4 methylcatechol

MDPV and MDPV-d8 were obtained from Cayman Chemical Company (Ann Arbor, MI, USA) and Cerilliant (Round Rock, TX, USA). 3,4-Catechol-PV and 4-OH-3-MeO-PV were synthesized and purified by the Drug Design and Synthesis Section of the National Institute on Drug Abuse (NIDA) Intramural Research Program (IRP), Baltimore, MD, USA (Anizan et al. 2014 (link)). Formic acid, methanol, LC-MS grade acetonitrile (ACN) and water, ethylenediaminetetraacetic acid (EDTA) and sodium metabisulfite (SMBS) were acquired from Fisher Scientific (Fair Lawn, NJ, USA). 4-Methylcatechol and sodium hydroxide were purchased from Sigma (St Louis, MO, USA) and JT Baker (Phillipsburg, NJ, USA), respectively. β-glucuronidase from Red Abalone was obtained from Kura Biotec (Culver City, CA, USA). Water for EDTA, 4-Methylcatechol and SMBS solution preparation was purified-in-house by an ELGA Purelab Ultra Analytic purifier (Siemens Water Technologies, Lowell, MA, USA).

MDPV and MDPV-d8 were obtained from Cayman Chemical Company and Cerilliant. 3,4-catechol-PV and 4-OH-3-MeO-PV were synthesized and purified by the Drug Design and Synthesis Section of the National Institute on Drug Abuse (NIDA) Intramural Research Program, Baltimore, MD, USA. Formic acid, methanol, acetonitrile, water, ethylenediaminetetraacetic acid (EDTA) and sodium metabisulfite (SMBS) were acquired from Fisher Scientific. 4-methylcatechol and sodium hydroxide were purchased from Sigma and JT Baker, respectively. β-glucuronidase from Red Abalone was obtained from Kura Biotec. Water for EDTA, 4-methylcatechol and SMBS solution preparation was purified-in-house by an ELGA Purelab Ultra Analytic purifier.

Caterpillars from each day of the fifth instar (days 0–4) were injected with 10 μL of Manduca saline to induce the production of PO. Hemolymph was collected 24 h later and centrifuged at 7,000 g at 4°C for 10 min to separate the hemocytes from the hemolymph plasma. A 10-μL fraction of the hemolymph plasma was mixed for 1 h with E. coli lipopolysaccharide (Sigma-Aldrich), an effective pro-PO to PO activator (Laughton and Siva-Jothy 2010 ). The reaction was started by adding 20 mM 4-methylcatechol (Sigma-Aldrich). The conversion of 4-methylcatechol to colorimetric quinone by PO was marked by a change in absorbance (at 490 nm), which was measured every 15 min for 1 h at room temperature by means of a microplate reader (Bio-Rad, Hercules, CA). The change in absorbency over time (mOD/min) is directly related to PO activity (Eleftherianos et al. 2008 (link)). Slopes from each hemolymph sample were calculated and compared. PO activity for each caterpillar was divided by the average hemolymph volume collected from the appropriate caterpillar age to account for increases in hemolymph volume during fifth-instar development.