Epinastine

Isoprotenerol, salbutamol, prazosin, clonidine were obtained from an adrenergic ligand library commercialized by Biomol International (cat. No. 2811 Plymouth Meeting, PA, USA). Epinastine, tyramine, amitraz, and octopamine were obtained from Sigma chemical company, St. Louis, MO, USA.

Octopamine, octopamine antagonists (epinastine and phentolamine) and dopamine were purchased from Sigma-Aldrich. Octopamine, epinastine, phentolamine and dopamine were dissolved in phosphate-buffered saline (PBS) and sterilized with 0.2-μm membrane filters. PBS was prepared at pH 7.4 with 50 mM sodium phosphate buffer and 0.7% NaCl. Various concentrations of octopamine (0.2, 2, 20 μg/μl), epinastine (0.01, 0.1, 1 μg/μl), phentolamine (0.01, 0.1, 1 μg/μl), and dopamine (0.2, 2, 20 μg/μl) solutions were injected into haemocoel through the abdominal proleg of fifth-instar larvae in a volume of 2 μL, using a 10-μL micro-syringe after surface sterilization with 70% ethanol. Injected larvae were placed in Petri dishes with leaves of corn under normal rearing conditions for 24 h.
Haemolymph samples were collected from groups of 30 larvae injected with octopamine, an octopamine antagonist (epinastine or phentolamine) or dopamine, as indicated. Haemolymph from larvae from each treatment were pooled as one biological replicate for the assays of PO and lysozyme activity and for haemocyte counts. Three biological replicates were established and used for three technical replicates. The haemolymph was collected and then frozen at −80 °C for the assays of phenoloxidase activity, total haemocyte count and lysozyme activity.

Animals were injected with 3 μl of saline containing 2 μM epinastine (Sigma-Aldrich, Tokyo) into the head hemolymph. The estimated final concentration after circulation is 7.0 nM30 (link).