Identification and functional validation of glutathione S‐transferase genes involved in detoxification of sulfoxaflor, afidopyropen and lambda‐cyhalothrin in Aphis glycines

Abstract

BACKGROUND

Glutathione S‐transferases (GSTs) play important roles in the detoxification of insecticides in insects. However, genome‐wide identification and functional characterization of the GST gene family in the soybean aphid Aphis glycines have not been performed.

RESULTS

A total of 17 AgGST genes were identified in the A. glycines genome and classified into five classes. Phylogenetic analysis and chromosomal mapping showed that delta and epsilon class genes experienced significant expansion. Exposure to LC₅₀ concentrations of sulfoxaflor, afidopyropen and lambda‐cyhalothrin strongly induced several AgGST genes with AgGSTd5, AgGSTd6 and AgGSTe2 displaying the highest expression levels. RNA interference of AgGSTd5 significantly increased aphid mortality following exposure to all three insecticides. Knockdown of AgGSTd6 significantly elevated mortality under sulfoxaflor, while knockdown of AgGSTe2 significantly increased mortality under both sulfoxaflor and lambda‐cyhalothrin. In contrast, silencing of AgGSTt1 and AgGSTt2 showed no significant effect on aphid mortality under the tested insecticides.

CONCLUSION

This study provides comprehensive characterization of the GST gene family in A. glycines and demonstrates that AgGSTd5 plays a central role in the detoxification of sulfoxaflor, afidopyropen and lambda‐cyhalothrin, while AgGSTd6 and AgGSTe2 contribute to tolerance against specific insecticides among the three compounds. These genes represent promising molecular targets for monitoring insecticide detoxification responses and for the development of strategies based on GST inhibitors to enhance insecticide efficacy in integrated pest management. © 2026 Society of Chemical Industry.

This study identified 17 GST genes in Aphis glycines and demonstrated that AgGSTd5, AgGSTd6, and AgGSTe2 play central roles in the detoxification of three distinct insecticides.

1. INTRODUCTION

The soybean aphid, Aphis glycines, is a phloem‐feeding insect pest that causes substantial damage to soybean through direct feeding on phloem sap as well as by transmitting major plant viruses, including soybean mosaic virus and alfalfa mosaic virus. ^{1 , 2} As one of the most important pests of soybean worldwide, A. glycines causes substantial yield losses of 8–35%. ³ Chemical insecticides remain the primary method for controlling A. glycines. ² Pyrethroid insecticides such as lambda‐cyhalothrin have been widely used in spray programs for many years. ⁴ North American field populations have developed significant pyrethroid resistance since 2014. ⁵ This resistance is conferred by voltage‐gated sodium channel mutations L1014F and M918I. ⁶ Asian laboratory strains exhibit resistance levels as high as 76‐fold and cross‐resistance to other insecticide classes. ⁷ To reduce cross‐resistance risks, selective insecticides with novel modes of action such as sulfoxaflor and afidopyropen are now included in rotation strategies. ^{8 , 9} These three insecticides induce significant xenobiotic stress in aphids owing to their distinct modes of action. Insects have evolved complex defense systems against xenobiotics, of which metabolic detoxification is the most common pathway. ¹⁰ Therefore, it is important to understand the early detoxification responses of A. glycines.

Glutathione S‐transferases (GSTs, EC 2.5.1.18) constitute a large multifunctional enzyme superfamily widely distributed in almost all organisms. ¹¹ In insects, GSTs play important roles in the detoxification of both endogenous and exogenous compounds. ¹² As one of the major phase II detoxification enzymes, GSTs catalyze the nucleophilic conjugation of reduced glutathione (GSH) with electrophilic substrates of endogenous and exogenous origin. ¹¹ This reaction increases the hydrophilicity of the xenobiotics, facilitating their excretion and mitigating oxidative stress. ¹¹ Consequently, GSTs can efficiently metabolize a wide variety of insecticides and play critical roles in the detoxification response to insecticide stress. For example, BdGSTd2 in Bactrocera dorsalis is strongly induced by malathion and contributes to insecticide detoxification by directly metabolizing the compound. ¹³ Similarly, SfGSTe9 in Spodoptera frugiperda is significantly upregulated following exposure to β‐cypermethrin, lambda‐cyhalothrin, chlorpyrifos and malathion and confers tolerance to these insecticides. ¹⁴ In insects, most GSTs belong to the cytosolic group, which is further divided into six principal classes: delta, epsilon, omega, sigma, theta and zeta. ¹² Among these, the delta and epsilon classes are most frequently implicated in insecticide detoxification and resistance. ¹⁵ While, the genome of A. glycines has been available since 2020, annotation of the GST gene family remains limited with only 14 GST genes identified to date. ¹⁶

