DOI: 10.5061/dryad.4f4qrfjht
title: SlGSTE8 in Spodoptera litura participated in the resistance to phoxim and chlorpyrifos

description: Glutathione S-transferases (GSTs) were reported to participate in
 insecticides resistance by metabolic and antioxidant activities. In our
 previous study, an ε class gene of GSTs, SlGSTe8 in Spodoptera litura, was
 screened out to be upregulated in a population resistant to pyrethroids
 and organophosphates. SlGSTe8 was highly expressed in the larvae stage,
 and the digestive tissue, foregut, midgut and hindgut. While the relative
 expression level was low in the pupae stage and other tissues. To further
 explore its role in the resistance to pyrethroids and organophosphates,
 the metabolic activity to insecticides by its recombinant protein was
 determined by Ultra Performance Liquid Chromatography, and its antioxidant
 enzyme activity was evaluated by disc diffusion assay. The recombinant
 protein showed significantly metabolic activity to phoxim and
 chlorpyrifos, but not to fenvalerate, cyhalothrin or β-cypermethrin. After
 incubation, the depletion rate of chlorpyrifos is 85.3%, higher than that
 of phoxim (17.5%). Also, the inhibition zone around filter discs decreased
 significantly after exposure to cumene hydroperoxide in recombinant
 plasmid than vector only, suggesting significant antioxidant activity of
 SlGSTE8. Further modeling and docking analysis indicated that the 3D
 structure of SlGSTE8 were well shaped for phoxim and chlorpyrifos, with
 the binding energy -5.58 and -5.15 kcal/mol, respectively. Our work
 provides evidence that SlGSTe8 in S. litura plays important roles in
 phoxim and chlorpyrifos resistance.
description: <strong>1. Insects and
 chemicals</strong> <em>Spodoptera
 litura</em> populations used in this study were
 described in Li et al. (2021a). Briefly, GX population of <em>S.
 litura</em>, kindly provided by Guangxi Tianyuan Biochemistry Corp
 Ltd, served as a susceptible population. QJ population of<em> S.
 litura</em>, collected from the field in 2019, served as a resistant
 population to pyrethroids and organophosphates. Compared with GX
 population, QJ population showed 3659-, 295- and 207-fold resistance to
 β-cypermethrin, phoxim and chlorpyrifos, respectively. And the resistance
 level to fenvalerate and cyhalothrin were higher than 25000- fold (Li et
 al., 2021b). The insects were reared in the insectary at 26±1℃, 60-70%
 relative humidity, 14:10 h of light:dark photoperiod, without exposure to
 insecticides. Fenvalerate (93.4%, Jiangsu Changlong
 Chemicals Co., Ltd, China), β-cypermethrin (95.0%, Beijing Huarong
 Biochemical Co., Ltd, China), cyhalothrin (98.4%, Beijing Huarong
 Biochemical Co., Ltd, China), phoxim (91.0%, Beijing Huarong Biochemical
 Co., Ltd, China) and chlorpyrifos (95.0%, Beijing Huarong Biochemical Co.,
 Ltd, China) were used as the tested insecticides. 
 <strong>2. Characterization of the relative
 expression of <em>SlGSTe8</em> in the QJ population
 </strong> The spatiotemporal expression of
 <em>SlGSTe8<strong> </strong></em>in the QJ
 population and the comparison of its expression<strong><em>
 </em></strong>in the third instar larvae from QJ and GX
 populations were determined by quantitative RT-PCR (qRT-PCR). The first,
 second, third, fourth, fifth, sixth instar larvae and pupae, tissues
 including head, cuticle, foregut, midgut, hindgut, malpighian tubules, fat
 body from the fifth instar larvae were sampled for RNA extraction by
 TaKaRa MiniBEST Universal RNA Extraction Kit (Takara, Dalian, China).
 qRT-PCR reaction system was conducted using SuperReal PreMix Plus (SYBR
 Green, Tiangen, China) on ABI Prism 7500 Real-Time PCR System (Applied
 Biosystems by Life Technologies, Foster, CA, USA). Each reaction was
 carried out in triplicate with 3 independent mRNA samples. Primer pairs
 used in this study were listed in Table 1. <em>EF1α</em> and
 <em>RPL10</em> were used as housekeeping gene (Lu et al.
