Centrifugal evaporator

Fifth-instar nymphs were collected at 24, 48, and 72 h post-ecdysis from Wt, NlPTTH, and NlTorso mutants. Ecdysteroids were subsequently extracted from the entire bodies of N. lugens according to Nakaoka et al. with some modifications [38 (link)]. Nymphs were homogenized and sonicated for 20 min in 200 μL sonicated buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl and 2 mM EGTA. A quantity of 400 μL of 1-butanol was added to the samples to extract ecdysteroids by vortexing for 5 min and centrifugation at 1000× g for 10 min. For complete extraction of ecdysteroids from the nymphs, three repetitions of the 1-butanol treatment were performed. The supernatants were collected and subsequently evaporated in a centrifugal evaporator (Eppendorf, Hamburg, Germany). ELISA was performed using a commercially available Insect 20-Hydroxyecdysone Enzyme Immunoassay Kit (Arbor Assays, Ann Arbor, MI, USA) according to the manufacturer’s instructions.

Total malondialdehyde (MDA) (free and protein bound) in liver and brain homogenates was determined by a previously described chromatographic method [25 (link)], with slight modifications, using an ACQUITY UPLC system coupled with an ACQUITY PDA detector (UPLC-PDA) (Waters, USA). In brief, the homogenate samples (each analyzed in duplicate) were submitted to an alkaline hydrolysis step (in the presence of NaOH) at 60 °C in a water bath. After protein removal with perchloric acid, MDA was derivatized with 2,4-dinitrophenylhydrazine (DNPH) and the obtained derivative was extracted in n-hexane, followed by the evaporation of the organic layer in a centrifugal evaporator (Eppendorf, Germany). Then, the residue was dissolved in a mixture of 1% formic acid/acetonitrile and injected into the UPLC–PDA system. Chromatographic separation was realized on a BEH C18 column (50 mm × 2.1 mm i.d., 1.7 μm) (Waters, USA), using gradient elution (1% formic acid/acetonitrile). The flow rate was 0.3 mL/min, the absorbance of the eluent was monitored at 307 nm, and the total chromatographic runtime was 7.5 min. Diacetone alcohol was used as internal standard. The data processing was completed using Empower 2 software (Waters, USA), and total MDA levels were expressed as nmol/mg protein.

HpHRP was performed using an Agilent high performance liquid chromatography (HPLC) 1200 system (Agilent, Santa Clara, California). Reversed-phase chromatography buffers (buffer A; 0.1% (v/v) ammonium hydroxide in HPLC grade water and buffer B; 0.1% (v/v) ammonium hydroxide in acetonitrile) were made fresh. Each iTRAQ-labelled pool sample was re-suspended in 900 μL of 3% (v/v) buffer B and loaded onto a HpHRP column (ZORBAX 300Extend-C18 4.6 × 150 mm 3.5 μm, Agilent) for 40-min with a flow of 1 mL/min at 3% (v/v) buffer B. The peptides were then eluted using the gradient as follows (minutes:%B); 0:3, 5:3, 30:27, 35:50, 36:100, 41:100, 42:3. A total of 86 fractions were collected in a 96-well plate, which was dried in a centrifugal evaporator (Eppendorf) and stored at –20 °C prior to LC-MS/MS analysis.