In this study, we performed a genome‐wide identification of the GST gene family in A. glycines. We identified 17 AgGST genes and systematically analyzed their phylogenetic relationships, chromosomal distribution, and physicochemical properties. We examined the transcriptional responses of these genes to LC₅₀ concentrations of sulfoxaflor, afidopyropen and lambda‐cyhalothrin using qRT‐PCR. We further evaluated the functional roles of key candidate genes through RNAi and molecular docking simulations. This study reveals the molecular basis of GST mediated detoxification of multiple insecticides in A. glycines and identifies promising candidate genes for monitoring insecticide detoxification responses and for sustainable pest management strategies.

2. MATERIALS AND METHODS

2.1. Insect culture and chemicals

The A. glycines was collected from soybean fields in Harbin, Heilongjiang Province, China, in 2023. The colony was reared on soybean seedlings (cv. Heinong 306) at 25 ± 1 °C, 65–70% relative humidity and a 16:8 h (L:D) photoperiod without insecticide exposure. Three insecticides were used: sulfoxaflor (97.9% TC, Dow AgroSciences), afidopyropen (97.3% TC, Shenyang Sinochem Agrochemicals R&D Co., Ltd.) and lambda‐cyhalothrin (97% TC, Jiangsu Changlong Chemical Co., Ltd.). Stock solutions were prepared in 1% acetone and diluted with 0.05% Triton X‐100 in distilled water.

2.2. Bioassays

Baseline toxicity was determined by leaf‐dip bioassay. Soybean leaves were dipped in insecticide solutions for 25 s, air‐dried and placed in Petri dishes lined with moist filter paper. ¹⁷ Twenty adult aphids were placed on each leaf and mortality was scored after 48 h. LC₅₀ values were calculated with PoloPlus v2.0. For gene expression assays, aphids were exposed to LC₅₀ concentrations via leaf dip and sampled at 6, 12, 24 and 48 h. For RNAi bioassays, aphids were treated with dsRNA for 48 h and then exposed to LC₅₀ concentrations of the corresponding insecticides.

2.3. Genome‐wide identification and phylogenetic analysis of the AgGST gene family

The genome sequence of A. glycines (GenBank accession GCA_046127445.1) was downloaded from NCBI. Hidden Markov Model profiles of the GST N‐terminal domain (PF02798) and C‐terminal domain (PF00043) were obtained from Pfam and used to search the proteome with HMMER 3.1 (E‐value ≤1e‐5). Protein sequences of GSTs from Drosophila melanogaster, Nilaparvata lugens, Bactrocera dorsalis, Apis mellifera, Cydia pomonella, Myzus persicae, Acyrthosiphon pisum and Bemisia tabaci were downloaded and used as queries for local BLASTP (E‐value ≤1e‐10, identity ≥50%). Phylogenetic analysis was performed using the amino acid sequences of GST proteins from A. glycines and other insect species (Table S1). All candidate sequences were validated for conserved GST domains using SMART and NCBI CDD. After removal of redundant and incomplete sequences, 17 putative AgGST genes were retained.

The 17 AgGST proteins together with the above outgroup sequences were aligned with MAFFT v7.525. Ambiguous regions were trimmed with trimAl v1.5.0. A maximum‐likelihood phylogenetic tree was constructed using RAxML v8.2.13 with 1000 bootstrap replicates and visualized in iTOL. Genes were named according to insect GST nomenclature (delta, epsilon, sigma, theta, omega). Chromosomal locations and exon‐intron structures were extracted from the GFF3 file and plotted with TBtools v2.2.7. Molecular weights and isoelectric points of the deduced proteins were calculated with ExPASy ProtParam.

2.4. RNA extraction and quantitative real‐time PCR

Total RNA was extracted from adult aphids using TRIzol reagent (Vazyme, China) according to the manufacturer's protocol. Genomic DNA was removed with RQ1 RNase‐Free DNase (Promega, USA). First‐strand cDNA was synthesized from 1 μg total RNA using the PrimeScript RT reagent kit with gDNA eraser (TaKaRa, Japan). Quantitative real‐time PCR was performed on a QuantStudio 5 Real‐Time PCR System (Thermo Fisher Scientific, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). Each 10 μL reaction contained 5 μL 2× Master Mix, 0.5 μL each primer (10 μM), 1 μL cDNA and 3 μL ddH₂O. The thermal cycling conditions were 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. Melting curve analysis was performed from 60 °C to 95 °C to confirm specificity. EF1α and 18S were used as the reference genes. ¹⁸ Their Ct values were combined using the geometric mean for normalization. Relative expression levels were calculated by the 2^−ΔΔCt method. All primers used for qRT‐PCR and dsRNA synthesis are listed in Table S2. Three biological replicates, each consisting of 20 aphids, were analyzed for each treatment and time point (6, 12, 24 and 48 h after LC₅₀ insecticide exposure).

