Intermolt crayfish were endocrinologically induced to enter premolt as described below. The crayfish were held in individual cages, and the progression of the molt cycle was monitored daily by measuring gastrolith MMI. Two alternative molt induction methods were applied: (1) Removal of the XO–SG complex by a bilateral surgical removal of the eyestalk with a surgical blade; or (2) administration of 20E, by repeated injections into the sinus at the base of the fifth walking leg. Different concentrations of 20E (from a stock solution: 1·g·l–1 of a saline buffer containing 10% ethanol) were injected twice a day, 1000·pg·l–1 was calculated as the
maximum physiological level (Fig.·1B), assuming that hemolymph volume was 30% of the wet mass of the crayfish. Injections of 20E, at the maximal concentration, were continued until an MMI value of 0.1 was obtained. Control animals were injected with comparable amounts of 10% ethanol in saline.