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\begin{document}

\title{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}



\author[1]{Masahiro Yamaguchi}%
\author[2]{Kouichi Soga}%
\affil[1]{National Institute of Technology, Suzuka College}%
\affil[2]{Osaka City University Graduate School of Science Faculty of Science}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Na+ and Cl-- are the most abundant dissolved ions in seawater,
constituting \textasciitilde{}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 SO42--, 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.%
\end{abstract}%



\sloppy


\textbf{1\textbar{} INTRODUCTION}

Dissolved ions in seawater play a crucial role in maintaining the
osmolality of body fluids of marine invertebrates.
Na\textsuperscript{+}and Cl\textsuperscript{--} are the most abundant
ions in seawater, constituting \textasciitilde{}85\% of total ions, in
addition to minor ions such as SO\textsubscript{4}\textsuperscript{2--},
Mg\textsuperscript{2+}, Ca\textsuperscript{2+}, and
K\textsuperscript{+}. If dissolved ions are required only for
maintaining the osmolality of body fluids, minor ions should not be
essential and their replacement by Na\textsuperscript{+} and
Cl\textsuperscript{--} should have no effect on the life of marine
invertebrates. However, the euryhaline prawn \emph{Penaeus mondon}cannot
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\textsuperscript{+}, Mg\textsuperscript{2+},
and SO\textsubscript{4}\textsuperscript{2--}increases the survival rate
of euryhaline prawns \emph{Litopenaeus vannamei} and \emph{Melicertus
latisulcatus} (formerly called \emph{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\textsuperscript{+}/K\textsuperscript{+} ATPase (NKA) in the
basolateral membrane, which transports Na\textsuperscript{+} from the
cytoplasm to the hemolymph and maintains intracellular
Na\textsuperscript{+} concentration relatively low. Another key enzyme
in chloride cells is cytoplasmic carbonic anhydrase (CAc), which
catalyzes the formation of H\textsuperscript{+} and
HCO\textsubscript{3}\textsuperscript{--} from H\textsubscript{2}O and
CO\textsubscript{2}, and the derived H\textsuperscript{+} supports
Na\textsuperscript{+} uptake through activation of the
Na\textsuperscript{+}/H\textsuperscript{+} exchanger (NHE) located in
the apical membrane. Other molecules, such as V-type
H\textsuperscript{+} ATPase and
Na\textsuperscript{+}/K\textsuperscript{+}/2Cl\textsuperscript{--}cotransporter
(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 Mg\textsuperscript{2+},
Ca\textsuperscript{2+}, and K\textsuperscript{+} (hereafter called
``ambient minor cations'') on survival, hemolymph ionic composition, and
gene expression in the gills of three euryhaline crabs: \emph{Helice
tridens} , \emph{Macrophthalmus japonicus} , and \emph{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.

\textbf{2\textbar{} MATERIALS AND METHODS}

\textbf{2.1\textbar{} Animals and evaluation of survival rates in
various bathing media}

\emph{H. tridens} and \emph{M. japonicus} were collected from the Tanaka
river estuary in Tsu City. \emph{C. 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 \selectlanguage{ngerman}× 300 × 95 mm
plastic tanks with 1000 mL of bathing media (for \emph{H. tridens}
and\emph{C. dehaani} ), or in 177 × 195 × 100 mm plastic tanks with 500
mL of bathing media (for \emph{M. japonicus} )\emph{.} The compositions
of bathing media used in this study were summarized in Table 1. The
tanks were kept in an incubator in a 16 h/8 h light/dark cycle at 21°C.
The survival rate of crabs was assessed at 16, 24, 40, and 48 h, and
carcasses were removed at each time point. The survival rate at 48 h was
calculated and presented unless otherwise noted. The experiments were
performed at least in triplicate.

\textbf{2.2\textbar{} Carbonic anhydrase and Na\textsuperscript{+}}
/\textbf{H\textsuperscript{+} exchanger inhibitors}

Acetazolamide and amiloride hydrochloride hydrate, which have already
been used in crustacean studies (Burnett and Towle, 1990; Henry et al.,
2003; Genovese et al., 2005; Tresguerres et al., 2008), were used as
inhibitors for carbonic anhydrase (CA) and the NHE, respectively.
Acetazolamide was dissolved in dimethyl sulfoxide (DMSO) to prepare a
200 mg/mL (1.11 mol/L) stock solution. In order to assess the effect of
acetazolamide on survivals, four individuals of the crabs were reared in
natural seawater containing 1 mg/mL (5.6 mmol/L) of acetazolamide 2 days
prior to experiments and then transferred to the 3\% NaCl solution
containing 1 mg/mL of acetazolamide. As the control group, the same
number of the crabs were also reared in natural seawater and then
transferred to the 3\% NaCl solution supplemented only with DMSO.
Amiloride hydrochloride hydrate was dissolved in DMSO to prepare a 50
mg/mL (187.9 mmol/L) stock solution. In order to assess the effect of
amiloride on survivals, four individuals of the crabs were reared in
513.3 mmol/L NaCl solution containing 0.25 mg/mL (0.9 mmol/L) of
amiloride hydrochloride hydrate. The control group was reared in 513.3
mmol/L NaCl containing only DMSO. The survival rate was calculated 24 h
after transfer. The experiments were performed at least in triplicate.

\textbf{2.3\textbar{} Hemolymph Na\textsuperscript{+}} ,

\textbf{K\textsuperscript{+}, and osmotic concentrations}

The crabs were anesthetized and dissected, and \textasciitilde{}300 \selectlanguage{greek}µ\selectlanguage{english}l
(for \emph{H. tridens} and \emph{C. dehaani} ) or \textasciitilde{}150
\selectlanguage{greek}µ\selectlanguage{english}l (for \emph{M. japonicus} ) of hemolymph was collected from each crab.
They were centrifuged at 2390 \selectlanguage{ngerman}× g for 5 min at 4°C and the supernatant
was collected to be used as the sample solution.
Na\textsuperscript{+}and K\textsuperscript{+} 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).

\selectlanguage{greek}\textbf{2.4\textbar{} Amplification and isolation of
Na\textsuperscript{+}/K\textsuperscript{+} ATPase α subunit, cytoplasmic
carbonic anhydrase, Na\textsuperscript{+}/H\textsuperscript{+}exchanger,
and
Na\textsuperscript{+}/K\textsuperscript{+}/2Cl\textsuperscript{--}cotransporter}\selectlanguage{english}

Amplification and isolation of complementary DNAs (cDNAs) encoding NKA \selectlanguage{greek}α
\selectlanguage{english}subunit, CAc, NHE, and NKCC in \emph{H. tridens} , \emph{M. japonicus} ,
and \emph{C. dehaani} were performed, as previously described (Yamaguchi
and 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 resultant cDNAs served as
templates for polymerase chain reaction (PCR) amplification. Degenerate
primers were designed on the basis of the amino acid sequence of the
gene products in \emph{Callinectes sapidus, Carcinus maenas}
,\emph{Portunus trituberculatus} , and \emph{L. vannamei.} The primer
sequences used for PCR were as follows: NKA \selectlanguage{greek}α \selectlanguage{english}subunit: forward,
5'-ATGACNGTNGCNCAYATGTGG-3'; reverse, 5'-GGRTGRTCNCCNGTNACCAT-3'; CAc:
forward, 5'-TAYGTNTTRGARCARTTYCA-3' or 5' -TAYGTNCTNGARCARTTYCA-3';
reverse, 5'-CKRAANGCRTCYAAYTG-3' or 5'-CKRAANGCRTCNAGYTG-3'; NHE:
forward, 5'-AARATHGGNTTYCAYATGAC-3'; reverse,
5'-TGYTCNACRTARTTYTTCAT-3'; NKCC: forward, 5'-AAYATHTGGGGNGTNATG-3';
reverse, 5'-GCRAARCANCCNGCRTADAT-3'.

