterpenoid compound isolated from the Indian neem tree (Azadirachta indica), is one of the most prominent commercial biopesticides, exhibiting strong insect antifeedant properties as well as growth- and reproduction-regulating effects.38, 39 Besides, triterpene glycoside compounds play crucial roles in the defense of tobacco (N. attenuata) against tobacco hornworm (Manduca sexta) larvae.40 Although betulin exhibits various pharmacological activities,14–17 reports on its insecticidal activity are limited. Encouragingly, the effects of betulin and its derivatives on pests have attracted increasing attention. Betulinic acid and its derivatives showed larvicidal activity against Aedes aegypti larvae.41 Betulin-cinnamic acid-related hybrid compound 5b exhibited strong aphicidal activity, and compound 2l could destroy the ultrastructure of midgut cells and significantly inhibit the activity of α-amylase in diamondback moth (Plutella xylostella L.) larvae.42 Our previous studies also indicated that betulin possesses potent insecticidal activity and is a key endogenous secondary metabolite related to the defense of peach against M. persicae.5 Elucidating the insecticidal mechanism of betulin against aphids will provide a basis for the development of novel aphicides and sustainable strategies for aphid control.
GABA receptors have been confirmed to be targets of terpenoids that impair insect neuronal function in herbivores.19 GABRs are heteropentameric ligand-gated ion channels in the central nervous system that conduct chloride and bicarbonate ions. These receptors are targets of numerous drugs for the treatment of neuropsychiatric disorders.20 A variety of terpenoids act as positive allosteric modulators or noncompetitive antagonists of GABRs, such as diterpenoids (isopimaric acid and miltirone), sesquiterpenoids (picrotoxin, bilobalide and ginkgolides) and monoterpenoids (α-thujone and thymol).19 In this study, both GO and KEGG enrichment analyses revealed that the DEGs identified by RNA-seq were enriched in GABAergic signaling-related pathways (Fig. 2F and G). Additionally, the expression of the DEGs related to GABRs, particularly MpGABR (Table S4), in the betulin group was significantly lower than that in the CK group (Fig. 2H). Besides, the relative expression of MpGABR, MpGABRAP and MpGABRB decreased gradually after M. persicae was exposed to the LC30, LC50, and LC70 of betulin for 48 h (Fig. 3B-D). Among them, MpGABR was the most sensitive to betulin, and its expression was reduced by 82.91% after exposure to the LC30 of betulin for 48 h. Furthermore, compared with the control group, the M. persicae group with MpGABR silenced by RNAi presented a significant increase in mortality, by 30.44%, after 48 h of exposure to the LC50 of betulin (Fig. 3I). Collectively, these results suggest that betulin may have insecticidal effects on aphids by inhibiting MpGABR expression.
Additionally, betulin has been reported to be able to bind to GABRs.43 We further investigated the interaction of betulin with the MpGABR protein. The MST assay revealed that betulin was able to bind to MpGABR (Kd = 2.24 µM) (Fig. 5D), which is consistent with previous findings showing that betulin binds to GABA receptors in mouse brains in vitro.44 Voltage-clamp-based electrophysiological recordings indicated that betulin acted as an inhibitor (EC50 = 20.66 µM) for MpGABR (Fig. 5E and F). Subsequent molecular docking analysis suggested that four key amino acid residues (ARG224, ALA226, PHE227 and THR228) interact with betulin in the MpGABR binding pocket (Fig. 6A), among which only ALA226 and THR228 interact with betulin via hydrogen bonding (Table S7). The results of the sequence alignment revealed that only THR228 was conserved in Hemiptera (Fig. 6B). Furthermore, evaluation of the ability of betulin to bind to MpGABR mutants with mutations at those four sites revealed that the ability of betulin to bind to T228R was significantly weaker than its ability to bind to the WT (Fig. 6D and E, Table S8), indicating that THR228 is an essential specific site for the binding of betulin to MpGABR. Moreover, to further prove that THR228 is the specific binding site for betulin in MpGABR, the binding affinities of betulin with the WT and mutant (R122T, equivalent to THR228 in MpGABR) Drosophila DmGABR proteins were assessed using MST. The results showed that betulin was able to bind to DmGABRR122T (Kd= 342.4 µM) but not DmGABRWT (Fig. 7B). Additionally, after exposure to different concentrations of betulin, the mortality rate of DmGABRR122T Drosophila was significantly greater than that of DmGABRWT Drosophila (Fig. 7E and Table S9). Similarly, a previous study indicated that the R122G amino acid site substitution, generated via RNA editing, affects the sensitivity of Drosophila to fipronil.45 Together, these findings suggest that betulin binds specifically to MpGABR via THR228, acting as an inhibitor of MpGABR and causing aphid death.
