4.3| Role of ambient minor cations under hypo-osmotic conditions
This study revealed that ambient minor cations, especially K+, caused lethal damage to M. japonicus under hypo-osmotic conditions (4.3 mmol/L NaCl solution) (Fig. 7), showing a striking contrast to isosmotic conditions (Fig. 2B). Ambient K+ was essential in isosmotic conditions, but harmful in hypo-osmotic condition. Thus it appears that ambient K+ has an opposite influence on the survival ofM. japonicus in an osmotic condition–dependent manner. The hemolymph K+ concentration in M. japonicusincreased when the crabs were bathed in 4.3 mmol/L NaCl+1.2 MCK solution compared to 4.3 mmol/L NaCl solution (Fig. 8F). Because the hemolymph Na+ concentration is the same in both solutions, the Na+/K+ ratio in hemolymph decreases in 4.3 mmol/L NaCl+1.2 MCK solution. It is possible to assume that a decreased Na+/K+ ratio accounts for the lethality in M. japonicus. NKA and NKCC have been identified as molecules involved in K+ transport in chloride cells in the gills of euryhaline crustaceans (Freire et al., 2008; Charmantier et al., 2009; Henry et al., 2012; Griffith, 2017). NKA transports K+ from hemolymph to the cytoplasm in the opposite direction to Na+. On the other hand, NKCC is distributed in the apical membrane of chloride cells and incorporates K+ from the environment into cells, concomitant with Na+ and Cl. Northern blotting showed that NKA α subunit expression was attenuated in all three species in the presence of ambient minor cations under hypo-osmotic conditions (Fig. 9). This decreased NKA α subunit expression might decrease K+ transport from hemolymph to chloride cells, possibly resulting in increased hemolymph K+concentration. However, it is unclear whether this downregulation of NKA α subunit gene accounts for the increased hemolymph K+concentration in M. japonicus , since this gene was also attenuated in C. dehaani in which ambient minor cations did not affect the hemolymph K+ concentration (Fig. 8G). In addition, NKCC gene expression also decreases in the presence of ambient minor cations under hypo-osmotic conditions in both H. tridensand M. japonicus , indicating that NKCC does not contribute to increased hemolymph K+ concentration, because decreased expression of this gene should lead to decreased hemolymph K+ concentration. It is possible that unidentified transporters and/or channels involved in K+ transport are activated by ambient minor cations to accelerate K+ uptake under hypo-osmotic conditions.
Interestingly, in H. tridens , ambient minor cations increased the hemolymph K+ concentration in hypo-osmotic condition (Fig. 8E) although they caused no mortality (Fig. 7A). Therefore, it is possible that H. tridens is more tolerant to elevated K+ concentrations and low Na+/K+ ratio in hemolymph compared to M. japonicus. It is noteworthy that the average hemolymph K+ concentration in M. japonicus bathed in 513.3 mmol/L NaCl+1.2 MCK is 15.3 ± 1.28 mmol/L, which is significantly higher than the nominal K+ concentration in bathing media (13.4 mmol/L), although the average hemolymph K+concentration in H. tridens is 11.5 ± 1.79 mmol/L, still less than the nominal K+ concentration in bathing media. Thus ambient minor cations may affect the K+ transport system differently in H. tridens and M. japonicus. Another possibility is that some unidentified damage occurs only in M. japonicus but not in H. tridens in the presence of ambient minor cations under hypo-osmotic conditions. The fact that osmotic concentration increased significantly in the presence of minor cations in M. japonicus but not in H. tridens (Table 3) suggest that transport of some ions other than Na+ and K+ was affected only in M. japonicus . The mechanism by which ambient minor cations cause mortality to M. japonicus under hypo-osmotic conditions should be investigated in the future.