For amplification of CAc and NKCC cDNA, we performed nested PCR using
part of the products from the first amplification as a template. The
primer sequences used for nested PCR were as follows: CAc: forward,
5'-CAYTGGGGNAARACNAAYGA-3'; reverse, 5'-CANGGNGGNGTNGTNA-3'; NKCC:
forward, 5'-ATHTGGGGNGTNATGYT-3'; reverse, 5'-ARYTCCATNACYTGRAARCT-3'.

In every case, conditions for PCR were as follows: 40 cycles for 30 s at
94\selectlanguage{ngerman}°C, 30 s at annealing temperature, and 30 s at 72°C. The annealing
temperature for NKA \selectlanguage{greek}α \selectlanguage{english}subunit, CAc, NHE, and NKCC was 50\selectlanguage{ngerman}°C, 43°C, 45°C,
and 45°C, respectively. Amplified PCR products were electrophoresed and
extracted, and then inserted into a pTA2 cloning vector (TOYOBO), and
finally, their nucleotide sequences were determined. The sequences of
isolated cDNAs were deposited to GenBank (Accession numbers of NKA \selectlanguage{greek}α
\selectlanguage{english}subunit, CAc, NHE, and NKCC are LC214855, LC375964, LC572286, and
LC572287 for \emph{H. tridens} , LC214856, LC375965, LC572288, and
LC572289 for \emph{M, japonicus} , and LC572290, LC572291, LC572292, and
LC572293 for \emph{C. dehaani} ). In addition, amino acid sequences were
predicted from the cDNA fragment sequences of \emph{H. tridens}
,\emph{M. japonicus} , and \emph{C. dehaani} and compared with those of
the shore crab \emph{C. maenas} (GenBank Accession numbers are AY03550,
EU273943, U09274, and AY035548 for NKA \selectlanguage{greek}α \selectlanguage{english}subunit, CAc, NHE, and NKCC,
respectively) (Towle et al., 1997; Serrano and Henry, 2008).

\textbf{2.5\textbar{} Northern blotting}

Northern blotting was performed, as previously described (Yamaguchi et
al., 2000). Briefly, using cDNA fragments for genes encoding NKA \selectlanguage{greek}α
\selectlanguage{english}subunit, CAc, NHE, and NKCC as templates, digoxigenin-labeled RNA probes
were synthesized according to the manufacturer's instructions for the
10x DIG RNA labeling mix (Roche, Basel, Switzerland). Total RNA was
extracted from the most posterior pair of gills using Isogen reagent
(Nippon Gene), and 8.5 \selectlanguage{greek}µ\selectlanguage{english}g of total RNA was electrophoresed in 1\%
agarose gel containing 1x 3-(\emph{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 5x saline-sodium
citrate (SSC), 50\% formamide, 5x 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 1:2000. 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 (Fig. 5) or 4.3 mmol/N NaCl solution (Fig. 9) was 1.0.

\textbf{2.6\textbar{} 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 1x 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 1:2000. 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.

\textbf{2.7\textbar{} Statistical analysis}

All rearing experiments to evaluate survival rates were performed at
least in triplicate, and Na\textsuperscript{+}, K\textsuperscript{+},
and osmotic concentrations were measured in at least three crabs in each
group. Error bars in the figures show standard errors. Mean values
comparisons among more than three groups were performed using analysis
of variance (ANOVA), followed by Williams' test, Student's \emph{t}
-test with Bonferroni correction, or Tukey's test. Mean values
comparisons between two groups were performed by Student's \emph{t}
-test. Results were considered statistically significant if the \emph{P}
value was less than 0.05.

\textbf{3\textbar{} RESULTS}

\textbf{3.1\textbar{} Effects of ambient minor cations on survival and
hemolymph composition of euryhaline crabs under isosmotic conditions}

To investigate effect of minor cations on survivals of euryhaline
crabs,\emph{H. tridens} , \emph{M. japonicus} , and \emph{C. dehaani}
were reared in 8.0, 16.0, 32.1, 64.2, 128.3, 256.7, and 513.3 mmol/L
NaCl solution (i.e., 513.3 mmol/L NaCl solution was geometrically
diluted with a factor of 2.0) and natural seawater. The survival rate of
all of three species was 100\% in natural seawater and relatively high
in 8.0 mmol/L NaCl solution (Fig. 1), indicating that all three species
are strongly euryhaline and are hyperregulators in low salinity.
However, the survival rate significantly decreased in \emph{H. tridens}
and\emph{M. japonicus} at higher NaCl concentrations, and the majority
of crabs (most of \emph{H. tridens} and all of \emph{M. japonicus)} died
in 513.3 mmol/L NaCl solution, although its salinity was comparable to
that of seawater (Fig. 1A, B). The few \emph{H. tridens} individuals who
survived in 513.3 mmol/L NaCl solution were moribund. These results
showed that 513.3 mmol/L NaCl solution without minor cations caused
severe damage in crabs. Profit analysis revealed that median lethal
concentrations (LC\textsubscript{50}) for \emph{H. tridens} and \emph{M.
japonicus} were 431.57 \selectlanguage{ngerman}± 37.1 mmol/L and 164.37 ± 22.3 mmol/L,
respectively. In contrast, \emph{C. dehaani} had a high survival rate
(\textgreater{}80\%), even in 513.3 mmol/L NaCl solution (Fig. 1C),
although we observed a few signs of damages, such as slowed movements,
indicating that \emph{C. dehaani} responds to ambient solutions
differently compared to \emph{H. tridens} and \emph{M.
japonicus.}Because \emph{C. dehaani} showed high survival rates even in
high concentrations of NaCl solutions, Profit analysis could not
determine LC\textsubscript{50} in this species.