2.5. RNA interference

Double‐stranded RNA (dsRNA) targeting AgGSTd5, AgGSTd6, AgGSTe2, AgGSTt1 and AgGSTt2 was synthesized in vitro using the T7 RiboMAX expression system (Promega, USA). For topical delivery, dsRNA was mixed with a star polycation (SPc) nanocarrier at a 1:1 mass ratio (final concentration 500 ng μL⁻¹ each), and 0.3% (v/v) alkyl polyglycoside (APG) was added to enhance penetration. ¹⁸ A 0.3 μL aliquot of the mixture was applied to the dorsal thorax of each adult aphid. dsGFP was used as the negative control. Knockdown efficiency was verified by qRT‐PCR at 48 h treatment. For bioassays, aphids were exposed to LC₅₀ concentrations of the corresponding insecticides 48 h after dsRNA treatment, and mortality was recorded after an additional 48 h. Three biological replicates with 20 aphids per replicate were performed for each gene and insecticide combination.

2.6. Molecular docking analysis

The three‐dimensional structures of AgGSTd5 were predicted using the AlphaFold server. The 3D structures of sulfoxaflor, afidopyropen and lambda‐cyhalothrin were downloaded from the PubChem database in SDF format and converted to PDBQT format using AutoDock Tools v1.5.6. Protein structures were prepared by removing water molecules and adding polar hydrogens in PyMOL v2.6.0. Molecular docking was performed with AutoDock Vina v1.1.2. The grid box was centered on the predicted active site pocket. The binding pose with the lowest binding energy was selected for each ligand‐protein pair and visualized in PyMOL. Key interacting residues and hydrogen bonds were analyzed and recorded.

2.7. Statistical analysis

All data were analyzed using SPSS Statistics 26.0 (IBM, USA). Differences in gene expression levels among treatments and time points were analyzed by one‐way ANOVA followed by Tukey's HSD test. Differences in knockdown efficiency and bioassay mortality between dsGFP control and dsAgGST treatments were analyzed by Student's t‐test. Results are presented as mean ± standard error (SE) of three biological replicates. Statistical significance was set at p < 0.05.

3. RESULTS

3.1. Identification and phylogenetic analysis of the AgGST gene family in A. glycines

A total of 17 putative AgGST genes were identified in the genome of A. glycines. Based on phylogenetic clustering and sequence similarity with known insect GSTs, these genes were assigned to five classes. These included three sigma class genes (AgGSTs1, AgGSTs2, AgGSTs3), one omega class gene (AgGSTo1), four epsilon class genes (AgGSTe1, AgGSTe2, AgGSTe3, AgGSTe4), two theta class genes (AgGSTt1, AgGSTt2), and seven delta class genes (AgGSTd1, AgGSTd2, AgGSTd3, AgGSTd4, AgGSTd5, AgGSTd6, AgGSTd7). The open reading frames (ORFs) of the AgGST genes ranged from 516 to 1650 bp, encoding proteins with molecular weights ranging from 20.07 to 62.68 kDa and theoretical isoelectric points (pI) between 4.68 and 9.32 (Table 1). The open reading frames (ORFs) of the 17 identified AgGST genes are provided in Table S3.

Table 1.

Summary of 17 AgGST Genes Identified from the A. glycines Genome

Class	Gene name	ORF(bp/aa)	M(kDa)	pI
Sigma	AgGSTs1	612/203	23.64	6.22
Sigma	AgGSTs2	516/171	20.07	5.41
Sigma	AgGSTs3	612/203	22.30	5.31
Omega	AgGSTo1	933/310	35.46	8.74
Epsilon	AgGSTe1	930/309	36.11	5.67
Epsilon	AgGSTe2	1161/386	44.31	9.32
Epsilon	AgGSTe3	1143/380	43.15	5.89
Epsilon	AgGSTe4	807/268	31.27	6.61
Theta	AgGSTt1	699/232	27.31	8.20
Theta	AgGSTt2	702/233	27.49	8.68
Delta	AgGSTd1	807/268	31.08	6.51
Delta	AgGSTd2	666/221	24.90	6.90
Delta	AgGSTd3	651/216	24.18	6.51
Delta	AgGSTd4	651/216	24.34	4.68
Delta	AgGSTd5	1650/549	62.68	6.84
Delta	AgGSTd6	1293/430	49.22	5.91
Delta	AgGSTd7	810/269	31.21	5.70

