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First published online June 15, 2007
Journal of Experimental Biology 210, 2320-2332 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.005041
Salinity-stimulated changes in expression and activity of two carbonic anhydrase isoforms in the blue crab Callinectes sapidus
Department of Biological Sciences, 101 Life Science Building, Auburn University, Auburn, AL 36849, USA
* Author for correspondence (e-mail: henryrp{at}auburn.edu)
Accepted 16 April 2007
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-subunit of the
Na+/K+-ATPase is similar to that for CasCAc, and both
precede the establishment of the new acclimated physiological state of the
crab in low salinity. A putative `housekeeping' gene, arginine kinase, also
showed about a threefold increase in expression in response to low salinity,
but only in the posterior gills. These results suggest that for studies of
expression in crustacean gill tissue, a control tissue, such as the anterior
gill, be used until an adequate control gene is identified.
Key words: carbonic anhydrase, ion regulation, crustacean, Callinectes sapidus
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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-CA, ß-CA and 
-CA), have been identified and studied in a
wide variety of animals, plants, and bacteria (reviewed in
Hewett-Emmett, 2000
). Their
physiological roles vary according to the tissue and cell type in which they
are found, their subcellular localization (e.g. cytosolic, membrane-bound,
mitochondrial or secreted) and to the specific physiological or biochemical
process to which they are linked (for reviews, see
Dodgson et al., 1991;
Henry, 1996;
Geers and Gros, 2000). Despite
being ubiquitously distributed throughout the animal kingdom, most of the
existing work on molecular structure and phylogeny has been focused on
mammalian CAs: to date, 16 different isoforms have been characterized, all of
them being members of the -CA gene family (reviewed in
Esbaugh and Tufts, 2006). More
recent work has shed light on the molecular structure and function of
-CA isoforms of non-mammalian vertebrates, such as fish
(Peterson et al., 1997;
Lund et al., 2002;
Tufts et al., 2003;
Esbaugh et al., 2004;
Esbaugh et al., 2005;
Boutet et al., 2006;
Tohse et al., 2006) and, to a
lesser extent, of invertebrates including some mollusks, worms and insects
(reviewed in Hewett-Emmett,
2000). Nevertheless, little information on the molecular structure
of these isoforms is available for crustaceans, a group in which CA function
has been extensively studied (reviewed by
Henry, 2001;
Henry et al., 2003).
Euryhaline crustaceans can survive wide variations in environmental
salinity by maintaining relatively high and constant hemolymph osmolality and
ion concentrations, compared to those in the external medium. This process
involves the active uptake of salts (primarily Na+ and
Cl–) across the posterior gills, which are highly specialized
for active ion transport (Mantel and
Farmer, 1983; Gilles and
Péqueux, 1985;
Péqueux and Gilles,
1988; Péqueux,
1995; Taylor and Taylor,
1992). These gills also contain high levels of activity and/or
expression of known ion transport proteins and transport-related enzymes such
as the NaK2Cl co-transporter, the Na/H exchange protein (NHE), the
Na+/K+-ATPase, and carbonic anhydrase (CA)
(Towle and Weihrauch, 2001;
Henry et al., 2003;
Luquet et al., 2005).
Furthermore, the activity and/or expression of these proteins are
salinity-sensitive, increasing when crabs are exposed to low salinity water
(Towle et al., 2001;
Genovese et al., 2005;
Luquet et al., 2005;
Chung and Lin, 2006;
Henry et al., 2006;
Li et al., 2006). Anterior
gills are typically unspecialized, having thin epithelia that function
primarily in diffusive gas exchange.
Some of the largest reported changes in activity (8–14-fold) in
response to low salinity have been for branchial CA in two euryhaline species
of crabs, the green crab Carcinus maenas, and the blue crab
Callinectes sapidus (Henry,
2005). Isolation of subcellular fractions of branchial CA activity
has shown the cytoplasmic pool of CA to be the most highly sensitive to low
salinity. However, there are multiple CA isoforms in the gill, and
membrane-associated CA is also induced by low salinity, although to a much
lesser degree (2–3-fold) (Henry,
1988a; Henry et al.,
2003). Recent evidence from C. maenas for one CA isoform
strongly suggests that CA induction is under transcriptional regulation.
Relative abundance of CA mRNA increased at 24 h after transfer to low
salinity, and CA activity increased shortly thereafter
(Henry et al., 2006).
