Gibco distilled water

Pharmaceutical grade MEL (Wako Pure Chemical Industries, Osaka, Japan) was dissolved in a minimal volume of ethanol and then diluted with Gibco distilled water (Thermo Fisher Scientific, MA, USA), 0, 0.03, 0.1, 0.3, and 1 mM MEL concentrations, with less than 0.01% EtOH. Cockroaches were treated with MEL per os. 29 (link) The EtOH did not influence the amplitude, acrophase, and daily activity of the locomotor rhythm (data not shown). Luzindole, a competitive antagonist of MEL receptor type 2 (MT 2 ), was purchased from Sigma-Aldrich (St. Louis, USA). It was dissolved in 10 µl phosphate-buffered saline (PBS) at 100 nM (=30 pg). Luzindole was injected into each cockroach with a Hamilton 701 syringe equipped with a 26s gauge needle (Hamilton Co., Reno, Nevada, USA). The injury to the cuticle was sealed with an instant adhesive, Aron-alpha (Toagosei Co. Ltd., Tokyo, Japan). Locomotor activity was examined in 10 male cockroaches injected with Luzindole, and an equivalent control group was injected with MEL at concentrations reported in Results. Locomotor activity in LD 12:12 was recorded for 10 days before injection and for 10 days after injection. The activity was recorded for a further 10 days in DD from the last lights-off.