AMER. ZOOL., 41:770–780 (2001)
Osmoregulation by Gills of Euryhaline Crabs:
Molecular Analysis of Transporters1
DAVID W. TOWLE2
AND
DIRK WEIHRAUCH
Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672
and Department of Biology, Lake Forest College, Lake Forest, Illinois 60045
SYNOPSIS. The physiological mechanisms by which aquatic animals regulate the
osmoconcentration of their body fluids remain unclear despite many excellent studies of tissue and cell function. This review summarizes the current status of an
ongoing molecular biological approach to investigating transporters and transportrelated enzymes in ion-transporting gills of osmoregulating crustaceans. We have
identified cDNAs coding for six candidate proteins in gills of the blue crab Callinectes sapidus and the green shore crab Carcinus maenas, including a Na1 1 K1ATPase a-subunit, a V-type H1-ATPase B-subunit, a Na1/H1 exchanger, a Na1/
K1/2Cl2 cotransporter, two isoforms of carbonic anhydrase, and arginine kinase.
Although our account is far from complete, examination of mRNA abundance by
quantitative reverse transcription/polymerase chain reaction (RT/PCR) has identified candidates that are preferentially expressed in gill epithelium, including the
Na1 1 K1-ATPase a-subunit and Na1/H1 exchanger. The osmoregulatory response
to salinity reduction includes enhanced mRNA expression of at least one form of
carbonic anhydrase.
INTRODUCTION
Many of the papers in the symposium
Osmoregulation: An Integrated Approach
are focused at the cellular level, asking
questions about mechanisms by which cells
regulate their intracellular osmotic condition. At the organismal level, we can ask
similar questions about regulation of extracellular osmolytes, focusing on tissues that
are specialized for osmolyte uptake, retention, or excretion. This review addresses
such an organismal level of osmoregulation, with an emphasis on applications of
molecular biology to studies of osmolyte
transporters and transport-related enzymes
in gills of euryhaline crustaceans.
Among the brachyuran crabs, some species (e.g., the blue crab Callinectes sapidus)
are capable of osmoregulating and thriving
in freshwater (Mangum and Amende, 1972;
Cameron, 1978). Others (e.g., the shore
crab Carcinus maenas) are less tolerant of
freshwater but nevertheless maintain high
extracellular osmolyte concentrations well
1 From the Symposium Osmoregulation: An Integrated Approach presented at the Annual Meeting of
the Society for Integrative and Comparative Biology,
4–8 January 2000, at Atlanta, Georgia.
2 E-mail: dtowle@mdibl.org
into brackish water (Siebers et al., 1982).
Still others (e.g., the lesser blue crab Callinectes similis) appear less capable of osmoregulation over their entire range (Piller
et al., 1995; Guerin and Stickle, 1997) (Fig.
1). The range of osmoregulatory capacities
expressed in aquatic crustaceans thus affords the possibility of a comparative physiological approach to the identification of
the specific genetic capabilities that contribute to osmoregulatory capacity.
Which organ is most involved in organismal osmoregulation in aquatic crustaceans? The antennal glands of brachyuran
crabs are incapable of producing urine that
is anisosmotic to the hemolymph (Cameron
and Batterton, 1978) and thus cannot be
considered as important organs in osmoregulation. Rather, the loss of osmolytes via
the urine presents a stressful condition in
dilute salinities that must be met by active
ion uptake. The crustacean hepatopancreas
expresses a variety of transporters (Ahearn
et al., 1992), but little attention has been
paid to their possible involvement in osmoregulation by euryhaline species. Although the crustacean intestine is known to
transport ions (Mantel and Farmer, 1983)
and appears to be the major site of water
770
MOLECULAR ANALYSIS
OF
GILL TRANSPORTERS
771
FIG. 1. Osmoregulatory capacity of three brachyuran crab species, represented by blood Na1 concentration as
a function of external Na1 concentration in the blue crab Callinectes sapidus (V) (Mantel and Farmer, 1983),
the lesser blue crab Callinectes similis (n) (Piller et al., 1995), and the green shore crab Carcinus maenas (m)
(Siebers et al., 1982). The straight line represents isoionic conditions.
uptake at molt (Neufeld and Cameron,
1994), its overall contribution to osmoregulatory ion uptake is believed to be minimal
(Chu, 1987).
