CYP6AB328‐mediated spinetoram resistance via N‐demethylation in Tuta absoluta : A potential resistance monitoring marker

Jingang Xie; Furong Hai; Guorong Yan; Xiaoning Liu; Ning Liu

doi:10.1002/ps.70849

. 2026 May 3;82(8):7883–7892. doi: 10.1002/ps.70849

CYP6AB328‐mediated spinetoram resistance via N‐demethylation in Tuta absoluta : A potential resistance monitoring marker

Jingang Xie ¹, Furong Hai ¹, Guorong Yan ², Xiaoning Liu ^1,^✉, Ning Liu ^2,^✉

PMCID: PMC13352221 PMID: 42071273

Abstract

BACKGROUND

Tuta absoluta, a major pest of solanaceous crops, has rapidly evolved resistance to spinosyn insecticides, causing significant damage to global crops. Our previous study demonstrated that CYP6AB328 plays an important role in the development of spinetoram resistance. However, the direct metabolic role of this detoxification enzyme in spinetoram metabolism remains unclear.

RESULTS

In this study, we conducted a docking analysis between CYP6AB328 and spinetoram. Our docking analysis showed a good binding affinity between CYP6AB328 and spinetoram, with an energy of less than −6.0 kcal mol⁻¹. Metabolomic analysis showed that recombinant CYP6AB328 exhibited substantial metabolic activity toward spinetoram‐J (16.62%) and spinetoram‐L (12.42%), which were metabolized into two products, N‐desmethyl spinetoram J and N‐desmethyl spinetoram L, respectively. We also found that laboratory‐selected spinetoram‐resistant strain of T. absoluta displayed 34‐fold cross‐resistance to spinosad, but little to no cross‐resistance to imidacloprid (1.26‐fold), chlorantraniliprole (1.12‐fold), deltamethrin (1.76‐fold) and avermectin (1.34‐fold. Meanwhile, the expression level of CYP6AB328 was positively correlated with spinetoram resistance across four field populations of T. absoluta.

CONCLUSIONS

These findings provide valuable insights into the molecular mechanisms underlying detoxification of spinetoram and support the development of a candidate molecular marker for monitoring spinetoram resistance in the field populations, thereby contributing to improved pest management strategies. © 2026 Society of Chemical Industry.

Keywords: CYP6AB328, field resistance, metabolism, N‐demethylation, spinetoram

CYP6AB328 metabolic spinetoram into two distinct products, and CYP6AB328 expression level exhibited a clear linear relationship with the resistance level from the field populations.

graphic file with name PS-82-7883-g004.jpg

1. INTRODUCTION

Tomato leafminer (Tuta absoluta, Lepidoptera: Gelechiidae) is a major agricultural pest, affecting tomato and other solanaceous crops. It was first recorded in the Xinjiang Uygur Autonomous Region in China, in 2017. ¹ , ² , ³ It is a highly destructive invasive pest, and with damage of 80–100% tomato yield loss. ⁴ , ⁵ , ⁶ To effectively control T. absoluta, insecticides such as spinetoram have been globally applied owing to its high efficacy and security properties. However, T. absoluta has developed resistance to spinetoram owing to frequent overuse. ⁷ Thus, it is necessary to elucidate the mechanisms underlying spinetoram action and resistance in T. absoluta, to be able to develop effective strategies for controlling resistant strains.

Metabolic resistance to insecticides in insects is a complex process that involves multiple metabolic reactions. ⁸ , ⁹ , ¹⁰ Spinosyns undergo extensive metabolism by multiple enzymes in vivo, with substituent variation contributing to differential metabolic reactions and metabolites. ¹¹ , ¹² For example, Spinetoram‐J mainly metabolizes into N‐demethyl‐175‐J, N‐formyl‐175‐J, 3‐O‐deethyl‐175‐J, C9‐pseudoaglycone‐175‐J and aglycone‐175‐J, and spinetoram‐L also undergoes demethylation, formylation and succinylation. ¹¹ However, the metabolic pathway of spinetoram in insects remains unclear.

Insect detoxification enzymes metabolize endogenous and exogenous substrates, thereby supporting growth and detoxification. ¹³ , ¹⁴ , ¹⁵ Overexpression of detoxification enzyme genes which resulted in increased detoxification enzyme activities, have been shown to contribute to pest resistance to insecticides. For instance, cytochrome P450 monooxygenase and glutathione S‐transferase activities increased in a tetraniliprole‐resistant strain of T. absoluta. ¹⁶ Overexpression of four CYP genes resulted in significant increase in cytochrome P450 activity in cyantraniliprole‐resistant strain of T. absoluta. ¹⁷ In our previous study, the in the had no mutations and we showed that three detoxification enzymes (cytochrome P450 monooxygenases, glutathione S‐transferases and carboxylesterases) activities were enhanced in spinetoram‐resistant strain of T. absoluta, owing to the absence of mutation in the nicotinic acetylcholine receptor α6 subunit gene. ¹⁸ We also found out that the expression level of CYP6AB328 positively correlated with an increasing level of spinetoram resistance in laboratory resistant strains of T. absoluta, suggesting its important role in promoting development of spinetoram resistance. ¹⁹ However, the metabolic roles of CYP6AB328 to spinetoram in T. absoluta remain unclear.

Insecticide resistance is positively correlated with the expression level of detoxification enzymes such as P450 genes. For instance, the expression levels of CYP4CE1, CYP4CS5 and CYP6DW3 were shown to positively correlate with the resistance ratios to nitenpyram, thiamethoxam and imidacloprid, indicating their potential as molecular markers for monitoring insecticide resistance in pest populations. ²⁰ , ²¹ , ²² Also, owing to the complexity of pesticide use in the field, resistance in field populations of T. absoluta is often monitored using bioassay methods. Therefore, it is necessary to develop rapid and precise molecular markers for monitoring spinetoram resistance in field populations of T. absoluta.

