Animals

Adult males of the American crayfish, P. clarkii, were used in our experiments. They were reared at 25°C and fed a pellet diet every other day in the laboratory. The molt stages of the crayfish were determined based on the changes in the volume of gastrolith in the stomach and ecdysteroid titers in the hemolymph (Nakatsuji et al., 2000). The gastrolith volume was monitored by X-ray photography (Nakatsuji et al., 2000). The ecdysteroid titer in the hemolymph was determined by radioimmunoassay (RIA) according to the method described previously (Sonobe et al., 1991).

Preparation of samples for TR-FIA

The hemolymph (250–500 μl) was withdrawn from the arthro-dial membrane between the coxa and the base of cheliped using a 1-ml syringe with a 23-gauge needle and immediately mixed with two volumes of 60% acetonitrile on ice. The mixture was centrifuged (20,000g, 20 min), and the supernatant was evaporated under reduced pressure. The dried residue was suspended in the sample buffer for TR-FIA (see below), then centrifuged (20,000g, 5 min), and the supernatant was subjected to TR-FIA. Samples (50 μl) were assayed in duplicate.

In order to evaluate the recovery of Prc-MIH from the hemolymph in the extraction procedure, 0.5, 5 and 50 fmol of authentic Prc-MIH (Kawakami et al., 2000; Sonobe et al., 2001) were added to 500 μl of the hemolymph, which was collected from the crayfish whose eyestalks were removed 7 days before hemolymph sampling in order to eliminate endogenous Prc-MIH in the hemolymph. Authentic Prc-MIH added to the hemolymph was extracted with acetonitrile as described above and quantified by TR-FIA.

TR-FIA for Prc-MIH

Antibodies were raised against Prc-MIH(1-7) and Prc-MIH(55-75)-NH₂ fragments conjugated with bovine serum albumin, and purified using affinity columns conjugated with Prc-MIH(1-7) and Prc-MIH(55-75)-NH₂ fragments, respectively (Nakatsuji et al., 2000). The amino acid sequences of these fragments show no similarity with any segment of the hyperglycemic hormone in P. clarkii (Yasuda et al., 1994), which belongs to the same family as Prc-MIH (Van Herp, 1998). Anti-Prc-MIH(55-75)-NH₂ IgG was biotinylated (Bayer and Wilchek, 1990) and used as the secondary antibody in the TR-FIA.

Wells of a polystyrene microtiter plate (Costar, USA) were filled with 80 μl of a solution of the primary antibody, anti-MIH(1-7) IgG (4 μg/ml of 0.1 M phosphate buffer, pH 7.5, containing 0.1% NaN₃) and incubated for 16 hr at 4°C. Subsequently, the wells were washed five times with the washing buffer (0.01 M phosphate buffer, pH 7.0, containing 0.1 M NaCl, 0.01 M MgCl₂ and 0.05% Tween-20) with shaking, then incubated with 250 μl of the blocking buffer (0.01 M phosphate buffer, pH 7.0, containing 0.05% casein, 0.1 M NaCl, 0.01 M MgCl₂ and 0.1% NaN₃) for 1 hr at 25°C or overnight at 4°C. Authentic Prc-MIH was dissolved in the sample buffer (0.01 M phosphate buffer, pH 7.0, containing 0.05% casein, 0.4 M NaCl, 0.01 M MgCl₂ and 0.1% NaN₃) and assayed in triplicate. The test solutions (50 μl) were added to the wells coated with the primary antibody and incubated for 6 hr at 25°C with shaking. The wells were washed five times with the washing buffer, filled with 50 μl of the biotinylated secondary antibody solution (200 ng/well) diluted with the blocking buffer, and incubated for 1.5 hr at 25°C with shaking. After the wells were washed five times with the washing buffer, 50 μl of europium-labeled streptavidin (Wallac, Finland) solution diluted to 0.1 μg/ml with DELFIA assay buffer (Wallac) was added, and the wells were incubated for 30 min at 25°C with shaking. After washing five times with the washing buffer, DELFIA enhancement solution (Wallac) was added and the mixture was allowed to react for 5 min with shaking. The fluorescence intensity of europium chelates that developed was measured with a time-resolved fluorometer (Wallac).