GSTD1 Mediates the Tolerance to Abamectin and Beta-Cypermethrin in the Fall Armyworm Spodoptera frugiperda

Simple Summary

Spodoptera frugiperda is a notorious pest native to subtropical and tropical regions of the Americas, and it can cause serious harm to many economic crops. Although the use of chemical insecticides is the most effective and quickest way to control S. frugiperda, it inevitably leads to the development of insect resistance. GSTD1 has been reported to be involved in the detoxification of insects towards insecticides. In this study, we cloned and identified the SfGSTD1 gene in S. frugiperda. To verify whether SfGSTD1 mediates the tolerance of S. frugiperda to insecticides, we firstly used RNAi technology to knock down the SfGSTD1 gene, and then analyzed the changes in insecticide tolerance of S. frugiperda to two insecticides (abamectin and beta-cypermethrin). Subsequently, we further validated the gene function of SfGSTD1 by overexpressing it in Sf9 cells and Drosophila melanogaster. Finally, the inhibition assay was carried out to explore the potential interactions between SfGSTD1 and insecticides (abamectin and beta-cypermethrin). Our results showed that SfGSTD1 is closely related to the detoxification of abamectin and beta-cypermethrin, and suggested that the abamectin and beta-cypermethrin resistance of S. frugiperda may be related to SfGSTD1 overexpression.

Abstract

Glutathione S-transferase (GST) is a class of detoxifying enzymes in the second stage of insect metabolism and plays a key role in insecticide resistance. In this study, based on the transcriptome sequences of S. frugiperda, the full-length cDNA of SfGSTD1 was cloned and characterized. The temporal and spatial expression pattern showed that SfGSTD1 was highly expressed in Malpighian tubules, which are key excretion organs. Knocking down SfGSTD1 reduced S. frugiperd tolerance to abamectin and beta-cypermethrin. The overexpression of SfGSTD1 enhanced the viability of Sf9 cell under abamectin and beta-cypermethrin treatment. Furthermore, SfGSTD1 was overexpressed in Drosophila melanogaster using the GAL4/UAS binary expression system, and this overexpression strain was also less susceptible to abamectin and beta-cypermethrin. The enzyme activity of recombinant SfGSTD1 could also be significantly inhibited by abamectin and beta-cypermethrin. Taken together, our findings indicate that SfGSTD1 might be involved in the tolerance of abamectin and beta-cypermethrin in S. frugiperda. And these results provide theoretical foundations for understanding the resistance mechanism of S. frugiperda to abamectin and beta-cypermethrin.

1. Introduction

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is one of the most devastating pests worldwide and can cause severe crop and economic loss. It is a highly polyphagous pest, and its hosts range over 350 species across 76 plant families including maize, sorghum, soybean, cotton, rice and wheat [1,2,3,4]. The chemical control of S. frugiperda has a long history, and a variety of insecticides have been used for its prevention and control, with field populations in many countries developing varying degrees of resistance to traditional pesticides. The first report on the chemical resistance of S. frugiperda was in 1979; a field population in Georgia (USA) exerted a certain level of resistance to carbaryl [5]. Its resistance to trichlorfon, pyrethroids, carbamates and organophosphorus insecticides was reported soon thereafter [6,7]. Since then, S. frugiperda has evolved resistance to a minimum of 47 active ingredients in different chemical classes from the arthropod pesticide resistance database (APRD, 2025, https://www.pesticideresistance.org/). Due to past selection by insecticides, a single S. frugiperda population could evolve multiple resistance; for example, a population in Puerto Rico was found to have resistance against 10 commonly used pesticides [8]. The study of resistance mechanisms of S. frugiperda is the basis of field-integrated pest management. Metabolic resistance mediated by detoxification enzymes is one of the most important mechanisms of multidrug resistance generation.

Glutathione-S-transferase (GST) is a multifunctional enzyme family present in most living organisms, and plays a very important physiological roles including immune response, antioxidation, odorant degradation, xenobiotic resistance and ecdysteroid biosynthesis [9,10,11,12]. GST is one of the most important families of detoxification enzymes. The research on insect GSTs mainly focuses on the detoxification of electrophilic substances such as insecticides, toxic secondary metabolites and endogenous toxic substances. GST can participate in the formation and development of insecticide resistance through glutathione conjugation, noncatalytic binding, antioxidant stress response and directly catalyzing the metabolism [13]. The overexpression of GST mediates enhanced detoxification, which has been implicated in the formation and development of insecticide resistance. For example, in Helicoverpa armigera, the overexpression of HaT_119 and HaGST_121 were significantly linked to lambda-cyhalothrin resistance, whereas the upregulation of HaGSTS1 and HaGSTD1 participated in malathion resistance [14,15]. In Nilaparvata lugens, the overexpression of NlGSTS1 and NlGSTS2 was associated with organophosphates and synthetic pyrethroids resistance [16]. In addition, qualitative changes in GST were also reported to have evolved in insecticide resistance, such as a single mutation V128A in the BdGSTE8 which confers malathion resistance in Bactrocera dorsalis [17].