In this study, we used molecular docking to predict the binding of CYP6AB328 to spinetoram and further demonstrated by in vitro metabolism assays that CYP6AB328 is involved in the metabolism to spinetotam. Then we evaluated cross‐resistance of the laboratory‐selected spinetoram‐resistant strain to other insecticides commonly used to control T. absoluta, and analyzed the correlation between the expression level of CYP6AB328 and the resistance ratio across the field populations from the four Xinjiang regions. These findings provide insight into the P450‐mediated metabolic detoxification of spinetoram in T. absoluta and serve as a basis for the rapid monitoring of spinetoram resistance in field populations of the pest.

2. MATERIALS AND METHODS

2.1. Insects

The susceptible strain (SPI‐S) was collected in 2021 from commercial tomato field in Yining, Xinjiang, China, and were maintained without exposure to pesticides for 2 years. The laboratory‐selected spinetoram‐resistant strain (SPI‐R) was established from the SPI‐S strain and successively selected with spinetoram. Four field populations of T. absoluta were sampled from Xinjiang, China, in 2025 and named Yining (YN), Kashi (KS), Luntai (LT) and Anningqu (ANQ), based on their collection sites. All populations were reared on 4‐week‐old tomato plants at 25 °C, under a 14 h:10 h, light:dark photoperiod and 65% relative humidity. Insects were maintained in the laboratory, in plastic cages (50 × 50 × 50 cm) covered with insect mesh.

2.2. Insecticides

All chemicals utilized in the study were analytical grade. Spinetoram (CAS: 935545–74‐7, purity ≥98.0%), was purchased from Corteva Agriscience China Holding Co., Ltd (Shanghai, China). Spinosad (CAS no. 131929–60‐7, purity 93.2%) was purchased from WeiShi reagent Co., Ltd (Hubei, China). Chlorantraniliprole (CAS no. 500008–45‐7, product code DRE‐C11145000, purity 95%), broflanilide (CAS no. 1207727–04‐5, purity 98.2%), Imidacloprid (CAS no. 105827‐78‐9, purity 95.3%) and Avermectin (CAS number 9036‐19‐5, purity 95%) were obtained from Shandong United Chemical Co. Ltd. Triton X‐100 (CAS no. 9036‐19‐5, product code X100) was obtained from Sigma Aldrich (Shanghai, China).

2.3. Protein and ligand preparation of molecular docking

The tertiary structure of CYP6AB328 was predicted using homology modeling by AlphaFold 3 (https://alphafoldserver.com/). The P450s heme cofactor and spinetoram molecule were downloaded from PubChem (https://pubchem.ncbi.nlm.nih.gov/). First, we constructed conjugated protein model by docking CYP6AB328 with heme (CYP6AB328–Heme); then, the heme was docked into the surrounding region of I‐helix and the β‐bulge segment to construct conjugated protein model, which was used as the receptor. ²³ Subsequently, the substrate‐entry channel was predicted using MOLE 2.0. ²⁴ Molecular docking between the P450 protein structures and spinetoram was performed using AutoDock Vina. ²⁵ The docking site was defined as a cubic box (80 × 80 × 80 Å) centered on the heme iron. PyMOL (http://pymol.org/) was used to generate and visualize the molecular modeling and docking figures.

2.4. Recombination of P450 protein

The CYP6AB328 sequence of T. absoluta and the NADPH cytochrome P450 reductase (CPR) gene sequence of S. frugiperda ²⁶ were synthesized and inserted into pFastBac1 by Sangon (Shanghai, China) Co., Ltd. The recombinant plasmids were transformed into competent cells of Escherichia coli DH10 Bac. The recombinant bacmid containing the CYP6AB328 and CPR proteins were then extracted using an EasyPure® EndoFree Pro Plasmid MaxiPrep Kit (TransGen Biotech, Beijing, China), following the manufacturer's instructions. Sf9 cells from Wuhan Procell (China) were then transfected with the baculovirus plasmid utilizing the Fugene transfection reagent from Promega (Madison, WI, USA) following the method described by Shi et al. ²⁷ To express the proteins, Sf9 cells were infected with a mixture of CYP6AB328 and CPR baculovirus and incubated in SF‐SFM Serum‐free medium (Suzhou Womei, China) at 27 °C. After a 10 min incubation at room temperature (RT), the cells were maintained at 27 °C for 5 days in six‐well plates. The supernatant (P1 virus) was collected by centrifugation at 800 × g for 5 min and then added to Sf9 cells, which were incubated at 27 °C for 48 h. The P2 virus supernatant was collected, and 40 μL of the P2 virus supernatant was added to 20 mL of Serum‐free medium in a conical flask. After 3 days later, CYP6AB328 and CPR of P3 virus were added to Sf9 cells, which were cultivated at 27 °C and 130 rpm in a shaking incubator with Serum‐free medium. The recombinant protein was detected by western blot. Total protein concentrations were determined using the bicinchoninic acid assay method, with bovine serum albumin as the standard. The enzymatic activity of the recombinant CYP6AB328 protein was determined as described previously. ¹¹

