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+ ,