Hemolymph composition, gene expressions in the gills, and thus the survival of euryhaline crabs are controlled by ambient minor cations according to osmotic condition‐dependent manner

Abstract Na+ and Cl− are the most abundant dissolved ions in seawater, constituting ~ 85% of total ions. They significantly affect the osmolality of body fluids of marine invertebrates. Seawater also contains minor ions such as Mg2+, Ca2+, K+, and SO4 2‐ , but their effects on marine organisms are unclear. This study analyzed the effects of Mg2+, Ca2+, and K+ (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. Ambient minor cations were required for survival of H. tridens and M. japonicus under isosmotic conditions with seawater. The ambient minor cations also affected the osmolality and ionic composition of hemolymph by regulating expressions of specific genes in the gills required for Na+ uptake, such as Na+/K+ ATPase, cytoplasmic carbonic anhydrase, and Na+/H+ exchanger. Administration of carbonic anhydrase and Na+/H+ exchanger inhibitors increased the survival rate even if ambient minor cations did not exist. In contrast, under hypo‐osmotic conditions, ambient minor cations had different effects on crabs, a lethal effect on M. japonicus, and an increase of the hemolymph K+ concentration in H. tridens and M. japonicus. It is thus concluded that the effects of ambient minor cations are osmolality‐dependent. In contrast, in C. dehaani, the hemolymph ionic composition and survival rate were hardly affected by ambient minor cations, probably reflecting the habitat of this species. These results strongly indicated that C. dehaani is less susceptive to ambient minor cations compared to H. tridens and M. japonicus.


| INTRODUC TI ON
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 SO 4 2-, Mg 2+ , Ca 2+ , 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 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 & 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 HCO 3 from H 2 O and CO 2 , 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 + /2Cl − cotransporter (NKCC), are also involved in regulating salinity and ionic composition of hemolymph (Charmantier et al., 2009;Freire et al., 2008;Griffith, 2017;Henry et al., 2012). Transcriptome analysis has identified numerous genes whose expression levels are modified by changes in ambient salinity (Lv et al., 2013;Towle et al., 2011), 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 Mg 2+ , Ca 2+ , 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.

| Animals and evaluation of survival rates in various bathing media
Helice tridens and M. japonicus were collected from the Tanaka river estuary in Tsu City. Chiromantes dehaani was collected from the Suzuka river estuary in Yokkaichi City, Mie Prefecture, Japan.
In order to examine the effects of ionic composition of bathing media on survival rate, four individuals of each species were reared in 210 × 300 × 95 mm plastic tanks with 1,000 ml of bath-

| Hemolymph Na + , K + , and osmotic concentrations
The crabs were anesthetized and dissected, and ~300 μl (for H. tridens and C. dehaani) or ~150 μl (for M. japonicus) of hemolymph was collected from each crab. They were centrifuged at 2,390 g for 5 min at 4°C and the supernatant was collected to be used as the sample solution. Na + and K + concentrations in 1:10 diluted and undiluted sample solutions, respectively, were measured using sodium (model C-122) and potassium ion meters (model C-131) (HORIBA, Kyoto, Japan), respectively. The osmotic concentrations were measured with a vapor pressure osmometer (Model 5600, Wescor, Logan, UT, USA). japonicus, and C. dehaani were performed, as previously described (Yamaguchi & Wakahara, 2001). Briefly, the most posterior pair of gills was isolated from crabs, and total RNA was extracted using Isogen reagent (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. cDNAs were synthesized using an oligo (dT) primer and ReverTra Ace (TOYOBO, Osaka, Japan), and

| Northern blotting
Northern blotting was performed, as previously described (Yamaguchi et al., 2000). Briefly, using cDNA fragments for genes encoding NKA α subunit, CAc, NHE, and NKCC as templates, digoxigenin-labeled RNA probes were synthesized according to the manufacturer's instructions for the 10× DIG RNA labeling mix (Roche, Basel, Switzerland). Total RNA was extracted from the most posterior pair of gills using Isogen reagent (Nippon Gene), and 8.5μg of total RNA was electrophoresed in 1% agarose gel containing 1× 3-(N-morpholino)propanesulfonic acid (MOPS) and 5.5% formalin. Next, RNA was transferred from the gel to a positively charged nylon membrane (Roche) overnight. The resultant membrane was prehybridized using a solution containing 5× saline-sodium citrate (SSC), 50% formamide, 5× Denhardt's solution, 0.5% sodium dodecyl sulfate (SDS), and 0.005% transfer RNA (tRNA) and then hybridized with digoxigenin-labeled RNA probes overnight. Finally, the membrane was washed, blocked, and treated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche) at a dilution of 1:2,000. Signals were detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. The intensity of detected signals of Northern blotting was normalized with amount of electrophoresed ribosomal RNA, both of which were quantified using image processing program, ImageJ (National Institute of Health, Maryland, USA). The relative expression level was calculated as the expression in 513.3 mmol/L NaCl + MCK ( Figure 5) or 4.3 mmol/L NaCl solution ( Figure 9) was 1.0.