GABRs belong to the cysteine loop (Cys)-loop superfamily of neurotransmitter receptors and are essential heteropentameric ligand-gated ion channels in the central nervous system. After GABA binds to the extracellular Cys-loop of a GABR,46 the GABR is activated and opens up its central pore to allow chloride ions to pass through, thereby hyperpolarizing neurons and attenuating excitatory neurotransmission.47 In insects, GABRs play an important role in circadian rhythms,48 sleep,49 movement,50 olfactory memory,51 and so on. Additionally, GABR is a critical target for a variety of insecticides and ectoparasiticides. Insecticides targeting GABRs are categorized into noncompetitive antagonists (NCAs) and competitive antagonists (CAs) according to their different binding sites. NCAs block chloride channels in nerve cells by interacting with amino acid residues of the GABA-gated chloride channel, causing a conformational change in the receptor, which interferes with the normal function of the central nervous system and ultimately leads to insect death.19, 52 A series of first- and second-generation NCA insecticides have been successfully developed. Picrotoxinin, isolated from Anamirta cocculus fruit, is the oldest natural NCA and is toxic to houseflies.53 Among the first-generation NCAs, polychlorocycloalkanes, including dieldrin and lindane, were commonly used as pesticides in the middle and late 20th centuries.54, 55 As second-generation NCAs, fipronil and its derivatives are commercially available phenylpyrazole insecticides that have been widely used for agricultural pest control.56, 57 Additionally, CAs bind to orthogonal binding sites, such as the GABA recognition site in the extracellular region, competitively inhibiting the binding of GABA to its receptor, leading to toxic effects in insects. Given the lack of interference from existing insect resistance mechanisms, CAs hold promise for the development of efficient new insecticides. Novel 1, 6-dihydro-6-iminopyridazine-derived insecticides, as CAs, have insecticidal properties against common cutworms and houseflies.58 Besides, nootkatone, a sesquiterpenoid, also acts as a CA to induce insect mortality.59 Fluralaner, an isooxazoline ectoparasiticide, inhibits both parasites and Aedes aegypti by acting as a CA.60, 61 Our results revealed that the binding site (THR228) for betulin in MpGABR was located in the extracellular neurotransmitter-gated ion-channel ligand-binding domain (Fig. S2), implying that betulin acts as a CA of MpGABR. Our work may have provided a novel target for developing insecticides. In this study, betulin, on the one hand, inhibited the expression of MpGABR and, on the other hand, specifically bound to MpGABR through THR228, acting as an inhibitor of MpGABR and causing aphid death (Fig. 8).

Proposed model for the mechanism of action against Myzus persicae by targeting GABAA receptors (GABR).
After exposure to betulin, the expression of MpGABR was inhibited the level of MpGABR protein is decreased, resulting in a decrease in the channel of chloride ion influx. Besides, betulin directly and specifically binds to the amino acid residue THR228 of MpGABR, thereby disabling it.
5. Conclusion
Betulin, a key metabolite in the aphid-resistant wild peach P. davidiana, possesses potent aphicidal effects on M. persicae. This study confirmed that betulin exhibited excellent control efficacy against M. persicae in both greenhouse and field experiments. RNA-seq, qRT‒PCR and western blotting assays revealed that betulin significantly inhibited the expression of MpGABR in aphids. In addition, RNAi-mediated silencing of MpGABR markedly increased the sensitivity of aphids to betulin. Moreover, MST and voltage-clamp-based electrophysiological recording assays indicated that betulin was able to bind to MpGABR (Kd = 2.24 µM) and acted as an inhibitor (EC50 = 20.66 µM) of MpGABR. Molecular docking analyses suggested that the amino acid residue THR228, which is highly conserved in Hemiptera, might be a critical specific binding site for betulin in MpGABR. Mutagenesis and genome editing assays revealed that betulin bound specifically to this amino acid residue in aphids but not in Drosophila, resulting in aphid death. Collectively, the results suggest that the aphicidal effects of betulin on aphids occur in a two-pronged manner: on the one hand, betulin inhibits MpGABR expression; on the other hand, it specifically binds to MpGABR via THR228 and acts as an inhibitor of MpGABR. Elucidating the insecticidal mechanism of betulin against aphids will provide a basis for the development of novel insecticides and sustainable strategies for aphid control.
Acknowledgements
This research was supported by the National Natural Science Foundation of China (32402492, 32302394), Special Funding for Chongqing Postdoctoral Research Project (2312013543265057), Sichuan Science and Technology Program (2025ZNSFSC1120), and Fundamental Research Fund for the Central Universities of China (SWU-KR25018).
Additional files
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