The blue crab, C. sapidus, is one of the strongest osmotic and
ionic regulators, having the highest rates of ion uptake and maintaining the
largest ionic gradients between its hemolymph and the surrounding medium
(Cameron, 1978). An essentially
marine species, it may extend into brackish and full freshwater in nature and
can cope with large step changes in salinity
(Péqueux, 1995;
Henry, 2001). CA activity is
induced more quickly in C. sapidus than in C. maenas; the
initial measurable increase occurs at 24 h after low salinity exposure
(Henry and Watts, 2001), and
the magnitude of CA induction is also greater than in other species. Because
of these characteristics, the blue crab is potentially a more powerful model
system in which to study CA induction and its regulation. Furthermore, a large
expressed sequence tag (EST) cDNA library from the gills of C.
sapidus has been recently produced
(Coblentz et al., 2006) and is
now easily accessible (a collection of almost 12 000 ESTs, representing more
than 2000 putative transcripts, are available), facilitating the
identification of the multiple CA isoforms within the blue crab gill. CA is
also an ideal transport-related protein for this type of study, as it is a
very tractable enzyme to work with: activity, protein concentration and mRNA
expression can all be reliably measured, typically in the same animal.
The aim of the present study was to expand upon the mechanism and regulation of low salinity-stimulated CA induction by identifying the multiple CA isoforms in the crustacean gill and elucidating their potential role in the process of low salinity adaptation in euryhaline decapod crustaceans, using the strong hyperosmoregulating crab C. sapidus as model organism. Two isoforms were fully sequenced: one putative cytoplasmic form and one putative membrane-bound form. The relative levels of abundance of these two isoforms were then monitored in crabs transferred from high to low salinity for up to 28 days, a period that encompasses both the acute and acclimated stages of salinity adaptation. This report represents a coordinated study of changes in hemolymph osmolality, and CA branchial activity, mRNA expression, and protein abundance. Because other authors have employed arginine kinase as a highly expressed control transcript, we also examined its expression.
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Experimental protocol
Crabs that were collected from salinities above 28 p.p.t. were kept in the
laboratory at 35 p.p.t. for 1 week prior experimentation. Crabs collected at
lower salinities, were held at 35 p.p.t. for 3 weeks in order to ensure
complete acclimation with respect to hemolymph osmolality and levels of
transport-related enzymes (Henry and
Cameron, 1982; Henry and
Wheatly, 1988). Before beginning any experimental protocol,
branchial CA activity was measured in a subset of four crabs to confirm that
CA activity was at low and uniform baseline levels typically found in
high-salinity acclimated animals. To follow the time course of CA induction,
crabs were directly transferred to 120 L aquaria of 15 p.p.t. water equipped
with undergravel biological filters. Crabs were sampled before transfer
(t=0), and then at 2, 6, 12, 24, 48 h and 4, 7, 14 and 28 days after
transfer. As several physiological parameters, including branchial CA activity
(Henry and Kormanik, 1985),
change over the molting cycle, only specimens in intermolt stage C,
established by observation of the edge of the fifth pereiopod, were retained
for analysis at the end of each experimental time period
(Drach and Tchernigovtzeff,
1967). Hemolymph was sampled from the infrabranchial sinus at the
base of the fifth pereiopod using a hypodermic 22 gauge needle mounted on a 1
ml syringe and frozen at –20°C until osmolality determination. Prior
to dissection, crabs were chilled on crushed ice for 10 min. Anterior (G3) and
posterior (G7) gills from the left and right sides of the crab were dissected
out and used for measurement of CA activity and mRNA expression,
respectively.
A second group of animals were acclimated to 35 p.p.t. and 15 p.p.t. for 7
and 14 days in order to determine the total concentration of free enzyme in
gills (E0). Anterior (gills 1–4) and posterior
(gills 6–8) gills were dissected out, pooled, and frozen in liquid
nitrogen and stored at –80°C. Gill number 5 in blue crabs is a
transitional gill, containing both respiratory and ion-transporting lamellae
(Henry and Cameron, 1982), and
so it was excluded from this study.
Osmolality
Hemolymph samples were thawed on ice, sonicated for 15 s at 25 W (Heat
Systems Microson, Farmingdale, NY, USA) and centrifuged 14 000
g for 1 min to separate out clot material. Osmolality was then
measured on 10 µl samples using a Wescor 5100C vapor pressure
osmometer.