To determine whether administration of some ambient minor ions besides
Na\textsuperscript{+} and Cl\textsuperscript{--} restore survival rate
of \emph{H. tridens} and \emph{M. japonicus} in 513.3 mmol/L NaCl
solution, 513.3 mmol/L NaCl solution was supplemented with 27.4 mmol/L
MgSO\textsubscript{4}, 25.2 mmol/L MgCl\textsubscript{2}, 9.9 mmol/L
CaCl\textsubscript{2}, and 10.7 mmol/L KCl (the resultant solution was
referred to as 513.3 mmol/L NaCl+MMCK solution) to replicate the ionic
composition of artificial seawater, and \emph{H. tridens} and \emph{M.
japonicus} were reared in this solution. The survival rate significantly
increased and reached 100\% in both species (Fig. 2A, B), indicating
that 513.3 mmol/L NaCl+MMCK solution contains sufficient ambient minor
ions for both species to survive in the presence of 513.3 mmol/L NaCl.
To investigate which ambient minor ions are required for
survival,\emph{H. tridens} and \emph{M. japonicus} were reared in
several kinds of bathing media of isosmotic condition as follows; 513.3
mmol/L NaCl, 513.3 mmol/L NaCl+MMCK, and 513.3 mmol/L NaCl+MMCK minus
any one of the four additional salts. The survival rate decreased in
both species in the bathing medium lacking KCl (Fig. 2A, B), indicating
that K\textsuperscript{+} is indispensable for survival of both species
in the presence of 513.3 mmol/L NaCl. To determine the specific role of
K\textsuperscript{+}, the 513.3 mmol/L NaCl solution was supplemented
only with 10.7 mmol/L KCl (referred to as the 513.3 mmol/L NaCl+K
solution), and \emph{H. tridens} and \emph{M. japonicus} were reared in
this solution. The survival rate did not fully recover in both species
in 513.3 mmol/L NaCl+K solution (Fig. 2C, D), suggesting that addition
of K\textsuperscript{+} alone is insufficient. Thus the 513.3 mmol/L
NaCl+K solution was supplemented with either 25.5 mmol/L
MgCl\textsubscript{2} or 9.9 mmol/L CaCl\textsubscript{2} and \emph{H.
tridens} and \emph{M. japonicus} were reared in these solutions. Both
513.3 mmol/L NaCl+K+25.5 mmol/L MgCl\textsubscript{2} and 513.3 mmol/L
NaCl+K+9.9 mmol/L CaCl\textsubscript{2} restored survival rate of both
species (Fig. 2C, D), indicating that the presence of both
K\textsuperscript{+} and a divalent cation (either
Mg\textsuperscript{2+} or Ca\textsuperscript{2+}) is necessary and
sufficient for the survival of \emph{H. tridens} and \emph{M. japonicus}
.

Next step of the study was to examine the hemolymph ionic composition
and osmotic concentration of these crabs, \emph{H. tridens} , \emph{M.
japonicus} , and \emph{C. dehaani} incubated in the 8.6 mmol/L NaCl,
513.3 mmol/L NaCl supplemented with 25.5 mmol/L MgCl\textsubscript{2},
9.9 mmol/L CaCl\textsubscript{2}, and 10.7 mmol/L KCl (referred to as
513.3 mmol/L NaCl+MCK solution), and 513.3 mmol/L NaCl solution.
Hemolymph Na\textsuperscript{+}, K\textsuperscript{+}, and osmotic
concentrations were determined at 6 h after incubation. The hemolymph
Na\textsuperscript{+} concentration in both \emph{H. tridens}
and\emph{M. japonicus} held in 513.3 mmol/L NaCl solution was
significantly higher compared to 513.3 mmol/L NaCl+MCK solution (Fig.
3A, B). In addition, the hemolymph K\textsuperscript{+} concentration in
crabs held in 513.3 mmol/L NaCl solution was significantly less compared
to 513.3 mmol/L NaCl+MCK solution and was comparable to the hemolymph
K\textsuperscript{+} concentration in crabs held in 8.6 mmol/L NaCl
solution (Fig. 3D, E). Moreover, hemolymph osmotic concentration
in\emph{H. tridens} held in 513.3 mmol/L NaCl solution was significantly
higher compared to that in 513.3 mmol/L NaCl+MCK solution, but not
in\emph{M. japonicus} (Table 2). In contrast, differences in ionic
compositions of bathing media did not change hemolymph
Na\textsuperscript{+}, K\textsuperscript{+}, and osmotic concentrations
in \emph{C. dehaani} , which had a high survival rate in 513.3 mmol/L
NaCl solution (Fig. 3C, F; Table 2).

\selectlanguage{greek}\textbf{3.2\textbar{} Isolation and expression analysis of genes
encoding Na\textsuperscript{+}/K\textsuperscript{+} ATPase α subunit,
cytoplasmic carbonic anhydrase, and
Na\textsuperscript{+}/H\textsuperscript{+} exchanger}\selectlanguage{english}

To analyze the possibility that changes in the expression of genes
involved in Na\textsuperscript{+} uptake disturbed hemolymph ionic
composition and ultimately led to the death of crabs in rearing
experiments, cDNA fragments of NKA \selectlanguage{greek}α \selectlanguage{english}subunit, CAc, and NHE were
amplified by PCR and isolated, and gene expression was analyzed in the
most posterior gills of \emph{H. tridens} , \emph{M. japonicus} ,
and\emph{C. dehaani.} The lengths of isolated cDNA fragments for the NKA
\selectlanguage{greek}α \selectlanguage{english}subunit, CAc, and NHE genes were 700, 300, and 800 bp, respectively.
Sequencing results showed that the cDNA fragment sequences of these
genes were highly similar to those in other crab species. The deduced
amino acid sequences of the NKA \selectlanguage{greek}α \selectlanguage{english}subunit of \emph{H. tridens} ,
\emph{M. japonicus} , and \emph{C. dehaani} had 95\%, 95\%, and 94\%
similarity, respectively, to that of \emph{C. maenas} ; those of CAc of
\emph{H. tridens} , \emph{M. japonicus} , and \emph{C. dehaani} had
76\%, 78\%, and 75\% similarity, respectively, to that of \emph{C.
maenas;} and those of the NHE of \emph{H. tridens} , \emph{M. japonicus}
, and \emph{C. dehaani}had 94\%, 93\%, and 92\% similarity,
respectively, to that of \emph{C. maenas} (Fig. 4A--C) (Towle et al.,
1997; Serrano and Henry, 2008).

The expression of these genes in the most posterior gills of crabs held
in the 8.6 mmol/L NaCl, 513.3 mmol/L NaCl+MCK, and 513.3 mmol/L NaCl
solutions for 6 h were analyzed by Northern blotting. However, the
expression levels of CAc gene was not sufficiently high in \emph{M.
japonicus} to be detected by Northern blotting; therefore, RNase
protection assay was carried out to detect the expression of this gene
in \emph{M. japonicus} . These analyses revealed that the expression of
all genes in the most posterior gills in \emph{H. tridens} and \emph{M.
japonicas} was higher in 8.6 mmol/L NaCl solution compared to 513.3
mmol/L NaCl+MCK solution except CAc in \emph{M. japonicus} .
Furthermore, the expression of all genes in the most posterior gills was
also higher in 513.3 mmol/L NaCl solution compared to 513.3 mmol/L
NaCl+MCK solution as in 8.6 mmol/L NaCl solution (Fig. 5). On the other
hand, the expression of these genes was different in \emph{C. dehaani.}
The expression of genes encoding NKA \selectlanguage{greek}α \selectlanguage{english}subunit was enhanced in 513.3
mmol/L NaCl solution but was less drastic compared to \emph{H. tridens}
and\emph{M. japonicus.} In addition, the expression of CAc gene was not
induced in 513.3 mmol/L NaCl solution but was in 8.6 mmol/L NaCl
solution. In contrast, the expression of NHE gene was enhanced in 513.3
mmol/L NaCl solution but not in 8.6 mmol/L NaCl solution (Fig. 5).