The majority of experimental evidence
suggests that it is the gills of euryhaline
crustaceans that serve as the primary site of
osmoregulatory ion transport. Although
varying widely in morphology, gills and associated structures of osmoregulators typically contain cell types that are reflective of
their transporting capabilities (Taylor and
Taylor, 1992). Typical characteristics of
these epithelial cells include a greatly elaborated membrane surface, particularly in
the basolateral region facing the body fluid.
Numerous mitochondria fill such cells, providing the ATP and related phosphagens
that power transport processes (Fig. 2). Gill
epithelial cells that are believed to function
principally in gas exchange are much thinner, with limited membrane elaboration and
few mitochondria (Copeland and Fitzjarrell,
1968; Goodman and Cavey, 1990).
Studies of intact animals, isolated gills,
gill lamellae, and membrane vesicles have
produced a variety of models of osmoregulatory ion transport in euryhaline crustaceans (Lucu, 1990; Towle, 1990; Taylor and
Taylor, 1992; Pequeux, 1995; Onken and
Riestenpatt, 1998). Nearly universal, however, is the assumption that the major driving force for ion transport across gills is
provided by the sodium pump or Na1 1
K1-dependent ATPase (Towle, 1990). In
isolated perfused gills of the blue crab, for
example, the Na1 1 K1-ATPase inhibitor
ouabain blocks more than 50% of the Na1
uptake from external medium to the hemolymph side (Burnett and Towle, 1990)
(Fig. 3). Ouabain also blocks ammonia excretion across isolated gills, supporting the
idea that a functional sodium pump is essential for this important process as well
(Lucu et al., 1989; Weihrauch et al., 1998).
A second ATP-utilizing pump, the vacuolar-type H1-ATPase, is considered to play
an important role in ion uptake by a variety
of animal species that successfully tolerate
freshwater (Wieczorek et al., 1999). The Vtype ATPase is a highly conserved multisubunit protein similar to the F-type ATP
synthase of mitochondria and chloroplasts.
Found in eukaryotes, eubacteria, and archaea, the V-type ATPase transports H1
ions against an electrochemical gradient
across vacuolar, vesicular, or plasma membranes (reviewed by Nelson et al., 2000).
772
D. W. TOWLE
AND
D. WEIHRAUCH
FIG. 2. Ultrastructure of two major cells types in gill epithelium of the blue crab Callinectes sapidus (Towle
and Kays, 1986). (A) Ion-transporting-type cell, showing extensive membrane proliferation and numerous mitochondria. (B) Respiratory-type cell, much thinner with less developed membrane surface area compared with
A. a: apical membrane; b: basolateral membrane; bl: basal lamina; c: cuticle; h: hemolymph space; m: mitochondrion; n: nucleus; w: water space. Bar indicates 1 mm.
A plasma membrane form of the V-type
ATPase has been implicated in energizing
NaCl uptake by frog skin, freshwater teleost
gill, and gill of the freshwater-tolerant Chinese crab Eriocheir sinensis (Lin and Randall, 1995; Onken and Putzenlechner, 1995;
Ehrenfeld and Klein, 1997), where generation of a membrane potential may link its
action to current flow through apical Na1
channels or basolateral Cl2 channels. The
significance of the V-type ATPase in brackish-water-tolerant species is less clear.
Although these ATPases likely provide
the electrochemical gradients that drive osmoregulatory ion transport, they must be
accompanied by other transporters to complete the process. The Na1 1 K1-ATPase is
exclusively located in the basolateral membrane of gill epithelial cells (Towle and
Kays, 1986), where it likely pumps three
MOLECULAR ANALYSIS
FIG. 3. Effects of ouabain (Na1 1 K1-ATPase inhibitor) and amiloride (inhibitor of Na1/H1 exchanger and
epithelial Na1 channel) on Na1 uptake across isolated
perfused gills from the blue crab Callinectes sapidus,
expressed as percentage of control perfusions without
ouabain or amiloride. Ouabain was provided at the indicated concentrations in the internal medium (black
bars) or the external medium (white bar). Amiloride
was provided at the indicated concentrations in the external medium (grey bars). Data taken from (Burnett
and Towle, 1990).
Na1 ions outward toward the hemolymph
and two K1 (or NH41) ions inward for each
ATP hydrolyzed (Towle and Hølleland,
1987). Thus the net movement of Na1 mediated by Na1 1 K1-ATPase is from cytosol
to hemolymph, resulting in osmoregulatory
accumulation of Na1 (and Cl2, by other
mechanisms). Other transporters must mediate movement of Na1 ions from the environment across the apical membrane to
the cytosol.
