**4. Materials and Methods**

#### *4.1. Insects and Insecticide*

The susceptible laboratory (Sus-Lab) strain of WBPH (*S. furcifera*) established in our laboratory was obtained from the research group of Prof. Li, College of Plant Protection of Hunan Agricultural University (Changsha, China) in 2016, where the strain had been reared in the laboratory without exposure to any insecticide since 2009. All stages were maintained on rice seedlings under standard conditions of a temperature of 27 ± 1 ◦C, relative humidity (RH) of 70–80% and a light/dark cycle of 16:8 h. Sulfoxaflor (95%, technical grade) was purchased from Dow AgroSciences (Shanghai) Co., Ltd. China (Shanghai, China).

#### *4.2. Selection of the SF-Sel strain with a Sublethal Dose of Sulfoxaflor*

The toxicity of sulfoxaflor to *S. furcifera* was performed using the rice seedling dipping method, with some modifications [42]. First, technical grade sulfoxaflor was dissolved in acetone, and a series of suitable concentrations (i.e., 1, 2, 4, 6, and 8 μg/mL) were prepared with 0.1% Triton X-100; the 0.1% Triton X-100 solution alone was used as the blank control. Four to five leaves of rice seedlings were cleaned with water and air-dried at room temperature. Fifteen rice seedlings were bundled together, immersed in the diluted solution for approximately 30 s, and then dried at room temperature. Second, moistened cotton was wrapped around the rice roots, which were immobilized in a 500 mL plastic cup. Then, fifteen 3rd-instar nymphs were transferred to each plastic cup, and all treatments were set up in triplicate. All treatments were performed under standard environmental conditions (26 ± 1 ◦C, 85 ± 10% R.H., 14:10 L: D), and mortality was recorded after 96 h of treatment. Individual nymphs were considered dead if they did not show movement after being slightly nudged with a #26 soft brush. Probit analyses were conducted using a Statistical Analysis System (SAS) software to calculate the slope, LC50, 95% CI, and χ2 values of sulfoxaflor or sulfoxaflor plus synergistic agents after 96 h of treatment [16]. Then, continuous selection with the sublethal LC25 dose of sulfoxaflor was performed for six generations in the SF-Sel strain.

#### *4.3. Test for Synergism*

The synergism bioassays for the Sus-Lab and SF-Sel strains of *S. furcifera* to sulfoxaflor were performed as described by Mu et al. [13] with some modifications. Three synergistic agents, DEM, TPP and PBO, were dissolved with acetone and diluted with Triton X-100 to the highest possible concentrations showing no adverse effect on the tested insects (PBO, 30 μg/mL; TPP, 160 μg/mL; DEM, at 300 μg/mL), after which rice seedlings into the synergistic treatment solutions for 30 s and naturally dried them. Then, approximately 300 of 3rd-instar nymphs were transferred to the rice seedlings treated with each synergistic agent for approximately 2 h. The remaining procedures were similar to the rice seedling dipping method as described above.

### *4.4. Enzyme Assays*

To evaluate the potential role of the detoxification enzymes of *S. furcifera* in resistance to sulfoxaflor, the activities of CarEs, GSTs and P450s in the 3rd-instar nymphs of the Sus-Lab and SF-Sel strains treated with synergistic agents (TPP or DEM or PBO) were determined.

CarE activity was determined according to the method described by van Asperen [43]. Twenty 3rd-instar nymphs were placed in a centrifugal tube and stored in liquid nitrogen as quickly as possible, then homogenized on ice in 2 mL of homogenization buffer (0.04 mol/L phosphate buffer, pH 7.0) using a 5 mL glass homogenizer and centrifuged at 4 ◦C, 10,000× *g* for 15 min using a 5417R centrifuge (Eppendorf, Germany). The supernatant was subsequently transferred to a clean Eppendorf tube as the crude enzyme solution. A mixture of 0.45 mL of phosphate buffer (0.04 mol/L, pH 7.0), 1.8 mL of <sup>3</sup> <sup>×</sup> <sup>10</sup>−<sup>4</sup> mol/<sup>L</sup> <sup>α</sup>-NA solution (containing 3 <sup>×</sup> <sup>10</sup>−<sup>4</sup> mol/L physostigmine) and 50 <sup>μ</sup>L of diluted enzyme liquid was added to each tube, followed by mixing and then incubation in a water bath at 30 ◦C for

15 min, after which the process was stopped with 0.9 mL of staining solution (0.2 g of fast blue-B salt in 20 mL of distilled water plus 50 mL of 5% sodium dodecyl sulfate). The absorbance values were recorded at 600 nm after 5 min in a UV 2000-Spectrophotometer (Unic [Shang Hai] Instruments Incorporated, Shanghai, China).

GST activity was determined using 1-chloro-2, 4-dinitrobenzene (CDNB) as a substrate according to the method of Wang et al. [16] with minor revisions. Twenty 3rd-instar nymphs were homogenized on ice in homogenization buffer (0.1 mol/L phosphate buffer containing 1.0 mmol/L EDTA, pH 6.5) and centrifuged at 10,800 rpm at 4 ◦C for 10 min, after which the supernatant was used as an enzyme source. A mixture including 2470 μL of phosphate buffer (0.1 mol/L, pH 6.5), 90 μL of CDNB (15 mmol/L), 50 μL of the enzyme source and 90 μL of reduced GSH (30 mmol/L) were added to a 5 mL centrifuge tube, which was promptly shaken. The OD value was recorded at 340 nm for 2 min and calculated as ΔA340/min.

P450 activity was assayed using the method of Rose et al. [44] with some modifications. One hundred and fifty 3rd-instar nymphs were homogenized on ice in 2 mL of homogenization buffer (0.1 mol/L, pH 7.6, containing 20% glycerol, 0.1 mmol/L EDTA, 0.1 mmol/L DTT, and 0.4 mmol/L PMSF). The homogenates were centrifuged at 4 ◦C at 10,000× *g* for 10 min using a 5417R centrifuge (Eppendorf, Germany) to obtain the supernatant, which was used as the crude enzyme. Then, 100 μL of 4-nitroanisole (2 <sup>×</sup> 10−<sup>3</sup> mol/L) was added to the cell culture plate and mixed with 90 <sup>μ</sup>L of crude enzyme liquid, followed by incubation for 3 min at 27 ◦C in a water bath kettle and the addition of <sup>10</sup> <sup>μ</sup>L of NADPH (9.6 <sup>×</sup> <sup>10</sup>−<sup>3</sup> mol/L) for reaction. The changes in the OD value were recorded at 405 nm (Model 680 Microplate Reader, Bio-Rad) every 20 s for 2 min. A standard curve was generated using p-nitrophenol, and the specific activity of P450s was finally calculated as nanomoles of p-nitrophenolper minute per milligram of protein.

All treatments were set up with three samples (tubes) as biological repetitions, and each enzyme sample was individually prepared. Each assay of enzymatic activity was replicated three times as mechanical repetitions for each enzyme sample. The total protein content of the enzyme solution was determined by the Bradford method [45] using bovine albumin as a standard. The activities of CarEs, GSTs and P450s were analyzed using unpaired Student's t-tests, and the significance level of the results was set at *p* < 0.05.