\textbf{3.3\textbar{} Effects of carbonic anhydrase and
Na\textsuperscript{+}/H\textsuperscript{+} exchanger inhibitors}

Increased expression of genes involved in Na\textsuperscript{+} uptake
could be a possible cause of the death of \emph{H. tridens} and \emph{M.
japonicus} in 513.3 mmol/L NaCl solution. To examine this
possibility,\emph{H. tridens} and \emph{M. japonicas} were reared in
513.3 mmol/L NaCl solution supplemented with acetazolamide or amiloride,
an inhibitor of CA and NHE, respectively. Amiloride significantly
increased the survival rate of \emph{H. tridens} and \emph{M. japonicus}
, and acetazolamide increased the survival rate of \emph{M. japonicus}
reared in 513.3 mmol/L NaCl solution (Fig. 6B--D). The survival rate was
also higher in \emph{H. tridens} reared in 513.3 mmol/L NaCl solution
supplemented with acetazolamide compared to 513.3 mmol/L NaCl solution,
but the results were not statistically significant (Fig. 6A).

\textbf{3.4\textbar{} Effects of ambient minor cations on survival and
hemolymph composition of euryhaline crabs under hypo-osmotic conditions}

To investigate the effects of ambient minor cations on the survival of
euryhaline crabs at low ambient salinity, \emph{H. tridens} , \emph{M.
japonicus} , and \emph{C. dehaani} were reared in 8.6 mmol/L NaCl
solution supplemented with 25.2 mmol/L MgCl\textsubscript{2}, 9.9 mmol/L
CaCl\textsubscript{2}, and 10.7 mmol/L KCl (referred to as 8.6 mmol/L
NaCl+MCK solution), 8.6 mmol/L NaCl solution, and 17.1 mmol/L NaCl
solution without additional salts. Additional salts did not have any
effect on the survival of \emph{H. tridens} and \emph{C. dehaani} ,
showing 100\% survival in both species (data not shown). In contrast,
the survival rate considerably decreased in \emph{M. japonicus} in 8.6
mmol/L NaCl+MCK solution (data not shown). Because some \emph{M.
japonicus} individuals still appeared intact in 8.6 mmol/L NaCl+MCK
solution, further experiments were conducted using 4.3 mmol/L NaCl, 4.3
mmol/L NaCl supplemented with 30.5 mmol/L MgCl\textsubscript{2}, 11.7
mmol/L CaCl\textsubscript{2}, and 13.4 mmol/L KCl (referred to as 4.3
mmol/L NaCl+1.2 MCK solution), and 17.1 mmol/L NaCl+1.2 MCK solution.
The survival rate of \emph{H. tridens} and \emph{C. dehaani} was 100\%,
but that of \emph{M. japonicus} significantly decreased to 17\% in 4.3
mmol/L NaCl+1.2 MCK solution (Fig. 7). A few \emph{M.
japonicus}individuals survived in this solution but were moribund. In
contrast, most \emph{M. japonicus} individuals survived in 4.3 mmol/L
NaCl and 17.1 mmol/L NaCl+1.2 MCK solution (Fig. 7B). These results
indicated that higher concentrations of additional salts in combination
with a lower NaCl concentration caused high mortality in \emph{M.
japonicus} in 4.3 mmol/L NaCl+1.2 MCK solution.

To examine which minor cations have lethal effect on \emph{M. japonicus}
, 30.5 mmol/L MgCl\textsubscript{2} and 11.7 mmol/L
CaCl\textsubscript{2} (1.2MC) and 13.4 mmol/L KCl (1.2K) were added to
4.3 mmol/L NaCl solution separately. In \emph{M. japonicus} reared in
4.3 mmol/L NaCl solution supplemented with 13.4 mmol/L KCl (referred to
as 4.3 mmol/L NaCl+1.2 K solution), the survival rate significantly
decreased to a comparable level as that in 4.3 mmol/L NaCl+1.2 MCK
solution (Fig. 7D). Even after diluting KCl to 2.7 mmol/L (almost
one-fourth of the concentration in seawater), the survival rate did not
hardly recover (Fig. 7E), indicating that K\textsuperscript{+} accounts
for the lethality in 4.3 mmol/L NaCl+ 1.2 MCK solution. In contrast,
supplementation of 4.3 mmol/L NaCl with 30.5 mmol/L
MgCl\textsubscript{2} and 11.7 mmol/L CaCl\textsubscript{2} did not have
a lethal effect on \emph{M. japonicus} ; instead, these divalent cations
attenuated the lethal effect of K\textsuperscript{+} 15 h after
initiation of rearing, although it did not block the lethal effect at
all 48 h after initiation of rearing (Fig. 7D, F).

Possible effects of ambient minor cations under hypo-osmotic conditions
on hemolymph ionic composition and osmotic concentration were examined
in \emph{H. tridens} , \emph{M. japonicus} , and \emph{C. dehaani.} They
were incubated in the 4.3 mmol/L NaCl and 4.3 mmol/L NaCl+1.2 MCK
solutions, and hemolymph Na\textsuperscript{+}, K\textsuperscript{+},
and osmotic concentrations were determined 6 h after incubation. There
were no significant differences in the hemolymph
Na\textsuperscript{+}concentration between crabs incubated in 4.3 mmol/L
NaCl and 4.3 mmol/L NaCl+1.2 MCK solutions in all three species (Fig.
8A--C). In contrast, the hemolymph K\textsuperscript{+} concentration in
crabs incubated in 4.3 mmol/L NaCl+1.2 MCK solution was significantly
higher compared to 4.3 mmol/L NaCl solution in \emph{H. tridens} and
\emph{M. japonicus}but not in \emph{C. dehaani} (Fig. 8E--G). Moreover,
addition of minor cations increased osmotic concentration significantly
in \emph{M. japonicus} , but not in \emph{H. tridens} and \emph{M.
japonicus} (Table 3). Additional experiments were conducted to know
specific role of K\textsuperscript{+} on the hemolymph ionic composition
in \emph{M. japonicus.} Administration of KCl increased the hemolymph
K\textsuperscript{+} concentration as combined administration of
MgCl\textsubscript{2}, CaCl\textsubscript{2}, and KCl (Fig. 8H).