Transporters that may be involved in the
apical uptake of Na1 and other ions from
the environment include several likely candidates (Fig. 4). One of these is the Na1/H1
exchanger, a transmembrane protein found
in many animal cells, where it moves Na1
across the plasma membrane into the cytosol in exchange for H1, depending on a
chemical gradient of one of the ions (reviewed by Wakabayashi et al., 1997). Six
isoforms of the Na1/H1 exchanger have
been described in mammalian species,
varying in their tissue and cellular locations
and responses to regulatory factors. In gills
of teleosts, Na1/H1 exchangers appear to be
similar to those of other vertebrates, namely
exchanging one Na1 for one H1 (Claiborne
et al., 1999). In crustacean gills, however,
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GILL TRANSPORTERS
773
FIG. 4. Hypothetical model of candidate ion transporters in gills of osmoregulating crabs, based on studies of whole organisms, isolated gills, split gill lamellae, and membrane preparations. ATP levels may be
maintained through the action of arginine kinase (not
shown), providing ATP for the Na1 1 K1-ATPase and
the V-type H1-ATPase. The latter ATPase may be
linked to basolateral Cl2 channels or apical Na1 channels (not shown). Apical transporters may include a
Na1/H1 exchanger, a Cl2/HCO32 exchanger, or a Na1/
K1/2Cl2 contransporter. Counterions for the Na1/H1
exchanger and Cl2/HCO32 exchanger may be provided
through the activity of intracellular carbonic anhydrase.
a Na1/H1 exchanger has been identified that
is electrogenic, apparently exchanging two
Na1 for one H1 (Shetlar and Towle, 1989).
A similar electrogenic exchanger has been
described in lobster and prawn hepatopancreas (Ahearn and Clay, 1989; Ahearn et
al., 1990). Amiloride, an inhibitor of the
Na1/H1 exchanger and epithelial Na1 channels, is known to reduce Na1 uptake across
isolated crab gills (Burnett and Towle,
1990) (Fig. 3). Thus the Na1/H1 exchanger
(or epithelial Na1 channel) represents one
candidate possibly participating in the
transfer of Na1 ions across the apical membrane. Because no evidence exists for epithelial Na1 channels in crustacean gills, we
have focused our attention on the exchanger.
A second candidate for an apical medi-
774
D. W. TOWLE
AND
ator of Na1 uptake is the Na1/K1/2Cl2 cotransporter, a membrane protein that has
been identified in basolateral membranes of
salt-excreting epithelia and in apical membranes of salt-absorbing epithelia (Haas,
1994; Russell, 2000). This electroneutral
transporter moves Na1, K1, and 2Cl2 ions
from extracellular fluid to the cytosol, depending usually on an electrochemical gradient for Na1. Experiments with gills isolated from the shore crab Carcinus maenas
have indicated that a portion of Na1 uptake
from the medium may be coupled to Cl2
uptake, although little effect of the Na1/K1/
2Cl 2 cotransporter inhibitor furosemide
could be demonstrated, possibly due to inhibitor interference by the cuticle (Riestenpatt et al., 1996). Although it is unlikely
that the electrochemical Na1 gradient would
be sufficient to drive ion uptake from fresh
water via the cotransporter, the cotransporter may play a role in brackish environments
inhabited by species like the shore crab.
Two intracellular enzymes that may support active ion transport across crustacean
gills are carbonic anhydrase and arginine
kinase. Carbonic anhydrase is believed to
provide the counterions H1 and HCO32 for
Na1 and Cl2 exchange by catalyzing the hydration of CO2 within gill cells (reviewed
by Henry, 1996). The activity of the cytoplasmic form of carbonic anhydrase increases dramatically in gills of euryhaline
crabs upon transfer from high to low salinities (Henry, 1988), apparently enhancing
the supply of counterions for NaCl uptake.
In the tissues of many invertebrates, arginine kinase catalyzes the hydrolysis of
phosphoarginine, phosphorylating adenosine diphosphate to form ATP. ATP-utilizing processes such as active ion transport
may depend on the ATP buffering function
of arginine kinase or on its mitochondrionto-cytosol shuttle function (Ellington and
Hines, 1991). Following transfer of crabs
from high to low salinities, the enzymatic
activity of cytosolic arginine kinase approximately doubles in gills of the strong osmoregulator Callinectes sapidus, but does
not change in the more limited osmoregulator Carcinus maenas (Kotlyar et al.,
2000).