Insect GSTs are mainly divided into seven subfamilies, including delta, epsilon, omega, sigma, theta, zeta and microsomal, among which the delta and epsilon are the most abundant and are unique to insects. Extensive studies have documented the detoxification activity of GSTs in various insect species, with particular emphasis on the detoxification ability of the Delta family to insecticides. In Bradysia odoriphaga, BoGSTD2 exhibits peroxidase activity, preventing oxidative stress caused by exposure to malathion and thiamethoxam [18]. In Cydia pomonella, lambda-cyhalothrin could be significantly metabolized by recombinant CpGSTD1, indicating that CpGSTD1 may be involved in the formation of cyhalothrin resistance [19]. TuGSTD01 in Tetranychus urticae was significantly inhibited by abamectin but could not detoxify abamectin, which indicates that it may undergo noncatalytic binding with abamectin to reduce its toxicity [20]. TcGSTD2 from Tribolium castaneum has been reported, which can detoxify insecticides by combating oxidative stress [21]. In terms of the GST having various mechanisms of interaction with insecticides, exploring their important roles in detoxifying insecticides helps in understanding the mechanisms of insecticide resistance and cross resistance.

To elucidate whether GSTs are involved in abamectin and beta-cypermethrin detoxification in S. frugiperda, a delta class GST gene SfGSTD1 was cloned for functional verification in this study. The expression pattern of SfGSTD1 in different tissues and developmental stages was analyzed via quantitative real-time PCR. The susceptibility of S. frugiperda to abamectin and beta-cypermethrin after knocking down and SfGSTD1 was further analyzed to investigate the role of SfGSTD1 in insecticide detoxification. Moreover, the function of SfGSTD1 was also determined through a GAL4/UAS binary expression system in D. melanogaster and the piggyBac overexpression system in the Sf9 cell line. We successfully purified and validated the recombinant proteins through a prokaryotic expression system and then characterized their biochemical properties, including kinetic parameters and insecticide inhibition, using spectrophotometric microplate assays. These results demonstrate that SfGSTD1 plays a crucial role in the detoxification of S. frugiperda to abamectin and beta-cypermethrin.

2. Materials and Methods

2.1. Insects and Insecticides

The S. frugiperda strain used in this research was kindly supplied by Dr. Shao-Hua Gu from the School of Plant Protection, China Agricultural University, and was originally collected from a maize field (Kunming, China) in June 2019. The adult was fed with 10% sucrose water and laid eggs on cotton gauze. The newly hatched larvae were kept on a semisynthetic diet at 27 ± 1 °C and relative humidity at 75 ± 5% with a 16 h:8 h light/dark photoperiod. The pupae were disinfected with 0.25% sodium hypochlorite solution and placed under the same environmental conditions for emergence.

Abamectin (96.8%) was procured from Hebei Weiyuan Biochemical Co., Ltd. (Shijiazhuang, China). Beta-cypermethrin (95%) was procured from Jiangsu Gongcheng Biotechnology Co., Ltd. (Nanjing, China). Abamectin and beta-cypermethrin technical-grade insecticides were dissolved in dimethyl sulfoxide and acetone, respectively, to form the solution of 50,000 mg/L.

2.2. RNA Isolation and cDNA Synthesis

Total RNA was extracted by employing the RNAiso Plus reagent (Takara, Dalian, China) according to the instructions. The integrity and concentration of the obtained total RNA were determined through the use of agarose gel electrophoresis, along with a Nanodrop2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). First-strand cDNA was synthesized by using a HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China). The cDNA and RNA samples were stored at −40 °C and −80 °C, respectively.