The number of identified GST genes across the selected representative insect species is summarized in Table 2. To resolve the evolutionary relationships of the 17 AgGST genes, a phylogenetic tree was constructed using amino acid sequences from A. glycines and eight other insect species (Drosophila melanogaster, Nilaparvata lugens, Bactrocera dorsalis, A. mellifera, Cydia pomonella, Myzus persicae, Acyrthosiphon pisum, and Bemisia tabaci). The 17 AgGST genes were resolved into five major clades corresponding to the sigma, omega, epsilon, theta and delta (Fig. 1). Bootstrap support was strong at most nodes. The delta and epsilon classes formed the two largest clades.

Table 2.

Comparative analysis of the number and class composition of GST genes in different insect species

Order	Species	Delta	Epsilon	Omega	Sigma	Theta	Zeta	Unclassified	Microsomal	Total	Reference
Coleoptera	Tribolium castaneum	3	19	3	7	1	1	2	5	41	19
	Leptinotarsa decemlineata	3	10	5	4	4	1	2	1	30	20
Diptera	Drosophila melanogaster	11	14	5	1	4	2	0	1	38	21
	Anopheles gambiae	12	8	1	1	2	1	3	3	31	22
Hemiptera	Nilaparvata lugens	2	1	1	3	1	1	0	2	11	23
	Bemisia tabaci	14	0	1	6	0	2	—	2	25	24
	Diaphorina citri	2	2	0	3	0	0	1	2	10	25
	Aphis glycines	7	4	1	3	2	0	0	0	17	This study
	Myzus persicae	8	0	0	8	2	0	0	2	20	26
	Acyrthosiphon pisum	10	0	2	6	2	0	0	2	22	26
	Aphis gossypii	2	0	1	3	2	0	1	2	11	27
Hymenoptera	Apis mellifera	1	0	1	4	1	1	0	2	10	24
Lepidoptera	Spodoptera frugiperda	5	21	3	7	1	5	3	2	47	14
	Plutella xylostella	5	5	5	2	1	2	2	‐	22	28

Note: Data for A. glycines were obtained in this study. ‘‐’ indicates data not reported.

Figure 1.

Phylogenetic analysis of GSTs from A. glycines and representative insect species including D. melanogaster, N. lugens, B. dorsalis, A. mellifera, C. pomonella, M. persicae, A. pisum, and B. tabaci. Different classes are indicated by colored ranges.

3.2. Chromosomal distribution of the GST gene family in A. glycines

Chromosomal mapping revealed that the 17 AgGST genes are localized across four distinct chromosomes within the A. glycines genome (Fig. 2). Chromosome 1 contained eight genes. Five of them formed a tight cluster near the distal end. Mapping analysis assigned five genes to chromosome 2 and positioned three additional members at the distal extremity of chromosome 3. Only AgGSTd1 was found on the X chromosome. All 17 AgGST genes were successfully mapped to their chromosomal positions.

Figure 2.

Chromosomal distribution of GST genes in A. glycines. The physical locations of AgGST genes on chromosomes X, 1, 2, and 3 are illustrated. Different colors represent different classes.

The exon‐intron structures of the 17 AgGST genes in A. glycines exhibited substantial variation in genomic length and architectural complexity (Fig. 3). The majority of genes possessed relatively simple organizations, typically comprising 2–4 exons separated by short introns and spanning less than 5 kb. In contrast, AgGSTd7 displayed the most extended structure, exceeding 20 kb with multiple exons distributed across long intronic regions. AgGSTe3 also featured a distinctive extended architecture characterized by a large intron separating distant exons. Several delta class genes, including AgGSTd2 and AgGSTd3, showed moderately dispersed exon arrangements, whereas sigma, omega, theta, and most epsilon class members generally maintained compact, clustered organizations.

Figure 3.

Exon‐intron structures of AgGST genes in A. glycines.