D. WEIHRAUCH
MOLECULAR ANALYSIS
OF
TRANSPORTERS
Our approach to characterizing candidate
transporters and transport-related enzymes
depends on the application of the techniques of molecular biology. To qualify for
inclusion in a working model of osmoregulatory ion transport, the following questions should be answered:
(1) Is the candidate cDNA identifiable, in
our case by reverse transcription and polymerase chain reaction (RT/PCR)? Although
this question may seem simplistic in relation to model building, a clear absence of a
particular transporter cDNA would lead to
eliminating that transporter from the model.
(2) Is the candidate mRNA expressed
preferentially in ion-transporting gills?
Comparisons with tissues less specialized
for osmoregulatory ion transport may identify transporters that are highly expressed in
gills.
(3) Is candidate mRNA and/or protein
expression sensitive to osmoregulatory
challenge? Although transporters may be
regulated at a variety of levels, including
cell signaling cascades altering phosphorylation levels of the transport protein, an osmoregulatory response at the transcriptional
or translational level could help to identify
components of a model.
To address the first question, we have focused initially on transporters and transportrelated enzymes for which nucleotide or
amino acid sequence information exists
from other species and tissues. Conserved
regions of these sequences are identified
and used as the basis for designing degenerate oligonucleotide primers for PCR in attempts to amplify the transporter of interest
from cDNA prepared by reverse transcription of gill mRNA. If a related transporter
is expressed in the tissue being examined,
then a distinct product of the polymerase
chain reaction should be amplified and can
then be sequenced directly without subcloning. Analysis of the sequence via open
reading frame detection and an alignment
search of GenBank (Altschul et al., 1997)
may identify the transporter of interest. If
such experiments fail to reveal the target
MOLECULAR ANALYSIS
transporter, either the primers are not effective or the target template does not exist in
the tissue being examined. If the target is
identified, then we can proceed with further
analysis.
Answers to the second and third questions require an analytical tool that measures the relative level of transporter
mRNA in the tissue. Northern blots or
RNAse protection assays can be used in
these applications but suffer from lack of
sensitivity. We have chosen to use quantitative RT-PCR for these purposes, demonstrating that the method can detect less than
a two-fold difference in the level of a specific mRNA between two tissue samples
(Towle et al., 1997; Kotlyar et al., 2000).
In this method, the polymerase chain reaction is performed using gene-specific primers and a concentration of template that limits the amount of product formed. To ensure
that template is indeed limiting, PCR is carried out for a limited number of amplifications (18–22), requiring product detection
that is considerably more sensitive than the
conventional ethidium bromide staining.
We have found that incorporating biotinylated dUTP into the product (replacing
some of the dTTP) provides a tag that can
be easily detected (Towle et al., 1997).
One major hypothesis rests on the prediction that levels of mRNA coding for osmoregulatory transporters may be higher in
the tissues that are specialized for such
transport compared to those that are specialized for other functions. Moreover, we
predict that osmoregulatory stress may lead
to an increase in the transcription rate of the
relevant genes, most likely leading to enhanced mRNA levels. Quantitative RT-PCR
by itself cannot measure transcription rates
or mRNA stability but rather provides a
snapshot of mRNA availability for translation. It is clear that post-transcriptional regulatory mechanisms, including translational
and cell-signaling controls, may be essential components of the osmoregulatory response, as summarized elsewhere in this
symposium. The focus here, however, lies
in the initial identification of candidate
transporters using primarily molecular approaches.
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CANDIDATE TRANSPORTERS AND TRANSPORTRELATED ENZYMES
Na1 1 K1-ATPase
Originally described enzymatically in homogenates of nerves from the shore crab
Carcinus maenas (Skou, 1957), the Na1 1
K1-ATPase is known to increase in activity
in gills of aquatic animals when they are
osmotically stressed. For example, transferring blue crabs (Callinectes sapidus) from
35% salinity to 5% leads to a doubling of
Na1 1 K1-ATPase activity in the ion-transporting posterior gills (Towle et al., 1976).
Similar results are seen in the shore crab
Carcinus maenas (Siebers et al., 1982) and
many other species. These results imply
that the energetic costs of osmoregulation
in stressful conditions are met, at least in
part, by enhanced activity of the Na1 1 K1ATPase.
It has not been clear whether the salinityrelated changes in Na1 1 K1-ATPase activity result from enzyme activation or from
enzyme (and perhaps mRNA) synthesis.