2.3. Sequence Analysis and Phylogenetic Tree Construction

Based on the S. frugiperda transcriptome, the sequence of SfGSTD1 (GeneBank: XP_050562830.1) was amplified using 2 × Phanta Flash Master Mix (Vazyme, Nanjing, China) and specific primers (Table S1). The PCR product was then linked to the pMD™19-T Vector (TaKaRa, Beijing, China) for sequencing validation. The open reading frame (ORF), amino acid sequence, and sequence alignment were performed on the obtained SfGSTD1 clones using DNAMAN version 8.0 and ESPript 3.0. The protein isoelectric point (pI) and molecular mass were predicted using the Expasy Proteomics web tool https://web.expasy.org/protparam/, accessed on 5 March 2024). The phylogenetic tree was generated using the maximum likelihood (ML) method implemented in MEGA 7.0 software [22]. Jones–Taylor–Thornton matrix-based mode with 2000 bootstrap replicates was implemented for phylogenetic tree analysis. Bootstrap values higher than 50% and the GenBank accession numbers of sequences used in the phylogenetic analyses are given in Figure 1.

2.4. Real-Time Quantitative PCR Analyses (RT-qPCR)

The gene expression pattern at different development stages and various tissues was investigated. In different development stages, eggs, 1st- to 6th-instar larvae, pupae and adults (male and female) were sampled separately. Various tissues, including the head (HD), midgut (MG), Malpighian tubules (MT), fat body (FB), hemolymph (HE) and integument (IN), were dissected from 6th larvae (n = 60). These dissected samples were temporarily kept in cold RNAiso Plus reagent (Takara, Dalian, China) and then subjected to RNA extraction according to the instructions. The cDNA was synthesized using the HiScript II Q Select RT SuperMix for qPCR kit (Vazyme, Nanjing, China) in a 20 μL reaction volume with 1 μg of RNA. Specific RT-qPCR primers (Table S1) were designed using Primer3 software (http://bioinfo.ut.ee/primer3/, accessed on 10 June 2024). The qPCR analyses were carried out using the QuantStudio 5 system (Applied Biosystems, Foster City, CA, USA) with UltraSYBR Mixture (with ROX) Kit (CWBIO, Beijing, China). Each qPCR reaction contained 4 μL of cDNA sample, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 4 μL of RNase-free water and 10 μL of UltraSYBR mixture. The program parameters were set as follows: initial denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 40 s. A final melt curve stage was implemented to assess the specificity of primer amplification. Each primer pair’s amplification efficiency was counted by a serial dilution of cDNA (0.01 ng to 100 ng). Each sample had at least three biological replicates. The ribosomal protein L32 (RPL32) and glyceraldehyde-3-phosphate dehydrogenase (GADPH) were selected as housekeeping genes for the normalization of target gene mRNA levels. Relative gene expression was calculated using the 2^−ΔΔCT method [23].

2.5. Preparation of dsRNA and RNA Interference

An approximately 442 bp fragment of SfGSTD1 was amplified (Table S1) and ligated into the pET-2P vector constructed from pET-30a (+) [24]. A 428 bp fragment of EGFP was also amplified and ligated into pET-2P as a negative control. The recombinant plasmids were transferred into Escherichia coli HT115 (DE3) competent cells to synthesis dsRNA. The positive transformants were cultured in 500 mL Luria broth medium containing 50 μg/mL kanamycin at 37 °C with shaking at 220 rpm. The bacteria was induced with a 0.5 mM final concentration of isopropyl-β-D-thiogalactopyranoside (IPTG) when OD₆₀₀ reached 1.0, followed by shaking at 220 rpm for an additional 4 h at 37 °C. The bacterial cells were harvested by centrifugation at 8000 rpm for 10 min, and the pellet was weighed and resuspended in 0.05 M phosphate-buffered saline (pH = 7.4). The expressed dsRNA was extracted from E. coli and validated by electrophoresis on a 1% agarose gel.

A diet incorporation bioassay was implemented to knock down the expression level of SfGSTD1. Firstly, the E. coli expressing dsRNA were mixed for an artificial diet at a concentration of 3 mg/g. The dsRNA diet was added to 24-well plates and fed to 3^rd-instar larvae of S. frugiperda. To verify the RNAi efficiency, the SfGSTD1 transcription levels in the whole larval body were analyzed daily by qPCR. Subsequently, 200 mg/L abamectin or 25 mg/L beta-cypermethrin were added to an artificial diet that contained dsRNA. The mortality of S. frugiperda was determined at 72 h after insecticide treatment. The bioassays were performed with three biological replicates, and 20 larvae were used in each bioassay.