3.3. Developmental expression patterns of AgGST genes

To investigate the developmental expression profiles of AgGST genes, transcript levels across all life stages were quantified using the 2^−ΔΔCt method, with first‐instar nymphs (1 N) serving as the calibrator. Expression analysis demonstrated that transcripts for all candidate genes were detectable across every developmental phase. (Figure 4). A total of six genes (AgGSTd2, AgGSTd3, AgGSTd4, AgGSTd5, AgGSTd6, AgGSTs3) are highly expressed at the adult stage. In contrast, AgGSTd1 and AgGSTs2 exhibited low expression levels across all developmental stages. These results indicate that most AgGST genes display stage specific expression patterns, with peak expression in adults for several delta class and sigma class members.

Figure 4.

Expression profiles of AgGST genes at different developmental stages in A. glycines. Heatmap showing log₂‐transformed relative expression levels of AgGST genes in first‐instar to fourth‐instar nymphs (1 N–4 N) and adults (Ad). Relative expression was calculated using the 2^−ΔΔCt method with first‐instar nymphs (1 N) as the calibrator (set as 1). The color scale indicates relative expression intensity (red: high expression; blue: low expression; white: intermediate expression).

3.4. Toxicity baseline of three insecticides against A. glycines

The baseline toxicity of three insecticides against A. glycines adults was determined using the leaf‐dip bioassay (Table 3). Sulfoxaflor exhibited the highest toxicity, with an LC₅₀ value of 1.501 mg/L (95% CI: 1.141–1.958). Afidopyropen showed moderate toxicity (LC₅₀ = 6.919 mg/L, 95% CI: 5.404–8.653), while lambda‐cyhalothrin was the least toxic (LC₅₀ = 13.950 mg/L, 95% CI: 11.314–17.221).

Table 3.

Toxicity of three insecticides to A. glycines adults

Insecticide	Slope (±SE)	LC50 (95%CI) (mg/L)	df	χ2
Lambda‐cyhalothrin	2.117 ± 0.273	13.950 (11.314–17.221)	13	1.712
Afidopyropen	1.459 ± 0.138	6.919 (5.404–8.653)	19	6.350
Sulfoxaflor	1.702 ± 0.138	1.501 (1.141–1.958)	22	33.781

Note: SE, standard error; LC, lethal concentration; CI, confidence interval; df, degrees of freedom; χ², chi‐square value.

3.5. Response of AgGST genes to exposure to three insecticides

To investigate the response of AgGST genes to insecticides, aphids were exposed to LC₅₀ concentrations of sulfoxaflor, afidopyropen and lambda‐cyhalothrin (Fig. 5, Tables [Link], [Link]). Multiple genes showed an increase in transcript levels. AgGSTd5, AgGSTd6 and AgGSTe2 were strongly induced under sulfoxaflor treatment. The expression of AgGSTd5 increased from 3.54‐fold at 6 h to a peak of 11.09‐fold at 24 h. AgGSTd6 reached its highest level of 10.14‐fold at 12 h. AgGSTe2 maintained high expression under both sulfoxaflor and lambda‐cyhalothrin, reaching 9.65‐fold at 12 h and 9.42‐fold at 48 h, respectively. In contrast, AgGSTt1 and AgGSTt2 showed different response patterns. AgGSTt1 was strongly induced by afidopyropen, reaching 9.27‐fold at 6 h, while AgGSTt2 was highly expressed under lambda‐cyhalothrin at 48 h. Several genes, including AgGSTd1, AgGSTd7 and AgGSTe1, showed relatively weak response to all three insecticides.

Figure 5.

The expression of AgGST genes in A. glycines adults at different time points following LC₅₀ concentrations of sulfoxaflor, afidopyropen, and lambda‐cyhalothrin induction. The color scale represents relative expression (log₂‐transformed fold change; dark blue: high expression; white: low expression). Bubble size indicates statistical significance (−log₁₀ (P‐value)), with larger bubbles representing more significant differences compared with control.

3.6. RNA interference validation of selected AgGST genes and bioassay results

To evaluate the functional roles of candidate AgGST genes in insecticide detoxification, dsRNA‐mediated gene silencing was performed. RNAi‐mediated silencing of AgGSTd6, AgGSTt2, AgGSTe2, AgGSTt1 and AgGSTd5 achieved knockdown efficiencies of 56%, 58%, 59%, 67% and 48%, respectively (Fig. 6).

Figure 6.

RNAi knockdown efficiency of selected AgGST genes in A. glycines adults. Relative expression levels of (A) AgGSTd5, (B) AgGSTd6, (C) AgGSTe2, (D) AgGSTt1, and (E) AgGSTt2 in adults 48 h after dsRNA treatment compared with the dsGFP control. Data are presented as means ± SE. Asterisks indicate significant differences between dsAgGST treatment and dsGFP control (*P < 0.05, *** P < 0.001, Student's t‐test).