2.5. Spinetoram metabolism assay by recombinant CYP6AB328

In vitro metabolism was performed with the recombinant P450 as described by Shi et al. ²⁷ The reaction mixture in 1 mL contained 5 mg/mL recombinant protein, 1.2 mm NADPH‐regenerating system and 0.5 mg mL⁻¹ insecticide (spinetoram‐J and spinetoram‐L). Samples without NADPH regenerating systems were used as control. The spinetoram‐J, spinetoram‐L, N‐demethyl‐175‐J and N‐demethyl‐175‐L were purchased from Altas scientific (Tianjin, China). Reaction was incubated at 30 °C on a shaking table (150 rpm) for 2 h. Thereafter, reactions were centrifuged at 12 000 × g for 10 min to obtain the supernatant that was filtered through 0.22‐μm syringe filters (Keyilong, Tianjin, China) and collected into HPLC vials for analysis. Based on the concentration–peak area standard curves, the amounts of spinetoram‐J, spinetoram‐L, N‐demethyl‐175‐J and N‐demethyl‐175‐L (Supporting information, Table S2), were quantitatively determined using ultra‐high performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS). Chromatographic analysis was conducted using reversed‐phase UPLC on a 3.5 mm Eclipse Plus C18 column (2.1*150 mm; Agilent Technologies, Santa Clara, CA, USA), with 1 mL reaction solution extracted to determine the concentration of the tested insecticide and its primary metabolite. The chromatographic conditions utilized were as follows: a mobile phase composed of methanol/water (8:2, v/v) was delivered isocratically at a flow rate of 250 μL min⁻¹, with an injection volume of 5 μL and run time of 14 min. A similar UPLC‐MS/MS condition was adopted with positive ion mode for all analyses as described. ¹¹ The MRM transitions 734.5 > 84.3 and 734.5 > 128 were monitored for the quantification and identification of N‐demethyl‐175‐J, whereas 746.4 > 84 and 746.4 > 128 were monitored for N‐demethyl‐175‐L.

The Agilent 4500‐AB SCIEX Triple Quad 4500 triple quadrupole tandem mass spectrometer was configured with an electrospray ionization (ESI) source operating in positive ion mode. Essential instrument parameters included an ion source temperature of 550 °C, an ion emission voltage of 5500 V, and nitrogen (N₂) as the carrier gas. Q1 MS was used for full scans, and product ion scans (MS2) were performed for secondary ion analysis. Quantitative detection was carried out using MRM mode. Data acquisition and subsequent analysis were carried out using Analyst 1.6.3 and MultiQuanta 3.03.

2.6. Cross‐resistance of spinetoram‐resistant strain to insecticides

The resistance of T. absoluta larvae to spinetoram was evaluated using the IRAC leaf‐soaking bioassay with slight modifications. ¹⁸ Briefly, different concentrations of spinetoram and the control (using distilled water with Triton X‐100) were used for the bioassay. Fresh tomato leaves were collected, and leaf samples measuring 1.5 cm × 1.5 cm were prepared. Leaf samples were dipped for 10 s in each concentration (1 g spinetoram‐L 1 of acetone) diluted in Triton‐X 100 (0.01%). After the leaf samples were dried at RT, they were placed in disposable plastic containers containing 2% agar. Each concentration was evaluated in three biological replicates, with each replicate conducted in an independent plastic container with one treated leaf sample and 10 2^nd‐instar larvae of T. absoluta.

2.7. Evaluation of the relationship between CYP6AB328 relative expression level and spinetoram resistance ratio of four field populations

The 2^nd‐instar larvae from the YN, KS, LT and ANQ populations were collected into 2‐mL Eppendorf tubes and immediately stored at −80 °C. Subsequently, total RNA was extracted from the collected samples using the TransZol Up Plus RNA Kit (TransGen Biotech) following the manufacturer's instruction and stored at −80 °C. First‐strand complementary DNA (cDNA) was synthesized from 1 μg of total RNA using the TransScript One‐Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech) following the manufacturer's instructions. Relative expression level of CYP6AB328 was analyzed by quantitative real‐time (qRT)‐PCR, using the QuantStudioTM 3 Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA) with the following protocol: 5 min at 95 °C, followed by 40 cycles of 10 s at 95 °C and 30 s at 58 °C, and a final dissociation step. Primers demonstrating high amplification efficiency (≥ 95%) were used. Three genes were tested as references for qRT‐PCR (Table S1), including large subunit 5 ribosomal protein (RpL5), large subunit 23 A ribosomal protein (RpL23A) and rRNA, which were used as internal references in recent studies. ²⁸ , ²⁹ Each sample was run in triplicate. The 2^−ΔΔCt method was used to analyze the expression level of CYP6AB328 based on standard and fluorescence curves. ³⁰

2.8. Statistical analysis

Bioassay data were analyzed using Polo plus (LeOra Software, Berkeley, CA, USA). For the analysis of mRNA expression levels, significant differences between groups were assessed using one‐way ANOVA followed by Tukey's multiple comparisons test with Spss 22.0.

3. RESULTS

3.1. Homology modeling and docking

In order to determine the involvement of CYP6AB328 in the metabolism of spinetoram, we performed molecular docking between CYP6AB328 and spinetoram. The docking results revealed that CYP6AB328 formed a stable complex with Spinetoram‐J and Spinetoram‐L, with a binding free energy of −6.45 and − 6.26 kcal mol⁻¹ (Fig. 1). Spinetoram‐J and Spinetoram‐L were positioned within 3.2 Å and 1.5 Å of the heme iron, primarily stabilized by hydrophobic interactions. Additionally, 3 Pi‐Pi interactions were identified between Spinetoram‐J and CYP6AB328, including PRO‐105 (4.31 Å), ALA‐315 (4.09 Å) and PHE‐118 (4.29 Å). Three Pi‐Pi interactions were identified between Spinetoram‐L and CYP6AB328, including ALA‐219 (3.9 Å), PHE‐119 (4.0 Å) and PHE‐118 (3.9 Å). These interactions indicate the binding of CYP6AB328 to spinetoram, and also suggest key residues involved in ligand stabilization.

Molecular docking of CYP6AB328 homology model with spinetoram (spinetoram‐J and spinetoram‐L). Spinetoram is indicated by sticks and blue nitrogen atoms; heme is displayed as red carbon.

3.2. Metabolism of spinetoram‐J by recombinant CYP6AB328

In order to investigate the metabolic activity of CYP6AB328 on spinetoram, we recombinantly expressed CYP6AB328 protein (Fig. S1; Table S3). HPLC analysis revealed that recombinant CYP6AB328 metabolized spinetoram‐J and spinetoram‐L to yield a corresponding metabolite product, with retention time of 9.29 min and 9.71 min, respectively [Figs 2(A) and 3(A)]. The concentration of metabolic substrates was determined by quantifying the ion peak areas of the respective compounds using standard curves established from the relationships between concentration and ion peak area for spinetoram‐J and spinetoram‐L [Figs 2(B) and 3(B)]. The results showed that, in the presence of NADPH, the concentration of spinetoram J and spinetoram L decreased by 16.62% and 12.42%, respectively, compared with the control [Figs 2(C) and 3(C)]. These results indicate that spinetoram was metabolized by the recombinant CYP6AB328.

