4.2| Role of ambient minor cations under isosmotic
conditions with seawater
Under hypo-osmotic conditions, euryhaline crustaceans incorporate
ambient ions, mainly Na+ and Cl–,
actively through chloride cells in the gills to maintain their hemolymph
osmolality higher compared to the environment. Chloride cells in the
posterior gills play a prominent role in the ionic regulation of
hemolymph, and NKA, CAc, and NHE expressed in chloride cells are
required to incorporate Na+ from environmental water
to hemolymph. NKA is distributed in the basolateral membrane of chloride
cells, which transports Na+ from the cytoplasm to the
hemolymph and keeps the intracellular Na+concentration relatively low, generating driving force of
Na+ uptake. CAc in chloride cells catalyzes the
formation of H+ and
HCO3– from H2O and
CO2, and the derived H+ supports
Na+ uptake through activation of NHE located in the
apical membrane (Freire et al., 2008; Charmantier et al., 2009; Henry et
al., 2012; Griffith, 2017). In addition, the expression of genes
encoding NKA, CAc, and NHE in euryhaline crabs increases under
hypo-osmotic conditions (Lucu and Flik, 1999; Henry et al., 2003; Lucu
and Towle, 2003; Lovett et al., 2006b; Liu et al., 2015; Pan et al.,
2016). This study revealed that the expression of these genes was also
enhanced in H. tridens and M. japonicus under hypo-osmotic
conditions (8.6 mmol/L NaCl solution) (Fig. 5), consistent with previous
studies. In addition, the expression of these genes in posterior gills
also increased in both species in 513.3 mmol/L NaCl solution as in 8.6
mmol/L NaCl solution, strongly indicating that ambient minor cations
decrease the expression of these genes under isosmotic conditions with
seawater. The enhanced expression of these genes could account for the
increased Na+ concentration, and concomitant with
decreased K+ concentration, increased
Na+/K+ ratio in hemolymph in 513.3
mmol/L NaCl solution. An imbalance between Na+ and
K+ concentrations in hemolymph can cause an increase
in the mortality rate in crustaceans (Sowers et al., 2006). Thus the
increased expression of the genes encoding NKA, CAc, and NHE in the most
posterior gills might be the cause of death of H. tridens andM. japonicus reared in 513.3 mmol/L NaCl solution through an
increased Na+/K+ ratio. The
following findings support this view: (1) administration of
acetazolamide and amiloride, inhibitors of CA and NHE, respectively,
increased the survival rate of H. tridens and M. japonicusin 513.3 mmol/L NaCl solution (Fig. 6), and (2) incubation of C.
dehaani in 513.3 mmol/L NaCl solution, showing a high survival rate
compared to H. tridens and M. japonicus, induced only
limited increase of the expression of NKA α subunit and CAc and does not
increase the Na+/K+ ratio (Fig. 3).
It has been reported that the ambient minor cations in seawater are
indispensable for the survival of euryhaline crustaceans. However, the
physiological and ecological role of these ambient minor cations in
euryhaline crustaceans is not completely understood.
Mg2+ is involved in regulation of more than 300
enzymes, including NKA (Apell et al., 2017). In fact, activity of NKA in
gills of crab is modulated by ambient Mg2+ (Masui et
al., 2005, 2009; Antunes et al., 2017). Moreover, Ca2+plays an important role in various biological processes, particularly in
stabilizing biological membranes, increasing the tightness of
intracellular tight junctions, and thereby controlling ion permeability
across the gill epithelium in fish (McDonald et al., 1983; McDonald and
Milligan, 1998). Lowering the concentration of ambient
Ca2+ reduces hemolymph Na+concentration, irrespective of environmental pH in crayfish Cherax
destructor (Ellis and Morris, 1995). In addition, when ambient pH is
low, decreased ambient Ca2+ concentration reduces
plasma or hemolymph Na+ concentration in teleost
species, Salmo gairdneri and Oryzias latipes , and
crustaceans, Daphnia magna and D. middendorffiana , and
leads to increased mortality (Jozuka and Adachi, 1979; McDonald et al.,
1980; Haves et al., 1984). In this study, however, hemolymph
Na+ concentrations in 513.3 mmol/L NaCl solution
increased both in H. tridens and M. japonicus compared to
that in 513.3 mmol/L NaCl+MCK solution (Fig. 3). This suggests that the
lethality in the 513.3 mmol/L NaCl solution was not because of an
inhibition of NKA by removal of Mg2+. In addition, we
showed that the role of Ca2+ for survival of H.
tridens and M. japonicus was substitutable by
Mg2+ (Fig. 2). Furthermore, both these divalent
cations could not overcome the lethality caused by removal of
K+ (Fig. 2), which is a striking contrast toOryzias latipes , in which only the addition of
Ca2+ increases their survival rate in low pH
conditions (Jozuka and Adachi, 1979). These results strongly suggest
that minor cations have different roles on these species from those
described above, although it is possible that cell membrane integrity
and cell junction formation in gills would also be affected by the 513.3
mmol/L NaCl solution also in these species.
This study suggests that ambient minor cations regulate the expression
of specific genes in the gills, thereby affecting ion transport across
the gills and hemolymph ionic composition. It might be necessary for
euryhaline crustaceans inhabiting estuaries to sense subtle changes of
ambient minor cation concentrations to adapt environmental salinity
fluctuation. Future studies should be addressed to know whether these
ambient minor cations directly affect gene expression in the gills. A
comprehensive identification of the genes that are involved in ion
transport and whose expression is regulated by ambient minor cations is
also an important challenge.