2.6. Construction of Sf9 Cell Lines Stably Expressing SfGSTD1

S. frugiperda ovarian cells (Sf9 cells) were cultured in SF-900TM II medium (Gibco, Gaithersburg, MD, USA) supplemented with 100 units/mL penicillin, 100 mg/mL streptomycin and 10% heat-inactivated fetal bovine serum and maintained at 27 °C. In this study, the piggyBac vector system was adopted to construct stable expression Sf9 cell lines that overexpressed SfGSTD1 according to the method we previously used [25]. The piggyBac:SfPub/P2009-target:hr5/OpIE1-EGFP-PuroR vector was constructed using piggyBac vector (Miaoling Biology, Wuhan, China), as described in Chen’s research [26]. Then, 1 μg of constructed piggyBac vector and 1 μg transposase plasmid were mixed in equal proportions and transfected into cells using the FuGENE^® HD reagent (Promega, Madison, WI, USA) in a 6-well plate. Next, 10 µg/mL puromycin was used to selected recombination Sf9 cells, and the expression level of the SfGSTD1 was validated using RT-qPCR after three weeks selection. The EGFP overexpressed cell line was used as the control.

2.7. Cell Viability Assay

The Cell Counting Kit-8 (CCK-8, Yeasen Biotechnology, Shanghai, China) was used to determine the viability of cells according to the instructions. Firstly, 5 × 10⁴  Sf9 cells were seeded into each well of the 96-well plate. The final concentration of 20 mg/L abamectin and 60 mg/L beta-cypermethrin diluted with culture medium (10 μL) was added to each well after 2 h. The Sf9 cells treated with insecticide were then cultivated for 2 d. Finally, CCK-8 solution (10 μL/well) was added to the culture medium and incubated for 2 h again.

The absorbance at 450 nm was determined using a Spark multimode microplate reader (Tecan, Switzerland). Each treatment had at least three biological replicates. The calculation formula for relative cell activity is as follows:

$$\mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y} (\mathrm{\%}) = {\displaystyle \frac{\mathrm{O}\mathrm{D} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}-\mathrm{O}\mathrm{D} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}{\mathrm{O}\mathrm{D} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}-\mathrm{O}\mathrm{D} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{g}\mathrm{r}\mathrm{o}\mathrm{u}\mathrm{p}}}\times 100$$

2.8. Construction of Transgenic D. melanogaster and Bioassay

The ORF of SfGSTD1 was ligated to pJFRC2-10XUAS-IVS-mCD8::GFP plasmid (Addgene: 26214) for the UAS-SfGSTD1 construction of transgenic flies. The eggs containing PhiC31 transposase and the attP sequence were subsequently collected for injection by the Qidong Fangjing Biotechnology Co., Ltd. (Nantong, Jiangsu, China). After microinjection, F0 male flies were first hybridized with the wild-type strain (W¹¹¹⁸), and F1 male flies were selected again to hybridize with the balancer (W⁻;;MKRS/TM6B) to remove PhiC31 transposase. Homozygous strains of UAS-SfGSTD1 were obtained by self-crossing. The Actin-GAL4 strain (Bloomington Drosophila Stock Center: 30558) was employed to drive the expression of SfGSTD1 throughout the whole body of D. melanogaster. The expression level of the SfGSTD1 in transgenic D. melanogaster was verified by RT-PCR.

A bioassay was used to determine the susceptibility of female adults that had emerged for 3-6 days to insecticides. Here, 100 mg/L abamectin and 10 mg/L beta-cypermethrin were prepared with 5% sucrose and 1% agar, and air-dried overnight. Fifteen female flies were transferred to each vial containing the aforementioned toxic diet, and the mortality rate was counted after 72 h. At least four biological replicates were carried out for each genotype of D. melanogaster.

2.9. Expression and Purification of Recombinant Proteins

The verified SfGSTD1 ORF was amplified using primers (Table S1) containing homologous sequences and subsequently cloned into the pCold II expression vector (Takara, Dalian, China). The recombinant plasmid was then transformed into BL21 (DE3) competent cells. Positive transformants were selected and cultured in 500 mL of LB medium supplemented with 100 μg/mL ampicillin at 37 °C with constant shaking at 220 rpm. The bacterial culture was maintained at 15 °C for 30 min to allow temperature adaptation and then induced with isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.75 mM, followed by incubation at 15 °C with shaking at 160 rpm for 12 h. The bacterial cells were harvested by centrifugation at 10,000 rpm for 10 min at 4 °C, and the pellet was resuspended in 10 mL of ice-cold lysis buffer supplemented with imidazole (10 mM) and lysozyme (1 mg/mL). After sonication and centrifugation (12,000 rpm, 30 min, 4 °C), the supernatant was purified using Ni-NTA columns (Sangon, Shanghai, China). The recombinant protein was then purified according to the protocol and detected using 8% SDS-PAGE. The purified target protein was dialyzed overnight in Tris-HCl buffer (25 mM, pH 7.4). The concentration of purified SfGSTD1 protein was quantified by the BCA assay [27].