Spinetoram‐J metabolism by recombinant CYP6AB328. (A) Detection of spinetoram‐J metabolized by CYP6AB328 *in vitro* in the presence and absence of NADPH. (B) Electrospray ionization mass spectra of spinetoram‐J. (C) Ion peak areas of spinetoram‐J and shown as column charts (D). and shown as column charts (D). Values are means ± SD (n = 3), and asterisks represent significant differences between the treatment and control groups (***, P < 0.001).

Spinetoram‐L metabolism by recombinant CYP6AB328. (A) Detection of spinetoram‐L metabolized by CYP6AB328 *in vitro* in the presence and absence of NADPH. (B) Electrospray ionization mass spectra of spinetoram‐L. (C) Ion peak areas of spinetoram‐L and shown as column charts (D). Values are means ± SD (n = 3), and asterisks represent significant differences between the treatment and control groups (*** P < 0.001).

3.3. Identification of spinetoram metabolites catalyzed by recombinant CYP6AB328

Spinetoram metabolites generated by CYP6AB328 were analyzed using mass spectrometry, which revealed that the metabolic site is located on the forosamine sugar moiety, with N‐demethyl XDE‐175‐J and N‐demethyl XDE‐175‐L identified as the primary metabolites of spinetoram‐J and spinetoram‐L, respectively [Fig. 4(A)]. Based on ion peak area‐based quantification, in the presence of NADPH, spinetoram‐J and spinetoram‐L generated 8.2% and 7.6% N‐demethyl XDE‐175‐J and N‐demethyl XDE‐175‐L, respectively [Fig. 4 (B)]. These results indicate that CYP6AB328 catalyzes the demethylation of spinetoram‐J and spinetoram‐L to form N‐demethyl XDE‐175‐J and N‐demethyl XDE‐175‐L, respectively.

Identification of spinetoram metabolites by recombinant CYP6AB328. (A) Detection of the main metabolite (retention time: 9.29 min) and electrospray ionization mass spectrum of spinetoram‐J metabolized by CYP6AB328. (B) Detection of the main metabolite (retention time: 9.71 min) and electrospray ionization mass spectrum of spinetoram‐L metabolized by CYP6AB328.

3.4. Relationship between CYP6AB328 expression level and spinetoram resistance in field populations

3.4.1. Cross‐resistance of spinetoram‐resistant strain of T. absoluta to insecticides

Results on the potential cross‐resistance to various insecticides of laboratory‐selected spinetoram‐resistant strain (SPI‐R), showed a notable cross‐resistance of SPI‐R to spinosad (34‐fold), but minimal to negligible cross‐resistance to imidacloprid (1.26‐fold), chlorantraniliprole (1.12‐fold), deltamethrin (1.76‐fold) and avermectin (1.34‐fold), respectively (Table 1).

Table 1.

Concentration–mortality response (LC₅₀ ± 95% FL) of resistant and susceptible strains exposed to different insecticide

Insecticides	N ^*	Slope±SE ^†	χ² ^‡	LC₅₀ (mg L⁻¹) (95% CL ^§ )	RR^**
Spinetoram
SPI‐S	150	0.955 ± 0.272	3.34	0.044 (0.035–0.056)	‐
SPI‐R	150	2.229 ± 0.273	0.082	14.706 (7.933–42.910)	334.232
Spinosad
SPI‐S	150	2.060 ± 0.305	0.447	0.036 (0.027–0.129)	‐
SPI‐R	150	1.912 ± 0.304	0.403	1.224 (0.873–1.612	34
Avermectin
SPI‐S	150	1.774 ± 0.297	0.254	0.096 (0.066–0.201)	‐
SPI‐R	150	2.045 ± 0.307	0.361	0.129 (0.097–0.168)	1.344
Imidacloprid
SPI‐S	150	2.391 ± 0.330	0.547	0.194 (0.153–0.246)	‐
SPI‐R	150	2.250 ± 0.320	0.770	0.244 (0.192–0.317)	1.258
Chlorantraniliprole
SPI‐S	150	2.530 ± 0.492	1.369	0.041 (0.033–0.052)	‐
SPI‐R	150	2.214 ± 0.340	1.873	0.046 (0.034–0.059)	1.122
Deltamethrin
SPI‐S	150	3.476 ± 0.469	0.702	0.153 (0.127–0.185)	‐
SPI‐R	150	2.171 ± 0.322	1.233	0.316 (0.246–0.424)	1.756

Open in a new tab

Numbers of 2^nd‐instar larvae used in each bioassay.

^{^†}

Slope ± SE of the concentration–mortality regression line.

^{^‡}

Chi‐square.

^{^§}

SE.

^**

RR (resistance ratio) = LC₅₀ of the resistant strain/LC₅₀ of the most susceptible strain.

3.4.2. Evaluating the relationship between CYP6AB328 expression level and sensitivity to spinetoram of field populations of T. absoluta

The concentration–mortality responses of the four field populations of T. absoluta to spinetoram revealed significant variation (Table 2). The median lethal concentration (LC₅₀) ranged from 0.080 [95% confidence limit (CL) 0.061–0.101] mg L⁻¹ to 0.436 (95% CL 0.156–0.701) mg L⁻¹. Resistance ratio (RR) for these strains varied from 1.8‐ to 9.9‐fold, with two strains exhibiting RR > 5.0‐fold, suggesting low susceptibility among the four field populations.

Table 2.

