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.