Western blot analysis of Na1 1 K1-ATPase
a-subunit protein in plasma membranes
prepared from Carcinus maenas gills indicated that crabs acclimated for 3 wk to low
salinity showed twice the amount of a-subunit protein compared with crabs acclimated to high salinity or to low salinity for just
4 hr (Lucu and Flik, 1999).
Recent molecular data from our laboratory have revealed a higher abundance of
Na1 1 K1-ATPase a-subunit mRNA as
well as protein in posterior gills of Callinectes sapidus compared to anterior gills.
However, we observed little effect of salinity change on either a-subunit mRNA or asubunit protein (Paulsen et al., 2000; Towle
et al., 2001). Thus our evidence suggests
that tissue differences in Na1 1 K1-ATPase
activity in gills of the blue crab are the result of gene regulation, leading to enhanced
mRNA and protein levels. Salinity-related
changes in Na1 1 K1-ATPase activity, perhaps exerted by hormone-sensitive translational or cell signaling processes, may be
superimposed upon the likely differences in
gene transcription. In conclusion, molecular
properties of the Na1 1 K1-ATPase fulfill
two of our criteria for inclusion in a trans-
776
D. W. TOWLE
AND
D. WEIHRAUCH
FIG. 5. Quantification of mRNA coding for arginine kinase (AK) and V-ATPase B-subunit in anterior (AG)
and posterior (PG) gills of the shore crab Carcinus maenas acclimated to 35 or 10‰ salinity. Total RNA (2 mg)
was treated with RNAse-free DNAse and reverse transcribed to cDNA using oligo-dT as primer. Five percent
of the resulting product was used as template for polymerase chain reaction in the presence of biotinylateddUTP. Biotin-containing amplification products were separated by gel electrophoresis, blotted, and visualized
with streptavidin-conjugated alkaline phosphatase. The number of amplification cycles is indicated. M 5 biotinylated DNA marker. Exerpted from Figure 7 in Weihrauch et al. (2000).
port model, suggesting a central role of the
Na1 1 K1-ATPase in osmoregulatory ion
transport.
Vacuolar-type H1-ATPase
cDNAs coding for the B subunit of the
V-type ATPase have been identified and at
least partially sequenced in gills of five
brachyuran crab species (Weihrauch et al.,
2000). A detailed study of the expression of
B subunit mRNA in gills of Carcinus
maenas suggests that the V-type H 1 ATPase is not expressed preferentially in
posterior ion-transporting gills, nor is its expression enhanced by osmoregulatory challenge (Fig. 5) (Weihrauch et al., 2000). The
V-type H1-ATPase thus satisfies the first
criterion for inclusion as a candidate transporter in C. maenas, but not the second and
FIG. 6. Quantification of mRNA coding for Na1/H1
exchanger and actin in tissues of the shore crab Carcinus maenas, using the method described in the legend for Figure 5 with 24 cycles for actin and 26 cycles
for exchanger. The right-most lane represents a control
for genomic DNA contamination, omitting reverse
transcriptase (Towle et al., 1997).
third. On the other hand, in the freshwatertolerant Chinese crab E. sinensis, V-type
H1-ATPase B subunit mRNA is preferentially expressed in ion-transporting posterior gills of osmotically challenged animals
(Weihrauch et al., 2000), thus meeting all
three of the proposed criteria for inclusion
in transport models. The shore crab C.
maenas may not survive in freshwater because it is incapable of activating membrane-specific V-type ATPase gene transcription.
Na1/H1 Exchanger
A Na1/H1 exchanger cDNA has been
amplified and sequenced from gills of two
crab species, Carcinus maenas and Callinectes sapidus (Newton et al., 1996; Towle
et al., 1997). Closely related to the vertebrate isoforms previously sequenced, it is
not known whether the cloned exchanger is
identical with the electrogenic exchanger
described in membrane vesicles from crustacean epithelia (Shetlar and Towle, 1989).
Nevertheless, the cloned exchanger is expressed very strongly at the mRNA level in
gills, showing much lower abundance in all
other tissues examined (Fig. 6). Thus the
Na1/H1 exchanger meets the first two criteria for retention as a candidate transporter
involved in osmoregulation. Whether exchanger mRNA levels respond to osmoregulatory challenge is the subject of current
work.