Relative toxicity of spinetoram among field‐collected T. absoluta populations from Xinjiang

Strain	N ^*	Slope±SE ^†	χ² ^‡	LC₅₀ (mg L⁻¹) (95% CL ^§ )	RR^**
SPI‐S	150	0.955 ± 0.272	3.34	0.044 (0.035–0.056)	‐
YN	150	1.484 ± 0.338	1.595	0.436 (0.156–0.701)	9.909
ANQ	150	2.269 ± 0.323	0.952	0.080 (0.061–0.101)	1.818
LT	150	2.038 ± 0.306	0.485	0.085 (0.065–0.111)	1.932
KS	150	1.664 ± 0.259	1.069	0.235 (0.138–0.342)	5.341

Open in a new tab

Numbers of 2^nd‐instar larvae used in each bioassay.

^{^†}

Slope ± SE of the concentration–mortality regression line.

^{^‡}

Chi‐square.

^{^§}

SE.

^**

RR = median lethal concentration (LC₅₀) of the field population / LC₅₀ of the susceptible strain (SPI‐S).

We also measured the expression level of CYP6AB328 in the four field populations of T. absoluta. Compared with the SPI‐S, the gene expression level of CYP6AB328 in four field populations significantly increased to 5.44‐fold for YN (P < 0.001), 1.59‐fold for ANQ (P = 0.421), 2.07‐fold for LT (P = 0.030) and 3.94‐fold for KS (P < 0.001) [Fig. 5(A), (B)]. Linear regression analysis showed that the CYP6AB328 expression level was positively influenced by the resistance level (spinetoram: P < 0.01) [Fig. 5(C)]. Taken together, the results show that the overexpression of CYP6AB328 resulted in an increase in spinetoram resistance ratio of the field populations of T. absoluta.

Relationship between *CYP6AB328* expression level and sensitivity to spinetoram of field populations. (A) Map of Xinjiang showing sample locations of field populations. The administrative boundary data of provinces in China are from http://www.resdc.cn/ (last access: November 3, 2024) © Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences. (B) Quantification of *CYP6AB328* expression level in the experimental populations. Bars represent mean ± SD (n = 3). The expression patterns of populations were analyzed with one‐way ANOVA followed by Tukey's multiple range test. Different lowercase letters indicate significant differences (P < 0.05) among all samples. (C) Linear regression analysis between the spinetoram resistant ratio and the relative expression level of *CYP6AB328*.

4. DISCUSSION

The overexpression of cytochrome P450 genes is widely recognized as a major mechanism underlying metabolic resistance to insecticides in pests. In T. absoluta, several P450 genes, including CYP321C40, CYP6AW1, CYP4M116, CYP9A307v2, CYP15C1, CYP339A1 and CYP6AB327, have been reported to be significantly upregulated following spinosad exposure, suggesting a potential involvement of P450‐mediated detoxification in spinosyn resistance. ³¹ , ³² In our previous studies, CYP6AB328 was consistently overexpressed in a laboratory‐selected spinetoram‐resistant strain of T. absoluta, and its expression level showed a significant positive correlation with resistance development. ¹⁹ Moreover, increased P450 enzyme activity was observed in the laboratory‐selected spinetoram‐resistant strain, whereas no mutations were detected in the nAChR α6 subunit, indicating that metabolic resistance rather than target‐site insensitivity predominates in spinetoram resistance. ¹⁸ Based on these findings, the present study predicted stable binding of spinetoram‐J and spinetoram‐L within the active pocket of CYP6AB328 by molecular docking. We also provided direct biochemical evidence that CYP6AB328 metabolizes spinetoram. In vitro metabolism assays demonstrated that CYP6AB328 metabolized both spinetoram‐J (16.62%) and spinetoram‐L (12.42%). LC–MS/MS analysis further identified N‐desmethyl spinetoram‐J and N‐desmethyl spinetoram‐L as the primary metabolites, indicating that N‐demethylation is the dominant catalytic reaction mediated by CYP6AB328. Notably, N‐demethylated spinetoram derivatives have also been reported as major metabolites in plants, animals, soil and aquatic environments, suggesting that this metabolic pathway is conserved across biological systems. ²⁹ , ³⁰ Similar detoxification roles of individual P450 enzymes have been reported in other insects. For example, CYP6DW3 catalyzes the conversion of imidacloprid to imidacloprid‐urea in Bemisia tabaci. ²¹ CYP4CE1 mediates hydroxylation and N‐demethylation of nitenpyram in Nilaparvata lugens. ²² Additionally, CYP3002B2 from Varroa destructor exhibits catalytic activity toward multiple acaricides, including amitraz and flumethrin. ³³ Taken together, these results strongly suggest that CYP6AB328 plays an important role in the metabolic detoxification of spinetoram in T. absoluta, with N‐demethylation representing a predominant metabolic pathway. However, the involvement of additional P450 enzymes and their potential synergistic effects cannot be excluded and warrant further investigation.

Cross‐resistance is a key factor influencing the efficacy and utility of insecticides. ³⁴ Rotating insecticides with varied mode‐of‐action (MoA) is a practical strategy to mitigate the selective pressure exerted by a single insecticide. ¹⁷ Spinetoram is a highly efficient second‐generation product of spinosyns. ³⁵ It is widely used in diverse crop systems globally. ³⁶ However, its extensive use has resulted in cross‐resistance in pests. Previous studies showed no significant correlation between resistance to spinosyns and resistance to avermectins, neonicotinoids, or pyrethroids. ³⁷ , ³⁸ , ³⁹ However, spinosyn resistance showed weak negative correlation with cross‐resistance to diamides and neonicotinoids. ³⁴ In another study, a spinosad‐resistant strain of T. absoluta showed some cross‐resistance to spinetoram. ⁴⁰ Likewise, our study showed the laboratory‐selected spinetoram‐resistant strain developed a 34‐fold cross‐resistance to spinosad, with little cross‐resistance detected for deltamethrin, avermectin, imidacloprid and chlorantraniliprole in T. absoluta. Hence, resistance to spinetoram in T. absoluta does not a priori confer resistance to other classes of insecticides, which is likely to be associated with the novel MoA of spinosyns. This evidence indicates that, in areas where significant resistance is detected, spinosyns should be rotated with chemical insecticides of other classes, such as avermectin, imidacloprid, chlorantraniliprole and deltamethrin. Furthermore, it is necessary to intensify systematic resistance monitoring of T. absoluta field populations across regions to inform timely, targeted management strategies.

