Methoprene

Precocene 1 (Sigma-Aldrich, catalog no. 195855-1G) was diluted in acetone with final concentrations 6 μg/μL and 20 μg/μL and applied on the abdomens of newly eclosed virgin flies with a small brush (5 μL was applied to each abdomen). After precocene treatment flies were checked for recovery (locomotion, wing expansion and flipping). For 96 h treatment Precocene 1 was applied a second time after 48 h. Control flies were exposed to acetone alone. As a second approach newly eclosed virgin flies were fed Precocene 1 mixed into food medium (final concentration 0.5 μg/μL). Control flies were fed food medium alone.
Methoprene (Sigma-Aldrich, St Louis, MO, PESTANAL 33375, racemic mixture), a JH analog50 (link), was diluted in acetone with final concentrations of 1.5 mM or 1 mM. For topical application 0.5 μL of 1 mM Methoprene was quickly applied on the abdomens of virgin flies after three weeks of diapause and flies were transferred back into vials and the diapause incubator as soon as possible to minimize the interruption of diapause. Flies after one week in diapause were taken for immunohistochemistry. For feeding Methoprene 2S:1Y food medium supplemented with 1.5 mM Methoprene was given to newly eclosed virgin flies kept for two weeks in diapause.

Methoprene (Sigma Chemical Co., Ltd.,cn, United States, weight, 100 mg, Purity, 99.5%) was dissolved in 1 mL acetone to produce a 100 μg/μL stock solution, and stored at -20°C (Ayoade et al., 1999 (link)). The stock solutions were diluted to concentrations indicated in Results with acetone and topically applied to the backs of newly emerged, RNAi-treated (100 ng/μL) females using a syringe (Terumo), and a micro applicator (Burkard). The BPH were transferred into glass jars described above and maintained on rice plants at 26 ± 2°C, RH 90%, and 16L:8D photoperiod. The insects were collected at 24 and 48 h, and NlVg and NlUGT12 expression levels, and Vg protein synthesis post Methoprene administration were determined. Each treatment was performed in biologically independent triplicates.

At the first day after ecdysis (day 0), 60 male nymphs were divided in two groups. An experimental group of 30 nymphs was topically treated with the JH III hormone mimic, methoprene (Sigma Aldrich), dissolved in acetone, while a control group received only acetone. The locusts were treated with a daily dose of 100 μg for four consecutive days. During the experiment, locusts were fed with grass ad libitum. On day 4, midguts were collected 3 hours post feed for total RNA extraction. Tissues for RNA extraction were dissected in 5 pools of at least 4 individuals.

Methoprene (Sigma-Aldrich, catalog number 40596-69-8) was dissolved in 95% ethanol (1 μg/μl) and added to warm fly food medium (in a liquid state) to produce a final concentration of 1.04 μl/ml^{23 ,68 (link)}. Flies were fed this food containing 1.04 μl/ml Methoprene or 1 μl/ml 95% ethanol (vehicle control) individually for 3 days after eclosion.

For hormone mimic treatments, methoprene (Sigma-Aldrich, St. Louis, United States) and 20E (Sigma-Aldrich, St. Louis, United States) were dissolved in acetone. 20E or methoprene was topically applied to the dorsal abdomen of 5th-instar larvae (2 μg/larva), and controls were treated with the same dose of acetone.
When RNAi and JH mimic treatments were to be combined, newly emerged moths after dsRNA treatment were further treated with methoprene (5 μg per moth) 1 day after the dsRNA treatment. At 2 days after the methoprene application, the tissue of fat body was dissected and subjected to qRT-PCR and western blot analysis.

JH-III, methoprene, and pyriproxyfen were purchased from Sigma-Aldrich (St. Louis, MO, USA), and the three plant diterpenes (kanakugiol, methyl linderone, and methyl lucidone) were isolated from L. erythrocarpa as described previously [29 (link)]. Each reagent was prepared as a stock solution in dimethyl sulfoxide (DMSO) for the Y2H assays, and in ethanol for the larval development and RNA-seq tests.