\selectlanguage{greek}\textbf{3.5\textbar{} Isolation of the gene encoding
Na\textsuperscript{+}/K\textsuperscript{+}/2Cl\textsuperscript{--}cotransporter
and expression analysis of the gene and
Na\textsuperscript{+}/K\textsuperscript{+} ATPase α subunit under
hypo-osmotic conditions}\selectlanguage{english}

To analyze the possibility that changes in the expression of genes
involved in K\textsuperscript{+} transport disturbed hemolymph
K\textsuperscript{+} concentration and ultimately led to the death
of\emph{M. japonicus} in 4.3 mmol/L NaCl+1.2 MCK solution, cDNA fragment
of NKCC was amplified by PCR and isolated, and gene expression of it and
NKA \selectlanguage{greek}α \selectlanguage{english}subunit was analyzed in the most posterior gills of \emph{H.
tridens} , \emph{M. japonicus} , and \emph{C. dehaani.} The deduced
amino acid sequence of NKCC of \emph{H. tridens} , \emph{M. japonicus} ,
and\emph{C. dehaani} had 81\%, 79\%, and 77\% similarity to that
of\emph{C. maenas} (Fig. 4D). Northern blotting revealed that NKA \selectlanguage{greek}α
\selectlanguage{english}subunit expression in the most posterior gills of crabs incubated in 4.3
mmol/L NaCl+1.2 MCK solution for 6 h decreased compared to 4.3 mmol/L
NaCl solution in all three species. In \emph{H. tridens} and \emph{M.
japonicus} , NKCC expression also decreased in the presence of ambient
minor cations, although the expression was relatively low (Fig. 9). In
contrast, NKCC expression in \emph{C. dehaani} was less affected by
ambient minor cations (Fig. 9).

\textbf{4\textbar{} DISCUSSION}

In this study, three species of euryhaline crabs, \emph{H. tridens}
,\emph{M. japonicus} , and \emph{C. dehaani} were used. Habitats
of\emph{H. tridens} and \emph{M. japonicus} are restricted within
estuaries, but \emph{C. dehaani} is a species which invaded more
upstream regions and is adaptive to fresh water (Irawan and Kijima,
1993; Kobayashi, 2000). These species belong to three different
families: Varunidae (\emph{H. tridens} ), Ocypodidae (\emph{M.
japonicus} ), and Sesarmidae (\emph{C. dehaani} ), although \emph{H.
tridens} and \emph{C. dehaani} belong to the same superfamily,
Grapsoidea. The different responses to environmental water of these
species are possibly due to the differences in the microenvironment of
their habitats or phylogeny.

\textbf{4.1\textbar{} Effect of ambient} \textbf{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 \emph{P.
mondon} reaches 100\% during the first 48 h 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\textsuperscript{+} and Cl\textsuperscript{--}, 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\textsuperscript{+}, Mg\textsuperscript{2+}, and
SO\textsubscript{4}\textsuperscript{2--} increases the survival rate of
euryhaline prawns \emph{L. vannamei} and \emph{M. latisulcatus} (Saoud
et al., 2003; Davis et al., 2005; Prangnell and Fotedar, 2005, 2006; Roy
et al., 2007). Furthermore, removal of either Ca\textsuperscript{2+} or
Mg\textsuperscript{2+} or both, but not K\textsuperscript{+}, from
seawater causes lethal damage to the shore crab \emph{C. maenas} ,
showing that both divalent cations, but not K\textsuperscript{+}, are
required for its survival (Lovett et al., 2006a).

This study revealed that both \emph{H. tridens} and \emph{M.
japonicus}can survive even in 8.6 mmol/L NaCl solution but not in 513.3
mmol/L NaCl solution. The high mortality of \emph{H. tridens} and
\emph{M. japonicus} in 513.3 mmol/L NaCl solution indicates some ambient
minor ions besides Na\textsuperscript{+} and Cl\textsuperscript{--} are
required for these crabs to survive under isosmotic condition. For
complete recovery of the survival rate in 513.3 mmol/L NaCl solution,
both K\textsuperscript{+} and divalent cations, either
Mg\textsuperscript{2+} or Ca\textsuperscript{2+}, are required (Fig. 2).
In addition, the required ambient minor ions for both \emph{H.
tridens}and \emph{M. japonicus} are different compared to other
euryhaline crustaceans previously examined: (1)
SO\textsubscript{4}\textsuperscript{2--} is not needed for survival
of\emph{H. tridens} and \emph{M. japonicus} unlike \emph{L.
vannamei}(Saoud et al., 2003), (2) K\textsuperscript{+} and either
Mg\textsuperscript{2+} or Ca\textsuperscript{2+} are essential for
both\emph{H. tridens} and \emph{M. japonicus} , although both
Mg\textsuperscript{2+} and Ca\textsuperscript{2+}, but not
K\textsuperscript{+}, are required for \emph{C. maenas} (Lovett et al.,
2006a), and (3) while \emph{P. mondon} cannot survive 48 h in 0.17\%
NaCl solution (Cawthorne et al., 1983), both \emph{H. tridens}
and\emph{M. japonicus} can survive in NaCl solution with similar
salinity (32.1 mmol/L, corresponding to 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, \emph{C. dehaani} has a
significantly high survival rate, even in 513.3 mmol/L NaCl solution, in
striking contrast to \emph{H. tridens} and \emph{M. japonicas} (Fig.
1)\emph{. C. dehaani} is more adaptive to lower salinity compared
to\emph{H. tridens} and \emph{M. japonicus} (Irawan and Kijima, 1993),
which would account for the robustness of this species in 513.3 mmol/L
NaCl solution.

\textbf{4.2\textbar{} Role of ambient minor cations under isosmotic
conditions with seawater}