Carbonic anhydrase activity
Branchial carbonic anhydrase activity was measured electrometrically by the
delta pH method previously described
(Henry, 1991). Freshly
dissected gills were homogenized in 2 ml of cold homogenization/assay buffer
(225 mmol l-1 mannitol, 75 mmol l-1 sucrose, 10 mmol
l-1 Trizma base, adjusted to pH 7.4 with 10% phosphoric acid) using
an Omni TH115 hand-held homogenizer (Omni Instruments, Warrenton, VA, USA) and
sonicated on ice at 25 W for 30 s (Heat Systems Microson). Homogenates were
centrifuged at 10 000 g for 20 min at 4°C (Sorvall RC5-B,
Wilmington, DE, USA). For the assay, 25–75 µl of supernatant were
added to 6 ml of homogenization/assay buffer, refrigerated at 4°C and
stirred vigorously. The enzymatic reaction was initiated by the addition of
100 µl CO2-saturated water. The drop in pH (approximately 0.25
units) was monitored using micro pH and reference electrodes (World Precision
Instruments, Sarasota, FL, USA) and a null-point pH meter. Protein
concentration in the supernatant was measured by Coomassie Brilliant Blue dye
binding (Bio Rad laboratories, Hercules, CA, USA). CA activity was expressed
in µmol CO2 mg protein-1 min-1.
Titration of free enzyme concentration
Changes in CA protein concentration were measured using a method previously
described (Henry et al.,
2006). From each pool of gills stored at –80°C (anterior
gills 1–4 and posterior gills 6–8), 4 g of tissue were placed in
20 ml of cold homogenization/assay buffer (see above) and homogenized by 15
passes of a motor-driven, Teflon-glass homogenizer. The homogenate was then
subjected to differential centrifugation. First, the crude homogenate was
centrifuged at 1750 g (Sorvall RC5-B, Wilmington, DE, USA) for
20 min at 4°C, producing a pellet of intact cells, nuclei and large cell
fragments, and a supernatant containing cytoplasm, mitochondria and
microsomes. The resulting supernatant was then centrifuged at 7500
g for 20 min at 4°C (Sorvall RC5-B) to eliminate the
mitochondria. Then, to separate the microsomal fraction from the cytoplasm,
the supernatant was centrifuged at 100 000 g for 90 min at
4°C (Beckman L8-70M ultracentrifuge). The cytoplasmic fraction
(supernatant) and the microsomal fraction (pellet, washed twice and
resuspended in 2 ml cold homogenization/assay buffer) were assayed for CA
activity while being titrated with increasing volumes of a 5 µmol
l-1 acetazolamide solution (Az; a CA inhibitor). The data were
transformed and graphed as a double reciprocal plot, according to the
following relationship:
I0/I=Ki/(1–I)+E0,
where E0 is the total concentration of free enzyme,
Ki is the inhibition constant, and I is the
fractional inhibition of enzyme activity at an inhibitor concentration of
I0 (Easson and
Stedman, 1937). CA concentrations from the inhibitor plots were
adjusted for differences in sample volumes used in the assay.
Total RNA purification
Total RNA was extracted from gills by phenol–chloroform extraction,
using RNAgents® Total RNA Isolation System (Promega, Madison, WI, USA).
All dissecting equipment and homogenizers were cleaned with RNAse-Zap (Ambion,
Austin, TX, USA) and rinsed in RNAse-free water in order to work under
RNAse-free conditions. Total RNA concentration, as well as the integrity and
purity of each sample were determined with a Bioanalyser 2100 (Agilent
Technologies, Wilmington, DE, USA). No genomic DNA contamination was
observed.
Polymerase chain reaction and specific primers
All polymerase chain reactions (PCR) mentioned in this study were carried
out in a MJ Research PTC 200 thermocycler (Global Medical Instrumentation,
Inc., Ramsey, MN, USA) using REDTaqTM ReadyMixTM PCR Reaction Mix
kit (Sigma-Aldrich, St Louis, MO, USA). Template DNA (1 µl), 0.5 µl of
each primer at 25 µmol l-1 and 25 µl REDTaq Ready Mix PCR
reaction mix (20 mmol l-1 Tris-HCl pH 8.3, 100 mmol l-1
KCl, 3 mmol l-1 MgCl2, 0.002% gelatine, 0.4 mmol
l-1 dNTP mix, stabilizers, 0.06 units/µl Taq DNA polymerase)
were used in a final volume of 50 µl. After an initial denaturing step at
92°C for 5 min, 30 cycles of 1 min at 92°C, 1 min at 55°C and 2
min at 72°C were performed, followed by a final extension step at 72°C
for 5 min. PCR products were tested for purity and molecular size by agarose
gel electrophoresis (1.2% agarose in 1xTAE buffer). DNA bands were
stained with Ethidium Bromide and visualized over an ultraviolet light
source.
All specific primers were synthesized by Integrated DNA Technologies Inc. (Coralville, IA, USA) and tested for specificity by PCR on gill cDNA and confirmed by sequencing of the resultant PCR products.
Determination of carbonic anhydrase cDNA sequences
3'end
Expressed Sequence Tags (ESTs) from blue crab gill and hypodermis
normalized cDNA libraries were produced by Coblentz et al.
