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).