Under hypo-osmotic conditions, euryhaline crustaceans incorporate
ambient ions, mainly Na\textsuperscript{+} and Cl\textsuperscript{--},
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\textsuperscript{+} from environmental water
to hemolymph. NKA is distributed in the basolateral membrane of chloride
cells, which transports Na\textsuperscript{+} from the cytoplasm to the
hemolymph and keeps the intracellular Na\textsuperscript{+}concentration
relatively low, generating driving force of Na\textsuperscript{+}
uptake. CAc in chloride cells catalyzes the formation of
H\textsuperscript{+} and HCO\textsubscript{3}\textsuperscript{--} from
H\textsubscript{2}O and CO\textsubscript{2}, and the derived
H\textsuperscript{+} supports Na\textsuperscript{+} 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 \emph{H. tridens} and
\emph{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\textsuperscript{+} concentration, and concomitant with decreased
K\textsuperscript{+} concentration, increased
Na\textsuperscript{+}/K\textsuperscript{+} ratio in hemolymph in 513.3
mmol/L NaCl solution. An imbalance between Na\textsuperscript{+} and
K\textsuperscript{+} 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 \emph{H. tridens}
and\emph{M. japonicus} reared in 513.3 mmol/L NaCl solution through an
increased Na\textsuperscript{+}/K\textsuperscript{+} ratio. The
following findings support this view: (1) administration of
acetazolamide and amiloride, inhibitors of CA and NHE, respectively,
increased the survival rate of \emph{H. tridens} and \emph{M.
japonicus}in 513.3 mmol/L NaCl solution (Fig. 6), and (2) incubation of
\emph{C. dehaani} in 513.3 mmol/L NaCl solution, showing a high survival
rate compared to \emph{H. tridens} and \emph{M. japonicus,} induced only
limited increase of the expression of NKA \selectlanguage{greek}α \selectlanguage{english}subunit and CAc and does not
increase the Na\textsuperscript{+}/K\textsuperscript{+} 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.
Mg\textsuperscript{2+} 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 Mg\textsuperscript{2+} (Masui et
al., 2005, 2009; Antunes et al., 2017). Moreover,
Ca\textsuperscript{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 et al.,
1983; McDonald and Milligan, 1998). Lowering the concentration of
ambient Ca\textsuperscript{2+} reduces hemolymph
Na\textsuperscript{+}concentration, irrespective of environmental pH in
crayfish \emph{Cherax destructor} (Ellis and Morris, 1995). In addition,
when ambient pH is low, decreased ambient Ca\textsuperscript{2+}
concentration reduces plasma or hemolymph Na\textsuperscript{+}
concentration in teleost species, \emph{Salmo gairdneri} and
\emph{Oryzias latipes} , and crustaceans, \emph{Daphnia magna} and
\emph{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\textsuperscript{+} concentrations in 513.3 mmol/L
NaCl solution increased both in \emph{H. tridens} and \emph{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
Mg\textsuperscript{2+}. In addition, we showed that the role of
Ca\textsuperscript{2+} for survival of \emph{H. tridens} and \emph{M.
japonicus} was substitutable by Mg\textsuperscript{2+} (Fig. 2).
Furthermore, both these divalent cations could not overcome the
lethality caused by removal of K\textsuperscript{+} (Fig. 2), which is a
striking contrast to\emph{Oryzias latipes} , in which only the addition
of Ca\textsuperscript{2+} 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.

\textbf{4.3\textbar{} Role of ambient minor cations under hypo-osmotic
conditions}

This study revealed that ambient minor cations, especially
K\textsuperscript{+}, caused lethal damage to \emph{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\textsuperscript{+} was essential in isosmotic conditions, but harmful
in hypo-osmotic condition. Thus it appears that ambient
K\textsuperscript{+} has an opposite influence on the survival
of\emph{M. japonicus} in an osmotic condition--dependent manner. The
hemolymph K\textsuperscript{+} concentration in \emph{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 (Fig. 8F). Because the
hemolymph Na\textsuperscript{+} concentration is the same in both
solutions, the Na\textsuperscript{+}/K\textsuperscript{+} ratio in
hemolymph decreases in 4.3 mmol/L NaCl+1.2 MCK solution. It is possible
to assume that a decreased Na\textsuperscript{+}/K\textsuperscript{+}
ratio accounts for the lethality in \emph{M. japonicus.} NKA and NKCC
have been identified as molecules involved in K\textsuperscript{+}
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\textsuperscript{+} from hemolymph to
the cytoplasm in the opposite direction to Na\textsuperscript{+}. On the
other hand, NKCC is distributed in the apical membrane of chloride cells
and incorporates K\textsuperscript{+} from the environment into cells,
concomitant with Na\textsuperscript{+} and Cl\textsuperscript{--}.
Northern blotting showed that NKA \selectlanguage{greek}α \selectlanguage{english}subunit expression was attenuated in
all three species in the presence of ambient minor cations under
hypo-osmotic conditions (Fig. 9). This decreased NKA \selectlanguage{greek}α \selectlanguage{english}subunit
expression might decrease K\textsuperscript{+} transport from hemolymph
to chloride cells, possibly resulting in increased hemolymph
K\textsuperscript{+}concentration. However, it is unclear whether this
downregulation of NKA \selectlanguage{greek}α \selectlanguage{english}subunit gene accounts for the increased
hemolymph K\textsuperscript{+}concentration in \emph{M. japonicus} ,
since this gene was also attenuated in \emph{C. dehaani} in which
ambient minor cations did not affect the hemolymph K\textsuperscript{+}
concentration (Fig. 8G). In addition, NKCC gene expression also
decreases in the presence of ambient minor cations under hypo-osmotic
conditions in both \emph{H. tridens}and \emph{M. japonicus} , indicating
that NKCC does not contribute to increased hemolymph
K\textsuperscript{+} concentration, because decreased expression of this
gene should lead to decreased hemolymph K\textsuperscript{+}
concentration. It is possible that unidentified transporters and/or
channels involved in K\textsuperscript{+} transport are activated by
ambient minor cations to accelerate K\textsuperscript{+} uptake under
hypo-osmotic conditions.

Interestingly, in \emph{H. tridens} , ambient minor cations increased
the hemolymph K\textsuperscript{+} concentration in hypo-osmotic
condition (Fig. 8E) although they caused no mortality (Fig. 7A).
Therefore, it is possible that \emph{H. tridens} is more tolerant to
elevated K\textsuperscript{+} concentrations and low
Na\textsuperscript{+}/K\textsuperscript{+} ratio in hemolymph compared
to \emph{M. japonicus.} It is noteworthy that the average hemolymph
K\textsuperscript{+} concentration in \emph{M. japonicus} bathed in
513.3 mmol/L NaCl+1.2 MCK is 15.3 \selectlanguage{ngerman}± 1.28 mmol/L, which is significantly
higher than the nominal K\textsuperscript{+} concentration in bathing
media (13.4 mmol/L), although the average hemolymph
K\textsuperscript{+}concentration in \emph{H. tridens} is 11.5 ± 1.79
mmol/L, still less than the nominal K\textsuperscript{+} concentration
in bathing media. Thus ambient minor cations may affect the
K\textsuperscript{+} transport system differently in \emph{H. tridens}
and \emph{M. japonicus.} Another possibility is that some unidentified
damage occurs only in \emph{M. japonicus} but not in \emph{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 \emph{M. japonicus} but not in \emph{H.
tridens} (Table 3) suggest that transport of some ions other than
Na\textsuperscript{+} and K\textsuperscript{+} was affected only in
\emph{M. japonicus} . The mechanism by which ambient minor cations cause
mortality to \emph{M. japonicus} under hypo-osmotic conditions should be
investigated in the future.

\textbf{4.4\textbar{} Distinct response of \emph{C. dehaani} to ambient
minor cations}

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



\textbf{5\textbar{} CONCLUSION}

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 \emph{H. tridens}
and\emph{M. japonicus.} In contrast, in \emph{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\emph{.} 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.

\textbf{References}

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\textbf{TABLE 1.} The composition of bathing media used in this study.
Concentrations of dissolved salts are shown by mmol/L and measured
osmotic concentration are given by mOsm/kg. 513.3 mmol/L NaCl is
corresponds to 3\% NaCl. N. D. means not determined.