| RNase protection assay
RNase protection assay was performed according to Sugiyama et al. (Sugiyama et al., 2001). Briefly, total RNA was extracted from the most posterior pair of gills and hybridized with digoxigenin-labeled RNA probes overnight. Next, RNA was treated with RNase A and RNase T1 to digest single-stranded RNA and electrophoresed in 1% agarose gel containing 1× MOPS and 5.5% formalin. Subsequently, RNA was transferred from the gel to a positively charged nylon membrane (Roche) overnight. Finally, the membrane was blocked and then treated with alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche) at a dilution of 1:2,000. Signals were detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. The intensity of the signals was quantified and the relative expression levels were determined as in Northern blotting.

| Statistical analysis
All rearing experiments to evaluate survival rates were performed at least in triplicate, and Na + , K + , and osmotic concentrations were measured in at least three crabs in each group.     (Table 2). In contrast, differences in ionic compositions of bathing media did not change hemolymph Na + , K + , and osmotic concentrations in C. dehaani, which had a high survival rate in 513.3 mmol/L NaCl solution (Figure 3c,f; Table 2).   supplemented with acetazolamide compared to 513.3 mmol/L NaCl solution, but the result was not statistically significant ( Figure 6a).

| Effects of ambient minor cations on survival and hemolymph composition of euryhaline crabs under hypo-osmotic conditions
To  (Table 3).
Additional experiments were conducted to know specific role of K + on the hemolymph ionic composition in M. japonicus. Administration of KCl increased the hemolymph K + concentration as combined administration of MgCl 2 , CaCl 2 , and KCl ( Figure 8h).

| Isolation of the gene encoding Na + /K + /2Cl − cotransporter and expression analysis of the gene and Na + /K + ATPase α subunit under hypoosmotic conditions
To analyze the possibility that changes in the expression of genes involved in K + transport disturbed hemolymph K + concentration and  (Figure 9). In contrast, NKCC expression in C. dehaani was less affected by ambient minor cations (Figure 9).

| D ISCUSS I ON
In this study, three species of euryhaline crabs, H. tridens, M.
japonicus, and C. dehaani were used. Habitats of H. tridens and M.
japonicus are restricted within estuaries, but C. dehaani is a species which invaded more upstream regions and is adaptive to fresh water (Irawan & Kijima, 1993;Kobayashi, 2000

| Effect of ambient minor cations in seawater on the survival of euryhaline crabs
It has been reported that not only salinity but also ionic composition of the environment significantly affects the survival of euryhaline crustaceans. For example, the mortality of the euryhaline prawn P. mondon reaches 100% during the first 48 hr in 0.17% NaCl solution, although it remains low in diluted artificial seawater of the same salinity (Cawthorne et al., 1983), indicating that some ambient minor ions, besides Na + and Cl − , are essential for its survival. In addition, analysis of ionic profiles of inland well water for cultivation of prawns shows that the presence of K + , Mg 2+ , and SO 4 2increases the survival rate of euryhaline prawns L. vannamei and M. latisulcatus (Davis et al., 2005;Prangnell & Fotedar, 2005Roy et al., 2007;Saoud et al., 2003). Furthermore, removal of either Ca 2+ or Mg 2+ or both, but not K + , from seawater causes lethal damage to the shore crab C. maenas, showing that both divalent cations, but not K + , are required for its survival (Lovett et al., 2006a).

TA B L E 3
Osmotic concentrations (mOsm/kg) of hemolymph bathed in 4.3 mmol/L NaCl and 4.3 mmol/L NaCl + 1.2 MCK solutions 0.19%). These data show that the required minor ions for survival differ among species of euryhaline crustaceans, and the osmotic condition itself in which the ambient minor ions are functioning also differ, respectively. This difference might, in turn, reflect the difference between the microenvironments of their habitats and/or phylogeny. On the other hand, C. dehaani has a significantly high survival rate, even in 513.3 mmol/L NaCl solution, in striking contrast to H. tridens and M. japonicas (Figure 1). Chiromantes dehaani is more adaptive to lower salinity compared to H. tridens and M. japonicus (Irawan & Kijima, 1993), which would account for the robustness of this species in 513.3 mmol/L NaCl solution.