2.10. Enzyme Kinetic Properties and Enzyme Inhibition Assays

The kinetic parameters of recombinant protein were evaluated using 1-chloro-2-dinitrobenzene (CDNB) and GSH as substrates, and the absorbance was monitored using a microplate reader SpectraMax PLUS384 (Molecular Devices, San Jose, CA, USA). The 200 μL reaction solution contained 1 μg of recombinant protein, CDNB (0.125 to 4 mM) and 4 mM GSH. The absorbance of the reaction mixture was detected at 340 nm. The heated protein was used in place of the recombinant protein as the control, and each assay was performed in triplicate. The K_m and V_max values were determined using GraphPad Prism 9 (Graphpad, San Diego, CA, USA).

The half maximal inhibitory concentrations (IC₅₀) were assayed by pre-incubating 1 μg recombinant protein with a range of concentration of 0.0018 to 0.114 mM for abamectin and 0.0038 to 0.240 mM for beta-cypermethrin that included seven concentrations. The residual activity of the protein was determined according to the method mentioned above.

2.11. Statistics

Statistical analysis was performed using a two-tailed unpaired Student’s t-test and one-way ANOVA with Tukey’s multiple range test for post hoc comparisons. Significance was set at * p < 0.05, ** p < 0.01 and *** p < 0.001. GraphPad Prism 9.0 software (San Diego, CA, USA) was employed to draw the histograms containing the mean and standard error of the mean (SEM).

3. Results

3.1. Molecular Characterization of SfGSTD1

The cDNA sequence of SfGSTD1 (GeneBank: XP_050562830.1) was characterized from S. frugiperda. The complete ORF of SfGSTD1 with 735 bp was amplified by PCR, which encodes a 244 amino acid residue protein. The molecular weight of SfGSTD1 was predicted as 27.89 kDa and the theoretical pI point as 5.48. No signal peptide, transmembrane domain and N-glycosylation site were predicted in SfGSTD1. The GSH-binding site (G-site) and the hydrophobic substrate-binding site (H-site) of SfGSTD1 were predicted, which included 6 and 10 amino acids separately (Figure S1). According to the secondary structure of protein prediction results, SfGSTD1 has nine α-helixes and four β-sheets. The alignment between SfGSTD1 and the genome sequence of S. frugiperda showed that SfGSTD1 has six exons and four introns, located in chromosome 30 (JAKUHG020000066.1) with the reverse orientations (Figure 1A). To analyze the evolutionary relationship of SfGSTD1, a phylogenetic tree was constructed with orthologs from 15 insect species. The phylogenetic tree comparison showed that SfGSTD1 clustered with GSTD1 from other Lepidoptera insects, and has a slightly distant homologous relationship with Diptera insects (Figure 1B).

Figure 1. Sequence analysis of SfGSTD1 gene. (A) Gene structure of SfGSTD1 gene. The exons are depicted as blue boxes, while the introns are represented by black lines connecting the boxes. The intron-to-exon ratio was determined to be 20:1. The predicted start codon (ATG), stop codon (TAG) and the chromosomal location of gene locus (with “-” indicating the reverse orientation with chromosome) are annotated at their respective positions. (B) Phylogenetic tree of the SfGSTD1 gene. The phylogenetic tree is constructed using maximum likelihood method. The number above the branch indicates support for the phylogenies and only displays values greater than 50. Accession numbers of the used sequences are listed after the corresponding genes. Aa, Aedes aegypti; Ag, Anopheles gambiae; Am, Apis mellifera; Bm, Bombyx mori; Bt, Bemisia tabaci; Cm, Cnaphalocrocis medinalis; Cs, Chilo suppressalis; Dm, Drosophila melanogaster; Ha, Helicoverpa armigera; Mp, Myzus persicae; Nl, Nilaparvata lugens; Nv, Nasonia vitripennis; Se, Spodoptera exigua; Sf, Spodoptera frugiperda; Sl, Spodoptera litura.

