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.