(Coblentz et al., 2006). Clones
matching contig sequences identified as carbonic anhydrase in that project and
archived at the University of North Carolina at Wilmington were used to obtain
full nucleotide sequences of the gill CA isoforms. Five cloned inserts were
obtained and sequenced from both ends using SP6 and T7 primers. Alignment of
all the sequences obtained resulted in two different contigs (ORF followed by
3'UTR) both identified as CA sequences by BLAST analysis.
5'end
The 5'ends of the CA isoforms were determined using the GeneRacer
protocol (GeneRacerTM kit, Invitrogen, Carlsbad, CA, USA) designed for
full-length RNA ligase-mediated amplification of 5'end (RLM-RACE).
Briefly, truncated mRNA and non-mRNA were eliminated from total RNA by
dephosphorylating treatment with calf intestinal phosphatase. The 5'cap
structure from intact full-length mRNA was removed and a GeneRacer RNA-oligo
was ligated to the 5'end. Ligated mRNAs were reverse transcribed using
GeneRacer oligo-dT primer and Superscript III reverse transcriptase
(Invitrogen). The 5'end was then amplified by PCR using GeneRacer
5' Primer and reverse gene specific primers CasCAg-Race and CasCAc-Race
designed according to 3'end CA cDNA sequences described above
(Table 1).
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PCR products were purified from 1% agarose gel (MinElute Gel Extraction kit, Valencia, CA, USA), subcloned into a pCR®4-TOPO and transformed into chemically competent TOP10 Escherichia coli cells (TOPO TA Cloning® kit, Invitrogen). After selection on LB-ampicilin agar, transformed cells were screened for appropriate size inserts by PCR using forward GeneRacer 5' Primer and reverse gene specific primers CasCAg-Race and CasCAc-Race. Recombinant plasmids were purified by QIAprep Spin Miniprep (Qiagen) and individual clones were sequenced using T7 and M13 reverse primers.
Sequencing and sequences analysis
A CEQTM 8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA,
USA) using CEQ dye terminator chemistry was used for bidirectional sequencing.
The resulting automated traces were edited with Chromas software and
identified by comparison with published sequences in GenBank using the BLAST
algorithm (Altschul et al.,
1997)
(http://www.ncbi.nlm.nih.gov/BLAST/).
Multiple alignments were produced with Multalin software
(Corpet, 1988)
(http://prodes.toulouse.inra.fr/multalin/)
and GeneDoc software
(http://www.psc.edu/biomed/gendoc/).
Quantification of mRNA expression by real-time quantitative PCR
Poly-A mRNA in 2 µg of total RNA per sample was reverse transcribed
using an oligo-dT primer and Superscript II® reverse transcriptase
(Invitrogen, Madison, WI, USA). As such, the samples were normalized to total
RNA levels in each preparation (Bustin,
2002). The resulting cDNAs were checked by PCR and stored at
–20°C. The mRNA levels were assessed by real-time quantitative
polymerase chain reaction (qPCR) with a MiniOpticon Real-Time PCR detection
system using iQTM SYBR® Green Supermix kit (Bio-Rad laboratories,
Hercules, CA, USA). Reactions (25 µl) were run containing 12.5 µl iQ
SYBR Green mix (2x), 10.9 µl nuclease-free water, 0.3 µl of each
specific primer (25 µmol l-1 and 1 µl template cDNA. The
thermal profile consisted of an initial step at 95°C for 3 min and 35
cycles of denaturing at 95°C for 10 s and annealing–elongation at
55°C for 60 s. Each sample was analyzed in 1 µl duplicate or triplicate
aliquots. A standard curve, representing the threshold cycle
(Ct, cycle at which the fluorescent signal is detected)
data plotted as a function of template availability (Ct
vs log10 cDNA volume), was generated by serial dilution of
one sample containing high CA activity. Furthermore, absence of non-specific
PCR products and primer dimers was confirmed by examination of dissociation
curves generated after the amplification cycles were completed (from 55°C
to 95°C with a heating rate of 0.5°C every 5 s).
Specific primer pairs CasCAgF/CasCAgR and CasCAcF/CasCAcR were designed based on CasCAg and CasCAc nucleotide sequences obtained in this study to discriminate between both isoforms expression patterns (Table 1).
Na+/K+-ATPase -subunit mRNA expression was
studied over low-salinity adaptation using previously described primers
NAKSP12F and NAKSP16R specific to ion transport -subunit sequence
(Table 1)
(Li et al., 2006).
Arginine kinase (AK) mRNA expression was also monitored, as a putative
internal control, using specific forward AKF5 and reverse AKCALLR1 primers
(Table 1) previously described
(Towle et al., 2001).