Subsequently, the RNAi‐treated aphids were exposed to LC₅₀ concentrations of sulfoxaflor, afidopyropen and lambda‐cyhalothrin, and mortality was recorded after 48 h (Fig. 7). Knockdown of AgGSTd5 significantly increased mortality under all three insecticides. Mortality rose from 43.3% to 73.3% under sulfoxaflor, from 46.7% to 76.7% under afidopyropen, and from 58.3% to 68.3% under lambda‐cyhalothrin. Similarly, silencing of AgGSTd6 resulted in a marked increase in mortality under sulfoxaflor, from 40.0% to 78.3%. Knockdown of AgGSTe2 also significantly elevated mortality under both sulfoxaflor (from 50.0% to 80.0%) and lambda‐cyhalothrin (from 55.0% to 76.7%). In contrast, silencing of AgGSTt1 and AgGSTt2 did not significantly affect aphid mortality under afidopyropen or lambda‐cyhalothrin treatments.

Figure 7.

Effect of AgGST gene silencing on sulfoxaflor, afidopyropen, and lambda‐cyhalothrin toxicity in A. glycines adults. (A) AgGSTd5 with sulfoxaflor, (B) AgGSTd5 with afidopyropen, (C) AgGSTd5 with lambda‐cyhalothrin, (D) AgGSTd6 with sulfoxaflor, (E) AgGSTe2 with sulfoxaflor, (F) AgGSTe2 with lambda‐cyhalothrin, (G) AgGSTt1 with afidopyropen, (H) AgGSTt2 with lambda‐cyhalothrin. All data are indicated as the mean ± SE of at least three biological replicates. (*P < 0.05 Student's t‐test).

These results indicate that AgGSTd5, AgGSTd6 and AgGSTe2 play important roles in the detoxification of multiple insecticides in A. glycines, while AgGSTt1 and AgGSTt2 appear to have limited contribution to resistance against the tested compounds.

3.7. Molecular docking analysis

To investigate the binding mechanisms of AgGSTd5 with sulfoxaflor, afidopyropen and lambda‐cyhalothrin, molecular docking simulations were performed. All three insecticides were successfully accommodated within the binding pocket of AgGSTd5 (Fig. 8). The calculated binding free energies were −8.5 kcal/mol for afidopyropen, −8.0 kcal/mol for lambda‐cyhalothrin and −6.3 kcal/mol for sulfoxaflor. For afidopyropen, the ligand was positioned in the binding cavity. It was stabilized by hydrogen bonds and hydrophobic interactions with key residues Ser130, Asp197, Glu198, Ile177, Phe117 and Tyr273 (Fig. 8(A),(B)). In the lambda‐cyhalothrin complex, the ligand formed hydrogen bonds primarily with Ser123, Ala124 and Ser129. It was also supplemented by interactions with Ile177 and Tyr273 (Fig. 8(C),(D)). In contrast, sulfoxaflor adopted a distinct binding mode. It interacted mainly with Glu198 (Fig. 8(E),(F)). These results provide structural evidence that AgGSTd5 can bind chemically diverse insecticides through both shared and insecticide‐specific amino acid residues.

Figure 8.

Molecular docking analysis of AgGSTd5 with three insecticides.(A) General view of the binding site of AgGSTd5 with afidopyropen. (B) Analysis of the binding site of the AgGSTd5 protein with afidopyropen. (C) General view of the binding site of AgGSTd5 with lambda‐cyhalothrin. (D) Analysis of the binding site of the AgGSTd5 protein with lambda‐cyhalothrin. (E) General view of the binding site of AgGSTd5 with sulfoxaflor. (F) Analysis of the binding site of the AgGSTd5 protein with sulfoxaflor. The insecticides are represented by yellow sticks. Key amino acid residues are highlighted and hydrogen bond distances (Å) are shown.

Molecular docking analysis was also performed for AgGSTd6 and AgGSTe2 to provide structural support for their functional roles observed in RNAi experiments. AgGSTd6 exhibited a binding energy of −5.3 kcal/mol with sulfoxaflor (Fig. S1(A),(B)). For AgGSTe2, the binding energies were −8.4 kcal/mol with lambda‐cyhalothrin (Fig. S2(A),(B)) and −6.4 kcal/mol with sulfoxaflor (Fig. S2(C),(D)). These values indicate that both proteins can accommodate their respective interacting insecticides in the binding pocket.