Figure 1. Sequence analysis of SfGSTD1 gene. (A) Gene structure of SfGSTD1 gene. The exons are depicted as blue boxes, while the introns are represented by black lines connecting the boxes. The intron-to-exon ratio was determined to be 20:1. The predicted start codon (ATG), stop codon (TAG) and the chromosomal location of gene locus (with “-” indicating the reverse orientation with chromosome) are annotated at their respective positions. (B) Phylogenetic tree of the SfGSTD1 gene. The phylogenetic tree is constructed using maximum likelihood method. The number above the branch indicates support for the phylogenies and only displays values greater than 50. Accession numbers of the used sequences are listed after the corresponding genes. Aa, Aedes aegypti; Ag, Anopheles gambiae; Am, Apis mellifera; Bm, Bombyx mori; Bt, Bemisia tabaci; Cm, Cnaphalocrocis medinalis; Cs, Chilo suppressalis; Dm, Drosophila melanogaster; Ha, Helicoverpa armigera; Mp, Myzus persicae; Nl, Nilaparvata lugens; Nv, Nasonia vitripennis; Se, Spodoptera exigua; Sf, Spodoptera frugiperda; Sl, Spodoptera litura.

3.2. Temporal and Spatial Expression of SfGSTD1

SfGSTD1 was expressed in different developmental stages and various tissues. In different developmental stages of S. frugiperda, the expression level of SfGSTD1 was the lowest during the egg stage, gradually decreasing from 1st to 5^th instar, and then reaching the highest in the adult stage (Figure 2A). Tissues expression results showed that SfGSTD1 was predominately expressed in Malpighian tubules and head, followed by midgut and integument, while the fat body was the tissue with the lowest expression level (Figure 2B).

3.3. Silencing of SfGSTD1 Increased Larval Susceptibility of S. frugiperda to Insecticides

To evaluate whether SfGSTD1 was involved in the tolerance of S. frugiperda to insecticides, we performed RNAi technology to knock down the expression level of SfGSTD1. These results showed that compared with larvae feeding with the artificial diet containing dsEGFP, the expression level of SfGSTD1 did not significantly change after feeding with an artificial diet containing dsSfGSTD1 for 1 d, but significantly decreased after 2–3 d (Figure 3A). Subsequently, a diet incorporation bioassay was implemented to evaluate the tolerance of S. frugiperda to two insecticides after SfGSTD1 was knocked down. The mortality of S. frugiperda larvae fed on dsSfGSTD1 was significantly increased to 40.0% after being exposed to abamectin compared to larvae fed on dsEGFP (21.7%) (Figure 3B). Similarly, the silencing of SfGSTD1 could increase the mortality of S. frugiperda exposed to 25 mg/L beta-cypermethrin (Figure 3C).

3.4. Overexpression of SfGSTD1 Decreased Toxicity of Insecticides to Sf9 Cells

We utilized the piggyBac transposon to stably express SfGSTD1 in Sf9 cells to investigate its function. The qPCR results showed that compared with the negative control group that only overexpressed EGFP, the expression level of the SfGSTD1 gene was significantly upregulated in stable overexpressing SfGSTD1 cell lines (Figure 4A). The relative viability of SfGSTD1 and EGFP overexpressing cells was subsequently measured after treatment with abamectin and beta-cypermethrin. Compared to EGFP overexpressing Sf9 cells, the overexpression of SfGSTD1 significantly enhanced the cell viability of Sf9 cells exposed to abamectin and beta-cypermethrin, by 29.2% (from 46.0% to 75.2%) and 22.9% (from 60.1% to 83.0%) (Figure 4B,C).

3.5. Expression of SfGSTD1 in D. melanogaster Leads to Decreased Susceptibility to Insecticides

To further verify the function of the SfGSTD1 gene in the insecticide’s detoxification, we constructed a UAS-SfGSTD1 transgenic fly strain. The Actin-GAL4 strain was employed to drive the overexpression of SfGSTD1 in D. melanogaster, and this result was validated through semi-quantitative analysis (Figure 5A). The mortalities of female adults treated with abamectin and beta-cypermethrin were then recorded. Bioassay results showed that the mortality of transgenic flies overexpressing SfGSTD1 was significantly reduced by 40.0% after exposure to abamectin compared to the control flies (SfGSTD1/+) (Figure 5B). Moreover, the overexpression of SfGSTD1 could significantly decrease the susceptibility of D. melanogaster exposed to 10 mg/L beta-cypermethrin (Figure 5C).