 2013). The relative expression was quantified according to the
 2<sup>−ΔΔCt</sup> method.  <strong>3. Heterologous expression and the
 determination of enzyme activity of the recombinant protein
 SlGSTE8</strong> The cDNA full length of
 <em>SlGSTe8</em> was cloned and expressed in a prokaryotic
 expression system<em> E. coli </em>BL21 (DE3) (Tiangen). The
 heterologous expression of SlGSTE8 was conducted as described in Li et al.
 (2021a). Briefly, restriction enzyme sites Nde I and Xho I were introduced
 to the cDNA full length of <em>SlGSTe8. </em>Plasmid
 pET-26b(+) and the cDNA of <em>SlGSTe8</em> were both digested
 by Nde I and Xho I (Takara), and the recombinant plasmid
 pET-26b(+)/SlGSTE8 was constructed using T4 DNA ligase (Tiangen).
 pET-26b(+)/SlGSTE8 was transformed in BL21 (DE3). Then, IPTG (Tiangen) at
 a final concentration of 1.0 mmol mL<sup>-1</sup> was added in
 LB liquid medium to induce the expression following incubation at 37 °C,
 160 rpm for 3 h. The recombinant protein was extracted using the following
 procedure: centrifuging at 4 °C, 10,000 g for 10 min; resuspending in
 potassium phosphate buffer (pH 7.0, 20 mmol L<sup>-1</sup>);
 lysing on ice by ultrasonic (Sonics and Materials, Inc., USA); final
 centrifuging at 4 °C, 20,000 g for 30 min. The supernatant was collected
 and purified by HisPur<sup>TM</sup> Ni-NTA Purification Kit
 (Thermo Fisher Scientific Inc., Shanghai, China) with a linear gradient of
 0-300 mmol L<sup>-1</sup> imidazole, desalted by Zeba™ Spin
 Desalting Columns (Thermo Fisher Scientific Inc.). 
 The extracts of pET-26b(+), crude
 recombinant protein and purified recombinant protein were detected by
 SDS-PAGE (12.5%). Briefly, a gel containing 12% seperation gel and 5%
 stacking gel was prepared. Samples were loaded in the gel and run in
 buffer at 80 v for 15 min and 150 v for 1 h. The gels were then
 transferred to Coomassie Blue R-250 (Tiangen) for staining. To detect the
 enzyme activity, <em>K<sub>m</sub></em> and
 <em>V<sub>max</sub></em> values of recombinant
 protein SlGSTE8 were determined using 2,4-dinitrochlorobenzene (CDNB) as
 the model substrate (Li et al., 2021a).  <strong>4 . <em>in vitro</em> metabolism
 activity of SlGSTE8 to pyrethroids and
 organophosphates</strong> Mixture containing purified recombinant
 protein SlGSTE8 (180 μL, 1 mg L<sup>-1</sup>) and PBS (250 μL,
 0.1 mol L<sup>−1</sup>, pH 6.8) was preheated<em>
 </em>at 37 °C and added with insecticides (20 μL, 500 mg
 L<sup>-1</sup>), freshly prepared GSH (50 μL, 100 μmol
 mL<sup>-1</sup>, pH 7.0) following incubation at 37 °C for 3
 h. The reaction was stopped by adding acetonitrile at equal volume and the
 residual insecticides were extracted by adding saturated sodium chloride,
 shaking at 200 rpm for 2 h, centrifuging at 3,000 rpm for 2 min. The
 residual insecticides from the upper organic phase passed through a 0.22
 μm membrane and quantified by Ultra Performance Liquid Chromatography
 using the detection method described in Li et al. (2021a). Briefly, the
 injection volume was 3 μL, and acetonitrile/water (80:20, v/v) was used as
 the mobile phase with flow rate of 0.4 mL min<sup>−1</sup>
 using Acquity UPLC BEH C18 analytical column (2.1 mm × 100 mm, 1.7 μm,
 Waters Acquity UPLC system, USA). The residue of fenvalerate,
 β-cypermethrin, cyhalothrin, phoxim and chlorpyrifos were detected at 220,
 220, 230, 280 and 290 nm, respectively using a PDA detector. The boiled
 recombinant protein SlGSTE8 was used as control.