4. DISCUSSION

GSTs play a major role in insecticide tolerance in insects. ¹² However, genome‐wide identification of the GST gene family and characterization of key detoxification genes have not been performed in A. glycines. In the present study, we identified 17 AgGST genes and demonstrated their involvement in the detoxification of three insecticides with different modes of action. Among hemipteran insects, the number of GST genes in A. glycines (17) is higher than in the closely related Aphis gossypii (11), but lower than in the highly polyphagous B. tabaci (25) and M. persicae (20). ^{24 , 26} This difference is likely due to differences in host range and feeding habits. ²⁴ B. tabaci is a highly polyphagous species that exploits numerous plant families whereas A. glycines is primarily a specialist on soybean. Consequently, polyphagous insects gain a greater evolutionary advantage than oligophagous species, leading to the expansion of a larger GST gene repertoire to cope with diverse xenobiotics. ²⁹ Notably, the delta and epsilon classes, two GST classes that are specific to insects comprise 11 of the 17 AgGST genes, accounting for approximately 65% of the family. This expansion suggests that these classes experienced more frequent gene duplication events during the evolutionary history of A. glycines thereby enhancing its adaptability to plant allelochemicals and synthetic insecticides. In contrast, A. mellifera possesses only a single gene belonging to the delta and epsilon classes, which appears adequate for adaptation in an environment with low insecticide pressure. ²¹

Developmental expression profiling revealed that all 17 AgGST genes were expressed in both nymphal and adult stages, consistent with the ubiquitous expression patterns reported in other hemipteran species, including M. persicae and Acyrthosiphon pisum. ^{26 , 30} In this study, AgGSTd2, AgGSTd3, AgGSTd4, AgGSTd5, AgGSTd6, and AgGSTs3 exhibited significantly higher transcript levels in the adult stage compared with the nymphal stages. This expression pattern is consistent with findings reported in N. lugens. ²³ These differences in expression between nymphal and adult stages indicate that AgGST genes have additional physiological roles beyond detoxification. These GSTs may also contribute to the detoxification of endogenous oxidative byproducts during metamorphosis and adult maturation. ¹⁵

In insects, GSTs are essential for the metabolic clearance of both synthetic pesticides and host plant secondary metabolites. ³¹ Previous studies have shown that North American field populations of A. glycines have developed significant resistance to lambda‐cyhalothrin since 2014, mainly due to voltage‐gated sodium channel mutations. ^{5 , 6} Therefore, it is important to identify candidate AgGST genes involved in insecticide detoxification of both pyrethroids and the newer selective insecticides sulfoxaflor and afidopyropen used in rotation. In this study multiple AgGST genes were upregulated following exposure to LC₅₀ concentrations of all three compounds. The transcriptional responses differed among the three insecticides. Sulfoxaflor significantly induced AgGSTd5, AgGSTd6 and AgGSTe2. Afidopyropen significantly induced AgGSTe2 and AgGSTt1. Lambda‐cyhalothrin significantly induced AgGSTt2. This induction pattern is consistent with the inducible expression patterns of delta and epsilon GSTs reported in other hemipteran species such as Aphis craccivora and Spodoptera exigua. ^{32 , 33} However, the response intensities differed among genes. GSTs have been shown to contribute to insecticide resistance not only through direct metabolism but also via sequestration. ³⁴ Future experiments will be needed to determine whether the AgGSTs identified in this study function through sequestration or direct metabolism RNAi experiments confirmed the roles of individual GST genes in insecticide tolerance. ^{25 , 35} Knockdown of AgGSTd5 significantly increased aphid mortality under all three insecticides. Silencing of AgGSTd6 resulted in a marked increase in mortality under sulfoxaflor, and knockdown of AgGSTe2 significantly elevated mortality under both sulfoxaflor and lambda‐cyhalothrin. In contrast, silencing of AgGSTt1 and AgGSTt2 did not significantly affect aphid mortality under afidopyropen or lambda‐cyhalothrin. These results indicate that AgGSTd5 plays a broad role in detoxification of multiple insecticides in A. glycines, whereas AgGSTd6 and AgGSTe2 contribute to tolerance against specific compounds. These results are consistent with previous RNAi studies in other hemipteran pests. Silencing of NlGSTs1, NlGSTs2, NlGSTe1 and NlGSTd1 in N. lugens significantly increased susceptibility to multiple insecticides. ³⁶ Consequently, AgGSTd5, AgGSTd6, and AgGSTe2 play a central role in insecticide tolerance to sulfoxaflor, afidopyropen, and lambda‐cyhalothrin in A. glycines.