\textbf{Hosted file}

\verb`image1.emf`  available at \url{https://authorea.com/users/351272/articles/475958-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}

\textbf{TABLE 2.} Osmotic concentrations (mOsm/kg) of hemolymph bathed
in 8.6 mmol/L NaCl, 513.3 mmol/L NaCl+MCK, and 513.3 mmol/L NaCl
solutions. Values represent means (n=3) \selectlanguage{ngerman}± standard errors. Significant
differences were observed among different bathing media in \emph{Helice
tridens} and \emph{Macrophthalmus japonicus} , (ANOVA,
\emph{P}\textless{} 0.05) but not observed in \emph{Chiromantes dehaani}
(ANOVA,\emph{P} \textgreater{} 0.05). Different letters indicate
significantly different values within each species (\emph{P} \textless{}
0.05; Student's \emph{t} -test with Bonferroni correction).

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/351272/articles/475958-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}

\textbf{TABLE 3.} Osmotic concentrations (mOsm/kg) of hemolymph bathed
in 4.3 mmol/L NaCl and 4.3 mmol/L NaCl+1.2 MCK solutions. Values
represent means (n=3) ± standard errors. Different letters indicate
significantly different values within each species (\emph{P} \textless{}
0.05; Student's \emph{t} -test).

\textbf{Hosted file}

\verb`image3.emf`  available at \url{https://authorea.com/users/351272/articles/475958-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}

\textbf{FIGURE LEGENDS}

\textbf{FIGURE 1} Survival rates of (A) \emph{Helice tridens} ,
(B)\emph{Macrophthalmus japonicus} , and (C) \emph{Chiromantes dehaani}
at various NaCl concentrations and in natural seawater 48 h after
initiation of rearing. The survival rate was determined in 8.0, 16.0,
32.1, 64.2, 128.3, 256.7, and 513.3 mmol/L NaCl solutions and seawater.
Horizontal axes are scaled logarithmically. Values represent means of
three experiments, and error bars represent standard errors. Significant
differences were observed among different ambient NaCl concentrations in
all three species (ANOVA, \emph{P} \textless{} 0.05). *Significantly
different values compared to seawater (\emph{P} \textless{} 0.05;
Williams' test). Different letters indicate significantly different
values among three species within the same NaCl concentration
(\emph{P}\textless{} 0.05; Tukey's test). (SW), seawater.

\textbf{FIGURE 2} (A, B) Survival rates of (A) \emph{Helice tridens} and
(B) \emph{Macrophthalmus japonicus} in 513.3 mmol/L NaCl solution, 513.3
mmol/L NaCl+MMCK solution, and 513.3 mmol/L NaCl+MMCK solution minus any
one of the four additional salts. Values represent means of three
experiments, and error bars represent standard errors. Significant
differences were observed among different compositions of bathing media
in both \emph{H. tridens} and \emph{M. japonicus} (ANOVA,
\emph{P}\textless{} 0.05). Different letters indicate significantly
different values (\emph{P} \textless{} 0.05; Tukey's test). (C, D)
Survival rates of (C) \emph{H. tridens} and (D) \emph{M. japonicus} in
513.3 mmol/L NaCl, 513.3 mmol/L NaCl+K, and 513.3 mmol/L NaCl+K
solutions supplemented with MgCl\textsubscript{2} or
CaCl\textsubscript{2}. Values represent means of three experiments, and
error bars represent standard errors. Significant differences were
observed among different compositions of bathing media in both \emph{H.
tridens} and \emph{M. japonicus} (ANOVA, \emph{P} \textless{} 0.05).
Different letters indicate significantly different values (\emph{P}
\textless{} 0.05; Tukey's test). (MMCK), 27.4 mmol/L MgSO4, 25.2 mmol/L
MgCl\textsubscript{2}, 9.9 mmol/L CaCl\textsubscript{2}, and 10.7 mmol/L
KCl; (K), 10.7 mmol/L KCl.

\textbf{FIGURE 3} Hemolymph Na\textsuperscript{+} and
K\textsuperscript{+} concentrations in (A, D) \emph{Helice tridens} ,
(B, E) \emph{Macrophthalmus japonicus} , and (C, F) \emph{Chiromantes
dehaani} 6 h after initiation of incubation. Hemolymph (A--C)
Na\textsuperscript{+} and (D--F) K\textsuperscript{+} concentrations in
crabs held in 8.6 mmol/L NaCl, 513.3 mmol/L NaCl+MCK, and 513.3 mmol/L
NaCl solution. Values represent means (number of analyzed crabs shown
above each bar), and error bars represent standard errors. Significant
differences of both Na\textsuperscript{+} and
K\textsuperscript{+}concentrations were observed among different
compositions of bathing media in \emph{H. tridens} and \emph{M.
japonicus} (ANOVA, \emph{P}\textless{} 0.05), but not in \emph{C.
dehaani} (ANOVA, \emph{P}\textgreater{} 0.05). Different letters
indicate significantly different values (\emph{P} \textless{} 0.05;
Student's \emph{t} -test with Bonferroni correction). (MCK), 25.2 mmol/L
MgCl\textsubscript{2}, 9.9 mmol/L CaCl\textsubscript{2}, and 10.7 mmol/L
KCl.

\textbf{FIGURE 4} Amino acid sequences of (A) NKA \selectlanguage{greek}α \selectlanguage{english}subunit, (B) CAc,
(C) NHE, and (D) NKCC of \emph{Helice tridens} , \emph{Macrophthalmus
japonicus} , and \emph{Chiromantes dehaani} deduced from cDNA fragments
and alignments with those of the shore crab \emph{C. maenas.}

\textbf{FIGURE 5} Expression of genes encoding NKA \selectlanguage{greek}α \selectlanguage{english}subunit, CAc, and
NHE in the most posterior gills of \emph{Helice tridens}
,\emph{Macrophthalmus japonicus} , and \emph{Chiromantes dehaani} 6 h
after initiation of incubation in 513.3 mmol/L NaCl, 513.3 mmol/L
NaCl+MCK, and 8.6 mmol/L NaCl solutions. All gene expression was
detected using Northern blotting, except the expression of CAc
in\emph{M. japonicus} , which was detected using RNase protection assay.
The relative expression level was shown in each band as the expression
in 513.3 mmol/L NaCl+MCK solution is 1.0. (NKA),
Na\textsuperscript{+}/K\textsuperscript{+} ATPase; (CAc), cytoplasmic
carbonic anhydrase; (NHE),
Na\textsuperscript{+}/H\textsuperscript{+}exchanger; (MCK), 25.5 mmol/L
MgCl\textsubscript{2}, 9.9 mmol/L CaCl\textsubscript{2}, and 10.7 mmol/L
KCl.