X-statistics
Statistics were performed using Sigma Stat version 3.1 and figures were
plotted using Microsoft® Office Excel 2003.
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-CA family when
compared to the GenBank database using the BLAST program
(Fig. 1). They were named
CasCAc (denoting a presumptive cytoplasmic localization) and CasCAg (denoting
the presence of a membrane glycosyl-phosphatidylinositol (GPI) link). The
complete sequence of the CasCAc (GenBank EF375490) contained a 50 bp 5'
untranslated region, an 816 bp open reading frame (including the stop codon),
and a 227 bp 3' untranslated region [excluding the poly(A)-tail]. The
complete sequence of the CasCAg (GenBank EF375491) contained a 108 bp 5'
untranslated region, a 927 bp open reading frame (including the stop codon),
and a 518 bp 3' untranslated region [also excluding the poly(A)-tail].
According to Kozak rules, sequences surrounding the first methionine in CasCAc
and CasCAg displayed, respectively, 3 and 8 out of the 9 nucleotides expected
before a translation initiation codon. Both sequences included the highly
conserved puridine in position –3, and both sequences maintained the G
following the ATG (Kozak,
1987). The CasCAc nucleotide sequence contained two putative
N-glycosylation motifs (translating as Asn-anything but
Pro-Thr/Ser-anything but Pro) in the coding region and a possible
polyadenylylation cleavage signal (AATAAA) starting 55 bp upstream from the
poly(A)-tail. Only one putative N-glycosylation motif was identified
in the coding region of CasCAg.
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Two variations in the nucleotide sequence were noted in the CasCAc isoform, and ten variations were present in the CasCAg. Except for three variations in CasCAg (one in the 5'UTR and two in the 3'UTR), all variations were found in open reading frames and were in the third codon position. As none of them led to amino acid substitution, and as most of them were found in two or more sequenced products, they are unlikely to be taq errors. It is possible that variations in cDNA sequence could have existed in the different individual organisms from which the tissues for sequencing were taken.
The deduced amino acid sequences for CasCAc and CasCAg were different in
length (271 and 308 amino acid residues, respectively) and shared only 30% of
identity (Fig. 2). Both
sequences were submitted to DGPI analysis
(Kronegg and Buloz, 1999):
CasCAg was diagnostic as a GPI-anchored protein with an N-terminal signal
(Met1 to Ala18), a potential cleavage site at 284
(Asp284-Ser285-Ser286) and the presence of
hydrophobic and hydrophilic tails. The amino acid sequences of CasCAc and
CasCAg were aligned and compared with previously characterized -CA
sequences from invertebrates and vertebrates
(Fig. 2). The -CA
peptides are highly variable both in length and in sequence, even though some
features essential to the catalytic mechanism appear to be common to nearly
all family members. Among the presented enzymes, 15 out of the 36 active site
residues are identical. The three zinc-binding histidine residues annotated
His-94, His-96, His-119 in HosCAII, and the proton-binding network annotated
Glu-106 and Thr-199 in HosCAII are conserved. All but CasCAc, AngCA, HosCAIII
and HosCAV have the highly conserved histidine residue implicated as a proton
shuttle group defined as His-64 in human CAII.
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). After transfer to low salinity (15 p.p.t./450 mOsm kg
H2O-1), the hemolymph osmolality sharply decreased
during the initial 12 h post-transfer (from 1110 to 820 mOsm kg
H2O-1) and by 24 h stabilized at around 780 mOsm kg
H2O-1, corresponding to the new acclimated level. After
28 days, the hemolymph osmolality value was 285 mOsm kg
H2O-1 higher than the 450 mOsm kg
H2O-1 ambient medium.
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Expression of CasCAg mRNA was not significantly different (P=0.49, t-test) between G3 (0.12±0.05, N=6) and G7 (0.16±0.03, N=8) in crabs acclimated to 35 p.p.t. (Fig. 5A). There were no significant changes in G3 mRNA expression at any times after low salinity transfer (P>0.995, ANOVA, Tukey). Changes in expression of CasCAg mRNA in G7 were pulsatile. There was an initial increase in CasCAg mRNA relative expression of approximately 3.5-fold and 5.5-fold at 2 h and 6 h post-transfer, respectively (t=6 h: P<0.001, ANOVA, Tukey). Expression then started to decrease at 12 h to reach a value at 24 h that was not significantly different from the 35 p.p.t. controls. There was a second increase in CasCAg mRNA expression starting at 48 h followed by two time periods (4 days and 28 days after transfer) at which expression decreased. By the end of the 28 days acclimation, the level of CasCAg mRNA (0.46±0.17) was threefold higher, but not significantly different compared to the level in crabs acclimated to high salinity (P=0.85, ANOVA, Tukey).