Molecular docking is an important computational tool for revealing the binding interactions between proteins and insecticides. ³⁷ In previous studies, SfGSTe9 from S. frugiperda metabolizes pyrethroids, with docking analyses indicating a good binding fit of the pyrethroid with its active site. ¹⁴ Similarly, direct metabolic resistance to lambda‐cyhalothrin in P. xylostella is largely governed by PxGSTD5, which captures the insecticide utilizing specific hydrogen bonds. ³⁸ In the present study, docking analyses of AgGSTd5 revealed that this protein accommodates all three insecticides within its active‐site pocket through specific hydrogen‐bond and hydrophobic interactions involving conserved residues such as Ser130, Asp197, Glu198, Ile177, Phe117, and Tyr273. Collectively, the docking results indicate that AgGSTd5 can accommodate structurally diverse xenobiotics within its active site. However, it should be noted that functional validation in this study was mainly based on RNAi and molecular docking, both of which have limitations. More definitive evidence could be obtained through gene knockout approaches. For example, CRISPR/Cas9‐mediated knockout of a GST gene cluster in the cotton bollworm has demonstrated its critical role in insecticide resistance. ³⁹ Future application of gene editing in A. glycines would help provide stronger functional validation of the AgGST genes.

In summary, this study provides a systematic, genome‐wide characterization of the AgGST family, detailing their structural organization, chromosomal distribution, developmental expression dynamics, and physiological functions. Expression profiling following exposure to sulfoxaflor, afidopyropen and lambda‐cyhalothrin combined with RNAi validation and molecular docking demonstrates that AgGSTd5, AgGSTd6 and AgGSTe2 are key contributors to multi‐insecticide detoxification. While RNAi results demonstrated that AgGSTd5, AgGSTd6, and AgGSTe2 contribute to insecticide detoxification and tolerance in A. glycines, it should be noted that other mechanisms, such as cytochrome P450‐mediated detoxification or target‐site insensitivity, may contribute to the overall response to insecticides. The laboratory population used in this study was maintained without prior insecticide exposure and exhibited baseline sensitivity to the three tested insecticides.

5. CONCLUSION

This study provides the comprehensive characterization of the GST gene family in A. glycines and demonstrates that AgGSTd5 plays a central role in the detoxification of sulfoxaflor, afidopyropen and lambda‐cyhalothrin, while AgGSTd6 and AgGSTe2 contribute to tolerance against specific insecticides among the three compounds. These genes represent promising molecular targets for monitoring insecticide detoxification responses and for the development of strategies based on GST inhibitors to enhance insecticide efficacy in integrated pest management.

AUTHOR CONTRIBUTIONS

Jixing Xia and Naichen Guan conceived the study. Naichen Guan and Shaolong Qiu performed the genome‐wide identification, gene expression analysis, RNAi experiments and molecular docking. Fujie Dang assisted with insect rearing and bioassays. Naichen Guan, Fujie Dang and Shaolong Qiu analyzed the data. Naichen Guan and Jixing Xia wrote the manuscript. All authors edited and approved the final manuscript.

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Supporting information

Figure S1. Molecular docking analysis of AgGSTd6 with sulfoxaflor. (A) General view of the binding site of AgGSTd6 with sulfoxaflor. (B) Analysis of the binding site of the AgGSTd6 protein with sulfoxaflor.

Figure S2. Molecular docking analysis of AgGSTe2 with lambda‐cyhalothrin and sulfoxaflor. (A) General view of the binding site of AgGSTe2 with lambda‐cyhalothrin. (B) Analysis of the binding site of the AgGSTe2 protein with lambda‐cyhalothrin. (C) General view of the binding site of AgGSTe2 with sulfoxaflor. (D) Analysis of the binding site of the AgGSTe2 protein with sulfoxaflor.

Table S1. Accession numbers of amino acid sequences in other insect species used in phylogenetic analysis.

Table S2. Primers sequences used in this study.

Table S3. The ORF sequences of 26 AgCCE genes.

Table S4. Fold changes in expression of GST genes in Aphis glycines at 6, 12, 24 and 48 h after sulfoxaflor exposure.

Table S5. Fold changes in expression of GST genes in Aphis glycines at 6, 12, 24 and 48 h after afidopyropen exposure.

Table S6. Fold changes in expression of GST genes in Aphis glycines at 6, 12, 24 and 48 h after lambda‐cyhalothrin exposure.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon request.