Buprofezin

L-menthol (natural source, food grade, ≥ 99% purity), BHT (food grade, ≥ 99% purity), formic acid, elevated purity grade solvents (acetonitrile, ethanol), as well as analytical standards of azoxystrobin, boscalid, buprofezin, chlorpyrifos, chlorpyrifos-methyl, clofentezine, dodine, fludioxonil, hexythiazox, methoxyfenozide, myclobutanil, penconazole, propiconazole, pyraclostrobin, pyriproxyfen, pyridaben, spirotetramat, tebuconazole and tebufenpyrad were purchased from Sigma Aldrich-Merck S.r.l. (Milan, Italy). A Milli-Q Plus apparatus (Millipore, Bedford, MA, USA.) was used for obtaining ultrapure water.
Weighted amounts of the analytical standards (OhausDV215CD Discovery semi-micro and analytical balance, 81/210 g capacity, 0.01/0.1 mg readability, Ohaus Corporation, Pine Brook, NJ, USA) were dissolved in methanol or toluene (clofentezine and pyraclostrobin) in volumetric flasks, in order to obtain individual stock solutions at a concentration of 1 mg mL⁻¹.The last ones were diluted in methanol for preparing the multi-standard working solutions at 0.02, 0.5, 1.5, 2.5 and 4 ng μL⁻¹ used for the method validation.

The insecticides technical of imidacloprid (96.70%), deltamethrin (98.00%), pymetrozine (98.00%), and buprofezin (97.50%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The four insecticides were dissolved in dimethyl sulfoxide (DMSO) and diluted to 3.50 mg/L, 1.00 mg/L, 9.27 mg/L, and 1.35 mg/L in acetone according to LD₅₀ or LC₅₀. The VF 3rd-instar nymphs were reared on RB rice seedlings for two days and collected as RB L. striatellus. Each insecticide (100 nL) was injected into thirty RB nymphs in the conjunction between prothorax and mesothorax by FemtoJet microinjector (Eppendorf, Hamburg, Germany). Acetone was injected as a control. The injected nymphs were transferred to healthy rice seedlings for three days. The effects of insecticides on virus abundance were determined by changes in the relative expression levels of S5-1, S6, S9-1, and S10 by RT-qPCR. The RT-qPCR experiments were conducted on a iQ5 real-time PCR system (Bio-Rad, Hercules, CA, USA) using SYBR PrimeScript^TM RT-PCR Kit (Takara, Kusatsu, Japan). The experiments were repeated three times independently, and each sample contained thirty insects.