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CasCAg was the more highly expressed isoform in either G3 or G7 at any of the sampling times during the experiment (Fig. 6). In crabs acclimated to 35 p.p.t. salinity, CasCAg was expressed at levels 9000 and 500 times greater than CasCAc in G3 and G7, respectively. After transfer to 15 p.p.t., the ratio of CasCAg to CasCAc transcript fell to between 2450 and 147 in G3 and between 2 and 17 in G7, indicating that CasCAc was the isoform with the highest degree of salinity sensitivity.
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; Henry et al.,
2006). In crabs acclimated to 35 p.p.t., the protein
concentrations (E0) of both CA isoforms were in the same
value range when comparing AG with PG, and CAg was the most abundant isoform
in both gills. After transfer to 15 p.p.t., E0 of both CA
isoforms almost doubled in anterior gills, but with the small sample size it
is not possible to say whether this increase is real. In posterior gills,
however, a large (45-fold) increase in CAc E0 resulted
after 7 days of low salinity acclimation compared to the lesser 1.5-fold in
CAg E0. After 2 weeks, the CAc concentration was even more
enhanced, reaching a 65-fold induction.
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In crabs acclimated to 35 p.p.t., Na+/K+-ATPase
-subunit (NaK) and arginine kinase (AK) mRNA levels were fourfold
significantly higher (P<0.05, t-test) in G7
(0.15±0.04, N=7 and 0.12±0.04, N=5,
respectively) than in G3 (0.04±0.01, N=5 and 0.03±0.01,
N=5, respectively) and they remained relatively stable in G3,
exhibiting no significant variation (P>0.2, ANOVA, Fisher LSD)
(Fig. 7 and
Fig. 8). Six hours after the
crabs were transferred to 15 p.p.t. Na+/K+-ATPase mRNA
expression in G7 had significantly increased by fourfold (P<0.001,
ANOVA, Fisher LSD). Then, the NaK level remained significantly higher
(fluctuating between 3 and 4.8-times) in G7 of crabs acclimated to low
salinity than in G7 of crabs acclimated to 35 p.p.t.. This pattern of
expression was similar to that seen for CasCAc in response to low salinity.
After transfer to 15 p.p.t., AK expression in G7 exhibited a progressive
increase from 6 h (0.23±0.04, N=5, P<0.05, ANOVA,
Fisher LSD) to 7 days (0.40±0.05, N=5, P<0.001,
ANOVA, Fisher LSD) then it decreased at 28 days.
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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).
This study also presents data on the different responses of the two
isoforms to changes in environmental salinity that link their regulation and
expression to their specific physiological roles in the gill. Relative to the
other major gill CA isoform, CasCAg, the CasCAc isoform is expressed in
extremely low levels in crabs acclimated to 35 p.p.t., being nearly
undetectable in both anterior and posterior gills. Cytoplasmic CA is believed
to be the isoform that is directly involved in osmotic and ionic regulation
(Henry, 1988a;
Henry, 1988b), and so it is
not surprising to find very low levels of expression in the gills when the
crab is acclimated to a salinity at which it is an osmotic and ionic
conformer, and at which the major ion transport mechanisms are silent.
The degree of salinity-sensitivity of the cytoplasmic isoform, however, was
very large. The relative abundance of CasCAc mRNA in the posterior,
ion-transporting gills, increased by 100-fold in the initial 12 h after
transfer from 35 to 15 p.p.t. This is the largest magnitude of induction
reported for any transport-related protein in the gills of euryhaline
crustaceans, and this could be a conservative estimate. A recent study on
another euryhaline species of crab, Chasmagnathus granulatus,
reported that the relative abundance of the
Na+/K+/2Cl– co-transporter mRNA
increased a maximum of 20-fold, and that of the
Na+/K+-ATPase -subunit increased a maximum of
55-fold in crabs given a salinity transfer from 30 to 2 p.p.t.
(Luquet et al., 2005). That
was a much larger magnitude salinity transfer than was used on blue crabs in
this study (35–15 p.p.t.); therefore, for comparable salinity transfers,
the degree of cytoplasmic CA mRNA induction could be much greater. The
relative abundance of the Na+/K+-ATPase -subunit
in the posterior gills of blue crabs increased by only fivefold by comparison
(Fig. 7), but in this species
the relative levels of expression in crabs acclimated to 35 p.p.t. were
already high. High levels of expression of the
Na+/K+-ATPase most likely represent the molecular basis
for correspondingly high levels of the Na+/K+-ATPase
activity in gills in crabs at high salinity
(Henry et al., 2002). Another
recent study reported a 2.5-fold increase in
Na+/K+-ATPase expression in posterior gills of blue
crabs but only after 5 days of low salinity (10 p.p.t.) exposure
(Lovett et al., 2006a). The
differences in baseline levels of expression and activity between CA and the
Na+/K+-ATPase at high salinity, and the different
degrees of salinity sensitivity, further support the idea that the two
proteins are regulated differently in response to low salinity exposure.