 <strong>5. The antioxidant activity of SlGSTE8
 determined by disc diffusion assay
 </strong> The antioxidant activity of SlGSTE8 was
 determined by disc diffusion assay using cumene hydroperoxide (CHP,
 J&K, Beijing, China) as the ROS inducer referred to Labade et al.
 (2018). A total of 0.2 mL of pET-26b(+)/SlGSTE8 expressed in <em>E.
 coli</em> with OD<sub>600</sub>=1 was spread evenly on
 the LB plate containing kanamycin (50 mg L<sup>-1</sup>) and
 IPTG (1 mmol L<sup>−1</sup>). Then the same volume of culture
 of empty vector pET-26b(+) in <em>E. coli</em> only was used
 as control. The plates were incubated at 37 ℃ for 1 h to make the
 bacterial resuscitate. Then, filter discs around 5 mm soaking with CHP at
 0, 50, 100, 200, 300 mmol L<sup>−1</sup> were laid on the
 surface of LB plate. After incubation at 37 ℃ for 48 h, the inhibition
 zone around each filter disc was measured twice by cross. Each treatment
 was repeated three times. <strong>6. Homology modeling and molecular
 docking</strong> The 3D protein structure of SlGSTE8 was
 modeled online by Swiss model server (Bienert et al., 2017; Waterhouse et
 al., 2018, https://swissmodel.expasy.org/) using GSTs isozyme E7 from
 <em>Drosophila melanogaster</em> (PDB ID: 4png.1.A) as the
 most suitable modeling template. Their sequence identity was 36.15%. The
 conserved domains of SlGSTE8 were analyzed by CD-search in NCBI
 (https://www.ncbi.nlm.nih.gov/cdd/?term=). The molecular structures of
 chlorpyrifos and phoxim were sketched in ChemDraw 20.0 (PerkinElmer,
 Shelton, CT, USA) and imported to Chem3D (PerkinElmer) to obtain its 3D
 structure. Molecular docking of SlGSTE8 with these insecticides were
 conducted using Autodock 4.2 (Morris et al., 2009; Autodock, San Diego,
 CA, USA) with the default parameters. The possible insecticide
 ligand-binding pockets and binding free energies were predicted. The
 interaction of optimal conformation of SlGSTE8 and ligand were further
 predicted with Protein-Ligand Interaction Profiler (PLIP,
 https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) online. To
 ensure a stable conformation of the complex, a Ramachandran plot of
 SlGSTE8 and insecticides were conducted using Procheck of SAVEs
 (Laskowski, et al., 1993; https://servicesn.mbi.ucla.edu/SAVES/). The
 results of modeling and docking were visualized by PyMOL (New York, NY,
 USA). <strong>7. Statistical
 analysis</strong> All the data were analyzed by SPSS 16.0
 (SPSS, Chicago, IL, U.S.A.). The spatiotemporal expression of
 <em>SlGSTe8</em> was analyzed by One-way analysis of variation
 (ANOVA) followed by Tukey’s HSD multiple comparisons test. Expression of
 <em>SlGSTe8</em> in the QJ and GX populations, the
 <em>in vitro</em> metabolism activity of SlGSTE8 and the data
 from disc diffusion assay were evaluated by Students’
 <em>t</em>-test. <em>P </em>&lt; 0.05 was
 regarded to be statistically significant.
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