3.1| Effects of ambient minor cations on survival and
hemolymph composition of euryhaline crabs under isosmotic conditions
To investigate effect of minor cations on survivals of euryhaline crabs,H. tridens , M. japonicus , and C. dehaani were
reared in 8.0, 16.0, 32.1, 64.2, 128.3, 256.7, and 513.3 mmol/L NaCl
solution (i.e., 513.3 mmol/L NaCl solution was geometrically diluted
with a factor of 2.0) and natural seawater. The survival rate of all of
three species was 100% in natural seawater and relatively high in 8.0
mmol/L NaCl solution (Fig. 1), indicating that all three species are
strongly euryhaline and are hyperregulators in low salinity. However,
the survival rate significantly decreased in H. tridens andM. japonicus at higher NaCl concentrations, and the majority of
crabs (most of H. tridens and all of M. japonicus) died in
513.3 mmol/L NaCl solution, although its salinity was comparable to that
of seawater (Fig. 1A, B). The few H. tridens individuals who
survived in 513.3 mmol/L NaCl solution were moribund. These results
showed that 513.3 mmol/L NaCl solution without minor cations caused
severe damage in crabs. Profit analysis revealed that median lethal
concentrations (LC50) for H. tridens and M.
japonicus were 431.57 ± 37.1 mmol/L and 164.37 ± 22.3 mmol/L,
respectively. In contrast, C. dehaani had a high survival rate
(>80%), even in 513.3 mmol/L NaCl solution (Fig. 1C),
although we observed a few signs of damages, such as slowed movements,
indicating that C. dehaani responds to ambient solutions
differently compared to H. tridens and M. japonicus.Because C. dehaani showed high survival rates even in high
concentrations of NaCl solutions, Profit analysis could not determine
LC50 in this species.
To determine whether administration of some ambient minor ions besides
Na+ and Cl– restore survival rate
of H. tridens and M. japonicus in 513.3 mmol/L NaCl
solution, 513.3 mmol/L NaCl solution was supplemented with 27.4 mmol/L
MgSO4, 25.2 mmol/L MgCl2, 9.9 mmol/L
CaCl2, and 10.7 mmol/L KCl (the resultant solution was
referred to as 513.3 mmol/L NaCl+MMCK solution) to replicate the ionic
composition of artificial seawater, and H. tridens and M.
japonicus were reared in this solution. The survival rate significantly
increased and reached 100% in both species (Fig. 2A, B), indicating
that 513.3 mmol/L NaCl+MMCK solution contains sufficient ambient minor
ions for both species to survive in the presence of 513.3 mmol/L NaCl.
To investigate which ambient minor ions are required for survival,H. tridens and M. japonicus were reared in several kinds
of bathing media of isosmotic condition as follows; 513.3 mmol/L NaCl,
513.3 mmol/L NaCl+MMCK, and 513.3 mmol/L NaCl+MMCK minus any one of the
four additional salts. The survival rate decreased in both species in
the bathing medium lacking KCl (Fig. 2A, B), indicating that
K+ is indispensable for survival of both species in
the presence of 513.3 mmol/L NaCl. To determine the specific role of
K+, the 513.3 mmol/L NaCl solution was supplemented
only with 10.7 mmol/L KCl (referred to as the 513.3 mmol/L NaCl+K
solution), and H. tridens and M. japonicus were reared in
this solution. The survival rate did not fully recover in both species
in 513.3 mmol/L NaCl+K solution (Fig. 2C, D), suggesting that addition
of K+ alone is insufficient. Thus the 513.3 mmol/L
NaCl+K solution was supplemented with either 25.5 mmol/L
MgCl2 or 9.9 mmol/L CaCl2 and H.
tridens and M. japonicus were reared in these solutions. Both
513.3 mmol/L NaCl+K+25.5 mmol/L MgCl2 and 513.3 mmol/L
NaCl+K+9.9 mmol/L CaCl2 restored survival rate of both
species (Fig. 2C, D), indicating that the presence of both
K+ and a divalent cation (either
Mg2+ or Ca2+) is necessary and
sufficient for the survival of H. tridens and M.
japonicus .
Next step of the study was to examine the hemolymph ionic composition
and osmotic concentration of these crabs, H. tridens , M.
japonicus , and C. dehaani incubated in the 8.6 mmol/L NaCl,
513.3 mmol/L NaCl supplemented with 25.5 mmol/L MgCl2,
9.9 mmol/L CaCl2, and 10.7 mmol/L KCl (referred to as
513.3 mmol/L NaCl+MCK solution), and 513.3 mmol/L NaCl solution.
Hemolymph Na+, K+, and osmotic
concentrations were determined at 6 h after incubation. The hemolymph
Na+ concentration in both H. tridens andM. japonicus held in 513.3 mmol/L NaCl solution was significantly
higher compared to 513.3 mmol/L NaCl+MCK solution (Fig. 3A, B). In
addition, the hemolymph K+ concentration in crabs held
in 513.3 mmol/L NaCl solution was significantly less compared to 513.3
mmol/L NaCl+MCK solution and was comparable to the hemolymph
K+ concentration in crabs held in 8.6 mmol/L NaCl
solution (Fig. 3D, E). Moreover, hemolymph osmotic concentration inH. tridens held in 513.3 mmol/L NaCl solution was significantly
higher compared to that in 513.3 mmol/L NaCl+MCK solution, but not inM. japonicus (Table 2). In contrast, differences in ionic
compositions of bathing media did not change hemolymph
Na+, K+, and osmotic concentrations
in C. dehaani , which had a high survival rate in 513.3 mmol/L
NaCl solution (Fig. 3C, F; Table 2).