Salinity-mediated CA induction appears to be under transcriptional
regulation. The timing of the induction of CA activity was most closely
correlated with that of CasCAc mRNA. Increases in CasCAc mRNA and CA activity
occurred as stair-step patterns that were slightly out of phase. The initial
increase in CasCAc mRNA occurred first, at 6 h post-transfer and peaked at 48
h, while the initial measurable increase in CA activity occurred at 24 h
post-transfer and peaked at 7 days. The pattern of induction of CA activity
was similar to that already reported
(Henry and Watts, 2001). After
reaching peak values, both mRNA and activity remained high for the remainder
of the experimental time course. CasCAc mRNA levels were elevated first in
response to low salinity exposure, and synthesis of new CA protein followed
thereafter, giving rise to higher levels of CA activity. In support of this
relationship, the concentration of cytoplasmic CA protein in posterior gills
was approximately 60-fold higher in crabs after 7 days exposure to low
salinity. This is a similar pattern to that seen in another euryhaline species
of crab, Carcinus maenas (Henry
et al., 2003; Henry et al.,
2006), suggesting that transcriptional regulation of CA induction
may be a common mechanism in euryhaline crustaceans. In contrast, there does
not appear to be any mechanism of CA induction in stenohaline, strictly
osmoconforming crabs, such as Cancer irroratus
(Henry and Campoverde,
2006).
The timing of induction of both CA and the Na+/K+-ATPase corresponds to the timing of the establishment of the new, acclimated physiological state of the crab after transfer to low salinity. These changes occur rapidly. In the transition from osmoconformity to osmoregulation, hemolymph osmotic concentration in blue crabs becomes stabilized by 24 h post-transfer. The transition to osmotic and ionic regulation is preceded by the up-regulation of two of the central transport-related proteins required for active ion transport, which is the mechanistic basis for hemolymph osmotic and ionic regulation.
There also appears to be a difference in the baseline levels and the
magnitude of up-regulation of cytoplasmic vs membrane-associated
proteins. The membrane-associated proteins appear to be expressed at higher
levels in high salinity, but the cytoplasmic proteins appear to have a greater
degree of `inductive scope' (magnitude of difference between baseline and
maximal levels of expression). For CA, the levels of mRNA for the
membrane-associated isoform, CasCAg, are as much as 2–4 orders of
magnitude higher than those for CasCAc in crabs at 35 p.p.t. The differences
can be linked to isoform-specific physiological function. The
membrane-associated isoform is believed to function in the mobilization of
hemolymph HCO3– to molecular CO2 in
order to facilitate CO2 excretion across the gill
(Henry, 1987). The blue crab
is highly active, regardless of salinity, and it is possible that the
relatively high abundance of CasCAg mRNA is needed to maintain the levels of
the membrane-associated CA protein necessary to provide the degree of
catalytic activity required for branchial CO2 excretion. The degree
of salinity-mediated induction in CasCAg, however, is small by comparison
(about 5- vs 100-fold for CasCAc), and this also corresponds to a
smaller degree of CA activity induction in the membrane vs
cytoplasmic fractions of the gill (about 2- vs 14-fold)
(Henry, 1988a). Induction of
the membrane-associated isoform is most likely a result of the proliferation
of the population of ion-transporting `chloride cells' that takes place in the
posterior gills in response to low salinity exposure. This cell type is
characterized by a highly infolded basal membrane
(Compere et al., 1989), where
CasCAg is localized, and the area that this cell type occupies in the lamellae
of the posterior gills of the blue crab expands significantly after transfer
to low salinity (Neufeld et al.,
1980; Lovett et al.,
2006b). So, essentially, there is more membrane-associated CA
because there is simply more basolateral membrane in the gill lamellae at low
salinity.
Interestingly, the timing and degree of the induction of the single CA
isoform reported previously for C. maenas
(Henry et al., 2006) is most
similar to that of CasCAg in C. sapidus. That isoform, which was
termed CAI (Henry et al.,
2003), was expressed in higher amounts than a second isoform,
termed CAII, and so was presumed to be the dominant, cytoplasmic form. At that
time, only partial nucleotide sequences were available for the CA isoforms
from C. maenas. More complete sequence data and relative levels of
expression have indicated that in C. maenas the CAI isoform is most
likely membrane-associated, and CAII is the cytoplasmic form (R.P.H. and L.S.,
unpublished data), and as result of this new information a more thorough
investigation of both C. maenas isoforms is under way.