Monitoring insecticide resistance is an important prerequisite for insecticide resistance management (IRM), which can ensure the long‐term effectiveness of chemical control. ⁴¹ Compared to traditional bioassay, molecular diagnostics utilizing reliable markers, either independently or alongside bioassay, can significantly enhance the efficiency of IRM. ⁴² For instance, the insecticide resistance of field populations was determined by measuring the frequency of mutant alleles in nAChR of Frankliniella occidentalis. The expression levels of CYP4CE1, CYP4CS5 and CYP6DW3 positively correlated with the resistance ratios of various insecticides in the field, indicating their potential as molecular targets for monitoring pest insecticide resistance. ²⁰ , ²¹ , ²² Our previous study found no mutations in nAChR α6 in the laboratory spinetoram‐resistant strain, which exhibited a resistance ratio of 410.86‐fold. In this study, a significant positive correlation was observed between CYP6AB328 expression levels and LC₅₀ in the different generation of laboratory resistant strains. ¹⁸ This was further confirmed by the absence of cross‐resistance between resistance to spinetoram and other classes of insecticides. Besides, the CYP6AB328 expression level exhibited a clear linear relationship with spinetoram resistance in the four field populations of T. absoluta. Therefore, we recommend the development of quantitative detection techniques using the CYP6AB328 gene. Analysis also showed that among spinosyns, spinosad and spinetoram both contain the forosamine sugar moiety. ⁴³ Therefore, further investigation is required to determine whether CYP6AB328 can also directly metabolize spinosad through N‐demethylation and to assess its potential utility as a molecular diagnostic marker for monitoring spinosad resistance in field populations of T. absoluta. These techniques could serve as tools for monitoring resistance level in field insect populations, thus reducing the number of test insects required and enhancing the precision of resistance assessment.

In this study, we found that CYP6AB328 exhibits metabolic activity that converts spinetoram into two distinct metabolites. Also, CYP6AB328 expression level exhibited a linear relationship with the resistance level of field populations of T. absoluta. In conclusion, our study provides insight into the mechanism underlying of spinetoram resistance and application of the knowledge to tackle resistance development. It also provides new diagnostic markers derived from CYP6AB328 for detection and monitoring of spinetoram resistance.

CONFLICT OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supporting information

Table S1. Primers used for full‐length sequencing of CYP6AB328 and qRT‐PCR analysis genes.

Table S2. Concentration–peak area standard curves for spinetoram J, spinetoram L, N‐demethyl XDE‐175J and N‐demethyl XDE‐175L.

Table S3. Metabolism of two model substrates by CYP6AB328–CPR.

Fig. S1. Expression and identification of recombinant CYP6AB328 and CPR. (A) SDS–PAGE analysis of recombinant CYP6AB328 and CPR. (B) Western blot detection of recombinant CYP6AB328 and CPR. M, protein marker; L1, loading sample with cell lysis mixture; L2, loading sample with cell lysate supernatant; L3, loading sample with microsomal fraction; L4, loading sample with postmicrosomal supernatant.

PS-82-7883-s001.docx^{(263.3KB, docx)}

ACKNOWLEDGEMENTS

This work was supported by the Graduate Student Research and Innovation Projects in Xinjiang Autonomous Region (XJ2025G026) and the “Tian Shan Talent” Training Program, “Agriculture, Rural Areas, and Farmers” Backbone Talent Project (2024SNGGGCC011).

Contributor Information

Xiaoning Liu, Email: liuxn0103@xju.edu.cn, Email: liuxn0103@sina.com.

Ning Liu, Email: 13579401853@163.com.

DATA AVAILABILITY STATEMENT

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

REFERENCES

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28. Li Y, Huang A, Li X, Edwards MG and Gatehouse AMR, RNAi targeting Na _ν and CPR via leaf delivery reduces adult emergence and increases the susceptibility to λ‐cyholthin in Tuta absoluta (Meyrick). Pestic Biochem Physiol 204:106089 (2024). [DOI] [PubMed] [Google Scholar]
29. Askew WT, Edwards MG and Gatehouse AMR, Ex vivo delivery of dsRNA targeting ryanodine receptors for control of Tuta absoluta . Pest Manag Sci 80:6400–6408 (2024). [DOI] [PubMed] [Google Scholar]
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32. Li W, Yang W, Shi Y, Yang X, Liu S, Liao X et al., Comprehensive analysis of the overexpressed cytochrome P450‐based insecticide resistance mechanism in Spodoptera litura . J Hazard Mater 461:132605 (2024). [DOI] [PubMed] [Google Scholar]
33. Mavridis K, Tsakireli D, Vlogiannitis S, Charamis J, Siden‐Kiamos I, Osabutey AF et al., Identification and functional characterization of CYP3002B2, a cytochrome P450 associated with amitraz and flumethrin resistance in the major bee parasite varroa destructor . Pestic Biochem Physiol 210:106364 (2025). [DOI] [PubMed] [Google Scholar]
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35. Sparks TC, Crouse GD, Dripps JE, Anzeveno P, Martynow J, DeAmicis CV et al., Neural network‐based QSAR and insecticide discovery: spinetoram. J Comput Aided Mol Des 22:393–401 (2008). [DOI] [PubMed] [Google Scholar]
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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1. Primers used for full‐length sequencing of CYP6AB328 and qRT‐PCR analysis genes.

Table S2. Concentration–peak area standard curves for spinetoram J, spinetoram L, N‐demethyl XDE‐175J and N‐demethyl XDE‐175L.

Table S3. Metabolism of two model substrates by CYP6AB328–CPR.

