2.1| Animals and evaluation of survival rates in various bathing media
H. tridens and M. japonicus were collected from the Tanaka river estuary in Tsu City. C. dehaani was collected from the Suzuka river estuary in Yokkaichi City, Mie Prefecture, Japan. In order to examine the effects of ionic composition of bathing media on survival rate, four individuals of each species were reared in 210 × 300 × 95 mm plastic tanks with 1000 mL of bathing media (for H. tridens andC. dehaani ), or in 177 × 195 × 100 mm plastic tanks with 500 mL of bathing media (for M. japonicus ). The compositions of bathing media used in this study were summarized in Table 1. The tanks were kept in an incubator in a 16 h/8 h light/dark cycle at 21°C. The survival rate of crabs was assessed at 16, 24, 40, and 48 h, and carcasses were removed at each time point. The survival rate at 48 h was calculated and presented unless otherwise noted. The experiments were performed at least in triplicate.
2.2| Carbonic anhydrase and Na+ /H+ exchanger inhibitors
Acetazolamide and amiloride hydrochloride hydrate, which have already been used in crustacean studies (Burnett and Towle, 1990; Henry et al., 2003; Genovese et al., 2005; Tresguerres et al., 2008), were used as inhibitors for carbonic anhydrase (CA) and the NHE, respectively. Acetazolamide was dissolved in dimethyl sulfoxide (DMSO) to prepare a 200 mg/mL (1.11 mol/L) stock solution. In order to assess the effect of acetazolamide on survivals, four individuals of the crabs were reared in natural seawater containing 1 mg/mL (5.6 mmol/L) of acetazolamide 2 days prior to experiments and then transferred to the 3% NaCl solution containing 1 mg/mL of acetazolamide. As the control group, the same number of the crabs were also reared in natural seawater and then transferred to the 3% NaCl solution supplemented only with DMSO. Amiloride hydrochloride hydrate was dissolved in DMSO to prepare a 50 mg/mL (187.9 mmol/L) stock solution. In order to assess the effect of amiloride on survivals, four individuals of the crabs were reared in 513.3 mmol/L NaCl solution containing 0.25 mg/mL (0.9 mmol/L) of amiloride hydrochloride hydrate. The control group was reared in 513.3 mmol/L NaCl containing only DMSO. The survival rate was calculated 24 h after transfer. The experiments were performed at least in triplicate.
2.3| Hemolymph Na+ ,