1| INTRODUCTION
Dissolved ions in seawater play a crucial role in maintaining the osmolality of body fluids of marine invertebrates. Na+and Cl are the most abundant ions in seawater, constituting ~85% of total ions, in addition to minor ions such as SO42–, Mg2+, Ca2+, and K+. If dissolved ions are required only for maintaining the osmolality of body fluids, minor ions should not be essential and their replacement by Na+ and Cl should have no effect on the life of marine invertebrates. However, the euryhaline prawn Penaeus mondoncannot survive in 0.17% NaCl solution, although it can survive in diluted artificial seawater of the same salinity (Cawthorne et al., 1983). In addition, the presence of K+, Mg2+, and SO42–increases the survival rate of euryhaline prawns Litopenaeus vannamei and Melicertus latisulcatus (formerly called P. latisulcatus) (Saoud et al., 2003; Zhu et al., 2004; Davis et al., 2005; Prangnell and Fotedar, 2005, 2006; Roy et al., 2007). Therefore, minor ions are required for the survival of marine invertebrates, at least for some euryhaline crustaceans. However, the mechanisms by which minor ions affect the survival of marine organisms are unclear.
Most crustacean species inhabiting estuaries are osmoregulators; they can regulate hemolymph osmotic and ionic concentrations in response to ambient salinity changes to some extent. Typical osmoregulation in estuarine crustaceans is hyper-isosmotic, with hyperregulation in low salinity and isosmotic regulation in salinity close to or higher than seawater (Charmantier et al., 2009; Lignot and Charmantier, 2015). Such osmoregulatory pattern allows crustacean species in estuaries to be euryhaline and tolerate a wider range of ambient salinity. Many studies have investigated the mechanisms by which euryhaline crabs exposed to fresh/brackish water maintain their hemolymph osmolality higher compared to the environment, and chloride cells in posterior gills play an important role in hemolymph osmotic and ionic regulation (Freire et al., 2008; Henry et al., 2012). Chloride cells express Na+/K+ ATPase (NKA) in the basolateral membrane, which transports Na+ from the cytoplasm to the hemolymph and maintains intracellular Na+ concentration relatively low. Another key enzyme in chloride cells is cytoplasmic carbonic anhydrase (CAc), which catalyzes the formation of H+ and HCO3 from H2O and CO2, and the derived H+ supports Na+ uptake through activation of the Na+/H+ exchanger (NHE) located in the apical membrane. Other molecules, such as V-type H+ ATPase and Na+/K+/2Clcotransporter (NKCC), are also involved in regulating salinity and ionic composition of hemolymph (Freire et al., 2008; Charmantier et al., 2009; Henry et al., 2012; Griffith, 2017). Transcriptome analysis has identified numerous genes whose expression levels are modified by changes in ambient salinity (Towle et al., 2011; Lv et al., 2013), and these genes might also contribute to hemolymph osmotic and ionic regulation. However, it is unknown at present what factor(s) in environmental water are used as key signal of ambient salinity, and what factor(s) induce changes of the gene expressions in the gills.
This study analyzed the effects of Mg2+, Ca2+, and K+ (hereafter called “ambient minor cations”) on survival, hemolymph ionic composition, and gene expression in the gills of three euryhaline crabs: Helice tridens , Macrophthalmus japonicus , and Chiromantes dehaani. The different response to ambient minor cations between euryhaline species could be due to the difference in the microenvironment of their habitats or phylogeny.