PS-82-7883-s001.docx^{(263.3KB, docx)}

Data Availability Statement

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

[ps70849-bib-0001] 1. Zhang G, Ma D, Wang Y, Gao Y, Liu W, Zhang R et al., First report of the South American tomato leafminer, Tuta absoluta (Meyrick), in China. J Integr Agric 19:1912–1917 (2020). [Google Scholar]

[ps70849-bib-0002] 2. Zhang G, Xian X, Zhang Y, Liu W, Liu H, Feng X et al., Outbreak of the South American tomato leafminer, Tuta absoluta, in the chinese mainland: geographic and potential host range expansion, Pest . Manage Sci 77:5475–5488 (2021). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0003] 3. Wang M, Ismoilov K, Li H, Zhang X, Lu Z, Feng L et al., Polygyny of Tuta absoluta may affect sex pheromone‐based control techniques. Entomol Gen 41:357–367 (2021). [Google Scholar]

[ps70849-bib-0004] 4. Cocco A, Serra G, Lentini A, Deliperi S and Delrio G, Spatial distribution and sequential sampling plans for Tuta absoluta (lepidoptera: Gelechiidae) in greenhouse tomato crops. Pest Manag Sci 71:1311–1323 (2014). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0005] 5. Li X, Li D, Zhang Z, Huang J, Zhang J, Hafeez M et al., Supercooling capacity and cold tolerance of the south American tomato pinworm, Tuta absoluta, a newly invaded pest in China. J Pest Sci 94:845–858 (2021). [Google Scholar]

[ps70849-bib-0006] 6. Wang M, Ismoilov K, Liu W, Bai M, Bai X, Chen B et al., Tuta absoluta management in China: progress and prospects. Entomologia 44:269–278 (2024). [Google Scholar]

[ps70849-bib-0007] 7. Ullah F, Güncan A, Gul H, Hafeez M, Zhou S, Wang Y et al., Spinosad‐induced intergenerational sublethal effects on Tuta absoluta: biological traits and related genes expressions. Entomol Gen 44 :395–404 (2024). [Google Scholar]

[ps70849-bib-0008] 8. Xu L, Zhao J, Xu D, Xu G, Peng Y and Zhang Y, New insights into chlorantraniliprole metabolic resistance mechanisms mediated by the striped rice borer cytochrome P450 monooxygenases: a case study of metabolic differences. Sci Total Environ 912:169229 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0009] 9. Lu K, Song Y and Zeng R, The role of cytochrome P450‐mediated detoxification in insect adaptation to xenobiotics. Curr Opin Insect Sci 43:103–107 (2021). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0010] 10. Xu Y, Du J, Zhang K, Li J, Zou F, Li X et al., The dual resistance mechanism of CYP325G4 and CYP6AA9 in Culex pipiens pallens legs according to transcriptome and proteome analysis. J Agric Food Chem 72:27150–27162 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0011] 11. FAO , Spinetoram http://www.fao.org/fileadmin/templates/agphome/documents/Pests_Pesticides/JMPR/Evaluation08/Spinetoram.pdf. [accessed 25 January 2026].

[ps70849-bib-0012] 12. Zhao L, Chen G, Zhao J, Zhang Y, Zhu Y, Yang T et al., Degradation kinetics of the insecticide spinetoram in a rice field ecosystem. Chemosphere 119:1185–1191 (2015). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0013] 13. Qiu Y, Tittiger C, Wicker‐Thomas C, Le Goff G, Young S, Wajnberg E et al., An insect‐specific P450 oxidative decarbonylase for cuticular hydrocarbon biosynthesis. Proc Natl Acad Sci 109:14858–14863 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]

[ps70849-bib-0014] 14. Li W, Zangerl AR, Schuler MA and Berenbaum MR, Characterization and evolution of furanocoumarin‐inducible cytochrome P450s in the parsnip webworm, Depressaria pastinacella. Insect Mol Biol 13:603–613 (2004). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0015] 15. Li H, Zhao P, Li S, Guo J and Hao D, Trial and error: new insights into recombinant expression of membrane‐bound insect cytochromes P450 in Escherichia coli systems. Int J Biol Macromol 273:133183 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0016] 16. Qu C, Yao J, Huang J, Che W, Fang Y, Luo C et al., Tetraniliprole resistance in field‐collected populations of Tuta absoluta (lepidoptera: Gelechiidae) from China: baseline susceptibility, cross‐resistance, inheritance, and biochemical mechanism. Pestic Biochem Physiol 203:106019 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0017] 17. Ullah F, Gul H, Chen L, Wang J, Zhang Z, Huang J et al., Cyantraniliprole resistance in Tuta absoluta: selection, comparative transcriptomes, and nanocarrier‐mediated RNAi studies. Entomol Gen 45:565–575 (2025). [Google Scholar]

[ps70849-bib-0018] 18. Xie J, Turak R, Liu N, Zhuang Z, Liu X and Song Y, Spinetoram resistance in Tuta absoluta: selection, inheritance, biochemical mechanism and fitness cost. Pestic Biochem Physiol 215:106659 (2025). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0019] 19. Xie J, Wang S, Zhuang Z, Wang X, Lin M and Liu X, Exploring the role of CYP6AB328 in spinetoram resistance and growth and development of Phthorimaea absoluta . Pestic Biochem Physiol 208:106316 (2025). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0020] 20. Hu J, Fu B, Liang J, Zhang R, Wei X, Yang J et al., CYP4CS5‐mediated thiamethoxam and clothianidin resistance is accompanied by fitness cost in the whitefly bemisia tabaci . Pest Manag Sci 80:910–921 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0021] 21. Xue H, Fu B, Huang M, He C, Liang J, Yang J et al., CYP6DW3 metabolizes imidacloprid to imidacloprid‐urea in whitefly (Bemisia tabaci). J Agric Food Chem 71:2333–2343 (2023). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0022] 22. Zhang H, Zhang Z, Zhang Y, Zhang X and Liu Z, CYP4CE1 metabolized nitenpyram through two types of oxidation reaction, hydroxylation, and N‐demethylation. J Agric Food Chem 72:20122–20129 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0023] 23. Hlavica P, Insect cytochromes P450: topology of structural elements predicted to govern catalytic versatility. J Inorg Biochem 105:1354–1364 (2011). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0024] 24. Sehnal D, Svobodová Vařeková R, Berka K, Pravda L, Navrátilová V, Banáš P et al., MOLE 2.0: advanced approach for analysis of biomacromolecular channels. J Chem 5:39 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]