From the limited data in this and two related studies, it appears that
transport-related proteins have different degrees of induction depending on
their subcellular localization. Specifically, cytoplasmic CA mRNA has a
2–20-fold larger degree of induction than that of any of the
membrane-associated proteins. This may be related to potential diffusion
limitations within the gill itself. The boundary layer on the inside of the
apical surface of the gill acts as a separate fluid compartment, and the large
excess of CA activity, a result of the high degree of CasCAc mRNA induction,
may be necessary to maintain a large enough intracellular gradient of
H+ and HCO3– to drive their diffusion
into the boundary layer, especially when ion transport is stimulated by low
salinity. Cytoplasmic CA activity in gills of crabs acclimated to 35 p.p.t. is
still present in approximately 1000-fold excess over what is needed to meet
the ion transport needs of crabs in low salinity
(Henry, 2001), and yet it is
induced in this subcellular compartment by up to 15-fold. This suggests that
the excess catalytic enhancement of the cytoplasmic CO2 hydration
reaction is needed to prevent intracellular diffusion limitations of
H+ and HCO3–. Conversely,
trans-membrane ion transport takes place within the confines of the boundary
layer where presumably, smaller changes in transport protein expression would
be effective.
As discussed above, for crabs acclimated to 35 p.p.t., the relative levels of CasCAg mRNA are between 500 and 10 000 times higher than that for CasCAc (in G7 and G3, respectively). Despite this large difference in expression, cytoplasmic CA activity is high, being just about equal to that of membrane-associated CA. The reason behind this may be the low turnover rate of the CA protein in the gill. Once synthesized, cytoplasmic CA appears to have an extended biological half-life. For C. sapidus or C. maenas acclimated to low salinity and transferred back to high salinity, high levels of CA protein concentration and activity persist for 3–4 weeks, while mRNA levels decline rapidly (N. Jillette and R.P.H., unpublished data).
One observation that should be noted is that traditionally accepted
`housekeeping' genes (i.e. genes that are expressed constitutively and do not
change in response to the treatment) were not applicable to the posterior gill
of the blue crab. Actin expression has already been reported to change in
response to low salinity (Lovett et al.,
2003). Furthermore, arginine kinase, whose activity and expression
do not change in the posterior gills of C. maenas, increased by
threefold after 7 days of low salinity exposure
(Fig. 8). AK activity has also
been shown to double under similar conditions
(Kotlyar et al., 2000). The
posterior gill of the blue crab is a metabolically active tissue, and the rate
of oxygen uptake and the concentrations of oxidative enzymes both increase as
a result of low salinity exposure (Pillar
et al., 1995), so it is not surprising to find that the expression
of metabolic genes such as AK also increases. At the current time there is no
known gene in the blue crab gill that is completely unresponsive to
environmental salinity. This is not surprising, given the physiological,
biochemical and ultrastructural changes that the posterior gill undergoes in
response to low salinity. Ion-transporting gills in both invertebrates and
vertebrates undergo complete re-modeling in response to salinity changes,
including cellular differentiation and proliferation, membrane and
cytoskeletal re-organization, and up-regulation of a variety of transport and
transport-related proteins (Perry,
1997; Luquet et al., 2002;
Evans et al., 2005). On the
other hand, neither the activity nor the expression of the proteins examined
in this study was significantly affected by salinity in the anterior gills. It
may therefore be more accurate in crustaceans to use the anterior gills as a
control tissue. Anterior and posterior gills are anatomically similar,
homologous tissues that differ only in their functional responses to
environmental salinity. This pattern appears to hold for other euryhaline
species of crustaceans as well. There were no changes in CA activity, or CA or
AK mRNA expression in anterior gills of C. maenas in response to low
salinity (Henry, 2006) and no
change in expression of the V-type H+-ATPase or the
Na+/K+/2Cl– co-transporter in anterior
gills of Pachygrapsus marmoratus
(Spanings-Pierrot and Towle,
2004). In another species, Chasmagnathus granulatus,
expression of both the co-transporter and the
Na+/K+-ATPase changed in posterior and anterior gills,
but the changes in the anterior gills were much smaller, and they were not
consistently correlated with low salinity exposure. But at this time it
appears that using the anterior gill as a control tissue is more reliable than
trying to identify a control gene.
In summary, two isoforms of CA have been identified and sequenced in the blue crab gill that are different in cellular localization, physiological function, expression and regulation. Changes in expression of the cytoplasmic isoform appear to be the basis for low salinity-mediated induction of CA activity, and the cytoplasmic isoform displays the largest inductive scope of any known transport-related protein.
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