Although chemical pesticides are effective in pest control, the long-term unreasonable application of these substances has led to the emergence of resistant pests, environmental deterioration, and deleterious effects on nontarget organisms, including humans, raising widespread and intense concerns (Devi et al., 2022). 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 (Ling et al., 2022; Ayilara et al., 2023). Our previous study revealed that P. davidiana, a wild relative of cultivated peach, strongly resists M. persicae by accumulating high contents of betulin (Wang et al., 2022a; Wang et al., 2024). 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 (Figure 1A–G). These results indicate that betulin has a similar control effect to pymetrozine 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 (Wang et al., 2025a) and indirect (Wang et al., 2025b) defense mechanisms. In direct defense against herbivores, triterpenes play important roles in diverse biological activities, including antiparasitic, insecticidal, and antifeedant activities (Tian et al., 2021; Kuzminac et al., 2023). 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 (Dawkar et al., 2019; Bae et al., 2022). Besides, triterpene glycoside compounds play crucial roles in the defense of tobacco (Nicotiana attenuata) against tobacco hornworm (Manduca sexta) larvae (Yang et al., 2024). Although betulin exhibits various pharmacological activities (Amiri et al., 2020; Li et al., 2022; Yan et al., 2022; Lv et al., 2023), 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 A. aegypti larvae (da Silva et al., 2016). 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 (Pl. xylostella L.) larvae (Huang et al.). 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 (Wang et al., 2022a). 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 (Guo et al., 2023). 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 (Thompson, 2024). A variety of terpenoids act as positive allosteric modulators or NCAs of GABRs, such as diterpenoids (isopimaric acid and miltirone), sesquiterpenoids (picrotoxin, bilobalide, and ginkgolides), and monoterpenoids (α-thujone and thymol) (Guo et al., 2023). In this study, both GO and KEGG enrichment analyses revealed that the DEGs identified by RNA-seq were enriched in GABAergic signaling-related pathways (Figure 2F and G). Additionally, the expression of the DEGs related to GABRs, particularly MpGABR (Figure 2—source data 1), in the betulin group was significantly lower than that in the CK group (Figure 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 hr (Figure 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 hr. Furthermore, compared with the control group, the M. persicae group with MpGABR silenced by RNAi presented a significant increase in mortality (p<0.001), by 30.44%, after 48 hr of exposure to the LC50 of betulin (Figure 4E). Collectively, these results suggest that betulin may have insecticidal effects on aphids by inhibiting MpGABR expression. The regulation of gene expression is sophisticated and delicate (Pope and Medzhitov, 2018). The regulatory network controlling GABR expression remains unclear. In adult rats, epileptic seizures have been reported to increase the levels of brain-derived neurotrophic factor, which in turn prompted the transcription factors CREB and ICER to reduce the gene expression of the GABR α1 subunit (Lund et al., 2008). In Drosophila, it has been demonstrated that WIDE AWAKE, which regulated the onset of sleep, interacted with the GABR and upregulated its expression level (Liu et al., 2014). In the Drosophila brain, circular RNA circ_sxc was found to inhibit the expression of miR-87-3-p in the brain through sponge adsorption, thereby regulating the expression of neurotransmitter receptor ligand proteins, including GABR, and ensuring the normal function of synaptic signal transmission in brain neurons (Li et al., 2024). However, it remains unclear how betulin reduces MpGABR expression, and further research is needed.