[ps70849-bib-0025] 25. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS et al., AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30:2785–2791 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]

[ps70849-bib-0026] 26. Amezian D, Mehlhorn S, Vacher‐Chicane C, Nauen R and Le Goff G, Spodoptera frugiperda Sf9 cells as a model system to investigate the role of detoxification gene expression in response to xenobiotics. Curr Res Insect Sci 2:100037 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[ps70849-bib-0027] 27. Shi Y, Wang H, Liu Z, Wu S, Yang Y, Feyereisen R et al., Phylogenetic and functional characterization of ten P450 genes from the CYP6AE subfamily of Helicoverpa armigera involved in xenobiotic metabolism. Insect Biochem Mol Biol 93:79–91 (2018). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0028] 28. Li Y, Huang A, Li X, Edwards MG and Gatehouse AMR, RNAi targeting Na _ν and CPR via leaf delivery reduces adult emergence and increases the susceptibility to λ‐cyholthin in Tuta absoluta (Meyrick). Pestic Biochem Physiol 204:106089 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0029] 29. Askew WT, Edwards MG and Gatehouse AMR, Ex vivo delivery of dsRNA targeting ryanodine receptors for control of Tuta absoluta . Pest Manag Sci 80:6400–6408 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0030] 30. Livak KJ and Schmittgen TD, Analysis of relative gene expression data using real‐time quantitative PCR and the 2^−ΔΔCT method. Methods 25:402–408 (2001). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0031] 31. Ullah F, Murtaza G, Li X, Gul H, Wang Y, Zhao S et al., Selection‐induced spinosad resistance and associated fitness costs in Tuta absoluta: a key invasive tomato pest. Agronomy 15:358 (2025). [Google Scholar]

[ps70849-bib-0032] 32. Li W, Yang W, Shi Y, Yang X, Liu S, Liao X et al., Comprehensive analysis of the overexpressed cytochrome P450‐based insecticide resistance mechanism in Spodoptera litura . J Hazard Mater 461:132605 (2024). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0033] 33. Mavridis K, Tsakireli D, Vlogiannitis S, Charamis J, Siden‐Kiamos I, Osabutey AF et al., Identification and functional characterization of CYP3002B2, a cytochrome P450 associated with amitraz and flumethrin resistance in the major bee parasite varroa destructor . Pestic Biochem Physiol 210:106364 (2025). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0034] 34. Sparks TC, Wessels FJ, Perry T, Price MJ, Siebert MW and Mann DGJ, Spinosyn resistance and cross‐resistance – a 25 year review and analysis. Pestic Biochem Physiol 210:106363 (2025). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0035] 35. Sparks TC, Crouse GD, Dripps JE, Anzeveno P, Martynow J, DeAmicis CV et al., Neural network‐based QSAR and insecticide discovery: spinetoram. J Comput Aided Mol Des 22:393–401 (2008). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0036] 36. Sparks TC, Insecticide mixtures—uses, benefits and considerations. Pest Manag Sci 81:1137–1144 (2025). [DOI] [PubMed] [Google Scholar]

[ps70849-bib-0037] 37. Geng C, Watson GB and Sparks TC, Chapter three ‐ nicotinic acetylcholine receptors as spinosyn targets for insect pest management, in Advances in Insect Physiology, ed. by Cohen E. Academic Press, Oxford, pp. 101–210 (2013). [Google Scholar]

[ps70849-bib-0038] 38. Tabuloc CA, Carlson CR, Ganjisaffar F, Truong CC, Chen CH, Lewald KM et al., Transcriptome analysis of Drosophila suzukii reveals molecular mechanisms conferring pyrethroid and spinosad resistance. Sci Rep 14:19867 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]

[ps70849-bib-0039] 39. Lira EC, Bolzan A, Nascimento AR, Amaral FS, Kanno RH, Kaiser IS et al., Resistance of Spodoptera frugiperda (lepidoptera: Noctuidae) to spinetoram: inheritance and cross‐resistance to spinosad. Pest Manag Sci 76:2674–2680 (2020). [DOI] [PubMed] [Google Scholar]

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PERMALINK

CYP6AB328‐mediated spinetoram resistance via N‐demethylation in Tuta absoluta : A potential resistance monitoring marker

Jingang Xie

Furong Hai

Guorong Yan

Xiaoning Liu

Ning Liu

Abstract

BACKGROUND

RESULTS

CONCLUSIONS

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Insects

2.2. Insecticides

2.3. Protein and ligand preparation of molecular docking

2.4. Recombination of P450 protein

2.5. Spinetoram metabolism assay by recombinant CYP6AB328

2.6. Cross‐resistance of spinetoram‐resistant strain to insecticides

2.7. Evaluation of the relationship between CYP6AB328 relative expression level and spinetoram resistance ratio of four field populations

2.8. Statistical analysis

3. RESULTS

3.1. Homology modeling and docking

Figure 1.

3.2. Metabolism of spinetoram‐J by recombinant CYP6AB328

Figure 2.

Figure 3.

3.3. Identification of spinetoram metabolites catalyzed by recombinant CYP6AB328

Figure 4.

3.4. Relationship between CYP6AB328 expression level and spinetoram resistance in field populations

3.4.1. Cross‐resistance of spinetoram‐resistant strain of T. absoluta to insecticides

Table 1.

3.4.2. Evaluating the relationship between CYP6AB328 expression level and sensitivity to spinetoram of field populations of T. absoluta

Table 2.

Figure 5.

4. DISCUSSION

CONFLICT OF INTEREST

Supporting information

ACKNOWLEDGEMENTS

Contributor Information

DATA AVAILABILITY STATEMENT

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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