Spinosad

Metaflumizone (240 g/L SC) was obtained from the BASF Chemical Co., Ltd. (Shanghai, China). Indoxacarb (95%), abamectin (95%), beta-cypermethrin (96.1%), chlorfluazuron (95%), chlorfenapyr (95%), diafenthiuron (98%), and chlorantraniliprole (95%) were purchased from Hubei Kangbaotai Fine-Chemicals Co., Ltd. (Wuhan, China). Spinetoram (60 g/L SC) and spinosad (25 g/L SC) were purchased from the Dow AgroSciences Co., Ltd. (Shanghai, China). BT WG-001 (16000 IU/mg) was supplied by the Hubei Biopesticide Engineering Research Center. The following were obtained from the Sigma Chemical Co., Ltd. (St. Louis, MO, USA): triphenyl phosphate (TPP, reagent grade); diethyl maleate (DEM, reagent grade); piperonyl butoxide (PBO, reagent grade); 1-chloro-2,4-dinitrobenzene (CDNB); reduced glutathione (GSH); fast blue B salt; sodium-dodecyl sulphate (SDS); dithiothreitol (DTT); eserine; phenylmethanesulfonyl fluoride (PMSF). Alpha-naphthol acetate (α-NA) and ethylene diamine tetra-acetic acid (EDTA) were purchased from the Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was obtained from Roche Molecular Systems, Inc. (San Francisco, CA, USA). The protein-assay dye reagent was provided by Bio-Rad Laboratories, Inc. (Shanghai, China).

Five to seven days old, adult male and female flies were subjected to a non-choice feeding test with spinosad (88%, 76.1% spinosyn A and 11.9% spinosyn D, DOW AgroSciences). The insecticide were diluted with analytic-grade acetone and impregnated on. Susceptible male and female flies were given spinosad in dose 0.063 mg per g sugar and male and female flies from the field populations 766b and 845b were given spinosad in dose 0.25 mg and 0.5 mg per g sugar, respectively. Males and females of multi-resistant strain 791a were given respectively 0.5 mg and 2 mg spinosad per g sugar [17] .
All flies had access to water, milk and sugar ad libitum before trials. Flies, which were alive at day 5 (130–500 flies) were placed in cages with full access to water and were given excess of spinosad-treated granular sugar in a small petri-dish as the only food. The feeding tests were carried out at 25–26°C, 60–65% RH in continuous light. Twenty-four hours upon test start living and fresh looking flies were collected by vacuum suction, immediately sedated by cold and killed at –20°C. The flies were hereafter kept on –80°C until RNA extraction.
Parallel experimental set-ups were performed with resistant and susceptible flies, with the one exception that the sugar offered were left acetone-coated. These flies served as reference for examinations of constitutive gene expression levels.

Feeding bioassays were performed with lambda-cyhalothrin (KarateZeon 10% p/v (100 g/L), Syngenta Limited, Surrey, UK) and spinosad (Dow AgroSciences 88% p/p, Indianapolis, IN, USA). Adult flies 3–5 days old were tested. Five concentrations of each insecticide were tested in concentration–response assays. They were prepared by adding 1 mL of the diluted insecticide to 0.9 g of the rearing diet without water and homogenizing in a mortar. Dilutions of insecticides were prepared with water in the case of lambda-cyhalothrin, and with a buffer composed of acetic acid: sodium acetate (1:3, pH 4.7) in the case of spinosad. These solvents (water or buffer) were mixed with the rearing diet for the non-treated controls. Three replicates of 10–15 flies per replica were performed at each concentration. During the assays, flies were kept in ventilated plastic dishes (89 mm in diameter and 23 mm in height) at standard conditions. Flies were starved for 24 h before the exposure to insecticide. Mortality was recorded after 48 h and flies were considered dead if they were ataxic.