Additionally, betulin has been reported to be able to bind to GABRs (Manayi et al., 2016). 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) (Figure 6B), which is consistent with previous findings showing that betulin binds to GABA receptors in mouse brains in vitro (Muceniece et al., 2008). Voltage-clamp-based electrophysiological recordings indicated that betulin acted as an inhibitor (EC50=20.66 µM) for MpGABR (Figure 6C and D). Subsequent molecular docking analysis suggested that four key amino acid residues (ARG224, ALA226, PHE227, and THR228) interact with betulin in the MpGABR binding pocket (Figure 7A), among which merely ALA226 and THR228 interact with betulin via hydrogen bonding (Figure 7—source data 1). The results of the sequence alignment revealed that only THR228 was conserved across 11 species in the Aphididae family of Hemiptera (Figure 7B). 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 (Figure 7D and E, Figure 7—source data 2), 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 (Figure 8B). Additionally, after exposure to different concentrations of betulin, the mortality rate of DmGABRR122T Drosophila was significantly greater than that of DmGABRWT Drosophila (Figure 8E, Figure 8—source data 1). Similarly, a previous study indicated that the R122G amino acid site substitution, generated via RNA editing, affects the sensitivity of Drosophila to fipronil (Es-Salah et al., 2008). Together, these findings suggest that betulin binds specifically to MpGABR via THR228, acting as an inhibitor of MpGABR and causing aphid death. Studies on key amino acids that are crucial for GABR function have primarily focused on transmembrane regions. For instance, based on the mutational research and Drosophila GABR modeling approach, multiple key amino acids have been identified as insecticide targets in the transmembrane domain (Nakao and Banba, 2021). Guo et al., 2023 proposed that amino acid substitutions in transmembrane domain 2 contribute to terpenoid insensitivity during plant-insect coevolution. However, these studies have neglected the extracellular domain. Our study signified that betulin targets the THR228 site in the extracellular domain of MpGABR, which is conserved only in the Aphididae family. Therefore, betulin is speculated to be a specific insecticidal substance evolved by plants in response to aphid infestation. Besides, further verification is needed to determine whether betulin is toxic to other insect species.

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 (Ashby et al., 2012), the GABR is activated and opens up its central pore to allow chloride ions to pass through, thereby hyperpolarizing neurons and attenuating excitatory neurotransmission (Tremblay et al., 2016). In insects, GABRs play important roles in circadian rhythms (Schellinger et al., 2022), sleep (Chaturvedi et al., 2022), movement (Eick et al., 2022), and olfactory memory (Yamagata et al., 2021). Additionally, GABR is a critical target for a variety of insecticides and ectoparasiticides. Insecticides targeting GABRs are categorized into 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 (Nakao and Banba, 2021; Guo et al., 2023). 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 (Tong et al., 2023). Among the first-generation NCAs, polychlorocycloalkanes, including dieldrin and lindane, were commonly used as pesticides in the middle and late 20th centuries (Hainzl et al., 1998; Tanaka, 2019). As second-generation NCAs, fipronil and its derivatives are commercially available phenylpyrazole insecticides that have been widely used for agricultural pest control (Sheng et al., 2018; Li et al., 2021). 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 (Liu et al., 2022). Besides, nootkatone, a sesquiterpenoid, also acts as a CA to induce insect mortality (Norris et al., 2022). Fluralaner, an isooxazoline ectoparasiticide, inhibits both parasites and A. aegypti by acting as a CA (Wang et al., 2022b; Asahi et al., 2023). Our results revealed that the binding site (THR228) for betulin in MpGABR was located in the extracellular neurotransmitter-gated ion-channel ligand-binding domain (Figure 7—figure supplement 1), implying that betulin acts as a CA of MpGABR. Although the mechanism by which betulin competes with GABA for binding to MpGABR requires further experimental validation, 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 (Figure 9).

Proposed model for the mechanism of action against M. persicae by targeting GABAA receptors (GABR).

After exposure to betulin, the expression of MpGABR was inhibited, and the level of MpGABR protein 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.

The development of bioinsecticides should not only focus on the toxic effects of active substance on target organisms, but also on their influence on the ecosystem (Haddi et al., 2020). Although our results indicate that betulin has specific toxicity to aphids, previous studies have reported that betulin and its derivatives had effects on P. xylostella L. (Huang et al., 2025), A. aegypti (de Almeida Teles et al., 2024), and D. melanogaster (Lee and Min, 2024). Therefore, further research is needed to determine whether there are other insecticidal mechanisms or off-target effects of betulin. Additionally, betulin exhibits a wide range of pharmacological activities (Amiri et al., 2020), which have been used to treat various diseases, such as cancer (Lv et al., 2023), glioblastoma (Li et al., 2022), inflammation (Szlasa et al., 2023), and hyperlipidemia (Tang et al., 2011). Before applying betulin in the field, it is necessary to fully verify and consider whether betulin has any impact on farmers’ health. Furthermore, will betulin cause residue or diffusion during the field application process? Will long-term application promote the evolution of resistance to aphids or other insects? These issues also need further experimental verification. In summary, before any field application, further research on the environmental behavior, degradation process, and safety of betulin is needed.

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 across 11 species in the Aphididae family of 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.