Na1/K1/2Cl2 Cotransporter
A putative Na1/K1/2Cl2 cotransporter
has been identified and sequenced in gills
of blue crab Callinectes sapidus (GenBank
MOLECULAR ANALYSIS
OF
GILL TRANSPORTERS
777
FIG. 7. Ethidium bromide gel of PCR-amplified cDNAs for Na1/K1/2Cl2 contransporter and actin in tissues of
the blue crab Callinectes sapidus after 30 cycles of amplification. Degenerate primers for arthropod actin or
specific primers for Callinectes cotransporter were employed. The negative control omitted reverse transcriptase,
checking for contamination with genomic DNA (Towle and Burnett, 2001).
Accession Number AF190129) (Towle,
1998). Gills of the shore crab C. maenas
also express the Na1/K1/2Cl2 cotransporter
(Weihrauch and Towle, unpublished), contrary to earlier reports from our lab. Preliminary evidence suggests that the cotransporter is expressed preferentially in posterior gills of C. sapidus (Towle, 1998) (Fig.
7). Current work is examining whether gills
of C. maenas express the cotransporter
strongly and also whether osmoregulatory
challenge enhances mRNA and protein expression.
anterior gills. In addition, the levels of carbonic anhydrase mRNA increase dramatically when crabs are transferred from high
to low salinity (Fig. 8). Our evidence suggests therefore that carbonic anhydrase satisfies all three criteria for inclusion as a
transport-related enzyme critical for osmoregulation by crab gill, likely enhancing the
availability of counterions for transporter
proteins.
Carbonic anhydrase
We have successfully amplified two different isoforms of carbonic anhydrase
cDNA from gills of Callinectes sapidus and
Carcinus maenas (Gehnrich et al., 2000a,
b). Messenger RNA coding for one of the
isoforms is much more abundant than the
mRNA coding for the second isoform. Both
are more abundant in posterior gills than in
In the course of molecular work on transporters, we serendipitously amplified a
cDNA coding for arginine kinase, producing a complete cDNA sequence for Carcinus maenas and a complete open reading
frame for Callinectes sapidus (Kotlyar et
al., 2000). Although the enzymatic activity
of arginine kinase is enhanced following
transfer of C. sapidus from high to low salinity, mRNA abundance is not affected by
this osmoregulatory challenge. In the weaker osmoregulator C. maenas, both enzyme
activity and mRNA abundance were not responsive to salinity reduction (Fig. 5). We
can conclude that arginine kinase expression is not tightly coupled to osmoregulatory processes in these two species. The enzyme may, however, mediate buffering of
ATP levels that are generally required for
active ion transport and other processes.
FIG. 8. Quantification of carbonic anhydrase mRNA
and arginine kinase mRNA in posterior gills of the
shore crab Carcinus maenas acclimated to 35 or 10‰
salinity, using the method described in the legend for
Figure 5 (Gehnrich et al., 2000b).
Arginine kinase
778
D. W. TOWLE
AND
CONCLUSION
Four of the six candidate transporters and
transport-related enzymes so far analyzed
meet two of the criteria for inclusion in a
working model of NaCl uptake across gills
of hyperosmoregulating crabs. These are
the Na1 1 K1-ATPase a-subunit, the Na1/
H1 exchanger, the Na1/K1/2Cl2 contransporter, and at least one form of carbonic
anhydrase. Our preliminary data indicate
that the transcription of carbonic anhydrase
is enhanced following salinity reduction.
The V-type H1-ATPase B-subunit seems
not to be expressed preferentially in iontransporting gills of the brackish-water-tolerant Carcinus maenas, but it may be important in freshwater adaptation by the Chinese crab Eriocheir sinensis.
Much remains to be investigated regarding candidate transporters and transport-related enzymes in gills of osmoregulating
crustaceans, particularly with regard to localizing the mRNA and proteins within the
epithelial cells. The existence of multiple
splicing products and protein isoforms must
be investigated more completely. Molecular
cloning and complete sequencing of the
cDNAs coding for candidate transporters
will allow the in vitro expression of each
protein, which can then be used to generate
antibodies for immunocytochemistry, permitting further exploration of osmoregulatory ion transport mechanisms.
ACKNOWLEDGMENTS
This review is dedicated to the memory
of Charlotte P. Mangum, mentor, collaborator, and friend. Her early encouragement
and constructive criticism formed the basis
of the work summarized here. The author’s
research is supported by the National Science Foundation (IBN-9807539) and the
Foster G. McGaw Foundation. The symposium was supported by the National Science Foundation (IBN-9904521) and by the
Divisions of Comparative Physiology and
Biochemistry, Invertebrate Zoology, and
Comparative Endocrinology of the Society
for Integrative and Comparative Biology.
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