\textbf{FIGURE 6} Survival rates of (A, C) \emph{Helice tridens} and (B,
D) \emph{Macrophthalmus japonicus} in 513.3 mmol/L NaCl solution with or
without acetazolamide, an inhibitor of CA (A and B), or amiloride
hydrochloride hydrate, an inhibitor of NHE (C and D), 24 h after
initiation of rearing. Values represent means of three (for \emph{H.
tridens} ) or four (for \emph{M. japonicus} ) experiments, and error
bars represent standard errors. Different letters indicate significantly
different values (\emph{P} \textless{} 0.05; Student's t-test). (CA),
carbonic anhydrase; (NHE),
Na\textsuperscript{+}/H\textsuperscript{+}exchanger.

\textbf{FIGURE 7} Survival rates of (A) \emph{Helice tridens} ,
(B)\emph{Macrophthalmus japonicus} , and (C) \emph{Chiromantes
dehaani}reared in 4.3 mmol/L NaCl, 4.3 mmol/L NaCl+1.2 MCK, and 17.1
mmol/L NaCl+1.2 MCK solutions. Values represent means of four (for
\emph{H. tridens} and \emph{M. japonicas} ) or three (for \emph{C.
dehaani} ) experiments, and error bars represent standard errors.
Significant differences were observed among different compositions of
bathing media in \emph{M. japonicus} (ANOVA, \emph{P} \textless{} 0.05),
but not in\emph{H. tridens} and \emph{C. dehaani} (ANOVA, \emph{P}
\textgreater{} 0.05). Different letters indicate significantly different
values (\emph{P} \textless{} 0.05; Student's t-test with Bonferroni
correction). (D) Survival rate of \emph{M. japonicus} in 4.3 mmol/L
NaCl, 4.3 mmol/L NaCl+1.2 MCK, and 4.3 mmol/L NaCl solutions
supplemented with 30.5 mmol/L MgCl\textsubscript{2} and 11.7 mmol/L
CaCl\textsubscript{2} (1.2 MC) or 13.4 mmol/L KCl (1.2 K). Values
represent means of three experiments, and error bars represent standard
errors. Significant differences were observed among different
compositions of bathing media (ANOVA, \emph{P} \textless{} 0.05).
Different letters indicate significantly different values
(\emph{P}\textless{} 0.05; Tukey's test). (E) Survival rate of \emph{M.
japonicus} in 4.3 mmol/L NaCl solution supplemented with 0, 2.7, 5.4,
and 10.7 mmol/L KCl 24 h after initiation of rearing. Values represent
means of four experiments, and error bars represent standard errors.
*Significantly different values from that in 4.3 mmol/L NaCl solution
without KCl. (\emph{P} \textless{} 0.05; Williams' test). (F) Survival
rate of \emph{M. japonicus} reared in 4.3 mmol/L NaCl (circles), 4.3
mmol/L NaCl+1.2 MCK (triangles), and 4.3 mmol/L NaCl+1.2 K (squares)
solutions 15, 24, 39, and 48 h after initiation of experiments. Values
represent means of three experiments, and error bars represent standard
errors. Different letters indicate significantly different values in
each time point (\emph{P} \textless{} 0.05; Tukey's test).
*Significantly different values from the beginning point
(\emph{P}\textless{} 0.05; Williams' test). (1.2 MCK), 30.5 mmol/L
MgCl\textsubscript{2}, 11.7 mmol/L CaCl\textsubscript{2}, and 13.4
mmol/L KCl; (1.2 MC), 30.5 mmol/L MgCl\textsubscript{2} and 11.7 mmol/L
CaCl\textsubscript{2}; (1.2 K), 13.4 mmol/L KCl.

\textbf{FIGURE 8} Hemolymph (A--D) Na\textsuperscript{+} and (E--H)
K\textsuperscript{+} concentrations in (A, E) \emph{Helice tridens} ,
(B, D, F, H) \emph{Macrophthalmus japonicus} , and (C, G)
\emph{Chiromantes dehaani} incubated in (A--C, E--G) 4.3 mmol/L NaCl and
4.3 mmol/L NaCl+1.2 MCK solutions or in (D, H) 4.3 mmol/L NaCl, 4.3
mmol/L NaCl+K, and 4.3 mmol/L NaCl+MCK solutions 6 h after initiation of
incubation. Values represent means (number of analyzed crabs shown above
each bar), and error bars represent standard errors. Significant
differences were observed among different compositions of bathing media
in (D, H) (ANOVA,\emph{P} \textless{} 0.05). Different letters indicate
significantly different values (\emph{P} \textless{} 0.05; Student's
\emph{t} -test for A--C and E--G, and Student's \emph{t} -test with
Bonferroni correction for D and H). (1.2 MCK), 30.5 mmol/L
MgCl\textsubscript{2}, 11.7 mmol/L CaCl\textsubscript{2}, and 13.4
mmol/L KCl; (MCK), 25.2 mmol/L MgCl\textsubscript{2}, 9.9 mmol/L
CaCl\textsubscript{2}, and 10.7 mmol/L KCl; (K), 10.7 mmol/L KCl.

\textbf{FIGURE 9} Expression of genes encoding NKA \selectlanguage{greek}α \selectlanguage{english}subunit and NKCC in
the most posterior gills of \emph{Helice tridens} , \emph{Macrophthalmus
japonicus} , and \emph{Chiromantes dehaani} 6 h 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\textsuperscript{+}/K\textsuperscript{+}ATPase; (NKCC),
Na\textsuperscript{+}/K\textsuperscript{+}/2Cl\textsuperscript{--}cotransporter;
(1.2 MCK), 30.5 mmol/L MgCl\textsubscript{2}, 11.7 mmol/L
CaCl\textsubscript{2}, and 13.4 mmol/L KCl.

\textbf{DATA ACCESSIBILITY STATEMENT}

The sequences of isolated cDNAs in this study were registered in GenBank
(accession numbers: LC214855, NKA of \emph{H. tridens} ; LC375964, CAc
of\emph{H. tridens} ; LC572286, NHE of \emph{H. tridens} ; LC572287,
NKCC of\emph{H. tridens} ; LC214856, NKA of \emph{M. japonicus} ;
LC375965, CAc of \emph{M. japonicus} ; LC572288, NHE of \emph{M.
japonicus} ; LC572289, NKCC of \emph{M. japonicus} ; LC572290, NKA of
\emph{C. dehaani} ; LC572291, CAc of \emph{C. dehaani} ; LC572292, NHE
of \emph{C. dehaani} ; LC572293, NKCC of \emph{C. dehaani} ). Raw data
are publicly available on figshare (DOI: 10.6084/m9.figshare.12788132).

\textbf{CONFLICT OF INTERESTS}

The authors declare that there are no conflicts of interests.

\textbf{AUTHOUR CONTRIBUTIONS}

\textbf{Masahiro Yamaguchi:} Conceptualization (lead), Formal Analysis
(lead), Funding Acquisition (lead), Investigation (lead),
Writing-Original Draft Preparation (lead), Writing-Review \& Editing
(lead). \textbf{Kouichi Soga:} Investigation (supporting),
Writing-Review \& Editing (supporting).

\textbf{ACKNOWLEDEMENTS}

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.\selectlanguage{english}
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