Xanthotoxin

The following compounds were tested: LPS (Sigma-Aldrich, St. Louis, MO, USA), xanthotoxin (8-metoxypsoralen), and umbelliferone (7-hydroxycoumarin). xanthotoxin and umbelliferone were extracted from the dichloromethane extract of the fruits of Pastinaca sativa L. and methanol extracts of the fruits of Heracleum leskovii Grossch. (Apiaceae), respectively, according to previously published methods [20 (link)].
LPS was dissolved in saline solution (0.9% NaCl). xanthotoxin and umbelliferone were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) and dissolved in saline solution. Drugs were administered intraperitoneally (i.p.) at a volume of 10 mL/kg. New drug solutions were prepared daily. The control group received saline injections of the same volume and via the same route of administration. The doses of umbelliferone, xanthotoxin, and LPS were chosen based on literature data [56 (link)], our recently published articles [20 (link),22 (link)], as well as preliminary studies.

The following compounds were tested: (–)-nicotine hydrogen tartrate (expressed as a salt, 0.1 and 0.2 mg/kg, Sigma–Aldrich, St. Louis, MO, USA), xanthotoxin [8-methoxyfuro[3,2-g]chromen-7-one] (15 mg/kg), bergapten [5-methoxyfuro[3,2-g]chromen-7-one] (25 mg/kg), and umbelliferone [7-hydroxychromen-2-one] (25 mg/kg). Coumarins were isolated as described below. Nicotine was dissolved in saline solution (0.9 % NaCl) and administered subcutaneously (s.c.) at a volume of 10 mL/kg. The pH of the nicotine solution was adjusted to 7.0. Coumarins were suspended in a 1 % solution of Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in a saline solution and administered intraperitoneally (i.p.) at the volume of 10 mL/kg. Fresh drug solutions were prepared on each day of experimentation. Control groups received saline injections of the same volume and through the same route of administration.

A laboratory colony of H. zea, generously provided by Dr. May R. Berenbaum (Department of Entomology, University of Illinois at Urbana-Champaign), was maintained in an insectary kept at 28 °C with a photoperiod of 16 h light: 8 h dark on a semi-synthetic control diet containing wheat germ [25 ]. Induction treatments were performed as described by Li et al. [9 (link)]. The analytical grade plant allelochemicals, xanthotoxin, chlorogenic acid, indole-3-carbinol, flavone, rutin, gossypol, 2-tridecanone, quercetin, coumarin and plant signal molecules jasmonate and salicylate, used in this study, were obtained from Sigma (Sigma-Aldrich, St. Louis, MO, USA) (Figure 1). In brief, 30 newly molted 5th instar larvae were allowed to feed on control diets or control diets containing 0.1% plant xenobiotics for 48 h. Three independent biological replicates of the control diet or each plant xenobiotic treated diet were prepared for subsequent RNA extraction. Midguts and fat bodies were then dissected out, flash-frozen in liquid nitrogen, and stored at −80 °C for subsequent RNA extraction.