3.6. Enzymatic Properties and Inhibitions of Insecticides on Recombinant SfGSTD1

To assess the catalytic activity of SfGSTD1, the recombinant protein with an N-terminal His-tag was expressed in E. coli BL21 (DE3) and purified through the Ni-NTA spin column. SDS-PAGE analysis revealed a single band at approximately 27 kDa (Figure S2), corresponding to the predicted size of SfGSTD1 (27.89 kDa) plus the His-tag (0.97 kDa). Kinetic analysis using CDNB and GSH as substrates revealed that recombinant SfGSTD1 exhibited a K_m value of 0.421 ± 0.109 mM and a Vmax value of 0.719 ± 0.100 μmol/min/mg to CDNB (Figure 6A).

Subsequently, the inhibition assay was determined to explore the potential interactions between SfGSTD1 and two insecticides. The result revealed that the enzyme activity of SfGSTD1 was significantly inhibited by abamectin and beta-cypermethrin (Figure 6B,C). Low concentrations of abamectin (0.002–0.057 mM) showed relatively weak inhibitions to SfGSTD1, while the inhibition was remarkably increased to 54.17% when 0.458 mM of abamectin was used. The inhibitions of beta-cypermethrin on SfGSTD1 activity were raised with the increase in insecticide concentrations, and relative inhibition reached 56.39% when 0.960 mM beta-cypermethrin was used. The abamectin and beta-cypermethrin showed the inhibition on SfGSTD1 with the half-inhibitory concentration (IC₅₀) of 0.208 mM and 0.171 mM, separately.

4. Discussion

GST, as an important detoxifying enzyme, is widely involved in the detoxification of insects towards insecticides and plant secondary metabolites. This metabolic mechanism ensures that insects can adapt to exposure to external insecticides [28]. In this study, a GST gene was cloned from S. frugiperda, which belongs to the delta subfamily. Similar to the GST family genes of other insects [29], SfGSTD1 has two conserved GST-signature motifs; one is the GSH-binding site (G-site) and the other is the hydrophobic substrate-binding site (H-site) (Figure S1). These play a critical role in insecticide metabolism [30]. In addition, SfGSTD1 has nine α-helixes and four β-sheets, according to the predicted protein secondary structure. The homologous gene of SfGSTD1 has also been found in multiple other insects, including Lepidoptera, Hemiptera, Diptera and Hymenoptera. Evolutionary tree analysis showed that SfGSTD1 was closely clustered with homologous genes in the Lepidoptera of S. exigua and S. litura (Figure 1B). BLASTP analysis showed that SfGSTD1 shares 96.5%, 95.8%, 73.3%, 51.3%, 48.9%, 48.2% and 44.1% sequence identity with the SfGSTD1 of S. litura, S. exigua, C. medinalis, A. gambiae, B. tabaci, D. melanogaster and N. lugens, respectively. Thus, the SfGSTD1 was characterized and bioinformatic analysis performed from different levels and perspectives.

The temporal and spatial expression pattern showed that SfGSTD1 was expressed in different developmental stages and various tissues, suggesting that SfGSTD1 may be involved in many physiological functions. During the developmental stages from 1^st-instar larvae to adult, the expression level of SfGSTD1 showed a trend of first decreasing and then increasing, and there were significant differences between male and female adults (Figure 2A). This result indicates that SfGSTD1 may also have other physiological functions, such as regulating the reproductive behavior of S. frugiperda, but more work is needed to uncover these physiological roles. The Malpighian tubules are well known as the essential excretory tissues in most insects [31], and we found that SfGSTD1 was predominantly expressed in these tissues (Figure 2B). Consistent with our results, BdGSTD1 and BdGSTD10 also exhibited high expression levels in the Malpighian tubules of B. dorsalis [32]. In addition, SfGSTE9-mediated beta-cypermethrin tolerance in S. frugiperda was observed to be the most abundant in the head, early larval stages and pupae [33]. Based on these research results, it could be inferred that some insects’ GST associated with insecticide resistance are not only most abundant in detoxifying tissues and larvae stages, but also highly expressed in other developmental stages such as pupae and adults.