| 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 HCO 3 -HCO from H 2 O and CO 2 , and the derived H + supports Na + uptake through activation of NHE located in the apical membrane (Charmantier et al., 2009;Freire et al., 2008;Griffith, 2017;Henry et al., 2012).
japonicus under hypo-osmotic conditions (8.6 mmol/L NaCl solution) ( Figure 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). 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. Mg 2+ is involved in regulation of more than 300 enzymes, F I G U R E 9 Expression of genes encoding NKA α subunit and NKCC in the most posterior gills of Helice tridens, Macrophthalmus japonicus, and Chiromantes dehaani 6 hr after initiation of incubation in 4.3 mmol/L NaCl and 4.3 mmol/L NaCl + 1.2 MCK solutions. All expressions were detected using Northern blotting. The relative expression level was shown in each band as the expression in 4.3 mmol/L NaCl solution is 1.0. NKA, Na + /K + ATPase; NKCC, Na + /K + /2Cl − cotransporter; 1.2 MCK, 30.5 mmol/L MgCl 2 , 11.7 mmol/L CaCl 2 , and 13.4 mmol/L KCl including NKA (Apell et al., 2017). In fact, activity of NKA in gills of crab is modulated by ambient Mg 2+ (Antunes et al., 2017;Masui et al., 2005Masui et al., , 2009. Moreover, Ca 2+ 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 & Milligan, 1988;McDonald et al., 1983). Lowering the concentration of ambient Ca 2+ reduces hemolymph Na + concentration, irrespective of environmental pH in crayfish Cherax destructor (Ellis & Morris, 1995). In addition, when ambient pH is low, decreased ambient Ca 2+ 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 (Havas et al., 1984;Jozuka & Adachi, 1979;McDonald et al., 1980). In this study, however, hemo-  (Figure 2). Furthermore, both these divalent cations could not overcome the lethality caused by removal of K + (Figure 2), which is a striking contrast to Oryzias latipes, in which only the addition of Ca 2+ increases their survival rate in low pH conditions (Jozuka & 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.

| Role of ambient minor cations under hypoosmotic 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) (Figure 7), showing a striking contrast to isosmotic conditions (Figure 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 of M. japonicus in an osmotic condition-dependent manner.
The hemolymph K + concentration in M. japonicus increased when the crabs were bathed in 4.3 mmol/L NaCl + 1.2 MCK solution compared to 4.3 mmol/L NaCl solution (Figure 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 (Charmantier et al., 2009;Freire et al., 2008;Griffith, 2017;Henry et al., 2012). 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 (Figure 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 (Figure 8g). In addition,

| Distinct response of Chiromantes dehaani to ambient minor cations
The response of C. dehaani to ambient minor cations is different from that of H. tridens and M. japonicus. First, under isosmotic conditions, the decrease of survival rate of C. dehaani in 513.3 mmol/L NaCl was less drastic (Figure 1). Consistent with this result, there was no significant difference in hemolymph Na + and K + concentrations between crabs bathed in 513.3 mmol/L NaCl and 513.3 mmol/L NaCl + MCK solutions (Figure 3c,f). In addition, NKA expression was enhanced but less drastic compared to H. tridens and M. japonicus, and CAc expression was not activated in 513.3 mmol/L NaCl solution ( Figure 5). Furthermore, ambient minor cations did not affect the survival rate ( Figure 7c) and hemolymph Na + and K + concentrations under hypo-osmotic conditions (Figure 8c,g). These results strongly indicate that C. dehaani is less susceptive to ambient minor cations compared to H. tridens and M. japonicus. Compared to H. tridens and M. japonicus, C. dehaani is adaptive to fresh water and habitats of this species are not restricted within estuaries but extend more upstream regions (Irawan & Kijima, 1993;Kobayashi, 2000). Susceptibility to ambient minor cations would be less adaptive for such species and lost in C. dehaani. Alternatively, the difference of sensitivity to ambient minor cations is due to phylogenetic difference of these species.
Comparative studies using other fresh water-adaptive crabs, such as Eriocheir japonica and E. sinensis (Varunidae), will elucidate the relationship between sensitivity to ambient minor cations and adaptation to fresh water.

| CON CLUS ION
Ambient minor cations regulate the expression patterns of specific genes involved in ion transport and thereby affect the hemolymph ionic composition and, ultimately, the survival of H. tridens and M.
japonicus. In contrast, in C. dehaani, which lives in more upstream region, ambient minor cations hardly affect the hemolymph ionic composition, likely because of less susceptibility of gene expression to ambient minor cations. The different response to ambient minor cations between euryhaline species could be due to the difference in the microenvironment of their habitats or phylogenetic difference.

ACK N OWLED G M ENTS
I would like to thank members of research group of crabs in NIT, Suzuka College for technical supports, and Dr. M. Wakahara for critical reading of the manuscript and constructive advice. I would like to thank also Enago and Dr. K. Ishida for English language review. This work was supported in part by the grant from the Okasan-Kato Foundation (18-1-66), and president of NIT, Suzuka College.

CO N FLI C T O F I NTE R E S T S
The authors declare that there are no conflicts of interests.

O PE N R E S E A RCH BA D G E S
This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://doi.org/10.6084/ m9.figsh are.12788132.

DATA AVA I L A B I L I T Y S TAT E M E N T
The sequences of isolated cDNAs in this study were registered in