GST genes in delta and epsilon subfamilies have been widely reported to mediate insecticide resistance in various insects such as B. dorsalis, S. litura and S. exigua [32,34,35,36,37]. Among numerous members of the delta class, GSTD1 has been shown to be involved in the resistance of insects to various insecticides. In Periplaneta americana, PaGSTD1 has been verified as an important detoxifying enzyme with high metabolic efficiency against chlorpyrifos-methyl [38]. In C. pomonella, CpGSTD1 was not only upregulated after treatment with lambda-cyhalothrin, but also significantly metabolized lambda-cyhalothrin [19]. Metabolism assays showed that malathion could be significantly depleted by BdGSTD1 of B. dorsalis [32]. Moreover, SlGSTD1 is associated with the resistance of S. litura to cyhalothrin and fenvalerate by antioxidant capacity and detoxication [37]. In C. suppressalis, CsGSTD1 might confer tolerance to abamectin through the noncatalytic passive binding and sequestration [39]. Here, we confirmed the SfGSTD1-mediated tolerance of S. frugiperda to two insecticides (abamectin and beta-cypermethrin) through the silencing of SfGSTD1 in S. frugiperda in vivo (Figure 3), the overexpression of SfGSTD1 in Sf9 cells in vitro (Figure 4), and the expression of SfGSTD1 in transgenic fruit flies in vivo (Figure 5), respectively, although there are also results indicating that SfGSTD1 (GeneBank: XP_035440580.2) has a strong binding ability to chlorantraniliprole, thereby regulating the tolerance of S. frugiperda to chlorantraniliprole [40]. However, through evolutionary tree analysis, we found that this gene is closely clustered with SeGSTD3 (Figure S3), not the SfGSTD1 (GeneBank: XP_050562830.1) in this study. In summary, we only found that SfGSTD1 is responsible for the tolerance of abamectin and beta-cypermethrin. As the homologous gene of GSTD1, whether SfGSTD1 could mediate the tolerance of S. frugiperda to other insecticides (such as chlorpyrifos, malathion, cyhalothrin, lambda-cyhalothrin and fenvalerate) needs further verification.

In insects, GST can not only directly metabolize insecticides, but also enhance their antioxidant stress activity or conjugation with insecticides, thereby mediating insect resistance to insecticides. For example, recombinant SeGSTo could protect super-coiled plasmid DNA from damage in the metal catalyzed oxidation system, indicating its involvement in the oxidative stress process of S. exigua [41]. GST genes detoxify organophosphorus insecticides by forming O-dearylation or O-dealkylation [42]. BdGSTD1 had the capacity to directly metabolize malathion, and could also catalyze the malathion reaction with the conjugation of reduced glutathione [32]. Although beta-cypermethrin was not directly metabolized by SlGSTD1, SlGSTE9, SlGSTE12 or SlGSTO2, these GST genes might play an important role in the tolerance S. litura to beta-cypermethrin through antioxidant and sequestration capabilities [37]. Similar to SlGSTD1, SlGSTE9, SlGSTE12 or SlGSTO2, CpGSTD1 also had glutathione peroxidase activity against substrate cumene hydroperoxide [19,37]. On the other hand, PaGSTD1 did not exhibit this activity, indicating that PaGSTD1 mainly promotes insecticide detoxification through metabolism rather than antioxidant capacity [38]. Furthermore, it was found that lambda-cyhalothrin could be quickly metabolized, but no new metabolites were discovered, suggesting that CpGSTD3, CpGSTE3 and CpGSTS2 are associated with the resistance of C. pomonella to lambda-cyhalothrin via sequestration [43]. Through inhibition experiments, we also found that the activity of SfGSTD1 could be significantly inhibited by abamectin and beta-cypermethrin (Figure 6), indicating that SfGSTD1 might regulate the tolerance of S. frugiperda to these two insecticides through sequestration. Based on these previous research results, it can be inferred that even homologous genes (such as GSTD1) have different detoxification metabolic pathways in different insects. Therefore, whether SfGSTD1 could exhibit antioxidant activity or directly metabolize insecticides such as abamectin and beta-cypermethrin remains to be verified through subsequent experiments.

5. Conclusions

In conclusion, we have cloned and characterized SfGSTD1, which is associated with the tolerance of S. frugiperda to abamectin and beta-cypermethrin. Temporal and spatial expression patterns showed that SfGSTD1 was predominantly expressed in the Malpighian tubules, which were key tissues for insects to detoxify and metabolize insecticides. Functional analyses showed that the silencing of SfGSTD1 reduced the tolerance of S. frugiperda to abamectin and beta-cypermethrin, the overexpression of SfGSTD1 increased the viability of Sf9 cells to abamectin and beta-cypermethrin and the overexpression of SfGSTD1 in D. melanogaster decreased abamectin and beta-cypermethrin susceptibility. Moreover, the enzyme activity of SfGSTD1 was significantly inhibited by abamectin and beta-cypermethrin. All these results support the finding that SfGSTD1 plays an essential role in the abamectin and beta-cypermethrin tolerance of S. frugiperda.